{SQiATéOfiN SWDIES 01" THE fxdlCRORC‘RA OF EARLS-Y KERNELS AND THE UTILIZATfON OF SELECTEE ORGANIC SUBSTRATE EY CERTAEN OF THESE [SOLATED ORGANISMS Thesis for the Degree of M. S. MICHEGAN STATE UNIVERSETY Evan Harold Pepper, Jr. 1958 mmu;{filmmmuwlmwl{I11um: ISOLATION STUDIES OF THE MICROFLORA OF BARIEY KERNELS AND THE UTILIZATION OF SELECTED ORGANIC SUESTRATES BY CERTAIN OF THESE ISOLATED ORGANISHS By EVAN HAROLD PEPPER JR. An Abstract Submitted to the College of Science and Arts Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Botany and Plant Pathology 1958 Approved An Abstract This study was concerned with the isolation and identification of barley microflora, the chemical com— position of the barley kernel, and the utilization of certain major constituents of the kernel by selected field fungi. A total of thirty-six barley samples consisting of ten varieties from 1956 and 1957 crops were used in the investigation. The kernels were surface-sterilized and placed on potato-dextrose agar plates. Fungal and bac- terial colonies growing from these kernels were counted and identified. The composition of an ungerminated barley kernel was determined from a survey of the literature and cer- tain of the major compounds were selected for use in the utilization studies. Isolates of Helminthosporium, Fusarium, and Altern- aria were grown on Czapek's sucrose-nitrate solution with substituted carbon and nitrogen sources. Alternaria isolates utilized amylose, cellulose, sucrose, raffinose, L-glutamic acid, edestin, L-aspartic acid, DL—d-alanine, and L—proline as the sole carbon source but were unable to use L—asparagine in this manner. Nitrogen sources utilized by Alternaria were: L-aspara— gine, L-glutamic acid, edestin, L—aspartic acid, DL—KE alanine, and L-proline. Helminthosporium isolates used the same nutrients as the cultures of Alternaria. The isolates of Fusarium utilized all carbon and nitrogen sources except cellulose. Not only did the amino acids employed in this in— vestigation promote growth of the fungi studied without additional carbon being supplied but, with the exception of DL-«Ealanine, the amino acids induced heavy sporu— lation of the Fusarium isolates when serving as the only carbon source. This effect is tentatively explained in terms of a reduced carbon-nitrogen ratio. The enzymatic abilities of the fungi tested in this study, their utilization of major barley kernel con- stituents, spore production, pH changes, and pigmentation indicate that the quality of malt processed from ”weather— ed barley” may be strongly influenced in either a bene— ficial or an undesirable manner. Selected references Anderson, J. A. and A. V. Alcock. 1954. Storage of cereal grains and their products. American Assoc- iation of Cereal Chemists. St. Paul, Minnesota. 515p., illus. Gottlieb, D. 1946. The utilization of amino acids as a source of carbon by fungi. Arch. Biochem. 9: 341-551. Hopkins, R. H. and B. Krause. 1937. Biochemistry applied to malting and brewing. D. Van Nostrand Co., New York, N. Y. 342p., diagr. Mead, H. W. 1943. Seed—borne moulds of barley. Wall ISOLATION STUDIES OF THE MICROFLORA OF BARLEY KERNELS AND THE UTILIZATION OF SELECTED ORGANIC SUBSTRATES BY CERTAIN OF THESE ISOLATED ORGANISMS By EVAN HAROLD PEPPER JR. A THESIS Submitted to the College of Science and Arts Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Botany and Plant Pathology 1958 ACKNOWLEDGEMENTS "If I have seen farther than others, it is by standing on the shoulders of giants”.......... Sir Isaac Newton Although I may not have seen farther than others, I certainly have stood on the shoulders of giants; my fellow workers, friends, and family. Specifically I wish to express my gratitude to my major professor, Dr. R. L. Kiesling, who not only suggested this line of endeavor but has been a continual source of inspir- ation, encouragement, and sound counseling. My thanks also go out to Dr. C. C. Kuehner of the University of Detroit, whose advice and assistance have been invalu- able. The H. W. Rickel Company and the Stroh Brewery Company of Detroit, Michigan have been extremely generous in furnishing samples, literature, equipment, and advice. Especially helpful were R. E. Spahn and O. Krohn of the Rickel Company and F. Roberts and H. Rosenbush of Stroh's. II. III. IV. V. VI. VII. TABLE OF INTRODUCTION . . . . . . . REVIEW OF LITERATURE . . . MA ERIALS AND METHODS . . EXPERIMENTAL DATA . . . . DISCUSSION AND CONCLUSIONS SUMMARY . . . . . . . . . LITERATURE CITED . . . . . CONTENTS Table II. III. IV. VI. LIST OF TABLES Source of isolates . . . . . . . . . . . Growth measures . . . . . . . . . . . . Substrates tested . . . . . . . . . . . Isolation of microflora . . . . . . . . Chemical composition of an ungerminated barley kernel . . . . . . . . . . . . . Mean utilization of substrates . . . . . a. Amylose (carbon source) . . . . . b. Cellulose (carbon source) . . . . c. Sucrose (carbon source) . . . . . d. Raffinose (carbon source) . . . . e. L—Asparagine (nitrogen source) . . f. L-Asparagine (carbon and nitrogen source) . . . . . . . . . . . . . g. L-Glutamic acid (nitrogen source). h. L-Glutamic acid (carbon and nitrogen source) ... . . . . . . . i. Edestin (nitrogen source) . . . . Page 14. 15. 16. 20. 22. 27. 27. 28. 29. 30. 31. 32. 53. 34. LIST or TABLES (Cont.) Table Page j. Edestin (carbon and nitrogen source) . . . . . . . . . . . . . . . . 36. k. L-Aspartic acid (nitrogen source) . . . 37. 1. L-Aspartic acid (carbon and nitrogen source) . . . . . . . . . . . . 38. m. DL- -A1anine (carbon and nitrogen source) . Q . C 0 O . O C O O O O O O . 39. n. L-Proline (carbon and nitrogen source) . . . . . . . . . . . . . . . . 40. . 41. VII. Utilization of barley growth medium . . . . LIST OF PLATES Plate Page I. Weathered and bright barley kernels ............ 19. II. Structure - composition of the barley kernel ... 26. I. INTRODUCTION Barley is grown in the United States mainly for feed and for commercial purposes. The manner of utilization determines the variety and agronomic type grown by the farmer. The major barley growing areas for commercial use are the Red River Valley, the states bordering the Great Lakes, and regions along the Pacific coast. The principal commercial utilization of barley is made by the maltster who, by germinating the kernels under carefully controlled conditions, produces malt, which serves as the basic raw material in the brewing process and in many food and beverage industries. Malting barley is carefully selected season after season by the maltster, who chooses special malting varieties which provide those qualities that will, in all probability, yield predict- able and consistent batches of malt. On the strength of many years of experience, the malting barley buyer chooses a crOp that shows natural color, plumpness, high germi- nation, and endosperm mellowness. Barley grown in regions of moderate to heavy rain— fall, is subject to staining of the kernel. This staining is caused by a number of factors; chiefly by moisture, heat, soil-contact, and associated microflora (1). The major cause of discoloration of barley kernels in the Mid— west is the presence of various microorganisms on the surface of, and within the kernel. The dominant kernel invaders are fungal parasites and saprOphytes which are l. customarily referred to as "field fungi", if they become associated with the kernels from flowering time until harvest; and as ”storage fungi" if they attack moist im- properly stored grain (7). It is common knowledge that maltsters do use "weath- ered" barley, in fact may not be able to buy anything but discolored grain in an extremely moist year. The problem then is this; is discolored barley comparable in quality to bright barley in the malting process? If this question could be resolved it would provide a more equitable basis upon which the farmer and the grain dealer could negot- iate, by replacing artistic with scientific selection methods. The farmer would not plant a crop of malting barley were it not for the high premium that is paid for acceptable malting barley which justifies the additional expense and care. Unfortunately for the barley grower in the Midwest, the maltster frequently rejects stained bar- ley lots as "unacceptable" without being able to point out just why a discolored kernel will not malt as well as, or better than, a kernel of "bright" barley. Any changes in kernel quality caused by "field fungi" must ultimately be traced back to the original chemical composition of the grain, the utilization of certain or- ganic constituents of the kernel by these fungi, and the various metabolic gains, losses, by-products. and other biochemical modifications resulting from this barley— fungal relationship. Working on this hypothesis, it was 2. decided to divide the problem into three main divisions; the isolation and identification of barley microflora from a number of different varieties grown at widely separated locations, the determination of the chemical composition of the ungerminated malting barley kernel, and assimilation studies using selected, frequently- occurring, fungal isolates on the major organic fractions of the barley kernel. II. EVIEW OF LITERATURE Isolation and identification of barley microflora A few years prior to the twentieth century, several investigators isolated fungi and bacteria from the sur- face of cereal grains. Unlike their predecessors, they recognized the true microfloral nature of these isolates. In earlier investigations seed-borne microorganisms had been regarded as enzymes in living form (1). Further observations not only confirmed the presence of associat- ed microflora on the surface of seeds, but demonstrated the occurrence of mycelium below the pericarp as well (1, 3, 6, 7, 24, 26, 27, 43, 44). As early as 1892, Zobl (47) concluded that discolor- ation was caused by a fungus, Cladosporium herbarum, which he had obtained from badly stained barley kernels. A few years later, Duggeli (11) found large numbers of bacteria, mainly Pseudomonas trifolii Huss, on apparently healthy grain kernels. Helminthosporium sativum and species of Alternaria were also identified as kernel- staining fungi (lO). Orton, in 1931, listed fourteen species of parasitic fungi and bacteria known to have been isolated from barley kernels (29). More than nine hundred samples of barley were examined by Christensen and Stakman (9) who identified species from more than twenty-five genera. Species of Alternaria, Helminthosporium, and Fusarium comprised the dominant fungal isolates with 4. Alternaria spp. occurring more frequently. They ob- served that species of Helminthosporium and Fusarium were usually isolated together in a given sample with Fusarium graminearum Schwabe and Helminthosporium sativum occurr- ing most frequently. A summation of the work through 1937 was given by Machecek and Greaney in their investigation of "kernel smudge" of cereals in Manitoba (24). These workers concluded that "black-tip" of grain was caused by bacterial infection while l'kernel smudge" was incited by fungi especially members of the form-family Dematiaceae. The predominant microflora in this study were, in des— cending order, Helminthosporium teres — 73 per cent, Hel- minthosporium sativum - 12 per cent, Alternaria spp. — 11.2 per cent, and Fusarium spp. — 3.8 per cent. The greater percentage of Helminthosporium spp. in this bar- ley sample was tentatively explained by the repressive action of Helminthosporium sativum on Alternaria tenuis, as exhibited on Petri plates. Spore counts were conducted during the summer months which were correlated with in— fection and penetration of the deveIOping florets. Studies on the microflora of wheat (20) increased the number of associated bacteria and fungi to some forty-four separate species, some of which had not been previously isolated. In a further study of seed-borne fungi in cereals Greaney and Machacek (16) isolated and identified species of more than fifty genera. Alternaria spp. were by far the most common, with Helminthosporium spp. and Fusarium spp. next 5. in order of abundance. Other commonly ocurring fungi were species of Nigrospora, Curvularia, Hormodendron, Epicoccum, Septoria, Cephalothecium, Penicillium, and smuts. Infected kernels commonly had as many as five different species associated with them. Comparatively few workers have discussed the presence of yeasts on cereal grains (1) but Lund (23), using selective media, found large numbers of pink yeasts of the genera Sporobolomyces and Rhodotorula on Danish barley. The plating techniques employed by most of these investigators to isolate cereal saprOphytes and parasites have utilized various surface-sterilizing sol— utions which are evaluated by Mead (25). Because of the greatly increased interest in storage deterioration of grain, a number of recent review articles are available which summarize the information on both "storage fungi" and the "field fungi" dealt with in this investigation (1, 2, 6, 7). The chemical composition of the ungerminated barley kernel Data on the chemical composition of the barley kernel has been presented by investigators in brewing and allied industries. Both qualitative and quantitative determin- ations of kernel constituents are often difficult to obtain. Therefore, this work is selective, scattered, and con- stantly revised with the advent of newer and more sensitive techniques. General tables of composition of barley ker— nels are available which give accurate determinations of the major compounds comprising the barley kernel (19, 42). 6. Utilization studies The nutritional requirements of microorganisms have been studied intensively since the pioneer work of Raulin, Pfeffer, and others in the late nineteenth century (45). These investigations have considered the mineral, organic, and growth factor requirements of various fungi and bac- teria. While some organisms have been subjected to num- erous nutritional studies, for example, Aspergillus niger (36, 37), others have been almost completely neglected. The essential organic compounds comprise two general clas— ses, those which provide assimilable carbon and those which contribute nitrogen. The standard procedure employed in the determination of organic compounds which can be util— ized by microorganisms has been outlined in a number of standard references (17, 22, 45). A basal medium is chosen which includes the essential minerals and a carbon and nitrogen source. Various carbon and nitrogen containing compounds are then substituted for the standard carbon and nitrogen sources in the basal medium. Growth of the or— ganism on the modified medium is then compared with growth on the standard medium. Steinberg (36, 37) has reviewed the requirements of fungi for carbon, nitrogen, minerals, and various other growth substances. The heavy metal re- quirements of filamentous fungi are summarized by Foster (13). A number of studies concerning the utilization of cellulose have been stimulated by degradation of fabrics by certain microorganisms (35, 39). 7. Starch is utilized by the majority of the higher fungi according to Thaysen and Galloway (40). Brown (5) demonstrated that when glucose was replaced by starch in growth media Fusarium fructigenum conidia production in— creased. Moore showed that Fusarium coeruleum could utilize starch in addition to maltose, sucrose, glucose, fructose, arabinose, xylose, glycerol, and various or- ganic acid salts as carbon sources (28). Alternaria tenuis has been shown to produce amylase, lipase, inver- tase, raffinase, maltase, cellulase, and various other enzymes (38), with cellulase produced in greatest abund- ance. Cellulose, like starch, is hydrolyzed by many fungi and bacteria. Many species of Alternaria, Helmin- thosporium, and Fusarium are reportedly cellulose decom- posers (35). Wolf (46) grew Fusarium oxysporum var. Electianae on Richard's medium with thirty compounds as the only source of carbon. He found that maltose and xylose promoted the greatest amount of growth while raffinose, sucrose, and some others were excellent carbon sources. Glucose was rated as good and starch as fair. Certain nitrogen sources were also tested, the fungus using nitrate, ammonium, and amino nitrogen. Certain amino acids were superior nitrogen sources, namely as— partic acid, glutamic acid and alanine, while nucleic acids were used to a lesser extent. Some fungi are known to utilize certain amino acids both as a source of carbon and nitrogen. In 1948, 8. Schultz gt El° (33) used isolates of yeasts on assorted amino acids serving as a source of nitrogen and obtained growth responses which indicated a possible taxonomic method. This study was followed in 1949 by an investi- gation of amino acids as the sole carbon source for certain yeasts (34). Using nine yeasts and eighteen amino acids, it was found that a few isolates were able to obtain their carbon requirements from alanine, argin- ine, aspartic acid, asparagine, glutamic acid, glycine, proline, and serine. Those yeasts that were not able to utilize glutamic acid or proline were unable to use any of the remaining amino acids as a carbon source. It was shown that a small amount of dextrose added to the med— ium acted as a "starter", aiding in both initiation and continuation of growth. Lewis (21) studied the inhibi- tory action of amino acids on Alternaria solani and found that most of the amino acids employed in the study supported rather than inhibited growth. He also found that combinations of amino acids gave results which dif— fered from those obtained when the amino acids were supplied singly. Gottlieb (15) tested twenty-one amino acids and four related compounds as a carbon source using Penicillium roguefortii and Fusarium oxysporum var. lycopersici. The fungi utilized seventeen of these com— pounds with similar growth on each compound. The Eusarium grew poorly on both lysine and norleucine. Cysteine and methionine were not used by these fungi nor did they support the growth of six other species in— 9. cluding Alternaria solani, Helminthosporium sativum, and Fusarium moniliforme. The effects of vitamins on the growth of selected species of Helminthosporium and Fusarium were investi- gated by Elliot (12) who found that Fusarium culmorum, F. avenaceum, F. moniliforme, E. poae, Helminthosporium gramineum, and H. victoriae were self-sufficient with regard to biotin, thiamine, inositol, and pyridoxine. H. avenae was somewhat stimulated by thiamine while one isolate of E. avenaccum was depressed by added vitamins and thiamine was suspected as being the repressant. The mineral, trace element, nitrogen, and vitamin require- ments of several species of Helminthosporium were studied by Peterson and Katznelson (30, 31) who found that zinc, manganese, and iron were required in small quantities for growth. Growth of the isolates was mod— erate using nitrate, ammonium, or amino nitrogen but was greatly improved upon addition of yeast extract or a mixture of trace elements. Addition of vitamins did not increase growth. Certain amino acids were utilized more favorably in the presence of trace elements, notably L- proline and DL-serine. Treggi studied the utilization of some amino acids as nitrogen and carbon sources, em— ploying a number of fungi which included Alternaria solani and Fusarium lini (41). He found that in general only DL—aspartic acid was used as both a carbon and a nitrogen source. 10. III. MATERIALS AND METHODS Isolation and identification of barley microflora The isolation of microorganisms from barley kernels was begun in January, 1957 while the author was a grad— uate student at the University of Detroit. A total of thirty-six samples of malting and seed quality barley was furnished by the H. W. Rickel Company of Detroit, Michigan. These barley samples were selected from 1956 and 1957 crops grown in North Dakota, South Dakota, Washington, Idaho, Minnesota, Ohio, Michigan and Manitoba. Ten barley varieties were used in this plating study. A surface-sterilizing solution consisting of two parts of five per cent commercial sodium hypochlorite and one part seventy per cent ethyl alcohol was prepared at the time of plating. Ten kernels were selected at ran- dom from the sample and placed in a Gooch crucible which was in turn immersed in a large glass finger bowl con- taining the sterilizing solution. After remaining in the solution for one minute the crucible was removed and the kernels were transferred aseptically with forceps to a Petri plate containing sterile growth medium. The kernels were plated ten to a plate which facilitated later percentage calculations. Potato dextrose agar, with and without acidification, was used as a general medium. Inoculated plates were incubated at room tem— perature and were observed at daily intervals for 11. bacterial and fungal growth from the kernels. The plates were observed microscopically from three to six days after plating. Colonies around each kernel were identified and transferred. Gross microsc0pic identi- fication was made in most cases by direct observation with the low power objective of the microsc0pe. Ad- ditional plates and prepared slides were used to identify yeasts, bacteria, actinomycetes, and non- sporulating fungi. Pure cultures were obtained in most cases by making mycelial transfers before the colonies had extended too far from the plated kernels. All pure cultures were transferred to potato dextrose agar slants after identification and placed in refrigerated storage. Chemical composition of the ungerminated barley kernel The literature dealing with the chemical make—up of the ungerminated barley kernel is scattered through many journals and restricted in its nature. Therefore, in order to determine the relative abundance of com— pounds which comprise the barley caryopsis and to establish the location of these constituents, an ex- tensive survey of the literature was undertaken. Approxi— mately one hundred and sixty—five articles, many with analyses made possible only with recently developed techniques, were reviewed in this study. By converting the assembled information into uniform percentages, in 12. terms of an ideal malting variety kernel, the data pre- sented later was obtained. Those compounds occurring in greatest abundance were subsequently selected for use in the utilization studies. Utilization studies Six isolates of three genera; Alternaria, Helmin- thosporium, and Fusarium were selected at random from the original stock cultures isolated from barley kernels. Potato dextrose agar plates were inoculated from the stock culture tubes and the colonies were inspected in order to verify identifications. The identity, stock numbers, and plating sources of the isolates used in the study are presented in Table I. Since semi-qualitative results were desired, it was necessary to devise methods for the inoculation of growth media and for the recording of growth. A "bisquit cutter” type of inoculating needle was constructed of nichrome wire which would cut a plug of mycelium and agar one- quarter inch in diameter and with a volume of approxi— mately eighty cubic millimeters. A table of growth measures was established on the basis of colony diameters, ranging from one to ten with one equaling "no growth", i.e. the size of the initial inoculum, and ten equaling maximum growth, i.e. the colony covering the bottom of the flask. The growth measures are given in Table II. 13. 'Il' has omma anopHamE unmaxnwm flmmqmm a Hmchflm Amxov mfiaamhmo.m afiomoh.h.MI¢H14 hflS mmmH MQOPflQNS dngMHwW Homfidm % Hmvhgm AmMOv mflfimmfiwon ESQmOH.W_NI¢H|4 as: mmma mpopaqm: enmagnwm nmmqam a seesaw onov maawmwmo.m admmosnm disasa HHHQ¢ mmmfi .Q.z .Ponflz concsfix nomamm @ Hmchsm Amxov mflawmkww.m admmoh.h mid Hanna mmoa .m.z .omwam emweqam gmmqmm s panama Amaov maammamo.a summon.w mu< Scam: wmma .Q.Z .omhmm mwmpnm> Qmmfimm E Hmohsm Amxov mflammhmo.u BSmmoH.m N14 .mm afiflhwmsm .poo Amos .noaz .mpoamwawm my :a a . was . mssam aspapmm.m sans .poo omma smfiaa> nm>am emm Wmanww was m a .s a .oomMImmmmwum H19 .mem PmmH OHSO .mdbmaamm umhdfiflm mxxwm % mfiHM.Hmafiwm afibflvmw.m mHlo .pamm bmmfl hoHHw> uw>Hm dom woncsflm mxxmm a mnfim.amaamm sabflpmm.m_ mlo HHHQ¢ wmmfi .m .z .fiocmflmH HHHNHB mxxmm a mflflM.Hmafiwm afibflva.m HHI¢ an: mmma .m .2 .qoomamg Hammae msxmm a wsam.amsswm sabflpmm.m HI< .mm adflhommoflpnfiaamm .Pmom mmmfi .m .z coacafim .mmmz madfimp.H N10 .ms« 8 w paw: vmnHHM .mmmz madnmp.H. mum hm: mmma wDo#Hfidz Hm % O14 .mm mflhwnhmpad Abmmavumfima hmmw nadhw M#mHHd§WI mmpwaomH IomH .sz mono soapmoog hmanwm Hmwnsm m0#dH0mH MO ovhfiom .H mewe 14. Table II. Growth Measures Colony diameter Amount of growth 1/4 inch 1 (no growth) 3/8 inch 2 1/2 inch 3 7/8 inch 4 1-7/16 inch 5 1-5/8 inch 6 1-7/8 inch 7 2-1/8 inch 8 2-5/8 inch 9 3-1/16 inch 10 (maximum growth) The basic medium employed in the utilization studies was Czapek's sucrose nitrate solution (32). Various car- bon and nitrogen sources were substituted as shown in Table III. All chemicals used in the study were ”reagent grade" unless otherwise specified. Two considerations determined which compounds were to be used; the quantity of the constituent found within a barley kernel and the availability of the compound by chemical suppliers. A barley medium which promoted excellent growth of the isolates was used as a check. This growth control 15. .cmmeHHmca Po: mmpmaomH H m.o msHQmH Hc>Hm chm ccnesHm o o m o o o H o o o mH emmH :cmanHz ccncsHs H o H o o o o o m 0 OH smmH scmHscH: ccncsHm a H H H o o o H o O on cmmH mpcsnm Hancz ccncsHH m o H o o o o H o o o ommH nomeHnmmz smfloqsmm o o H o H o o o o o e cmmH «Honcsst cchsHs m m s o m o H o o o o cmmH smmHscHz concha m H o o o o o o o o mH cmmH chHmH cpncz echequ m H m m m o H H o 0 AH cmmH HcHHn> nc>Hm ecm echegHs m _ a m n _ m w.: hnmam Ham on S . . . mHHm a. r r . . r Hmmw nzohw p . > n .Hmim m 1%.m m m wmwm 3 ep 1 M "EbWbHS PYHE M P HUSHd M HS A 20. Apsmo Hem same as Ucmmmhmxmv mHOHmoHOHa Mo HOHPMHomH .>H mHQmB mined only for those isolates employed in the utili- zation studies. The following genera, not listed in Table IV, were identified on plated kernels; Clado- sporium, Aspergillus, Cephalothecium, Curvullaria, Rhizopus, Chaetomium, Phoma, Nigrospora, Stemphylium, Monilia, Botrytis, Verticillium, Helicostylum, Gonato- botrys, Actinomycetes, and white yeasts. The "pink yeasts" listed in the table were species of Sporobolo- myces and Rhodotorula. The ”yellow bacteria" were tentatively identified as Pseudomonas trifolii Huss. The "white bacteria" were not identified, nor was the identity of the smuts or Actinomycetes established. Plating checks were made to determine the efficacy of the surface-sterilizing solution. These unsterilized kernels frequently showed a completely different dis- tribution of surface microflora than did sterilized kernels from the same barley sample. Penicillium, fig— pergillus, bacteria, and members of the Mucorales ap~ peared in much greater numbers on unsterilized kernels. These organisms are the ”storage fungi" mentioned earlier (6, 7) which apparently contact the kernels even under approved storage conditions. The chemical composition of the ungerminated barley kernel The results of a survey of the literature referring to the chemical make-up of the barley kernel are pre- sented in Table V. Since the data gathered from these 21. 000.0 IV 0 ”H00 Ital-[all- sco o no.0 IIIII cWmecwwwchm . . . mcpw m0 0 m.m 0.0 I 0.H "msHmHHHaoo m m mm00.0 mm00.0 lllll p m mooo.o moo.o IIIII chanaH dH00.0 mm00.0 IIIII aH>mHHonHm 0H00.0 $00.0 IIIII QHKOUHHHA #0000.0 H000.w 0H 0 IIIII mo 0 6H08 OHQHvoon :HPOHm mome UHo¢ OHQHoom< mmo.0 mH.0 IIIII "mQHwHHaaoo mQHampH> >m0.0 000.0 m00.0 I #:0 0 . mmood no.0 #w0.0 000.0 I 000 0 mmoprm 30.0 000.0 HH.0 I 000 0 mmopossm HH.0 mnm.0 Hm.0 I am 0 mmopoSHHHooofiHw mm.0 00.0 mm.0 I dm.0 mmousmsm ms 0 NH.H MH.H I HH H mmoHoSm «mSHmHHmaoo No.0 m.m m.m I H.m nncmsm ccnm 0m.m mo.m m.» I m.¢ mmOHsHHmo m.H 0.m .m I m.m QHsmHH o.Hm m.mm mo I as scnspm 0.: NH 0H I OH AUmHopm mmv depmHoE H pgmHmR “Xv osHm> sac: ARV omsmm pamdepmsoo HmQHmH mmHHmp uopmsHEHmmsd Sm mo QOHpHmomaoc HHOHEcQO .> mHQma 22. 0H00.0 00.0 00.H 00.0 0H.0 00.m 00.m $H0.0 0H0.0 HH0.0 000.0 000.0 0H.0 00.0 00.0 0$.0 00.H 0$H.0 00$.0 000.0 $00.0 00$.0 $$0.0 50.0 0H.H 00H.0 H0.0 000.H $0.0 'Ill' mm 000m HOOO O IN A.onoV .> |o~® 0 oQHsmH< "mmwflflmpmoo AcHQSHomQHm QHHoPSHw AchmHH v :chhom HnHHscchv sHpmccm AflHademv mHmooSoH “mfiHmHHmEoo Ammamssm mmczHosHv QHmHOHm Hausdoaaoo OHQmonSHHSHV mfidmovnmm .sN .50 .sz .H< .cm “Ho mmomHP msHm cHom OHHSHH30 mcHNo OHHHmm msHHOHao mEHH muom mHmeme gmmpom UHom OHOHHHm mcHom OHHonmmoflm "msHmHHmEoo Azmmv mHmHoflHz wHom OHmHodz GHHHQQHom fianHomH :Hpmmm Hoppms mHanmHsOammab cHow OHsmHosHH cHom OHHosHH cHom oHmHo mHQmB 23. |1l ll l..l II \F fh a .h\ 0 0000.0 0000.0 0000.0 0000.0 0000.0 00000.0 H000.0 H000.0 H000.0 0000.0 0H00.0 0H00.0 0000.0 0000.0 .808 PM mHmmnpszm sHmPon Ho mQOHpommH hHm8HHH mHmmgpsHm aHopon meHQ .808 0$I00 mm noxmp HmmHoH mmHHm "Ho mpfldoEm HHm8m msHmHHm8oo o IIIII 0000.0 0000.0 0000.0 0H00.0 0000.0 00000.0 0000.0 0000.0 0000.0 0000.0 000.0 IIII lllll lllll IIII ''''' """" ''''' IIII 0 A.psoov .> meme 8H umbHobnw an op 0m8dmm< .HO.H Hwflpfimmmm mar OP H3089. n msHHHcs assHa Ho HHchz mHHo Hmesmmmm mem8mHm aname HNM mdomsmHHmomHz 0 mfiHmmHmmm< 0 mQHHmm msHHm> msHmloa qusomHQa msHHon mSHsmHmHmficflm mnHmmH msHode czHosmHomH mchHpmHm msHoHHw cHom OHSmpus msHpmmo wHom OHPHmmm< manHOH< ech cHnapsnnccHsHIx 0 0 24. articles was expressed in various quantitative units it was necessary to reduce all information to some uniform system of measurement. All quantities were therefore calculated on the basis of dry weight in milligrams. A standard malting variety barley kernel was chosen which would weigh between thirty—five to forty milligrams and this "standard kernel" served as the basis for numerical calculations. The constit- uents are listed in the table with their respective percentage ranges of total kernel weight, their mean percentages, and the individual compound weights in milligrams. Many of the compounds known to exist in the kernel have not been assigned percentage or weight values because of the difficulty in obtaining accurate quantitative measurements. Plate II shows a diagrammatic representation of a barley caryopsis and the locations in which the major chemical constituents are known to occur in com— paratively large amounts. This information is neces- sarily incomplete since histochemical investigations have not kept pace with other chemical studies of barley. Utilization studies The results of the utilization studies are Sum- marized in Tables VI (a) through (n). Table VII lists the growth of all isolates on the barley medium which served as the growth standard. 25. Plate II. Structure - composition of the barley kernel Compound Sucrose Glucose Raffinose Protein Fats Minerals Cellulose Pigments Tannin Lignin Suberin Resins Hemicelluloses Starch Enzymes Vitamins Gums { .J . 9. - ‘0 ‘ U U ‘0 ‘ ‘0 . ‘0 mwwm>>0P>w>>>>bflN Location C. D. E, F, G, H, I, J, K D, E, K (LINE. F, G, H, I,J,K Plate 11. Structure - Composition of the barley kernel (longitudinal section) Hull (A) ( Pericarp (B) '/ Testa (C) A 4 1 Area below aleurone (F) Outer endosperm (G) Aleurone layer (E) Aleurone Perisperm - ’ ”H layer (B) éééé?\\ ~Inner endosperm (H) Scutellar epith- elium (I) Scutellum (J) Embryo (K) Only locations of larger quantities of compounds are shown. 26. Table VI a. Amylose (initial pH - 7.3) Mean utilization of substrate (Carbon) Amount pH of the Color Appearance Isolate media after of of media .no. fungus after growth 01_ co A—7 3 Gray—black no change E; A-9 3 Gray-black pink-cloudy f3 A-lO 4 Tan-gray cloudy £3 A—l3 5 Brown-gray no change ’4 B—D 3 Black no change {i C—2 3 Gray—black no change U) E A-l 4 Black no change 1 g A-ll 4 Black no change 1 if C-9 4 Black no change 1 ii C-l6 4 Black no change 1 .8 13-1 4.5 Gray-black cloudy TE) E-l4 A Black no change 0 A-2 4 Pink cloudy . A-3 4 Pink cloudy E A-8 4 Pink cloudy S Ar14(1) 4 Pink cloudy I: A-l4(2) 4 Pink cloudy (U - a o g A-l4( 5) 4. 5 White-pink cloudy All isolates formed as many bead-like colonies. Table VI. b. Mean utilization of substrate (Carbon) Cellulose (initial pH - 7.4) Isolate Amount pH of Color Appearance no. of media after of of media Growth growth fungus after growth A-7 3 7.6 gray-white no change A A-9 4 7.7 gray pink m A-lO 4.5 7.9 gray—white no change .3 A913 4 7.? white no change g B_D 3 7.7 gray-white no change 5 C-2 3.5 7.7 gray-white no change :3 («q o‘. m A-l 4 7.5 gray—black no change ,é A-ll 4 7.7 gray—black no change 8 C-9 3.5 7.6 gray-black no change g C-l6 4 7.6 gray-black no change ii D-l 3 7.5 gray-white no change f3 E—14 4 7,5 gray-black no Change .5 CD A-2 l 1 7.4 white—red no change A-3 1 7.4 white-red no change ° ' —red no change g A-8 l 7.3 white m A-l4( 1) 1 7 .4 white—red no change hite-red no change ,g A-14(2) l 7. W . 0 Chan 8 H A-l4(3) l 7.5 white-red n g U) :3 _.__ a, 1 No growth of fungus after agar plug material was used. 28. Table VI. 0. Mean utilization of substrate (Carbon) Sucrose (initial pH - 7.4) Isolate Amount pH of Color Appearance no. of media after of of media Growth growth fungus after growth . A-7 4 7 4 black no change a. m A-9 4 7 6 brown-black pink .3 A-lO 4 7 7 white-gray no change g A-l3 4 7 8 brown-gray no change 3*, B-d 4 7 7 brown-black no change :1 C-2 4 7.8 black-white no change < d. U} A-l 4 6.9 black no change .§ A-ll 3.5 7.6 black no change g C-9 4 7.5 black no change 8 C-16 4 7.7 black no change i3 D-l 1+ 7.3 gray-black no change Q 'g E—l4 4 7.6 black no change H :13 A-2 4 7.8 pink cloudy A-3 3 7.5 pink CiOUdY E A—8 3 7.3 pink cloudy g A-14(1) 3 7.6 pink no change I: A-l4(2) 5 7.4 pink Cloudy E A-l4(3) 4 7.4 pink yellowed 29. Table VI. d. Mean utilization of substrate (Carbon) Raffinose (initial pH - 8.0) Isolate Amount pH of Color Appearance no. of media after of of media growth growth fungus after growth 5 .A-7 3 4.9 black no change m .A-9 3 7.9 black no change ~§ .A—lO 4 8.0 white—gray pink g3 A—13 4 7.9 brown yellowed é: B—D 5 7.9 black no change 4.) :3 C-2 3.5 7.9 brown—white no change 54 ‘0 A—l 3 7.9 black no change 1 g A-ll 3 8.0 black no change a C-9 3 7.9 black no change 3 C-l6 3 7.9 black no change i3 D-l 2.5 7.8 black no change :3 E-l4 3 7.9 black no change .5 (D A-2 4 7.8 pink cloudy 2 A-3 5 6.9 pink cloudy ' - loud % A-8 4 7.5 pink 0 y g A-l4(l) 4 7.0 pink cloudy - ° , loud g A-l4(2) 4 7.5 pink 0 dY 3 A-14(3) 4 8.4 plnk clou y Many colonies as small beads Loose hyphal strands dispersed through media. 50. Table VI. e. Mean utilization of substrate (Nitrogen) L—Asparagine (initial pH - 7.3) Isolate Amount pH of Color Appearance no. of media after of of media growth growth fungus after growth 54 A-7 4.5 6.5 brown—black no change m A-9 4 7.0 black yellowed E A-lO 4 6.2 brown—white no change g A-13 4 6.4 black pink E B-D 4.5 6.4 brown-white yellowed Q C-2 5 7.2 brown-white no change 5?; 5 A-l 4 7.4 black-gray no change 2 A-ll 4 7.3 black—white no change 84 C-9 4 7.1 black no change g C-16 4.5 7.5 gray—black no change 2 D-l 4 7.9 gray—black no change E E-l4 3.5 7.1 black-white no change (1) A-2 4 7.1 pink cloudy l , A-S 4 7.0 pink cloudy $‘ A-8 4 6.6 pink cloudy a A-l4(l) 4 7.2 pink clear 2 A-l4(2) 4.5 7.1 pink cloudy g A-l4(3) 4 6.5 pink cloudy &. All media with yellow tinge. 31. Table VI. f Mean utilization of substrate Carbon and Nitrogen I-Asparagine (initial pH - 7.3) Isolate Amount pH of Color Appearance no. of media after of of media growth growth fungus after growth f§ A-7 1 8.1 black no change Cg A-9 1 7.9 black no change E; A-10 l 8.3 white no change if A-l3 l 8.2 brown no change :3 B-D l 8.0 brown—white no change H 4: C-2 1 8.2 gray no change Ag.‘ U) a A-l l 8.2 black no change {1 A-ll l 7.9 black no change E; C-9 1 7.9 black no change is C-l6 l 7.7 black no change :3 D-l l 7.8 brown—white no change 2; E-l4 l 7.7 black no change 73 a: A-2 4 8.7 white no change A-3 3.5 8.8 white no change . - ' cloud 3* A-8 3,5 8.7 white y g Arl4(l) 4.5 8.9 white no change - ° l ud g Arl4(2) 4 8.8 white C O hy e ‘g Ayl4(3) 3.5 8.8 white no 0 ang 32. Table VI. g. Mean utilization of substrate (Nitrogen) L-Glutamic acid (initial pH - 4.1) Isolate Amount pH of Color Appearance no. of media after of of media growth growth fungus after growth g§ A-7 4 4.3 black-white no change G3 A—9 4 4.2 white—brown no change I: A—lO 4 4.5 white no change e E3 A~l3 3.5 4.4 brown-white no change :3 B-D 4 4.8 white-brown no change :2 C-2 3.5 5.4 white—brown no change 3‘ 5 Ari 1+ 4.2 gray no change {i A-ll 4 .2 gray no change 3 C-9 3 . 5 . 2 gray no change 3 C—l6 3 4. gray no change :E ILJ_ 3 4.2 white no change ;§ EpflA. 3.5 in gray no change .2 A—2 3 5.9 pink yellowed l A-3 4 6.1 pink pink l 3 A48 4 5.6 pink yellowed 5 Ayl4(l) 4.5 5.6 orange yellowed .3 Ael4(2) 5 6.0 orange yellowed F2 Arl4(3) 5 5.3 yellow yellowed l Isolates gelled media. 33. ‘ Table VI. h. Mean utilization of substrate Carbon and Nitrogen L—Glutamic acid (initial pH - 4.1) Isolate Amount pH of Color Appearance no. of media after of of media growth growth fungus after growth 5. A-7 4 8.1 brown no change 1 m A-9 4 7.3 brown yellowed .2 A—lO 4 8.3 brown yellowed g A-l3 4 8.1 brown yellowed 8 B-D 4 7.9 brown no change 33 C-2 4 8.0 brown yellowed :2 m A-l 2.5 4.6 black no change .3 A-ll 2.5 4.2 black no change Q C-9 2.5 4.1 gray no change g C-16 2.5 4.1 gray no change :fi D—l 2.5 4.2 gray no change '2 E_14 2.5 4.2 black no change 3:: A-2 3 8.5 pink cloudy 2 . A-3 4 8.6 pink cloudy % A-8 3 8 . 6 pink cloudy S .A-l4(l) 4 8.3 pink cloudy g A-l4(2) 4 8.5 pink cloudy é A-l4(3) 4 8.6 pink Cloudy All isolates as beads. H Heavy sporulation as sediment in flask bottom. 34. Table VI. 1. Mean utilization of substrate Nitrogen Edestin (initial pH - 7.5) Isolate Amount pH of Color Appearance no. of media after of of media growth growth fungus after growth ;; A-7 4.5 7.2 black yellowed (a A—9 6.5 7.0 black yellowed E3 A—lO 5 7.1 brown-black yellowed g A—l3 4 7.2 black no change 3; B—D 4 7.3 black yellowed :3 C-2 4.5 7.1 black yellowed U) A-l 4 7.3 black no change 1 E A—ll 4 7.3 black no change i: C—9 4 7.4 black no change :5; 0'16 5 7.3 black no change fl D-l 3 7.3 gray-black cloudy E ZE-l4 5 7,} black no change 23 :1 A-2 4 7.0 pink no change . A-3 4 7.4 pink yellowed 3* A—8 4.5 7.3 pink pink g .A-l4(l) 4.5 6.9 pink pink '3 A-14(2) 4 6.7 pink Pink g ‘A-l4(3) 4 6.7 pink cloudy All colonies as beads. 55. Table VI. 3. Mean utilization of substrate Carbon and Nitrogen Edestin (initial pH — 7.5) Isolate Amount pH of Color Appearance no. of media after of of media growth growth fungus after growth . A-7 2.5 7.9 black no change 5? A-9 3 7.8 brown—black no change .33 A—lO 2.5 7.8 black no change E: A—l3 3 8.2 black no change :2 B-D 3 7.8 brown no change 23 C-2 3 8.1 black no change '3‘; A—l 3 7.7 black no change :3: A—ll 3 7.8 black no change é; C-9 3 8.0 black no change El C-l6 3 7.9 black no change :E ILJ_ 2.5 7.9 brown no change E§ E—l4 3 7.8 black no change (1) £1: A-2 3.5 8.0 pink yellowed l A-3 4 8,3 pink yellowed :3. A-8 3.5 8.2 pink yellowed U) A-l4(l) 4 8.4 pink yellowed 2 A—l4(2) 3 8.0 pink yellowed § Arl4(3) 2.5 7.8 pink yellowed r3 1 All Fusarium media-cloudy. 36. Table VI. k. Mean utilization of substrate Nitrogen L—Aspartic acid (initial pH - 6.4) Isolate Amount pH of Color Appearance no. of media after of of media growth growth fungus after growth 34 A-7 4.5 6.6 black pink m A-9 5 7.1 black pink 'g A—lO 5 7.0 brown—gray pink a A-l3 5 6.9 black yellowed 3 B-D 6.5 7.1 brown pink H < C-2 4.5 7.1 gray-brown no change A U) 5 A-l 3.5 6.8 black no change '2 A-ll 4 6.7 black-gray no change a C—9 3 6.6 black no change ,§ C—l6 4 6.7 black no change E D-l 5 7.0 green—gray no change '5 E-l4 6 7.0 black no change 3 t1: A-2 8 orange cloudy A-3 7 orange cloudy . cloud 3* A-8 8 orange y 5 A-l4(l) 8 pink cloudy 'g A—l4(2) 8 yellow Cloudy 3 A-l4(3) 8 yellow cloudy Table VI. 1. Mean Utilization of substrate Carbon and Nitrogen L—Aspartic acid (initial pH - 6.4) Isolate Amount pH of Color Appearance no. of media after of of media growth growth fungus after growth :i A—7 2 6.6 brown no change :: A-9 2 6.6 black no change I: A-lO 2.5 7.0 gray-black no change Si A-l3 2.5 7.0 black no change :3 B—D 2 6.7 black no change :2 C-2 2.5 6.8 gray no change a; A-l 2.5 6.8 black no change r5 A-ll 3 6.8 black no change 84 C-9 2 6.7 black no change 8 C-l6 2 6.7 black no change *2 D-l 2.5 6.7 gray no change '3 E-l4 2 6.7 black no change #4 fl A-2 2.5 8.2 pink cloudy l . A-3 3 8.2 pink cloudy 5; A-8 3 8.1 pink cloudy 5 A—l4(1) 3.5 8.1 pink cloudy g A-l4(2) 3.5 8.2 pink cloudy é A—l4(3) 3 8.1 pink cloudy Heavy sporulation as sediment on flask bottom. 38. Table VI. m. Mean utilization of substrate Carbon and Nitrogen DL-OC-Alanine (initial pH — 7.5) Isolate Amount pH of Color Appearance no. of media after of of media growth growth fungus after growth 5‘ A-7 3 7.9 red—black no change m A-9 2.5 8.0 black no change a '8 A-lO 2.5 8.1 gray no change g A-13 3 8.2 gray no change § B—D 2.5 7.9 gray no change 2 C-2 3 7.9 gray no change :3. U} 5 A-l 2.5 8.0 black no change '2 A—ll 2 7.9 black no change 8+ C—9 2.5 7.8 black no change g C-l6 2 7.9 black no change 'E D-l 2.5 7.8 gray no change Ei E-l4 2.5 7.9 black no change 0) :13 A-2 4 8.4 salmon no change A-3 4 8.3 salmon no change 3‘ A-8 4 8.3 salmon no change 5 A414(l) 4 8.4 salmon no change I: A-l4(2) 4 8.3 salmon no change g: A-l4(3) 4 8.3 pink cloudy 39. Table VI. n. Mean utilization of substrate Carbon and Nitrogen L-Proline (initial pH - 7.6) Isolate Amount pH of Color Appearance no. of media after of of media growth growth fungus after growth d A—7 3 7.6 black—brown no change : A-9 2.5 7.7 black—brown no change '3 A-lO 2.5 7.7 black-brown no change S A-l3 3 7.8 black-brown no change in _g B-D 2.5 7.8 black-brown no change 2 C—2 2.5 7.7 black—brown no change m A-l 2 7.8 black no change -§ A-ll 2.5 7.8 black no change a C—9 2 7.7 black no change 8 C—l6 2 7.8 black no change 2g D—l 2.5 7.8 gray—black no change 8 'g E-l4 2 7.7 black no change #1 CD A-2 4 8.3 pink cloudy l A-3 4 8.3 pink cloudy E‘ A-8 3.5 8.2 pink cloudy 5 A—l4(l) 3.5 8.3 pink cloudy .3 A-l4(2) 3 8.2 pink cloudy § A-l4( 3) 3 8. 3 pink cloudy m 1 Heavy sporulation as sediment on flask bottom. 40. Table VII. Utilization of Barley growth medium (initial pH - 6.2) Isolate Amount pH of Color Appearance no. of media after of of media growth growth fungus after growth 5‘ A—7 10 5.3 black ? m A-9 7 6.6 black ? '2 A-lO 8 5.6 white-gray ? S A-l3 10 6.2 gray-black ? E B—D 8 6.7 gray-black ? Q C-2 6 6.1 gray-black ? l 54 m A-l 10 6.4 black ? .3 A-11 9 6.4 black ? 8 c_9 7 6.1 black ? g C-l6 9 6.3 black ? fi D—l 9 5.9 gray—black ? '§ E-l4 9 6.6 black ? #1 £7 A—2 10 6.4 red 7 . 11,3 10 6.1 red ‘? 2* A-8 8 4.5 red ? g A-l4(l) 10 6.7 red ? ‘2 A-l4(2) 10 5.9 red ? ‘2 A-l4(3) 6 4,7 yellow-pink ? 2 1 Media gelled. 2 Contaminated. 41. Carbon sources Amylose (soluble starch): Amylose was added to the basal medium as the sole carbon source. Growth ranged from three to five with Helminthosporium and Fusarium growing slightly more than Alternaria. All isolates of Helminthosporium formed numerous bead—like colonies. (K—l-Cellulose (technical): Cellulose served as the only carbon source in the medium. Growth ranged from 3 to 4.5 for both Alternaria and Helminthosporium. All isolates of Fusarium failed to grow after exhaustion of the nutrients in the agar—plug inoculum. Sucrose (saccharose): Each isolate was grown with- out replication with sucrose as the only carbon source. Growth ranged from 3 to 4 with slightly better growth displayed by Alternaria and Helminthosporium. Raffinose (trisaccharide): Pusarigg utilized raf— finose better (4 to 5) than either Alternaria or Hglgin— thosoorium (2.5 to 4). The colonies of Helminthosporium were small and bead-like. The figsgrium colonies were composed of loose hyphal strands dispersed through the media. Nitrogen sources (with sucrose added) L—Asparagine: This acid amide was substituted for sodium nitrate as the nitrogen source and growth was greater for all isolates (3.5 to 5) than the nitrate nitrogen. All isolates of Fusarium gave a yellow color 42. to the media. L-Glutamic acid: This amino acid was utilized by all isolates and gave growth rates approximately equal to that of the nitrate nitrogen. A considerable pH shift (1 to 2 pH units) towards alkalinity was made by all isolates of Eusarium. Three isolates of Eggggigm and one isolate of Alternaria which also showed an alkaline pH shift gelled the media. Edestin (barley globulin): Edestin, as a nitrogen source, promoted growth with all isolates in excess of nitrate nitrogen. Alternaria grew especially well ranging from 4 to 6.5. The Helminthosporium colonies formed as beads. IfAspartic acid: Growth was good with both A1333- naria and Helminthosporium isolates (3.5 to 6.5). All isolates of Fusarium grew extremely well with growth measures of 7 to 8. The Fusarium media was cloudy and sporulation was moderately heavy. Carbon and nitrogen source (no additional carbon source) L-Asparagine: No growth was obtained with the Alternaria and Helminthosporium isolates. All Fusarium isolates grew moderately well (3.5 to 4.5). All isolates showed a pH shift towards alkalinity of from 0.4 to 1.6 pH units. L-Glutamic acid: Moderate growth was made by Alternaria and Fusarium cultures (3 to 4) while Helmin- thosporium showed only slight growth (2.5). Alternaria 43. colonies were numerous and bead—like and the pH of the Alternaria media was raised from 3.2 to 4.2 units. A pH increase of from 4.2 to 4.5 units was made by the Fusaria. No significant pH rise was noted for Helmin- thosporium. A dense deposition of spores on the bottom of the flask was made by the Eggggggm isolates. Edestin: Growth of all isolates was reduced as compared to the series with edestin serving as only a nitrogen source. All cultures ranged from 2.5 to 4 with Fusarium giving slightly better growth. Sporulation was stimulated in the Fusarium group and the media was slightly cloudy. L-Aspartic acid: The Alternaria and Helminthos- porium isolates gave only slight growth (2 to 3) while Fusarium growth was greater (2.5 to 3.5). Epsarium sporulation was heavy with spore-sediment deposited on the flask bottom and the cultures also showed a pH in- crease of about one and three-quarters of a pH unit. D—LdQAlanine: Alternaria and Helminthosporium growth was slight (2 to 3). Fusarium showed consistent growth of 4.0 with only a small pH increase. L-Proline: Growth was slight for Helminthospprium (2 to 2.5) and for Alternaria (2.5 to 3). Fusarium growth was fair (3 to 4) with a slight pH rise and pro- fuse spore deposition on the flask bottom. The barley medium was run with each of the above series and was inoculated with the various isolates 44. employed in the study. All isolates grew very well on this medium with growth values ranging from 7 to lO. Changes in the appearance of the media were impossible to detect because of the dense growth of the cultures. Pigmentation of the fungi was pronounced in all cases. 45. V. DISCUSSION AND CONCLUSIONS Isolation and identification of barley microflora A significant increase in the number of microflora present was noted for the 1957 barley crop as compared with the harvest of 1956. The high humidity which pre- vailed throughout the barley producing areas of the Midwest, not only aided head infection, but severe lodging was responsible for the invasion of soil—in— habiting fungi not normally found associated with the barley kernels. The majority of the genera identified on plated kernels has been reported previously in the literature. However, the writer could find no reference to Helicostylum sp. or Gonatobotrys sp., both of which were isolated in the course of this study. Various yeasts have been isolated by previous investigators from cereal grains, but a review of the literature revealed only one reference to the "pink yeasts" and that inves— tigation was conducted in Denmark (23). Many pink yeasts isolated from kernel platings in this study were subsequently identified as species of the "wild yeasts", Sporobolomyces and Rhodotorula. One barley sample con- sistently showed a higher percentage of Penicillium, Aspergillus, and BhizOpus species which, coupled with a germination of forty per cent, indicated a heat damaged barley sample. Green and kilned malt samples taken at various stages in the malting process were plated and showed a reduced microorganism population which was 46. similar in the number of species to the barley samples. The presence of certain viable graminicolous fungi on kilned malt demonstrates the high temperature resist- ance of these species since the kiln temperature often reaches 170° F. for four hours (42). Chemical composition of the barley kernel The compilation of chemical compounds which comprise the barley kernel is far from a complete one. Qualita- tive and quantitative determinations of the carbohydrates, proteins, amino acids, minerals, and fats need to be com- pleted. In addition to the incomplete information avail- able concerning these constituents, the pentosans and vitamin contents of barley remain ill—defined. Little is known about the relative proportions of kernel constit- uents in relation to anatomical position within the grain. Further analysis is needed to investigate the precise chemical composition of the barley kernel before the bio- chemical changes induced in a barley grain by its as— sociated microflora can be fully determined. Utilization studies The major constituent of the barley kernel is starch which occupies approximately seventy-five per cent of the volume and comprises about fifty per cent of the dry weight. Starch was utilized by all isolates in a manner comparable to that of sucrose. This amylytic ability 47. could conceivably confer either a harmful or a bene- ficial effect in the malting process, depending upon whether the starch hydrolysis products were fermentable sugars or deleterious synthetic products. The utilization of cellulose and the pentosans is requisite for the penetration of the microorganisms into the interior of the kernel. In addition to cell wall dissolution various breakdown products might, as in the case of starch, impart beneficial or undesirable qual— ities to the barley in malting. The inability of the Fusarium isolates to utilize cellulose in this study in- dicates further investigation in the light of previous investigations (35). Possible explanations might in- clude a loss of cellulytic ability after prolonged growth on nutrient media, the necessity for additional growth factors, co—enzymes, or co-factors. The possible inability for certain species or strains of Fusarium to hydrolize cellulose must not be overlooked. The free sugars are found in greatest abundance in the barley embryo. Since all isolates tested were able to assimilate both sucrose and raffinose, the most fre- quently ocurring sugars, two possible effects upon malting quality are indicated if the fungus penetrates as far as the germ. First, germination may be inhibited by an up— setting of the necessary sugar balance and second, as before, metabolic products produced by the fungus might produce desirable or unwanted effects. 48. Protein is found throughout the kernel in both active protoplasm and stored forms. The globulin, edestin, is capable of providing both carbon and nit- rogen for the isolates tested. Speculations as to the effects on malting quality rendered by the utilization of edestin by these fungi must take into account the formation of fungal products, the enhanced manner of penetration, dissolution of the matrix which contains the starch granules, and effects on enzyme production by the kernel. In addition, the conversion of insoluble protein to soluble products and the stability of the wort in the mashing process may be affected by fungal proteolytic enzymes. The acid amide, L-asparagine, is found largely in the barley embryo and is known to be essential for plant protein synthesis. The compound was utilized by all iso- lates as a nitrogen source and only by the Epsarium isolates as a combined carbon and nitrogen source. By comparison L-aspartic acid, which has one less amino group than its acid amide, supported growth of all iso— lates as both a nitrogen source and as a combination carbon-nitrogen source. The nonutilization of aspara- gine, as the sole carbon—nitrogen source, by Helmintho- sporium and Fusarium isolates and growth by all isolates on aspartic acid under similar experimental conditions is tentatively explained in terms of the presence of the ad- ditional amino group. Apparently this radical exerts an 49. adverse influence on the carbon or nitrogen availability perhaps merely by a positional effect. The reaction of the media would not seem to be responsible for the growth differences since both initial and final pH readings were well within the growth range of all isolates (17). The Fusaria were stimulated to sporulate heavily when grown on aspartic acid without additional carbon being supplied. The spores were deposited on the bottom of the flasks as a visible, heavy sediment layer. This stimulatory effect on spore production was noted also with all Fusarium colonies grown on L—proline and L-glutamic acid, each serving as the complete carbon and nitrogen source, but not with L—glutamic acid when additional carbon was sup- plied. Dqu—Alanine as a combined carbon—nitrogen source did not stimulate Epggglgm sporulation. It is postulated that this increased sporulation is brought about by greatly decreasing the carbon—nitrogen ratio since the same amino acids did not induce this effect when sucrose was added to the media in relatively large quantities (30 grams/liter). The increase in sporulation was not observed in either the Alternaria or Helminthosporium series which sporulated sporadically and never in large amounts on the synthetic media. The shifting of pH by the Fusarium cultures towards alkalinity occurred fre— quently; this change in medium reaction has been ex- plained by a number of workers (l4, 18) who attribute the shift to a building up of ammonia from more complex 50. nitrogen compounds in the absence of sufficient carbon compounds. Presumably the carbon-containing substances, when present, combine with the ammonia produced and hence prevent these radical pH changes. The barley media promoted excellent growth of all isolates with more intense pigmentation of the cultures and heavier sporulation being the only other observable phenomena. The fungal pigmentation and media color changes were recorded for various reasons. One labora— tory procedure employed in malt quality evaluation is the color of the malt extract. The pigments of the fungi and of the synthesized compounds by them might, in all pro- bability, promote radical color changes in the extract and consequently influence quality evaluation. On the basis of the results obtained in this study further studies are indicated in certain major areas. Histological investigations of stained kernels would be of great value in assessing the nutritional relationship between microorganism and host in £313. Chemical com— position studies of grain before, during, and after fungal or bacterial establishment has taxen place would further clarify the effects of such a relationship. More complete utilization studies, employing other abundant constituents would amplify the results already presented. Especially valuable would be a synthetic "barley medium” which would approach the natural substrate in nutritional quality but would be reproducible in a standard manner. 51. Finally the Fusarium sporulation phenomenon discussed above should be run not only with other amino acids, but with varying levels of additional carbon supplied, from O to 30 grams/liter, in order to test the hypo- thesis stated previously. 52. Summary The preceding results are summarized below, cog- nizant of the restricted nature of the investigation when contrasted with the complexity of any biological relationship. 1. In all "weathered" kernels Alternaria spp. were predominant with Helminthosporium spp. and Fusarium sp‘. occurring in lesser abundance. LP 0 Some microflora not previously reported on barley in the United States were identified. These in- cluded the pink yeasts, Ehodotorula sp. and §po§g— bolomyces sp., and the filamentous fungi, Helicos- £112m_sp. and Gonatobotrys sp. Qualitative, quantitative, and locational chemical composition information concerning the barley kernel constituents were obtained from the litera— ture. This data provided the basis for the as— similation studies. The carbon sources tested were; amylose, sucrose, raffinose, cellulose, L—asparagine, L—glutamic acid, edestin, L-aspartic acid, L-proline and DL-KL alanine. Alternaria and Helminthosporium isolates utilized all carbon sources except L-asparagine. Fusarium isolates used all carbon sources except cellulose. The nitrogen sources tested were; L—asparagine, 53. L—glutamic acid, edestin, L—aspartic acid, L-proline, and DL-x—alanine. These nitrogen sources supported growth with all isolates of Alternaria, Helminthos- porium, and Eugarigm. Abundant sporulation was induced in the Egsarggg cultures by all amino acids except DLax-alanine when no additional carbon source was provided. It is postulated that this effect was due to a low carbon- nitrOgen ratio. Cultures of Fusarium when grown in low carbon media showed pronounced pH shifts toward alkalinity sup- porting the observations of previous investigators. The enzymatic abilities and growth phenomena of the isolates tested are indications that malting quality may be affected, either adversely or beneficially by the microflora of stained barley kernels. Further investigations are indicated by this study, namely histological studies of stained kernels, further utilization studies with synthetic ”barley" media, more complete barley chemical studies, and biochemical analyses of the synthetic products of associated barley microorganisms. 54. 10. 11. 12. 13. Literature cited Anderson, J. A. and A. W. Alcock (edited by). 1954. Storage of cereal grains and their products. American association of Cereal Chemists. St. Paul, Minnesota. 515 p. illus. Blum, P. H. 1954. Grain Spoilage: a review. Wall. Bolley, H. L. 1915. Wheat: Soil Troubles and Seed Deterioration. N. Dak. Agr. Exp. Sta. Bull. 107. Brown, W. and A. S. Horne. 1924. Studies of the genus Fusarium. I. General account. Ann. Bot. Lond. 58: 579-585. Brown, W. 1925. Studies of the genus Egsarium. II. An analysis of factors which determine the growth forms of certain strains. Ann. Bot. Lond. 59: 575—408. Christensen, C. M. 1956. Deterioration of stored grains by molds. Wall. Lab. Comm. 64: 51—46. Christensen, C. M. 1957. Deterioration of stored grains by fungi. Bot. Rev. 25: 108-154. Christensen, J. J. 1922. Studies on the parasitism of Helminthosporium sativum. Minn. Agr. Exp. Sta. Tech. bull. 11: 1—52. Christensen, J. J. and D. C. Stakman. 1955. Re— lation of Pusarium and Helminthosporium in barley seed to seedling blight and yield. Phytopathology 25: 509—327. Drechsler, C. 1925. Some graminicolous species of Helminthosporium. J. Agr. Res. 24: 641—759. Dfiggeli, H. 1904. Die Bakterienflora gesunder Samen und daraus gezogener Keimpflanzchen. Zentr. Bakt. Parasitenk. lnfekt. Abt. 12: 602-614; 15: 56-65, 198-207. Elliott, E. S. 1949. Effects of vitamins on growth of some graminicolous species of Helminthosporium and Fusarium. Pro. W. Va. Acad. Sci. 20, 9-11: 65-68. Foster, J. W. 1959. The heavy metal nutrition of fungi. Bot. Rev. 5: 207—259. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. Foster, J. W. 1949. Chemical activities of fungi. Academic Press Inc., New York, 648 p., illus. Gottlieb, D. 1946. The utilization of amino acids as a source of carbon by fungi. Arch. Biochem. 9: 341-551. Greaney, F. J. and J. E. Machacek. 1942. Prevalence of seed-borne fungi in cereals in certain seed- inspection districts of Canada. Sci. Agr. 22: 419—437. Hawker, L. E. 1950. Physiology of fungi. Univ. of London Press, London. 560 p., illus. Hawker, L. E. 1957. The physiology of reproduction in fungi. Cambridge Univ. Press, 128 p. Hopkins, R. H. and B. Krause. 1957. Biochemistry applied to malting and brewing. D. Van Nostrand Co., New York. 542 p., diag. James, N.,J. Wilson, and E. Stark. 1946. The micro- flora of stored wheat. Can. J. Res. C. 24: Lewis, B. W. 1957. Amino acid nutrition of Alter- naria solani. Phytopathology. 47: 121-125. Lilly, V. G. and H. L. Barnett. 1951. Physiology of the fungi. McGraw—Hill Book Co., New York, Toronto, and London. 464 p., illus. Lund, A. 1956. Yeasts in nature. Wall Lab. Comm. Machacek, J. E. and F. J. Greaney. 1958. The "black— point" or "kernel smudge" disease of cereals. Can. J. Res. 16 C: 84-115. Mead, H. W. 1955. Studies of methods for the iso— 1ation of fungi from wheat roots and kernels. Mead, H. W. 1942. Host—parasite relationships in a seed—borne disease of barley caused by Helmin— thosporium sativum Pammel, King, and Bakke. Can. J. Res. 20 C: 501-525. Mead, H. W. 1945. Seed—borne moulds of barley. Wall. Lab. Comm. 17: 26-52. Moore, E. S. 1924. The physiology of Fusarium coeru- leum. Ann. Bot., Lond. 149: 157—161. 56. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. Orton, C. R. 1951. Seed-borne parasites - a biblio- ggaphy. V. Va. Univ. Agr. Exp. Sta. Bull. 245, p. Peterson, E. A. and H. Katznelson. 1954. Studies on the nutrition of Helminthosporium sativum and certain related species. Can. J: Microbiol. 1: 190—197. Peterson, E. A. and H. Katznelson. 1956. The effect of trace elements on growth of Helminthosporium sativum and several related species. Can. J. Microbiol. 2: 441-446. Riker, A. J. and R. S. Riker. 1956. Introduction to research on plant diseases. Planographed by John S. Swift Co. Inc. St. Louis, Mo. Schultz, A. S. and S. Pomper. 1948. Amino acids as nitrogen source for the growth of yeasts. Arch. Biochem. 19: 184-192. Schultz, A. S., D. K. McHanus, and S. Pomper. 1949. Amino acids as carbon source for the growth of yeasts. Arch. Biochem. 22: 412—419. Siu, R. G. H. 1951. Microbial decomposition of cel- lulose with special reference to cotton textiles. Reinhold Publishing Corp., New York. Steinberg, R. A. 1959. Growth of fungi in synthetic nutrient solutions. 1. Bot. Rev. 5: 527-350. Steinberg, R. A. 1950. Growth of fungi in synthetic nutrient solutions. 11. Bot. Rev. 16: 208-228. Tandon, R. N. and J. P. Srivastava. 1950. Proceed- ings of the 57?11 Indian Science Congress, Poona, Section of Agric. Sci. Abstr. pp 56-59, 75—92. Thaysen, A. C. and H. J. Bunker. 1927. The micro— biology of cellulose, hemicelluloses pectin and gums.' Oxford Univ. Press, London. 565p. illus. Thaysen, A. C. and L. D. Galloway. 1950. The micro— biology of starch and sugars. Oxford univ. Press, London. 556pp. illus. Treggi, G. 1954. Sulla utilizzazione di alcuni aminoacidi da parte di funghi, fitopatogeni. Annali della sperimentazione agraria. N. b. i: 1955-1965 (with English summary). E. H. Schwaiger, H. G. Leonhardt, . *. Jr. Vogel, E d ’ The practical brewer; and J. A. Merten. 1946. 57. 43. 44. 45. 46. 47. a manual for the brewing industry. Master brewers Association of America, St. Louis. Mo. Weniger, W. 1925. Pathological morphology of durum wheat grains affected with ”black point". Phyto- pathology 15: 48-49. Whitehead, d. D. 1949. Studies on some seed—borne microorganisms of cereals. PhD thesis, Univ. of Wisconsin. Wolf, F. A. and F. T. Wolf. 1947. The fungi. Vol. II. John Wiley and Sons, Jew York. 558 pp. illus. Wolf, F. T. 1955. Nutrition and metabolism of the tobacco wilt Fusarium. Pull. Torr. Bot. Club. 82: 545—554. 25b1, A. 1892. Braunspitzige Gerste. Allgem. Brauer, HOpfen. Ztg. 106. 58. . r4. ‘2‘; .u 1 41 .I 1+1 14F Ilrl.lIIi|¢[lll A C TBKTY 11111 A- 7L1: £11 :1 I L 1 1r I] JrLIifi er\I ‘48} O 7 _L a -¢' ‘ (J ‘ __ ,' -4. [flgflfiY EL‘t Staff :3" 'l ..fl 2 T S w. ##1gflfid' “#1 L 1; La c» ‘1! .l‘ 1 115.1 “fl?“ RUU: 1;. Lhunco-293 HICHIGQN STRTE UNIV. LIBRRRIES 11 1111111111 11111 31293101301574