PLACE IN RETURN lOXto romanthb chookout from your record. TOAVOID FINESrduuonorbdonddoduo. —— — \ DATE DUE DATE DUE DATE DUE WM 51 LA! . T7 I; =_______J _TT—1| MSU Is An Affirmdivo Adlor'VEquol Opportunity Inflation emano-m NATUPE or 3.15515me TO PENETRATION AM) nmomor or USTILAC—O HOPmI (13333.) LAGBRH. IN SEEDLINGS OF FOUR HULL—LESS SPRING BILRIEY VARIETIES PARUL CI‘LATTERJEE An Abstract Submitted to the School of Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botanar and Plant PathOIOgy 1956 An Abstract An investigation of the nature or resistance to penetration by Ugtilago hordei (Pers.) Lagerh. on four hull-less spring barley varieties was made. Seedlings were inoculated with.§; hordei race 6 and an.Alaskan isolate, fixed and sectioned two to four days after inoculations. Prepared slides were observed for the penetration and development of the pathogen and for the possible anatomical barriers produced by the host. The nature of the host response to artificial wounding in the coleOptile of resistant and susceptible varieties was also studied. Differences between the anatomical changes of the coleoptile attacked by the pathogen and the coleOptile wounded an- tificially were noted. Spore germination and penetration of the host by the pathogens was siiilar on all resistant and susceptible varieties. Some ana— tomical changes deve10ped in the resistant varieties after penetration. A sheath formed in the epidermal cells of the resistant varieties around the penetrating hyphae which prevented the further penetration of the byphae into the deeper tissue of the coleoptiles. Collapsed zones were observed very frequently in both resistant and susceptible varieties where there was a heavy inoculum on the surface of the cole- optiles. The size of the collapsed areas increased with an increase in the amount of inoculum on the surface of the coleoPtiles. Penetration Parul Chatterjee of the cells by the pathogen in or near the collapsed areas was not always seen in the development of these necrotic areas. In some cases the hyphal growth was retarded in the host without any visible sign of the anatomical changes in the nature of the cytoplasm of the host cells. In these cases the fungal hyphae decreased in diameter and finally ceased to grow. A very specific reaction was found in all varieties in response to artificial wounding. F0 sheath formation.was observed in this case. Suberin was revealed by microchemical test in the dark stained arses produced by wounding, but it was absent in the necrotic areas produced by hyphal penetration. Microchemical tests revealed cellulose in the sheath~like structures. The effect of several amino acids on the penetration and growth of the hyphae of race A in the coleoptiles of resistant varieties was studied in seedlings grown on mineral nutrient agar,to which the respective amino acids were added separately. Four of the ten amino acids tried aided the fungus in establishing tself in the tissues of the resistant host. However, amino acid treatments did not change the host-pathogen relationship for the susceptible variety. NATURE or RESIST won TO Pmmrnarmn AND INFECTION or USTILAoo HORDEI (ma) means. In 5123aner or FOUR HULL-LESS SPRIB’G HARLEY VARIETIES PARUL CHATTBRJEE A.THESIS Submitted to the School of Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 1956 ACKNOWLEDGMENTS The author wishes to express her sincere thanks to Dr. B. In. Riesling. under whose inspiration. constant supervision. and guid-v- snco this investigation was undertaken. She is also ghostly indebted to Drs. W. B. Drew, C. J. Alempoulos, L. I. Msricle, and H. J. Stafseth for the criticism of the manuscript. Achowledgement is also made to Dr. R. L. Riesling for the photographic work of this manuscript. TABLE OF CONTENTS I. ImmCTIONQOOOOOOOO0.00.00.00.00OOOOOOOOOOOOOOOO... 11. “mama AND WTHODSOQOCOOOOOO...OOOOOOOOOOOOOOOOOOO III. mmmm RESULTS....C.00.00.0.000000.00....00000. 1 u 8 Iv. DISWSSION...OOOOOOOOOOOOOOOOO0.0.00.0O..0000.0.00... 22 v. Wosooooocsooosoooooooooo.ooooooooooocooooososos n. LIMTUBE CITEDOOOOOOOOOOOO0000000000000000000000000 25 26 LIST OF ILLUSTRATIOI‘J'S Page Figure 1. Nepal seedling 48 hours after inoculation w th race A. LILhora-8100000000000...0000000....000.000.000.010 Figure 2. Nepal seedling #8 hours after inoculation with I‘O'Ce 6.LIL1‘01.C:ei.......'......0..OIOOOOOIOCOOOOOOOOOOOlO Figure 3. Excelsior seedling #3 hours after inoculation with ra'ce A,LL-Eloj‘clei-OOOOOOODIOO0...0.0.0.0...OOOOOOOOOOOOO11 Figure #. Excelsior seedling #8 hours after inoculation with race 6’E.-lloré-ei..............0....000000.OOOOCOOQOOICIII Figure 5. Lompa seedling #8 hours after inoculation with race A, L hordei.........0.000....0O..0....................13 Figure 6. Lonpa seedling 48 hours after inoculation with race 6’ U4 hordei......0000000000...0..0....................13 Figure 7. Brachytic seedling #8 hours after inoculation with race A,p4-hordei.............OO00.0.00...0....0000000015 Figure 8. Brachytic seedling #8 hours after inoculation.with race 6’ E'-horde-10.essences-0000000000000.-coosoooosOQQIS Figure 9. Nepal seedling #8 hours after inoculation with race A, U;.hordei showing collapsed areas in COIeoptileOOCOCOOO.OOOOOCIOCOOCOOOOCOOOOOO00.000.000.001? Figure IDs-Nepal Seedling Vfith PericarpoossosoooooooocsssssoOOosoozo Figure ll.Lompa seedling 96 hours after inoculation with race A, and treatment with.phenylalanine....................20 Figure 12.Lompa seedling 96 hours after inoculation with race A, and treatment ‘With prOJ-inessososooooosssoossoooooooozl Figure 13.Lompa seedling 96 hours after inoculation.with race ‘A, and no treatment with an amino acid.................21 INTROIIJCTION Covered smut of barley caused by Ustilgo m (Perm) Lagerh. is world wide in its distribution. Covered smut is more widely dis- tributed than either of the other two smuts on barley and decreases the value of the barley crop both qualitatiwa and quantitatively. the disease is controlled by resistant varieties of barley and/or chemical seed-treatment. Several studies of the seedling-infecting smuts, including covered smut of barley, have been raported and demonstrate the general systemic development of these pathogens in their respective hosts (2.6.8.9,10. 19.20.22). Western (20) studied the invasion of some susceptible and resistant varieties of cats by ‘4 biolcgical forms of Ustilagg M (Pers.)Jens. . m cat varieties tested by Western differed in their resistance to y_,_ avenue, and in the smut-resistant Markton variety the infection tubes failed to pass beyond the epidermal cells. The resistance of oat varieties to E... M was demonstrated by the prevention of pen- etration in the epidermal cells. retardation of the growth of the m- celiun within the host cells, or by necrosis of the invaded host cells. ' Riesling (8) studied the pathological histology of covered smut race 6 on It barley varieties. The host-pathogen relationship was very compatible in the susceptible variety studied. less compatible in the intermediate varieties, and incompatible in the resistant variety. The mechanism for resistance to covered sent in the four barley varieties studied varied with the different varieties. In the highly resistant 2 Pannier (G. I. 1330'), as well as in the intermdeiate type, Chevron (c. I. 1111). smutted heads failed to develop because incompatible host-pathogen interactions occurred at the points of penetration. However, in the variety Jet (O. I. 967) the coleoptile escaped in- fection by remaining under the tough pericarp during the initial elongation of the sprout which prevented the spores from contacting the growing shoot (8). Biological specialization of the covered smut isolates and va- rietal reactions are reported by several authors (1,151,516.18). Vari- able results were obtained in the percent of infected heads under field conditions after inoculation with different strains of the barley cov- ered smut pathogen (h). Vigorous growth did not enable a susceptible variety to escape this disease (Dr). Bodenhiser (11+) described 7 phys‘ iological forms of §_._ M23. and 2 of these forms could be recognized by their differences in pathogenicity on 2 barley varieties. Infection of barley seedlings growing from kernels which still retained their hulls was poor, but dehulled seed gave a significant increase in the percent of infected heads (1). Tapke (18) isolated 8 races of y; m from 200 field collections. These 8 isolates of covered smut differed from one another in their reaction on 5 named varieties of spring bar- ley. Race 6 was the most widely distributed race. The races of y" m demonstrated a high degree of heterozygosity with respect to their pathogenicity (16). i'he studies reported in this paper were undertaken to compare the host-pathogen relationships for 1+ naked barley varieties to two iso- lates of E: hordei. These it naked barley varieties were chosen for 3 this study because of their resistance to race 6 and the naked condition of their kernels which eliminated hand-dehullihg before inoculation. The purpose of this comparison was to determine if any histological or morphological differences existed which could explain the differences in pathogenicity of the two strains of covered smut on four naked barley varieties. MATERIALS AND LETHODS roar naked.barley varieties, Nepal (0. I. 595). Excelsior (c. 1. 12nd). Lcmpa (a. I. 1312), and Brachytic (o. I. 6572) were inoculated with spores of race 6 and an isolate from Alaska of Ustilgg £1331; (rero.) Legerh. . The smut isolate fromtllaska‘will be designated as race A throughout this paper. The varieties and their average smut percentages are given in Table I. Seed of these It barley varieties was obtained from the barley nursery grown at East Lansing in 1955. Spores ”M71. The percentage of mature barley plants showing smutted heads after inoculation and growth in thejreenhouse. 195M. Variety O. I. No. Pegsntage of smttegplants Race F Base A Feral 595 0 30 Excelsior 12% O 60 Lamps 1312 0 O Drac£§ic 6512 O '- ef race A, P; hands; were obtained from smutted heads of Excelsior and spores of rade 6 from Odessa (G. I. 9334) grown at East Lansing in 1953. Race A. produces no smutted heads on Odessa, but rather stunts and kills the infected plants before the smutted heads develop. The viability of the spores of the two isolates was tested on 2 perdent agar plates prior to each inoculation. Kernels of the 1% barley varieties were pro-soaked in water for 2 hours and blotted on paper towels to remove the excess water. In some trials the pericarps were removed over the embryos after the kernels were 5 moistened. Inoculations were made on these kernels with the pericarps removed to evaluate the roll of the pericarp in preventing early seed- ling infections. After the kernels were moistened, they were dipped in spores and placed in moist chambers (8). Ten inoculated kernels were placed in each moist chamber. and the chambers were stored in a desic- cator enter a saturated calcium sulfate solution to maintains constant 98 percent relative humidity required for good smut germination (3). This high relative humidity also promoted good seed germination. The desiccator with the moist chambers was then placed in a temperature con- trolled incubator at 20°C.. tbchanical wounds were made on the coleoptile of “8 hour old seed- lings in order to compare the histological symptoms produced by mechan- ical injury and fungal penetrations. In some seedlings the coleoptile was pierced with bristles, vegetable fibers, and human hair. These latter objects were left in the host tissue throughout the sectioning process. The seedlings with wounded coleoptiles were incubated fi 2000. for 3 and 5 days. Free-hand sections and microtome sections of healthy, diseased and mechanically wounded tissues were made for histochemical tests (7. 13). ' Odessa, Excelsior and Lamps were inoculated with different con- centrations of spores of both race 6 and A to determine the reaction of the coleoptile tissue to different amounts of inoculum. Seedling were inoculated when they were 7-8 mm. long. Relative amounts of spores were applied to the coleoptile with a dissecting needle by dipping the needle in the spore powder and smearing the spores on one area of the surface of the coleoptile from one to five times. 6 Oultures of race 6 andi of§_.__h_g_l;d_e_i_were grown in 50 ml. of po- tate dextrose liquid media in 250 ml. flasks for 3 days at 20°C.. The cultures were then filtered through Beitz filters using a 1/10 mm. fil- ter pad. Seedlings of Excelsior and Lompa were treated with the culture filtrate on the surface of the coleoptile. In a few seedlings the col- eoptiles were pierced in several places and the culture filtrate was applied to the injured areas. The treated seedlings were inoculated at 20°C. for 1&8 hours. Glycine, l-glutamic acid, 1-leucine, dl-lysine, dl-methionine, d1- phenylalanine, l-proline, dl-treonine, dl-tryptophane, and dl-valine were used in a study of the effect of several amino acids on the pehetration and infection of Excelsior and Lompa seedlings by race A. Kernels of these two barley varieties were soaked in each of the amino acids for 148 hours. After soaking, the seedlings were removed and inoculated with race A and incubated. The amino acids were tested in this manor at concentrations of 25, 50, and 100 p.p.m.. in untreated control wee also run. In the second trial the germinating embryos were excised from the endosperms and then grown on agar containing nutrient salts. sucrose, and the amino acid under test (21). The amino acid was added to the media before sterilization at a concentration of 25 p..p.m.. The en- bryos were sterilized for half a minute in equal volumes of 70 percent ethyl alcohol and sodium hypochlorite solution. rinsed with distilled water. and then placed in the Petri dishes on the medium containing a single amino acid. The coleoptiles of the embryos were inoculated with race A when the sprouts started to elongate (’48 hours after being placed on the agar plates). After the coleoptiles were inoculated, the T seedlings were incubated at 20°C. for us and 96 hours. An inoculated control with sucrose but without any additional amino acids in the mineral agar was also run; The seedling materials for anatomical studies were killed and fix- ed in 1.1.1.. for 18 hours, then placed under vacuum to remove air from the sections. Thematerials were dehydrated by dioxane and ter- tiary butyl alcohol (7) and embedded in Fisher's tissue mat (56°C.- 58°O. ). Seedlings were not allowed to stay in the paraffin oven more than 21! hours. All of the seedlings were sectioned at 12 er. 1’4 microns and mounted on glass slides with Haupt‘s adhesive. A modification of Conant's Quadruple stain (s) which substituted thionin for crystal violet was used to stain the slides. mam RESULTS Epore germination and penetration of the coleoptiles of all it varieties by both of the races of covered smut were similar. Spores germinated on the surface of the coleoptile and formed promycelia and . conjugation tubes. but no sporidial development was observed. After diploidization, the promcelial cells formed dicaryotic hyphae which“ penetrated the coleoptile at any point on its surface. Penetrating hyphae upon contacting the surface of the cuticle became somewhat thickened, and then produced a thin peg-lino structure which pierced the epidermal wall. After penetration the hypha passed through the col- eoptile perpendicular to the long axis of the coleoptile and penetrated the walls of the host cells directly. m:- cells of the coleoptiles of some varieties often collapsed at points on the coleoptile covered by large masses of smut spores (Fig. 9). These collapsed areas were found in all varieties inoculated with ace 6 and 1. except the variety Brachytic inoculated with race 6 (Fig. 8). Penetration of the coleoptile by the pathogen was not always associated with the development of these collapsed areas. Cases were found where these areas did not show any penetrations even in the first layer of the coleoptile. In the sons of collapsed tissm the cells were plasma- lysed and the staining reactions of the host tissues were changed. 1‘19 walls of the collapsed cells were thickened. has collapsed areas stained deeply with safranin. but no traces of hyphal fragments were detected in some of these regions. Ihe reaction of the coleoptilar tissues to different amounts of inoculum on their surfaces showed more extensively 9 collapsed areas in the varieties Odessa. Excelsior, and Lompa when the amount of inoculum was increased on the surface of the coleoptile. Collapsed zones in the coleoptile also developed when the culture fil- trate from the smut organism was applied to the punctured spots of the coleoptile, but no collapsed areas developed whenthe filtrate was applied on the unbroken surface of the coleoptile. About 90 percent of the hyphae of ace 6 penetrating the coleoptiles of Repal were enclosed in sheaths which prevented further penetrations of the hyphae into the coleOptile. The rest of the hyphae penetrated further into the interior of the coleoptile and died without branching (Fig. 2). About 60 percent of the hyphae of lace 1 which penetrated the coleOptiles of Nepal were enclosed by sheaths. The remainder of the intection-hyphae penetrated across the coleoptile (Fig. 1). some of the hyphae penetrated into the coléeptile and formed lateral branches that frequently ran vertically for some distance (Fig. 1). Ninety percent of the penetrating hyphae of race 6 on Excelsior were enclosed by sheaths (Pig. 14). The remaining hyphae which penetrated further into the coleoptile did not have a vigorous appearance. The hyphae did not stain deeply or produce any branches. Frequently only the tips of these hyphae stained and. the remining parts appeared faded. Occasionally hyphae were seen in close association with host nuclei in the epidermal cells without any effect. However, in the base of the coleoptile some of the host nuclei were distorted and stained deeply with safranin when their neighboring cells were penetrated by the 13th- ogen. he hyphae of race ‘ branched abundantly in the coleOptiles of the variety Excelsior ( lie. 3). These branches grew vertically for Iigure 1.50pal (0.1.595) seedling us hours after inoculation with race A. EL'hordei. Mycolium at the inner edge of the coleoptile. 11 seedling EB ESurs after inoEEIa tion with race . u hordei. The hyphae failed to penetrate very far into the coleoptile.— figure 3. heelsior (6.1.1213) seedling 1&8 hours after inoculation with race A, p_._ hordei. Branchéd. healthy mycelium in coleoptilar tissues. rig-me 1+. Incelsior (6.1.1248) seedling 1&8 hours after inoculation with race 6, IL hordei. Sheaths formed around penetrating hypha.‘ 12 short distances but eventually grew’across the coleoptile. In Excelsior the healthy, branched, red-stained hyphae contrasted sharply with the hyphae of’race 6. VOnly about 8 percent of the infection hyphae of race A.were enclosed in sheaths. About 88 percent of the hyphae of race 6 which penetrated the col- eoptiles of Lompa were enhlosed by sheaths. The rest of the hyphae pen- etrated three or four cell layers deeper into the coleoptile and then stopped. In some cases the cytoplasm of the host cells in the epidermis formed layers surrounding the penetrating'hyphee. These layers of the host cytoplasm differed in some degree from the sheath formation as- sociated with the other varieties studied (Fig. 6). About 90 percent of the penetrations of Lompa'by race.L were enclosed in sheaths. Hew- ever. there was a tendency for the penetrationrhyphae to escape from the sheath and to grow deeper into the host tissues of the coleoptile without branching (Fig. 5). In these cases the sheath formation did not totally prevent the growth of the pathogen into the host. The hyphae that excaped from.the sheath.penetrated two or three cells further into the coleoptile and then failed to progress further. The cells of the host which.pere penetrated were disorganized. In a few of these disorganized cells swollen, red-stained fragments of hyphae were detected. These disorganized.cells were not grouped together, but they developed singly in the third and fourth layers of the coleoptilar tissue. Oc- casionally the hyphae escaped from the sheath. penetrated deeper into the coleoptilar tissue without being stopped by disorganized cells. but these hyphaelalso failed to continue to grow. The response of the variety Brachytic to the penetrating hyphae of 13 ‘5} “~“ rigure 5. Lompa (C. I. 1312—Yieedlingfi hours after inoculation with race A. U_. hordei. Sheaths formed around penetrating hyphae. D a A "E _ - i, ii figure 6. Lompa (G. I. 1312) seedling 48 hours eater inoculation with race 6, 1°. hordei. Sheaths formed around penetrating hyphae 1’4 race 6 differed from the other varieties studied. Although a few of the hyphae from the areas of heavy inoculum on the surface of the col- eoptile penetrated three or four cells deep into the coleoptile, most of the hyphae. penetrated only half or two thirds of the distance through the layer of epidernml cells (Fig. 8). No collapsed cells were ob- served under areas of the coleoptile covered by large numbers of spores (Fig 8). Spores of race A germinated and infected the coleoptile of the variety Brachytic (Fig 7). Infection hyphae from areas of the col- eOptile covered by moderate amounts of inoculum appeared to grow faster although these hyphae did not branch (Fig. 7). Many hyphae were seen protruding from the inner edge of the coleoptile, between the coleoptile and the tightly whirled leaves. Sixty percent of the hyphae of race A which penetrated the coleoptiles of Brachytic were sheathed. The hyphae of race 6 in the coleoptiles of Repel, Excelsior, Lompa. and Brachytic showed no further deve10pment at the end of 96 hours. The hyphae of race A. in Lompa and Brachytic had grown no further at the end of 96 hours. At the end of this longer incubation period the hyphal fragments were often seen in discolored host cells. However, in the more compatible reaction between race A and Nepal more hyphal fragments were seen at the end of 96 hours. In the susceptible variety Excelsior inoculated with race A branched hyphae and intercellular chelium was found at the end of 96 hours. ' Very little change was observed after 1+8 hours in the coleoptiles of the seedlings pierced with a microneedle. A hole in the coleoptile was located where the wound was made, but only the cytoplasm of the cells immediately surrounding the wound stained with safranin. No sheath or rigors s. Brachytic (c. I. 657257seed1ingiis hour; at}; inoculation with race 6, Pa. hordei. Penetrating hyphae restricted to the epidermal cell layer. 16 .other growth was formed around the wounded areas. Wounds in the coleop- tile of the seedlings in which the piercing object was left in the wound stained deeply with safranin. Walls of the intact cells adjacent to the foreign body-were thickened and stained more intensely. The cell walls, intercellular spaces and the discolored cytoplasm gave a positive reaction to histochemical tests for suberin (Table II). Penetrations of the coleoptiles of resistant barley varieties by hyphae of covered smut were accompanied by certain anatomical changes. “istoéhemical tests of these invaded areas were made (Table II). All of the resistant varieties gave similar results. The sheaths formed Table II. Histochemical tests of the penetrated and wounded areas of three day old barley seedlings. Materials Cell Walls Sheaths Outicular Collapsed Wounded Layers Areaa Areas Cellulose «I «#3) - - - Pectin ‘ - - - - Lignin - - -(b) - - Guberin - - -‘b) - ‘ Callgpe - - - - - e. fiid not disolve in 50 percent chromic acid. b. Slightly positive in five day old seedlings. around the penetrating hyphae in the resistant varieties gave a positive reaction for cellulose. The wounded areas gave a positive reaction for suberin.. However, suberin was not detected in the collapsed zones deve10ped by the attacks of the smut pathogen. A few penetrating hyphae of race A were observed in the seedlings of the variety Lompa after the seeds were soaked in various amino acids at Figure 9. Nepal (0. I. 595) seedling #8 hours after inoculation with race A. E: hordei. collapsed areas in the coleoptile under large masses of spores. 17 18 50 p.p.m.. However, these hyphae did not penetrate beyond the epidermal layer of the host coleoptile and were not surrounded by sheaths. Histological examinations of the inoculated seedlings grown from the kernels soaked in various amino acids at 25 p.p.n. showed the same re- action as untreated seedlings. The penetrating hyphae did not reach the deeper layers of the coleoptiles of these treated seedlings and no sheath formation was observed. However, collapsed areas were frequently seen. Spore germination and penetration of the coleoptile of seedlings grown.from.excised embryos of the variety Lompa on mineral agar con- taining sucrose and one amino acid occurred in the usual manner; The progress of the hyphae in the coleoptiles was extensive 96 hours after inocuIation in the l-glutamic acid, l-leusine, dlaphenylalanine, and l-proline treatments (Table III, Fig. 11, 12). Healthy, stained hyphae with branches were seen horizontally and occasionally parallel to the long axis of the coleoptile. .A new disorganized cells in the deeper tissues of the coleoptile were observed. but these cells did not prevent the pathogen from advancing through.the host tissues. Examination of the inoculated seedlings of Excelsior showed that amino acids did not prevent the growth.of”the pathogen in this susceptible variety. Hest- pathogen relationships in the sucrose treated checks did not diffler from.those reactions feund in the inoculated materials for these two varieties (Fig. 13). ‘ Table III. 19 The penetration and growth of race A, E. hordei in the coleoptilar tissues of Lompa barley seedlings grown on a mineral- sucrose agar containing amino acids at 25 49.9.!!!" Status of the penetration hyphae 7+8 and 96 hours Amino Acid after inoculation. Treatment 118 hours 96 hours Glycine Few penetrations, no No change. l-Glutamic Acid l-Leucine dl-Lys ins Methionine dl-Phenylalanine l-Proline dl-Treonine dl-Tryptophane dl-‘Vaiine Sucrose Control sheaths. Sheaths formed, necrosis in areas with penetrations. Sheaths formed, collapsed areas present. Few sheaths, collapsed areas present. Sheaths formed, collapsed areas present. Sheaths formed, collapsed areas present. Ho sheaths around pene- trating hyphae. Few penetrations. Few penetrations. collapsed areas present. Sheaths formed, collapsed areas present. Sheaths formed, collapsed areas present. Hyphae branched and growing. Heal thy, branched hyphae in coleoptile. Unbmnched hyphae, disorganization ahead of hyphae. I'ew, long. unbranched hyphae. Healthy, branched mycelium. Healthy, brancm mycelium. No change. NO Changes Unbranched hyphae, disorganized cells. Unbranched hyphae. disorganized cells. rig-use“. Nepal (0. I. 595) seedling 1&6 hours after inoculation with race A, L hordei. Peric‘arp serving as a barrier between spores and coleoptile. l'igure ompa (c. I. 1312) seedling 96 hours fami- inoculation with race A. 2:. hordei and incubation on mineral-sucrose agar containing 25 p.13... phenylalanine. Branched, healthy nycelium in the coleoptile. Figure Lamps. (0. I. 1312) seedling 96 hours after inoculation with race A, y_._ hordei and incubation on mineral-sucrose agar containing 25 p.p.n. prolins. Branched. healthy nycelium in the coleoptile. (1 ~ I ' v ,7. , ,7,, Figure 13. Lompa (G. I. 1312) seedling 96 hours after inoculation with race A, U. hordei and incubation on mineral-sucrose agar containing no added-amino acids. Penetrating hyphae stopped by disorganization of host cells. 22 DISCUSSION The dicaryotic mycelium of both races of the covered smut organism penetrated the coleoptiles of the four barley varieties directly. The penetrating hyphae upon contacting the surface of the coleoptile be- came thickened and produced a.peg-like structure which.penetrated the epidermal wall. The cell walls at the point of penetration did not stain differently than unpenetrated.areas. Histochenical tests of the cell wall at the point of penetration were made but no change was observed in the nature of the cuticle or cell wall surrounding the point of penetration. When the culture filtrate of the smut organism was placed on the surface of the coleoptile, no reaction in the coleoptilar tissues was observed. However. the culture filtrate altered the host tissues when it was applied at the site of injury on the coleoptile. The lack of a dectsctable change in the cuticle and cell walls at the point of penetration indicated that the penetration of the cuticle and cell wall is mechanical in nature. Penetration of resistant varieties was usually followed by demon- strable host-pathogen interactions. The most common types of incompatible host-pathogen interactions were the formation of a sheath and the death of the penetrated cells. Several authors have reported similar develop- ment of sheaths around.penetrating mycelium in their studies of'various parasite relationships (5.17.20.23.2h). However. a few sheaths were also formed around the penetrating hyphae in the susceptible varieties. but they are completely absent in the coleoptiles of all varieties wmunded 23 artificially. Therefore, sheath formation is not a wound response to mechanical injury but rather a specific reaction to the penetrations of fungal hyphae. When there was a heavy inoculum on the surface of the coleoptile, host tissues in the coleoptile collapsed. Similar collapsed zones in“ the host coleoptile were also demonstrated by other authors (8.20) in studies of seedling-infecting smuts. Collapsed zones in the coleoptiles were found in both resistant and susceptible vari§ties. In the resistant varieties this area of dead cells developed in the second and third cell layer of the coleoptilar tissues when the first layer was penetrated. However. some cases were found where no penetration was detected. and no hyphal strands were seen in the necrotic area of these resistant vari- eties. In those resistant seedlings where no penetration was detedted. collapsed areas developed from the epidermal cells inward into the in- terior of the coleoptile. Discoloration of the cell cytOplasm and the thickening of the cell walls were the main features of the areas of collapsed cells. These histological data indicate that there are host- parasite reactions in advance of actual fungal penetration. The fail“ ure of the culture filtrate to produce necrotic areas on the uninjured coleoptile does not exclude the possibility of diffusable substances being produeed by the contact of the parasite and the host. Failure of the penetrating hyphae to gow deeper into the host coleoptilar tissues was the common expression of incompatibility be- tween the host and pathogen. No branching of the fungal hyphae in the host was observed in the seedlings of resistant varieties after 96 hours. Some hyphal fragments were found which had grown across the coleoptile 2h without branching. These hyphae did not thrive in the host as is demon- strated by the diminishing diameter of the mycelium. its lack of branchr ing. and.pale staining reactions. The incompatible host-paraiite reac- tion is most fully demonstrated by the variety Brachytic inoculated with race 6 in which none of the penetrating hyphae grow beyond.the epidermal cells. Howefer. no collapsed areas were formed by these pen- etrations. Rapid growth of the nycelium of race A of I}; 3.12.1311. in the coleoptiles of a resistant variety was observed when the inoculated seedlings were grown from.excised embryos on mineral agar to which certain amino acids were added. This development was not observed.when the whole caryopsis was treated prior to inoculations with the several amino acids tested. However. while resistance was altered in the seedling stage. tests were not conducted which carried the mycelium through to the head smut stage. Resistance as found in these four barley varieties when inoculated with two races of smut seems to be the result of several factors. The hypersensitive type of readtion is encountered when heavy inoculum is ‘used upon some varieties. In those cases where the fungus penetrated andgrew but finally perished in the coleoptilar tissues the idea of starvation on the part of the parasite is indicated. Some of the pen- etrations were walled off by the host in a direct response to fungal penetration. Tests indicate that this sheath reaction is more than a wound reaction. .thhough treatment of resistant seedlings withsnertain amino acids altered the seedlingreaction to penetration by race A. sue” ceptibility was not altered by any of the amino acid treatments tried. 1. 2. 3. 5. 25 SUMMARY Spore germination and penetration of the mycelium of 2; 9.213229 races 6 and A. into the coleoptiles of resistant and susceptible varieties were studied. The resistant varieties frequently developed sheaths around the hyphae penetrating into host tissues. However. in the variety Iempa inoculated with rade A the penetrating hyphae often escaped from these sheaths. In this case the further progress of the hyphae was prevented by disorganimtion of the host cells in the third and fourth layers ofthe coleoptile. Sheath formations were less fre- Quently seen in the susceptible varieties. Recrotic areas in the coleoptiles of both susceptible and resistant varieties was observed under areas of hoavy inoculum on the surface of the coleoptiles. Brachytic. wien inoculated with race 6. did not develop necrotic areas in association with large spore concentrations. Retardation of hyphal growth in the host tissues was found in re- sistant varieties. Anatomical comparisons of diseased and wounded tissues were made and differences were noted. listochemcial tests were performed on injured, uninjured. and penetrated coleoptilar tissues. l'our amino acids were found to alter the seedling reaction to race A of covered smut. However. similar treatment of a susceptible variety failed to change its susceptibility to race A. L. 2. 3. 5. 9. LITERATURE CITED Aamodt‘. O. 8.. and W. H. Johnston. 1935. Reaction of barley varieties to infection with covered smith (Ustilego £93311). Gan. Jour. Res. 128590-613. Allison. C. C. 1937. 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