‘vpl, . -I:l 1-... .. ‘ Lle‘n ziw‘vlicralil‘ ‘II. n quéit-fikl’ iti533‘ . ,— ¢ ilk L .. .1..!tu|ll;llin.llluIIII|IP-.t . :I . ABSTRACT THE MICROFLORA OF BARLEY KERNELS; THEIR ISOLATION, CHARACTERIZATION, ETIOLOGY, AND EFFECTS ON BARLEY, MALT, AND MALT PRODUCTS by Evan Harold Pepper The various methods used in the isolation of the microflora of barley were evaluated. Eighteen different nutrient media, eleven surface-disinfectants, and six disinfection-time periods were compared. The dilution— plate method was also evaluated by using several kernel- aggregate sizes in the preparation of the dilution mate erial. A differential series of nutrient media is des- cribed for the isolation and/or estimation of microfloral populations on barley kernels. The series employs Cyclo- heximide for the suppression of fungal growth and Chloram- phenicol for the inhibition of bacterial growth. Barley kernels of several varieties, grown in var- ious locations, were plated and their microfloral popula— tions were assessed. Eighteen bacterial species have been isolated and identified from barley foliage and/or kernels. Eight of these species have not been previously reported from barley kernels. Two previously unreported fungal spe- cies have also been isolated from barley kernels. The location of the kernel microflora, as well as the form in ‘mnch they exist, were also studied. Field studies on the etiology of barley kernel ‘1' u‘ -.1 v.» :v' 2" infection were carried out, using plating and histol- ogical procedures, and exposed slides and culture plates. While varietal differences in resistance were noted, the patterns of infection were similar. Bacterial infection occurred earlier than fungal infection; with the bacterial populations declining as the fungal populations increased above fifty percent. The organisms isolated from exposed slides and culture plates were similar to those infecting barley kernels. Significant increases in infection were made as the harvest period was prolonged. Single applications of five chemical compounds failed to reduce microfloral infec- tion. Barley kernels, infected by both fungi and bacteria, usually become discolored. The common field fungi cause more severe discoloration than do the kernel-infecting bacteria. Under certain conditions, barley kernels may support large populations of bacteria without being dis- colored. Several species of bacteria and fungi have been shown to be effective germination-inhibitors; while other common kernel microflora have no effect on barley germination. The culture filtrates of three fungal spe- cies were inhibitory to barley germination. The inhibi- tory principle of two culture filtrates was heat stable. Evidence is presented for the possible storage deteri- oration of barley by several bacterial species under laboratory conditions. Barley, infected with a large .., y.- . .u ‘I. . ..‘ bacterial population, proved to be unpalatable to swine when offered as feed. Three varieties of bright barley were inoculated with several isolates of bacteria, fungi, and yeasts. These barleys were pilot malted in a pilot scale appam ratus constructed for this purpose. Several malt defects were obtained with inoculated grain although the only consistently defective malts were obtained with isolates of known pathogenicity. The progress of microfloral population changes during malting was also studied. Sevm eral fungal and bacterial species were found capable of surviving the kilning process. Circumstantial evidence was presented for the consistent infection of green malt by bacteria carried in the humidified air of germinating equipment. Disinfected kernels of barley have been shown to exert an inhibitory effect on a species of Bagillgg. This effect varied with the barley variety. Chromatograms of bright and stained barley husk extracts indicated that the discoloration principle is a melanin-like pigment. This brown pigment found in barley husks is waterwand alcohol- soluble. Other evidence is presented to show that these pigments are produced as kernel-microorganism interaction products. The steeping of disinfected barley kernels in an aqueous solution of L-tyrosine showed promise as a possible measure of microfloral content. Brown pigments produced in this solution varied in intensity with the barley variety as well as with the microfloral popula» tion of the barley lot. Hypotheses are presented which assert that the melaninalike pigments are produced by the kernel as an inefficient resistance mechanism. This pigment production is enhanced by the presence of kernel microflora. It is also suggested that these polyphenolic materials may be of importance in the produ:tion cf nonwbiom logical beer hazes. A further hypothesis states that the gas-stability of beer may be reduced by the use of malt prepared from barley which is severely infected by micron flora. This speculation is based on information concerning the physiological potential of the kernel microflora. THE MICROFLORA OF HARLEY KERNELS; THEIR ISOLATION, CHARACTERIZATION, ETIOLOGY, AND EFFECTS ON HARLEY, MALT, AND MALT PRODUCTS By Evan Harold Pepper A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 1960 .s ’c. ///.2.2 fé/ ACKNOWLEDGEMENTS No enterprise, of even the most meager value, is carried to completion by any one person. So it is with this thesis. I therefore wish to sincerely thank every- one who has assisted me in this investigation. To Dr° R. L. Kiesling, my major professor, who gave unceasingly of his time and energies, to the members of my guidance com— mittee: Dr. G. P. Steinbauer, Dr. C. Ro Olien, Dr. J. Lockwood, Dr. L. W. Mericle, and Dr. E. S. Beneke for their appraisal of this effort, and to the Michigan Agri- cultural Experiment Station for the research assistant- ship, I extend my sincere appreciation. This program has been supported in part by research monies granted by the Malting Barley Improvement Associa- tion. This financial aid for the years 1958, 1959, and 1960 is gratefully acknowledged. A number of friends in the malting and brewing industry have contributed their time, counsel, and technical assistance. Specifically I wish to thank R. E. Spahn and R. Bonner of the H. W. Rickel Malting Company, F. Roberts of the Stroh Brewery Company, A. J. Lejeune of the Malting Barley Improvement Associa- tion, and R. Lacey and J. H. Ladish of the Ladish Malting Company for their kind assistance. The technical assistance of J. L. Clayton, Grace A. Hill, and E. Akinimrisi is appreciatively acknowledged. ii The understanding and benefices of my mother, Mrs. Evan H. Pepper, Sr., and my wife's parents, Mr. and Mrs. Anton Poznanski, were a major source of encouragement to me. Their help and understanding is acknowledged with a warm feeling of pride and gratitude. To Marie, my wife, this thesis is dedicated with greatest affection. iii TABLE OF CONTENTS CHAPTER Page INTRODUCTION ................................ 1 I THE ISOLATION OF MICROFLORA ................. 5 Literature review ......................... 5 Materials and methods ..................... 15 Experimental results ...................... 22 Discussion and conclusions ................ 30 II THE CHARACTERIZATION OF MICROFLORA .......... 42 Literature review ......................... 42 Materials and methods ..................... 55 Experimental results ...................... 57 Discussion and conclusions ................ 62 III THE ETIOLOGY OF HARLEY KERNEL INFECTION ..... 68 Literature review ....... ..... ............. 68 Materials and methods ..................... 81 Experimental results ...................... 85 Discussion and conclusions ................ 105 IV THE EFFECTS OF MICROFLORA ON HARLEY KERNELS.. 111 Literature review ......................... 111 Materials and methods ..................... 125 Experimental results ...................... 129 Discussion and conclusions ................ 156 V THE EFFECTS OF MICROFLORA ON MALT AND MALT PRODUCTS .00....00°0......OOOOOOOOOCOOOOQOOOO140 Literature review ......................... 140 Materials and methods ..................... 150 Experimental results ...................... 167 Discussion and conclusions ................ 189 VI OTHER STUDIES 00.00..OOOOOOOOOOOOOOOOOOOOOOOO1% Literature review ......................... 194 Materials and methods ..................... 206 Experimental results ...................... 211 Discussion and conclusions ................ 227 iv TABLE OF CONTENTS — Continued CHAPTER VII GENERAL DISCUSSION AND SUMMARY ........... LITERATURE CITED .. ................. .............. APPELVDICES O O O O O 0000000000000 O O o 0 000000000000 O O O O O A. Some methods commonly used in the deter- mination of barley microflora ....... B. A list of microorganisms reported as oc— curring on and within barley kernels. C. The orgin, variety, color, germination, and microflora of barley samples fur- nished by the Malting Barley Improve— ment Association and representing barley lots being malted and brewed cooperatively by industry ........... Page 257 250 285 285 289 509 TABLE 10 11 12 LIST OF TABLES Page Culture media employed in the study of in— fluences on barley microflora exerted by the type of plating—media employed........ 17 Surfacewdisinfecting treatments employed in the study of the influences exerted by surface disinfection method on barley mim croflora.... . ...... ....... ....... ........ 19 The influence of culture medium on micro- flora isolated from barley kernels........ 25 The effect of various surfaceedisinfectants on the isolation of microflora and on bar- ley germination. .... ............. ........ 25 The effect of varying disinfection times with two surfaceudisinfection treatments on the isolation of microflora and bar- ley germination... ....... ................. 27 Percentages of microflora isolated from whole kernels and from “pearls“ of the same barley sample ............ ............ 52 The isolation of microflora from barley kernels of differing variety and orgin.... 58 Morphological and physiological character- istics of bacteria isolated from the air, barley foliage, and barley kernels........ 61 Bacterial species isolated and identified from barley kernels and barley leaves..... 65 The occurrence of microflora in the air about the grain plots at East Lansing as determined by exposed petri plates........ 87 The effect of variety, chemical treatment, and harvest time on several properties of barley 0.000.0.00000000000000000000COOOOOO 105 The percentage of microfloral infection of four barley varieties harvested at two different times 0.0.0000 00000 000.00.00.00. 104 vi LIST OF TABLES w Continued TABLE 15 l4 15 16 17 18 19 2O 22 25 Page Kernel discoloration and germination in- hibition of surfacewdiSinfected barley kernels plated on nutrient agar plates inoculated with five bacterial isolates... 150 The germination of barley kernels steeped in culture filtrates of selected fungal SpeCieSOOOQOOOOOOOUOOOOOOOOOGOOOOIOOOOOOOO 152 The germination of inoculated barley ker- nels after storage in sealed bottles at room tempel‘atureSOO O. 000000 O 00 000.000.. 0.0 1.55 Temperature and humidity readings of ger- mination chamber as a function of spray- water temperature and refrigeration unit temperatures ............................. 156 The microfloral contents of the three bar- ley varieties used in the pilot malting studies as determined by whole~kerne1 platings on agar plates................... 161 The source and identity of organisms used to inoculate barley processed in the pilot malting apparatus ........................ 162 Physical characteristics and chemical analysis of Montcalm pilot malts ......... 168 Physical characteristics and chemical analysis of Hannchen pilot malts ......... 169 Physical characteristics and chemical analysis of Traill pilot malts ........... 170 A summarization of the effects of six inoculated organisms on the quality of pilot malts prepared from Montcalm barley ........ ..... ...................... 172 A summarization of the effects of six inoculated organisms on the quality of pilot malts prepared from Hannchen barley ................................... 175 vii LIST OF TABLES - Continued TABLE 24 25 26 27 28 29 30 51 Page A summarization of the effects of eight inoculated organisms on the quality of pilot malts prepared from Traill barley .................................. 175 The amount of original inoculum, initial infection level, increase of original inoculum, and the increase of alien microflora during the pilot malting of three barley varieties .................. 177 Bacterial colony counts obtained from dilution plates of steep liquor taken at intervals during the steeping of Hannchen and Montcalm barleys ........... 182 Analyses of two pilot malts prepared from a bright Montcalm and a severely weathered Montcalm barley lot ........... 184 Biological and physical analyses of bar- leys used in the 'rate of steeping' experiments 000.000.000.000...0.00.060... 185 The inhibition of a Bacillus cereus var- iant by surface—disinfected barley ker- nels plated on seeded agar, and the sup- pression of barley germination by these bacteria ................................ 214 Association of the production of melanin pigments by surface-disinfected barley kernels steeped in a solution of L-tyro- sine for six days and the microfloral populations of these kernels as deter— mined by standard kernel-plating tech- niques .................................. 217 The effects of barley kernels, barley grist, culture filtrates, and inoculated barley on the production of "melanin" pigments in culture media, with and with- out the addition of L-tyrosine .......... 222 viii I‘N ”ft ‘1'. I \.~l-A—. ‘ 33‘ 'Waszes » r; V,:‘ ‘34‘ +‘l‘eJ‘rx .. .LJ ~.v _' .J- .3. \J . 0'" If}? C- a ’3. €- u ‘1'.“ -- myioi -- m——.—.‘ . WHQ learn VA. .IJ. \/ x; .‘_ I. \.I K.” ano yea Luintiiuied. Clhfl - r Sinfe te .r -t. the br I . ._ Page nitrogen compounds 7 pigments . u '- + "r (‘ ‘1 ’ V) ‘, dc du'. u L J. 9 L11 ‘ uh? Traill kernels, o. ”“rc‘ if nocuw CW g (I C ‘3 5 S . . ,- Q . ._, u .. o H. J .. O u lor and re mtion ' and without fie 0 0 O Q l‘ D) [U OW 006000000 .1 J F CHER. [: \J ‘ LIST OF FIGURES The effect of culture media and size of kernel plated on microfloral population. a (whole, half, and quarter kernels).... b (1/52” and l/'#" aggregate size) 0.... The location of microflora as determined by plating transverse sections of bar— ley kernels on culture medium ........ Infection by microflora at progressive stages of kernel development. a (Kindred barley) ....,.............. b (Traill barley) .....o.............. c (Montcalm barley) .................. d (Hannchen barley) ...a.o..o......... Steeping rate of barley. a (Bright Kindred barley) o........... b (Bright Hannchen barley) ..o..e..... o {Montcalm barley) ..o.,...o.o....... A proposed mechanism for melanin formation Page 28 29 51 9O 91 92 95 186 187 188 by microflora on barley kernels ......... 251 g PLATE II III IV VI VII VIII IX XI XII LIST OF PLATES Page The use of differential media in the plating kernels ...... ..................... 56 Fungi in the glume tissues of barley kernels, 94 Spores and mycelial fragments on the surface of barley kernels ........................0 95 Spores and mycelial fragments on the surface . OfbarleYKerneJ-S .00.. ..... 00.000.00.00... 9b Characteristic "dormant" fungal mycelium in glume and pericarp tissues ................ 98 Characteristic "dormant" fungal mycelium in glume and pericarp tissues ................ 99 Fungal mycelium in glume tissues of mature, weathered Hannchen barley (aniline—blue - lactic acid method) ....................... lOO Fungal mycelium in glume tissues of mature, weathered Hannchen barley (aniline—blue - lactic acid method ....... ........ ......... 101 Bacteria in the glume tissues of barley kernels OOOOOOOOOOOOOOOOOOO ...... 0.000.000.102 Pilot malting equipment used in the study of the effects of microflora on the quality of malt. (Steeping and germinating equipment) ...... 152 Pilot malting equipment used in the study of the effects of microflora on the uality of malt. Germinating and kilning equipment) ....... 157 The apparatus used in the inoculation of bright barleys with bacteria, yeasts, and fungi for use in the pilot malting studies ................................... 165 1L Th m‘ J..- ’ + O .4 . :t AFES m COnolndEO :finalm barley, inoculated with three spew cies of bacteria and an uninoculated check sample, plated after two and chew half days in the pilot malting germm 1.9-}- "LLK'J-(OI! ooooooooeooooooooooooooooeooooooooo e rljbi ion of bacteria by barley kerw 11613 and the inhibition of barley germinm dIALWJDD .by 128.01.91’13 ooooocoaoooacooooooooooo ea inhibition of bacteria by barley ker» ne1s and the inhibition of barley germin- ation bsr baCteI'ia oooooooooooooooooooooooo Page 180 215 INTRODUCTION Barley has traditionally been the raw material of choice in the preparation of malto Suitable malting varieties, grown under favorable environmental and cliw matic conditions and meeting rigid quality standards, command a substantial premium if accepted by the maltstero In addition to meeting the official grade standards for barley (6), acceptable malting barley must possess cerw tain other quality characteristicso These include: (1) strong and powerful germination, (2) a moisture con» tent below 14 percent, (5) plump and mellow kernels, (4) absence of skinned and broken kernels, (5) a clean bright color (166)° Malting is a carefully controlled process whereby the barley is: (1) cleaned and sized, (2) steeped in water until the moisture content reaches 45 to 47 percent, (5) germinated under exacting temperature and humidity conditions, (4) dried on kilns to stop germination, pre= serve enzymatic activity, and to reduce moisture content to four percent or less, (5) cleaned and stored (526), The major malting barley producing areas in the United States are the North Central states, the Pacific Coast states, and certain irrigated lands west of the Rocky Mountains (255)o Barleys grown in the North Central 1 2 states are subject to kernel staining when moderate to heavy rainfall occurs during the final stages of ripening and harvesto This staining condition is frequently termed "weathering" and may be caused by a variety of factors incluw ding: (1) moisture, (2) genetic composition of the barley variety, (5) microflora and microfaunao Barleys grown in the arid regions of the west, under irrigation, are usually free of kernel stain and are bright in appearance, The "bright" western barleys while possessing desirable color may, however, be subject to other undesirable features such as steeliness of the kernel and high percentages of skinned and broken kernels, The annual consumption of barley in the production of malt for food and beverage purposes is in excess of 100,000,000 bushels (255)° Even if "brightness” alone were the only quality criterion employed in the marketing of malting barley, the western areas could never produce an adequate supply of the grain (256)o Hence, the barley» buyer and the maltster must rely on the barley supplied by the North Central states, and in an unseasonably moist year the barley may be heavily stained° As a direct result of the climatic conditions generally existing in the Great Lakes region and the concommitant "weathering" of grain crops, barley culture has been pushed westward to the drier areas found in the Red River valleyo This westward shift in barley culture has been largely due to the reluctance of growers to plant malting barley which might prove 5 unsalable because of kernel staining (256, 557, 577). While several factors play a part in the staining of grain kernels, microflora, and especially fungi, are most influential in the weathering process. If "weathered" barley truly represents a decrease in malting quality, cri- teria other than aesthetic appeal must be formulated in order to properly and scientifically evaluate a specific lot of barley. A search for such criteria must, at least in part, begin with an investigation of the weathering pro- cess; how, when, and why it occurs. With this assumption as a basis for the overall investigation of kernel staining and its effect on malting quality, it is possible to sub- divide the problem into a number of other related investi- gations. Most previous studies of barley microflora have overlooked, or have deliberately excluded, the bacteria and yeasts commonly found associated with the barley ker- nel. Consequently the isolation and identification of these organisms, as well as the filamentous fungi, has been undertaken in this study. In order to both qualita- tively and quantitatively distinguish between these various microorganisms, it was necessary to carry out extensive methodological studies on the determination of seed-borne microflora. The techniques developed in this phase of the program were additionally employed in the study of the weathering process. The time and mode of infection by staining organisms was studied, using histological tech- niques. Barley inoculated with pure cultures of bacteria 4 and fungi were pilot—malted and analyzed in order to deter- mine what effect these organisms might have on the quality of malt. Finally, the physiology of staining and its effect on both malt and malt products were examined with respect to both the barley kernel and the staining micro- flora. CHAPTER I THE ISOLATION OF MICROFLORA "In some sorts of seeds the goodness cannot be known by the eye". . . Jethro Tull LITERATURE REVIEW "Seed—borne disease” is in actuality a misnomer. As Groves and Skolko have pointed out, the disease is not seedu borne; the bacteria and fungi which cause the disease are (150). Seed~microflora may exist in a number of forms with respect to the seed. Dorogin has listed five forms of mi» crofloral contamination: (l) the admixture of sclerotia or spore masses with the seed, (2) the mummification of the seed by fungal stromata or by internal spores, (3) the presence of mycelium in definite parts or organs within the seed, (4) fructifications‘:and mycelial fragments and bacterial cells] on the surface of seeds, (5) spores[:on the surface of and within seeds:](105). The seedwborne viruses must be appended to such a classification. Chen has also categorized the manner in which seedaborne or- ganisms exist (64). The way in which seed—flora occur determines which method will be employed for the isola- tion and/or the identification of the organism. Some gen- eral methods of seed analysis, based on the form in which the organisms occur, have been listed by Neergaard (284), Doyer (106), and Dorogin (105). These methods fall into 5 ..~ f4§ the following tread catacarées: 1. Visual methods a. macro b. micro 2. Washing methods and the use cf tne centrifuge 5. Assorted histological and staining techniques 4. Soil and blotter tests and the use of moiS' chambers 5. Various disinfection and plating methods Barley grains are examined visually for several qual» ity characteristics which may be affected by the presence of microflora. The intensity and location of the hull col— or is frequently indicative of the occurence of certain oru ganisms. Smudged kernels may indicate the presence of field organisms. Blackmpoiuted kernels may harbor sev- eral bacterial and fungal species, including species of Pseudomonas and Helminthospcrium. Shrunken, whitened ker~ nels often with a pinkish discoloration are characterm istic of scabbed grain, a condition caused by Engaging spp. (144, 226, 555). Skinned, broken, and harvest dam» aged barley kernels are also suspect because of the ease with which storage organisms attack the exposed embryo (140, 188, 226). The removal of the hulls from barley grains, using a barley pearler, is another valuable diag- nostic procedure. Heatudamaged barley characteristically possesses a dark-red, pearled kernel which is the result of heat generated by microflora in storage (226). Microw scopic examination of whole barley kernels may reveal other sicns of m.:r;:;‘:ai ::va;:on, 7H the form of fructifi~ cations, mycelial masses, etc. (69, 105). Surface contamination of seed by microorganisms can method and tie use of the cenu t- . be determired by the wash trifuge. The washing method is carried out by either piac~ ing the sample of grain under a stream of water or by shak~ ing the seed in a container or sterile water. The washings are plated on suitasge 1;*~1unt mesLa after which The numn ber of Viable mieroflora are determined from the devel~ oping colonies (lee, 19>). Populations can also be ass sessed, without plating, by the use of a hemacytometer or counting chamber. A sariation of the washing method is used in which the ksxueis are plated in weter, washed with agitation, and *ne an Minus ventrifa ed rm concenw tzratze tl1= 9::1 Lee, sguoi‘s--, 11a51 kl}; hgjl f, WLiNhrlltf? vmiiviri fiaxze been remove} from the gr 1: {lfif_ 39%, éwf . These methe , - —-.--~ ,- ,—. . . .-., l . . . __‘ y g A _' '7 , ' . . ,.v . ~ _. cds are oI greates' taint 'u the ind, 1.0u Jul identixl» .-‘ l 4 cf 101‘ of tile erxtellial-5*-buxuie sanits. h d 9. h19f'42gical erfi other staining te:Luiques are of great value in (seesSing the location and amount of micro“ flora present in seeds, but these methods provide little or no information about the identity of the microflora involved (4L6). One notable exception to this statement is. or course, the mycelium of loose~smut, which is located in the tissues of the barley embryo, chiefly in the scu~ tellum. Methods for the detection of this mycelium are constantly being modified and developed (278). Simmonds 8 has reviewed the techniques employed in the detection of this parasite and has outlined two methods which give rea- sonably satisfactory results (557)° The whole embryo method employs a dehydration and clearing process with no staining or sectioning iequiredo The sectioned embryo method necessitates embedding9 sectioning9 and staining of the germ° A preceduret similar to the whole embryo method, has been described for use with sick wheat and corn grains by Christensen and Qasem (72)o Hyde and Gale leymore have described a simple and rapid method for dem- onstrating the presence cf fungal mycelium in the outer tissues of cereal grains fi9l)o A similar procedure3 in“ volving the use of aniline blue to stain the inner pericarp layers of grain was presented by Oxley and Jones (295)o A method for the determination of Eusaripmmdamaged grain has been reported by Gadd and Kjaer \lQl) which employs the staining of longitudinaliyusectioned kernels with a selu enite and indigoecarmine dual staino Christensen has developed a methoa in which histological and plating techw niques are combined (68)° This procedure does provide information on species identification as well as location of the myceliumo The seeds are washed under running tap water with the addition of a small amount of detergent, and strips of pericarp tissue are removed and transferred aseptically to agar blocks held on coverslipso The cover slips are placed on van Tieghem cells in petri plates9 and the plated tissue is examined at intervals for several 9 days° The living and dead mycelium is then tabulated by observing the formation of new hyphae and sporulation by the funguso Other workers have suggested that surface— disinfected pericarp strips be plated on agar plates (191)9 or that the disinfected paleae and lemmas as well as the caryopsis be plated and population counts made separae tely (398)o The latter method has the additional advan— tage in that it provides some information as to the loca- tion of the seed-florao Seed microflora are determined9 in many cases9 in the course of routine germination testso These tests are ordinarily conducted in seedwlanoratoriesi using moist blotters or paper toweling containing the germinating seeds° The seedwborne organisms are subsequently identia fied as they grow out from the germinating seedling (246, 5069 3079 511)o While providing a limited amount of in» formation about the attendant microfloral the method is inadequate for the determination of bacteria and many slowegrowing or minute fungio Various soil tests have been employed in seedatesting worko These tests employ various combinations of sterile and nonasterile soil9 sand9 vermiculite, and brickwdust, as well as treated and unutreated seedo These methods are of great value when dealing with organisms which preferentially attack seedlings or older plants (144, 246, 2489 26‘:? 506, 507). Perhaps the most valuable diagnostic procedure in the determination of seed—microorganisms is the agar- 10 plating method. In general terms, the technique consists in the placing of seed, either with or without surface- disinfection, on a suitable nutrient medium and observing the microflora that grow out on the surface of the medium and/or the infected seed. Frequently, investigators em- ploy deep agar and submerge the seeds in the plating pro— cess to prevent the seed from lifting itself above the agar surface during germination. This method is some- times known as the “New Zealand” technique (288). Agar- plating has been in use in phytopathological work for at least 50 years. Some of the earlier papers dealing with the method are those of Bolley (47), Selby and Manns (343), and Chen (64). The so—called "Ulster" method was developed to measure the seed-borne organisms of flax- seed. In this procedure, the flax-seed are transferred without surfacewdisinfection or washing to malt agar with sterile forceps. This technique has been discussed in papers by Muskett (280), Muskett and Malone (281), and Noble (288). More frequently the plated seeds are dis- infected prior to plating in order to remove post-harvest surface contaminants. This procedure, termed the "Ottawa" method by Noble (288), employs numerous combinations of disinfecting materials and nutrient media. The choice of disinfectant and growth medium is dictated in each case by the information desired by the operator. Porter has indicated that, in spite of its usefulness, the .5 N. .11 disadvantages of the plating method are twoefold: (1) the method is timewconsuming and permits the examination of only a small seed population and (2) incorrect population counts may be obtained because of the selective destruc» tion of certain species (507). Simmonds and Mead eval» uated several methods used in the examination of diseased wheat seed and concluded that the plating method was in- ferior to direct examination of the seed and the use of sterile sand plantings and the moist chamber (556). Sev- eral workers have discussed the selective nature of the various disinfection, plating, and isolation techniques, as well as the importance of using proper culture media (70, 150, 162, 245, 247, 254, 564, 598). Although washing methods and the use of chemical compounds generally have been employed in the surface- disinfection of seeds, gaseous sterilants have been recw ommended and evaluated by several workers for the deter- mination of microorganisms or for the production of ster— ile plants (576, 400, 401). The use of gaseous sterili- zation has also been proposed for the preparation of cul- ture media composed of heat-labile biological materials (162). Evaluation of a number of seed-disinfection ma- terials have been made by Porter (507, 508) and deZeeuw (94). Wilson compiled a list of seed-sterilizing methods which had been previously used to remove microflora and/or to produce sterile plants (419). He found that the exist- ing methods were unsatisfactory and devised a technique 12 which employed calcium hypochlorite as the active agent. Levine and Schoenlien prepared a comprehensive listing of various types of culture media which is of great value in the proper selection of suitable plating media (251). Some commonly used media, disinfectants, and techniques are included in the manual prepared by Biker and Riker (522). In an effort to determine the most satisfactory culture medium for use in the isolation of cereal storage fungi, a number of various media were tested and evalun ated by Christensen (67), Christensen and Tuite (72), and Bottomley and his co-workers (51). In each case, malt-salt agar proved to be the most satisfactory iso- lation medium for species of Aspergillus. While the whole—kernel plating method has been valuable in the determination of relative percentages of microorganisms present on and within cereal grains, it does not provide an absolute population figure. Con- sequently, several investigators have suggested that the seed be crushed or ground and dilution plates be prepared from a suspension of the ’flour' (50, 51, S2, 67, 70, 367, 398). A serious problem in the plating of seeds and other plant tissues has been the suppression of certain organisms by seed flora and the masking of developing colonies by other, faster—growing microorganisms. Only partially satisfactory methods for the selective inhibi- tion of certain types of organisms were available until 15 recently. Bacterial colonies were usually inhibited by the use of acidified media; the suppression of the fila- mentous fungi was not as easily accomplished. The advent of antibiotics has made it possible to selectively sup- press one class of organisms to the advantage of another (109). Martin has described an antiwbacterial medium containing streptomyCin and rosebengai which has proved useful in the isolation of soilufungi (254).. Cyclohexi- mide (Actidione) was incorporated into culture media in a study by Vaughn and his co-workers (405). They demon- strated that the growth of ten phytopathogenic fungi was strongly inhibited at a concentration of l00 p.p.m. in media. Phillips and Hanel obtained effective control of twelve species of fungi with the addition of Actidione to solid culture media at a rate of 0.1 mg. of the com- pound to one m1. of the media (502). Actidione was em- ployed by Green and Gray in a selective medium for use in the enumeration of contaminating bacteria in brewery yeast (146, 147). This medium has received wide accept- ance and is now available commercially in de-hydrated form (Difco WwL differential medium). The same compound has also been employed in plant pathological practice by Jeffers (198) as a fungal-contaminant suppressant, and by Schneider (559) as a selective medium for ggaph- gum Elm; Schw. which apparently is tolerant of the com- pound. Streptomycin was also incorporated in this se— lective medium to suppress bacterial contamination. 14 Other techniques have been developed to evaluate the seed-borne organisms of barley and other seeds. Skolko has described a technique for determining the presence of certain bacterial pathogens of bean seed by inoculating suspensions of the comminuted seed with bacteriophages specific for the pathogens (559). An increase in the num- ber of phage particles determined by plaque formation is presumptive evidence that the bacterialwhost cells are pre— sent in the seed sample. Such a method might be employed with certain of the bacterial pathogens of barley since phages specific for Xanthomonas translucens f.spp.hordei, secalis, and hordei-avenae have been isolated from barley and oat kernels (580). Danckwortt has reported that bar- ley seed infected with Gibberella saubinetii (Mont.) Sacc. displays a green discoloration at the tips of the seed when exposed to ultra-violet irradiation (87). Simmonds has de- signed an apparatus to isolate fungi from the seeds of cereals which employs an aseptic-washing principle (554). This device was constructed in order to avoid damage to the seed-tissues and retardation of fungal growth as a result of chemical-disinfection injury. The plating of large numbers of clover seeds was facilitated by the use of a disinfecting apparatus, aseptic seed-drier, and vac— uum counters (220). The methods outlined above were not generally suited to the determination of obligate parasites. Consequently the seed-borne viruses of barley must be determined in a 15 different fashion. McKinney has outlined methods for de— tecting these pathogens in developing barley seedlings (260). Several authors have reviewed the many papers dealing with the isolation and identification of seedwborne organ~ isms (85, 247, 265, 509). Comparisons of seedetesting re- sults, using samples of the same seed-lot, have been made by various investigators (7, 267). In general, agreement between participating laboratories was consistent although certain testing procedures yielded greater uniformity than others. Some of the isolation and identification proce- dures, which have been successfully used with barley micro- flora, are tabulated in Appendix A. MATERIALS AND METHODS The methods used in any given laboratory for the determination of seed~microflora have largely been devised to meet the needs of the research program carried on in that laboratory. It must be emphasized that no one method is wholly satisfactory for the determination of fill micro— flora in either a qualitative or a quantitative sense. The purpose of this study was to evaluate the various methods employed in barley-microflora research in order to deter— mine the best method for assessing the microbiological population of stained barley kernels. The effectiveness and selectivity of various nutrient media, surface-disin- fectants, disinfection-times, and plating techniques were investigated as well as some preliminary work on the loca- tion of microflora on and within stained kernels. 16 Two barley lots were employed throughout the study: a moderatelymstained lot of Hannchen barley grown at East Lansing in 1959, and a sample of Hudson feed barley of ex- tremely poor quality grown in Michigan in 1958. Eighteen types of nutrient culture media were used in the study (Table 1). Eleven surface-disinfecting treat- ments were also employed as well as a non—disinfected check (Table 2). Various periods of disinfection ranging from zero minutes to ten minutes were employed in conjunction with two surface-disinfection methods. In the evaluation of the various types of media the standard surface-disinfecting procedure of the author was employed. This method is carried out as follows: One hun- dred kernels are counted out at random. The kernels are placed ten at a time in a Gooch crucible and immersed in a finger-bowl containing the surface-disinfectant. The disinfecting material is a mixture of one part 70% ethyl alcohol to two parts of commercial laundry bleach (5.25% available chlorine). The kernels remain submerged for a period of one minute with agitation of the container to prevent the kernels from floating on the surface. At the termination of the disinfecting period the crucible is re- moved from the finger-bowl, and the kernels are transferred aseptically to suitable culture media with a pair of sterile forceps. The kernels are placed uniformly in the petri plates, ten kernels to a plate, and ten plates to a series. The plates are incubated at room temperature (ca. 25°C.) 17 Table 1.-Cu1ture media employed in the study of in- fluences on barley microflora exerted by the type of plating- media employed. Source pH of Name of medium of medium medium Ref. Potato-dextrose agar (PDA) Difco 5.6 522 Acidified PDA (lactic acid) do 4.0 522 IPDA plus actidione (100 p.p.m.) do /g 5.6 —-- .PDA plus chloromycetin (50 p.p.m.) do /p 5.6 --— 'Water agar self 5.6 522 Barley agar } do 5.5 500 Malt-extract agar Difco 4.7 82 Malt-rootlet agar self /9 5.0 --- Malt-salt agar (10% NaCl) do 5.8 70 Lima-bean agar Difco 5.6 82 V-8 agar self 7.2 82 Prune agar do 5.8 522 Cornmeal agar do 6.0 522 Nutrient agar do 7.4 82 Nutrient-dextrose agar do 7.4 82 Czapek-Dox agar do 7.5 522 W-L agar Difco 5.5 82 W-L differential agar do 5.5 82 a. Actidione made available by the Upjohn Co., Kalamazoo, Mich. b. Chlormycetin made available by Parke, Davis & 00., Detroit, Mich. 0. Prepared by heating 70 grams of dehydrated malt rootlets 18 Table l. - Continued in 500 m1. of distilled water, strain through cheesecloth, add 15 grams of agar and 15 grams of dextrose, heat to dissolve agar, bring up volume to 1,000 ml. with distilled water, autoclave and bottle. 19 Table 2.- Surfacewdisinfecting treatments employed in the study of the influences exerted by surface-disin- fection method on barley microflora. Type of surfacemdisinfecting treatment /§ References None ("U1ster" method) 280, 281 Sterile water--no agitation —-— A.S.B.C. method 5 Modified method of author /2 500 NaOCl and sterile water (129) 507, 508, 522 70% ethyl alcohol 522 5% hydrogen peroxide 507, 508, 522 5% formalin 522 2% KMnO4 322 1% carbolic acid 522 HgCl2 w water (1:1,000) 522 HgCl (121,000) - two rinses in s erile water 508, 522 a. All treatments disinfected for two minutes and plated on PDA. b. Modified by increasing disinfection time to two minutes. 20 for 4m6 days. After the incubation period the microflora are identified, the number of colonies are counted, and the percentage of seed germination is ascertained. Identifica- tion of the fungi is made by direct examination of the col- ony with the microscope or after remisolation of the fungus in pure culture. The latter procedure is necessary in the event that the fungus fails to sporulate. With few excep- tions identification of fungi is made only to genus; bac- teria and yeasts are routinely recorded on the basis of their gross colony color (500). In the evaluation of the various surface-disinfec— tants, a standard disinfection period of two minutes was employed with all treatments. In this series of experi- ments all barley samples were plated on potato-dextrose agar (Difco). The two methods of surfacewdisinfection employed with the varying disinfection times were: (1) the method of the author described above, and (2) the method recommended by the American Society of Brewing Chemists for the determination of barley microflora (5). The use of ground barley 'flour' and dilution plates for the quantitative determination of seed microorganisms is a commonly employed method. The accuracy of the method was studied by plating portions of barley kernels decreas- ing in size from the whole kernel to aggregates held on a V64" sieve. Surface-disinfected kernels were aseptically sectioned with a sterile razor blade to provide half— and quarter-kernels. Aggregate sizes of V52" and V64" were 21 obtained by grinding surface‘disinfected kernels in a LabConCo burr-type mill. Dilution correction-factors were employed in order to accurately compare the population counts obtained With each reduction in aggregate size. The dilutionwplate technique employed in this phase of the study was carried out by suspending the lflour“ in sterile water and plating 0.1 ml. aliquots at three dilutions in agar maintained at 40°C. in a water bath. Four separate media were used in conjunction with the dilution-plate procedure: potato—dextrose agar (PDA), PDA plus actidione (100 p.p.m.), PDA plus chloromycetin (5O p.p.m.), and nutrient agar (Difco). The location of microflora associated with barley kernels was studied. Transverse sections of surface—dis- infected kernels were plated on PDA, and the developing colonies were counted separately for three regions of the kernel; embryo end, middle, and awn end. Surface-disin- fected barley kernels were also pearled for one minute in a barley pearler which had been previously cleaned by passing a large quantity of disinfected grain through the machine. The pearls were surface sterilized briefly and plated on PDA. A corresponding sample of whole barley kernels of the same seed lot was plated at the same time, and the microfloral contents of pearls and whole kernels were compared. Histological procedures were also employed in the study of the location of microflora in barley ker— nels. This work is discussed in another section (Chapt. 5). 22 EXPERIMENTAL RESULTS The Choice of culture media used in the plating of barley kernels is of some importance in influencing the kinds and amounts of microflora isolated from the seeds (Table 5). The mode of surface-dieinfection also exerts a selective influence on type and amount of microflora re- covered from plated seeds (Tables 4 and 5). For the sake of simplicity the microflora detected in these experiments are categorized into broad groups. The "field" fungi in— clude speCies of Alternaria, helminthosporium, Eusarium, Epicoccum, Cladosporium, and several others. "Storage" fungi refer to species of Aspergillus, Penicillium and members of the Mucorales. The bacteria included white, yellow, creamy, pink, and various Other morphological types. The yeasts were largely pink in color, although some wnite yeast colonies were observed. The barley ger- mination figures are for the Hannchen sample only since the feed sample was excluded because of erratic germina- tion. Linear increases in the number of bacteria and fungi per kernel were obtained in the plating of dilu- tions of barley kernel aggregates of decreaSing size (Fig. 1). The various media also showed a selective ac- tion on the microflora of the kernel in the grinding-pla- ting tecnnique (Fig. l). The percentages of microorganisms detected on pla- ted transverse sections of barley kernels were plotted as Harm gpzonm Hmmddm .soom spzoem Haanmpoam mm m m we so a a ma me name emopuaaae copmSPm hso> Hmsdm wacww .Hame some amuse mmspopm o o o o 0 mm o m om mama seamless: doom upsosm Acme sewage sea Hmaempomm am as m us so - a as as psopemupaas dmpqdpm museum Hmmadm Hams .ccUSUOHQ ossaw Hmflsopomm mm d A on om m 0 mm mm pomspxospamz menace «mm op nsaseflm em as o as mm o 0 we no name smseam doapmqfissom measmn boom .npzomw Hmnofimosofla poom so a O n m o a m mm meme some: z, Ummtcsoqa museum HmmGSM \.9..sommmeoms spzosm assemposm we se m 2a m a H a; so soaoflesom «he possess Gesumdassom mosses macaw .speoem assess seesaw me a: o an on o a- as asses mean age commchoda npzomm Ahmed“ Cfipcoha \ .emmameome museum assemposm mm a 9 ea 0H s m mm om aoeosso mesa «mm noaposcosm A hpzoem assess on II N Is mas I 0 II on Incomes an: cospommn mmmnusaos mm>au on II N II mm I o a- us name an: soosoonm mafiam Hmwpop ammo swoop Icon .Hoom npzopw Hmmsdm or II a II mad I 0 II we Ixcolpnofiupdz woos spzoem assess Hm s a OmH mas s 0 mm me name pqmeepsz soom spzoem amuse mmewopm em H s cm om me o om mm news sonIsmomso Room gazesm HmHOHMOHOHz mm II 3 II we I 0 II 5: ammo accessoo bounces museum Hmeouomm om m 0 mm me Os 0 mm no news madam Moog npzosw HmmdSH .hbmon museum pesos one amanopomm om 8H H mm mm a o as mm name mus Amv D m D m D m n m soapwmflasom ammo“ “meow adfldoa mMHmSmm hoanmn mpmmow mssopomm ommpopm caoflm we came pdoomom macsomx OOH scam mowdoaoo oposomflo mo mmmeoosmm dmdoflpdoo I .m magma 25 decades soaps Inflancm hmanmn use .Hmeso .maempoan me a 0 mm o o 0 oh a Aooo.anav maomm mm m a moa or m 0 Ho om deem oaaonnmo fia m pews d Ipmmep mo m .oo cm s m :0 me e m mm mos ones em empespm smauam me o o o a o m be on assesses an UmpmadsHpm soap m m Imefleemm scheme mm m m my me a m 0: was 0 m am copsdpm mmeeamonoa mm m a son mm m 6 mm mm Hosooam asses son mauav Hopmz no m 0 mm or o O we mm oaasomeHoowz mm m m Hm mm o o w em scheme m.nonps< mmdmoso do 0 a ON da 3 a ma mm combos ommd Cuchm pmmch sea assempoam as me mm mm mm s m mm me nope: massmpm A=sonpme mm o a so om m 0 on boa empmsp=v meoz m n m p m p m n m womanhomp dospmssssmm I amasw HMQSM pumpocmqflmflc mthsom mmahmm mummow mascpomm mmmHOpm dacflm mommsdm mo came soapocmqfi mo ommpsmopmm cospmoflsumw hoasmn so one msoamosoas mo qofipmaomfl map so mpcmpoowsHmHUImommuSm moosnm> mo pommmo mze I .: manta 26 .meH .smmHQOHS as sebum madame boom oosflmpm haoso>mm .p .mmaa .mqfimqmq pmmm pm macaw camamm nmaoqsmm canMpm haopmsmwoz .m wmflnmpomn woods Ion op odd soap Imqfiasom hoasmn Hops: baseman qH momsws 03p hp dozoaaow mmmpeooemm swam cos m 0 we as o 0 mm ms Aooo.anav Naomm m D m D m n m D m pamspmomp Gospmqflssmw HMQSM amasm unspommdfimflu mxsmaom mmasmm mummow mflnopomm ommhopm uaofih comMHSm we came soapoomnfl mo cmepsoomom sodsflpaoo I .s mapme donpma omm< .0 Honpsm mo bonus: .0 mHQamm boom .9 mHQSMm sonoqdmm .w om I m II oH II H II H5 6 OH mm H H Hm mm m o m mm 0 mm I 0 II a II m II on e m mm o m on HB OH m m mm o no I m II 5 II H II mm s m mm H a mm mm N o 0 mm o 50 I 0 II om II 0 II mm 6 H mm m m om mm o H OH on o no I H II mH II H II my a we a m oHH ms m m 3 mm o m\H #0 o m 00 mm mm m NH QMH o m n m n m a m n m doapmsHaHmm _ pscapmosp a.:Ha de mosses qudm chdm wdeoquHde doapomdemHU ommecopom mummcw mecpomn owmpopm cHon we song we dOHpmssa mHospmx OOH aosw mquOHoo opcnoch mo swepmconom .GOHmeHBHom thst com MHOHwoooHs mo QOHumHomH one so mpsoapmmep GOHp IommchHonommpdm 03p :sz mosHp QOHpommsHch dempm> we pomwmm one I .m oHQme NUMBER OF COLONIES PER 100 KERNELS 28 l l _ 350.4. Media: 35 x - PDA / 1:! - PDA-Actidione . / O - PDA-Chloromycetin / tic Nutrient agar Organismszj / 500 a...~H-— Fungi / __l -—-— Bacteria / » 250 4— __j 200 1 i . i I I 1.. I. _.: I I 9 : I‘ 100 {H __. l. " i so i— ___, O _ j I - l. I l l— I Whole 1/2 1/4 SIZE OF KERNEL AGGREGATE . Fig. 1-a. The effect of culture media and size of kernel entity plated on microfloral population. 29 I ” I" " " " _, >(- PDA El- PDA-Actidione O - PDA-Chloromycetin /\ £5- Nutrient agar if _. 5x105-l" co I—l [I] 2: (I: ea :24 c3 2 2x105d— .. 4 it] o, (0 [fl H Z C) I4 I O I L) g .¢ H Cr: ! E l C, 5 I <1: In 1x10 "T" __ I i I I I6” I o L l --_____ -40 _ _- .3 1/52" 1/64" SIZE OF KERNEL AGGREGATE PLATED Fig. 1-b. The effect of nutrient media and the size of the kernel entity plated on microfloral population (bac- teria only). O I 50 functions of the location on the kernel (Fig. 2). The sec- tioning and plating of the Montcalm sample was performed approximately ten months after harvest, whereas these op— erations were performed with the Hannchen sample after it had been stored only two and one-half months (Fig. 2). While the fungal determinations in each case are very simi- lar in their patterns, the bacterial counts are consider- ably disparate. The degree of kernel infection determined by plating disinfected barley pearls differed considerably from that of whole kernel determinations (Table 6). The most radical difference in the two treatments is the large increase in the number of Candida colonies isolated from the plated barley pearls. DISCUSSION AND CONCLUSIONS Certain culture media employed in the plating of barley kernels exert a selective effect on the kernel— flora, and the type of media used may also affect the ger- mination of the plated kernel. Most of the media tested in this experimental series are useful for some phase of barley—microfloral work. For the author's purpose a me— dium (or media) must accurately demonstrate the presence of "field" fungi, bacteria, and yeasts on sound malting- type barley kernels. "Storage" fungi would not ordinar- ily present a problem in an investigation of this nature since barley carrying any appreciable amount of "storage" mold would not be acceptable to a maltster after even a cursory visual examination. For workers interested in PERCENT INFECTION 51 lOO' “[«~«—--H_-_V.WTH- I Montcalm-bacteria 75 T and yeasts Hannchen-fungi H Montcalm-fungi I ;\ 50— Hannchen- bac— ’ / teria and yeasts / / ’ / / / // I / // / . / I I 7 / O "- . . _Tl L : Embryo end Middle Awn‘end SECTION FROM: Fig. 2. The location of microflora as determined by plating transverse sections of barley kernels on cul- ture medium. 52 .mnHHHmmm Hop InoaHnomxo on» op HOHHQ annoma one smoopnp ads mm: mmHHmn oHHHcpm Ho onEmm m was pmmHQIHHm one among new: OodMoHo mos Honmmm hoHHmD one .deHpmHm :HHMmmz Odd =HodmoMImHon3= Avon now poms mmz onamm hmHHmn aHmopnoz UoQHMpm 4 m m mm 60H 1. m. a R Hemmm O O mm O m 0 Oh cHonz mummmw adHHomm . Hoqnox nmflpo MOHquO meopomm msHHHmnomm< adHnmmsm IonanaHom MHsmosmpHd Ho cake mHonHmM OOH Sosm moHGOHoo mpoHomHO mo owmuaoonmm .I\oHQEMm hoHHmp came on» mo .mHHmom. Scum dam chqscM mHonz Scum OmpmHomH mAOHmosta Ho mommpdoommm I .o oHpme 55 the storage organisms several media are useful in plating work; one of these, malt—salt agar, was employed in this study and with the feed-barley sample permitted growth of the Aspergillaceae while suppressing other microfloral growth (Table 5). The high salt content (10%) is the active inhibitory principle in this medium and not the re- action of the medium. Prune agar with the same reaction (pH - 5.8) supported other fungal growth and the germina- tion of the kernels was not as drastically affected (Table 5). The "storage" fungi are tolerant of high osmotic pressures and are not able to compete in growth rate suc- cessfully with other organisms whereas the converse is true of the "field" organisms. The possibility is also suggested that many of the Aspergillaceae are sensitive to various germination inhibitors released from germin- ating barley kernels. This hypothesis is lent credence by the observation that "storage" molds only become strongly Iestsblished on weak and damaged kernels, and the embryo :region of the kernel is more resistant to infection after injury than is the endosperm area (188). Potato-dextrose agar is a satisfactory medium for the crommon bacteria and fungi of barley kernels; however, the high sugar concentration (two percent) in this and certain other media, e.g. nutrient-dextrose agar, pro- moted-Iwolysaccharide slime production by several bacteria (Table £5). Barley agar, malt-extract agar, and malt-root- let agazr also prove to be very satisfactory media for the 54 detection of the "field" organisms. These media have the additional advantage of being cheap and readily available to barley and malt workers. Several media support bacterial growth at the expense of the filamentous fungi. These in- clude nutrient agar, nutrient—dextrose agar, V-8 agar, lime-bean agar, and certain differential media (Table 5). Other media such as water agar, prune agar, and cornmeal agar have limited usefulness and are used for the isolation of certain specialized organisms, for sporulation, kernel germination checks, etc. While a standard method for the determination of fungi in barley is proposed by the American Society of Brewing Chemists, no accepted method is adopted by the society for the detection of bacteria in barley. The Sub- committee on Methods of Barley Analysis for the society states that "the satisfactory determination of the bac- terial count of barley and malt is complicated by the :many types of bacteria present as well as the interfer- ence of certain very common molds, for example - A122;- naria . . . An accurate determination would require the development of specific nutritional conditions which would inhibit the development of molds and permit the growth of bacteria . . ."(5). In lieu of an accepted method the society refers the reader to the method for the total bacterial count in flour used in the milling industry (581). The "specific nutritional conditions" are realized in the use of actidione in culture media. .... 55 Consequently a differential series of plating media was developed to accurately assess the relative bacterial, yeast, and fungal populations of plated kernels (Table 5, Plate I). This series consists of a basal medium such as potato-dextrose agar, barley agar, or malt agar; the same medium plus chloromycetin (SO p.p.m.) used to suppress bacterial growth; and the basal medium plus actidione (lOO p.p.m.) which selectively suppresses the growth of yeasts and filamentous fungi. By plating a barley sample on this series of media one can estimate the numbers of bacteria, yeasts, and fungi while avoiding the problems of microfloral antagonism and the ”masking" of small or slow- growing colonies by more rapid-growing species. These ad- ditives are available in powdered form, are easily added to culture media, and are not greatly affected by subse— quent heat sterilization (575). The phytotoxicity of actidione inhibits the germination of barley kernels on plates, consequently the germination readings, if required, must be made from either the basal medium or the chloro- mycetin-medium (Table 5). The failure of certain surface-disinfectants to eradicate "storage” flora from the surface of the Hann- chen sample is an indication that the treatment fails to properly disinfect the surface of the kernel (Table 4). This interpretation is based on the assumption that the "storage" organisms are generally present on the surface of well—conditioned samples only as fortuitous post-har- vest contaminants. This assumption is not valid in the r, 56 PLATE I The use of differential media in the plating of barley kernels. In both pictures; 'A' represents the basal medium - potato-dextrose agar, ‘B' is the basal medium plus Actidione (lOO p.p.m.), and 'C' is the basal medium plus Chloromycetin (SO p.p.m.). Plates incubated at room temperature for five days. Upper - Discolored Hannchen kernels plated on the differential series of media after surface-disinfection. Lower - Discolored Montcalm kernels plated on the differential series of media after surface-disinfection. 37 case of poorlyuconditioned samples, as exemplified by the feed-sample employed in this study. All of the hypo- chlorite treatments were satisfactory in removing surface contamination without materially reducing the true micro- floral population or injuring barley germination (Table 4). The mercuric chloride treatment used without subsequent rinsing and the formalin method had too severe an effect on both microflora and barley germination. The mercuric chloride disinfectant followed by two rinses in sterile distilled water compared favorably with the hypochlorite treatments (Table 4). Certain compounds had a stimula— tory effect on barley germination, notably hydrogen per- oxide and potassium permanganate. This effect has been noted by other workers (584) and is probably the result of increased oxygen supplied by the disinfectant. Evidence for the mild action of the hypochlorite- disinfectant treatments is provided by barley germination figures (Table 5). While a steady decrease in both fun- gal and bacterial counts occurred as disinfection time increased, barley germination was not severely impaired. The added advantage in the use of chlorine compounds is the ommission of a rinsing step in the plating procedure. Hypochlorite germicides rapidly lose the free chlorine and thus possess little or no residual action (566). Fungal populations did not vary significantly between surface disinfectant treatments, but the A.S.B.C. method significantly reduced the bacterial counts in all cases 58 (Table 5). Surface-disinfecting solutions containing hypochlorite and ethyl alcohol have been used by several workers (5, 18, 56, 115, 500, 415). The alcohol serves as a wetting agent, dissolves lipoidal material, and is slightly germicidal (especially at a 70 percent concen- tration). In conjunction, however, with hypochlorite compounds the alcohol (reducing agent) combines with the hypochlorite (oxidizing agent) to yield several re— action products, viz. acetaldehyde and sodium chloride. Thus the efficacy of both compounds as surface-disin- fectants is somewhat lessened in combination. The ground barley flour and dilution plate method for bacterial determinations, unless rigidly standard- ized, is subject to criticism for several reasons: (1) the results cannot easily be correlated with the whole— kernel plating method used in fungal determinations, (2) the size of the flourmparticles will determine the number of colonies that develop on the dilution plates, (3) fungi that produce copious numbers of conidia and spores will yield population figures all out of propor- tion to their actual presence in terms of comparative area and weight figures. It is reasonable to suppose that as the kernel is subadivided into smaller and smaller portions the numbers of bacteria and fungi will increase to the point where all viable spores, mycelial fragments, and bacterial cells have been separated into discrete units. Thus a kernel containing the mycelium of Alter- .- 59 maria, for example, might yield one fungal colony with the whole-kernel method and 1,000 or more colonies if ground to a fine flour and plated. The data in Figure 1 demonstrates that this is indeed the case. Some prelim- inary work has also indicated that if the size of the aggregate is further reduced, a point is reached at which the populations rapidly fall off, or are even reduced in number, perhaps indicating that heating and/or mechanical disruption of the cells may take place in the grinding process (Figure 1). It is postulated that this tech- nique, if properly refined, might be used to determine a correlation factor which could be employed with whole kernel plating on the differential media described ear- lier. This would give a reliable index of the total num- ber of viable cells, spores, and mycelial entities as well as a relative percentage value. Some measure of the foci of invasion of barley kernels by microorganisms is given by the data in Figure 2 and Table 6. In the hannchen sample the percentage of infection by bacteria and yeasts was markedly lower than that of fungi at the germ end of the kernel. A steady increase in the number of organisms of all three types was also noted in transverse sections taken successively from the germ end to the awn end of the kernels (Fig. 2). Observations made on numerous platings of barley kernels confirms this thesis. Since bacteria and yeasts are gen- erally less efficient than fungi in the infection and 40 establishment of tissues in which natural openings and wounds are reduced in number, this is perhaps not sur- prising. It must be emphasized that we are dealing here primarily with the "weathering" or "field" organisms and not with kernels afflicted With basal glume rot, black point, or severe kernel blight in which case this gen- eralization does not necessarily hold true. The close similarity of the curves representing fungal infection in the Hannchen and Montcalm samples emphasizes not only the location of infection, but the decrease in fungal viability as well. The data in Table 6 reinforces the observations of many workers, viz. that the major portion of the fun- gal microflora are located within the pericarp and hull tissues of barley. The increase in bacterial and yeast colonies on the plated pearls, as contrasted with the whole kernel determinations, is perhaps only of apparent significance since the techniques used in the experiment were semimaseptic at best. Existing evidence also indi- cates that bacteria, yeasts, and heavilywsporulating fungi may yield disprOportionately large population counts when physical disruption of the kernel takes place. Thus the particles of dust from the pearling operation may have adhered to the pearls and brought about the increase. The decrease in the number of com- petitive fungi might also explain such an increase in these organisms, whose normal presence is in many cases 41 masked by the more rapidly—growing and/or inhibitory fungi. More experimental evidence will be presented in Chapter III, with respect to the location of microflora on and within barley kernels. CHAPTER II THE CHARACTERIZATION 9E MICROFLORA LITERATURE REVIEW It is a truism that microflora are usually to be found intimately associated with plants and plant parts. Thaysen and Galloway have stated that: All green plants possess an epiphytic microflora which normally subsists on the slight traces of carbohydrates, protein and inorganic salts which dissolve in the water extruding from, or conden- sing on, the epidermis of the host . . . . It is able to subsist under the most severe climatic conditions to which the host normally becomes ex- posed, and . . . at periods of damp and rain it develops luxuriantly, spreading over the whole of the epidermis, including the flower and the seed (590). This "epiphytic microflora" may be comprised of numerous species of bacteria, yeasts, actinomycetes, and higher fun— gi; and represents a symbiotic spectrum, ranging from sap— rophytism at one end of the scale to obligate parasitism at the other. In general, the subject of seed-borne microflora in the cereal crops has been approached from two direc- tions: (1) the storage organisms implicated in the deteri- oration of stored grain and (2) seed-borne pathogens which injure or destroy plants which deveIOp from diseased seed. Several excellent review papers are available which treat of these two categories. Semeniuk has dealt with the problems of microflora in the deterioration of stored grain (544). An extensive 42 45 list of research papers is reviewed in this article, inclu- ding reports on cereal microflora not implicated in storage problems. Other review papers, primarily concerned with the storage microflora of cereals, are those of Christen- sen (69, 70) and Blum (45). The subject of seed-borne pathogens has also been adequately treated in several publications. The lists of Doyer (106), LeClerg (225), and Noble and her co-workers (290) are especially noteworthy. Older compilations of seed-borne diseases have been authored by Chen (64), Gard- ner (125), and Orton (295). The references by Dickson (lOO), Fischer (117), and Sprague (568) are also valuable sources of information on the seed-borne diseases of cere— als. On the other hand, the microflora of the small-grains, and expecially of barley, which are neither storage pests nor obvious pathogens, have been generally neglected by authors of review papers. The literature dealing with bar— ley microflora, in the broad sense, is, hence, voluminous and scattered. As early as 1702, Lisle described the condition in barley kernels that we recognize today as 'blight' and 'scab' (257). It was not until the latter half of the nineteenth century, however, that significant progress was made in the isolation and identification of seed-borne mi— croflora. This as a result of the pure—culture techniques perfected by Pasteur, Koch, and others. 44 It was commonly believed by several early workers that microorganisms were always present within healthy vege— tative tissue (59, 125). This view was challenged by Buch— ner (57) and Fernbach (116) who demonstrated that the inte— rior of plant tissue was devoid of microflora. More recent— ly Schanderl (558) has adduced evidence which leads him to conclude that the interior of vegetative tissues is not free from microorganisms. He regards cell mitochondria as bac— terial symbionts which under certain conditions become free— living forms. Reichard, in a study of barley exposed to ex— cessive dampness, assigned the decreased germinative ability of the grain to an excessive microflora, chiefly the lower— fungi (521). The same conclusion was reached by Becker in his paper dealing with bacterial flora isolated from barley (29). Chrzaszcz considered the microflora of cereal kernels to consist of 'true epiphytic' organisms and 'secondary' flora; that is, an 'internal infection' as contrasted with those organisms which become mixed with the grain in the form of soil particles (79). 28b1, in 1892, described the isolation of several fungi from discolored ("Braunspitzige") barley kernels (425). He concluded that the kernel discol— oration was caused by Cladosporium herbarum (Pers.) Link. The bacterial flora of plant surfaces was studied by Burri (59). He described a bacterium, later named Bacterium herb- , ll . . icola aureum Duggeli as occurring most frequently on normal- appearing plants (108). This View was supported by Duggeli in a later paper (108)° The two workers found Bacterium 45 fluorescens Lehm. and Neum. (Pseudomonas fluorescens Migula] was next in order of occurrence, followed by Bacterium puti- dum Lehm. and Neum. [Rseudomonas putida (Trev.) Migula] , Bacillus megaterium deBary, Bacillus vulgatus (Flfigge) Migula.[Bacillus subtilis Cohn emend Prazm.?;], Bacterium coli Lehm. and Neum. [Escherichia coli (Migula) Casteii.] , and several others. The identity of Bacterium herbicola aureum Dfigg. has been questioned by Thaysen and Galloway (590) and Beijerinck (50), since many bacteria superficially resemble this organism. It is possible that this isolate is identical to Pseudomonas trifolii Huss (55). In another early study, Ravn isolated Helminthosporium teres Sacc. from discolored barley kernels and attributed the staining of the grain to this fungus (520). Much of the early work, dealing with kernel microflora has been reviewed in papers by Chen and Rettger (65), Atanasoff (21), Mead (264, 266), Hyde (190), and Machacek and Greaney (247). More recently, the microflora of barley has been investigated by a number of workers. The Fusaria of cereal grains, including bar- ley, have been studied by Bennett (54, 55, 56, 57, 58) and by Gordon (151, 152, 155, 154, 155). Fusarium, and other seed-borne fungi of barley, were studied by Christensen and Stakman (77). Both, in a study of fungicidal action on seed-borne organisms, isolated a number of fungal and bac- terial species from barley kernels (550). Greaney, Mach- acek, Wallace and other Canadian workers have contributed greatly to our knowledge of the flora of cereal grains, 46 including barley (142, 145, 144, 245, 246, 247). The micro- flora of barley was investigated by Tervet in two reports (585, 587). Whitehead has reported on the microorganisms found associated with barley kernels in his doctoral the- sis (415), as has Pepper in a master's thesis (500). The fermentation industries have, for some time, been interested in the microorganisms found in barley and in other brewing and distilling raw materials (150, 215, 255). Because of the possible effects on barley and its products, malt and beer, interest in barley microflora has recently been stimulated. At least three groups have been identi- fied with barley microflora and its possible effect on brewing materials; these groups include workers in Minne- sota, Wisconsin, and Michigan. Several progress reports dealing with this work have been released from Minnesota (8, 9), and Wisconsin (97, 516). Numerous other papers dealing with grain microflora have appeared during the last half-century. A list of microorganisms, reported in the literature as having been isolated from barley kernels, is presented in the Appendix, Table B. Several workers have reported the quantitative de- terminations of microflora found on the surface of, and within, barley kernels. Semeniuk has tabulated popula- tions from several papers ranging from 5,000 to 12,000,000 bacterial cells per gram of barley(544). The number of bacterial cells per gram of barley was estimated by Hoff- man to be 7,740,000 (181), while Dfigeiii's investigation 47 yielded an estimate of 1,600,000 cells per gram (108). In general, such determinations are made either by plating dilutions of kernel-washings (for external flora) or by grinding or macerating the kernel and plating the flour (to measure internal populations). It is clearly EH1 approxi— mation in either case, and the determination will depend upon the methods employed. This has been demonstrated in a paper by Adams and his co-workers in which the bacterial content of distillers‘ malt was estimated variously at 70,000,000 bacteria per gram or at 90,000,000 cells per gram depending upon the plating method used (10). In the plating of whole kernels of grain it has been observed that individual kernels may be infected by as many as five dif- ferent species of fungi and/or bacteria (142). The location of microflora on and within the barley kernel is determined, to a great extent, by the anatomy of the kernel. The barley kernel is, botanically speaking, a ‘ caryocist, i.e., a fruit and is enveloped by a persistent palea and lemma. The fruit with its enclosed seed is termed a caryopsis (40, 252). Collins has shown that the barley grain is, with the exception of the micropyle and chalazal tract, completely invested by a strongly outin- ized membrance which is impermeable to strong acids and dissolved salts (81). The same membrane is described in a paper by Dickson and Shands (101), by Pugh and others (518), and by Tharp (588). This membrane and the various layers of the testa have been shown to serve as a protec- 48 tive barrier against the invasion of wheat kernels by Gibberella saubinetii (Mont.) Sacc. (518). Mann and Har- lan presented a comprehensive treatment of the morphology of the barley kernel and the process of germination, espe- cially as it pertained to malting (252). The authors con- cluded that the aleurone layer served as a protective struc- ture against the inroads of bacteria and fungi and that un— der moist conditions fungi were invariably to be found in- vesting the kernel and feeding upon the hull tissues. While the hyphae of the organisms extended throughout the envel- ope, they were unable to penetrate the aleurone layer. It was not determined whether the heavily cuticularized walls or the nature of the prot0plasm of the aleurone tissue were responsible for the resistance to microfloral penetration. It has been generally observed that the microflora of cereals are confined to the surface of the kernel or within the tissues exterior to the testa and aleurone layer (42, 47, 48, 68, 88, 104, 126, 190, 191, 250, 255, 258, 264, 280, 295, 501, 546, 590, 597, 598, 415, 415, 416, 425). The endosperm and embryo are also invaded by various cereal fungi (118, 199, 414, 424). The mycelium of Helminthosporium gramineum Rabh. is especially profuse in the region of the germ end of the kernel (425). Hel- minthosporium sativum Pamm., King, and Bakke was found by Head to be located mainly at both ends of the barley ker- nel (264). The same distribution of Fusarium sp. mycelium was described on barley by Dounin (104). Hyde and Galley- 49 more stated that the mycelium of fungi found on wheat ker— nels was expecially abundant wherever the epidermis was loose, i.e. at the two ends and in the region of the 'crease‘ and the ibreast“ (l9l). The authors also reported that the mycelium was less profuse at the germ end of the kernel. The kernels of wheat and barley, infected with basal glumerot caused by Pseudomonas atrofaciens McCull., are stated to be more heaVily infected at the germ end of grain (258). The fungal flora of cereals exists mainly in the form of 'dor- mant' or 'resting‘ mycelium; only rarely are Spores found within the husk of the kernel (191, 264, 280). This 'dor- mant“ mycelium is characteristically irregular in shape, dark, and has thickened walls (264) (Plates II, III). In general, the microflora of cereal grains are cap— able of surviving for long periods of time in a dormant state. It was indicated by Burri (59) that the resistance of bacterial cells on and within grain kernels to dessica- tion was due to the production of mucous sheaths by the individual cells. This protective layer also served to fix the organism to the plant tissue. The bacterial blight bacterium, Xanthomonas translucens (J.,J., & R.) Dows., is reported to survive for at least two years in diseased barley seed (206, 207). The mycelium of the barley loose smut fungus, Ustilago nuda (Jens.) Rostr., remained viable in barley seed embryos for eleven years according to Por- ter (510). Christensen reported no living mycelium in wheat seed infested with storage molds after eight years of storage (68). Whitehead et al. reported living mycelium D) 1.1. :3 of Microascus trigonosporu; barley kernels stored in bot- tles for 25 years (416). Barley seed stored under proper conditions retains high germinability while the microflora more rapidly lose their Viability. Haferkamp obtained high germination percentages with barley stored for periods of 24 to 29 years (155). The kernels. with or without hulls, had little or no mold present after the termination of the storage period [species of fungi were not named]. Macha- oak and Wallace stated Similarly that barley seed retained its germinability and the associated microorganisms lost viability after a ten year storage period (249, 250). By testing at intervals over this period they reported that only Helminthosporium tereg Saca. was still present at the conclusion of the experiment, while Alternaria tennis sensu Wilts. and Helminthosporium sativ h P.. K., & B. were no longer present, haVing died out at a comparatively rapid rate. Russell compared the longevity of several lots of wheat seed with cracked seedecoats over a 17 year period (552). All of the seed lots germinated well until eight years had elapsed, th n Viability decreased rapidly. Near- ly all the seed was dead at the end of the testing period. Alternaria tenuis disappeared after about seven years while Helminthosporium sativum decreased more slowly, with eight percent of the original amount still present after 17 years. Christensen reported a longevity period of ap— proximately seven years for H. sativum in barley kernels 1'." ‘1 , .l. (75). Mead stated that the dormant mycelium of the same fungus remained viable in the barley grain for two to five years (264). Shands furnishes longevity data for several barley seed-borne organisms. He reported that Gibberella saub- inetii (Mont.) Sacc. g. zeae (Schw.) Petch survived for periods ranging from eight to twentynseven months and lost its viability completely after 50 months (546, 547). Alternaria sp. had completely lost its viability after 65 months and Helminthosporium spp. were apparently non- viable after 51 months (547). Aspergillus sp., Cephal- othecium sp., Trichothecium sp., Chaetomium sp., and Helminthosporium gramineum Rabh. all remained viable after periods ranging from 75 to 125 months, Alternaris sp. from 57 to 75 months; and Fusarium culmorum (W.G. Sm.) Sacc. and E. avenacium (Fr.) Sacc. were not found at the end of 28 months (546). The author indicated that longevity of the seedwflora appeared to be affected by storage condi— tions. The longevity of H. gramineum in barley kernels has been variously reported as being slightly over 17 months (120) or in excess of five years (250). Paxton obtained conidia and conidiophores of the same fungus from 16 year old herbarium specimens of barley placed in a moist chamber (299). Many investigators have suggested that the quali- tative and the quantitative nature of seed microflora may be strongly influenced by interactionueffects between 52 these various organisms. Hess isolated an antibiotic substance produced by H. gativum in culture and active P against Bacillus mesentericpg LBacillus subtilis Cohn emend Prazm?:1(l76). He also attributed plant injury primarily to this substance. Conversely, several work- ers have shown that certain bacteria display marked inhib— itory effects against H. §§£lKET and other seedufungi. Christensen and Daviis found that isolates of Bacillus mesentericus produced a heatestable substance which sup- pressed growth, inhibited or retarded germination, caused abnormal hyphal growth, and induced mutation in certain races of g. sativum (75). Ledingham and others found that treatment of wheat seed with formalin prior to inoc- ulation with H. saggypm, increased the amount of lesion- ing (225). They attributed the cause of increased infec- tion to the removal of the surface bacteria wnich other— wise interfere with the fungus. The existence of a bac- terial surfaceeflora on cereal seeds antagonistic to H. sativum was also demonstrated by Simmonds (555). Chinn and Russell controlled covered smut of barley[:Ustilago horde; (Pers.) Lagerh.) and loose smut of barley[:g. Q3g§:] by soaking the infected seed in brorh cultures of the bac- terium. Pseudomonas viscosa (Fr. & Fr.) Migula, or of an unnamed filamentous yeast (66). Soaking seed, under sim- ilar circumstances, in cultures of Bacillus subtilis or in water failed to give satisfactory control. Leben pt 31. examined the filtrates from cereal seed soaked in 55 water and found that they all contained several volatile acids (222). They concluded that these organic acids, an- aerobiosis, and perhaps antibiotic principles were involved in the control of grain diseases using the “watermsoak' method. Hearigm, primarily F. poae (Pk.) Wollenw —— -u Species of and H. gglmorum, are antagonistic to H. sgjivgm, and this antagonism may express itself in lower percentages of ker- nel-infection by the latter organism (224, 552). The path- ogenic activities of H. sativum are strongly inhibited by Phoma humicola Gilm. and Abb., Epicoccum purpurascens Ehr., and Trichoderma virigg (60). Alternaria geppis inhibits H. gramineum, H. sativum, Pullularia ppllulans, and Stem- phylium botryosum and is inhibited in turn by Chaetomium erectum, Curvularia trifolii, Hgsarium pipiliforme, H. poae, and Gibberella zeae (52). Christensen and Tuite isolated a slow-grOWing species of Hspergillus which proved to be strongly antagonistic to other subaspecies of the A. glau- gus group (72). The "storage" fungi, viz. species of .Aspergillus, Henicillium, uucor, and HHigppgg, have been observed by Wallace to invade cereal seed already infected with "field" fungi and to inhibit their growth (409). The epiphytic flora associated With the aerial por- tions of grain plants, and especially the kernels, may be termed.the "aerospnere" (182). Certain pronounced simi- larities exist among these organisms and have been noted by nmury workers. In general, most investigators are agreed 54 that the ubiquitous, small, gram-negative rods commonly isolated from grain kernels are not characteristic of the flora associated With fecal material (65, 525). The micro- flora of the aerosphere has been shown to contain many gram- positive cocci and gramwnegative rods (55). Some authors have emphaSized the similarity existing between kernel-flora and soilnflora (246). Others have concluded that this flora is distinct and separate from chance soil contamination (555, 410). Wallace and Lochhead found a greater Similarity be- tween seed bacteria and those of the "rhizosphere", which appeared to be intermediate in type between seed— and soil— bacteria (410). The similarity between the microflora of barley kernels and that of the air is welleknown and will be discussed in a later section. That a more or less con— stant bacterial and fungal population exists, with respect to species, has been suggested by many workers (222, 246, 555, 410). Some of the commonly-occurring microorganisms are obvious pathogens, others are indicative of suppressed or inhibited parasitism (255). According to the hypothe- sis formulated by Hollis (182), many of these organisms involved in "nonreactive” (i.e. not producing disease symptoms) relations with higher plants may become "reactive" (i.e. pathogenic) through the agenCies of mutation and nat- ural selection. Many of the associated microorganisms are undoubtedly fortuitous saprophytes and intergrading sym- biotic relationships of various degrees are probably in evidence. Little has been done in this research area and 55 the kernelwmicroorganism relationship awaits further eluci- dation. MATERIALS AND METHODS 0n the basis of information gained from the evalua— tion of various plating methods, routine plating procedures were ad0pted for the detection of barley microflora. In general the following methods were employed; 1. Plating whole kernels, ten kernels per plate, ten plates per sample on: a. Potato-dextrose agar b. Nutrient agar c. Barley agar d. Any of the above three plus actidione (100 p.p.m.) for bacterial determinations. e. Any of the above three plus Chlormycetin (50 p.p.m.) for fungal determinations 2. Plating suspensions of ground kernels with suit- able dilutions on any of the above-listed media All kernels were routinely surfaceedisinfected with the standard method of the author (500). Plates were incuba- ted for four to Six days, at room temperature (ca. 25°C.), and colonies were identified under low and high magnifica- tion. As indicated, isolates were transferred to other media and subsequently identified. In addition to the iden- tification and enumeration of microflora other factors were noted. These included: the association of microflora with injured or dead kernels, the location of organisms on the kernel, the interactions between organisms, and the ger— 56 minability of the plated kernels. Additional germination tests were carried out using the method outlined by the Amer- ican Society of Brewing Chemists (5). For routine population determinations, colonies of bacteria and yeasts were listed according to colony color and/or morphology. When these organisms were to be identi- fied, they were isolated in pure-culture using poured-plate, streaking, or Singlemcell techniques. Bacterial identifi- cation requires the use of a number of diagnostic tests. The following tests were routinely employed in the identi- fication of selected bacteria: Gramis staining, cell form, cell size, cell arrangement, motility, spores (location and size), indole production, hydrogenesulfide production, ac- tion on litmus milk, reduction of nitrates, Voges-Proskauer test, methylured test, utilization of citrate, catalase activity, hydrolysis of starch, liquefaction of gelatin, and growth on potato slants, agar slants, agar plates, and nutrient broth (82). Microflora for population counts and for identifi- cation were obtained from the following sources: (1) four varieties of malting barley grown at various locations in the United States in 1958, (2) four varieties of malting barley grown at East Lansing in replicated nursery plots in 1959, (5) five varieties of barley grown at two locations in Michigan in rod-row nursery plots in 1959, (4) 84 sam- ples of barley lots being malted and brewed in cooperative tests conducted by the Melting Barley Improvement Associa— -- \. -.. , 57 tion and the malting and brewing industry in 1960, (5) rough and smooth awned isowgenic lines of barley grown in 1955 and 1956 at various locations, (6) exposed plates in barley plots at East Lansing in 1959, (7) assorted barley samples collec- ted and received from various locations in Michigan. EXPERIMENTAL RESULTS The percentages of microflora obtained by plating barley kernels of several varieties from diverse locations and from different crop years are listed in Table 7. The germination capacities of these barley samples are also in- cluded in this table. The population determinations obtained for the Malting Barley Improvement Association samples are tabulated in Appendix C. The great number of bacterial isolates obtained in these plating studies precluded the possibility of making specific identifications except in a few cases. Some of the more significant reactions to stand— ard physiological tests are recorded for bacterial isolates from exposed agar plates, plated kernels, and barley leaves (Table 8). No attempt was made to establish the relative number of bacteria of varying morphological and physiologi- cal characters which are present in the air, on foliage, and infect barley kernels. The data in Table 8 merely dem- onstrates the bio-chemical potential of randomly-selected bacterial isolates. The various cultural tests performed with the bacterial isolates are not tabulated in this sec- tion since they are mainly of taxonomic value. Relatively mm o o o m mm H H m 0 mm mmoH op I\onooz o #0 o o o 0 mm H m d H mm mmmH op I\HHHmea o no 0 o o m mm m o H H mm mmaH op l\pquMHmm 0 90 H o o 0 mm o O mH H mm ommH op poprHM OCH 0 o o 3 mm o o mH H mm mmOH op HHHwHe mm o H o 0 em 0 0 mm o 50H ame 06 aHmopqO: mm H H o 0 an o H mH H HoH mmmH .neHz.mnHmneH.m nenonqem mm m o o 0 on o o m c an whoH .nOH: .onno nenenHm mm o o H H 0: o o o o OH mmaH qmsmnopmxmmm aHmopnoz mm m o o o d o d m o m mmmH pmmoo pmo3 madmm am m o o H mm m o H 0 so wan .non.npnsneatha eenenHm . no as V In Ha mi 0 an H V 1. q S 8 B d T... n... 8 TL U, K d B 0 I. B S Ti. Q a O a s 4 o D. e m e I O J 4 a O o J I. J m on 8 Ta 0 s r. u u K I. T. 0 Y0 n 4 e \ A&V m U n. m m m N U. hoHHmp m 1, n r. s e amok mmHHmn thomdmo % s m w Ho U mono mo QHmHHO SOHudesHow m mpdeopH l\.nHwHHo can hpoHHm> wQHHomeU mo mHoQHoM moHHnn Scam mHOHHouoHE mo COHpmHomH was I .5 opre 59 .mHquoM popde Scum pquaHopoQ .mHodHoM popme OOH Hog mmHQOHoo opomome mo gonads on» ma commommxm H mm o o o d 00 o o H H m wm®H op I\HHIHINd mm o o o o m: o o H m om wmoH op W\HHamam¢ me o o o 0 em 0 o m m 0H mmmH on W\HH:nam¢ no 0 o o 0 mm 0 o o d mH mmmH op W\HHImnmd do m o o o m o o O H mm mmmH op W\NHIHIH¢ mm o o o o e O o m m Hm mmmH op W\NHImnH¢ Hm H o o m me o o o s 0H mmmH on WxHH|nnmm to o o o m or o o o d mm mmmH .mH3 .somemz W\HHIMImm mm m m o H mm o H H H ma ammH op IWEHmopqoz mm o H o H mm H m w O on mmmH op ol\poequM Ho 3 H o H mm m m e H :5 mmmH on o I\enooz mm o H 0 BH on m o o m mm omOH op . IWHHHmHB um m o o d mm o o m 0 mo omaH .Q0H2.hpo mHoomde W\mmmfi&mfl@ mm m o o a Hm m o a 0 mm mmmH on /\9 oo H o o H mm m s m m am omoH .neHz .mnHmneq.a a He 6O .qzm npoosm mHmsvo :H: .dzm QmSOH mHmswo =m: I moaHH hoHHwn oHcmmomH ..mH3 Ho .>HQD .mpcmnm .w .m .Hm hmopsdoo mdeawm hoHHmm p .pmo>Hmn Hopmm mflpdos pano popmHm ”mpOHQ 59m 3095609 8099 ..>HQD opmpm .non .deHmno .m .m .Ha hmoppooo moHdswm m he .25 flflfl/ A nmnnepooo I . eh ".0. no. \ .. n ‘v 9 61 Table 8. ~ Morphological and physiological characteris- tics of bacteria isolated from the air, barley foliage, and barley kernels. Source:and total Humber ofgisolates tested Morphological and physiolog- Air (57) Foliage (52) Kernels (140)_ ical reactions (+) (_) (+) (-) (+) (_) Gram‘s stain/é 5O 25 25 9 57 105 Rod-shaped 45 5 26 2 156 4 Motility 48 5 28 2 101 54 Spores 6 47 15 17 18 105 Indole production 1 15 1 25 4 6O H23 production 10 6 7 2 49 11 Nitrate reduction -- —- 10 19 4O 9 Voges-Proskauer l2 5 25 5 45 19 Methyl red 15 4 6 22 25 59 Citrate utilization 59 9 25 4 69 25 Catalase activity -- -- 28 —- 4O 5 Starch hydrolysis —— -- l6 12 27 51 Gelatin liquefaction 18 -- -- -— 9 -- a Read Gram's stain as ositive or negative, with all other tests read yes for (+ and no for (-). Blank spaces in- dicate that an insufficient number of tests were performed. 62 few bacteria have isolated and identified from barley ker- nels (Appendix B). The bacteria which were identified to species in this study are listed below (Table 9). Other organisms which have either, not been previously reported as associated with barley kernels, or which have been rar- ely isolated are listed in Appendix B. DISCUSSION AND CONCLUSIONS With the exception of the obligate microorganisms, it would seem that any organism possessing a broad-spec- trum enzyme system is capable of infecting barley kernels. Successive qualitative investigations invariably reveal the presence of previously unreported microflora on bar- ley kernels. The mycoflora of barley grains are, by far, better characterized than the bacterial and yeast flora. Certain technical difficulties have prevented an equiva- lent advance in the knowledge of the bacterial flora of grain kernels. The extremely large populations of these organisms on a single grain, as well as their rapid repro- ductive and physiological potential, are difficult to ac- curately assess. This is in part due to a lack of suit- able methods for the study of bacterial populations. The ,identification of only a few bacterial isolates is time- consuming, the task becomes nearly impossible when the flora of a small sample of grain is considered. Various quantitative methods have been devised to circumvent a 3part of this difficulty (of. Chapt. 1). By using stand- 65 Table 9. w Bacterial species isolated and identified from barley kernels and barley leaves. Source of organism Name of organism Frequency of occurrencg (a) (b) From: Leaves & kernels *Bacillus cereus Rare Rare Fr. & Fr. do ‘H. mggaterium D°Bary Seldom Seldom do *H. ppmilus Gotth. Common Rare do *B. spptilis Cohn do Seldom gmend Prz. do 'Pseudomonas spp. Rare Common do *Xanthomonas translu- Common do cens (J.,J.,&R.) Dows. Leaves only ‘Bacillus coagulagg Rare —— hamm. Kernels only ‘Achromobacter sp. we Rare do ‘ngobacter aerogenes -- Seldom (Kr.) (Beijj) do ’A. pigggag (Jord.) -« Common Berso 22 sl do ‘H. gereug var. migoideg -- do (Fl.) Smith 33 g; do H. mggatheriumucereus -- Seldom intermediates do ’B. ppgymyxa (Prz.) -- Common Migula do ‘BreVibacterium linens -- Rare (Weigm.) Breed do *Kurthia zopfii (Kurth) -- do Trev. (?) do *Coryneoacterium sp. -- do do Erwinia rhizogenes -- do (Rk. g3 22) Dows. (?) 64 Table 9. - Continued Source of organism Name of organism Frequengy)of occurfggce do *Pseudomonas atrofa- -- Common ciens (McC) Stevens a On leaves b 0n kernels t Species accepted by Breed £3 E; (54) 65 ardized procedures an investigator can accurately deter- mine the number of viable single cells occurring in a given sample of grain. These methods, however, do not yield qualitative information, nor do they provide an in— dex to the biochemical potential of a bacterial population. The bacterial isolates identified in this study do not represent the dominant flora of barley kernels (Table 9). These identifications were dictated by two factors: (1) the common occurence of the organisms, and (2) the inherent morphological and/or physiological properties of the organisms which facilitated their identification. Thus the large number of chillus species, commonly found in soil and water, is not a true index to the predominant bacterial flora of grain kernels. Varietal resistance to kernel-infecting microorgan- isms is poorly understood. If such reSistance exists, it is probably manifested quantitatively rather than qualita- tively (cf. Chapt. 5 and Appendices B and C). The origin of the seed is of great importance with respect to both the numbers and kinds of microflora present on and within barley kernels. Barley grown under irrigated conditions and having a bright appearance is relatively free of fun— gal infection. The one exception to this statement is seen in the case of superficial infection by Cladosporium spp. High counts of this fungus may be made from bright barley kernels, showing no visible evidence of fungal in- fection (see Chapt. 5 and Appendix C). Generally, bacter- 66 ial infection is less severe in bright barley kernels than in discolored grains; however, occasional bright samples may show a higher percentage of bacterial infection than a comparable stained sample (see Appendix C). In the case of discolored kernels, a positive correlation exists be- tween the origin of the seed and the number of field fungi present on and in the seed. So much so that the origin of the kernels may be determined from a microfloral analysis of the barley sample (280, 281) (Appendix C). The field organisms, both fungi and bacteria, are more abundant in the middle and awn regions of the kernel. This has been established by observations made on many pla- ted kernels, as well as by the plating of transverse sec- tions of barley kernels (see Chapt. I) and the use of histo- logical procedures (see Chapt. 5). As indicated earlier, this generalization does not hold for certain bacterial and fungal pathogens, nor is it valid in the case of the stor- age fungi (Chapt. I). A The barley samples, provided by Dr. Shands, yielded low fungal and bacterial populations, when contrasted with the place of origin and the color of the kernels. It is very likely that this is a reflection of the age of the kernels, viz. four and five years old, since the viability of kernel-infecting fungi decreases rapidly after several years. The bacterial counts made from these samples did not, apparently, decrease in the same order as the fungal counts. No information exists on the longevity of the 67 bacterial flora of barley kernels. The data indicates that little or no differences exist between the bacterial species found in the air, on barley foliage, and on barley kernels (Table 8). This may be an unwarranted assumption inasmuch as the isolation methods are themselves selective and may, therefore, preferentially screen out the less-specialized bacteria. The ability of many of these isolates to utilize sodium citrate as the sole carbon source, to hydrolyze starch, and to liquefy gelatin indicates that these organ— isms are able to infect and colonize a wide range of host substrates (Table 8). The more commonly occurring fungi, found on barley kernels, have also been shown to possess broad enzymatic properties which suggest that the fungal flora are similarly capable of attacking a variety of host substrates (500). CHAPTER III THE ETIOLOGY OF HARLEY KERNEL INFECTION LITERATURE REVIEW Developing barley kernels are subject to infection, invasion, and establishment by microorganisms. These ker- nel invaders comprise four general classes: the parasitic fungi and bacteria, the "field" fungi, the "storage" fungi, and the nonaparasitic bacteria and yeasts. The "field" fungi are those that invade the developing and mature ker— nel prior to harvest. They include species of Alternaria, Helminthosporium, Fusarium, Epicoccum, Cladosporium, and several others. The "storage" fungi rarely, if ever, are found on well-conditioned kernels prior to harvest. These species of Aspergillus, Penicillium, and certain Phycomyce- tes invade the kernel after harvest, expecially in damaged grain stored at high moisture levels. The "field" and "storage" fungi have been treated by Christensen (69, 70) and Tuite and Christensen (598). While certain barley pathogens are included in the "field" fungi group, viz. Helminthosporium and Fusarium, other parasitic species are generally considered separately. These include Claviceps purpurea (Fr.) Tul., the bacterial pathogens Xanthomonas translucens (J.,J., & R.) Dows. and Pseudomonas atrofaciens (McC.) Stevens, and the three Ustilago species. General discussions of the life-cycles, symptomology, etiology, and control of these organisms are found in the works of 68 69 Dickson (99, 100), Leukel (229), and Fischer (117). The non-parasitic bacteria and yeasts have not been comprehen- sively dealt with and references to their etiology are widely scattered through the literature. Previous studies on the etiology of the kernel- and floretuinfecting organisms of barley and other cereals have employed several methodological procedures. Whitehead in a study of the seedeborne organisms of barley and other cer- eals employed spore-traps to determine the numbers and kinds of available airwborne inocula (415). He also used plating— and histological-techniques to determine and/or confirm the mode of infection by these organisms. Similar techniques were employed by Whitehead and his comworkers in a study of cereal and legume seed~infection by Microascus trigonosporus (416). Koehler used a combination of histological and pla- ting techniques to determine the developmental stage at which corn ears were infected by Fusarium moniliforme and other species (216). Natural field inoculum has provided infection in several of these studies (9, 216, 556, 415) while other workers have inoculated developing kernels with suspensions of the organisms under study (ll, 12, 174, 175, 192, 264, 554, 541, 596, 404, 416, 425). The time of infection, location and form of the mi- croflora, as well as the qualitative and quantitative dif- ferences in the microorganisms of barley kernels are condi- tioned by several factors: the anatomy and physiology of the 'host'~kernel or ~floret, the availability of inoculum, 70 the macro- and micromenvironment, and the nature of the microorganiSms themselves. Thaysen and Galloway stated that grain florets provide particularly good growth-centers in that they furnish exudations for the nourishment of the in- fecting organisms and the glumes provide protection against the dessication of the microflora (590). A further protec- tive function of‘the glumes is noted in cereals, such as barley, in which the glumes fuse with the caryopsis during ripening. The cementing of the barley hull effectively shields the enclosed microflora against the external envir- onment. The relation of kernel—anatomy to the microflora of cereal grains is also discussed by Hyde and Galleymore (191). Harlan has discussed the developmental anatomy of barley kernels in some detail (165) and has shown that the moisture content of barley grains decreases from flowering to maturity (167). Similar data are presented in a paper by McLean (261). At the time of flowering the floret has a moisture content of approximately eighty percent. A steady decrease in moisture results in a moisture content of fifteen percent or less at harvest. As the moisture content of the kernel decreases, a sticky substance is se- creted by the caryopsis which causes the glumes to adhere thereafter to the developing kernel. As the kernel increae ses in size the underlying tissues of the pericarp are sub- jected to severe compressive forces. These tissues become compressed and distorted and are greatly reduced in thick— ness at maturity (165, 167, 191, 261). All of these fac- tors influence the injection process by microflora. The microflora infecting barley kernels are predom- inantly those speCies found in the atmosphere as spores, cells, and mycelial fragments. The occurrence of air-borne inoculum in relation to plant disease has been discussed in a reView paper by Stakman and Christensen (572). Hirst has presented a general discussion of the liberation and the dispersal of fungal spores (179). The trapping and iden- tification of airmborne organisms generally is carried out by means of mechanical sporemtraps, coatedmglass slides, or by exposing petrieplates containing culture-media. These methods have been discussed and evaluated by Gregory (148). A large number of papers have been written in which the air- spora of various locations is described. Almost without ex- ception, these determinations have shown that the most com- monly—occurring fungal spores in the air are species of Cladosporium (148, 149, 189, 219, 296, 570), Alternaria (219, 296, 570, 571), Sporobclomyces (148, 149, 219). Helmintho- sporium (57C, 571). and Tilletiopsig (219). Other fre- quentlymoccurring air-borne Spores are those of Er si he, Ustilago, Penicillium, Aspergillus, Epicoccum, Fusarium, Pullularia. Stemphylium. Rhizopus, Mucor, and several other yeast and fungal species. Gregory and hirst (149) reported that fungal spores appeared to be more numerous in the air than bacterial cells. They concluded that soil-microorgan- isms did not appear to constitute a significant proportion of the air-spora. Fluctuations in the number of spores 72 present in the atmosphere have been reported for many of the commonlymencountered fungi. Diurnal periodicity has been noted for the spores of Helminthosporium (571), H;— ternaria (219, 571), Epicoccum, Stemphylium (571), Clado- sporium (189, 219). and several other species. Meteorol- ogical conditions may also affect the number of spores pre- sent in the air; e.g. Sporobolomyces (219) and Fusarium (165) appear to be favored by wet weather, while Clado- sporium (189) and Alternaria (219, 571) are more prevalent on dry days. Seasonal variation in spore numbers has been noted for most of the commonly occurring fungi (189, 219, 571). Spurr noted that conidia of H. sativum did not occur on exposed slides until late in the growing season (569). He concluded that air-borne inoculum originating from straw was the principal source of inoculum for barley kernel in— fection in Michigan. Simmonds and others (558) have re- ported that H. sativum spores were rarely present on spore— trap slides exposed in western Canada. The authors claimed that development of the fungus in the field was restricted by indigenous bacteria and that the spores were not well adapted for air dissemination. The use of disease-free seed was advocated by Steinkraus and his co-workers in order to produce diseasemfree plants which would in turn produce larger quantities of disease-free seed (576). In the case of several of the barley kernel-infecting organisms this supposition may not necessarily hold true since air-borne inoculum is capable of traversing large areas while re- 75 taining its viability (572). Additional inoculum sources exist also in the form of over-wintering organisms. Sev— eral of the barley organisms, including H. sativum, and H. culmorum, have been shown to overwinter readily under nat- ural conditions as far north as Edmonton, Alberta (119). Many workers have noted the correlation between hu- mid weather and the increased incidence of kernel-infection by both fungi and bacteria. Presumably high humidity and the presence of free moisture influence infection by alter- ing the composition of the airwspora and by creating a micro- {climate suitable for the invading microorganisms. The sub- ject of micro-climate and infection has been generally dis- cussed by Yarwood (422). Hyde has presented evidence that the degree of wheat kernel infection by "field" fungi is dependent upon the atmospheric humidity during the ripen- ing of the grain (190). Whitehead noted that drier cli- matic conditions reduced the percentage of barley infection by Alternaria Spp. (415). Machacek and his fellow-workers observed that harvested or threshed grain from eastern Canada possessed higher percentages of fungi, both para— sitic and saprophytic species, than did western grown seed (246). Greaney and Machacek found that severe infection by Helminthosporium and Fusarium was usually associated with wet years and more particularly with years in which wet weather had prevailed during the later portion of the growing season (144). Gordon has reported that Fusarium infection of the cereals is more prevalent in eastern areas 74 than in western Canada (155, 154). Hind has stated in his brewing text that bacteria are most abundant in dry weather, the fungi occur in greatest quantity in damp weather, and that yeasts are most prevalent during the time of fruit ripening (178). The etiology of infection by species of Helmintho- sporium has been intensively studied. Leukel and his co- workers have discussed the mode of infection of barley ker- nels by H. gramineum (250). The kernels were shown to be infected by air-borne Spores of the fungus. Infection oc- curred for a considerable period after flowering. According to Yu, the most susceptible period for infection by the fun- gus was the milk or green mature stage or at any intervening period (425). Andersen has shown that infection of wheat kernels by H. sativum is conditioned by the developmental stage of the host (12). Little infection occurred on inoc- ulated wheat heads prior to flowering. Susceptibility in- creased from the flowering to the past-flowering stages. Heavy infection during the flowering and after-flowering stages resulted in reduced yield while infection in the later stages of host development affected only quality. Henry found that H. sativum invaded wheat kernels chiefly during the milk stage after inoculating wheat heads with the fungus (175). Scott and Sallans also sprayed spore suspensions of H. sativum on developing wheat heads (541). They concluded that most infection occurred during the blossoming period of the florets but that infection could occur at any later stage in kernel development. Vendrig 75 reported that many seed of barley and wheat failed to de- velop following inoculation by H. sativum at the time of flowering and that susceptibility continued.unti1 twenty days after flowering (404). Mead intensively studied the infection of barley kernels by H. sativum (264). He showed that if inoculation of barley occurred at, or soon after flowering, the ovaries were killed. If inoculation occur- red from five to ten days later, the glumes, pericarp, and lodicules were invaded and areas of necrotic tissue occur- red in these organs. Usually there was some shriveling of the grain at this stage of inoculation. Some infection of fifteen to twenty day kernels was also obtained, but it was confined mainly to the pericarp and glume tissue at both ends of the kernel and no shriveling of the grain occurred at this stage. Atanasoff concluded that the greatest amount of in— fection of grain kernels by Fusarium species was obtained after inoculation of wheat heads during the period after flowering (21). In an early paper, Schaffnit showed that infection of grain kernels by H. nivale was caused by air— borne spores lodging between the grain and the glumes of the kernel (556). He reported that infection could occur from the late—dough stage until maturity of the kernel. Andersen found that wheat headblight infection, caused by Gibberella zeae, varied with the stage of seed develop- ment (11). Little infection was obtained on inoculated heads prior to flowering. Susceptibility increased from 76 the flowering to past flowering stages with decreasing sus- ceptibility at later developmental stages. Fusarium head- blight infection occurred from pre—flowering until the soft dough stage in small grains inoculated with spore suspen— sions of the fungus (595, 596). Haskett inoculated spring barley with g. 2232 and reported that the kernels were sus- ceptible at any developmental stage with the critical period for infection occurring from flowering to the milk stage (174). Dounin found that many of the scabbed kernels of barley and other cereals were infected with Fusarium in shocks after harvesting (104). The kernel-infecting spe- cies of Fusarium have been reported by several authors to be favored by hot, moist weather (11, 21, 201, 556). Cro— sier and Waters have attributed the high incidence of Fusarium infection of small grains in New York state to cool, humid climatic conditions during the growing season (86). Dickson (99. 100) and Haskett (174) have reported that kernel infection by Fusarium spp. is initiated by entry of the fungus through dehisced anthers. Atanasoff (21) asserts that the fungus invades the kernel at any point at which contact is initially made between the kernel and the pathogen. Tu concluded that the flower or anther was not necessarily the point of initial infection and that the glume seemed to be the more common site of infection by Fusarium (596). Wheat kernels were inoculated with spore suspen— sions of Alternaria by Sallans (554). He obtained kernel 77 discoloration most frequently when the kernels were inocula- ted during or after the soft dough stage. Whitehead found that approximately one—half of the infection of barley ker— nels occurred from the period of flowering to the time of the cementing of the glumes to the caryopsis (415). John- son and Hagborg (205) stated that the main course of infec- tion of wheat kernels by Alternaria tenuis was from spores entering the florets usually at the tip of the lemma. Dead floral parts such as anthers and stigmas favored the estab- lishment of mycelial growth. Fungal mycelium was rarely found in areas beneath the interior of the lemma. Macha- cek and Greaney reported that Alternaria spp. do not seem to I be as actively parasitic as H. sativum in infecting grain kernels since they do not usually attack the glumes until these tissues have withered and died (247). They specula- ted that Alternaria infection was a direct-contact process with infection occurring either during the blossoming per- iod, when the florets are expanded, or near kernel maturity, when the glumes are separated. These assumptions were based on the unavailability of inoculum during the early stages of development and the correlation between kernel-plumpness and the presence of "kernel-smudge" microflora. Whitehead and his co-workers observed that blossom-infection of bar- ley and other plants occurred with Microascus trigonosporus (416). The fungus apparently persists in the enveloping tissues of dormant seeds and caused no apparent damage to the development of the host (416). They indicated that ,- u ,. . .- a a u‘ "tol 78 this type of infection might be characteristic of the group of fungi commonly isolated from plated seed. Five varieties of barley were tested for microflora in the milk stage, early dough stage, and at maturity (9). Alternaria was shown to appear soon after the appearance of the heads. HH- sarium infection ranged from ten to eighteen percent in the milk stage and increased to forty to fifty percent at matur- ity. The greatest increase in bacteria and yeasts occurred at about maturity and varietal differences in the percen- tage of infection by several organisms was also noted. Varietal differences may exist in susceptibility to the various kernel-infecting bacteria, yeasts, and fungi isolated from barley (9). Five barley varieties were com- pared as to their relative microfloral populations. In all cases Kindred kernels gave the lowest percentage of infec- tion while Moore yielded the highest number of bacteria and yeasts. More Fusarium infection has been noted on the kernels of oats and barley than on wheat (155, 154). This difference was attributed to the presence of hulls on oats and barley. Greaney and Machacek found some varieties of wheat, oats, and barley were more susceptible to internal infection by species of Helminthosporium and Fusarium than were others (144). Immer and Christensen found that sus— ceptibility of barley varieties to kernel-infection by Helminthosporium and Fusarium species was usually corre- lated with the date of heading (192). In general, they observed that barley varieties that were resistant to .hP‘ o o... 79 Fusarium blight were also resistant to Helminthosporium blight. Wallace has asserted that Elite, Foundation, and Commercial seed of small grains is equally susceptible to fungus attack and that hulleless seeds are more suscep- tible to fungal infection than are seeds with hulls (409). A similar observation has been made by Smith (562). Links has suggested that smoothuawned varieties of barley are more susceptible to infection by the kernel-staining fungi (256). He attributes this difference to the more access- ible space between the kernel base and lemma to rain and dew, in turn facilitating infection by the fungi. Studies on the mode of kernel—infection by bac- teria and by yeasts are not as abundant as those dealing with the fungi. Lund isolated several species of both white and pink yeasts from barley grains by the use of special plating techniques (242, 245). He found that while many yeast cells were present on the kernels prior to harvest, only a few cells were isolated after thresh- ing had taken place. One of these yeasts, Sporobolomyces, has also been isolated in quantity from the surface of bar— ley leaves with the aid of special techniques by Last (218). Lund observed that Sporobolomyces and Rhodotorula were apparently favored by very hot and dry climatic conditions (242). Last concluded that the number of colonies of Sporobolomyces was correlated with high humidity (218). Bamberg has presented an intensive review of the litera- ture dealing with the black-chaff organism, Xanthomonas A . .uy 8O translucens (24). The bacterium typically enters the host- tissue through wounds or natural openings. Moderate tem- peratures, high humidity, light, and the age of the host all influence the infectivity of the pathogen. Continued per- iods of wet weather were reported to favor epidemics of the disease. Boosalis has stated that this bacterial patho- gen is also favored by the rootmrot and leaf—spotting fungi which apparently premdispose the host tissue to attack (49). Thomas and others have noted that the number of coli-aero- genes bacteria associated with developing grain kernels in- creased during the growing season (592). Various other factors have been shown to influence the invasion of barley kernels by microorganisms. The in- creased use of combineoharvesting of barley and other crops has increased the danger of high moisture contents of the harvested grain as well as the incidence of skinned, bro- ken, and otherwise mechanically damaged seed (140). Grain stored at moisture contents above twelve percent are sub— ject to invasion by the storage fungi which subsequently cause deterioration of the seed (45, 69, 70, 226). Hurd has shown that damaged kernels of wheat and barley permit the invasion of Penicillium and Rhizonus species (188). Exposed embryos of wheat are more susceptible to mold in- vasion by H. sativum and other species according to Mead and his couworkers (268). Weak-strawed varieties of bar- ley are subject to lodging. The heads of lodged grain may come in contact with the soil thus enabling the infection 81 of kernels by several species of soil-flora to take place. Swathed and windrowed grain may be infected in a like man- ner, especially when heavily rained upon (226). MATERIALS AND METHODS The progress of infection by "field" fungi, bacteria, and other organisms was studied at successive stages of de- velopment of the barley kernel. Four varieties of malting barley: Hannchen, Traill, Kindred, and Montcalm were planted in replicated rod~row nursery plots at East Lansing. The varieties were planted at the rate of 40 grams per plot (1.7 bushels per acre). All plots were planted, harvested, and threshed with standard experimental—nursery equipment and methods. Date of planting was April 16, 1959; date of har— vest was July 15, 1959 for Kindred and Traill, and July 20, 1959 for Hannchen and Montcalm. Glass slides, coated with glycerol, and petri plates containing either potato-dextrose or nutrient agar were exposed daily at five locations in the field. The slides were taped to the sides of upright 2x4's at the same height as the barley inflorescences. The ex- posed plates were placed on the t0p of a small platform affixed to the same 2x4°s. The slides were exposed for 24 hour periods, the plates for a period of approximately ten minutes. The numbers and kinds of available air—borne inoc- ulum were later determined from counts obtained from these exposed 'traps'. A scale was devised denoting progressive stages of 82 kernel development from preuflowering to maturity (i.e. harvest), patterned after the developmental stages of bar- ley discussed by Harlan (165). This scale is listed below: STAGE NO. 1 \OWQO‘W‘PWN H F’ »* NHO H \N 14 DESCRIPTION 9H STAGE Inflorescence in sheath, completely enclosed Inflorescence in sheath, flowering Inflorescence in sheath, awns emerging Inflorescence partly emerged from sheath Inflorescence emerged from sheath Early milk stage Early—intermediate milk stage Late-intermediate milk stage Late milk stage Early (soft) dough stage Intermediate dough stage Late dough stage Mature, one week prior to harvest Mature, at harvest Florets and kernels were harvested from each variety at each stage of development. Each sample was sub-divided; one half was placed in strong FAA ( 200 ) to fix and pre- serve it for subsequent histological work, the other half was transferred to the laboratory for microfloral deter- minations by means of kernel-plating techniques. Surface- disinfection time was shortened to onewhalf minute for stages one through four because of the delicate nature of 85 the tissues. The lemma and glume awns were not removed from the florets in either the plating or the histologi- cal procedures. The fixed florets and kernels were removed from the FAA, infiltrated, and embedded in a 1:1 mixture of embed- ding paraffin and "tissue—mat" (Fisher) using standard histological procedures (200). The embedded tissues were serially—sectioned with a rotary microtome at from 10 to 14 p, depending upon the structure of the tissues. A modifi- cation of Conant‘s quadruple-staining procedure was employed to differentiate the kernel tissue and the attendant micro- organisms (200). The staining schedule is listed below: 1. Place slides in xylene for 10 minutes (to re- move paraffin) . Transfer to xylene bath for five minutes . Transfer to absolute alcohol for five minutes 2 5 4. Transfer to 95 percent alcohol for five minutes 5. Transfer to 70 percent alcohol for five minutes 6 . Place slides in safranin (50 percent alcoholic solution) for two hours 7. Rinse in distilled water 8. Place slides in thionin (1% thionin in 4% aqueous phenol) for one minute 9. Pass slides quickly through three rinses of absolute alcohol 10. Place slides in clove oil plus fast green plus gold orange for two minutes 11. Pass slides through four baths of clove oil plus gold orange for two minutes each 12. Pass slides through two rinses of xylene and store in xylene until cover slips are applied -..H 84 Following this procedure the slides were cleaned and the cover slips were applied. A total of 56 sets of slides, each set containing an average of 15 slides, and each slide consisting of approximately 15 sections, were prepared. This large number of preparations made it nec- essary that only a representative sample be subjected to micrOSCOpic observation. Consequently a slide was se- lected from each end of the serial series and these two were read together with the middle slide in the series. The possibility of chemical control of the "field" organisms was also studied. Five chemical compounds and an untreated check were used in conjunction with the four varieties and three replications discussed above. The chemical compounds, manufacturer, and other information is presented below. Treatment Code name/number Manufacturer Rate of number application (per 4 gal. water) 1 Form 67 (Actidione) Upjohn 24.0 ml 2 Untreated check ---------- 5 CuP (copper omadine) 01in Matheson 1.6 gm 4 CR~5009 (Ni fungicide) Rohm & Haas 1.6 m1 5 0-5818 B ( do ) Rohm & Haas 1.6 gm 6 Wettable sulfur ————— 75.0 gm. It was decided that, if chemical control of the staining or weathering organisms was to be economically feasible, only one application of the compound could be made. This ,-. . '- 85 premise is dictated with field crops as a result of the relatively low cash return per acre. Consequently the application was made, using a portable field sprayer, on June 27, 1959. This date coincided approximately with the developmental stage of the four barleys designated number seven, i.e. the mid-point of kernel development. Analyses of variance were run with all four varie— ties in order to determine whether significant differences existed between 1,000 kernel weights, bushel weights, yields, treatments, varieties, replications, and dates of harvest. EXPERIMENTAL RESULTS The exposedwslide technique employed in this por- tion of the study was not refined enough to use as a quan- titative estimation of the air-borne inoculum present in the air around the experimental plots. Such a method is of great value in determining the diurnal and seasonal variations in airnspora but only when some procedure is used for measuring time intervals on the slides. The fol- lowing data were obtained from the exposed slides:_(1) H;- ternaria sp. spores were present in moderate amounts throughout the period of kernel development, (2) Helmin- thosporium spores were not observed on the coated slides until late in the season at a time corresponding to devel- opmental stage eleven (see p. 82), (5) large numbers of unidentifiable hyaline fungal spores (powdery mildew?) .‘s 86 were observed throughout the experimental period (Table 10). No significant differences were noted between plates exposed at the same time at different locations in the bar- ley plots. The chOice of media exerted a selective effect on the type and number of colonies developing on the exposed plates. For the determination of bacteria and yeasts by this method, nutrient agar plates gave the best results (Table 10). Nutrient agar was decidedly inferior to pota- to—dextrose agar for the trapping of fungal air-inoculum. Exposure periods in excess of ten minutes had two unwanted effects: (1) the culture media dried out rapidly, and (2) the number of deve10ping colonies became too numerous to accurately count. 1n general all of the species of micro- flora commonly isolated from barley kernels were present on the exposed plates. Differences in the amount of air- spora present in the field and the amount of infection obtained on kernels by certain of these organisms were pronounced in several cases. Cladosporium sp. was gener— ally the most prevalent fungus obtained on exposed plates and slides. However, the amount of infection obtained with this fungus on barley kernels was negliglible. Simi- lar differences were obtained with Dematium pullulans, Candida, and other fungi. In addition to platings of the fourteen develop- mental stages of kernel maturation, a plating determin- ation was made of samples which had remained standing for several days after harvesting (Fig. 5). A11 barley varieties 87 pdomohg 0.m o.H o.H 0.: 0.0 0.0 N.0 ¢.H 0.0 N.m .mm wsonm mml0ml0 astpwm .m 90 mean o.H o.o m.H 4.0 4.H 0.0 4.0 m.o m.m m.o newsman pnnHm omummuo o.H 4.0 o.m o.mH 4.0 4.0 s.H 4.0 4.0m 0.o In- mmuemum nepeanmpnoo nepeHm nun omummuo m.o o.o m.o H.mH 4.HH m.o 0.0 8.0 e.om 0.4 II. mmummuo nomomxo mopMHm 02 III mmuHml0 pnomomm HoMHq 0.0 e.# 0.0 ¢.¢H 0.H 0.0 m.0 m.0 0.: 0.H msHHHmnmmm< mmloml0 4.0 0.0 0.0 m.mm m.m a.o 4.0 0.0 m.mH m.m mmamHuo 0.0 0.0 ¢.0 0.0 0.m 0.H 0.0 0.m N.0m 0.m ill mmanl0 0.0 0.0 m.0 0.0 0.H 0.0 m.H m.m 0.0H 0.: an: mmIbHo0 O 73v 41 4M 1 a H A no V 4 efls nee ecu. a U. .0 n TL .L U. Una JHL 1.; n. .A I. s s n+ 8 _L. 9 .L. I T: 1. S O O B D. 8 J o J a o s e 4 o o J o J For n s m o it s u Tit. 0, pk o m .d e ITL o. e o m o J I. I e o e I I. m m w . m. r. e ondmom 3 “WI mxuwaom 1N0 opme m\doHnoQ onsmomxo opssHa 0H . Ho open Hog opMHm Hod moHsoHoo mo Hopasa owwno>< .mopmHQ anom domomxo hp uqusHopov mm qumsmH pmmm pm mpon nHmnw map pdonw HHw on» GH mnoHHOHOHs Ho coconsooo one I .0H oHpma 88 Ho gonads ”cmpos omHznmnpo mmqud .0omd Hmmm omompNoUIopmpom pmHmHUHowInoz .ommo Home sH mmpMHQ mwa 90 ommuobm Gm mpsmmmnmme moHHOHoo m pdmmomm 3.H m.0 3.0 0.0H m.H 0.0 0.0 0.0 3.5 m.m meneeseoanea 0mI0Is 3.0 0.0 0.m 0.mm 0.0 0.0 3.0 0.m 0.0H 0.0m III omIqu comOQXo mopmHm 02 III mmIBIm m.H 3.0 3.H 3.mm 0.H 3.0 0.0 3.0 3.0m 0.mH III mmI0I0 3.0 0.0 3.0 3.0H o.H 0.0 0.H 0.m 0.mm 0.0H III mmImle mopmHm 9000 use 0.3 0.H 0.0m m.mm 0.mH 0.0 0.0 0.0 m.mm 0.0m IHnnsn cheeses 0mI3Is pdomomd 080nm new 3.H 0.0 0.0 0.3m m.0 m.0 0.0 m.H 0.00H 0.0H mopmohsosHpo< mmIqu pnmmoem maonm 0.H 0.0 0.3 0.MH m.m 0.0 0.m 0.0 0.33 0.0H quH oHeoHuom mmImIB mmdon m msHooooon 0.H m.H 0.m m.0 o.H 0.0 0.0 3.0 0.0H 3.m I nHen s>eem 0mIHIe 0.H 0.3 0.3 0.m 3.m 0.0 m.H 3.0 0.5m 0.3 III 0mI0mI0 0.0 0J0 0.H 3.mH 0.H 0.0 m.0 3.m 0.0 m.0H III mmImmI0 3.H 0.0 3.m 0.HH m.H m.0 0.0 0.H o.H 3.0H III mmImmI0 demoaxo mmpmHm oz III mmI0NI0 wmanpcoo I .0H oHpne 89 performed similarly in their susceptibility to microfloral infection (Figs. 5a, 5b, 5c, 5d). The progress of fungal infection was very similar in all cases. Fungal infection began at a later stage than did bacterial infection. The shape of the curves for bacterial and yeast infection was more similar for the six—rowed varieties than for the two- rowed variety (Fig. 5d). In all cases, bacterial and yeast infection decreased after the milk stage in the six-rowed barleys and after the intermediate-dough stage in Hannchen. The optimum stage for bacterial and yeast infection in the six—rowed varieties was the intermediate- to late-milk stage and for Hannchen the intermediate-dough stage. Fun- gal infection increased in all varieties up to harvest. Little or no fungal infection was obtained prior to the early milk stage. The plating data were corroborated by the histologi- cal evidence. Slide preparations however, gave little in— formation as to the degree of infection. In almost every case, fungal mycelium was found in abundance only at the awn end of the kernels i.e., at the point of juncture be- tween the palea and lemma (Plate II). Hyphae were also observed in tangential sections of the glumes which indi- cated that infection and ramification of the mycelium takes place on the sides of the kernel as well as at the ends (Plates III & IV). In several cases mycelial frag- ments and/or spores were found lodged under the epidermal barbs of the hull in the mid-kernel region (Plate IV). 90 AN0 .0 .H00 pdeQOHm>00 Homemx Ho 000nm .JH mH NH HH OH m w B 0 m 3 m m H _ e tIl ---I-..7.II- _ _ _ _ _ _ _\ I. H .__ EH IT L 7 _. H 41.xu H 0 \.\ \ \ x I- II 0m % i n i m i m i s \ 1.. 03 m. \ r. U \ H... \ m. II 00 I \ m \ \ , mpmteh w mHumpomm \ om \ \\ FL I. I..- ‘ i _L _. I--_;,-.L:.-L-I( OOH AUQHUGHMV PC08QOH®>®U H®GH®M HO mmWMPm ®>Hmm®HmOHQ pm wHOHHOHOHE kn COHDO®HQHImm mhfimflm AN0 .0 .ro oanmonboc Hosnom no ommpm 3HMHmHHH0H0050m3mmH w. e PILI-» I» r H r FI»IPl’-l$llll+ wH H 1 HI H H H .H a H\\ .0 \\ H \_ \ \ \ lrdN .lI03 \ \ .lr00 \ mpmwoh a mHnopowm 1I00 P P p F in. I? b F L _ L _ .OOH AHHHmHBV \pdoaQOHonroHv HodHouH Ho mowmpm o>HmmonOHm pm mHOHHoHoHHH kn noeptoHdHlnm ondwfim uorioegur :0 eSequeoded AN0 .9 .HoV pdeQOHo>00 HdeoM Ho owwpm 3H MH NH HH OH 0 w B 0 0 +3 m N H i _ i _j._.._, _.i- i _ ti 1 \ O I IIom a 9 a 0 9 U n... B I. I4 03 Aw 0 H2 \.\.III .4 o, I. U I. 3 I l8 m. T... O U I -100 _ Ins-Ir - A e L H h h _ i. i 1 8H nSHwOHQOSv.MQmEQOHm>®U Hmflhmx HO memMPm mbfimmothHQ pm mHOHHOHOHfi ED GOHpomHGHIOM mHDMHh 95 30 cm {HOV pnoaHHOHoboc HonHoM no 00090 1.: NH «_H H_H 0H 0 w s 0 m 3 m m H H i _ _ L. h_ LH * _ I» w H_\\HI.TI H. 0 \ x \ x I \ lIom % \ a \ m \ \ e .I Henna \ I03 0 \ I. \ r. x m. \ .l \ 4100 M. u // \ \ / \ \ mpmwmh em mHnopomm I I ‘ I II II Il. \ \ \ .I I Iron 1 _ _ _ i i h _ _ e _ i _ 00H J \ . Adonodddmv pamEQOHmbmd Hodnmx Ho mommpm obflmmohmonm Pm MHOHMOHOHE hfl QOH#00HQHIUM whimflh 94 PLATE II Fungi in the glume tissue of barley kernels° Upper — Fungal mycelium ramifying through the glume tissue of a longitudinally—sectioned kernel of Kindred barley, developmental stage 13 (magnified ca. 1,000 X)o Lower - Fungal mycelium located in the glume tissue, at the distal juncture of the lemma and palea, of a longitudinally-sectioned kernel of Traill barley, developmental stage 11 (magnified ca. 1,000 X). 95 PLATE III Spores and mycelial fragments on the surface of bar- ley kernels. Upper - A two—celled spore on the surface of a longitu- dinally-sectioned kernel of Traill barley, de- velOpmental stage 12 (magnified ca. 1,000 X). Lower - Spores and hyphal fragments on the surface of a longitudinally-sectioned kernel of Traill barley, developmental stage 12 (magnified ca. 1,000 X). H n. PLATE IV Spores and mycelial fragments on the surface of bar- ley kernels° Upper — Fungal hyphae and/or spores on the surface of a longitudinally-sectioned kernel of Traill barley, developmental stage 5 (magnified ca° 1,000 X). Lower - Germinating (?) fungal spore lodged against a barb on a longitudinally-sectioned kernel of Traill barley, develOpmental stage 12 (magnified ca. 1,000 X)° 97 Fungal spores were rarely observed within the glumes (Plate V). The spores were identified as Alternaria and Helminthosporium conidia. Mycelium was never observed to penetrate the ker- nels deeper than the inner pericarp layer (Plate VI). Slides of glume-strips stained with aniline blue in lactic acid (191) also demonstrated that fungal infection by the field organ- isms was superficial and scattered over the kernel surface (Plates VII and VIII). Stained entities which morphologi- cally resembled bacterial cells in shape and in size were observed to increase in frequency with each progressive in- crease in kernel develOpment (Plate IX). The accurate as- sessment of bacterial numbers and location was impossible because of the similar artifacts obtained in the section- ing and staining process. In the chemical control experiments several proper- ties of the harvested barley were analyzed to determine whether significant differences existed between varieties of barley, replications, chemical treatments, and the time of harvest (Table 11). Varieties differed significantly at the one percent Level, with respect to 1,000 kernel weight, bushel weight, and yield. This difference is only an apparent one since one of the varieties, Hannchen, was rated against the six—rowed varieties. The five-day dif- ference in harvest time made an obvious difference in the percentage of kernel infection by fungi, bacteria, and yeasts (Table 12). Varieties differed significantly at the one percent level in their susceptibility to fungal -, w ray». fl. yea-bu-«H v 4‘. «411‘ ‘ .1 .. inflate We “' ’7“ 98 PLATE V Characteristic "dormant" fungal mycelium in glume and pericarp tissues. Upper - Fungal hyphae located in pericarp tissues Lower - of a longitudinally—sectioned kernel of Traill barley, developmental stage 14 (magnified ca. 1,000 X). Segmented spore or hyphal fragment lo- cated in pericarp tissue of a longitu- dinally—sectioned kernel of Traill bar- ley, developmental stage 12 (magnified ca. 1,000 X). 99 PLATE VI Characteristic "dormant" fungal mycelium in glume and pericarp tissues. Glume tissue injured at the point of fungal infection. Upper — Mycelium in glume tissue of a longitudin- ally—sectioned kernel of Traill barley, developmental stage 15 (magnified ca. 1,000 X). Lower - As above, different section of same series. PLATE VII Fungal mycelium in glume tissues of mature, wea- thered hannchen barley. Mycelium stained with aniline- blue and lactic acid method (191). Upper - Mycelium in glume tissues near middle por- tion of the kernel (magnified ca. 500 X). Lower - Detail of above (magnified ca. 1,000 X). 101 PLATE VIII Fungal mycelium in glume tissues of mature, wea- thered Hannchen barley. Mycelium stained with aniline- blue and lactic acid method (191). Upper - Mycelium in glume tissues near awn end of the kernel (magnified ca. 500 X). Lower - Detail of above (magnified ca. 1,000 X). 102 PLATE IX Bacteria in the glume tissues of barley kernels. Upper - Bacterial cells located at a natural opening of the hull of a longitudinally- sectioned kernel of Traill barley, devel- opmental stage 10 (magnified ca. 1,000 X). Lower - Bacterial cells located in the glume tis- sues, at the base of the lemma-awn, of a longitudinally-sectioned kernel of Traill barley, developmental stage 5 (magnified ca. 1,000 X). v.‘ ..| ‘\ i. ‘- 105 Table 11. - The effect of variety, chemical treat— b ment, and harvest time on several properties of barley. r :- a Variety Propggty . Variety l 2 Chemigal treatment é— 6 Total :égogtker- (Expressed in grams) (dry basis) Montcalm 52.04 50.89 51.75 50.27 51.05 51.68 187.68 Kindred 50.46 50.87 50.54 51.88 50.59 52.09 186.25 Hannchen 55.75 55.74 57.81 57.24 55.67 57.49 219.70" Traill 2. 51.72 50.51 52.25 1.4 1.61 189.71 150. 2 129.22 150.21 151.64 128.7 15 .87 Bushel wt. (Expressed in pounds) Montcalm 44.1 45.9 45.9 44.5 44.5 44.6 265.5 Kindred 46.5 46.5 46.7 46.5 45.7 45.8 277.5 Hannchen ‘50.5 49.8 51.1 51.4 50.6 50.7 504.1“ Traill 48.4 47.5 48.5 49.5 47.6 47.9 289.2 189.5 187.5 190.2 191.5 188.4 189.0 Yield (Expressed as grams/2 harvested rows) Montcalm 509 557 425 491 511 724 5017 Kindred 526 559 617 609 595 585 5489 Hannchen 549 581 681 605 659 612 5667 Traill 805 589 669 667 699 719 4148*‘ 2589 2086 2592 2572 2444 2658 a See page 84 for explanation of chemical treatments used. b Double asterisks indicate statistically significant dif- ferences at the 1 percent level between varieties. 104 mmoqonmmmfic pnmofimflnmflm hHHwOHpmewpm opmoHUqH mxmfinopmw mandon .moflpownm> noozpmp Hm>oa pdoonom H onp pm .mmflpmwnw>.nmozpmn Hoboa pdoonmm m on» no oodmhmmmfld pqwoamflamwm haawowpmflpmpm mopmowqu menopmm oawnwm .maoqnox dopmam on» ma Hmnsm :owmnopm: mo monomonm on» mopwoadnw .m. M .com: mpdoapwmnp HwOflEono Ho defipwqwamxo Hom_¢m omen 0mm 9 .onHpMOHHmoH eonwpaoo on» no owmnobw on» aonw mpdfioo HmnoamoHOHz m Ham mom 60m mom oom 30H bam mam mam mom Nam mam Ham 60H mm Hm mm mm am: moo mmm mmm mwmm «ml .moa mooa nononqem .mmm moH 00H maa OHH #HH ONH ..um® maa maa mmoa maa OHH oaa admopnoz Ammaomuuv pmmbnmn opmq mma mma Hwa mma 00H NNH mma med mma boa mma mma Hem mm: am mm am 66. mm: ..mmm mm: mm: mm: 66 mm .mm HH...B Ho¢ mm mm mm mm mm mm mm: mm 06 db up #8 mom convnwm mmm:maunv pmobnmn hanwm Hmpoe m m e m m a fleece m m e m m H M\pqmapwmnp Hwowaono mpmmoh d wwnmpomm M\flmnfim “hp doapoomafi no owprooHom hpownwb hoanwm pmmbnmn Ho mafia nuda mmfipmanw> moanmn snow mo soapoomqa HmnoamoQOHa no omen . I\ moaHp pnowommfld 03» pm Umpmob coupon one a .ma magma 105 infection in both early and late harvested material (Table 12). «The least susceptible varieties were Kindred and Hann- chen respectively. No significant differences were noted in bacterial and yeast infection on the early-harvested varie— ties. Hannchen showed significantly less bacterial and yeast infection at the five percent level in the late-har- vested material (Table 12). DISCUSSION AND CONCLUSIONS A positive correlation exists between the number and types of kernel-infecting microflora found in the air and isolated from barley grains. This correlation is further strengthened by the simultaneous appearance of certain or- ganisms, e.g. E. sativum, on kernels and exposed-plates at a particular stage in the growing season. The absence of typical soil-inhabiting microorganisms on barley kernels plated from the experimental plots and from other samples which have not suffered "ground—damage" is taken as evi- dence that the bulk of kernel infection arises from air- borne inoculum. In these trials only Montcalm lodged ex- cessively and also supported a much higher microfloral pop- ulation than did the other three varieties. Large numbers of white bacteria, primarily aerobic, gram—positive, spore- forming, rods (Bacillus spp.) were isolated from this vari- ety in the final stages of kernel maturation. These organ- isms are typically soil-inhabitants, indicating that bac- terial’soil-inoculum may be of importance in the infection 106 of lodged grain. It seems probable that Cladosporium sp. is an ineffectual "parasite" under the humid growing conditions encountered in the Midwest. The low percentages of infection obtained with this organism determined from a large number of plating of stained barley samples, indicates that this effect is primarily due to inhibition and/or suppression by the more efficient inhabitants of grain kernels. This hy- pothesis is supported by the plating data in Appendix C. In no case was Cladosporium found to be present in excess of four or five percent on samples grown east of the Rocky Mountains, i.e. samples which support moderate to large pop- ulations of other fungi. On the other hand, samples grown under irrigated conditions may show percentages of 95353- sporium as high as forty—five percent but are relatively free of other fungi. The data obtained from the plating and sectioning of barley kernels at progressive stages of develOpment support the observations made by previous workers. The ability of the bacteria to infect the maturing florets prior to or during flowering may be explained by the high moisture con- tent of these spikelets (about eighty percent) (167, 261), the opening afforded by the separated glumes, the succu- lent tissues of the inflorescence, the protruding awns, and the sheltered micro-environment within the enclosing sheath. Any or all of these factors may favor bacterial invasion and establishment of the immature spikelets. The bacteria isolated during the early stages of kernel growth 107 most frequently developed from the lemma awn or from the awn-end of the kernel. The slower invasion of developing kernels by the field fungi is thought to be primarily due to the preference shown by these organisms for a senes- cent nutritional substrate as well as for a less-moist en- vironment. The author is in accord with Atanasoff's in- terpretation of the foci of infection (21), viz, that the kernel—infecting organisms can attack any portion of the kernel, but are generally to be found in the most-freely exposed areas. The points which seem to furnish the most accessible infection loci for fungal spores and mycelial fragments are the separated glumes at the base of the lemma awn, the base of the kernel at the point of attach- ment to the rachis, the epidermal barbs on the exterior of the hull, and wounded glume tissue. The data have indi- cated that many of the field organisms preferentially at- tack and establish themselves in the awn end of the kernel. Infection frequently takes place along the ventral crease as well. With those organisms causing "black-point" and "basal glume—rot", i.e. g. sativum, P. atrofaciens, etc., this observation does not hold and the embryo end of the kernel is usually attacked. Why these organisms behave in this manner remains to be explained although there are some indications that these differences are reflections of varying responses of the pathogens to a higher concentra- tion of diffusible sugars and amino acids in the embryo region of the kernel (500). 108 The stage of kernel development at which suscepti— bility to bacterial and yeast infection declines seems to be related to three separate phenomena: (1) the adherence of the glumes to the caryopsis with resultant compression and destruction of the underlying tissues, (2) the decrease of the moisture content of the grain to about sixty percent or less, and (5) the increase of fungi on and within the kernel. That the fungi continue to infect the kernels up to the time of harvest is again a reflection of their pref- erence for less free moisture and for dead and dying tissue. The microfloral determinations and subsequent sta- tistical analyses have given some indication that varietal differences may exist with respect to kernel infection. This supposition has not been supported by data obtained from field observations and the plating of barley varieties over the last several years. Montcalm was the only smooth- awned variety employed in this experimental series. The microfloral content of Montcalm was also the highest of the three varieties. Shands has reported that smooth-awned varieties are more susceptible to "weathering" and gener- ally show more undesirable hull color than do rough-awned varieties (548). The high correlation between hull-color and microflora seem to indicate that if varietal suscepti- bility differences do exist, they may be in some way related to the smoothness or roughness of the awns. Rough- and smooth-awned isogenic barley lines were obtained from Dr. Shands, and were plated in an attempt to determine whether 109 the reported color differences were due to varietal dif- ferences in susceptibility to kernel-infecting flora. The results of the plating test have been listed in Table 7, (see Chapter II). The seed furnished by Dr. Shands was four to five years old and consequently the microfloral determinations were only an approximation. In all cases however, the germination capacity of the smooth-awned bar- leys was significantly lower than that of the rough-awned lines. This, barring a genetic difference in germinability, indicates that the smooth-awned lines may have had a con- siderably higher microfloral content than the rough-awned barleys at the time that the color differences were re- corded. The chemical control trials recorded in this sec— tion were of no measurable use in controlling the infec- tion of barley kernels by the field organisms. Other work- ers have reported some success in the use of chemical appli- cations, but these trials have employed more than one ap- plication of the material in the growing season (9). As is the case with most field crops, the cash return per acre will not permit the repeated use of chemical fungicides and/or bactericides. Further, the presence of chemical residues on barley kernels would render the grain unmar— ketable, if offered for use in the malting and brewing industries. Consequently it would seem that if control of kernel—infection, caused by those organisms which re- duce quality rather than yield, is desired it must be 110 sought, at present, in the direction of varietal resis— tance. The rapid development of improved pesticides may within the near future provide an effective and econom- ical control method for the field-infecting flora of bar- ley kernels. CHAPTER IV. THE EFFECTS OF MICROFLORA ON THE BARLEY KERNEL LITERATURE REVIEW Barley microflora may affect the associated kernels in several ways. The nature of these effects is, in most cases, determined by the use to which the kernels are put, i.e., whether the barley is to be malted, planted, or used as livestock feed. In general, these effects are manifes- ted as quality defects, seedling diseases, or as storage deterioration. The most obvious quality factor affected by grain microflora is the color of the kernel. The color of grain kernels is determined by their genetic composition, the temperature and moisture conditions under which the crop is grown, the way in which the harvested grain is stored, and the number and kinds of microorganisms associated with the kernels. Harlan studied the pigmentation of barley kernels and found that all kernel color was the result of two pigments; anthocyanin and a melanin-like pigment (164). The four basic barley colors; white or yellow, blue, pur- ple, and black, are the result of the absence or combina- tion of these pigments. These heritable colors may be present in the aleurone layer and/or the barley glumes. Wiggans has also discussed the genetic basis of barley coloration (418). The author indicates that coloration may also be influenced by immaturity, weathering, or pre- 111 112 mature harvesting of barley. That discoloration of grain kernels may arise as a result of genetic factors control- ling the production of melanin pigments is well known in the case of certain wheat varieties, e.g. Hope, H-44, and their derivatives (197, 205, 204). This form of glume dis- coloration has been termed "pseudoblack chaff" because of the resemblance to the condition caused by Xanthomonas translucens infecting wheat kernels (55, 197). Campbell has noted that wheat kernels of some varieties may take on a translucent pink appearance, without the presence of fungi, if harvested prematurely (61). The influences of temperature and moisture on the discoloration of grain kernels are difficult to separate from the influences of genetic composition and associated microflora. In both cases, suitable temperatures and suf- ficient moisture must be present for disColoration to oc- cur, regardless of its orgin. The term "weathering" has been much used and abused with reference to the staining of grain kernels. Authors have usually considered weath- ering as a process distinct from microfloral infection and discoloration; e.g. Preece has stated that ". . . a dark- ening or discoloring from the desired golden hue is fre- quently met with as a result of more or less unfavorable harvest weather with too much rain and too little sun" (515). Lejeune and Parker have indicated that "Severely weathered barley may be infected with molds and other microorganisms . . ." (226). The Official Grain Standards 115 also distinguish between "weather damaged" and "mold dam- aged" in the definition of "damaged Ebarleyi'kernels" (6). Hind has suggested ". . . that many barley corns described as weathered have been attacked by Helminthosporium teres (177). Several workers have reported that grain discolora- tion may be caused by suitable weather conditions, without the intercession of kernel microflora. Thorpe reported that samples of California-grown barley contained kernels with blackened ends and masses of a sugary, glutinous ma- terial. These kernels were not infected by the "black- point" fungi (594). The author suggested that the condi- tion was a result of the inability of the kernels to con- vert the translocated sugar to starch as well as the ef- fect of dew and rain collecting at the angle formed by the kernel and the rachis. Hagborg (156) and Johnson and Hag- borg (204) have reported that wheat kernels of some varie- ties may be discolored after exposure to high temperatures and high humidities under greenhouse conditions. The au- thors concluded that the staining resulted from a combina- tion of genetic composition and suitable environment and not as a result of pathogenic organisms. They did isolate "non-pathogenic" bacteria, however, but concluded that these organisms were simply secondary invaders favored by the discolored, "degenerate" tissue. Hagborg has also presented a thorough analysis of the various discolora- tion symptoms of wheat kernels caused by microflora and by 114 physiologic staining (158). Ledingham and his co—workers have also attempted the artificial weathering of wheat ker- nels by placing maturing wheat plants, previously grown under dry conditions, in moist chambers (225). The diffi- culty of growing plants under moist conditions, while avoid- ing microfloral infection, is emphasized in this paper since later platings of the kernels showed high bacterial concen— trations. Wheat kernels may also present a bleached ap- pearance due to severe weathering. According to Cayzer this is the result of the swelling of wet grain with concommitant formation of numerous air spaces between the endosperm cells (62). The author also states that badly-weathered wheat may frequently be dirty and grayish in color as a result of mold development. Another undesirable type of kernel discoloration is found in heat-damaged barley. The Official Grain Standards define heat damage as those ". . . kernels and pieces of kernels of barley, other grains, and wild oats, which have been materially discolored and damaged by heat." (6). This condition is usually caused by storage organisms which gen- erate heat in barleys stored at high moisture levels (226). It is a well—known fact that grain discoloration is frequently associated with the presence of microorganisms. Few workers have, however, indicated how closely kernel staining and the microfloral content of the grain are as- sociated. Hanson and Christensen reported that the preva- lence of wheat seed discoloration was generally correlated 115 positively with the prevalence of microorganisms (161). Greaney and Wallace found that in most years wheat sam— ples showed a higher percentage of internal fungal infec- tion than was indicated by the number of discolored ker- nels (141). In years when leaf rust was not a complica- ting factor the amount of internal infection was always positively correlated with the incidence of stained kernels. James and his fellow-workers found that when kernel wash- ings of wheat seed were plated, the number of bacteria and fungi increased proportionately with the visible increase in grain damage, from vitreous to weathered to immature to frosted kernels (195). Workers at the University of Minnesota have reported that fungi were more numerous in several stained samples of barley as compared to bright, western—grown lots al- though in one bright sample they isolated a much higher percentage of bacteria and yeasts than were present in light and heavy stained barleys (8). Investigators have employed a number of symptomol- ogical terms to describe the staining of grain kernels by specific organisms. "Blighted" barley usually refers to kernel discoloration caused by Helminthosporium spp. (226) although some workers have also used the term for barleys discolored by Alternaria and other fungi (78, 115). Bar- ley kernels infected by Gibberella and Fusarium species are frequently termed "scabbed", but some authors group these infected kernels together with those in the "blight— 116 ed" category. The term "blackpoint" has been applied to those grains which are discolored at the embryo end. This condition is typical of kernels infected by Helminthosporium sativum and Pseudomonas atrofaciens (47, 88, 110, 112, 158, 247, 258, 555). Zgbl applied the similar term "braunspit- zige" to discolored barley kernels (425). He attributed the cause of the discoloration to Cladosporium herbarum, but several other fungi were also isolated from the ker- nels. Peyronel concluded that Q. herbarum was not respon- sible for the basal blackening of grain kernels, a condi- tion he termed "puntatura" (501). This conclusion was further supported by Bockmann who found that g. herbarum, Alternaria spp., and other so-called "blackening fungi" did not cause the brown discoloration that is typical of black-point (48). He further suggested that these barley organisms are of little economic importance, except inso- far as they tend to lower the market value of the grain. The word "moucheture" has been used by Rosella to describe the discoloration of wheat kernels caused by two species of Alternaria (528), and barley staining caused by g. sativum and g. gramineum (529). Fomin and Nemlienko employed the term "black rad- icle" in a study of cereal grains showing basal discol- oration (118). The authors concluded that the condition was due to a number of factors, the chief causes being g. sativum and an undetermined species of Alternaria. Ziling found that "black germ" of wheat was produced by 117 g. sativum, Alternaria tenuis, and Fusarium Spp., and that the causal agent could be determined by the location and the intensity of the discoloration (424). Drechsler found that while both 3. gativum and Alternaria spp. caused cer- eal seed discoloration, the symptoms induced by these fungi differed slightly (107). Christensen and Stakman inocula— ted barley plants with 200 isolates of eight genera of fun— gi and found that many of these organisms caused kernel dis- coloration (77). They concluded that, while species of Fusarium and Helminthosporium were the most serious blight- ing fungi, other organisms were also capable of causing kernel defects. Canadian workers have coined the term "kernel smudge" to describe kernel staining brought about by a number of organisms. Machacek and Greaney have reviewed the liter- ature dealing with the types of cereal seed discoloration (247). They concluded that "black-point", "black-tip", and similar descriptive terms, while suitable for kernels infected by certain bacteria, were not appropriate for the symptoms produced by fungal infection. They suggested that the appellation "kernel smudge" be applied to the disease of cereal grains in which a dark or pale brown discoloration occurs on the kernel, particularly in the embryo region, and which is produced by fungi, expecially those members of the Form-family, Dematiaceae. Mead inoc- ulated developing barley heads with several commonly occur- ring barley organisms (262). "Smudged" kernels were ob- 118 tained from the inoculations made with Helminthosporium sativum, 84 percent; Gibberella saubinetii, 45 percent; Fusarium sp., 60 percent; and Alternaria sp., 55 percent. Symptomological differences in staining also were noted among the inoculations used. Russell described two types of smudge on wheat kernels which can be visually distin- guished: the "mild" type caused by Alternaria, and the "severe" type caused by H. sativgm (551). "Alternaria blotch" has been applied to wheat kernels discolored by Alternaria tenuis, in a paper authored by Johnson and Hag- borg (205). The authors found that the fungus incited a grayish-brown discoloration of the lemma. The microflora of barley and other grain kernels may adversely affect the germinability of the seed. The early work dealing with this effect has been reviewed by Thaysen and Galloway (590). Early workers thought that seed—germination was reduced by the excessive growth of the associated microflora which subsequently clogged the pores of the embryo and thereby prevented respiration. This explanation was questioned by Thaysen and Galloway who postulated a multiple-enzymatic inhibition of germin— ation by the kernel microorganisms. Bishop has discussed the problem of barley germination in a series of three memoranda (40, 41, 42). These memoranda include inten- sive reviews of the literature dealing with the effects of microflora on germination, as well as the results of the author's original researches. He found that the ear- 119 lier explanations of Reichard and Becker were at least partially correct, viz. that bacterial slime and fungal growth retarded the entrance of oxygen into the embryo (40). This type of dormancy was overcome by either re- moving the hull or by using various disinfectant treat- ments. Thiourea did not affect dormancy, but it did sup- press fungal growth on the germinating kernels (41). At- tempts were also made to determine the cause of germination inhibition "at a distance” by kernel microflora (42). While the effect resembled that caused by the evolution of cer- tain chemical vapors, no naturally produced lethal vapor was detected. The author also suggested the possibility of enzymatic inhibitors released by the microflora. The presence of a microfloral “gelatinous film" as a germina- tion inhibitor has been further noted in a discussion fol- lowing a paper presented by Crafa and his co-workers (84). According to this interpretation, the infected barleys may present a sound appearance and yet germinate as low as 60 percent. Leukel has stated that kernel microflora, even those not considered as pathogens, may prevent or inhibit germination by attacking the food material stored in the kernel (228). Helminthosporium sativum has frequently been impli- cated as an inhibitor of kernel germination, as have species of Fusarium (74, 506, 404, 424). Rosella reported that bar- ley kernels, discolored by E. sativum, germinated normally, but the author indicated that the lack of inhibition may 120 have been caused by the drastic surface—disinfection employed (529). Dastur attributed the normal germination of many dis— colored wheat grains to the freedom of the embryo from in- fection by g. sativum (88). Alternaria rarely, if ever, caused a reduction in germinability of infected kernels ac- cording to Christensen (74), Ziling (424), and Machacek and Greaney (247). Nilson, in 1905, stated that bacteria prevent normal germination in barley by their biochemical activity (287). Messinger reported that culture filtrates of Achromobacter sp. retarded barley germination, but the inhibitory mater— ial soon lost its power in contact with the kernels (275). Barley steep liquor was found to be inhibitory to barley germination by Gilbert and his fellow-workers (127). They concluded that this effect was caused by barley microflora which competed with the barley for dissolved oxygen and by the production of a bacterial exotoxin. This exotoxin was produced from pure cultures of certain of the bacteria, and its properties suggested that it was a salt of an organic acid of high molecular weight. In a later paper, Gilbert and Blum reported that barley, germinated for as little as twelve hours under conditions unfavorable for microfloral growth, was able to complete germination after inoculation with these organisms (128). Minnesota workers have also shown that barley steepwater is inhibitory to barley ger- mination (9). The effect was associated with the presence of large numbers of bacteria and yeasts and the inhibitory 121 material was at least partially heat-stable. The same workers have reported that certain bacteria isolated from steep-liquor produced a volatile germination-inhibitor which is especially active against pre-steeped barley. Toxic sub- stances were isolated from Cephalothecium sp. and species of Fusarium. These inhibitory materials were not recovered in subsequent experiments, and the authors attributed this loss to either a fungal mutation or a dependence on specific cultural conditions. Closely related to the tOpic of germination inhibi- tion and injury, are the categories of seedling and post- emergence diseases. Alternaria species, commonly found on barley kernels, have generally been absolved of guilt in these categories (202, 247, 262). Species of Cladosporium have also been shown to be non-parasitic in the sense that they do not initiate either seedling diseases (202) or head diseases (55). Stakman (575) and Bolley (47) have claimed that certain species of Alternaria isolated from grain ker- nels may cause seedling defects. Various species of Egg- ggigm and Helminthosporium, on the other hand, are impli- cated in the lesioning of emerging coleoptiles, the pro- duction of seedling—blight, and foot- and root-rots (54, 202, 262, 264, 511). Greaney and Machacek have stated that soil bed tests have shown a high positive correla- tion between the percentage of cereal seed infected by H. sativum and the incidence of seedling diseases (145). Mead has demonstrated that the greatest amount of seed- 122 ling blight caused by g. sativum occurs under environmental conditions that are unfavorable to the barley plant, i.e. extremes of temperature and moisture (265). General dis- cussions of the diseases caused by the seed-microflora of barley are also found in the references by Dickson (100), Sprague (568), and Leukel (229). Several workers have noted that cereal grains, dis- colored by microflora, are frequently plumper than normally- colored seed (161, 247, 501, 528, 408). Jodidi and Peklo have asserted that certain symbiotic fungi, identified as smuts, produce the aleurone layer in infected cereal grainsanmi penetrate the peripheral cells of the endosperm to produce the gluten (199). This observation is not supported by other reports in the literature. The subject of grain storage deterioration has been intensively investigated and several comprehensive reviews, dealing with this topic are available (45, 69, 70, 544). Early workers attributed storage losses to heating caused by the biological OXidation of carbohydrates, especially in the embryo of the kernel (22). Gilman and Barron, in 1950, suggested that certain speCies of Aspergillus were probably responsible for the heating of stored grain (129). Thaysen and Bunker Cited the work of Cohn who in 1889 re- garded the enzymatic action of Aspergillus fumigatus as responsible for the spontaneous heating of germinating barley (589). These authors stated that Cohn9s assump— tion was not very well substantiated. More recent evidence 125 indicates that the assumption was not only substantiated but was indeed a very far-sighted observation. Robertson and his co-workers noted that the viability of stored cer- eal seed decreased in storage with increased humidity (524). Heavy fungal and bacterial growth were observed, but the data did not indicate what influence these organisms had on the stored grain. Leach (221) and Denny (92) have shown that respiration rates of germinating grain kernels may be altered significantly by the attendant microflora. 0xley and Jones arrived at the same conclusion as Leach, viz. that the respiration of stored grain is almost entirely produced by microorganisms growing in the pericarp tissues (295). Roth reported that in barley stored under humid conditions the number of fungi and bacteria initially in— crease (550). Further increases in humidity caused a de- cline in the number of bacteria and an increase in the num- ber of certain fungi. The minimum humidity requirements for germination of the conidia of several grain storage fungi have been reported by Armolik and Dickson (1?). Hum— mel and others have shown that respiration is greatly in- creased by fungi on stored cereals (186). Loss of via- bility, increases in fat-acidity, and decreases in non- reducing sugar content were also noted by the authors. According to Geddes the increase in fat-acidity caused by fungal lipases is a measure of the soundness of stored grain (126). Armolik and his co-workers found that stor- age molds reduced the germination of stored barley and 124 that the variety of barley as well as the geographical lo- cation of the crop-producing area greatly influenced the amount of the grain deterioration (18, 19). Tuite and Christensen have stated that no significant amount of in- vasion of barley kernels by storage fungi takes place prior to harvest (598). Species of Aspergillus and Penicillium increased at moisture contents above 14 percent, and the field fungi disappeared at moisture levels above 15 or 16 percent (597, 598). Bacteria have not been considered of great importance in the deterioration of stored grain (70, 544), although bacterial decomposition of grain products is of some importance (544). Hofer and Hamilton have im- plied the possibility that bacteria, especially spore-form— ers, may be of importance in spoilage of food products in which ordinary processing would not destroy the bacteria (180). The effect of certain seed-borne organisms has been variously noted with reference to the feeding value of in- fected barley. The most intensively studied example is that of scabbed barley infected by species of Fusarium and Gibberella. When scabbed barley is fed to swine and certain other animals, an emetic principle in the infected grain induces vomiting or death in the animals (99). The principle remains active for at least 26 months after the fungus loses its viability (547) and is not produced in culture media by the fungus (76). If the percentage of Fusarium-infected grain is too high, the animals will fre- 125 quently refuse to eat the poisonous feed (76, 579). The feeding of scabbed barley has been discussed at length by Dickson (99). Several species of Aspergillus have been re- ported to cause deterioration of barley and to produce toxic effects in laboratory animals (550). Rhizopus nigricans rendered feed-barley unfit for use by cattle according to 'Wagn (405). Miessner and Schoop found that while only gig- berella-infected barley was toxic to swine, the animals showed some reluctance in accepting Alternaria-infected barley (274). Jones and his co-workers found that moldy wheat infected by species of Aspergillus, Penicillium, Alternaria, Cladosporium, Actinomyces, Rhizopus, Mesobotrys, and Trichoderma was more digestible by swine than non-moldy wheat (205). The infected grain was not unpalatable to either swine or sheep. German workers have shown that the presence of certain unidentified bacteria in barley pro- duced copious amounts of gas and caused a putrid smell (1). The investigators concluded that these bacteria caused pro- tein decomposition in the stomachs of animals and that the decomposition products were toxic to swine. MATERIALS AND METHODS Several approaches were used in the study of ker- nel discoloration and its relation to the associated micro- flora. The 84 barley samples furnished by the Malting Bar- ley Improvement Association, mentioned previously (cf. Chapt. II), were visually evaluated for color intensity. 126 The visual evaluation scale ranged from 1 to 10, with 1 representing extremely bright samples and 10 designating severely discolored samples. Additional notations were made with respect to: the number of "blackepointed" ker- nels, under-sized kernels, green kernels, and varieties with pigmented aleurones and glumes. Microfloral deter- minations for these samples are described in the aforemen- tioned section. Surface-sterilized kernels of bright Montcalm, Hann- chen, and Traill barleys were plated on nutrient agar which had been previously seeded with the following bacterial cultures: B-5, B-210, B—226, and B-529 (see Table 18 for identity of bacteria). The surface-disinfection treatment was the standard method of the author (cf. Chapt. 1). One hundred kernels of each variety were used with each bac- terial treatment. The plates were incubated for eight days at room temperature (ca. 25° 0.); and the degree of growth of kernels and the amount of kernel discoloration, were then determined. A check series consisting of the three barley varieties was also plated on nutrient agar without the addition of the bacteria. The degree of staining was rated on an arbitrary scale of one to five, where one de— notes no staining and five represents severe staining. Notations as to the location of the discoloration on the kernel were also recorded. The amount of barley growth was measured by removing and weighing the extended acro- spires, after drying to a moisture content of approximately [L1 1 '7 10 percent (by weight). Color differences in the inocu- lated barleys, both before and after germinating, were also noted. Several other methods were used in the over-all study of barley kernei~staining. These are treated in Chapter Vl. The inhibition of barley germination by associated microflora was investigated by means of three general methods: (1) the recording of germination and/or kernel injury in conjunction with the attendant microorganisms on agar plates, (2) the use of culture filtrates of a num- ber of barley organisms as the steeping medium in the rou- tine A.S.B.C. germination test (5), (5) the A.S.B.C. ger- mination procedure used With the inoculated barleys pre- pared for pilot-malting. In all cases the barleys were 1‘5 r J S—D C,» t) read at the approp time for germination energy and germination capacity (5). The first method has been rou- tinely employed by the author with all barley samples pla- ted in the years 1958-1960. The second method, outlined above, consisted of t.e culturing of fifteen fungal species on a reciprocating shaker for seven days. The fungi were grown in Czapek’s sucrose-nitrate solution, 50 m1. of medium per each 250 m1. Erlenmeyer flask. The details of this cultural procedure have been outlined elsewhere (500). At the concluSion of the growth period, the reaction of the media, color of the media, odor of the media, color of the fungi, growth form of the fungi, and the amount of growth were noted. The fungal mycelia and/or spores were removed 128 by coarse filtration and the liquid portion was passed through a Seitz filter. The filtered liquid was diluted 1:1 with sterile distilled water and divided into two por- tions. One portion was used without autoclaving for the steeping liquor; the other portion was autoclaved for 50 minutes at 15 pounds pressure before being used as a steep. The same procedure was followed with check tubes of sterile distilled water, Czapek“s solution without inoculum (sta- tionary), and Czapek“s solution without inoculum (on sha- ker). All cultures were replicated three times. The bar- ley used in connection with the above culture filtrates was a bright Kindred sample, 1958 crop, held on a 6/64" sieve. The third method used in this section of the study was em- ployed with the inoculated barleys after they had remained in storage for eighteen or more days. The inoculation and storage methods are presented in the section on pilot- malting (Chapt. V). This procedure also provided some in- formation as to the effects of certain barley organisms on storage deterioration of the inoculated kernels. A sample of feed barley, grown in Michigan in 1958, was submitted by a grower who stated that the grain had been refused by swine. The barley was plated with the routine procedure outlined previously. A sample of the grain was also sent to the Barley and Malt Laboratory for an evaluation of the abundance of Fusarium "emetic prin- ciple". 129 EXPERIMENTAL RESULTS The color and microfloral properties of the 84 bar- ley samples, received from the Malting Barley Improvement Association, are tabulated in Appendix C. Microfloral de- terminations have been made on most of the same samples by workers at the University of Minnesota (9). The color of these samples is correlated positively with the number of organisms isolated from the kernels. A positive correla— tion also exists between the number of microflora in a seed-lot and the climatic conditions which prevail in the location where the seed was grown. Barley kernels plated on agar, seeded with bacteria, were both discolored and inhibited by the bacteria (Table 15). In all cases the barley kernels were severely dis- colored by this treatment. The intensity and the loca— tion of the discoloration varied slightly between varie- ties and treatments. In general, the staining was more intense along the ventral crease and at the embryo end of the kernels. The staining was expecially pronounced in the region of the scutellum, forming a dark brown band. The inhibition of kernel germination by associated microorganisms was noted in several hundred barley lots plated during the past several years. In general, the following fungi have proved to be inhibitory to barley germination:' Helminthosporium sativum, g. teres, Fus- .2212! roseum f.sp. cerealis, Aspergillus spp., Egg;- cillium spp., and several members of the Mucorales. Many 150 .mopwam memo pdoflnpdq dopmadooaHIQon do maoanom oopoomqflmfioloowmudm mafipmam hp meme pamepmoup guano 6 .ooa hp wqflhamflpada one .mamnw GH mohwmmonom mo pnwflms Gama mqflpmadoamo .mmnflamouom emenopxo Mdflnmfioz hp ocnflapopoo npzonw o .wnflpmam mnomon AHV UmHHmSUm mmflpoahm> Ham mwmqfimpm haonm>mm on wdflpdommmhnoo Amv .panHQ hum» mcwaamdvo adv mm mouth Hoaoo D .mopmaomfl Hmfinmpomp mo mpwpdmow How ma capes com w mm.o m m¢.o m om.o m ommum em.a m mm.o m 03.0 m mmmlm ma.m m mm.© m mw.¢ m wmmlm mm.o m 00.0 m wa.o m oamum em.H m mm.fi m ee.a m mum Ha.m m eH.e m mm.m m |\someo U lo\npSon m\moaoo m\npzonw m\noaoo m\nuzonw m\Hoaoo nonoanmm Hawwme Samopnoz I\ad.mdooafl flu I. “O mmapmaum> mpapqoeH .mmpwaomfl Hwflnmpomn o>fim spa: Umpmasooafl mopmam Hmww psofimpdq so vmpmam maoapox hmanwn Umpomqumav noommHSm no QOHpflnfindH dofipmdfiammm 6am scapegoaoomwo HoaHoM I .ma manma 151 species of seed-borne bacteria, both white and yellow types have also proved to be virulent germination—inhibitors. Species of Alternaria, Cladosporium, Stemphylium, Epicoc- ‘ggm, Candida, and several less commonly occurring fungi and yeasts have not seriously affected the germination of the associated kernels. Frequently, the germination of the plated grains appears to be adversely affected by the "non-pathogenic" fungi, but closer inspection usually re- veals the additional presence of bacterial colonies. The effects of associated microflora on barley germination are tabulated elsewhere (Appendix C. and Table 7). In most cases, a negative correlation exists between bacterial and/or fungal populations and barley kernel germination. The reaction of the culture medium was significant- ly altered by Aspergillus niger and a Penicillium isolate (Table 14). The culture filtrates from only three fungal isolates exerted marked deleterious effects on kernel germination under the experimental conditions described above. These species; Aspergillus niger, Fusarium roseum f.sp. ggggglgg, and an unidentified species of Penicill- gpm; also proved to be inhibitory when the work was re- peated. While both the autoclaved and the unheated fil- trates from the first two isolates were toxic to the bar- ley grains, only the unheated medium was inhibitory in the case of the Penicillium isolate used in the study. The data indicate that the inhibitory principle produced by Aspergillus niger and Fusarium roseum f.sp. cerealis 152 Table 14. - The germination of barley kernels steeped in culture filtrates of selected fungal species/-. Percentage germination Source of Reaction . . Filtered Filtered & filtrate of filtrate only autoclaved b c b c Check (distilled 7.8 92 96 95 99 water) Check (Czapek's - 7.4 100 100 98 100 stationary) Check (Czapek°s - 7.5 89 100 99 99 shaker) Rhizopus sp. 6.5 97 98 97 98 Gliocladium sp. 7.5 97 98 98 99 Chaetomium sp. 7.4 99 99 98 99 Aspergillus niger 1.9 69 75 59 67 Nigrospora sp. 7.4 99 99 98 99 Cephalosporium sp. 7.5 97 98 99 99 Verticillium sp. 7.5 99 99 98 99 Aspergillus sp. 7.4 97 97 98 99 Helminthosporium 7.6 98 98 98 98 sativum Fusarium roseum 7.9 69 84 88 91 f.sp. cerealis Alternaria tenuis 7.5 98 99 98 98 Epicoccum sp. 7.5 97 98 98 99 Gloeosporium sp. 8.0 97 98 97 98 Curvullaria sp. 7.7 97 98 97 98 Cladosporium herbarum 7.0 98 99 98 99 155 Table 14. - Continued ‘ Penicillium sp. 4.8 77 94 96 98 All figures listed represent an average of three or more replications. Germination energy — A.S.B.C. method (5). Germination capacity _ A.S.B.C. method (5). Visible injury to germinating seedling, viz. stunting and necrosis. 154 is heatestable while that of the Penicillium isolate is either heat—labile or inactivated by combining with other substances. The acid reaction of the culture filtrates may also be responsible far germinationwinhibition al- though other data indicate that barley kernels will ger- minate at low pH levels (Table 5). Initial moisture contents and germination energies were determined for the inoculated barleys used in the pilot malting studies (see Chapt. 5). These barleys were dried at room temperatures and humidities prior to stor- age, and the initial moisture contents ranged from approx- imately 11.8 percent to 15.4 percent (Table 15). Moisture contents of the grain increased during storage to as much as 17.0 percent in the case of certain inoculated barley varieties after thirty days of storage. The pronounced decrease in germinability with several of the treatments is seen in Table 1‘. This loss of viability was directly reflected in the moisture increases of these tieated varie- ties. A slight increase in moisture content was noted in the stored check samples. No microflora were isolated from either the Traill or the Montcalm check lots after storage, nor was there any marked decrease in the germin— ability of these samples. Fungi and bacteria were iso- lated from both steeped and unsteeped Hannchen check sam— ples. The varieties responded differently to the various inoculations; and the two six-rowed varieties were more susceptible to storage deterioration in this experiment 155 Table 15. - The germination of inoculated barley a kernels after storage in sealed bottles at room temperatures/— Identity of b varieties inoculum /_ Montcalm _m , Traill Hannchen c d e c d e c d e Check (no 10.9 95 treatment) (3 \N I~‘.: O 0 \O 97 98 11.0 97 96 Check (steepwll.9 95 95 11.8 96 92 12.5 92 96 ed & stored) 345 12.5 9. -4 15.1 89 71 12.6 95 _- B~210 ; 15.2 65 -4 15.4 79 79 15.9 95 -- 84218/£ 12.4 5 T8 12.2 95 94 12.0 95 97 84226 14.5 85 44 12.5 59 58 12.8 80 -— 84522 15.5 84 42 15.4 15 0 15.8 94 91 B—529 15.7 86 82 15.7 47 2 15.9 78 87 14521 12.5 77 79 15.1 91 85 15.8 91 95 F=504 12.2 80 -4 12.5 96 —4 12.8 95 -— F4510 12.8 78 -- 12.7 85 44 12.4 91 —- F4521 12.4 81 a- 12.5 94 -4 12.5 98 -- F~524 12.2 89 1- 12.4 95 -4 12.2 97 -- a See Chapt. 5 for inoculation procedure. Storage periods of eighteen and thirty days unless otherwise noted. b See Table 18 for identity of species used as inoculum. C Percentage moisture content at time of storage. d Germination energy after eighteen days of storage. e Germination energy after thirty days of storage. f Germination energies determined after 51 and 65 days of storage. 156 than was the two~rowed variety, Hannchen. The analysis of the sample of feed barley, conducted by the Barley and Malt Laboratory, showed that the amount of emetic principle present in the sample was not high enough to render it either unpalatable or poisonous to livestock. The barley was however refused by swine. The high bacterial content of this barley lot (Tables 5, 4, and 5) may perhaps explain why the grain was not accept- able to swine when offered as feed. DISCUSSION AND CONCLUSIONS Almost without exception, the discoloration of bar- ley kernels signals the presence of fungi, bacteria, and yeasts on and within the kernel. Kernel discoloration is, however, neither a necessary or a sufficient condition for the occurrence of seed-borne microflora. The mechanism of kernel discoloration, either by microflora or by other factors, will be discussed in a later section (Chapt. VI). The results of numerous studies, especially in the area of storage deterioration, have shown that the loss of germinability of barley and other cereal grains is mainly due to the presence of microflora. The research reported in this section has supported this observation. The in- hibitory influence of the seed-borne pathogens of barley has been repeatedly observed on plated kernels. It must be emphasized that germination—determinations made on plated kernels are generally lower than those conducted 157 on moist blotters; since microflora are favored by the presence of abundant nutrients and moisture. Kernels in- fected with species of Helminthosporium and Fusarium fre- quently germinate, but the rootlets rapidly become necrotic and the seedling fails to continue its development. Ker- nels have repeatedly germinated, in a normal fashion, on agar plates when completely covered with certain fungi, e.g. Alternaria spp. On the other hand, kernels infected, in a similar way, with species of Helminthosporium show marked inhibition. The two genera (Alternaria and Hel- minthosporium) are phylogenetically related, cause similar staining effects, and infect kernels in somewhat the same manner; the pathological difference seems to indicate the production of a toxin and/or other physiological differ- ences. Bacterial colonies arising from plated barley ker- nels are frequently inhibitory to kernel germination. These organisms include white, mucoid, creamy, and yellow types. Not all bacterial isolates are germination inhib- itors, a few isolates have demonstrated stimulatory effects (Table 15). Various degrees of kernel~resistance and bac- terial-virulence, with respect to the inhibition of bar- ley germination, have also been demonstrated (Tables 15, 15, 54). Most of the commonly isolated bacteria have shown inhibitory effects, especially if the bacterial colony develops at, or near, the embryo end of the kernel. The inhibition effect by these organisms differs from that 158 of the seeduborne fungi. Kernels infected with bacteria usually show little, or no, evidence of germination. If germination does begin, the acrospire is usually affected less than the developing rootlets. The mechanism of ger- mination-inhibition is poorly understood. The inhibition of kernels plated on agar, seeded with various bacterial species, may take place without visible contact between the kernels and the bacterial cells. Such an occurrence seems to indicate that a volatile and/or diffusible inhib- itor may be produced by certain organisms, as has been sug- gested by other workers (9, 42). The effects of seedaborne bacteria on barley ker- nels have not been previously studied in great detail. In addition to their inhibitory properties, as shown by bar- ley plating studies, there is some evidence that bacteria, especially certain ngiggpg species, may be of importance in the deterioration of stored grain (Table 15). It has been generally assumed that bacteria are not involved in grain storage injury, since they require free water to grow (70, 544). The data presented above does not entirely support this contention. Traill and Montcalm samples, in- oculated with two Bacillus species, were stored at initial moisture contents of approximately 14 percent. The ger- minability of these samples steadily decreased in storage (Table 15). The moisture content of the samples was cal- culated, after sixty days of storage, at from 16 to 17 percent. The kernels proved to be non-germinable at this 159 time. All samples were stored in sealed mason jars. These preliminary observations are subject to several possible explanations: (l) the moisture increase was the result of bacterial respiration and kernel decomposition, (2) the moisture content of the inoculated grain was not homogeneous, (5) the bacterial content of the inoculated grain was not homogeneous, (4) certain bacterial species may be able to multiply and carry out storage deterioration at moisture contents at the "safe" level of 14 percent. The methods employed in moisture and bacterial determinations of these inoculated barleys require further refinement before any general conclusions may be validly drawn. The refusal of swine to eat barley infected with large numbers of bacteria, and possessing only slight amounts of the Fusarium-emetic principle, indicates that the seed-borne bacteria of barley may exert other impor- tant effects on the quality of the grain. .5 ... .‘ CHAPTER V. THE EFFECTS OF MICROFLORA ON MALT QUALITY LITERATURE REVIEW Barley damaged by microflora has long been impli- cated in the lowering of malt quality. Edward Lisle, in the early part of the eighteenth century, reported that ". . . Barley, reddish and stained at the germinating or sprouting end . . . W111 come away untowardly in malting, much of it lying behind on the maltingwfloor .1. ." (257). The same author also indicated that mownburnt and heat- damaged barley were unsuitable for malting. In a discus- sion of the malting process Lisle pointed out the neces- sity of changing the steep water to avoid “souring” and "sliminess" - undoubtedly the result of bacterial action. Matthews conducted an investigation on Fusarium- infected barley and malt (257). He concluded that the fungus reduced malt quality by lowering germination, re- ducing extract, and by imparting peculiar flavors to the malt. Gassow studied an organism, identified as Bacter- ium herbicolg rubrum Dagg., which seriously interfered with the malting process (152). The bacterium was re- ported by a British brewer to spread from the embryo end of the kernel over the surface of the husk. The husk and rootlets were discolored, the endosperm was converted to a slimy mass, and the rootlets later became necrotic. 140 141 The organism was effectively controlled by washing the infected grain with lime water prior to germination. Lott found that moldy wort gave less extract, less fermentable material (and consequently beer with less alcohol), and was more acid than non-moldy wort (240). He found that, contrary to other reports, fungus-infec- ted malt did not cause a moldy taste or smell in beer and wort. Nilson stated that bacteria lowered the enzymatic power of malt by their biochemical activity (287). Mason found that Egggglgm infection of green malt reduced ger- mination capacity and gave uneven growth on the malting floor (255). Hind has indicated in his brewing text- book that, in addition to the loss in germinability, bac- teria may cause beer instability by the putrefaction of infected barley kernels {177). Wahl and Henius recom- mended that an antiseptic material be added to the first steep of barley to destroy fungal growth which might have an effect on beer flavor (406). De Clerck states in his brewing reference that the heavy seed-borne flora will multiply in steep and in germination and bring about the asphyxiation of the barley embryos (90). Germination in— hibition by the microflora of severely weathered barleys has also been discussed by Preece (515). According to Leonhardt, weathered barley produces stained malt and off-flavored beer (227). ,Beaven has also indicated that barley over-exposed to bad weather may be rendered unfit for malting (28). ; .‘. 'J h. o- ‘L rb Fusariun~iure Lei tailey was found by Shands to injure the malting quality of the grain by lowering ger- mination, reducing vigor of growth, and altering chemical composition. Fungus growth during malting caused discolor- ation of the malt (547). Striulcic reported that the starch grains of barley infected by species of Fusarium presented an atypical corroded appearance, as the result of local fermentation: and this type of corrosion was dis— tinct from that observed in germinating kernels (579). Hopkins and Cooper haVe indicated that the betamamylase content of barley may be contaminated by variable amounts of alphauamylase secreted by the kernel microflora, eg. Bacillus subtilis, p. mesentericus, and Aspergillus spp. (184). Blackwood found that analytic alphawamylase, pro- duced by Bacillus subtil;a, also contained appreciable amounts of cytolytic enzymes active on barley gum (45). The author tested 114 species of Bacillus for cytase ac— tivity; and while all of the isolates produced some cytase, _p. subtilis gave the best yields of the enzyme. Meredith investigated the effects of peeling barley on malting quality (270). He found that the peeled kernels were subject to serious mold infection, and the finished malts were of poor quality, probably as a result of infection by Mgggg sp. The kernels germinated poorly and abnormally, modification was poor, extract yield and enzymatic activity were reduced and the malts had an unattractive appearance. A cooperative study of the influence of weather- l45 ing on malting and brewing quality of barley was carried out by the Malt Research Institute and several malting and brewing companies (5). Pilot scale and commercial maltings were made of paired bright and weathered barley lots from the 1951 and 1955 crops. Pilot scale brewing tests were also conducted with these malts by the par- ticipating laboratories. The barleys, malts, and beers differed markedly in their properties, depending on the degree of weathering. The following abnormal properties were, however, almost unanimously agreed upon by the co- operating laboratories for the combined 1951 and 1955 weathered barleys: l. Weathered barley steeped at a faster rate, had poorer germination energy and uniformity, and usually showed a prolonged postmharvest dormancy. 2. Malts prepared from weathered barley showed an abnormally high protein modification, higher wort color, and generally tended to be lower in ex~ tract. 5. Halts prepared from weathered barleys brewed and fermented abnormally only if weathering was severe. 4. Beers prepared from weathered barley malts were abnormally high in nitrogen and were dark in color. Taste and aroma were also less desirable. 5. Certain malt and beer abnormalities had a greater tendency to persist through de— creasing degrees of weathering than did others. Abnormally high beer and wort nitrogen and color, while diminishing in intensity with reduced weathering, were apparent with all weathered lots. This was true to a lesser degree of beer aroma lfifi and taste. Other :ctors, e.g. gas insta- bility and poor ferientihg and breWing response, while Obvious with severely weathered be rleys, vere is es apparent as weathering decreased. ‘3 i") The authors this repcrt acknowic,grl the ass: i‘ation of microorganisms with the complex of weathering, but pointed out that the microflora of the barleys studied in this program were not irwve tigated as a separate fac— tor. Sheneman and Hollenbeck have investigated the microbial patterns in malt, during the various stages of the malting proce ess (549). The authors state that char— acteristic groups of mwiaro Higanis us; including the aero- genes group, psychropn nilic grar negative rods, lactic- acid bacteria, aerobic sporeuformers, molds, and yeasts, can be distinguished in these various stages. The in- crease and decrease of lac mi acid bacterial populations during the malting process are described in detail by the authors. omyk has reported on the effects of microflora on the diastatic power of distillery and brewery malts (565). The author stated that brewery malts contained fewer’microflora, both qualitatively and quantitatively, than did the distillery malts. Preliminary studies in- dicated that dlS Llery mal ts, infected by fungi and other organisms, from l0 to 50 percent, showed a sig- nificant reduction in.diastatic power. Penicillium expansum, Fusarium herpgpum, and RhiZOpus nigricans all 145 reduced barley germination and lowered the enzymatic strength of the grain. Distillers object to high bacterial counts in grain because the alcoholic yield is lowered and bacter- ial metabolic products, such as acrolein, may be carried over with the alcohol distillate (50). The effect of the bacterial content of distillers' malt on fermentation ef— ficiency has been discussed by Adams and his co—workers (10). These workers have shown that the pasteurizing effect of conversion temperatures so greatly reduce the bacterial count of barley malt that fermentation efficiency is not lowered. The reduction of barley germination by steeping has been noted by several workers. It has been indicated by authors that certain chemical compounds, usually poly- phenolic in nature, may be removed from the barley kernels in the steeping process, and these compounds exert an in- hibitory effect on the germination of the grains (90, 214, 256). Gilbert and his co—workers have attributed the in- hibition of barley germination by steep liquor to an exo- toxin produced by the bacterial flora of the kernels (46, 127). Piratzky found that the growth of microorganisms was suppressed by the addition of small amounts of form- alin to the steep water (505). He also showed that the inhibitory materials present in the husks were not very active in reducing barley germination. Green and Sanger added both hydrogen peroxide and peracetic acid to steep 146 water to overcome inhibitory effects; they concluded that the action of microflora was of minor importance (145). Jansson and others have reported that "water-sensitivity" of steeping barleys is not due to the action of microflora, as was suggested by Gilbert, Piratzky, and others (196). The authors concluded that the phenomenon of "water-sen- sitivity" is an intrinsic property of certain barleys, a view shared by Kirscp and Pollock (211). Kudo and Yoshida have taken a stand between these two camps, and assert that "water~sensitivity” is related to both the state of the barley and its microbial flora (217). Workers at the University of Minnesota have re- ported that microflora on and in barley kernels may in- crease very rapidly under temperature and moisture con- ditions similar to those of commercial steeping and ger- mination (8). These investigators have also analyzed nine samples of barley for malt and beer properties, e.g. gas instability, reduced alphamamylase, and low wort nitrogen ratio; but the relationship to the microfloral populations of the samples was not completely clear (9). The malting process is subject to a number of inter-acting variables. Consequently researchers have designed facilities which permit rigid control and eval- uation of these variables in order to better understand the process. This has been best achieved by the use of pilot malting equipment, i.e. highly specialized equip- ment which, stepwbywstep, duplicates the commercial malt- 147 ing process. During the past thirty years such equipment has been increasingly employed by industrial, government, and university laboratories. Until recently pilot malting and pilot brewing facilities were most widely used in the quality evaluation of barley hybrids and varieties (96, 97). A more recent use of this equipment has been made in the study of microfloral irfluence on malting quality (97. 516). In 1957, Anderson described pilot malting equip- ment in use at the Grain Research Laboratory at Winnipeg (15). The duplicated components of the plant permitted the concurrent processing of eight ~55O gram samples. This equipment was modified to allow steeping of 250 gram samples along with several other improvements (l4, 15). The germination chamber was changed to accomodate twenty- four ~25O gram samples (272) and later a drum—type ger- minator was introduced into the system to permit greater flexibility (271). The pilot malting equipment of the United States Department of Agriculture Barley and Malt Laboratory permits the processing of 200 to 250 gram bar- ley samples for the evaluation of plant breeders' selec— tions, and ten to fourteen pound samples for pilot brew— ing studies (95). Davis and Pollock have described an experimental malting apparatus capable of malting sev- eral 800 gram barley samples under a wide range of con- trolled conditions (89). A description of an industrial pilot malting laboratory is given in a paper by Baker and 1.48 Burant (25). This facility was an outgrowth from exper- ience gained in the operation of an older pilot plant (154) and was capable of processing barley samples in excess of oneahalf bushel. Another industrial pilot malting system has been described which differs from con- ventional experimental equipment in that it provided an approximately adiabatic environment in both the germin- ator and the kiln (157). Banasik and his cowworkers have devised a micro- malting apparatus and method for the evaluation of barley selections developed by the breeder (25). The apparatus was capable of producing twentwaour microumalts, each weighing 60 grams. In general, the correlation coef— ficients for malt prOperties were high between micro and macro methods. Bendelow (51) and Whitmore and Sparrow (417) have also described micro-malting methods utilizing fifty and thirty grams of barley respectively. Burkhart and his co-workers have compared commercial and laboratory malts prepared from the same barley lots (58). They con- cluded that laboratory malting was apparently satisfactory for pilot brewing evaluation of barley. The influence of malting environment and the varietal response of barleys to this environment were studied in detail by Shands and others (545). This study emphasized the importance of temperature, moisture, and period of growth on the varie- ties Oderbrucker, Wisconsin Barbless, Peatland, and Chevron. Prentice and others (97, 516) have applied ninety- 149 seven microorganisms, commonly found on barley, to kernels during malting in a pilot plant. Micro—brews were also prepared from these malts. They found that with inocu- lated malts: (l) wortwnitrogen levels increased, (2) a1— phaamylase and diastatic power increased, and (5) beer gas- stability decreased. Although several types of organisms were associated with these effects9 Fusarium species were most commonly implicated in the production of undesirable characteristics. Methods of inoculating barley and other cereal grains with microorganisms have been reviewed by Sallans, who found that infectiVity of fungal inoculum decreased with the age of the culture (555). A method for inocu- lating barley with helpinghosporium gramineum Rbh° was devised by Fuchs (120}. The author used a needle to in- sert conidial suspens1ons of the fungus between the glumes of soaked ripe grains. With large quantities of inoculum, he found that placing the grains in a conidial suspension for twenty minutesp under vacuum, gave the highest per- centage of infection. It was recommended that the inocu- lated grain be stored in an incubator at high humidity (90 to 100%) for two days, followed by drying at room temperature and humidity in order to promote abundant mycelial growth beneath the glume surface. A modifica- tion of the “vacuum method“ has been described by Moore for the inoculation of wheat and barley with the loose smut fungus (27b). in C) MATERIALS AND METHQ§§I To study the influence of certain microorganisms on the quality of malt. a pilot malting unit capable of producing multiple malts under comparable malting con» ditions was designed and constructed. The unit was con— structed so that a maximum of four samples, of approxi- mately one kilogram each, could be run concurrently un- der conditions as nearly uniform as possible. These sam— ples were placed in the unit in separate compartments to avoid the danger of crossmcontamination. A final, but necessary, consideraticn was the cost of constructing such a unit. Insofar as possible, salvage materials or existing equipment was employed throughout the construc- tion of the pilot plant. Cleaning and grading All of the barley samples were cleaned and sized by passing the grain twice over the reciprocating sieves of a benchmtype grader. The sized grain was stored in canvas grain sacks until used in the malting runs. Steeping equipment A forty gallon aluminum steam cooker was mounted on a wooden platform and adjusted to a height sufficient to ensure water drainage into the adjacent laboratory sink. A plastic tubular hoop covered with a fine—mesh aluminum screening was placed near the bottom of the kettle. This screen served as a trap for spilled ker- nels, preventing their entrance into the drain outlet. l5l A wooden framework was constructed to support the indi- vidual steeping containers upon the screen. Four - 5 gallon metal fruit containers served as steeping chambers. Cylindrical baskets made of aluminum screening were placed in these containers. These baskets held the steep— ing grain and facilitated drainage, aeration, and manipu— lation of the steeped barley. Water was supplied from the university wellewarer system. Temperatures of the water varied from 5a°F.. in the winter to 59°F., in the summer. The water was distributed from the sink outlet in a one-half inch rubber hose to a four-way distribution manifold constructed of threeufourths inch galvanized pipe and fittings. Aeration was achieved by the use of a rigid aluminum tube at the end of a rubber hose leading to the laboratory compressed-air outlet. The steeping unit is pictured in Plate X. Germinating equipment Paired germinating chambers were constructed from two _ 8 cubic feet iceboxes placed backstomback. The bottom doors were sealed and the upper doors were gas— keted to ensure airutightness. An aluminum shelf was placed in each germinator and openings in the shelves permitted the insertion and removal of the individual germinating pans. The pans were prepared by drilling one—eighth inch holes on one inch centers in the bottom of stainless steel cake pans (2" x 9" x 15"). When the germinating pans were placed in position, all air passing 1.35%.,“ a? .. "7.3x...” mi. . 152 PLATE X Pilot malting equipment used in the study of the effects of microflora on the quality of malt. Upper - Aerial view of the steeping equipment; showing water supply, steeping tank, and individual steeping containers. Lower — Overall view of the germination equipment; showing the spray chamber, paired germina- tors, and refrigeration unit. 153 into the upper chamber of necessity, flowed through the pans and was carried out through the exhaust openings in the lower chamber. Two cooling coils were wound from one-quarter inch copper tubing and were placed separately along the rear of each germinator. Aluminum strips were placed under each coil to deflect the condensate away from the germinating pans. A "T"-fitting joined the two coils at each side of the two germinators. Three-eighth inch, flexible, plastic garden hose was connected from each "T"-fitting to a "force-flow liquid coolant circu- lator" (Daigger model R 5250) which had a cooling capacity, using water initially at 50°F., of 0.90 to 4.S°F. per gallon per minute and a temperature range of from 10° to 90°F. with an accuracy of 1 2°F. The coils, hoses, and refrigerator when connected formed a closed system and stabilized the temperatures within the germinating cham- bers. The formation of vapor-locks in the system was prevented by introducing a suitable venting line which also permitted the replenishing of lost coolant. Dis- tilled water was employed as the coolant which restricted operation of the unit to temperatures above 52°F. A spray chamber was constructed to attemperate and saturate the air passing into the germination chambers with water vapor. A stainless steel sink (17" wide x 17" deep x 36" long) was fitted with a one-eighth inch thick aluminum cover and served as the spray-chamber. The cover was 154 gasketed and secured to the sink by means of small ma- chine screws. A Redmond blower of 100 c.f.m. capacity was fitted to one end of the spray chamber top. The air leaving the spraymchamber passed through a system of rigid plastic piping (la%" diameter) which delivered the attemperated and humidified air to the chambers below. As originally executed, the air was saturated by passing it through a series of glasswwool battens which were in turn impregnated with water by means of a copper-tubing manifold connected to the Slnk outlet. This system was later discarded because of the heavy bacterial deposition of slime on the battens. A more satisfactory water screen was subsequently employed. This consisted of a brass spray nozzle (Chicago fog jet » Spraying Systems 00., 5/4 7G) which was conneCted to the water inlet by means of three~quarter inch galvanized piping. The excess spray water was carried by gravity to the laboratory sink be- low through a sink-drain placed in the bottom of the spray- chamber. A recording thermometer (Weksler with flexible tubing and bulbs) was fitted to the side of the germina- ting chambers. The two flexible lines from the thermom— eter were placed, one in each chamber, so as to record the temperatures within the two compartments. Tempera— tures of the green malt could also be recorded by placing the thermometer bulbs in the germinating pans. Since the temperature and tre relative humidity of the germinating chambers were functions of temperatures of both the spray- 155 water and the coolant systems it was necessary to provide for adjustments of the spray-water temperature. The tem— perature of the spray-water was varied by introducing a "temperator" (Central 00.), designed for use in reducing the condensate which forms on toilet tanks. Table 16 shows some selected temperatures and relative humidity readings, obtained by varying the temperatures of the two systems. The germination equipment is illustrated in Plate X and XI. Kilning equipment A standard gratitywconvection laboratory oven (Modernlab Model l7~;2, 800 watts, 180°C. temperature range) with interior dimenSions of 20" x 15” x 20" was modified for use as a kiln. A small blower (Redmond - 50 c.f.m.) was installed at the bottom of the oven, and the base of the oven was sealed to prevent air—leakage. An auxilliary blower (Redmond - 180 c.f.m.) was placed at the exhaust Opening at the top of the oven. A large container (20" x 15" x ;0%*) was assembled from one—half inch hardware cloth to hold the individual kilning con- tainers. These containers were fabricated from fine- mesh aluminum screening and measured 7" x 7%" x 8%". The basket assembly prevented physical contact between the various malt samples and at the same time provided for the uniform passage of the heated air through the drying malt. Sulfurndioxide was applied to the malt by releasing the vapor into the small blower at the bottom .10 .H/ _.Ix .pdoomem GH penance QOHPmQHEch mo thOHasn o>HpmHom 0 .mo QH nmpaono soapmaHBHom mo osdpmsomsoe m OOH cm OOH Ob OOH mm OOH Hm OOH we OOH mo m.Hm om ow OOH Oh OOH mu OOH mo OOH m0 OOH NO O.#u om om cos on OOH as ago we ooH mo 00H mm o.Hs mm as so nu OOH OB OOH or OOH mm OOH mm 0.50 mm mm to mm m Or 00 we no He OOH mm m.m© em as so as m or on mm em or mm mm o.Hm e: me an we am mm mm mm cm Hm no mm m.mm me an em mm mm mm mm mm mm mm 00H mm 0.0m we on em mu ow mo dw do Hm ow OOH cm m.wm we on an mu mm mm do mo om 00 OO om O.@& a w s m a m p a p a p a H.m.O om - 3w :1 on om .m u o: wasps apogee» Hops: A.mov #Hnd QOHpmHomthmH mo mhdpmnomaop mpoawxonmag inseam (II. m we ompsmno cOHpqusomm go quHommu mpHoHasn one encompasses a .mH oHpme .moHSpmommsop qus QOHpmommemoo one endomommsmp nmpmsswmomm mo QOHposdw PLEASE NOTE: This is not original copy. Some light and dark type throughout. This page is too light to be readable when filmed. UNIVERSITY MICROFJIMS, INC. 158 of the kiln. The kilning equipment is shown in Plate XI. At the termination of kilning the rootlets were removed by rubbing the kernels in a clean canvas seed sack and the malt was then stored in air~tight metal sample cans. A number of preliminary malts were run using var— ious steeping, germinating, and kilning procedures. The most satisfactory malting schedule, and the one employed in all of the subsequent runs with inoculated barley, was as follows. Four weighed lots of a single variety of bar- ley were used in each malting. The cleaned and graded barley which passed through the 7/64 inch sieve and was held on the 6/64 inch Sieve was used in all runs. The weights of each barley lot ranged from 500 grams to 1,000 grams depending upon the number of analyses desired. The four barley lots were placed in the steeping baskets and the flow of steeping water was adjusted to approximately seven gallons per hour per basket. The steeped barley was inspected periodically and the steep water changed at approximately four, twelve, and twenty-four hours aft- er the beginning of steeping. The water was changed by agitating the steep basket several times to mix the grain and to release adsorbed material, removing and draining the steep baskets, and pouring off the old steep water. As fresh water was being added to the grain the barley was aerated for thirty seconds. When the steeped barley satisfied several empirical tests for the determination of moisture content it was removed from the steep con— . . 159 tainers and placed in the germinators. These tests in- volved: (l) pinching the kernels lengthwise between the thumb and forefinger, (2) pressing the kernel with thumb— nail against the flat surface, and (5) sectioning the ker- nels lengthmwise and observing water infiltration with the aid of a binocular dissecting microscope (406). A two gram sample of each barley lot was taken as the steeped grain was placed in the germinators and tested for mois- ture content by the hot air oven drying method (5, 91). Whenever possible the moisture content by weight was brought to 45 percent, as recommended by Shands and his co-workers (545). If the moisture content was below 45 percent the green malt was watered to allow further water imbibition. The water temperatures of the germination equip- ment were adjusted to provide a relative humidity of 100 percent and a grain temperature of from 50° to 65° F. These adjustments varied with the temperature and relative humidity of the laboratory. The germinating grain was manually agitated daily to prevent matting of the rootlets, a common occurrence in pneumatic malting (90). The separ— ate samples were watered with a polyethylene spray bottle containing fresh tap water. The grain temperatures, rel- ative humidity of the germinators, temperature and rela- tive humidity of the laboratory, and any manipulations of the equipment and/or grain were noted several times daily. When germination had progressed to the desired stage the L60 green malt was transferred to the kiln. This stage was reached when the acrOSpires (plumules) had extended from threeofourths to the entire length of the kernels of the apprOpriate check lot. The germinated barley was placed in separate wire kilning baskets and dried according to the following schedule: (1) twelve hours at 57° 0., (2) twelve hours at 49° 0., (3) three and onewhalf hours at 77° 0./l. This schedule was approximate; the times and temperatures were adjuSted in accordance with the mois- ture contents of the drying malt. The malt was sulfured during the final drying stage as described above. Immed— iately after completion of kilning, the malt rootlets were removed and the malt was placed in sample cans and stored. Three varieties of barley (Hannchen, Montcalm, and Traill), grown in the west in 1959 under irrigation, were used in this study. The three varieties were exception- ally bright and, except for the Hannchen lot, were rela- tively free of microflora (Table 17). These varieties were variously inoculated with pure cultures of six bac- terial species, four fungal species, and one pink yeast (Table 18). A modification of Fuch's method (120) was used for the inoculation of the grain. The inoculum was prepared by growing three repli- cates of each organism for one week. The cultures (50 ml of medium in 250 ml Erlenmeyer flasks) were maintained on a reciprocating shaker at 25° C. The bacteria were cultured l R. Bonner - personal communication. 161 .pOH donondmm on» How pneonom mm I am one anopqu one HHHwHe mmeoHnmb esp no“ pdooeom OOH I mm mm: hpflommmo GOHpoannoO .mchcHHm> HHm Hon ApQwHez hnv pqoohom dcboHo one new deozpen who: quuw consonpqs on» no manopdoo onspmHoz .sam>opmpoooaso one sam>apmpaamso neon .osHHm thnopmHmsoo one: monaom Moose ocHHo one oomecpm HOM one Ao>onw ocpmHHv mmHm IEMm Moose oopomnpas mom mpqsoo HomoHMOHOfia one .oononwogmm dH pnMHHp hHoEoHp Ixe HHs one: was .nOHpmeHHH Hoods pmoz one as axonw HHw one: mercHHmb coma» one .AOOm .moe .mov .H .pmwno sH oonflnomoo Houses on» nH ompme one: mHonnoM s O H O O H O O HHHsHa O O N a O m mm nonoqdmm O O O O O O H chopno: Henpo mpmwmw mHneuowm adHHommoomHo anmooonm sdfiHMmsm «HumanepH4 hpoHHwb hoHHmm mHqumM OOH Hem meHGOHoo Ho Honasz I\mopme some do deHpmHm HedeMIoHonB hp conflsnopeo mm moHospm wdest pOHHm on» nH w oomd meroHnmb hoHusn mean» on» no meepnoo HwROHmoHOHE one I .oH cHnme .HOOMV Houses onodmpm map qH UoPMHQ mHoGHmM Sony omaHmHDo mmpmHomH HHH s .MH SSHmnHon adHHommooMHO .QoHE .o>0HwHHmm momHodHM mem cmmlm .mqmm H .omqm mHHmono .mm.m EdemoH SSHHmmdm HOQMHMHom Balm Hmmum .m a ..M .m mmflflwmw. aoHHommonpsHsHom maHmopso: mmIc OHMIH .mmoz mHsmop mHHmsHm +H4 mflopHHmz mHmIo¢O mem ¢OmIm .Qm mHSHopooonm oo oo OOI¢ Hmmlw IIII .HIm Ho noHsm HHS 9i oooHoINs .Hm> uchoo mJHHHotn oo oo owls Ommlm mu .am MNHHHHHH oo oo ooIe mmmum .mm adHHcpomochfl- no .Ewmopn Z omsw wmmam optHoosHoHHH mooHoo ESHHMHmmma NHWHHoMH oo .chofinmm mmsw meIm .Hw Hwozv odoHomHomHM mtfimmwmaamm .ncH: .msHmHmH .m MHHHoHH mm IO OHNIm .miz H.N.Hmv swaMHQH msHHHHmm HmmHsoaa moonsHm mm d mIm oHoHomH Ho HHHHsooH ix opmiomH Ho coHSOm oopmwmmm omwwflwm Ill I.Ill|l" c SIHHirdH QHi HcHHso oHoHSoosH op ooms msmHsomHo Ho HHHHsooH ode to Hsom onH 5 .DH moHsHos HOHHM esp nH ommomoon oHosH 165 in nutrient broth and the fungi in potato broth (522). The bacterial cells were harvested by combining the con- tents of the three replications, after establishing their purity, and centrifuging in a laboratory centrifuge for thirty minutes at 5,500 r.p.m. The nutrient broth was poured off and the cells were remsuspended in sterile dis- tilled water. The fungal inoculum was harvested by comb- ing the separate replicates, as above, filtering off the potato broth, and suspending the mycelium and spores in sterile distilled water. This suspension was placed in a Waring blendor and comminuted for one minute. The barley was prewtreated, to remove surface contamination and to increase moisture content, by steeping for one hour in the manner outlined above._ The size of the barley samples was, in every case, two and oneahalf pounds. The steeped bar- ley was placed in a modified pressure cooker after draining off the steep liquor. The suspension of inoculum, either bacteria, fungi, or yeasts, was added to the container and the volume of the liquid increased to one liter (of. Table 25). With this quantity of liquid the suspension just covered the surface of the grain. The cover was placed on the container and the air evacuated by means of a vacuum line. A manometer, attached to the apparatus, per- mitted a rapid appraisal of the amount of air removed from the kettle. After the air had been exhausted, a pinch- cock on the exhaust line was opened allowing air to flow rapidly back into the container. This procedure was 164 repeated three times. The inoculation apparatus is pictured in Plate XII. After inoculation the grain was removed from the kettle, drained and spread out in a thin layer on news— paper to dry. The grain was periodically mixed to ensure even drying. A single sheet of paper was placed over the grain to exclude airwborne contamination. The relative humidities encountered in the laboratory at this time ranged from thirty to forty percent. The grain usually reached a moisture content, by weight, of thirteen to fourteen per— cent after two or three days of drying under these condi- tions. At the completion of drying the grain was stored in sealed, two~quart mason jars until malted. Check lots of each variety were prepared in the same fashion, except that only sterile distilled water was placed in the vac- uum container. The malting of inoculated barley was carried out using the schedule presented above. In each case only a single variety was malted at one time. A check and three inoculated samples were processed, exercising caution to avoid cross-contamination. The bacterial inoculations were run separately from the fungal treatments. Kernels of the inoculated barleys and of the check lots were plated, both before steeping and after germination, to determine the amount of contamination and the effect of malting on the microfloral populations. Samples of steep liquor were taken periodically and dilution plates run with the steep liquor for the same purpose. _ _. -__ "II! a. .._<-_.—’ PLATE XII The apparatus used in the inoculation of bright Mnfleys with bacteria, yeasts, and fungi for use in the Lfilot malting studies. The modified pressure—cooker, used to.hold the steeped grain and the inoculum, is shown on the left; the manometer is supported on the upright board at the right. The tubing on the left is connected to a vacuum line, the center tube is used as an air inlet. .Hn 1“ .a-v- ,...,. y "y... ‘— .u. _ n... II [I I Lee At the time 91 storage the pilotwmalts were given a code number to avoid any subjective bias in their eval- uationo These malts were subsequently analyzed, by code number9 for physical and chemical prorpertieso The analyses were conducted partly by the author and in part by two commercial malting laboratories. The tests included both standard malt analyses (5) and evaluations dictated by a knowledge of the properties of the inoculated organismso Experiments were also conducted to determine dif— ferences in the rates oi steeping between varieties and samples differing in their microfloral contents. To this end a number of miniature steeping baskets, two inches in diameter and two and one half inches high, were construc- ted of fine—mesh aluminum screening. Weighed portions of grain were placed in each basket and the baskets covered with a cheesecloth square. All baskets were submerged in the steep tank and at measured intervals they were with- drawn. The excess water was removed and steeped grain was weighed to determine the amount of water inbibed in this time interval. The moisture content of the grain was com- puted with the use of De Ulerck's formula (90): (Ben x 100 x a lOO - H where X is the necessary increase in weight of 100 grams of barley, H is the desired moisture content to be achieved, and h equals the initial moisture content of the grain. .o- \; I67 EXPERIMENTAL RESULTS Pertinent malt analytic factors of inoculated bar- leys and the non-inoculated check samples are presented in Tables 19 through 2i. Although obvious differences exist between the check samples and the inoculated malts, these differences must be viewed with caution. In many cases these differences are the result of malting conditions rath- er than a reflection of microfloral effects. Daily records of each malting run were maintained; any malting condition which altered the analytic characters of the finished malt were recorded. This information is included in the tables under "Remarks". The malting information and the analytic data were examined for differences existing between the inoculated malts and the corresponding check samples (Tables 22, 25, 24). Some of the isolates consistently showed no effects with certain varieties, e.g. bacillus polymyxa (B-S) on Traill and Montcalm (Tables 22, 24). Alternaria tenuis (Fu304) on Montcalm (Table 22), CladOsporium herbarum (F-524) on Montcalm (Table 22), and flelminthosporium sativum (F-BlO) on Traill (Table 24). Traill and Hannchen malts inocula- ted with the Flavobacterium isolate (B-226) showed in- creased l,OOO kernel weight (Tables 25, 24). The kernels of Mantcalm malt, inoculated with the same organism, were reduced in weight (Table 22). Malt prepared from Traill barley, inoculated with Cladosporium herbarum (F~524), .GOpraHom sflpflonmama pamzvomQSm nuflz oedpmfloa 0>Hmmooxm pm cmqawx mamamm .Hmahoq I an .HMcHo I Ho .hamn I an .hapnmfiam no 30am How am .hnm> I b "mzoaaom 00 ohm poms macapmw>mpnps .0003 mmp0aomfl mo hpwpaopw pom ma magma mom Q .Amv moonpma .o.m.m.4 op mafivuooom cosmomnmm mmpdpmoonm cephams< .mfimmn hmw 0 do UmpHomon meopomnmno m 0:.: m0.: Hm.: Hm.: 5a.: 00.: 00.: ::.: 00.: nampoua mangaom II II 00.0 00.0 00.0 H0.0 00.0 00.0 m0.0 mpaeaom who: o.H: m.m: 5.:0 0.0: :.o: 0.m: H.m: o.H: :.m: campopm Hmpoe\.aom 0.50 0.0: :.mm 0.0: :.mm H.5m 0.0m 0.00 4.00 mmwasam magaa 5ma 0ma m: 00 m: 00H 05 00 05 nmzoa oapmpmwan Ha as am an an as am an an mafia qoapmnpaam mw Ho Ho am H0 Ho Ho an am am am no Ho hpflpmao pmo3 i 0.H 0.H :.HH 0.0 5.:H 0.H 0.0 a.m m.m noaoo ago: I 5Im 2,5 5Im 5Im C 5-0 .3 . 3 was mouths 0.a m.m 0.m m.a 0.H m.a 5.H 0.H 0.H AmoImmV moqmnmamam :.55 0.55 m.05 H.05 :.05 H.55 0.05 0.55 0.55 A.m.ev pomnpwm 0.0 0.0 H.m H.: 0.m 0.: m.: 0.: m.: Aav mesumaoz II II 05.0m H0.0m 00.0m No.0m o0.0m 0:.mm 00.0w .pz .an ooo.H mmmwm mIaomeo HmmIm. :omIm. mIaomeo.NMWMMMpwmmwmIm 0am HIaomap I\mn0pomumao m m owpmqu« ampams woaflm,sHmcpcoz Mm mflmwamcm H00H60no 0:0 mOHpmflp0p0090:MIamoflmmsm I .0H canes 330‘, .I .3 MWIIUAOnmv‘ImnIV .....III I. IIIII. ll I1.[.|II II M \Vnmw m 4% DUI-Inn, I“ difi Li! 9‘ 'HI~ III. \ .I I l‘ .l I‘YI AVA. .5. .|~.Io .L 169 umZOHHOH mm 09m 60m? mQOHPMH>®HQP4 .aOHpmanom afloaoqwaoa pdmswmmnnm neat ondpmwoa obfimmoowm p0 dodHHM mamamm .Hwanoq I an .meao I Ho .huwn I an .hapnwaam no team you an .hhmb I b .000: 00p0H00H no hpapdooa you ma wanna mom n wdfidnooow omanomnmm 00Hddmoonm oaphamnd .00009 and wAmW MWMMWMWH.wwmwme0MW 0 05.0 00.0 00.0 00.0 05.0 ::.0 00.0 05.0 0000000 ago: 00.: :0.0 00.: 5:.: 05.0 0.: 00.0 0a.: 0000000 0090000 :.0: 0.00 0.0: 0.0: 0.50 0.0: 0.00 0.0: 0000000 a0poa\.aom H.H0 5.0a 0.:0 0.00 0.:0 0.00 0.00 0.00 000a»00 000H4 H0 00 0: :0 00 H0 00 :0 00:00 000000000 H0 H0 H0 am 00 H0 00 Ha 0aap noap0npaam an an am an ac an am an Ho Ho hpflnmao pnos H.0 0.:0 :.0 0.0 0.0 :.H o.H 0.H noaoo ago: 0HI0H 00I0H 0HI00 0Uv 5I0 0I0 0I0 0I0 0aam qoamum>qoo 0.0 0.0 0.0 0.0 0.: 0.0 0.: 0.0 AmoImuv 0000000000 0.00 0.55 0.00 0.00 0.05 0.00 0.00 0.00 0.0.00 0000000 0.0 0.0 0.: 0.0 0.0 :.0 0.0 H.0 000 0000000: :0.50 00.:0 00.50 00.00 00.50 00.00 00.00 m:.0m .p: .qu 000.H H00I0 000I0. :00Im mIao000 0mmIm 0H0I0 0I0 HI00000 I\0m0po0n0no m\mpnmapwmpe 0 cath0Q4 000000 poaam 00000000 00 00000000 H0000000 000 000000000000000 H0000000 I .00 00009 AJIHWINIIE- 4Ul.flhl- IIIIJI | 1' I . .. 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II {...- 170 05.0 05.0 05.0 00.0 00.0 00.0 00.0 0:.0 0000000 0003 0.0: 0.0: 0.:: 0.0: 0.0: 0.00 0.50 0.0: 0000000 000oa\.000 00.: 00.: 00.: 00.: 00.: 00.: 00.: 00.: 0000000 0000000 :.00 :.00 0.50 0.0: 0.00 0.50 0.00 0.:0 0000000 00000 00 00 05 000 :00 00 00 00 00300 000000000 00 00 0a 00 00 00 0a 00 0000 0000000000 an 00 Ho 00 an an > an en > 0000000 9003 5.: 0.0 0.0 0.0 0.0 5.: 0.0 0.: 00000 0003 0v 0v V 5I0 5I0 5I0 0v_ 5I0 0000 0000000000 0.0 0.0 0.0 0.0 0.0 0.: 0.0 0.0 000I000 0000000000 0.55 0.05 0.05 5.05 0.55 0.:5 0.05 0.55 0.0.00 0000000 5.0 0.0 0.: 0.5 0.5 0.0 0.0 0.0 000 0000000: 0:.00 00.00 00.00 00.00 00.00 00.00 00.00 00.00 .0: .00 000.0 000I0 000I0 :00I0 0I00000 000I0 000I0. 0I0. 0I00000. I\0000000000 IM\mpdmapwmne 0 000000Q< .mpama poawm 000009 no 000N00n0 Hmofiamno 0am 0000000000000no 00000N£m I .HN 00906 171 .000008000 0000000008 pqoswmmnsm £003 00000008 obwmmmoxm pm canafix maaamm .008000 I 00 .00000 I 00 .0000 I an .hapnwwam 00 30am How am .0Hmb I b 00300000 00 000 0000 0000000>00994 .000: 00000000 00 00090000 non ma 00909 00m Q mdwcpooow 008000000 mmnnumoonm oaphamq¢ n .Amv moonpoa .o.m.m.¢ on .00009 000 0 do 00000000 0000000000 d 0000000 0003 0.0: 0.0: 0.0: 0.0: 0000000 00000\.000 00.: 00.: 00.: 0:.: 0000000 0000000 0.00 0.00 0.0: 0.:0 0000000 00000 :0 05 00 :: 00300 000000000 00 00 00 00 0000 0000000000 00 an do an Hm 0000000 0003 :.0 0.0 0.0 0.0 00000 0003 00-5 00-00 5.0 5.0 0000 0000000000 0.0 0.0 0.0 0.0 000-000 0000000000 0.55 0.05 0.05 :.55 0.0.00 0000000 0.: 0.0 0.: 0.0 000 00000000 I- II I- I- .0: .00 000.0 000.0 000-0 :00I0 0-00000. |\0000000000 M\mpnoapmmhe 0 00000004 000000000 I .00 00000 172 Table 22. — A summarization of the effects of six :inoculated organisms on the quality of pilot malts pre— jpared from Montcalm barley/é. Inoculated b ‘with Effects 'B-S None iB-2lO Reduced 1,000 kernel weight Erratic germination Increased number of glassy kernels Increased wort color Hazy wort Slow filtration Reduced diastatic power Reduced alpha-amylase B-226 Reduced 1,000 kernel weight Erratic germination Hazy wort F-304 None F-521 Slight reduction in extract Slow conversion time Slightly hazy wort Slow filtration F-324 None a Physical and chemical analytic data are listed in Table 19. b See Table 18 for identity of isolates. Inoculated b with Table 25. 175 - A summarization of the effects of six inoculated organisms on the Quality of pilot malts pre- pared from Hannchen barley./- _. ‘,_4 cf” “.. Effects /_ B-5 B-210 B-226 F—304 F-510 F-521 00'9-0-000 Reduced 1,000 kernel weight Reduced kernel plumpness Slight increase in wort color Reduced 1,000 kernel weight Reduced kernel plumpness Increased wort color Slow filtration Slightly reduced alpha-amylase Lower wort acidity (pH) Increased 1,000 kernel weight Slightly reduced kernel plumpness Slight increase in wort color Slow filtration Slightly hazy wort Slightly reduced alpha-amylase Increased 1,000 kernel weight Slightly reduced kernel plumpness Slow conversion time Increased wort color Hazy wort Slightly reduced diastatic power Slightly reduced alpha—amylase Reduced 1,000 kernel weight Reduced extract Slow conversion time Increased wort color Hazy wort Reduced diastatic power Reduced alpha-amylase Lower wort acidity (pH) Increased 1,000 kernel weight Reduced kernel plumpness Slow conversion time Increased wort color Hazy wort Reduced diastatic power Reduced alpha—amylase a The effects noted here may be due to natural micro- may F.) (W l x A I Table l7)o V t7 / -- .L‘,‘ r'.‘ ‘fi .LJ. “ted 1 :‘ t 11 1. L 119 11101 O v A . 1’1 .7 ‘L if! ‘p‘ 1 . V .‘ dflIH Ullijd I C H in) ~/ I Ch ‘f .L L) v G. I' 1'1 T: U , . , M g ‘ . y“ r: 1?; marks '\ “entent of 1 CI“: IT Us I'll l ‘0 a " ILJ. uULSt r .- C 3... ,. W1. 1.! . 4r: Wm; 175 Table 24. _ A sunmxrizatior of the effects of eight inoculated organisms on the quality of pilot malts prepared from Traill barley/é. Inoculated b with Effects B—S None B—2lO Reduced kernel plumpness Erratic germination Slow conversion time Hazy wort Reduced extract Reduced alph—amylase B-226 Increased 1,000 kernel weight Hazy wort F-504 Reduced 1%000 kernel weight Reduced diaStatic power F—le None (under—modified) F-521 Slight reduction in extract Increased wort color Hazy wort Slow filtration Reduced diastatic power Reduced alpha—amylase F—524 Reduced diastatic power Increased alpha—amylase Y~52l Reduced kernel plumpness Slow Conversion time Hazy wort slow filtration Reduced diastatic power Reduced alpha-amylase Physical and chemical analytic data are listed in Table 21. See Table 18 for identity of isolates. I76 demonstrated an increase in alpha~amylase over the cor— responding check sample (Table 24). The most consistent defects in the inoculated malts were: increased wort color, haziness of the wort, slower conversion and fil— tration times, reduced kernel weight and plumpness, and reduced enzymatic power (Tables 19 through 24). The organisms which were most consistently associated with these effects were: Pseudomonas atrofaciens, (B-2lO), Helminthosporium sativum (F-BlO), and Fusarium roseum f.sp. cerealis (F-52l) (Tables 22, 25 24). Surface~disinfected kernels were plated with all varieties and treatments, both from storage containers ’ and from the germinators after two to three days of germination. The microfloral content of the stored sam— ples remained relatively constant, i.e. at or near the original inoculum level. With only one exception, the bacteria increased to nearly 100 percent on all of the plated green malt kernels, including the uninoculated check lots (Table 25). The exception was the first series to be run through the pilot plant after the ap- paratus had remained idle for several months. The check lot of this Montcalm series remained free of both bac- terial and fungal infection, although the inoculated samples all showed an increase of the original bacter- ial inoculum to 100 percent on the plated kernels (Plate XIII). The foreign bacteria were mainly white or creamy in appearance. A number of randomly-selected cells 177 a- nu am oe HHHmna II II OH 06 nmnonnmm II II NH op aHmopqoz mHHmo 000.000.0H Immwum. w mm OOH OH op HHHmne w ms OOH ON ow donoaqwm N do OOH Om ow aHmopqOS mHHmo ooo.ooo.om Immmum w OH OOH Om on HHHmHa w m OOH m: 06 smnodqmm a mm mm am 0e samopqoz mHHoo ooo.ooo.om Immmum W cm no we 00 HHHmea w mm OOH mm 00 amaoddmm N do OOH mm co SHmopdoz mfiamo 000.000.0m Iwum \ mQHpHma \ wsHpHma \ SmHsmeo M quHSO mHOHH w mansw astOo m UmpdeoodH !\ Ammstn we “do aOHOHa QmHHm isH HmsHmHHo an GOHpoom D .mQH fiam sway Hm>eH I\adeoqu we no mmwmeosH mo mmmmnqu usH HmeHsH aszoosH HmsHmHuO w thpsmvH .mmemHsmb NmHhmo mohap we msHpHma pOHHm mnp Masada wAOHmoeoaa mmHHm mo ommmeosH map cam .asHSoqu stHwHHo mo mmmomqu .Hm>mH sOHpomqu HmeHsH .adH5002H quHmHeo mo panosm one I .mm mHnme 178 00 00H 50 00 am 00 OOH om mm ON mH #N mm NH 0m OH MH mm E\- d‘ (D H r"! mHHmo mHHmo mHHmo mHHmo oe co co 000.000.: oe 0e co 000.00m oe oe oe 000.000.0m oe ea 00 000.000.HH oe oe oe 000.000.mH HHHMHB monoddmm aHmopnoz IHMNHM HHHMHB donodnmm aHmOpsoz ImeHM HHHMHB sonosqmm aHmopaoz Immwum HHHMHB nonessmm aHmopqoz IHMNHM Haemne amnoqqmm aHmopqu m Im Omdandoo .mm mHQme I. 111‘: nudfinvnv OAuAuwoomo H. dumW-lfiH. ,.,.I[ :1 ..-.l,!fmmnr“ I4fll1.®lmw alw'nflmnuufl 1.3.11.2, IWnNimlfu 1\.~v 179 .AmondeHa hHOHoo Ho omswoon pHSoHHHHc mm: madewmno HMHHaHm ImHu no GOHpMOHHHpnocHV oHdeH opmaHNonmmm aw mmpmowdnH mums doHpmosd .mHod Inca OOH Hem moHnoHoo HwHHaHmmHv hHHwOHMOHonmnoa Ho nonadd on» mopoaoo ondmfih o .mHmnan OOH Hem moHnOHoo HwHHsHm hHHonmOHonmnoa Ho Hogans on» mmpodod onsmHm d .mHonpmM OOH Hon mmHaOHoo HwHHsHm hHHMOHMOHonmnoa Ho Renaud on» mmponou Masada o .dOHmsmmmsm adeoonH on» dH vquwpdoo mHHoo oHanb Ho nonadq cup mpsmmmnmon pndoo .m.H .mmHHom mpMHmIQOHpsHHd MPH: cmpdmaoo HoboH astoodH n .msmHnmmHo Ho hpwpdovH How mH oHnwa com a mm a w 00 HHHMHB OOH m ON 06 denoddwm OOH OH dH 06 aHdopdoz uaaoo 000.000.H emwum eosqfipaoo u .mm canes .,. u .n I. B C ”0 180 PLATE XIII Montcalm barley, inoculated with three species of bacteria and an uninoculated check sample, plated after two and one-half days in the pilot malting germinator. prove i to be gram pos1,ive, sporemfcrming, aerobic bacten ria, i.e. species CI b1_iil2§ (55, 54). Petri plates con- taining sterile nutrient media ere expo ' ed in the disin— fected, empt't ge: Linazors for ten minute periods. Two to five uacterial colonies appeared on each plan; after incu» bation. The colony color and staining :eacticn of these bacteria resemb ed these KLJ‘lIV'QIHLHA o ganisms which ap~ Feared on the green Hill. A smaller number of bacteria were also isolated rim the spray water source by Streaking lOODw fuls of the water on the surface of aver plate”. . —_.\ ,2- u~~ .-, .-:~ -4.” A, .; , “r “ , ,4 ins surface bac,ealc cf CLUprhg carley showed Slam H- i...) F“ ... ,< * r -- ‘4 + :7, '— 1. ’ ' ‘\ -', .- I} ’ ‘ ‘ " ‘ - ".‘“ ’ “f ‘-’ ‘ ( H C 'r Art 9.31.: lit}. ;::. Lil. V’ ‘77 1 :75, [7' 5 3 ‘3' d}: L":_T:‘J'11Jnil.‘:30s plating of the stee ll use at successive time int;rvais T (Table 26). These changes were extremely “ariah le. but generally T is number of bacneria increased on the anCUw lated barleys, reaching a peak with the twe_.re hour 52 mm ple. A pronounced population QQTFGESG took place from , - .-. 1 , fl - , -s...‘ .L' - .. p- " r“ V .L. - the tweire hour period to bfle linal sampling. The Steep ++ H ,v C‘ 5 ¥ \ L H , h.- H . ,‘ up ,. r..- f ,. .fi IJ v—v I P? ‘._J O “fl“ «4 r U L. ’ l I ‘D 0. CT $.‘4 ,— ([1 liquor ct: dine ed followed by a stead" deurease in viable cells in succee'ing platings. The pattern of po ulation hargci was similar th bot} Hannche en and Montcalm in'culated barleys. A few pilot malts, as w:+ l as e.gmc Cial samples, were plated in orde QC determine the effect cf kilning on the k rnel microflora. ”uartit tively the fungi were affected more severely than vhe seedwborne bacteria. The 182 Table 26. _ Bacterial colony counts obtained from dilution plates of steep liquor taken at intervals during the steeping of Hannchen and Montcalm barleys/2. ...“-.‘n- mum-p .....— Bacterial colonieswper mlo Sample _ Barley taken after Barley inoculated with: b variety ‘ hrso of / Steeplng Tone B~5 B 2lO B~226 Hannchen 4 figuOG BOO l$860 800 do 12 570 6,490 500 49360 do 24 50 6%500 550 100 do 56 9C 500 240 60 do 46 50 650 lo 100 Montcalm 12 50 10 100 40 do 24 19080 10 59600 50 do 56 O 28 2O 2O a Dilution plate series carried out by mixing 001 ml of the steep liquor with liquid nutrient agar (at 40° Co)° Steep- baskets were agitated before sample was takeno b See Table 18 for identity of organisms used to inoculate barleyso cterial CoL th ":1 I- obtained w: \4' U A. tings were ently in the range ial eclonies po r lOO plated Kernelso teria w small numbers of yellow b :re'ii were 4150 malt Kernels” The fungi isolated from W i; i.) i 80 to Thee . A 1 7. L T) _, Oi" -. p l. TlT {3 ,e-kernel pla- lUO bacter- e surviving bac— although :Jiated from i malt included: I" . t“ x‘ ' . T V ' - * r ~ ~ ‘ »- - ‘r': .' . r4 7‘ ,-\ -- Al ter Jlk’irl :1 thl L1 1 S‘ I-‘ll-t L¢111 I1 f.‘ .‘C)I.:").r 11.101 8 P‘ f .1. 'J :1 [1... g 1" ICIKA :1 2480.]. e 8 Of T1 r; v ! v v v“ T ' q . . ' x » , 1: 1,15- C ’ :11 \2' 'i -33 ..7 k - j. . . p ,1. I - C :.'1 f .7. _ .1- ‘ . L" .‘A n ...rv_..—_- -... --._ --.- -~-§-_.-...._t --.. -_ _ - L.— a.-. - ...-..- ...—... A ' r‘ ‘ ' f ’ - ”- ‘ ‘I 5"\ _ l -’-\ V ‘ R " ' V I. 7‘ ‘ h ' -\. ' ‘V‘ t " “ unityses oi piio' ii.ts pzepaied Izom bright and W ‘ .- Iv -, r- . 'r- ,- ’ .» _.‘., " | " I ~ r ' "' P- \ aturailywinleatel (seve1«,y “Edtnfifrd/ ‘were Henna by $139 Baixe' iih' Til? inabcrator‘ The difikuwuuwas LU tie TTT;'MT;'- are .haren brlght and weathered malts l Veral ways r- - . .— rv. r, I: ~ J r 1 ‘ - - r -. I T - r V‘ - Sampler as rompired with fun plight n.. p 19000 Kernel we1gnti yiezxe. ie~s txtza~ v‘~ w - g ‘ . V ‘ * 1 A ‘ r -‘ ' P» - ‘ ‘-\ - ' '~ darKar WoiLT ant Lhowea J hjwner Tfutéifl m '\ x - . ~ r‘ - v ‘ q '\ ‘ r ‘. Trxe p :IWLII uf1t iii: ,7 L 1 tax * 17 . h in thw *.:ea or ate «’ITLr3‘ Ijflhil-5 are TM 1" r‘ ' u x o —— f" -- n - t v. . ~ 1 -. ‘ - a , « 5 CI 0 lfl" L r“ 1 J l. n .t l Uil‘. ‘_. L1 r, r," f: l J. Hi“? if T. 1:: (ii E" K 7 F1“ ‘ \r.‘ ‘- ‘- “h” Trut n '7 1‘ \JJ "'1 N' o . LA '7‘ I '/ .\ J (J N- [115 "‘3‘ f \v‘ _ (\' AI : KlIfiUfiwl and.lmipylo ' . A .- 'J (.4 steeped at a slightly faster rate than the 2) the orde r of €t~€“lh ra ., 31.3., m- V I " ' " .' .5...‘ r» 0"." ~‘~ , ‘. ’fi"“‘v" Dar.) 3)" :5 W615: Phlfk iJ"-'(j 1. HT hi («AI Dd. isdilb ‘ [1631.2 7 weights taK en in The same \fc-i‘L'ricrtaxi. the wea tn“ Lllg lit Mont a I- t, .. ‘| ,. (34.111 order thered Montzalm steeped hontCalm barleys Table 27)o teiistic of The weathered 0 had a lower piviuT d a ‘ed in samples bright Hann~ tea for the bright e the 19UOO w i] '1 l N were ".522 \g a {4) the viabl Table 270 w Analyses of two pilot malts prepared from a bright Montcalm and a severely weathered Montcalm barley - a lot /_o _.- --.— -—-.—. «pr—“an" _. Bright Montcalm Weathered Montcalm Analytic data (1957 crop) (1958 crop) 19000 kernel wto, dob. (gm) 5000 25.5 Moisture (%) 404 606 Extract$ FOG09 d.b. 7jo5 7108 Conversion (min.) less than 5 less than 5 Color of wort (Lev. 52) 108 205 Clarity of wort clear clear Wort nitrogen (%) thfiw @0995 Malt nitrogen, total gm< 202% 2025 WC: t/mal t 113' trogen (7‘6) 73’? . l. 44 o l Diastatio power (°L°) 200 228 Betawamylase (Maltese deivol b64 775 Alpha amylase (20°00 dext. units) 4002 Alol Ratio; nefa.algha am5lqae 15.5 1808 a halt analyses performed by the United States Department of Ag'iculture Barley and Malt Laboratory, Madisons Wisconsin. oompmmammflp haopmaaaoo Goon 6mg moenw dawns maflpmhmm was .wQHdeHC “mopdqfla 3m 90% afiamaaog mo moapdaom meQMmQ 950% m aw mqfixmom hp Umaaflx moaamm Q . otbmflm :¢®\@ m do damn : mwwm op wmwmam mhmfimmp aam “cm cam on emu anm powdmh Hobos mompm mo thpmHmmame m um mm aw wuomm mom empmnpmmz admopqoz am 0 n moodm m.#a :\ doom Samopdoz n yam mm cm mmoam Noam anmanm Samopqoz awn mm mm emomm oooa pnmapm umgoqamm amm mm mm euomm rooa unmanm eageqflm unmfim3 mfi pump n&v mfiv flouowd m&d amoo was maos Mia adwwoz “do\Q>o . u _a p _ , - . . w. a ,14 1 : +m a “w w w rmammm we Ppmahdb are momma ow lemmmo Qpr bwnmma soap atnaox inspmaca , . no+nmm k _ maze: @mmmmAm ewsflaamo imnwaaom 0000a Hmfiwnnw anaema omen r \ om amafiammxo umnfimompm mo m mama. map GH vow: whoammp mo mommfimqm Hmoamhzm cam Hmowmomon : 0mm mficme J memo two 9 mdwmpwwm Hwam om :thde; mandamu owwwmwo »- _.1 . C? 0 u‘ by , . / ature content 11101 K- .' ‘ L' 5; Erll (36193:? .t C; L) .8 . i a n a . . . _ a _ _ #0 Mn \t\\l v \‘ \ .‘w\l\\ \‘xq‘It‘i \\.\ \i \\ wo mo no _u . . arnll. .Llifli. ll. 1!..-c n.-. 1'1r3:l:fl»1.i m Hm m0 an mwmwmma dMBw ND fistfim Percentage moisture content (by weight) wwmo Pdo a mammbwbm Hmdw ow :wwwmfid: mmbbofimb dmfiwmw. mom 34...... a _ _ i l i i l l i 3.0 Fl ...- wOVI I- NOYN. Ir \ l K Ho.l -Sl b _ Ll _ _.r m m .i w P m Hm do we NF mm wm :m to wamwmma de® HS wocwm 188 11:) weigl Percentage moisture contort (by WC #0 mo NO mama #0. I mammbwbm Hmdm ow Sobdomws deHmM° . _ fl _ a _ :mdemHmQ ;:2e_‘ ¢ m . S “5 mo mp mHmwmma deem Hb Uocum L $91... L's... I: ”I, -..Ilr tr. ii! mm wm Wm Vi.__,_u._.l PO 189 bright Montcalm steeped at a faster rate than did the dead Montcalm, (5) the overall order of steeping rates, by variety, was: weathered Montcalm: bright Montcalm: dead Montcalm: bright Kindred: bright Hannchen, (6) in the order of increasing 1,000 kernel weights the varieties were: bright Kindred: weathered Montcalm: bright Mont» calm: dead Montcalm: bright Hannchen (Figure 4). DISCUSSION AND CONCLUSIONS Several problems must be faced in the pilot maltn ing of inoculated barleys. The pilot malting plant must provide an identical malting environment for replicated samples. The apparatus described in this chapter was re-designed, mid—way through the experimental series, to correct such a defect. Condensate from the cooling coils and from the walls of the germinators was unevenly dis- tributed to the germinating pans. This excess moisture increased the moisture content of certain green malts. Consequently these malts were subjected to excessive kilning temperatures, with respect to their moisture contents. This combination of high temperatures and excessive moisture contents was responsible for a de- crease in enzymatic power, caramelization, and for the production of melanoidins (351). These compounds, in turn, impart characteristics to the finished malt which may be mistaken for properties engendered by kernel micro- flora. These include: increased wort color (27, 212, l 90 213, dip), mar: flavor .2, 505), and the aroma of the mesh (2, 505). There is some preliminary evidence that certain bacteria, e.g. Pseudomonas atrofaciens (B—2lO), may alsc increase the moisture content cf infected green malt. Elevated grain temperatures were also noted with the germinating barleys infected with this organism. If these effects are not eXperimental artifacts, it is pos» sible that barley inie;Led With large numbers of certain bacteria may show inzreased mOisture and temperature changes as a result of bacterial respiration. The pace terium cited above is a barley pathogen and is favored by high humidity and by temperatures in the range of 20° to 25° C. (Ej, 54). Such conditions are prQV1ded in germin» ating drums and compartments. The increases in grain m01sture and temperature were observed most frequently in the case of grain infected by E. atrofaciens. These conditions were never found in the case of barleys inoc— ulated, with fungi . Another problem, not completely resolved, is found in the incculation procedures used in this study. A greater degree cf kernel infection may be achieved by in_ cubating the grain at increased temperatures and humidi- ties after inoculation. This solution, however, is not entirely editable since the moisture content of the grain is dangerously elevated, and germination may be seriously affected. A minimal amount of bacterial inoculum is 191 required since these organisms rapidly increase during steeping and germination. The fungi do not, however, show this rapid increase (Tables 25, 26). The infection and subsequent increase of alien bacteria, presumably introduced by means of the humidi- fied air, creates another serious problem. To properly assess the influence of inoculated microorganisms on the quality of malt, these ubiquitous bacteria must be ex- cluded from the germinating compartments. This would nec- essitate the construction of an apparatus with a filtered- air system. There is evidence to show that the same types of bacteria are commonly introduced into commercial malts in a similar fashion. This evidence is adduced from ker- nel platings of commercial malts in which the white and creamy types (mainly species of Bacillus and Lactq_bacil— lug) predominate over the bacterial types commonly found on unmalted barley kernels, e.g. white and yellow, gram» negative bacteria. If this is indeed the case, the occur- rence of these bacteria in either the malting process or on the finished malt is perhaps not of great importance (from an analytic standpoint) since most, if not all, malts will contain a certain number of these organisms. It is also of interest to note that similar types of bac— teria, viz. BeS and B-226, showed no pronounced quality defects in inoculated malts (Tables 22, 25, 24). Indeed the only inoculated malts demonstrating consistent de- F fects were those Samples niected with pathogenic bac— teria and fungi, viz. E. atrofaciens, Helminthosporium sativum, and Fusarium roseum f.sp. cerealis. The occur— rence of bacteria in malt kernels is of great importance to the maltster, primarily because of the population standards for distillers’ malt. Evidence presented in this paper and elsewhere indicates that these bacterial populations may be the result of airm and watereborne bacteria, introduced into the malting process as a func- tion of the malting equipment, rather than as an increase of the seedmborne bacteria of malting barley kernels. These concluSions are based on the morphological and stain- ing characteristics of the maltuhacteria, as well as their resistance to high kilnilg temperatures. A few similar quality defects were found in both inoculated malts and ESYlffillelflfeCted malts (Tables 22, 25, 2%, 27). These defects included; reduced kernel nezghf and increased wort calar. Additional malt defects such as increased wort acidity, greater protein modifica~ tion, and reduced extract are frequently found in "weath» ered” malt, but were not present in the inoculated malts. The similarity of deleterious quality effects in the Hannchen samples (Table 25) and the weathered Montcalm sample (Table 27) suggests that the naturallywoccurring microflora of the Hannchen lot (of. Table l?) exerted a more pronounced effect on quality than the inoculated 195 organisms. It seems that the microflora of barley kernels may exert effects on malt and beer quality in several ways: (1) as a direct effect of microorganism on the kernels, e.g. the reduction of germination by toxins or other path- ogenic materials (2) as an indirect effect of the organ- ism on the kernel, e.g. reduction of kernel substance and production of degradation products by the attendant micro- flora, (5) indirectly in the production of hostwparasite interaction products, e.g. the pigmented material found in infected barley kernels (see Chapt. VI). To properly evaluate any or all of these possibilities, it would be necessary to infect developing barley kernels With pure cultures of specific organisms, to the exclusion of other microflora, and to rigorously malt these inoculated bar- leys. Such a procedure would permit the kernelmorganism interactions to take place over a period of several weeks, approximating the natural infection process which occurs with weathered or stained barley kernels. The rate of steeping experiments indicate that weathered barley steeps at a faster rate than does bright and non-viable barley of the same variety. The increased rate of steeping was not a function of kernel weight alone. The order of steeping rates suggests that steeping is not exclusively a physical process and that perhaps enzymatic solubilization of stored kernel materials may also be involved in the process. CHAPTER VI. glfimk smotiss LITERATURE REVIEW A number of workers have demonstrated that the seeds of cereals or their attendant microflora may exert an inhibitory effect on certain microorganisms. Smith found that aqueous extracts of wheat chaff were occasion- ally effective in both the inhibition of seed germination and in reducing the growth of Rhizopus nigricans on shelled seeds (362). Novotei”nov and Ezhov have reported that an antibiotic, isolated from barley grains, was anti~bacw terial and inhibitory to aspergillus niger and Penicillium ““m‘--- cm . glaucum (292). Ark and Thompson have shown that wheat and |r_J . barley seeds contain an antic otic principle that is ac~ tive against fungi and grammpOSitive bacteria when testm ed in vitro (16). Both secdwextracts and plated whole‘ kernels were used in this study. i,ner has postulated that ouinones, formed in the embryos of wheat or barley during fermentation, may be responsible for the elimina« H ) ioi of loose smut myceiiam in the water soak treatment of infected seed (599). Leben and others attributed the control of seedwborne pathogens, by the water—soak method, to the production of organinic acids by the normally—oc- curring microflora of the seed (222). The seeds of barley, as well as the seeds of other 194 cereals, contain substances which, when extracted, may inhibit the germination of these seeds (90). These sub- stances are polyphenolic compounds located, for the most part, in the tissues of he husk, pericarp, and aleurone (256, 304). A number of these inhibitory compounds have been isolated from the husks and rootlets of barley by Massart and his co workers (256). These phenolic acids and coumarin derivatives were present in a bound state, the nature of which was not entirely understood. Kockova- Kratochvilova and Lukasova»chotna isolated seven poly- phenolic deriV“tives from barley steep water (214). They found that a chlorogen;d said Like compound and a ”tannin" were inhibitory to barley germination. Other workers have isolated and identified p013“ phenolic compounds from barley kernels, not necessarily implicated in the inhibition of seed germination. Pollock and Pool found that the barley protein, hordein, was assoc— iated with anthocyanogens (leucoanthocyanins) in barley grist but not in whole barley kernels (504). Six antho~ cyanogens, including cyanidin and deiphinidin. were iso- lated from the crude hordein preparation. Mullick and others have studied the anthocyanin and anthocyanidin content of the aleurone and pericarp tissues of white, blue, purple, and black barley varieties (279). The looselyudefined group of polyphenolic compounds known as "tannins" have been discussed by Preece (514). The author 196 classifies the “tannins‘ as: (1) the hydrolyzable galloyl glucoses, (2) the non—hydrolyzable catechins and their‘ derivatives, (3) the related leucoanthocyanins. The oc— currence of these compounds in barley and other brewing‘ materials, is dealt with in another paper by Preece (315). Ivanyi (193) and Lfier and Stauber (241) have reported the presence of tannin in the husks of barley grains. "Test- inic acid", i.e. compounds of protein and polyphenolic substances, have been shown to occur in the husks of bar— ley (212, 213, 421). Stevens has indicated that these compounds may be artefacts derived from protein and part, or all, of the husk polyphenols (378). The isolation and identification of polyphenolic substances from beer and other brewing materials has been studied by McFarlane and others (259). The authors indicated that many of the tannin-like compounds isolated in studies of this nature may be artifacts because of their susceptibility to oxi— dative changes. A direct method for the determination of tannins in beer and brewing materials, using ultraviolet spectrophotometry, has been developed by Owades and others (294). The nature of the discoloration of cereal grains, caused by microfloral infection, is poorly understood. Few workers have attempted to explain this effect, and these with little success. It has been reasonably es- tablished, however, that the staining of the kernel is the natural to a me1 Lin~iigt rl‘m hf, u stance, isolat»: frwm .h--* k ‘1 1 — '7 ' .A. ' .- — ... chlorOpnyil de* imioaiiti 111 W :12? I’M =1; 7‘1»- -ie mf‘ucll “I 911 properties indicitti that it Plex. Lewicki nonouer:d a; tion oT whee; k-rne s 1"?) mzenith;tiea wa' Ju to ‘z exzd was not deters ieo ”i: i In 1 :Litf ..‘J 1V) T? ’1 1.1" 1 i'a' T; h L ‘ A ' y I. a r- ‘ n ’ . A. \ i ’- < ECLLXJfiL :‘m “. Cal : (2.131 Li\,’ R I J 1 , 3 - A . ’9» " -v a -‘ seemei pinoubgo ,nl d2 C’ 3'. . :1 ‘ i 5 v1 ‘ ('1 l I‘ - - I i; ii: 34¢ a p. nvnti .wu u: l. ’ I .- ‘ V ’7 “ ,~ < ‘1 " a' r‘ .I ' l. ‘ ' ‘,'~. Fifi-j 31"-.) 9.3L: iiLAJl Ii: T; tjl‘: i-('1tll();5"2'ifii 7"! -: V r ‘ \ 1 ' .‘ \- - r- T r +- Vt‘ n -_ Ar‘l 4 f 7“ u“. AIL»; (J( L "JV kl ’kL“ \ .LJ“‘JC)L_ ‘7' ' r -\ rr 13-! \‘vQ ' v ’ ‘r‘T .f) - . ~. .---l 1:“. ium, DJ L'vlL--'Jy CA.;1I.1. C gmentation of barley -." (Z; )4 ) " 1‘- xv ° “lifiéd, W El :3 both indicated that -L', r-VV . i . :1 JV: kernels is due ood descritei a brown subm ernels, whio interfered with ,— .‘ While the exact nature of its chemical C‘ A \ ‘. L) \JIL"; a proteinuflavone com— early study on the pigmenta- + U He found hat the black pig~ ation of catechol tannins and 'i by 'YidaSes. While it 9 some ~hemi'ai reaction was a «red TlSfiifig the cells were chars ed with a yellow or brown pigment: and mycelium was either absent9 or rarely presento This reorJation su'flg ated the action of toxins or enzymes exsreted by the lyphae and working at a distanceo bince glare d'scoioration may be caused by parasitic actiong by environment and/or genetic factors, the agrwk 1's su ugg ested tiat the production 01' melanistic pigments mig ht be due either to phlobaphene formation by the oxidation of catechol tannins, or to the action of tyrosinase on tyrosine and other amino acids° Attempts to produce melanism by the injection of phenylalanine and tyrosine were negative however. WorKers at the University of Minnesota have made water—extractions of brown to black, \ 4.. (’33 viscous outstances iron barley. stained under laboratory conditions (9). A yellow substance was extracted from a bright barley sample using the same extraction procedure. All of the pigmented materials were inhibitory to barley D germination. The degre, 3: inhibition decreasing with the decrease in darn color. oriferential adsorption of com» ponents cf the pigmentei eutstances. varying in their in- hibitory properties. We: a; ompiished with activated car» bon and ion exzhange resins. The role of giant tannins and other polyphenolic compounds in host resgsaan e to microfloral infection has been diecussed by many worxers. 000K and Taubenhaus dem» onstrated that: (l) tannin ret>rd Lb CU d or inhibited fungal r—sa growth. (2) parasitic dug; were more sensitive to the acticui11a3 L-‘-g> T p - r._J V“ ('5' U.) Q U) 6': ~ g Milli/‘11: I‘rlidhxj, C ase sysa by yQflflSlCiVé reactlon of stem p ( 'f (L a With the actlvity of fiystem KELQJO Jacarane has .. t. . "7, ' , , .' m - .. ,. -{ wth tum OAfldlZlng enzymes ."L ed by secerag workers that . .‘ - ’ , . .n i at ,-.' ~ .. A? , .-, uabd kgaijyhcnuiwxludSc) pdlezryi:iiaqizzge Ewnlan sseIWIe =m¥1ve Icrmatlon of various . V "a .2 :- .' , . - :- ~ ' .'W‘ I '\ c1. -',‘(-’ Lk. 34;. L L: r161}. Lf‘TJ’ fiéjr“ ~o.+~ A 3.441195; «11. .M: .1. IJ.<~1L\.' tlffiSUBS rt.) .1 q -x v , .. . ,~ ,. ,‘ t" w‘ . . , ‘JL cl {1.3?‘11‘_.r.: 'JL ily 10:3;[1d88 CI pnemtm 111 watsr {Jfix))o .JZ‘ .. ,.-..« w ~.'.. ' .- w". - — 0L 9%:L flJyd been b;UWfl to with the wait and/or the bar- 0 V (_‘f I J) I 1 " I ll ,q__ . :.,;. f ‘F no .1: .3 :4 den :.t’ .1 AL 0 a; ) béél and unstable or hazy lflpOIlmUK :n récenf years” 202 Beer is said to “gush” when the contents are violently ejected upon Opening the bottle (90). The phenomenon has been attributed to several factors, including; poor retention of carbon dioXIde, the presence of particles of high surface tenSion, agitation, and the presence of oxygen, metals, nitrites, and various protein degradation products (90, 158)o Nakamura analyzed wild beers and found higher values for gas pressure, for nitrogen—con- taining substances, and for acidity than for normal beers (282), The author found that some causes of gushing were related to the quality of the malt employedo halt pre- pared frcm weathered barley has been implicated in the production of beer with gushing tendencies (5, 159)° Gray and Stone reported that the widespread incidence of gush- ing which occurred in l948 and 1949 presumably ceased when new malt became available (159), Thorns and Helm have stated that the main cause of overfoaming is, in all probability, due to the vibration in transport acting together with certain abnormal constituents of the beer (595)o These abnormal constituents are presumably de- rived from the barley; and when the beer is agitated, cause the formation of innumerable minute nuclei which in turn bring about the violent evolution of carbon diox— ide immediately after the bottle is opened° The authors stated that overfoaming tended to occur in epidemic out- breaks restricted in both space and time; and they con— 205 cluded that these epidemics were probably due to some de- fects in the available barley. An example is furnished by the authors to support this conclusion. A severe outbreak of gushing occurred in EurOpe in 1955 and was undoubtedly due to the very poor quality of the 1954 crop of barley in the affected area. The poor quality of the barley was at- tributed to the unfavorable weather conditions which ex- isted at harvest. That other factors in addition to bar- ley quality were Operative was evidenced by the quantity of normal beer brewed from the 1954 barley crop. The au- thors arrived at the conclusion that the primary cause of overfoaming must be sought in the barley used in brewing (595). Beer instability has been generally categorized as haze of either biological or non-biological orgin (90). Hind has sub—divided non—biological haze in the following manner: (1) protein haze, (2) pasteurization haze, (5) chill haze, (4) metal haze, (5) oxidation haze, (6) starch or dextrin turbidity, (7) oxalate haze (178). According to this worker, chill haze and oxidation haze are normally— occurring turbidities, but hazes due to metals, starch and dextrins, and oxalates constitute beer defects. Beer in- stability has been dealt with in a number of review papers, including those authored by; Barton-Wright (26), Napier (285), Scriban (542), Schultze-Berndt (540), and Hopkins (185). The enzymatic treatment of beers, i.e. "chill- 204 proofing”, has been treated in a review paper by Waller— stein (412). Napier has discussed the anti-oxidant pro— perties of ascorbic acid and its use in reducing beer instability in his review article (285). Chill- and oxidation-hazes arise from the aggre- gation of colloidal particles in the beer, and these par— ticles represent complexes of polyphenolic compounds and fragmented proteins (185). According to Scriban the bulk of the polypeptides responsible for chill-haze formation arise from the malt and barley used in brewing although other sources are known (542). The polyphenolic fraction of chillwhaze derives mainly from the malt according to Schultze-Berndt (540), and Hopkins (185). Hopkins stated that the chill-haze of beer shows a tendency to darken on exposure to the air and speculated that the darken- ing was assoicated with the oxidation of tannin (185). Hartong has attributed the chemical variability of the components of chillmhaze to the variation occurring in barley and other brewing materials (175). Preece has indicated that a proportion of the tannins is essential in beer if certain desirable characteristics e.g. flavor are to be obtained (515). While certain polyphenolic ' materials e.g. the antocyanogens, are more responsible for haze formation, other less harmful tannins may be converted to the harmful anthocyanogens by oxidation pro- cesses (515). Kockova-Kratochvilova and Lukasova-Novotna 205 have also indicated that tannins, being strong reducing agents, are easily oxidized to form dark—colored phloba— phenes which in turn form haze complexes in beer (214). A comprehensive series of studies have been spon— sored by the Brewing Industry Research Foundation on the non—biological hazes of beer (52, 159, 168, lb9, 170, 171, 578). The chemical composition of chill~ and oxidation- hazes of beer was determined by Bengough and Harris (52). The material was composed mainly of protein and condensed tannins which were derived from both malt and hops. Harris has discussed the separation and identification of malt and hop tannins by means of paper chromatography (168). Harris and Ricketts isolated polyphenolic compounds from malt husks and suggested that the group of compounds known as anthocyanogens, isolated from m lt, make the major con- tribution to the formation of beer hazes (189). Stevens showed that the properties of testinic aCid extracted from barley and malt, depended to some extent on the physical state of the sample (578). The author also found that testinic acid recovered from whole barley resembled typ— ical beer hazes in composition. Hall and his co-workers confirmed an earlier suggestion that both malt and hop tannins participate in the formation of beer hazes (159). The authors also bore out earlier indications that the anthocyanogens, effective in promoting haze-formation, were possibly present in beer in bound form. Harris and 206 Ricketts found at least three separate groups of poly- phenols including anthocyanogens, methoxylated lignin—like compounds, and esters containing simple phenolic acids in representative chill~hazes (170). The same authors re— ported that insoluble polyamide resins served as selective adsorbents for the anthocyanogens in beer (171). Removal of the anthocyanogens, by means of these resins, enhanced the shelf-life of the beer. The problem of beer haze, and its treatment with powdered polyamide resins, has been re- viewed in a recent paper by Harris and Ricketts (172). MATERIALS AND METHODS Observations made on a number of barley kernels, plated on contaminated culture medium, suggested that the kernels of barley might contain a material, or materials, which exert an inhibitory effect on certain common lab- oratory contaminants. Consequently, the contaminant was isolated and identified as a variant of Bacillus cereus, a common soil-bacterium /‘l’o Potato—dextrose agar was seeded with three concentrations of the bacterium by add- ing bacterial suspensions to media cooled to ca. 40°C. in a water-bath. Plates were poured with the treated med- ium, approximately 15 m1 of medium per plate, and allowed to solidify. Bright barley kernels of four varieties; Hannchen, Traill, Kindred. and Montcalm, were surface- disinfected in the usual manner and were plated on the l Identified by Miss L. Neu, Department of Microbiology & Public Health, Michigan State University. 207 seeded agar. A check series of uninoculated media was also plated in the same manner. small discs, approximately 5/8 of an inch in diameter, were cut from paper toweling. These discs were saturated with the surface—disinfectant and were placed on the surface of the untreated and treated media to serve as additional checks. The plates were incubated for five days at room temperature and were examined, at the con- clusion of the incubation period, for both inhibition of barley germination and for suppression of bacterial growth. A series of experiments were conducted in an attempt to elucidate the nature of barley discoloration, especially staining caused by kernel microflora. Extractions of the pigmented material were made using distilled water and 50 percent ethyl alcohol. The material for extraction was prepared by pearling paired samples of bright and moder- ately-weathered barley for 50 seconds. The pearled husk material was then sieved in a nest of A.S.B.C. analytic sieves and subsequently divided into three portions: coarse, medium, and fine. Extracts of the coarse husk material were evaporated to dryness, re-suspended in 50 percent alcohol, and chromatographed uni-dimensionally on paper. Other extracts were also chromatographed in a like manner with the omission of the evaporation and concentration steps. The paper (Whatman #1) was cut into six inch strips and the husk extracts were streaked in 0.1 ml. amounts across the tOp of the paper strips. A descending 208 system was used and nmbutanol, glacial acetic acid, and water (5:4:1 by volume) was employed as the solvent (44). The chromatograms were removed from the jars at appropri- ate times, dried at room temperatures, cut into one-half inch strips, and sprayed with a number of developing sul- utions after visual and ultra—violet inspection. The de- veloping materials used were: FeClB, ninhydrin, phthalic acid and aniline, Benedict‘s reagent, Na2CO59 basic lead acetate, lead acetate, and alcoholic A1015. Color changes and values were variously noted with each paper chromato— gram. Evidence, obtained from several sources, indicated that the discoloration of barley kernels by microflora was an enzymatic process. A series of experiments was consequently devised to test this hypothesis. L—tyrosine was added to distilled water at the rate of 100 mgm of the amino acid to 1,000 m1 of the solvent. The solution was dispensed in small test tubes, 10 ml per tube, and autoclaved for 15 minutes at 15 pounds pressure. Ten surface-disinfected kernels of barley were then trans- ferred aseptically to each tube and the tubes incubated at room temperature for periods of from two days to one month. Check tubes were also prepared, either with or without the addition of kernels or microflora. Discolor— ation of the tyrosine solution and/or the steeping ker- nels was noted at appropriate intervals thereafter. The 209 solutions were read for qualitative and quantitative color changes, either visually or with a Coleman spectrophotometer (model 14) at 660 mp. This wave-length was chosen for maxi- mum absorption after preliminary tests were run over the entire absorption spectrum. Turbidity and precipitation was also noted in each case. This procedure was used with the 84 barley samples, furnished by the Malting Barley Im- provement Association, in an effort to relate the degree of enzymatic discoloration to the variables of barley variety and microfloral populations. Another experimental series was conducted to assess the mechanism of staining produced in the preceding experi- ments. Two steeping solutions were prepared; Czapek°s suc- rose-nitrate solution and the same medium With L-tyrosine substituted for the nitrate salt as a nitrogen source. To these solutions, surface—disinfected kernels of bright Traill barley, either with or without the presence of in— oculated organisms, and the organism alone, were added in all possible combinations. Cultures of the bacterium and solutions in which non-inoculated barley kernels had been steeped were filtered through sintered glass filters. The test bacterium was added to the filtered barley solution, and surface-disinfected barley kernels were added to the filtered bacterial culture medium. Inoculated and non-in- oculated kernels were ground in a laboratory burr—mill, and the grist was placed in the two test solutions. All 210 of the test combinations were replicated three times. 'At the termination of a three week incubation period, the tubes were examined for color changes, turbidity, and other effects, as noted above. In addition to the tyrosine solution, other nitrogen compounds were tested in a similar fashion to determine whether the discoloring was exclusively a "melaninogenic" type of reaction. Us1ng the same manipulative procedures stated above a number of organic and inorganic nitrogen com— pounds were tested for the production of pigmented material (Table 52). These compounds were dissolved in distilled water, in varying proportions (Table 52). Check of dis- tilled water and L-tyrosine were also run in the series. The quality and intensity of the color of the solutions, and the solutions plus the inoculated and non-inoculated kernels were measured and recorded as previously described. The organisms used in the above experiments as in— oculum were also grown in shake-cultures on Czapek's sucrose—nitrate solution and on Czapekis sucrose-tyrosine solution. The cultures were grown in 250 m1 Erlenmeyer flasks, containing 50 ml of medium per flask, on a recip— rocating shaker at 25°C. for 19 days. The mycelium, spores, and cells were removed by filtration and centrifugation, and the filtrates assessed for color changes and for re- action of the media. The intensity of color was measured as the percent transmission at 660 mp as described above. 211 The reaction of the media was determined with a Beckman laboratory pH meter. EXPERIMENTAL RESULTS The inhibitory effects of plated barley kernels on the Bacillus cereus variant are depicted in Plates XIV and XV. In two experimental trials, each with ten replications of the four varieties, the order of bacterial inhibition was Hannchen; Kindred) Trail£> Montcalm (Table 29). In all cases, the suppression of barley germination was negatively correlated with the ability of the barley variety to in- hibit bacterial growth. The surface—disinfectant markedly inhibited the growth of bacteria in every case. The anti- bacterial effect of Montcalm barley kernels was negligible, or non—existent in all experimental trials. A brown, pigmented material was extracted from bright and stained husk preparations of Traill, Hannchen, and Mont— calm barley with both water and 50 percent ethyl alcohol. The material obtained from the stained barley preparations was a darker brown color and was recovered in greater quan- tity than from the bright husk material. The alcohol ex— tracts gave greater yields of the pigmented substance; how— ever, tne gummy nature of the extracts suggests that other materials, soluble in alcohol and insoluble in water, may have been removed by this procedure. A visible band of yellow to brown pigmented material, with an Rf value of 0.95 to 0.99, was observed on paper chromatograms of bright PLATE XIV Four varieties of bright western barley, surface- disinfected and plated on potato-dextrose agar; and on potato-dextrose agar seeded with different concentrations of a Bacillus cereus variant. The plates demonstrate both the inhibition of bacteria by the barley kernels and the inhibition of barley germination by the bacteria. Upper - The four varieties plated on potato-dextrose agar check plates (no bacterial inoculum). Lower - The four varieties plated on inoculated agar at the highest inoculum level. 215 PLATE XV Four varieties of bright western barley, surface- disinfected and plated on potato-dextrose agar; and on potato—dextrose agar seeded with different concentrations of a Bacillus cereus variant. The plates demonstrate both the inhibition of bacteria by the barley kernels and the inhibition of barley germination by the bacteria. Upper - The four varieties plated on inoculated agar at the intermediate inoculum level. Lower - The four varieties plated on inoculated agar at the lowest inoculum level. tan-.cxn- ... ... 214 Table 29. - The inhibition of a Bacillus cereus var- iant by surface—disinfected barley kernels plated on seeded agar, and the suppression of barley germination by these a bacteria. E Bacterial 0 Bacterial ~d Barley Barley . Treatment/ growth /- inhibition/- inhibi- e germina- f _. tion /— tion PDA (check) 0 0 0 80 a “:3; Tube (high) 1 4 1 so a M-2 (med.) 1 4 1 so a: M-6 (low) 1 4 0 80 PDA (check) 0 0 0 100 2 Tube (high) 5 2 2 90 «F! :3. M—2 (med.) 3 2 1 100 M-6 (low) 2 2 1 90 PDA (check) 0 0 0 100 '3’, Tube (high) 2 5 2 90 g :3 M-2 (med.) 1 4 1 100 :A M—6 (low) 1 4 2 70 PDA (check) 0 0 0 100 E3 '3 Tube (high) 4 l 5 90 O *3 M-2 (med.) 4 o 5 80 o 2 M-6 (low) 4 o 3 so a Bacteria isolated from contaminated potato-dextrose agar. b See Plates XIV and XV for concentration of bacterial inoculum. c Rated on a scale of 0 equals no growth, 4 equals pro- fuse growth. d Rated on a scale of 0 equals no inhibition, 4 equals complete inhibition. e Rated on a scale of 0 equals no visible damage, 4 equals necrosis and stunting of shoots and radicle. f As percent germinated on agar plates. 215 and stained husk extracts, inoculated kernels in an aqueous solution of tyrosine, and of the tyrosine solution itself. The pigmented band fluoresced under ultra-violet illumina- tion in each case. The intensity of both visible color and fluorescence varied with the various extracts. Stained husk extracts showed greater intensity of brownness than did bright husk extracts. An unidentified material, with an Rf value of 0.25 to 0.50, was present in bright husk ex- tracts, but absent in stained husk extracts; this material was initially colorless but gradually turned light—brown in color on exposure to air for several days. Lysine was found, in relatively large amounts, in all husk extracts. A band of material, fluorescing with a blue color under U—V illumination and with Rf values of from 0.81 to 0.94 occurred in all husk extracts and tyrosine-steep liquor but not in the tyrosine-solution chromatograms. The brown- pigmented band, in each case, was apparently phenolic in nature, but occurred in a bound form (44). The blue- fluorescing band also appeared to be a phenolic compound (3), but was not amenable to identification under these experimental conditions. The production of a light to dark-brown discolor- ation of aqueous solutions of tyrosine by barley kernels, either with or without attendant microflora, was observed in a number of trials. The intensity of the brown color appears to be a function of both variety and microfloral 216 population. Attempts were made to correlate color pro— duction and the degree of micgcf‘ora‘ contamination. Re- peated trials with the same barley lot have indicated that a larger sample of a population of kernels is needed to ensure greater accuracy in the determination of micro— floral content with this method. The use of a check var- iety, free of microflora, is also requisite in order to separate the relative influences of barley variety and microflora. That the procedure holds some promise is ap- parent from the determinations made on the 84 barley sam- ples furnished by the halting Barley improvement Associa— tion (Table 50). A comparison of these values with the microfloral determinations obtained by plating these sam- ples (cf. Appendix C) shows, in many cases, a definite correlation between the two sets of data. Whenever bacterial turbidity occurred in the tyro- sine solution, the degree of pigment formation was minimal, and conversely, whenever the intensity of the pigmentation was pronounced, no bacterial turbidity was noted. Gener- ally barley samples showed uniform staining of the ker- nels in the tyrosine solution, with more intense staining of the ventral crease. In some samples, however, the ends of the kernels appeared darker than the rest of the grain. The period of incubation in the tyrosine test is also of importance since increases or decreases in color inten- sity may occur after some time has elapsed. The most 217 g Table 50. ~ Association of the production of melanin/ pigments by surface-disinfected barley kernels steeped in a solution of L-tyrosine for six days and the microfloral pop- 'ulations of these kernels as determined by standard kernel- plating techniques. _.7 ,_—‘ 11.3.1.1. Steep Planted/-62 sample 3 Bacter- Color Number of colonies/100 kernels number / iidi§;r72 :p:;n;g Fungi Bacteria Microflora 59-4 o 91.8 44 90 156 59-7 0 86.9 66 84 151 59~9 5 90.5 74 85 159 59-14 ' O 95.5 80 22 102 59-16 0 92.5 59 96 164 59-22 0 88.6 55 96 152 59—24 0 88.5 105 85 188 59-25 1 90.7 101 97 199 59-26 0 91.0 108 89 197 59—27 0 88.0 112 96 211 59'28 l 9100 97 79 176 59~29 0 89.0 88 80 168 59—50 2 89.9 95 82 178 59-51 0 88.9 107 19 126 59-52 2 91.0 105 5 108 59-54 2 87.1 f 115 27 140 59-55 0 88.8 7 0 7 59-57 0 89.1 7 0 59-58 0 91.4 5 1 6 59-59 0 89.0 15 4 17 59—40 0 89.1 6 0 6 218 Table 50. - Continued 59-41 0 92.5 7 O 7 59—42 0 85.0 5 21 26 59—56 0 89.0 152 26 158 59—57 0 76.1 ' 65 26 92 59-58 0 70.0 104 57 142 59-59 0 8705 85 45 127 59—60 0 81.0 87 26 95 59-61 0 76.6 78 17 95 59-62 0 85.1 69 9 78 59-65 0 88.9 75 85 157 59-64 0 91.6 85 86 169 59-65 0 89.2 45 89 152 59-68 2 90.2 155 55 168 59-69 1 8908 105 75 177 59—70 2 89.9 116 76 192 59_71 0 88.5 116 94 210 59—72 1 87.9 106 24 150 59-75 1 88.0 100 68 168 59-74 2 88.2 102 86 188 59-75 0 88.1 55 25 78 59-76 1 87.9 86 21 107 59-77 0 84.8 89 5 92 59-78 0 80.6 95 17 110 59-79 0 86.9 1 0 1 59-80 0 87.6 0 0 0 219 Table 50. - Continued 59-81 0 85.8 0 0 o 59-82 O 84.1 4 o 4 59-85 0 84.4 0 0 0 59-84 0 70.0 0 0 0 59—85 0 71.9 2 5 7 59-86 0 80.9 7 2 9 59-87 0 86.2 4 o 4 59~88 0 76.9 7 O 7 59-89 2 88.9 24 5 29 59—91 0 75.0 116 40 156 59—92 2 88.8 111 97 208 59-95 5 89.2 107 97 206 59-94 2 89.0 104 57 141 59-95 2 89.4 111 41 155 59—96 0 78.0 127 22 151 59~97 0 91.0 0 0 0 59—98 0 91.0 2 1 5 59-99 0 90.6 0 1 1 59-100 0 91.0 1 55 56 59-101 0 90.0 0 0 0 59-102 0 89.1 4 0 4 59-221 0 85.0 2 0 2 59-225 0 89.6 4 2 6 59-224 0 78.0 5 5 10 59—225 0 89.0 6 5 11 220 Table 50. ~ Continued 59-226 O 89.9 5 5 8 59-227 1 89.5 12 0 12 59-228 O 5.9 99 57 156 59-229 0 85.4 75 11 88 59m25l C 69.5 18 1 l9 59~252 0 82.9 7 9 l6 59‘255 0 88.2 5 1 4 59~254 0 84.8 6 0 6 59-255 0 89.9 0 0 0 59—256 1 89.7 6 l 7 59‘257 0 89o0 7 0 7 59—258 0 74.1 56 19 75 59—240 0 82.1 8 1 9 L—tyrosine — 99.0 - - - solution a See Appendix C for description of samples, germination, and microfloral determinations. See Appendix C for identity and orgin of samples. Rated as 0 equals n0 turbidity, 1 equals slight tur- bidity, 2 equals moderate turbidity, and 5 equals heavy turbidity. Color intensity expressed as percent transmission at 660 mp on a Coleman spectrOphotometer (Model 14). See Appendix C for microfloral data. 221 suitable incubation period appears to be six or seven days as determined from tests which ranged in duration from two to thirty days. Several variables were evaluated in order to deter- mine the mechanism of pigment production in the tyrosine test (Table 51). Some discoloration was obtained without the use of tyrosine in the steeping solution. The degree of discoloration did not, however, differ significantly from the value obtained elsewhere for kernels steeped in water (Table 52). Since the staining principle of barley kernels is somewhat water—soluble, it is not surprising that a certain amount of discoloration occurs in any aque- ous solution. In the tyrosine—supplemented solution, sig— nificant staining was obtained only when kernels, either with or without inoculated microflora, were present in the medium (Table 51). The addition of the organism to fil— tered steep liquor, or of kernels to the bacterial cul— ture filtrate, did not bring about a significant increase in pigment production, over the color produced by the leaching out of water-soluble pigments. The data obtained from the testing of various ni- trogen compounds for their pigment—inducing properties are presented in Table 52. None of the inorganic nitro- gen compounds demonstrated an ability to promote pigment formation. The decrease of light transmission in the ferric nitrate series is explained by the formation of 222 m mHoQHoM m macaw Mnmm m qzonp .602 oopwadoonH co maonmmx cop d dempm .80: # nmflndmp .60: Imadoonwlqoz op Amqflmonhp II II o hodoao .Hm Moono mdamv m.Mommuo madeoM oopmHSOOQH II II o Amazoaaow Bong pmwno 08 maodpox cop ImadoosHIqu I- II o saunas sown pawns on mommma puma mane II II o Imoooam opHQ3 amfldmmso ow maoanmx m dampm .60: m 08 oopwHSoodH 06 maoquom cop m aflmpm .Hm m awesome Imadoosfilsoz 06 AodflmOHmp II II o modoao .Hm Moono ocv m.Mommno \hpwmnoan Hoaoo I\hpflmdoan Hoaoo asficma m. . o \meapmmHB Hoqamm soapsHom m oHdeSU .osflmohhpIq mo noepflddm one pdonpaz cum Qua: .mfioma ondpado ca mpnmamflm :daqmaoa= mo moaposoomm can do hoanmn nonmadoodfi one I\.mopmnpaflm oHSpHSo .pmfinm moasmn .maoqsmx hoaomp mo mpommwm one I .Hm manna I.IIIII!,.I I, .0 all\(,\ I u‘. In. I I. Inn-h» 225 .doapwPQoamHm ozonn Mano mamsvo m use soapmnoaoomad on mamsvo 0 opens .m on 0 mo mamom 8 no oommm 0 .Ana manna .ro Ammmumv unewoowa .Hm> mdmsoo msHHHomm mm: snadooqfl Houses mm was macaw moms amflsmwpo one Q .mxmoz woman no“ mohdpbhmdfimp Econ 98 manna pmmp qH vopmndqu m mama opmnpafim Inmx doped maanSO a dflwpm .Hm anQGMp .Hm ISoonHIqu Hafihmpomm haqo Modded II II nmfladmu .Hm aqummmo mompm moammm maoqnox 8op8H300dH hodoao hedoao nnpunnaanam Bosh pmflnu maonnox , enema IdoodfiIqoq 809m pmflmu hado amfiqmmno co 06 Aoqfimoshp mSHmv m.Mmmmno ebsnwpuoo I .Hm manna 224 Table 52. - The effect of various nitrogen compounds on the formation of "melanin" pigments in solution after the ad- dition of surface-disinfected bright Traill kernels, with and without the presence of inoculated cells of Bacillus cereus 3 var. mycoides ggeggity ggggigh Quality 0010? Intensity/"12 pound (gm/100ml) c d e c d e Fe(N03)5 26.64 orange brown brown 70.7' 57.1 56.0 NHQNoa 7.91 clear tan tan 100.0 95.5 95.0 Ca(N05)2 25.55 do do do 100.0 95.0 95.2 KNO5 20.00 do do do 99.0 95.8 96.0 NaNO5 16.81 do do do 100.0 95.0 94.5 Mg(N03)2 25.57 do do do 100.0 96.9 95.0 Proline 0.10 do do do 99.5 95.2 94.0 Hydroxy- 0.10 do do cloudy 99.7 79.5 70.7 * proline Phenyl- 0.10 do do tan 99.8 92.0 91.0 alanine TryptOphane 0.10 do do do 100.0 85.0 88.2 "Dopa" 0.10 brown black brown 2.0 0.2' 15.4 * Histidine 0.10 clear tan tan 100.0 91.0 90.5 Tyrosine 0.10 do brown brown 99.4 72.5 66.0 Water -- do tan tan 100.0 96.5 95.8 a Stock number B-529, see Table 18. _ b As percent transmission at 660 mu on Coleman spectrOphotometer. C 0f solution alone. d 0f solution plus disinfected, non-inoculated kernels. 8 0f solution plus disinfected, inoculated kernels. Aster— isk indicates cloudiness or precipitate present. 225 dispersed flocculent material in those solutions contain- ing kernels. It is suggested that the flocculation may have been brought about by iron-complexes formed with the dissolved substances leaching out from the kernels. Sev- eral amino acids demonstrated the ability to promote pig- ment formation; viz. hydroxyproline, tryptophane, and 2,5- dihydroxyphenylalanine (dopa). The results obtained with histidine and phenylalanine are inconclusive. None of the compounds, with the exception of dopa, performed as well as tyrosine. The spectrophotometric readings of the dopa tubes were made after three weeks of incubation. Initially this solution was clear, but the compound was oxidized, non—enzymatically, thus giving the extermely low readings. The color intensity increased initially, and as flocculent masses of a melanin-like material formed and settled to the bottom of the tubes, the solution became lighter. The clearing of this solution, containing the inoculated ker- nels, was determined to be the result of a limited pigment- substrate. Microflora used in the several phases of this study were cultured in nutrient solutions with and without the addition of L—tyrosine. Since reports of melanin forma- tion in liquid culture are scanty, the organisms were cul- tured for extended periods of time. The results of these cultural studies are tabulated in Table 55. The color intensities and medium reaction obtained with the standard 226 Table 55. - The effects of selected bacteria, fungi, and yeasts on the color and reaction of Czapek's solution, a ‘with and without the addition of L-tyrosine. , Czapek's sucrose-nitrate Czapek‘s plus Identity solution tyrosine of b c d c d organism/ Color Intensity/ pH/_ Color Intensity/. pH/- Check Clear 98.0 7.4 Clear 98.0 7.5 B-5 Yellow 89.7 7.8 do 95.4 6.9 B-210 do 75.9 8.7 Cloudy 92.9 4.5 B-218 Clear 98.8 7.5 Pink 95.0 7.0 B-226 Tannish 79.4 8.6 Yellow 87.0 4.7 B-522 Clear 92.0 4.5 Tannish 87.2 4.1 B-529 do 98.0 7.5 Pink 88.0 6.8 Y-52l Yellow 89.4 4.8 Yellow 80.9 4.4 F-504 Pink 85.0 7.4 Tannish 90.5 6.5 F—510 Clear 96.5 7.8 Tan 88.7 6.6 F-52l Yellow 94.0 5.8 Red-brown 75.0 4.9 F-524 Tannish 88.0 5.8 Pink 92.4 6.0 a Organisms grown in flasks on a reciprocating shaker for 19 days. b See Table 18 for identity of organisms. C As percent transmission at 660 mp on Coleman spectropho- tometer. d Determined with Beckman laboratory pH meter. 227 Czapekgs sucrose-nitrate medium serve as a standard by which the effect of tyrosine in the other medium may be assessed. A tan to brown color and a markedly reduced reaction was taken as evidence for the possible produc- tion of melanin—like pigments in the tyrosine medium. According to this standard, the Alternaria (Fo504), Hel- minthosporium (F-510), and Fusarium (F-52l) isolates gave some evidence of poss1ble ”melanin" formation (Table 55). The bacteria, yeast, and the Cladosporium isolate did not satisfy the above criterion. DISCUSSION AND CONCLUSIOJS The cause of the suppression of the gram-positive9 spore‘forming bacteria, described in this study and else- where (16), is poorly understood. Evidence seems to in- dicate that such suppression may be the result of anti~ biotic production by the barley kernel. The less-inhib— itory nature of the variety Montcalm is of some interest, Since this variety also demonstrated a lesser degree of resistance to kernel microflora in the studies described earlier (cf. Chapt. III). It is possible that some re- sistance to kernel infection may be conferred by kernel antibiotics, and that the production of these antibiotics may be linked with other heritable characters, e.g. the smoothness of the lemma awn. Chromatographic studies and the use of the "tyro— sine test" have indicated that the brown discoloration 228 of barley kernels, usually associated with microfloral infection and/or weather-damage, is caused by brownish pigments. It further seems that these pigments are mem- bers of the somcalled class of polymerized, polyphenolic compounds known as "melanins". The "tyrosine test", de- scribed in this chapter, has demonstrated that polyphenol— oxidase (terSinase) is present in both barley kernels and in many of the commonly—occurring barley organisms. While some non—enzymatic oxidation of amino acids and various intermediates has been shown to occur, the conversion of these compounds to the "melanin" precursors is predomin— ately an enzymatic process. When kernels, rendered free of microorganisms by surface—disinfection, are placed in aqueous solutions of tyrosine and certain other amino acids a limited amount of the pigmented material is pro- duced. It appears that the discoloration results from both tyrosinase activity from the embryo and the leach- ing out of previously fcrmed water-soluble brown pigments. The production of brown pigments, i.e. melanin-type com- pounds, by several of the common microflora of barley kernels, is negligible or lacking when these organisms are grown on chemically-defined media. This may be due to the lack of certain nutritional factors, precursors, and other materials in culture media. The data indicate that the production of the brown discoloration of the tyrosine solution is mainly an additive process, i.e. 229 that increased colormproduction is proportional to the quantity of enzyme or substrate present in the kernel as well as the amount of microfloral enzyme present. While phenylalanine is known to serve as a substrate for melanin production by certain microorganisms, by enzymatic inter- conversion, the present data suggest that other amino acids, viz. hydroxyproline, tryptophane, and perhaps histidine as well, may also be oxidized and polymerized to form melanin pigments. The chromatographic data, with respect to the brown pigments of the various extracts, show a pronounced simi- larity to the evidence obtained from the tyrosine "model". The presence of an oxidizable material in bright husks, that is absent in the stained husk-extracts, indicates that a precursor (or precursors) exist in bright barley husks; and that this material may be oxidized and polymerized, either environmentally or by microflora, to yield the staining principle found in discolored kernels. The rela- tively small amount of tyrosine, present in mature kernel husks need not nullify the above conclusions. Other amino acids and oxidized intermediates, as well as a variety of phenolic compounds, each capable of further chemical con- versions, are present in sufficient quantity in the mature husk. Further, since staining naturally occurs some time prior to harvest, i.e. when the kernel is at a much higher moisture level and a larger quantity of free amino acids 250 are present in the still-living husk tissues, the problem of these materials being a limiting factor in mature ker- nels is obviated. The pathway of melanin formation, drawn from these investigations and from the existing literature is presented in Figure 5. The innumerable polyphenolic compounds en— gendered by the enzymatic and non—enzymatic conversion of tyrosine and other ring-compounds, as well as their labile nature, creates many methodological difficulties in the identification and isolation of these materials. Increased interest in the polyphenolic constituents of barley kernels, and expecially barley husks, will undoubtedly shed further light on these problems. It has been suggested by many investigators that the tannins, melanins, and other phenolic and polyphenolic compounds represent important defense mechanisms in plant tissues. With a few notable exceptions, this contention has received very little experimental support. In the work outlined in this section, some support has been obtained for the "polyphenolic theory of plant disease resistance". It was observed that the presence of bacteria and the de- gree of pigment formation in aqueous solutions of tyro— sine were negatively correlated. While a few bacteria could be recovered from the sharply-discolored steeping liquor, a much higher bacterial population was always dem- onstrated in equal quantities of a paler solution. Bac- 251 .msHHH ..mg mesa .smos smz .mnom a mess: egos .em .eqN .mmma .quOaEHm .m was dopdsm .m .6 an zhnpmHSmnoon Hmsmdmwa aonm puma GH momma .m mendomaoo Hmnpo w .opm onwamshe oflom oadoamonmahsmnahxosohmlm deem oaahhowahsonmhxouohmam deem oHpomaahuonahxondhmum mUQSomaoo smnpo n Hogans + HommHOIm + oflom oapmom Iamsmflmhxondhmnm a madmawfim qwnmaoz oaoqflsvnw.mioaoddH s mdoqflsvmxosohnwmnw.m n onoqwdvlm.m Ima0©fiaopdhfiwdlm.NIhfiODHmOIN » maoeqasxopesgae ne.muopesase-m.musxoppmoum a mqoafldwnd.muoqfldeMHKanm » Anmnpov oaHQmHmHManmhxosohnfloli.m omadflpmflm meQQOPQMHB enamonme quqmamH%QQO oqflaosghxonphm m V. Am w A N. V IV M .mamdpox hmanmn no \ mnoauoHOHa hp sowpmasom Gasmama mom smwsmnooa domomosm 4 s .m ohsmwm 252 terial turbidity was never observed in very-dark tyrosine solutions, but cloudiness due to the presence of bacteria was frequent in the solutions showing less discoloration. That this effect was brought about by the inhibitory in— fluence of the ”melanin” material is shown in those sam- ples which possessed comparable microfloral populations (of. Table 50), but differed in their degree of discolor- ation and in the presence or absence of bacterial turbid- ity. When strongly-discolored solutions of the tyrosine steep—liquor were decanted into sterile tubes, the "re- sistant” bacteria frequently continued to multiply at the expense of the pigmented material (and presumably the leached-out kernel nutrients). This was demonstrated by the increase in bacterial turbidity and the accompanying fading of the brown color in the solution. The rapid multiplication rate of bacteria would ensure solution tur- bidities in all samples in a short time were it not for the presence of some inhibitory material. Varietal dif— ferences in the ability to form the ”melanin" pigments may be important in this context. it is postulated that the staining of barley, and other cereal, kernels is brought about by an inefficient reSistance mechanism. The developing grain kernel pro- duces a limited amount of melanin pigments, by means of its inherent polyphenoloxidase and substrate content. This enzymatic process is favored by increased levels of 25,5 moisture and elevated temperatures. The same environmen- tal conditions favor the infection process by a great num- ber of kernel~infecting microflora; and given a suitable number of air-borne organisms, infection ensues. The in- teractions of favorable macro- and micro-environments, host nutrients and oxidizing enzymes, and the multiple enzyme systems of the microorganisms all favor an increased pro- duction of melanin, with concommitant staining of the husk. The greater prevalence of staining in the immediate area of the infection loci and the ramifying fungal mycelium supports this hypothesis. The inhibitory nature of the polyphenolic pigments, which seems to be nonmspecific, as well as certain other physical and chemical changes in the husk of the develop- ing kernel serve to suppress and destroy a certain portion of the invading microfloral population. The effect is, however, only partially effective since resistant strains of the microorganisms apparently exist, and the physiol- ogical condition of the host tissues, e.g. moisture con- tent and diffusibility of both nutrients and inhibitors, is also of importance. The reported blighting and death of inoculated florets and young kernels with the atten— dant production of extremely dark discoloration serves to illustrate the factor of physiological-age of the host tissue. Inoculation with a relatively innocuous organ— ism, such as Alternaria, produces an entirely different 254 hostnreaction on young kernels, as compared to more mature grains. To expand the hypothesis it would seem that this mechanism may have other "side-effects", viz. the inhibi— tion of host~kernel germination. Many of the polyphenolic compounds isolated from barley kernels and husks have been shown to be inhibitory to seed-germination and many of these compounds are soluble in water. The decrease in germination energy and capacity of plump, discolored kernels is commonly observed, even though the seedwborne organisms are not in close association with the embryo. While the inhibitory "action at a distance” has been variously explained as the result of tOXin~formation and the production of volatile inhibitory emanations from the kernel organisms, it is suggested that the pigmented materials and their precur- sors produced by the kernel, may also exert such an effect. In zitgg studies have confirmed the inhibitory effects of germination by extracts of the pigmented material (9, 44, l56, 205, 252, 50l). The production of "gushing" or "wild" beer has been circumstantially linked with "weathered” barley used in its manufacture. Weathered barley invariably has a high microfloral content as well as an increase in discolora— tion. Several features of microfloral action on the qual- ity of malt prepared from weathered barley and inoculated barley indicate that wildness in beer may be due primarily to this microbial action. Both increased proteolysis and 255 wort-acidity, as well as nitrate-reduction (to yield ni— trites) have been demonstrated experimentally with both malts prepared from inoculated barleys and/or physiolog- ical studies of the kernel microflora (of. Chapters II and V). The same properties have been shown to be typical of malts engendering the wildness in beer. Whether the poly- phenolic, pigmented materials of weathered barley play a role in beer wildness is questionable. If anything, they may bring about a reduction in gushing by precipitating out the proteinaceous "nuclei" and by reducing the amount of oxygen in the beer. That the latter is a possibility is demonstrated by the strong reducing nature of these compounds. Another beer defect, related to the microflora of barley, is suggested by the problem of beer instability. It has been amply demonstrated that the conditions known as ”chill—haze" and "oxidation~haze” are brought about by the presence of certain polyphenolic compounds and protein degradation products. These materials, if pres- ent in sufficient quantity in the beer, form aggregated complexes which in turn produce the aforementioned hazes. Several workers have pointed out that barley and barley malt are, to a large extent, implicated in the production of these hazes. Few, if any, of these investigators have discussed the possible role of barley microflora in this regard. While much experimental evidence, expecially 256 pilot-brewing studies, is lacking, some of the data re- ported in this thesis, as well as in other papers, indi- cate that beer instability may be indirectly influenced by the microflora of barley. It is postulated that many of the polyphenolic compounds, including the many inter- mediates in melanin formation, produced by the inter-ac- tion of kernel and infecting organism, may be carried over in the malt and subsequently into the beer. These com- pounds, by increasing the normal polyphenolic content of the beer, thus bring about an increase in haze formation. The increase in wort-nitrogen, brought about by weathered and inoculated barleys in the production of malt, also enhance this effect by providing more polypeptides, amino acids, and other proteolytic products with which the poly- phenolic materials might combine. CHAPTER VII. GENERAL DISCUSSION AND SUMMARY A number of commonly—used methods for the isola— tion of the microflora of barley kernels have been de- scribed (Appendix A). Several of these methods have been evaluated and new techniques have been devised (Chapt. 1). The purpose for which isolations of microorganisms are made will determine the procedure to be used by the in- vestigator. Qualitative assessments are most rapidly made by means of whole—kernel platings using a differential ser- ies of culture media. Existing quantitative methods are not completely satisfactory. The use of suspensions of ground or macerated kernels and dilution-platings will only provide an accurate population figure if the method is rig- idly standardized, by combining these techniques with an accurate qualitative method. Proponents of this method assert that it provides an accurate estimation of the total' number of organisms present on and within the kernel. This is undoubtedly true, with reference to the uni-cellular bacteria and yeasts, if one considers each cell, endospore, or sporangium as a separate individual. This is not nec- essarily the case, however, with the fungi located within and beneath the kernel glumes. The dormant fungal mycelium occurs as a network of knotted hyphae with few, if any, spores present. The question arises - is this dormant 257 258 mycelium one individual or a collection of many indiv- iduals? Since grinding the kernel into smaller aggre- gates will concommitantly separate the fungal mycelium in— to correspondingly smaller units, each fragment is capable, in principle, of initiating a new colony on the dilution plates. There is a need for a better quantitative measure of kernel microflora. Such 21 method should provide an estimate of potential kernel—organism biochemical activity. This might be achieved through the use of wet or dry weight organism/kernel ratios, or perhaps rapid biochemical tests might be devised. The tyrosine test, described in Chapter VI, shows some promise in this regard. A great number of microorganisms have been isolated and identified from barley kernels (see Appendix B). These include: 68 genera and 148 species of fungi, nine genera and six species of yeasts, l6 genera and 50 species of bacteria, and three strains of a barley virus. The fungal flora of barley kernels are better characterized than the bacteria. The bacterial flora have not been considered of great importance until recently. The difficulty of dealing with very large populations of these organisms and the arduous task of bacterial identification have impeded our understanding of the kernel bacteria and yeasts. Even a small bacterial population, e.g. 10,000 cells per gram of barley, would require some 200,000 or more sep- arate taxonomic procedures to establish specific identifi- 259 cations. Thus, it is obvious then that new isolation and identification techniques will be required to increase our knowledge of these organisms. The location of the kernel flora is related to the infection process. The greater number of organisms isola- ted from the middle and the awn~end of the kernel, as well as their position in the tissues of the husk and pericarp, suggests that infection and establishment of the kernel tissues is largely a fortuitous contact process. That is to say that cells, spores, and mycelial fragments will in- fect the developing kernel at the point that chance con- tact is made with physiologically-receptive kernel tissue by the air—spora. Three factors are of importance in this infection process: (I) he presence of potentially-infec— tive cells in the air, (2) floret or kernel tissues in a suitable physiological state, i.e. possessing optimal mois- ture and nutritive materials, (5) a favorable macro-environ— ment, viz. temperature and humidity (cf. Chapter III). The microflora of barley kernels are capable of surviving prolonged periods of storage at reduced moisture levels. The fungal and bacterial populations undergo a steady reduction after the first few months of storage. The ability of non—sporeoforming bacteria to withstand long periods of storage is surprising, and is perhaps re- lated to the presence of mucilaginous capsular material as well as the presence of entrapped moisture in the 240 micro—environment of the glume tissue. The infection process by bacteria and fungi was found to be similar with all varieties tested under field conditions. The time of infection, however, differed with the bacteria and fungi. Generally, the bacteria infected the developing kernels somewhat earlier than did the fungi. The increase of bacterial infection was initially rapid; with a reduction in bact,rial populations occurring as the number of fungi increased above the fifty percent level (calculated as the number of fungal colonies per 100 ker— nels of barley). It is suggested that this relationship is related to both the physiological state of the kernel (i.e. less moisture and reduced nutritive material) and to the inhibitory effects of the attendant Inycoflora. The most susceptible stage of barley kernels to infection by both bacteria and fungi has been shown to be in the late milk and early dough stages. There are indications that varietal differences to kernel infection exist in the bar- leys tested in these experiments. The smooth=awned vari- ety, Montcalm, was more susceptible to kernel infection than were the other rougheawned varieties. 0f greater im- portance is the length of time that the barley remains in the field before being harvested. Prolonging the time of harvest by even a few days may permit a significant in— crease in the number of infecting organisms, especially if rain falls during this period. 241 The most striking effect that microflora exert on infected kernels is the discoloration of the pericarp and glume tissues. Both fungi and bacteria are capable of inducing kernel discoloration; although the field fungi are more frequently associated with stained kernels than are the kernel-bacteria. Western grown, bright barley may harbor moderately large populations of bacteria and Cladosporium Without being significantly discolored. These organisms may be present in the air; and when brief periods of suitable microwenvironmental temperature and moisture occur, they are able to infect the developing kernels. It is further suggested that, since these suitable infection periods are snort, because of infrequent rain or varying irrigation applications, the infecting organisms are not permitted to ramify through the host tissue but remain at or near the point of infection in a dormant state. The location of these organisms near the base of the lemma awn on western barleys. and the absence of kernel discolora- tion, support this hypotheSis. The greater prevalence of Cladosporium on western bright barleys may also be due to the absence of the more aggressive fungal species, e.g. Epsarium and Helminthosporium, which may suppress the growth of the less-virulent Cladosporium in barleys grown in the more humid mid-west. (of. Appendix C). In the case of stained barleys a high positive correlation exists between the degree of microfloral infection and the sev- 242 erity of the kernel discoloration (of. Chapter IV and Ap- pendix C). Six-rowed, Manchurian type barleys are grown in the North Central United States. These barleys are extensively used by American maltsters for their desirable malting and brewing properties. Discoloration of these barley varie— ties increases concommitantly with an increase in climatic moisture, i.e. as barley cultivation moves eastward from the Dakotas (Appendix C). The correlation that exists be- tween microfloral content of barley and its place of orgin is striking, so much so that the orgin of a barley sample can usually be determined by the organisms isolated from plated kernels of the sample. The determining factors in this close relationship are the climatic conditions, i.e. temperature and rainfall, prevalent in the growing area. An unusual situation occurs in Michigan,,whereby barleys grown in the "Saginaw Valley" and “Thumb" areas of the state are typically bright, plump, and free of microflora. Malting barley produced elsewhere in the state invariably shows a high percentage of bacterial and fungal infection. That this isolated area in the state is ideally suited for the production of barley and is at the same time not favorable for kernel infection is explained by the lower day and night temperatures and lower annual precipitation prevailing there and not in other agricultural regions of Michigan/l. 1 Personal Communication, Dr. J. E. Grafius, Michigan State University. The prevalence of free moisture and high humidity, just prior to harvest, promotes the so~called "weathering" discoloration of barley and has been directly responsible for the westward shift in barley production. Maltsters and (T barley buyers have refused to buy stained aarley offered 2'.) for use in malting. Wn;;e it ha been shown that kernel n staining may result as an effect of genetic w environmenw \ \ tal interaction alone, ,n: moat influentiai cause of ker~ nel discoloration is the presence of associated microflora. This concluSion is inescapable for several reasons: (1) barley grown in humid areas invariably yields large num~ bers of bacteria, yeasts. and fungi if plated within a year after harvest, (2) large numbers of spores, cells, and mycelial fragments cf the same type of microflora en- countered on cereal grains are alwavs present in the air, during the heading period, (5) the prc‘uction of "germ« free” weathering has rarely, if ever, been accomplished with barley plants grown from seed to maturity, (4) the climatic conditions which favor “weatierirg“ are also U. favorable to the infectitn and esta lishment of plant tissues b microflora. (5) barley grown in arid regions I J x, C 9 under irrigation, typically are "bright” in appearance and possess little, or no, microflora. If the term ”wea" 1"} {3 F L h r... by '1 r. U C (0 1‘ L2,. Ci 0 Q) l; U) r) "5 p“: O” D V? (D ’3 F nel discoloration, without impizcitl; knowledging the presence of microflora. it is a misiomer. An alternative tern, e.g. ”kerneiasmndge’ or “microfloral stain”, should be used with ba arleys discolored by the field fungi or by bacteria. The Official Grain Standards define dam aged kernels as 5 . . . kernels and pieces of kernels of barley, ether g:a:nsi and tde rats which are dam» ed or materiaiiv eisoz‘ire: %‘;a i“; m new by blight , _ .. o b and/or mold, or which 3i" r n, I", 4 damaged, sprouted, malted, {Tr frosted. badly exca;l ; i-g:i, :adiy mea;ner damared. or J U x... ,_ . J otherwise ma teriailr snagged‘ ‘5}. Since most, if not all, naturally ebbir ”‘Tp kerne; d.qv “ation is massed by "m01dns I}; .3 (I! 1» k) H 2 4..- (D ( VJ 1..) f I r } "\ I 1' 2 what constitutes ”met. (I) :neis is probe lematic. Only 3y p ar;ng a Simpzé o; are aarley, can one accurately determine fh« a.o: is chi x:*;» a; microorganw isms present or, and w, ole in; rails? we nels. The diffIC“i+ es in wivsi *r to assessment of barley quality on a visaai~acicr basis are further mule \ tiplied by other observations. kernel; rat ogqiously "blackvpointed” or shriveied. may suitor: ;arge popula tions of Helminthosporium and Fusarium s eties and "blackw . L .. ___,. i pointed“ and/or shrLVeLeo kernels may 3:: :3ntain either "blighflfl‘ or ”scedfi‘ fungi. 'Wirodnd d6flh%§§j” giewfli. charm» acteriz ed by both stai ng and ground odor. has been gen» erally regarded as another form of environmental injury Murlif by soil :5 Li 1..) ‘4 ’ . ‘2 L.“ )—— }. ...“ ’ .1 0 \d (R :1 (l .\ _1’ per 3 . That this MfO microflora is easily demunstaatzd by the is: ation of . ‘1 these organisms from kernels wh: ch have CU’WaCf‘d the 24+. F) soil or soil—water and aparticleso The ”ground odor" of such grain is characteristic of the Actinomycetes which are rarely isolated from kernels not contacting the soil° The microflora of "heat~damaged” kernels frequently is made up of bacteria and members of the Mucorales and Aspergillales° Again? a reliable evaluation method for this type of damage is the kerneisplating teehnique used in conjunction with the pearling procedureo Barley infected by storage fungi rarely presents a problem to malting barley buyers since the storage or- ganisms are scarcely ever present on newly harvested and properly stored barley” While the ceier of barley grains gives some indication of the microflorai papulation presw ent in the sample? it is less reliahie as a qualitative measure. Bright barley‘ while possessing fewer numbers of the filamentous fungi, may eontain pepuiations of bac- teria and Clados.orium exual to; or greater than. the ... . O , number of these organisms in a severely stained sampleo Because of the arbitrary use, and the ambiguity? of many of the terms used with reference to barley discoloration9 the following nomenclature is proposed\ Io Bright A0 Westernmgrown, under irrigated conditions 10 Free of microflora 2o Microflora present, mainly bacteria and Qladosporium Bo Grown under enclosures9 egga greenhouse9 bo Iii CI"): .5 10 has " ‘71,.-. do 1‘ LA.‘ 7 *‘\;v ,' J ._ . a u ‘f c. u .3 KI.” Severa: ~ue ten germination e may also V) ; The ‘ “ -'" " ‘\' '- ' “ o” " 'V i I', ' ‘ ‘ C L Q Hi I ‘7‘ i. ilr’i :«ffi M‘ I- "J . perimental series h:: the Thin-1+ :11- T‘f ’337' I L l wni i did Cons r " 7.. ,!,.., w pared from niiieys ‘uJC Hlfi'Vhfler\( f‘r‘1-‘.:':‘_Jynl E—)'1T '.I _- -..—...... _...... ...—ab.--” ....- .. .. ..--.. ...- _- nd r"euuovo1a" 3 both pilot and c mravr} fi'ry/r. " {-n‘h‘h“ 1r) 'MuLC’i) Li; J L"::‘TJ P by 9 ~ ,. A. ‘ _. ,l .1 ,A ‘r‘- A A ~. 7 \ ’b ' _. I ‘P \ .1 1 (_ ... .. 1 ‘ ON 3. r | \ I t O l V ‘ x in k ... r ,~ I .1. o‘. , a L j p) p— a ‘- 1L Cd“ In. 5 x .' F;-" k.— .1 k1 v v x 1 4 . l‘. L; I " , .. q. w—a C‘J '1; .I}; cbhtr oiled Laisture and r‘- ‘0 r\ t “ W _coiora ion :Llyi eog, tisrl a ume rot t 6080 SEATS ergotg €7.00 ;1 ha:te-;a C rg an 1 ”.2: 1:%‘ ..L i~\ ‘... :acteriai t;zo xa tn monas spp K r « \ 1* TI ' ~u'qa' :i-, heir hthosp Iium C. ‘"‘. ~Ers “ _ ’1 1 “ _ ' eh“ -7; uitbeiella and arigm stpo -Cpbvt:i \r weaxly parasiticq ree< Lu e tftictjr' etc organ15ns eogo gggcr Pillus lilufl, end certain Phycomycetes dataxe erg Ar.tinomycetes a.“ T: .. i. ‘4. . “r? ~ 9;-;;-*5 *:P Ebk° ‘—, - \ , T‘\| Slater, t. t;. JIEJ/ri‘r {($31.20ng 'era. (" €111. siepieso , l , ., .4 - 49‘ r xi 1‘ . F a J glziiil'll bile V. - ".' ‘ .n V'—: l"r Unis effect a: v.;‘ 1; :.v<~5‘ (Chatter h l7 h » » ; arrwn- .~- 3: T, his 631% - r’“ , LI} {J ', . . J; I", (NJ-l \ 'O- r‘ ‘ ‘ 1.1 L':‘ 'f{ l" : melt? 131' M . 5 II A ~\ . —-. ‘ ‘V'l l 1 2’1 “1‘." L tr )- ‘S’ l "1 .‘ECI-Ab «'. 4 e :’ ’1’ : i”-e \J .I‘ I I a \ .f ‘ V ’T *i (It -‘ T - \ 7 1 “ L‘ "fltlflu 1". v: ‘. LJ‘J. tiono The t ... 1 V; 4' sms rapidly i germinationo better able to surVive . \ “ "‘ '5 ’7“. r \ ing pIOcess ins micro O 84 samples. furnished by Association tnnoend X C) sults from the cooperat; these barleys are publis i.“ g \ treiiminirv coloration by it lor btained fi»m per 3 u 11" . and alcoholic extracts C have estat V ered barley the brown szaining ngm barley kerieis in 34 sq shown that bailey k9Clfi kernel—infectina Fur~1 act vitya it: 1; hyperlu pigment 0) Infection by production9 apparently in an addit pigment po (0 ioeo it eems (1 Q anismd It is further nature of the “1' ) to repress The bacteria r r -~ - . l ‘J l 3" _ _ '\_I .J r 1 I) P; n0~ LLLU W -.. # fluaxPOil tertag is well as these alien r numbers diring steeping and hich infect the green malt are high tempeiatures in the kiln- ral analyses terfarmed on tie he halting Barley l nprovement if as of ihteiest when the re» malting anc brewing trials of do on the meta e o: barley disw ave been carried outo Evidence ogram prepirei from aqueous the nails of biight and weathw new the pug} "671'; .. :_ ('3 nature of 5 Ti; rteeg;np ~f i;s‘infected I woiit_ n oi L tyrosine has a: w—ll as m» j the ommon (gaff? 131 pt; 2w 1's :‘y'icsiriase zed that a -imltea amount of p.m( need hm :erti.n barley var~ ora enhance: this pigment ‘ ive fast lOIm The brown speculated that 13 sesses a limited anti~microfloral activity? elective resistanceumechw a polyphenolic may be oi importance in the produCLion of chil, hares in his ero Another hypothesis is presented to account for the production of gas- instability in beer by malts prepared from naturally~infected barleys. This explanation is ba:cd on the eVidenoe that the common- ly-occurring fungi and bacteria on barley kernels are cape I) able of pronounced physio l 31c.l activityfi such as the re_ \ J ,1 duction of nitrates to nitr tee ( and the degradation of barley proteinso The relationship oi barley microflora to the qualm ity of malt and malt products is as yet incompletely un~ derstoodi The mere presence of bacteria; yeasts? and fungi on ba ley does not satisfactorily eXplain many of the quality defects occurring in malt and beero That certain organisms constitute a menace to the maltster is a truismo host of these Carley pathogens and/or storage or organisms are not prayerfi htwev ve l bright or slightly t1 stained bail eyt Fro om these preliminary Studies it would seem that future research in this general area might cen- ter on the following aspects of the problem* (i) the ef- fects of the melanin-like pigments in infected barleys on the quality of beer. (2) the effects oi the foreign micro- flora; introduced during malting? on the quality of the malt and malt products? (5) the develOpment of rapid9 sim— ple9 and accurate tests for the quantitative determination of kernel microflorat especially those oiganis ms which are of commercial importance; (4) control methods for the pre« 249 vention or eradication o5 dazgerous barley flora, both in the field and in the malting process° 8. 90 10. 11° 12° Anonymouso 1928 Die Ursacne der GiftigKeit der amer- iKanischen Gerst e LTne cause of the toxicity of American barleyo; iit 3esellscho Vorratsschutz 42 ee- 67 lestro :1evA.pplo hycolo 8; 459;] Anonymouso ri9filo The flavour of malto brewer's douro V7gdjb LAbStro fisl.o Labo Comma 4; Zl4o] Anonymouso l95§o A study of the influence of weath- ering on the suitaoility of barley for malting and brewing r,:nd annual reports i9>l~929 1952a55, 1955—54. Malt Bess arch institute Publo la: Eul6o Anonymouso 1957, Grain grading primero UOSo Dept° Agra Misc° Publ° 740: l‘bbo Anonymous° i958 Methods of analysis of the American society of treqin chemistso Publo by the Associa- tion, Madison 53 W180, om editoq 209 ppo Anonymouso 1959J Official grain standards of the United Stateso U98o Depto Agro ADM.S. ~ 177: l~950 Anonymouso 9590 Report on the second international conference on seed pathologyo International Seed Testing Association3 Copenhagent Denmark9 22 ppo Mimeographedo Anonymous° Minnesota 0 Unpublished data l959 o Anonymouso Minnesota 0 Unpublished oata 1960 o Adams9 SOLOQ WOHO Sta k 3 Po Kolachtvo 194l° Effect of the bacterial co ontent of barle' malt on the fern mentation eH lCl ency of spirits masho Jour° Bacto p (AtStTo ) 49 8350 Andersen, AOLO 19480 The development of Gibberella zeae headblight of wheat: Phytopathology 58: 595-611° Andersen, AOLo 1952, Development of wheat headblight incited by Helminthosporium sativumo Phytopathology 42: 455‘4560 Anderson, Jvo 19370 Laboratorym ltin’ro Io Equip— mento Can. Jouro Research 150: 204~Elbo Anderson, JQAo & W.O°S° Meredithn 1940o Laboratory maltingo IIIO Steeping equipment and methodso Gero Chemo l7: bb~720 15. 16. 17. 18. 19. 20. 21. 22. 25. 26. 27. 25; Anderson, J.A. d H. ngland. 1957 Modified equipment and methods for the routine mal+ .ing test and a study of its precision. Sci. Agr. 17. 742- 751. ArK, P.A. & J.P. Thompson. 1958. Antibiotic properties of the seeds of wheat and barley. Pl. Dis. Rptr. 42: 959 962. Armolik, N. & J.G. DicKson. 1956. Minimum humidity requirement for germination of conidia of fungi associated With storage of grain. Phytopathology 46: 462wa65. Armolik, N., J.G. iicKson, & A D Dickson. 1956. De— terioration o: caries in storage by microorganisms. Phytopatholegy 46: 457~A61. Armolik, N.. J.G. Di Kson. & A.D. Dickson. 1956. The role of fungi in the rapid deterioration of barley in storage. Phytopatholcgy (Am .) 46: 7. Arny, D.C. & C. Leben. grain diseases by the Phytopathology A6 I 956. Control of several small water- soaK seed treatment. A 54 \ Vi Atanasoff, D. 1925. Fusarium blight of the cereal crOps. Mededeelingen van de Landbouwhoogeschocl9 Deel 27, No. 4: lelfii. Bailey. C.H. & A.M. Gurgar. l9i8. Respiration of stored wheat. Jour. Agr. Research 12; 685-715. Baker. D.L. & L.J. ixjo;.1nt. 1956 En: design of a pilot malting 1atcr3tn1"ty and the occurrence of strains wlthin the gen<1v “1.8;9 3111. L1nk and Alternaria Nees. J Angew Bofo 15 age j}; §E915850 {:Abstr° Re*vfi,11§plo 1;y:-; if. ‘ ‘ Iwns1 o; ,1 h if: l 490 Boos 3; is MOGO 19glo in; ep1d c1191 9y of Xanthomonas tr1nsr cens (J 1 and Re) Dowsom on cereals and J inSSBS 1nyfooathologf 4;: 587 $950 - ..-- 50° Boruff, 018,1 R910 Claassen & AOLO 2301163:o 193 a A study of the hav+e1 a} popu1at1or of grains used 1n a djst111erya Car, Chem» ll~ 4% 4180 510 Bottomleyg R.Ao 1M0 Fhristerseu & WOFD qeddeso 1952 Graln storage ru11eso ’ a i. ”ainzf 31aeratlon9 time; and mo1sture content on far 21:11:11,111};3 non re» ducinw -drae . and mold flora oi 3:11ed 3e11ow corno W 3. ~ 1‘ ’_ . Ce: Lhcm cfla 5, Me O A“) U) (‘1 p ,< U .3 ‘V‘ L_«' 1 l ( L..— 7‘ J L ‘3 52° 53° 54° 55° 56° 57° 58° 59° 60° 61° 62° 65° 640 1:: 5a Boyer9 MJGO 19551 Effect of Alternarja tenuis Auct° on other common seedaborne fungi, Procfi Assoco Off. Seed analysts 45: 55-540 Breed, R SM et a1oo 1948o Berggey 8 Manual of Determin- ative BdC+8T101§JVo hm Ed1tion1 The William & Wil— K1ns Coo9 Ba1t1more1 Koo, 1589 ppo Breed1 R 801 e: no 19570 Bergey’s Manual of Determin- 8 ,/ ative Bacte er1o119J Tm Edlthfid The W111iams & Wil- kins Cece Bait1more1 Eda; 1C9» ppo Broadfoot W,Co & HOT” Robertson, 19550 Pseudoablack chaff QI Renard wheat, 3111 Agra 15: 512w514o Bruehlg Go‘vJ195?o erhaloSpor11_m sfrlpe disease of wheat; Phytopatholcgy a 0+”-b490 Buchner. Ho 15580 Notiz. betreff end d1e Frage des Vor- Kommens von Bacierien 1m norma1en Pflanzengewebe. [Notice concern1ng the quest1on of the occurrence of bacter1a 1n norma1 plant tissuesoig Muncho medo Wochschro 55: 906w9070 Burkhart B A , AQDQ D1ckson & W J Huntu 1595 o A pilot Drew1ng comparison 01 C ..' 1 and labora« tory maltso Proco Amero 8001 B ewo Wk em 1955: 54-45 0 Burri‘ Ro 19C5 D1e Bak.r er flache normal en*v1cke1te f1 flora on the upper surface of r10 plantso 3 Centro Bakt; Pa 1 enreLHe at1on auf der Ober- arzeno 1 The bacterial r1. .a11y developing Campbell1 We?o 19560 The influence or as ssociated mlcroorganlsms on the pathogen1c1ty of Belmintho» sporium sativumo Canq Jouro Born 59‘ 665~874o Campbell, WCPO 19580 A cause of pink seeds in wheato Plo D180 Rptro 425 11.2 720 Cayzer, L081 19570 B1each1ng and weatheringo Effect on wheat gra1n qual1tyo Agra Gaza New South Wales 48: 665-6670 Chamberlain, E E 1954 In Report on the fifth Com- mor wealth Mycolog1cal Conference? 1954o 159° Comm monwealth Mycolo§1ca1 Institutes hew1 Surreyo Chen, Coco 19201 Internal fungous paras1tes of ag- riculture seedsa ng Agra Expo Stao Bull.o 240: 81-110 tenho 2 ADI:o 10: 756u765° 65. 66. 67. 68. 69. 70. 71. 72. 75. 74. 75. 76. 77. 78. 255 Chen. 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