MI I? l I IIIHWI \ MI I l ‘ 126 660 HTHS smmss ON ma mmsmsm 0? mm xv ms FUNGUS figggemmcsmgem smww Thasts Ear {‘52 Degree of M. S. MICHIGAN HATE UNWERSETY Harvey Wesiey Spun, Jr. 1958 Tifit’lb STUDIES ON THE PARASITISM OF PLAET BY THE FIMGUS ELIIIIWHOSPORIUE‘JI SATIVID-I By Harvey Wesley Spurr, Jr. A THESIS Submitted to the College of Science and Arts Michigan State University of Agriculture and Applied Science in partial ful- fillment of the requirements for the degree of MASTER OF SCIENCE Department of Botany and Plant Pathology ‘ 1958 ‘31.? ,2 -~ «J g QSV§A ACKIIOWIBDGIEJ NT S The author expresses appreciation to his committee: Dr. Donald J. de Zeeuw, Dr. Robert H. Fulton, and Dr. Richard L. Kiesling and to Joseph L. Clayton, agricul- tural technician. To a graduate student undertaking his first problem in scientific research, inspiration and insight play a vital role. This inspiration and insight were provided by my major professor Dr. Richard L. Kiesling. I am pleased to have been his student. *##***¢*#**** STUDIES ON THE PARASITISM OF PLANTS BY THE FUNGUS EBLKIKTHOSPORIUM SATIVUM By Harvey Wesley Spurr, Jr. Submitted to the College of Science and Arts Michigan State University of Agriculture and Applied Science in partial ful- fillment of the requirements for the degree of MASTER OF SCIEHCE Department of Botany and.Plant Pathology Year 1958 Approved ~ ABSTRACT Host range studies showed that gelminthosnorium sntivum Pam., King, and Bakke is capable of parasitizing a wide range of plant species outside of the grain and grass families. Several barley varieties were tested in three locations and showed no resistance to HL‘sativum. There was no correlation in the degree of susceptibility among these three barley nurseries. Smell grain and beans used in crop rotation increased the amount of infec- tion. Michelite 'can was shown to be a natural host of E; Sati‘jm. Sporulation of E. sativum on barley straw during the growing season is the main source of inoculum for kernel infection. Sporulrtion of H._srtivum on barley stra' appears where the straw is GXpOSGd to di- rect sunlight. Amylase, cellulase, and sucrase were present in acetone powder preparations of Hg atiyug. Barley plants infected with H, sativun A. show a higher amount of sucrase activity than plants not infected. The presence of alpha amylase, diastase, hemicellulase, and invertase separately correlates with the inhibition of germination of barley seeds. Culture filtrates from H, sativum cultures contained arylase, cellulase, and sucrase. The culture filtrate had a toxic effect on barley seeds (inhibited germination). The toxic effect correlated positively'with the presence of amylase and sucrase, and correlated negatively with the presence of cellulase. TABLE OF CO\-511 I. IL. .‘LnODUCTIOI‘T o o o o o o o o o o o o o o o o o o o a II. 141-..ququ UM FEVIEW o o o o o o o o o o o a o o o o 0 III. MATERIALS AND METHODS . . . . . . . . . . . . . . . Host ange Studies . . . . . . . . . . . . . . . Etiological Studies . . . . . . . . . . . . . . . 1. Reaction of Several Barley Varieties to Yatural Infectio . . . . . . . . . . . . . 2. The Natural Infection of Michelite Bean . . 3. Air-borne Inoculum in Relation to I.‘ iiection of Barley in hichigan . . . . . . . . . . . Enzyme Studies . . . . . . . . . . . . . . . . . l. The Preperotion and Reaction of Acetone Powders from H. setivum . . . . . . . . . . The Production of Sucrsse duri 1ng Paresitism The Effect of Extra.Enzymes on Barley Seed Germination . . . . . . . . . . . . . . . . a. Effect of purified enzymes . . . . . b. Effect of amylase, cellulasc, and sucrase produced by H. sativum . . . (0 Kn) IV. R: STETS o O I O O O O O 0 I o o o o o o o o 0 o o o 0 Host Range Studies . . . . . . . . . . . . . . . . tiological Studies . . . . . . . . . . . . . . . 1. Reaction of Several Barley Varieties to Natural Infection . . . . . . . . . . . . . 2. The Fe.turel Infection of Hichelite Bean . . 3. Air-borne Inoculum in Rel? tion to Infection of Be ley in Iiichigan . . . . . . . . . . . Enzyme Studies . . . . . . . . . . . . . . . . . . l. The Preparation and Reaction of Acetone Powders from H. sotivum . . . . . . . . . . 2. The Production of Sucrase during Parasitism 3. The Effect of Extra Enzymes on Barley Seed Germination . . . . . . . . . . . . . . . . a. Effect of purified enzymes . . . . . . b. EIfect of amylase, cellulase, and sucrase produced by g._sativun . . . . V. DISCUSSION AND COKCLUSIONS . . . . . . . . . . . . . PAGE O‘ 10 12 1 J. 13 lb 1 .4. l7 l7 17 q .L 23 O L.’ ' T‘BLE OF COFTEETS - CONTIEUED Host Range Studies Etiological Studies 1. Reaction of Several Barley Varieties to Natural Infection 2. Air-borne Inoculum in Relation of Barley in Michigan Enzyme Studies vi to Infection 29 29 29 29 30 31 33 II III IV VI VII LIST OF TABLES Cron Rotation at the Three Locations Used to Test the Resistance to Helminthosnorium sctivum of Several Rq‘rlezr ‘farieties O O O O O O O O O I O O O O D O O O O . Varieties of Barley Grown in Nurseries at Three Loon, tions in Michigan to Test Their Reaction to Naturally Occurring Inoculum of Helminthosnorium setivum . . . . . Preparation of Acetone Powder from a Mycelial Mat of Helminthosnorium sativufl . . . . . . . . . . . . . . . . Results of the Host Range Study Showing the Plants Inoculated in Each Trial, the Aopearance of Lesions, and the Reisolation of Helminthosgorium sativum from theLesions ...................... Reaction of Barley Varieties to Natural Infection by Helminthosnorium sativum. Comparisons Are made among Locations of Nurseries, Varieties, and Infected Plant Parts. Sporulation on the Straw of Infected Plants at East Lansing Is Compared to Plant Infection. . . . . . . The Effect of Extra Enzymes on the Germination of Barley Data Recorded from Liquid Culture of H, setivum . . . . vii 00 ll 26 27 LIST OF PLATES PLATE PAGE I Infection of Four Plant Species by Artificial Inoculation with Spores of Helminthosnorium srtivum . . 19 II Sporulation of Helninthosnorium sativum on Barley S t 1.8", O O O O O O O O I O O O O O O O O O O O O O O O 20 III Snorulation of Helminthosnorium sativum on Barley Straw Stimulated by Sunlight . . . . . . . . . . . . . 22 GRAPH PAGE I Amylase, Cellulese, end Sucrase Activity of Dialyzed Culture Filtrates . . . . . . . . . . . . . . 23 II Toxic Effect of Culture Filtrates on Barley Seeds . . 28 I. INTRODUCTION The fungus Helminthosoorium sativum Pam., King, and Bakke has presented perplexing problems to plant pathologists since it was first discovered to be a cereal pathogen in 1910. Since then an enormous amount of research has been conducted and hundreds of papers have been published on parasitism by E. sativum. In all, a great deal of know- ledge concerning H. sativum has been uncovered, but no solution or partial solution has ever been found. The variability of H, sativum is one of the most important and hardest to understand aspects of this problem. In approaching the problem in this study, an enzyme theory of parasitism was pronosed. It was postulated that in order for a coni- dium of H. sativum to invade a plant, it must first germinate and then produce exoenzymes to aid invasion by breaking down the plant constitup cuts. The breakdown products are then used for food by the fungus a- long with other constituents of the plant. Infection results when the composition of the plant and the enzyme system of the fungus are compatible, assuming environmental factors are favorable to enzyme reactions. In view of the theory outlined, host range, etiological, and en— zyme studies were made and are reported in this thesis. II. LITERATURE REVIEW Helminthosnorium sativug_was first described as a fungus disease of barley in 1910 by Pammel, King, and Bahke (?2). Since this first description, much of the disease damage found in barley and in wheat can be attributed to H._sativum. The fungus attacks mainly the foot of grain plants (foot rot) but is also important in many areas as a leaf parasite (snot blotch) and a kernel blight. During the period from 1920 to 1930 an extensive amount of re- search was carried on in an effort to understand and control parasi- tism caused by H. sativum (3, h, 5, 6, 9). This research produced some hope of finding resistance to the disease through a grain breed- ing progr(n. Tgvse hopes rare div nished when in l95b Wood, Christen— sen, and Lamoert published a paper entitled, "Helminthosroriun srtivtm becomes destructive on hitherto resistant varieties of barley" (35). Extensive host range studies have been made on grain and grass varieties (1, 2, 11, 12, 16, 23, 26, 28, 29, 33, 3a). The only plants, other than grains and grasses, reported as having been para- sitized by Eiisativum are cotton, Opium poppy, pea cotyledons, and seeding flax (8, 13, 21, 32). Natural inoculum is known to be present in the soil and in the air (18, 20, 2h, 25, 27, 31). The importance of the inoculum in the air and in the soil in relation to infection of grain plants during their development is not fully understood. Recent research indicates that a toxin produced by H.ksativum predisposes plant tissue to infection. Ludwig (lb, 15) evaluated the toxin produced by means of a barley germination test and suggested that the toxin limits the growth of E. srtivum as IJCll as host plants. Ludwig postulates tint the toxin produced by the fungus affects sus— ceptible hosts by conditioning them to infection. Hess (13) found that certain culture filtrates of R. SPtivum had a harmful effect on the development of barley and that the fil- trates were inactivated by a chanite in pH or by being heated. Bar- legr proved to be an unsuitable host in these tests because its sus- L ceptibility was altered by even slight changes in the nutrient solu- tion and bv re arded development. Earhart (7) found a positive cor— relation between pa t.ogenicity of H. ,ntivum and toxin in the culture filtrate e.s evaluated on seedlings of Victorgrain #Bn93 and Southland oats. III. MATERIALS AED {ETHODS The experiments reported in this work were all made using one isolate (H. S. 101) of H. sativum that was a result of single spor— ing through six generations. Because of the large number of types of the fungus and the indefinite limits of strains, no other method of insuring stability or reproducibility in tests was practical. Host Range Studies To obtain a better understanding of the capability of E, sati- 33m as a parasite, inoculations of 2h plant species and varieties were made. The plants were selected to give a range of plant fami- lies as well as structural and compositional differences. The seeds (Table IV.) were planted in four inch pots in the greenhouse. When the plants had developed sufficiently (several well developed leaves) they were placed in a moist chamber and sprayed with a water spore suspension of glpsativum (H. S. 101). The inoculum was prepared by scraping spores from dried potato dex- trose agar (P.D.A.) Petri plates into sterile distilled water. The spore suspension was filtered through cheesecloth to remove mycelial fragments. After inoculation of the plants the humidity in the cham- ber was kept relatively high (70-90 per cent) for two days. After this incubation period, the plants were returned to normal greenhouse conditions. Uninoculated controls were included in all cases. In nine days the plants were examined for lesions and where lesions were found, reisolations were made to recover H. sativum and verify the cause. Three separate inoculation trials were made in this experiment differing only in the plants used and the reisolation techniques em— ployed. The reisolation techniques used are as follows: Trial I: A small section of tissue was removed from the edge of a lesion and dipped in a solution of one part commercial sodium hypochlorite (Chlorox) to, one part 95 per cent ethyl alcohol for one minute. The section of tissue was then placed on a sterile P.D.1. plate and incubated. Trial II: Leaves containing lesions were removed and washed in runs ning tap water for #8 hours. The leaves were then dipped in a 20 per cent commercial sodium hypochlorite (Chlorox) solution for fifteen seconds followed by a rinse in ster- ile water. Small sections of the tissue were removed from the edge of the lesions and placed on acidified P.D.A. (5 drops of lactic acid per zoo ml of 9.13.11.) and incubated. Trial III: Leaves containing lesions were removed from the plant. A 3/8 inch cork borer was used to remove a plug of tissue from the edge of a lesion. The plugs were placed in a Gooch crucible and dipped in a solution of 1 part 95 per cent ethyl alcohol, 1 part commercial sodium hypochlorite (Chlorox), 1 part distilled water for five seconds. The plugs were then placed on acidified P.D.A. and incubated. Etiological Studies 1. Reaction of Several Barley Varieties to Natural Infection In the spring of 1957 a barley nursery to study g._sativum dis- tribution was established in Michigan at three separate locations. Most of the varieties used in this study were resorted by the Canadi- ans to have some resistance to E, sativum (Table II). The three 10- cations, East Lansing, Standish, and Tuscola represent three different areas in Michigan where barley is grown. The soil practices on the sites chosen were good; the crop rotation is shown in Table I. The varieties were read for infection by g, sativum on culms, leaveS, lower nodes and upper nodes after the plants had headed. In addition, sporulation on the straw was also read in the East Lansing nursery. The data are shown in Table V. 2. The Natural Infection of Michelite Bean Eight leaves were selected at random from a field of Michelite beans adjacent to the barley nursery at Tuscola. This was done in an effort to correlate the pathogenicity of H. sativum in the greenp house to the field. Fifty-eight isolations were made of lesions on the leaves using the technique of Trial III. The field in which the beans were growing had the same cron rotation as the Tuscola barley nursery (Table I). 3. Air-borne Inoculum in Relation to Infection of Barley in Michigan TABLE I Crop Rotation at the Three Locations Used to Test the Resistance to Helminthqucrium srtivufl of Several Barley Varieties Year East Lansing Standish Tuscola 1957 Barley Kursery Berley Nursery Barley Nursery 1955 Soy Beans Corn Spring Barley (Erie) 1955 Corn Corn Navy Beans 195A Small Grain Hay Oat and Brrley Plots 1953 Summer Fallow Hev Navy Beans 1952 --- Hey --_ o a o 1- o _ ' o O O O Varieties of Berley Grown In Lurseries at ance Locations in Michigan to Test Their Reaction to Naturally Occurring Inocuium of flglmintggsnoriyg; saiiiieb now I Row II 187 1245 1L8 1907 691 - 1517 711 L5“? 731 5369 73 691 12b5 2276 1367 19, 1517 £979 1907 7269 b578 13 4979 51e5 7269 69¢? 5105 7-1 (of 4'7 V7 59 13-11 acL variety is planted in an eight-foot row. {11 aEach variety is listed according to its United States Department of Agriculture Cereal Investigrtion (6.1.) number. To investigate the disease cycle a study of air-borne inoculum in a field of barley was made. Soil and seed-borne inoculum of E; sativum account for most of the foot rot and seedling blight infec- tion which occurs in Michigan. However, the source of inoculum which gives rise to kernel blight had not been determined in Michigan. A twenty acre field of barley which included at one end the East Lansing barley nursery was chosen for the site of this study. Six microscope slides were placed vertically on stakes six inches above the ground. Each slide had a thin coat of vaseline and was placed on the side of the stake facing the prevailing wind. The stakes were spaced in the field to obtain a random sample of the foreign spores which might pass through the field as well as those produced in the field. The slides were changed periodically during the season. After the slides were removed from the field, a one inch square cover slip was placed on the vaseline in the center of each slide. The slides were then read under the low power of the microscone (10:) by passing across the cover slip three times at 5 mm intervals. The average number of spores per slide for each group of six slides for each period was recorded. The experiment ran from April 27 to August 26, 1957. Enzyme Studies These studies were undertaken to determine if amylase, cellulase, and sucrase were produced by E. sativum and to correlate the produc- 10 tion of these enzymes with the pathogenicity of the isolate and the effects of such parasitism on the host. Amylase, celTulase, and sup crass, as referred to in this thesis, include any or all of the enzymes which could be present and could hydrolyze soluble starch, alpha cellu- lose, and sucrose respectively. 1. The Preparation and Reaction of Acetone Powders from H. sativum To determine the presence of amylase, cellulase, and sucrase, acetone powders were made in the following way: three Fernbach flasks containing 200 ml of potato dextrose solution were inoculated with H. S. 101. The flasks were then incubated a week at 28° C. The my- celial mat which had formed was harvested and made into an acetone powder (Table III). The acetone powder was then frozen and used as needed for reactions. To test the acetone powder for amylase, cellu- lase, and sucrase, a portion of the powder was added to #0 m1 of glass distilled water. The solution was then placed in a cellophane membrane and dialyzed for twelve hours at l0 C. against one liter of glass dis— tilled water. After dialysis, the solution was centrifuged to remove debris (a few minutes in a small centrifuge is sufficient). The supernatent is poured off and added to a substrate to test for enzymes. This is accomplished by adding 2 ml of the enzyme solution to 10 ml of substrate (1 per cent wt./ vol. soluble starch, alpha cellulose or sucrose) and incubating the reaction for four hours at 28° C. The re- actions are best if run in 100 ml volumetric flasks and contamination may be excluded by adding a small layer of toluene. At the end of the 1. 3. 7. 11 TABLE III Preparation of Acetone Powder from a Mycclirl Mat of T- w‘--._ "I‘ ~ g L. nelmintnpspprium SEAthm —.- Filter off the mycelial mat from the liquid media using a suction filter without filter paper. Place the mat in 200 ml of sterile glass distilled water in a beaker and shake a few seconds to dissolve particles from the media which may be adhering to the mat. Filter off the met with suction. Repeat the process three imes. Place the mat in 200 ml of cold acetone for 24 hours. Filter off acetone by means of suction and suck air through mat for 20 seconds (if desired, mat may be washed with 200 ml of C.P. ethyl ether before sucking air through the mat). a ‘ 0 o O "V 1 Dry mat in vacuo for two days at l C. (sulfuric ac1a or some other drying agent may be placed in the bottom of the dessicator). Grind mat in mortar and pestle machine for a minimum of two hours, add sterile sea sand and glrss distilled water as needed to facili- tate grinding. This Operation should be carried out at l0 C. if possible. Pour ground mat into an a'rtight pyrex container and freeze. The ground mat is the acetone powder. Remove portions of the acetone powder as needed for enzyme reactions. 12 four hour reaction period, 2 ml of Somogyi's reagent (30) are added and the flasks are placed in boiling water. The boiling water stops enzyme reaction and speeds the reaction between Somogyi's reagent and reducing sugars. Flasks in which sucrose is used as a substrate are boiled 10 minutes, soluble starch and alpha cellulose flasks 20 minutes. The flasks are cooled and 2 ml of Nelson's reagent (19) are added to bring out a stable color. The final solution is diluted to 100 ml with distilled water. Checks of 2 ml of enzyme solution and 10 ml of substrate solution are made at the same time. After the re- action solutions heve been diluted to 1C0 ml, they are read in a Klett—Summerson colorineter. The readings of all solutions are re- corded and then converted to equivalent mg of glucose which would pro- duce the same color under the conditions of the reaction minus the check values. 2. The Production of Sucrase during Parasitism If amylase, cellulase, and sucrase are necessary for parasitism by H. sativum, then possibly infected plants would contain a greater amount of such enzymes as compared to noninfected plants. This idea was tested on Odessa barley (0.1. 93k) grown in the greenhouse. Half of the plants were inoculated with H. sativum and half remained as uninoculated checks. Two days later lesions had appeared on the inp oculated plants and leaves of infected and uninfected plants were harvested. Samples were taken from leaves which were fairly uniform in size by cutting out sections approximately 1/2 cm square. From 13 this material .2 g of infected tissue and .2 g of uninfected tissue were weighed out. Each weighed sample was then placed in a sterile 200 ml Erlenmeyer flask. After freezing the snnules for 24 hours, 30 ml of glass distilled water was Faded to each flask. The flasks were placed on a reciprocal shaker for 17 hours at 25° C. The supernatent was filtered off and used in these studies. Bach solution was di- alyzed and tested for sucrese activity as described in the preceding section except that 2 ml of l per cent sucrose and 2 ml of enzyme solution were reacted at 37° C. Checks were made of the enzyme solur tions and the substrates. Results are recorded as equivalent mg of glucose which would give the same color minus the check vclues. 3. The Effect of Extra Enzymes on Barley Seed Germination a. Effect of purified enzfmes The effect of enzymes on the germination o: barley seed was tested using the method described by Ludwig (lb, 15). The puroose of this test was to determine whether enz5nes would actually inhibit the germination of barley seeds. Twentyefive seeds of a bright Montcalm barley sample (0.1. 71%?) were placed in a test tube. 1.0 and .l per cent wt./vol. solutions of enzymes were made using glass distilled water as a solvent. Five ml of solution (enzyme preparetion or c lture filtrate) were added and the seeds were allowed to soak in this solution for A hours. At tle end of the t hour soelirg period, the seeds were placed on moist filter paper in a Petri dish and incubated 14 at 23° C. for 3 days. Each treatment was repeated six times. After three days the seeds which had germinated were counted and the percentage germination was computed. Checks were made using water and water covered with toluene because the enzyme solutions used were preserved under toluene. h. Effect of amylase, cellulase, and sucrase produced oy H3 sativum This experiment was designed to show whether H. sativum produces amylase, cellulase, and sucrase as enoenzymes in cul- ture and whether these enzymes are related to the toxicity of the culture filtrate to barley seeds in the germination test. Five 250 ml Erlenmeyer flasks containing 25 ml of sterile potato dextrose solution were inoculated with 1 ml of a Spore suspension of H. S. 131 and incubated at 230 0. Starting on the first day and each successive day for b days, the following data were recorded: 1. pH of media after dialysis 2. ml of media after growth 3. weicht of the mycelia (dry weight) b. increase in weight of mycelia (growth increment) 5. toxicity of the media to barley seeds a. filtered media (1:0) b. media diluted 50 per cent(231) 0. media diluted 100 per cent (1:1) 15 d. dialyzed media 6. activity of enzymes in dialyzed media a. amylase b. cellulase c. sucrase The pH of the filtered media was determined by colorimetric methods after the media was dialyzed for b hours at l0 C. ‘ against 800 ml of glass distilled water. Each flask was harvested by filtering the mycelia on a pre-weighed filter paper. The volume of filtrate was measured. The mycelia and filter paper were dried in an oven for 2 days at 60° C., and then weighed. The toxicity of the filtered me‘ia was tested on 25 barley seeds as described in part a. above. The e“ tur. filtrrte wrs tested in series of 1:0, 2:1 and 1:1 dilutions. The toxicity of P" the filtrate was 9180 tests after dialysis. Results are re- corded as percentage germination. After dialysis the media was tested for amylase, cellulase, and sucrase activity. Enzyme r actions were incubated at 37° C. and reaction mixtures were protected from contamination by add- ing a small amount of toluene. Sucrase reactions were incubated for 4 hours while amylase and cellulase reactions were incubated for 138 hours. To imnrove the quantitative results in the cellu- lase reaction the solution was filtered after incubation to 16 remove the cellulose perticles which cause the solution to be turbid and to disturb the colorimetric readings. The filtered cel1ulose was washed with distilled wz‘ter to assure that all the reduction nroducts were present for the color test. Somogyi and Nelson reagents were added and the final volume diluted to 100 ml. The colorimeter readings for each solution are recorded as eouivalent mg of glucose which would give the same calor as the reduction products of the enzyme reaction. Four ml of enp yme solution (dialyzed media) was “un as a colorinetric check -. 1 and this value was suosrected from the value outeined in the enzyme reaction. Host Range Studies All of the species and varieties tested proved to he hosts 'ider the conditions of the experiment except for cabhage, rcdish, wild cu- cumber, and winter oets. Winter oats are known to be susceptible to E: srtivug under certain environnentel conditions. The lesions which appeared in Trial I and Trial II were not successfully reisolated in many cases because the sterilization techniques were too harsh and killed the pathogen (Table IV). The modified sterilization technioue used in Trial III was highly successful, Etioloeical Studies 1. Reaction of Several Barley Varieties to Eatural Infection The varieties tested showed no correlation in resistance from one location to another. hone of the varieties displayed a high amount of resistance to infection. In the East Lansing nursery sporulation of H, setivnm on the st‘ew seemed to correlate to an ex— tent with infectian of the culm. Generally, infection was in the form of culm rot. Nodes were infected in almost every variety. Al- though the leaves were usua‘ly infected with H. sativum, the damage was not extensive and soorulation did not occur on the leaves. In- fection wns (general?r heavier Ft the Tuscola location. 2. The Natural Infection of Michelite Bern Of the 58 isolations made, four (3.5 per cent) proved to be 17 TABLE IV Results of the Host Range Study Showing the Plants Inoculated in Bach Triel, the Anneernnce of Lesions, ngd the Reisolrtion of Hrlninthos*oriun S“tivum from the Lesions. Trial I Trial IT Trirl III Plant Host L“ Rb L R L R Beai: Blue Lake + + + - + + Fordhook No. 2&2 + - + _ + + Greet Torthern 4 _ + _ + 4 Idah + + + _ + + Michelite + + + _ + + Pencil Pod Black Wax + - + - + + Crucifers: Chinese Mandarin + _ - _ Danish Baldhead + — _ _ Premium Flat Dutch - — Wisconsin Hollander _ _ Early Scarlet Radish + — — — _ _ Corn: Golden Cross Bantam + f + + + Cow Pea: Black + + + + Cucurbits: National Pickling + + f f + Mammoth King Pumnhin + + + + + + Wild Cucnnber — - Horse Bean + — + a + + Peas: Aldermen + — _ _ + + Small Grail: Barley 93h + + Barley 5105 + + + + Winter Oats _ _ Sunflower 1 + Tomato: Bonnie Best + _ f f + + Rutgers 1 + a L (+) indicetes lesion produced by H. srtivum b R (f) indicates reisoletion of Y, S’tilmfl from lesions Infection of Four Plant Species by Artificial Inoculation with Snares of H817“. nthngporium setivu‘i. 1. Michelite Bean ?. Black Cow Pea 3. National Pickling Cucrmber h. Rutgers Tomato 1 .Lo ’3 Lo PLATE II Four isolations of Helminthosporium srtivum from arti- ficially inocclrtod larvae cf Kannoth King Pumpkin. Iota the variations amen" the colonies. Nine isolates of Kclninthosno ium catlvum from arti- ficially inoculoted plants. Reading left to right, tOp row, Black Cow Pea, Odessa Barley, and National Pickling Cucumber; Mifidle row, thmoth King Pumpkin, Golden Cross Sweet Corn, and Blue Lake Bean; Bottom row Nichelite Bean, Idaho Stringlecs Refugee Bean, and Michelite Been. 11 314.4» flaw“ Date Read: ‘ué' Hm I" TAB'B V Reaction of Barley Varieties to Natural Infection by Helninthosnor-um lr eativum. Conprrisonc Are Made anong Locations of Nureeries, Varieties, and Infected Plant Parts. Snorulation on the Straw of Infected Plents at East Lansing Is Concarcd to Plant Infection. Bprley East Lansing» Steniish Tuscqla East Lhngimg Sthniish Varieties bpo‘d' Culms Lvs Culms Lvs Culms Lvs *3 LR UN N lotion 69‘ 69 L M L L L M L M M 5105 L L M L H M L M ) ‘3' o -4 72 69 M H L M L M M II E M L578 L n L L M M M M H 2276 L L L n M H M H 1517 M M L M M M L M M 1367 L M L M M H M L M M 187 L M L L L M L H L L uly 15”- July 17, July 9 July 15 July 1?, l3 Aug. 5 Aug. 5L 731 L L L M L M L o M M J Heavy Infection 0 s No Infection UK = pror Hones Lvs = Lerveg Light Infection M‘= Medium Infection L3 = Lower Iogcs .P iniection rating in tnis table is the e'erege infection of each vnrict; C" C‘ S” d. ‘P p FJ O O ”l (7- Ho Sporulation of Helmintioeporium sativum on Barley Straw Stimulated by Sunlight 1. Heavy soorulation of Alterneria sp. and Helrintkosnoriua rotiv=1. 2. Moderate snorulntion of Helnirthosnoriun setivum. 3. Heavy snorulaticn of Eelninthosnorium sativnm. 23 H. sstivum. It was interesting to note that Alternnrin sn. was th most frequently isolated organism. 3. Air-borne Inoculum in Relation to Infection of Barley in Michigan Conidia of H, sntiyyg_did not appear on the spore slides until late in the growing season. The barley headed on June 20. Soorula- tion of H. setivum on the straw was first noted on July 31. The first slides which showed conidia of H. sativun were removed from the field on August 3. These slides avereged b.5 spores per slide. All slides thereafter, until the experiment terminated on August 26. averaged 2 soores per slide. ‘ It was noticed tlst socrulation of H. sntivnm in the field on barley straw was affected by sunlight. Snerulation alwnys appeared on the straw in view of direct sunlight. Careful expmination of the strew elimineted the possibility that the reaction was caused by humidity, gravity or temperature. Alterneria sp. was found sperup lating with E. setivum on the straw in many instances. Practice en— ables one to distingu'sh the two types of snorulation. 11. _ ru...,:. ° A $3.57 DU t‘~..li'€' , . Ln..- r '-.. .v .‘ .0. n 7* (4-: l, RtFCbluA oi nc‘tole POtcCrs lfCA n. s tlvum .0..- 7'. ,4-2.,.-.. "- ilOu.’-. r u.‘.... lb— Experiments with acetone powders Trepnred vealed that ?my1?SC, cellulase, and sucrese were present. While the reactions all showed the presence of the enzymes, no quantitative estimation of the enzvmes was made. A typical reaction gave the fol- lowing results: 2Q (Mg Equiv. of Glucose) Substrate Enzyme Tested For Reduction Products Soluble Starch Amylase 4.8 Alpha Cellulose Cellulase 5.2 Sucrose Sucrase 8.0 In every reaction using acetone powder as an nzyme source and a four—hour incubation period at 28° 0.: sucrose was hydrolyzed the most, soluble starch was usually second and alpha cellulose third. 2. The Production of Sucrase during Parasitism ) (Mg Equiv. of Glucose) Substrate Enzyne Source Reduction Products Sucrose Infected Barley Leaves 5.9h Voninfected Barley Leaves 3.6h These results indicate that the infected barley elants had a 63.19 per cent greater sucrese activity than the noninfected plants. 3. The Effect of Extra Enzymes on Barley Seed Germination a. Effect of purified enzymes Alpha amylase, diastase, hemicellulase, and inver Bee in— hibited germination (Table VI). None of the 0 her enzymes tested showed any appreciable inhibition of germination. b. Effect of amylase, cellulase, and sucrose produced by'flL .n4- 1 s,tivnm The data obtained in this experiment are shown in Table VII and are illustrated by Graphs I, II. The pH and mycelial weight increased each day while the volume of media decreased slowly. The filtered media had a toxic effect on barley seeds in the barley seed germination test. The 50 per cent (2:1) and 25 (1:1) 100 per cent dilutions as well as the dialyzed media were also toxic to barley seeds. Amylase, cellulase, and sucrase were present in the media rs exoenzymes produced by E. sativun. The results of the enzyme reactions day by day corre ate with the toxicity of the media in the barley seed germination test. The toxicit' of the media to barley seeds increased with an increase 0 0 F5 “3 t f.) I.) m ('D I) :23 p. m 8 ’— f: (D C) c,ivity in the media. The opposite ef- fect was observed for cellulase activity. F) C)». TABLE VI The Effect of Extra Enzymes on the Germination of Barley Solutions Tested Per Cent Germination Distilled Water Check 74 Dist lled Water—Toluene 62 P. Alnha Amylase Alnha Amylase Dihstrse Diestrse Hemicallulase hemicellulrse Invertrse Invertase Pectinnse Pectinase Protease Protease Proteinase Proteinase CA 0 \h to All of the above solutions rere made up on a weight/ volume basis and were preserved with a small amount of toluene. The enzymes were produced by the Nutritional Biochemicals Corporation. TABLE VII Data Recorded from Liquid Culture of H. Sgtiv-~ 2? Ml Grams Grams pH of of Mycelial Endyme Activity of Media Mycelial Dry (Mg Eouiv. of Glucose) Filtered after Dry Weight Day Media Growth Weight/Rep. Increase Amylase Cellulsse Sucrase 0 5.8 bl.5 0.0000 0.0000 0.00 0.00 0.00 1 6.0 u3.0 0.0030 0.0030 0.62 0.12 0.00 2 6.6 u1.5 0.0075 0.00u5 1.56 0.64 0.00 3 6.# 38.0 0.0630 0.0605 2.16 0.00 0.06 4 7.0 35.0 0.0315 0.0135 0.58 0.00 0.10 Toxicity of Culture Filtrate to Barley Seeds (Per cent Germination) Fllt‘led 2:1 1:1 Dialyzed Dav YC*1" l:C‘ D31 t-on Dilution hedia 0 no 52 36 60 l 60 AC 56 80 2 35 96 7o 88 1 23 88 6b 3 u 0 12 12 32 RAPH I 28 Amylase, Cellulase, and Sucrese Activity of Dialyzed Culture Filtrate 2.5 i --- Amylase (168 hr. Reaction) -u-~- Cellulase (168 hr. Reaction) 2.0 Sucrase (A hr. Reaction) 1.5 Mg Equiv. Glucose 1.0 .5 GRAPH II Toxic Effect of Culture Filtrateq on Barley Seeds 100 - fi\ 80 ' /\ \ /\ \ 60‘ \\ \ Per Cent \ Germi- \ \ nation 1&0 . 20 i ._————- Filtered Media (1:0) ___——- Dilution (2:1) __..... Dilution (1:1) ._...__ Dialyzed 0 1 2 DAYS (g1 :- V. DISCUSSION A33 COHCLUSICE Host Rar;:e Studies H. sativum is a capable parasite which can attack a wide range of plant species. In the case of the members of the Cruciferae which were not parasitized in this study, the reason may lie within the plant, i.e., perhaps the plant has a toxic compound which inhibits the fungus or nerhans the surf.ce of the nlant contains a compound which the enzymes nroduced during parasitism by H. shtivun cannot break down. This isolate (H. S. 101) may not be able to parasitize members of the Cr uciferse but some other isolate may be able to. 1. Susceptibility in Barley Varieties While none of the barley varieties tested was resistant enough to encourage a breeding project, the nurseries served a useful pur- pose in pointing out that some other method of control may be more desirable. The incidence of a heavier infection at the Tuscola nur- sery coupled with he rotation data indicate the irmoortance of crop rotation in relation to the bu ildyup of inoculum. This is also em- phasized by the isolation of E, tivum from Michelite oea nat Tus- cola. Environment and the genetics of H. sntivum, as they effect the important enzymes used in parasitism, are more immortnnt to the etiol Ogjy of this {is sense tlm n th e resista; _ce of barley. 2. Air-borne Inoculum in Relation to Infection of Barley in Michigan 30 The principal source of inoculum for kernel infection is air- borne inoculum which appears after the barley has headed. This was clearly indicated in this study and confirms a study which Machacek and Greaney made in Canada in 1935 (17). A good deal of the sire borne inoculum which is found during the time the head is Open to infection is from the barley straw which hosts abundant conidia of Hg sativum. Enzyme Studies Amylase, cellulase, and sucrase are produced by H. sativum and may be found in the acetone powder preparations or as exoenzymes in culture filtrate. This information c0‘pled with the increase in su— crase activity in infected barley plants and the toxic effect of amy- lase and sucrese on barley seeds support the proposed enzyme theory of parasitism. Of all the enzymes which H. sativum probably contains in its mycelia, none can be important or even useful without the en; zymes which provide the initial food. It is proposed here that amy- lase, cellulase, and sucrase are among the principal enzymes which provide food and establish parasitism of the plant. This research does not prove that the toxic effect which shows up in the barley seed germination test is the direct cause or toxi- city, but rather that the presence of enzymes correlates with the toxic effect. The actual toxicity may be due either directly to the reaction of an enzyme or indirectly to an enzyme reaction product. VI . SULULARY 1. H. sativum has a wide host range and is Ppable of parasi- tizing many species of plants. 2. No source of resistance to H; sativum has been observed in barley varieties. In barley the susceptibility of a particular vari- ety cannot be evaluated because of the wide range of variation in the isolates of H. sctivum in one location. 3. Sporulation of HL¥sativum on barley straw correlates with varietal infection. Sporulation is stimulated by sunlight. b. The isolation of H. sativum from Michelite bean and the general increase in infection where beans and grain are grown in suc- cession point out the importance of rotation. 5. Air-borne inoculum in Michigan is the principal source of inoculum for kernal infection. Host of this inoculum is from the sporulation of H. sativum on the straw of the current season. 6. Acetone powders prepared from H. sativum contain amylase, cellulase, and sucrase. 7. The activity of sucrase in infected barley is greater than in noninfected barley plants. The additional sucrase in infected plants is probably caused by H._sativum activity. 8. Alpha amylase, diastase, hemicellulase, and invertase have a toxic effect on barley seeds and thus inhibit germination to an extent. 31 9. Amylase, cellulase, and sucrase are present as exoenzymes in culture filtrate. The production of these enzymes can be corre- lated to the toxicity of the cu1t~“e filtrate to barley seeds. 8. 9. 10. ll. 12. VII. LITERATURE CITED Andersen, H. 1955. Species of Helminthosuorium on cereals and grasses in Denmark. Friesia 5: 60-89. Andrews, E. A. 1953. Seedling blights and root rot of forage grasses. Diss. 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