~A _. A tar-Iv -.‘ a? THE DEVELOPMENT OF APPRESSORIA, HAUSTORIA AND SECONDARY HYPHAE DURENG THE PRIMARY INFECTEON OF WHEAT AND HARLEY BY ERYSEPHE GRAMENES Thesis For His Degree of 535. D. MICHIGAN STATE UNIVERSITY Shawki S. Masri 1965 LIBRARY Michigan State University THESIS This is to certify that the thesis entitled ,. ‘ ’_ _ f L . ”Ac. cl CV'LU/l:hze;-,L (S (‘ flugau. a 4&4}.ng an“: c‘nz-‘l /jv(g‘77l'./&§lv/ /" ~ . . , . - " a . ' A - ' I I’ ,1 " i J”'/,-»L'M d W t WV ,1. hi. 1.9- ‘L l- J 4“,; ”i C Loci» o" 9/7,.7/1» 6 :n r/ 195;; Lc J l/ [I 7 ,0 m A .’ {j . ’f’ cf“ \' ”3 LL '5’)?" :E " 'l - . [try ‘30): . 5’1; I o ”'— -—‘—»_ ,. presented by Q ‘2 liq w LE; 3 . ‘V‘ (L.%&." has been accepted towards fulfillment i of the requirements for LL (3 degree in 93,11" 7’, 3';er Plant {3:}thin /# Major proféor Date 7774’»: /71 /?(\5‘ / _/ 0-169 ABSTRACT THE DEVELOPMENT OF APPRESSORIA, HAUSTORIA AND SECONDARY HYPHAE DURING THE PRIMARY INFECTION OF WHEAT AND BARLEY BY ERYSIPHE GRAMINIS By Shawki S. Masri The differential response of the various stages of pri- mary infection of powdery mildew (E. graminis) to different environmental conditions suggests that the primary infection is made up of several stages. These are: l) germination, 2) formation of appressorial initials, 3) maturation of appress- oria, 4) develOpment of the haustorial body, 5) the formation of secondary hyphal initials,and 6) the formation of function- al secondary hyphae. At the Optimum conditions of every phase, the parasite population was synchronized; that is, most units of the pOpulation reached each phase within a 4 hr period. The host was penetrated around the 10th hour (hr) after inocu- lation. The haustorial bodies appeared 16 hr after inoculation and full-sized haustoria were detected 34-36 hr after inoculation. Prior to infection, succinate dehydrogenase activity was detected only in the parasite. Two hr after penetration, succinate dehydrogenase activity was detected on the surface of the young deve10ping haustoria. This indicated that either the enzyme moved from the parasite to the host or its pro- duction was stimulated, by the parasite, in the host. Through the use of various strains of barley with major genes for resistance, it was demonstrated conclusively that only functional secondary hyphae should be taken as a crit- erion for the establishment of a successful host-parasite l 2 relationship. In susceptible, moderately resistant and highly-resistant hosts studied, the data showed that the number of functional secondary hyphae on the surface of the host equaled the number of haustoria formed inside the host. Hence, either the functional secondary hypha or the haustorium could be taken as a criterion for the establishment of primary infection. All major genes for disease resistance exhibited more than one effect in their mode of action and, therefore, these genes can be considered pleiotropic. The various ways and means by which these genes conferred resistance to their respective hosts were: 1) exclusion of the pathogen from the host; 2) delay in early haustorial develOpment; 3) distortion of some haustoria; 4) destruction and distor-' tion of the majority of haustoria 5 days after inoculation; and 5) suppression of fungus sporulation after the establish- ment of primary infection thereby preventing the occurrence of repeating disease cycles. THE DEVELOPMENT OF APPRESSORIA, HAUSTORIA AND SECONDARY HYPHAE DURING THE PRIMARY INFECTION OF WHEAT AND BARLEY BY ERYSIPHE GRAMINIS x)“ Shawki S. Masri A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 1965 ACKNOWLEDGEMENTS The author wishes to express his sincere appreciation to Dr. A.H. Ellingboe for his continuous encouragement and guidance throughout the author's graduate studies. Sincere appreciation is also due to Drs. R.P. Scheffer, J.L. Lockwood, E.C. Cantino, and W.M. Adams for their many helpful suggestions while preparing the manuscript. The writer is indebted to Dr. R.A. Fennel for his advise on the histochemical work; Janice McClelland for her help in preparing the manuscript; and Lidia Sicari for her help in some of the photography work. Special thanks are due to Mr. Joseph L. Clayton for his technical assistance. This investigation was supported by the National Institutes of Health for which the author is indebted. ii Dedicated To My Father and Mother iii TABLE OF CONTENTS INTRODUCTION 0 C O O O C O O C O O O O O O O O O O 0 LITERATURE REVIEW. . . . . . . . . . . . . . . . . . MATERIALS AND MTHODS O O O O O O O O C O O O O O O 0 Maintenance of Stock Culture . . . Inoculations of Host Plants . . Control of Environmental Conditions Examination of External Strucutres Examination of Internal Structures Histochemical Tests . . . . . Sample Size and Statistical Analysis. RESULTS 0 O O O O O O O O O O O O O O O O O O O O O Germination of conidia and Formation of Appressoria. . . . . . . . . . Effect of Light on Maturation of Appressoria. . Formation of Secondary Hyphal Initials. . . . . Effect of Temperature on Development of Secondary Hyphal Initials. . . . . Effect of Relative Humidity on DevelOpment of Secondary Hyphal Initials. . . . . Effect of Light on Development of Secondary Hyphal Initials . . . . . . Development of Haustoria . . . . . . . . Effect of Light on Development of Haustoria . . . . . . . . . . . . . Effect of Temperature on Development of Haustoria . . . . . . . . Morphological Development of the Haustorium . . . . . . . . . . . . . Comparative Development of Haustoria in 6 different Hosts . . . . . . . . . Relationship Between Haustoria and Functional Secondary Hyphae . . . . . . . . . . . . . Macroscopic Assay of Relative Resistance in the Barley Varieties . . . . . . . . . . . Brief Review of Sequential Events of Primary Infection . . . . . . . . . . . . . Histochemical Studies . . . . . . . . . . . . . Cross-Inoculation Experiments . . . . . . . . . iv Page 10 10 ll l3 l3 l6 l8 l9 19 2O 26 27 32 32 38 43 43 48 52 56 6O 62 69 TABLE OF CONTENTS (cont) Page DISCUSSION . . . . . . . . . . . . . . . SUMMARY . . . . . . . . . . . . . . . . . . . . . . 83 LITERATURE CITED . . . . . . . . . . . . . . . . . . 85 TABLE LIST OF TABLES_ Page The number of secondary hyphal initials, functional secondary hyphae and haustoria on the same inoculated leaf section. . . . . . 59 Effect of genes for resistance on lesion formation on leaves of barley inoculated with E. graminis f. sp. hordei . . . . . . . 64 vi LIST OF FIGURES FIGURE 1. IO. Conidia of E. graminis f. sp. tritici harvested A) 5 and B) 7 days after inoculation . . . . . . . . . . . . . . . . . Germination of conidia and formation of appressorial initials of E. graminis f. sp. tritici expressed as a percentage of the total population reaching these stages . . . . . . Effect of light treatments and time of application on maturation of appressoria of E, graminis f. sp. tritici_on wheat leaves . . . . . . . . . . . . . . . . . . . Structure of the population of parasite units in respect to time in the 3rd stage (maturation of appressoria) of primary infection of E, graminis f. sp. tritici on wheat leaves. . .‘. . . . . . . . . . . . . . Conidial germination, formation of appressorial initials and maturation of appressoria in a population of E, graminis f. sp. tritici on wheat leaves. . . . . . . . Effect of temperature on develOpment of secondary hyphal initials of Eb graminis f. sp. tritici on wheat leaves. . . . . . . . Percent of appressoria of E. graminis f. sp. tritici developing secondary hyphal initials at 100% RH 8—16th hr after inoculation . . . . Effect of light on development of secondary hyphal initials of E. graminis f. sp. tritici on wheat leaves. . . . . . . . . . . . . . . Comparison of the 4 curves representing percentage of the population in 4 stages of primary infection of E, graminis f. sp. _ tritiCi O O O O O O O O O I O O O O O 0 0 Infection pegs of E, graminis f. sp. tritici. vii Page 14 21 24 28 30 34 36 39 41 44 LIST OF FIGURES (cont) FIGURE Page 11. Effect of light on development of haustoria of E, grgginis f. sp. tritici . . . . . . . . 46 12. Morphological development of haustoria of E. graminis O O O O 0 O O O O O O O O O O O O 50 13. DevelOpment of haustoria in 5 different heats O O O O O O O O O O O O O O O O O O 54 14. Hypersensitivity reaction induced by the fungus in the presence of the Kwan gene 5 days after inoculation. . . . . . . . . . . 57 15. Succinate dehydrogenase activity on and in the haustoria of E graminis f. sp. tritici . 67 l6. Acid phosphatase activity on and in the haustoria of E, ggaminis f. sp. tritici . . . 7O 1?. Formation of secondary hyphal initials 0n non-susceptible hOStSO o o o o o o o o o o 72 viii INTRODUCTION.--Powdery mildew of wheat (caused by Erysiphe graminis E, gp. tritici) has been reported from nearly all wheatgrowing countries of the world (8,20,21). Although almost ubiquitous in distribution, powdery mildews cause less spectacular losses than those caused by several other important groups of plant pathogens (5}). Nevertheless powdery mildew is ranked second to rust in its economical importance (20,24,25). The purpose of this investigation was to: 1) define the various component phases that make up the process of primary infection; 2) synchronize the parasite population in its advance through the various stages of primary infection; 3) study the formation and development of haustoria in suscept- ible, moderately resistent and resistant hosts in an attempt to gain an insight to host-parasite interaction; 4) examine the histochemical changes that accompany the early stages in the development of haustoria in order to determine whether or not there is a detectable interaction between the host and the parasite in the initiation of infection; and 4) determine useful criteria for the establishment of a compatible host- parasite relationship. The ectoparasitic nature of most powdery mildews and the availability of different strains of barley with various levels of resistance made this host-parasite combination a very useful'system for investigating the several aspects of host-parasite interaction. LITERATURE REVIEW.--A rather voluminous literature is available on the various powdery mildews. A great deal of emphasis has been placed on the different environmental factors such as temperature, relative humidity (RH) and light as they affect conidial germination and the overall development of the disease. It was not until recently, however, that the infection process was considered as being made up of several distinct phases, each subject to experi- mentation, rather than a single process (27,30). For experimental purposes, these stages were considered to be: 1) germination, 2) formation of appressorial initials, 3) maturation of appressoria, 4) formation of haustoria, and 5) development of secondary hyphae (30). Yarwood g§_gl. (52) reviewed temperature relations of powdery mildews in general and concluded that the optimum ranges from about 11-28 C and averages about 22 C, while others (8, 39) reported maximum development at 17-19 C and 15-20 C. Dale (l2) and Cormack 53,2;. (10) noticed absence or scarcity of powdery mildew near the ground and it was proposed (53) that this was due to high temperatures. Delp (14) investigated rather extensively the effect of temperature on the grape powdery mildew fungus and reported that the minimum leaf temperature for conidial germination was below 6 C, but infection did not take place below 7 C. Growth was very slow at 7 C with more than 32 days from inoculation to sporulation. The growth rate increased with corresponding rise in temperature to a maximum of about 26 C. It was 2 3 concluded that temperature is the primary factor which limits the growth of powdery mildew of grapes. Nair and Ellingboe (33) reported that: "Germination of conidia was insensitive to light, but was dependent on tem- perature and relative humidity. Maximum germination was obtained by an amphidirectional change in relative humidity accompanied by a unidirectional change in temperature from 17-22 C." Although germination occurred at 17 or 22 C and 65 or 100% RH, the most favorable temperature for the maturation of appressoria was 22 C (30). These results were verified in a later study (27). The relation of RH to disease has been one of the most controversial aSpects of our knowledge of powdery mildews (53). Powdery mildews have been classified as dry-weather fungi (50). The germination of Erysiphe polyggni (5, 50) and Erysiphe graminis (8) occurred in the absence of water or at RH approaching zero. Other workers (9, 21) failed to get any germination of Erysiphe graminis below 85% RH. Free water may be necessary for grape mildew (Uncinula necator) development (18) and development of other mildews is limited by low RH (18). Yarwood (53) correlated the impOrtance of powdery mildews on the same host Species in different re- gions with the rainfall distribution. The powdery mildews of peach, apple, rose, cucurbits, beans, peas and grapes are commonly severe with rainfree summers. In fact, it was suggested (8, 51) that mildew development is checked by sprinkling infected plants with water, and Yarwood (51) even proposed control of powdery mildew with a water spray. Relative humidity close to 100% gave better germination of E. graminis f. sp. tritici than did 65% RH (32). A stimulatory effect of light on the germination and development of mildew has been reported (8, 38, 40, 48). Yarwood (48) observed that several powdery mildews gave higher germination percentages in light than in darkness. Light is essential for normal mildew development (46). With increase in day-length, vigorous host plants were produced and better development of mildew resulted (38). Work with the rusts also emphasized the importance of light on germination and overall development of the disease. Sharp 53,2l. (40) found that germination and appressorial development of urediospores of wheat stem rust (Puccinia graminis f. sp. tritici) occurred equally well over a range of 15.5-24 C and at light intensities below 300 foot-candles (ft-c). Post-appressorial develop- ment was favored by a higher temperature and light intensity than that which favored germination and appressorial forma- tion. Generally, a reduction in light intensity or total darkness after inoculation reduced the prevalence of rust infection (17, 22, 44). Post-appressorial development was greatest at 29.5 C and at light intensities greater than 500 ft-c (40). When appressoria were exposed to natural light of 1500 ft-c or more at about 29.5 C, they produced the substomatal vesicle and completed the infection process (40). Whereas the effect of light was emphasized by several workers, Rowell 31.3l. (37) also working with E. graminis tritici found that under conditions of dew at 21 C and little or no light, urediospores germinated during the first 4 hr and a majority of the appressoria developed in the next 4 hr. The effect of darkness and low light intensities on mildew development was studied by many workers. While Cherewick (8) reported slight or no effect on development of E, graminis f. sp. tritici when plants were put in darkness after inoculation, Yarwood (49) observed a definite reduction in development under the same conditions with Erysiphe polygoni. A more luxurious development of straw— berry mildew in shade than in full natural light has been observed (13). Part of the favorable effect of shade could be due to reduced temperature and increased RH and of the 2, temperature is considered more important (53). Yarwood (53) believed that mildew severity on plants increases with duration of light up to a point (which is not defined) and then decreases, and that light may affect powdery mildew through its effect on the host. The effect of light on wheat powdery mildew (E, graminis f. sp. tritici) varied with the stages of primary infection (30). Whereas germination and formation of appressorial initials were light independent, the maturation of appressoria was light sensitive. These findings were verified by a later study (27) where it was found that the maturation of appressoria was light sensitive and inhibited by darkness and high light. The optimum light treatment for this stage of infection was 240 ft-c applied 2 hr after inoculation. Powdery mildews, like rusts and many other pathogens, penetrate plants on which they will make no further growth (11, 47). A fine penetration tube emerges from the center of the appressorium and grows into the lumen of the cell (11, 40, 41). It has been repeatedly reported (1, 43, 47) that certain fungi penetrated resistant varieties as easily as susceptible varieties. The occurrence of Special stimuli for penetration in susceptible hosts can be questioned (1, 43). According to Corner (11), the mature appressoria of powdery mildews penetrate the host and produce haustoria in the host epid- ermal cell. Penetration begins 24 hr after inoculation at about 20 C and 100% RH. The penetration through the cell wall by a fine infection peg below an appressorium has been regarded as an entirely mechanical process (6). Failure to penetrate could have been due to: 1) the lack of infection pegs, 2) the mechanical toughness of the cuticle and epidermal cell wall, and 3) the poor attachment of the appressoria and consequent failure to take the back pressure of penetration (16). The resistance of older barley plants to E, graminis f. sp. hordei has been attributed to the abor- tion of the infection peg due to the hardness or thickness of the cuticle and epidermis (20). After penetration, a haustorium is formed which is restricted to the epidermal cells in most powdery mildews. A sheath has been shown to be present around the haustorium of E, graminis (4, 15, 42). Smith (42)*believed it represented cell-wall debris which accumulated around the haustorium at the time of penetra— tion. According to Ehrlich and Ehrlich (15), Hirata and Kojima in 1962 proposed that the body of the Sheath con- sisted of metabolic products of the host which accumulate around the haustorium via osmosis, and suggested that the sheath is important in establishing parasitic relationships and in preventing death of the host cell, but the mechanism of its action is uncertain. It was also observed (15) that the host plasma membrane invaginates during penetra- tion, but the sheath boundary is not Simply the host plasma membrane but is a membrane partly of fungal origin, or a special membrane produced by the host in response to the presence of the pathogen. A recent study (4) indicated that the Sheath membrane is continuous with the host ectoplast and the organization of the haustorial protoplast is similar to that of many plant and fungal cells. The formation of a functional haustorium is the crucial step in the development of a compatible host-parasite relationship. Corner(ll), working with resistant and immune hosts, observed that the development of powdery mildew is arrested at the papillae stage before the haustoria are formed. Physiological resistance was attributed to death of mesophyll cells adjacent to the epidermal cell in which the haustoria are formed (8, 11). White and Baker (47) used host plants with varying degrees of resistance. When susceptible varieties of barley were used, abundant development of mycelium on the leaf surface took place. There were 20-30 haustoria in the epidermal cells beneath each colony. With semi-resistant varieties, the infection site was surround- ed by a zone of dead host tissue. The mycelium was well developed in each colony, but was less dense than in the susceptible host. There were 10-15 haustoria in the epider- mal cells beneath each colony. In the resistant variety Goldfoil, the mycelium was restricted in development and a few conidial branches were produced by each colony. There were 5-12 haustoria in the epidermal cells beneath each colony. With highly-resistant varieties, mycelial develop- ment was very limited. Occasionally, l conidial branch was formed by a colony. Each infection court contained 1 or 2 haustoria in the epidermal cells. When rye, oats and certain wheat varieties were inoculated with E. graminis f. sp. hordei, germ tubes, appressoria and Short infection papillae were formed, but there were no haustoria. With almost every aspect of the powdery mildews, there is a great deal of controversy. Early work (8) with E, graminis indicated that the organism is homothallic, while later reports (35, 36) asserted heterothallism. Hiura (23) demonstrated hybridization between E. graminis f. Sp. hordei and E, graminis f. Sp. tritici on wheat, but ascopsores from such crosses failed to germinate. Plants infected with powdery mildews displayed pro- nounced increases in respiration, but these respiratory changes were not detected until 6 days after inoculation (2, 7). Infection with E. graminis stimulated respiration of areas of barley leaves adjacent to the cells invaded by the fungus (2, 7). A high concentration of acid phosphatase was Shown to be associated with the haustoria of powdery mildew on barley (3). It was suggested that these enzymes play a role in the utilization of phosphorylated intermediates by obligate parasites. An increased number of isozymes of acid and alkaline phosphatase, succinate dehydrogenase and malate dehydrogenase was reported in a recent study of rust-infected bean plants (45). The activity of acid phosphatase decreased after infection whereas a small increase in peroxidase activity was detected. Malca g£_§l. (26) observed that the activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconic dehydrogenase increased in mildewed barley leaves at the time of or shortly after the appearance of symptoms, and continued to increase as the disease developed. However, part of the increased activity was due to enzymes contributed by the fungus. MATERIALS AND METHODS.--Maintenance of stock culture.-— Two strains of mildew were used. The strain of E. graminis E, gp. tritici (cause of powdery mildew on wheat) was collec- ted in Michigan. The strain of E, graminis E. Sp. hordei (cause of powdery mildew on barley) was CR-3 (59.1), obtained from Dr. J.G. Moseman. Little Club wheat (Triticum compactum Host) and Manchuria barley (Hordeum vulgare L.), on which the mildew cultures were maintained, were grown in 4-in. pots and inoculated when 7-8 days old. Inoculations were made daily toward the end of the light period by shaking conidia onto the leaves of the host plants. Inoculated plants were kept under incandescent bulbs (50 ft-c) and cool, white fluorescent tubes (400 ft-c) with a total light intensity of 450 ft-c (at the leaf surface level) over a 15-hr day. The temperature was maintained at 18 1,2 C and the RH fluctuated between 60-70% during the day and near 100% at night. Mildew pus- tules were visible in 3 days, and an abundant supply of good quality conidia was available 6 days after inoculation (Fig. 1A). Inoculations of host plantS.--The "rolling method" (31) of inoculation, whereby conidia were applied by cotton swabs onto the host plants, provided a rather uniform distribution of single conidia on a leaf, and the means for the study of primary infection on a quantitative basis. All observa- tions of conidial germination, appressorial initials, and 10 11 mature appressoria were made on inoculated 5 or 6-day-old plants of wheat or barley, grown singly in 2-in. pots. About 2.5 cm of leaf surface was inoculated on each single plant with 40-130 conidia per cm. Heavier inoculations with as many as 500 conidia on 1-cm-leaf surface were needed in some experiments. Inoculations were made on either the upper or lower side of the leaf. The lower side was always inoculated for experiments in which the epidermal layer was peeled off for closer examinations, because of the ease with which the lower epidermis can be peeled. Mildew susceptible Little Club wheat and susceptible Manchuria barley were used. Also used were 4 additional barley strains each of which possessed a major gene for resistance backcrossed into the variety Manchuria. The pedigrees of the 4 strains were as follows: 1) Algerian C.I. 1179 (Mia) XéManchuria C.I. 2330 (crossed to Manchuria 4 times and then selfed 7 times); 2) Goldfoil C.I. 928 (Elg) x3 Manchuria C.I. 2330; 3) Kwan C.I. 1016 (Elk) xg Manchuria C.I. 2330; and 4) Psaknon C.I. 6305 (El?) x; Manchuria C.I. 2330. The genes conditioning resistance in these varieties have been designated Mla’ EEE, Elk’ and E; respectively (28). p’ Control of environmental conditions.--The various environmental conditions of temperature, RH and light were obtained with Sherer Gillett, Model No. CEL 512-37 growth chambers. In all experiments, inoculated plants were incubated 12 forl.hr at 18 C, 100% RH with or without light depending on the experiment. An accuracy of 1.1 C temperature and :_3% RH control was obtained in these chambers. Light was supplied by 60-watt incandescent bulbs and cool, white fluorescent tubes. The light treatments used and their respective intensities were: 1) darkness (0 ft-c) 2) three 60-watt incandescent bulbs (25 ft—c) 3) six 60-watt incandescent bulbs (50 ft-c) 4) three 60-watt incandescent bulbs (25 ft-c) and 2 fluorescent tubes (215 ft-c) - a total of 240 ft-c (designated as the low light treatment) 5) Six 60-watt incandescent bulbs (50 ft-c) and 22 fluorescent tubes (2750 ft-c) a total of 2800 ft-c (designated as the high light treatment) 6) six 60-watt incandescent bulbs (50 ft-c) and 11 fluorescent tubes (1375 ft-c) - a total of 1425 ft-c. 7) three 60-watt incandescent bulbs (25 ft-c) and 11 fluorescent tubes (1375 ft-c) - a total of 1400 ft-c. Temperature and RH treatments ranged from 60-90 C and 55-100% RH respectively depending on the kind of experiment and the effect investigated. However, the most frequently used temperatures and relative humidities were 65 or 72 C, and 65 or 100% RH. One hygrothermograph, calibrated with a Sling psychrometer 13 every 2 weeks, was installed inside each chamber. Examination of external structures.--Plants were re- moved from the growth chambers after receiving the desired treatment and at regular intervals, 1 plant at a time. The inoculated portion of the leaf was cut into sections appro- ximately 1 cm long and examined under a light microscope at 125x magnification. The various fungal structures were examined, with as little disturbance as possible, on the surface of the host plant. All conidia at the various stages of development were counted and recorded. Individual plants were then discarded. The external structures observed were: 1) germ tubes, 2) appressorial initials, 3) mature appressoria, 4) secondary hyphal initials, and 5) functional secondary hyphae. Examination of internal structures.--In most ectoparasitic powdery mildews, the only internal structure is in the epider~ mis. This structure, the haustorium, serves to absorb nutrients from the host. Since it was hard to examine this relative- ly transparent structure in epidermal strips, the haustoria were stained to aid in distinguishing them from the rest of the cell cytoplasm. The srtain used was acid fuchsin in lactophenol. Epidermal strips (lower epidermis) were re- moved directly into a drop of acid fuchsin on a glass slide, covered with a cover slip, and examined under the microscope. Low power (125x), high power (594x), bright field, and phase- contrast michSCOpeS were used. Haustoria stained a blue color. The time needed for examining a l—cm-section of the 14 Fig. l. Conidia of E, graminis f. sp. tritici harvested A) 5 and B) 7 days after inoculation. (575x) l5 16 lower epidermis varied from 8-20 minutes depending on the stage of development of the haustoria. For some experi- ments, it was necessary to examine secondary hyphae on the intact leaf and haustoria in the same leaf section after stripping the epidermis. This was more time-consuming and required 30-45 minutes. Eggtoehemical testS.--The activities of acid phosphatase, succinate dehydrogenase, and glucose-6-phosphate dehydro- genase were tested. Succinate dehydrogenase was chosen because it is involved in the Krebs cycle; glucose-6-phos- phate dehydrogenase was chosen because it is a part of the hexosemonophosphate shunt; and acid phosphatase was chosen because it is produced in the host in response to injury. Increased acid phosphatase activity was also reported (3) in and around the haustoria. I The histochemical methods used were: 1) Takeuchi and Tanoue modified lead nitrate method for acid phCSphatase (34). 2) Gomori improved histochemical technique for acid phosphatase (19). In this method, the enzyme reacts with its substrate and inorganic phosphate is released. Lead combines with the phosphate to form a precipitate which is subsequently converted into the brown sulfide of lead. 3) Nachals E; El. method for determining succinate dehydrogenase (34). The tetrazolium salt (Nitro-BT), 17 which is a hydrogen acceptor, is reduced in the reaction mixture, and turns blue. 4) Hess, Scarpelli and Pearse methods for testing glucose-6-phosphate dehydrogenase (34). In this case, the tetrazolium salt (MTT) was used which, upon reduction in the reaction, turns grey-black in color. All of these methods were designed to locate and qual- itatively determine enzymatic activity as reflected by a color reaction. They were originally used for work with animal tissues and needed modification in various ways when used on plant material. The modification of these methods was difficult because of basic differences between plant and animal tissues. In general, the time of exposure was prolonged sufficiently (more than doubled sometimes) to permit the various chemicals to penetrate the thick cell walls. It was also necessary to determine whether or not to fix, to prefix orrnstfix the tissue, and the kind of fixative to use. Most of these adjustments were made by trial and error. The lower epidermis of inoculated host plants was used in all these tests and special attention was focused on enzymatic activ- ities in and around the haustoria as revealed by a color reaction for a specific procedure. The epidermal strips were mounted in glycerine jelly or other synthetic media and ex- amined microscopically. Phase-contrast microscopy was very useful in locating young haustoria, especially when they 18 retained their relatively transparent appearance. The shortcomings and limitations of histochemical techniques in this study were numerous. These will be dis- cussed later. §§gple size and statistical analysiS.--When external structures were examined, the number of conidia observed var- ied from 40-130 with an average of around 60 on a l-cm-leaf section. When haustoria were examined, the sample size varied appreciably depending on the stage of development, the kind of experiment, and the type of host used. All experiments were replicated 3-12 times on successive days. Replication in days, although time-consuming, has the advantage of compensating for variability in the quality of conidia. Two-way analysis of variance was used and a level of 5 per cent was considered statistically Significant, where- as 1 per cent level was considered highly significant. RESULTS.--Germination of conidia and formation of appres- §g£;g.--Several experiments were conducted on the germination of conidia under the best environmental conditions found in a previous study (32). Inoculated wheat plants of the Little Club variety were incubated for 1 hr at 100% RH and 17 C followed by 3 hr at 22 C and 65% RH. Under these conditions, 94% of the applied conidia germinated in 4 hr (Fig. 2). Essentially identical results were obtained in polyethylene-covered wooden boxes in which RH was con- trolled with sulfuric acid solutions and in Sherer Gillett, Model No. CEL 512-37 growth chambers in which RH was con- trolled by mechanical means. The enlargement and partial elongation of germ tubes resulted in the formation of club-shaped appressorial ini- tials beginning 4 hr after inoculation. There was usually 1 appressorial initial per germinating conidium regardless of the number of germ tubes formed. The formation of these structures was observed at 22 C, 65% RH in darkness or in low light (50 ft-c) applied the 2nd hr after inoculation (30). There was no detectable difference between the 2 treatments; 91 and 96% of the applied conidia produced appressorial initials 7 and 8 hr, respectively, after inocu- lation. The curve representing the percxxm of units forming appressorial initials was almost identical to the curve of per cent conidial germination (Fig. 2). In a previous study (30) using the same environmental l9 20 conditions (except that wooden boxes were used as growth chambers) only 60% of the applied conidia produced initials 8 hr after inoculation. One basic difference between the wooden boxes and the Sherer Gillett chambers was the air circulation provided by the latter. This factor could account, at least in part, for the different results obtained in the 2 types of chambers. The enlargement, elongation and bending of an appres- sorial initial resulted in a conspicuous structure which was cut off from the rest of the germ tube by a septum; this structure was considered an indication of maturity of appressoria. Both length and bending (to form a hook-like structure) were taken as criteria for a mature appressorium (30). A fully-formed appressorium was approximately 30-35 u long as compared to 30 u for an average length of conidia. Mature appressoria were detected 5 hr after inoculation and over 90% of the applied conidia produced mature appressoria, in moSt experiments, 10 hr after inoculation. Effect oEflight on maturation of appressoria.--The effect of light treatments on this phase of primary infection was investigated next. With all light treatments, 22 C and 65% RH were used (30) beginning the 2nd hr after inoculation; only treatments of light were varied. High light (2800 ft-c) when applied 4 hr after inoculation delayed maturation of appressoria (Fig. 3). Although 89% of the appressoria were mature in 10 hr, yet the percentage was lower than 21 .mommpm omens mcfinomon :oHpmHsmom Hmvop esp mo omeSoo upon m we commoamxo .Hoflpflap .mm .m mflsflsmam «M 90 mHmeHsH Hmflsommohmmm mo Soapmahom cam mflcfisoo mo Goapmsflahow .m .mflm 22 O. 29.3.5002. mwhmd 3:5: 0 m h 0 n w n N d u - q d m4<_._._z_ 4<_m0mmwmaa<\ — u d - . zo_._.moa econ: no HOHPHHP .mm .m mfinfiaumw .M Mo manommoaqgw mo SowpmHSSSS So Scavmofiannm me asap eds neceapsenp smash he sesame .m .eaa 25 ZOE.moa noon: :0 Heapflmp .Qm .H mficfladam aw Ho cospoomcfi hamafinm Mo Amfiaommoaagm Ho SoapmHSPSEV ommpm can she SH oaflp op economy SH means spammawm Ho Schemazmom one Ho ohdpodhpm .e .mHm 29 l 0 CD 0 O O O 0 ID ¢ '0 N - HOOH 83d 9NIHDLVW VIHOSSBHddV JO 3W1N3083d HOURS AFTER INOCU LATION 30 .SOHHSHSSOQ on» SHSHHS mpHSS Ho SOHPSNHSoHSoSmm m mSHpmoHvSH .eoHHoS oaHp wopHSHH m SHSPHS ommpm Some Semen mpHSS oSP Ho woos pmSp opoz .mo>moH pwoSz So HOHHHHH .mm .m mHSHSme am No SOHHSHSQOS m SH mHHommoHSmw Ho SOHHSHSHSE SSS .mHmeHSH HmHHommoHSSw Ho SOHPmaHom .SoHHmSHEHom HmeHSoo .m .mHm 31 205.44 3002. mm._.m< 950: O. m m h m n V n N . HI 4 1 A q SmOmmwmn—ad magi: mn_<.._._2. 44—mommmmamd - J K 41. . \ \ \ \ 202.42.21uo L 0. ON on O¢ on 00 ON 00 cm 00. 39V1N3083d 32 after inoculation; and 5) 18 C during 8-16th hr and 22 C during 16-30th hr after inoculation. Results showed that the latter represented the best temperature treatment because 94% of the parasite units formed secondary hyphal initials 30 hr after inoculation (Fig. 6). Although this represented a significant improvement over other treatments, the parasite units were not as well synchronized as in the preceding stages (Fig. 5). These experiments indicated that development of secon- dary hyphal initials was temperature sensitive and that temperatures of 18 to 22 C are more favorable than 30 0. Effect of relative humidity on develgpment of secondary Eyphaiginit;§;§.--The effect of RH on development of secon- dary hyphal initials was not emphasized mainly because the parasite should not respond to such treatments since it was already in the host. The only treatment used was 100% RH between the 8-16th hr after inoculation. A temperature of 22 C and 240 ft-c was maintained starting the 2nd hr after inoculation and continued until 30 hr after inoculation. The RH before and after applying the treatment was kept at 65%. High RH had an inhibitory effect on development of secondary hyphal initials. With this treatment, 85% of the secondary hyphal initials were formed 30 hr after inoculation (Fig. 7). Effect of light on development of secondary hyphal initials.--With the optimum temperature treatment, ca. 90% 33 of the parasite units formed secondary hyphal initials between the 16-30th hr after inoculation. Formation of secondary hyphal initials took a longer time than the other preceding stages of primary infection, possibly because the optimum conditions for the formation of secondary hyphal initials were not yet reached. There- fore, light conditions were varied to see if this stage could be shortened. In all experiments, a temperature of 22 C and 65% RH was maintained starting the 2nd hr after inoculation. Before and after the light treatments were applied, a 240 ft-c intensity was maintained. High light treatments of 2800 ft-c applied between the 8-16th hr after inoculation seemed to have an inhibitory effect on the formation of secondary hyphal initials (Fig. 8). 0n the other hand, darkness (0 ft-c) during the same period gave Significantly more secondary hyphal initials than did high light (2800 ft-c). Darkness, although super- ior to high light, had an inhibitory effect on a portion of the conidial population as was evidenced by the level- ling of the curve between the 20-22nd hr after inoculation (Fig. 8). Identical results were obtained with very low light (25 ft-c) when applied the 8-16th hr after inoculation. When darkness was started at 6 hr, rather than at 8 hr after inoculation and continued until the 16th hr, a remark- able increase in the percentage of secondary hyphal initials 34 .m sea S Seam SHHsoapnapspn eonoHoHe 0 ans m Seem .m ens S gene eonoomae pan .0 sea m Sosa Haasoaanaaspn aoaoae so: eae a pSoapmoHB .SOHPSHSooSH Hopmm HS Sonnm 0 on Am .aS smH one hopes 0 mm as eoonHeo soHpsHSoosH seems as amHum e on Am .SoapsH nooosH norms SS somum 0 mm Ao .SoHPsHSeosH asses an sonum 0 ma Am .nh SSH one poems 6 mm as eoonHoS coassHSoosH noses as seaum o wH AS .mo>moH pmoSz So MMMMMMM .mm .H mmmmmmmm am Ho mHmHPHSH HmSShS hamcSooom Ho pSoEmoHo>oc So endpohomaov Ho poonmm .w .mHm 35 29.575002. awhud manor do. 3VHdAH AHVONOOBS so 39VIN3383d 36 .SoHpsHSeosH seems an Shaun one mSHHSc mm ROOH pm mo>moH pooSz So mHmHHHSH HoSth hamoSooom MSHSOHo>oc HOHPHHH .Sm .m mHSHsomw .m mo mHaommmHmSo mo HSoo Mom .S .maa 37 20:44:002. mwkud manor . 0» ON mN .VN NN ON 9 T A, 1 T . 4 3 H _ J L \o L c\0 . 1 O . ON On on 0m ON on 0m 00. 3VHdAI-l AHVONOOBS :lO 39V1N3383d 38 formed was detected, and a steep smooth curve was obtained (Fig. 8). This seemed to be the optimum light condition for the formation of secondary hyphal initials since 90% Secondary hyphal initials were formed in 6 hr (16-22nd hr). This was a significant improvement over all previous treat- ments because it shortened the period for the formation of secondary hyphal initials from 14 t0 6 hr. It was rather hard to conceive how a 2-hr increase in the dark period (as early as 6 hr after inoculation) could affect formation of secondary hyphal initials 10-16 hr later. One plausible explanation was that a portion of the conidia was inhibited by light between the 6-8th hr after inoculation and that this portion of the population was "released", so to Speak, when light (240 ft-c) was replaced by darkness (0 ft-c). These data suggested that the parasite population was progressing through the various stages of primary infection, namely germination, formation of appressorial initials, maturation of appressoria, and develOpment of secondary hy- phal initials in a synchronized manner (Fig. 9). Develepgent of haustoria.--Pri0r to the appearance of secondary hyphal initials on the surface of inoculated leaves, a series of host-parasite interactions took place during a seemingly quiescent period (between the 10-16m hr after inoculation). It was during this period that infection pegs were formed on the lower side of the appressoria (Fig. 10). Infection pegs penetrated the epidermis and developed 39 .S Sosa SHSseHansssn eonooaae S .S ens S Seam SHHsermHsssm eonoSSHe o ens S .S ens S .o .m Scum SHHmoHpmempm ooHoMHHo S pSoSpmoSe .SoHSsSSoocH nosus a: seHum House comma pSmHH wSoomoHoSHm SSS pSoomocSmoSH Am .SoassSSoosH seems as amS-m Henna osmv pseHS pSoomoHoSHm SSS pSoomocSooSH Am .SOHpmHSooSH Scams as amSum Sousa mmv armHS acoomoessesS As .SoaasSsoosH teams as amaum Housm ov mmoSMHmm Am .SOHPSHSooSH nopmm HS Serw Acne“ ov mmoSMSmQ AS .moemoH pmoSs So SHPHSP .Sm .M mHSHson .m no mHmeHSH HSSSSS mHmcSooom Se scosSoSosoe no pSmHH So pooSSS .m .mHS 40 20_._.<._ 3002. mural—4 mmDOI mm 3 ¢N H mm # ..oo_ 0 ON on or. on om 2. on on BVHdAI-I AHVONOOES :IO 39V1N3083d 41 .mHmHHHSH HSSSSS mamcSooom Ho SOHpmSSoS Am nSm oHnommoHSSo mo SSHHSHSPSS A0 mHoHSHSH HmHaommoHSSm mo SOHpoaHom Am SOHPSSHSSow AS one mommpm oSe .mo>ooH pmoS3 So HoHpHSH .Sm .m mHSHSde am Ho SOHpoomSH SHSEHHS Ho mommpm e SH SOHPSHSSOS oSu Ho ommHSooHoS mSHpSomoaSmH mo>SSo v mo SomHSmSSoo .m .mHm 42 20....415002. mmbmd mmDOI nN NN _N. ON m. m. h. m m h m n v n N u fi - J A — u‘ H H H . 1 4 _ q 1 h 1 m a: M552... >moQ .szm Bonn so: asp .ssanomH< ens SHoSeHem Soon Seam HHSsoHP ImHPSpm noHoMMHo SHHSSoSoz SH pSoaSOHo>on .mpmoS pSoHommHo m SH oHHopmSSS Ho pSoEmoHobon .mH .me on” 55 205... 302. mot? $50... on em mm 8 mm on em mm 8 . m. 1 a, q H _ . _ _ H _ >m.._mmnm m _ 44 'll. . \\.‘ m E z< mm .\ \ATA a\\\ >w4m< Hm S w mm om 0H NH Sm Hm mm SN or mm SN mm on so NH SH we Sozm mH was un-. nu a: a: mH HH mH om em m m mm MH SH SH omH w e m mm HHoHcHow m ass 6 mm 0H owe m mH mH 0mm m MH HH omN e NH. 0H HSM 4 OS . OS emH cospomHs am was nu in: an n: >w mH mH mm Sm mm mm mm mm mm SN mm SHHSSoSoz mm was on Hm ms - mm Hm mH mH Hm ow mm on mm mm on on em SSHo mm as we Hm oprHH wHHoP mHoHPHSH ImSSS SSH: mHmeHSH mHHovamm omSShH SHSSSooom HSASSS hnmoSooom mpmom HSSSSS ShocSooom mom HmSoHHommM, Hmpom SoHpoom HmoH oopoHSooSH osom oSp So oHnopmsoS SSS ooSSSS SamoSooom.HmSoHpoSSM .mHoHpHSH HSSSSS SnmcSooom Ho HomSSS one .H MHmSa 6O correlation occurred in all susceptible and resistant host combinations. Consequently, a safe conclusion is that for every functional secondary hyphae on the surface of the host, there is a corrSSponding haustorium in the epidermis. The formation of functional secondary hyphae clearly indicated a functional host-parasite relationship. Since the functional secondary hyphae were formed on the surface of the host and were easily detectable by microscopic examination, they were considered a useful criterion in studying host-parasite relationships. Macrgscgpic assay of relative resistance in the barley varieties.--To further test the relative resistance in the 5 strains of barley and correlate microscopic evidence of resistance with actual numbers of lesions formed on inocu- lated hosts, the following experiment was performed. Three 6-day-old plants of each variety were inoculated in the usual manner and kept under optimum conditions for germina- tion, appressorial maturation, and development of secondary hyphae. Four days after inoculation, a lesion count on l-cm-leaf sections were made on all plants. Each plant was considered as l replicate, and the 3 replicates of each variety averaged as shown in Table 2. Seventy-two lesions were formed on the susceptible variety Manchuria as compared to 0, 4, 36, and 39 lesions on the strains of barley with genes Algerian, Goldfoil, Kwan, and Psaknon, 61 respectively. These results were in agreement with the microscopic evidence of relative resistance of the various hosts. The percent of parasite units producing haustoria and functional secondary hyphae was 5, 15, 30, and 33 in the presence of the 4 genes (Algerian, Goldfoil, Kwan, and Psaknon) respectively (Table l). The count of the number of colonies on the leaf surface was done macroscopically in all cases. Microscopic examination of the strain with the Algerian gene revealed, however, some mycelial growth in a few places on the inoculated leaf sur- face. Microscopic examination of the epidermis of these same leaf sections revealed the formation of a few haustoria (4-5%). From this, it was inferred that the major gene for resistance in this variety suppressed the reproductive activity even though 4-5% of the parasite units had succeeded in establishing a successful, though short-lived, host- parasite relationship. The various mechanisms by which the Algerian gene con- ferred resistance to the host could be identified as follows: 1) Exclusion of 95% of the applied conidia from enter- ing the host 2) Delay in early haustorial development 3) Reduction of final size of haustoria 4) Distortion of a small portion of the haustoria 5) Suppression of fungus sporulation even after a successful host-parasite relationship was established. 62 Of all these, the first and the last mechanisms appeared to be of most importance in insuring a high level of macro- scopically observable resistance in the host. Brief review of sequential events ofyprimagyinfectio§.-- l) Germination of conidia--over 90% of the applied conidia germinated in 4 hr. 2) Formation of appressorial initials-over 90% of the applied conidia produced appressorial initials in 4 hr (5-8th hr after inoculation). 3) Maturation of appressoria--over 90% of the applied conidia produced mature appressoria in 4 hr, (6-9“ hr after inoculation). 4) Penetration--this most probably occurred around the 10th hr after inoculation. 5) Formation of haustoria--infection pegs were detected in epidermal cells 11 hr after inoculation. The haustorial body reached full Size 18 hr after inoculation. Finger-like projections on the haustorium elongated between the 18-36th hr. 6) Appearance of secondary hyphal initialS--over 90% of the applied conidia formed secondary hyphal initials in 6 hr (17-23rd hr after inoculation). 7) Development of functional secondary hyphae--ca. 90% of the applied conidia produced functional secondary hyphae by the 30th hr (in susceptible host-parastie combinations). When a successful host-parasite relationship was 63 established, functional secondary hyphae were formed which gave rise to secondary and tertiary appressoria, and subse- quently conSpicuouS pustules were formed on the inoculated leaf. Histochemical studies.--Few, if any histochemical stud- ies reported in the literature were on the very early interactions. Comparisons of healthy and diseased tissues are usually studied several days after inoculation. By this time, host-parasite relationships are established and external symptoms are apparent. There seemed to be a general agree- ment that histochemical changes in the host occurred simul- taneously with macroSCOpic symptom development and sporulation of the fungus. An attempt was made to detect early interactions between the host and the parasite before the onset of exter- nal visible symptoms. These early interactions are of prime importance in establishing parasitism. Since penetration of the host began around the 10th hr after inoculation and secondary hyphal initials began to form at the 16th hr, it was this 6-hr-period that was mainly investigated. However, histochemical changes beyond this period were also studied. As evaluated by the procedures used, there was an in- creased succinate dehydrogenase activity around the haustorium as early as 12 hr after inoculation, i.e. shortly after penetration. Tests made 12, l4, 16, 18, 24, 48, 72, and 144 hr after inoculation showed a marked increase of succinate 64- TABLE 2. Effect of genes for resistance on lesion forma- tion on leaves of barley inoculated with E, , graminis f. sp. hordei. Data are expressed as lesions per linear cm of inoculated leaf Manchuria Algerian Goldfoil Kwan Psaknon 110 O 3 33 56 55 0 0 29 49 51 0 6 46 42 Avg 72 Avg 0 Avg 4.5 Avg 36 Avg 49 Percent8 functional secondary hyphae formed on the various varieties are: Manchuria Algerian Goldfoil Kwan Psaknon 87 5 15 3O 33 a) Values obtained from another experiment. 65 dehydrogenase activity around the haustorium (Fig. 15). A positive test was characterized by numerous blue crystals of diformazan formed around the haustorium, while the lack of such crystals constituted a negative test. Three types of controls were run with every experiment. In one control, the tetrazolium salt (Nitro-BT) was not added to the reaction mixture. In another, no succinate was added. In the third, the succinate was replaced by substrates of other dehydrogenase systems, i.e. glucose-6—phosphate, lactate, malate, citrate, and acetaldehyde. In all controls where the tetrazolium salt was omitted, there was a com- plete absence of blue crystals, whereas when no substrate was added, very few and sporadic blue crystals formed most likely because of the occurrence of succinate in the host cells (Fig. 15). The increase in enzymatic activity was limited to the inside and around the haustoria. The only other places where such activity was detected were the chlor0plasts and both ends of the closed stomata (Fig. 15). These findings indicated that even 2 hr after pene- tration some kind of interaction between the host and the parasite occurred. Whether this increased enzymatic activity was stimulated in the host by the parasite or originated in the parasite was not determined. In either case, the results indicate an interaction between the host and the parasite at an early stage in primary infection. The activity of acid phosphatase was difficult to 66 determine. Results were not conclusive although 2 general methods for testing this enzyme and several other modifi- cations were tried. A major modification in fixing the tissue was tried. This consisted of fixing the intact inoculated leaf for 16-24 hr in Baker's solution (1.3% calcium chloride in formaldehyde) prior to peeling the epidermis and running the test. This eliminated the massive production of acid phoSphatase in the host cells caused by stripping the epidermis, and permitted the examination of any enzymatic changes around and in the haustoria. Controls consisted of omitting either the sub- strate or the eosin red. In some experiments, haustoria stained red-brown color (Fig. 16), but these results were erratic and hard to reproduce. What was more important was the continuous erratic results in critical controls for these tests. Although Atkinson and Shaw (3) reported increased acid phosphatase on and in the haustoria, all attempts to reproduce their work, using identical material and techni- ques, failed. Tests were run as early as 11 hr after inoculation to as late as 6 days after inoculation. No modifications of the original histochemical method for glucose-6-ph0Sphate dehydrogenase (34) were attempted other than increasing the time of exposure of the tissue to the various chemicals used. Controls consisted mainly of omitting the substrate, the tetrazolium salt (MTT), or the cobalt chloride. In some experiments, black-gray deposits 67 Fig. 15. Succinate dehydrogenase activity on and in the haustoria of E. graminis f. Sp. tritici. A) Blue diformazan crystals on haus- toria. B) Control (omitting the succinate from reaction mixture). C) Control (omitting tetra- zolium salt from reaction mixture.) D) Enzymatic activity in the chloroplasts. (370x) 68 69 were formed on and around the haustoria, but these results were not reproducible and similar deposits were observed in most control experiments. Cross—inoculatgon experimentS.--The purpose of these experiments was to determine if infection occurs in a supposedly incompatible system, and to further test the formation of functional secondary hyphae as a criterion for the establishment of a successful host-parasite relation— Ship. Manchuria barley was inoculated with EE_graminis E, g2. tritici and Little Club wheat was inoculated with E, graminis E. gg, hordei. Inoculated plants were kept under Optimum conditions for infection and the percentage of secondary hyphal initials formed was determined 18 hr after inoculation, and at 2-hr intervals thereafter. Even with these supposedly incompatible systems, secondary hyphal initials were formed. When wheat mildew was inoculated onto barley, 69% of the applied conidia formed secondary hyphal initials 23 hr after inoculation (Fig. 17). or the 69%, only 4% formed haus- toria and functional secondary hyphae. When wheat plants were inoculated with E, graminis E, sp. hordei, 24% of the applied conidia formed secondary hyphal initials and of these only 0.75% were capable of producing haustoria and functional secondary hyphae (Fig. 17). These results once again emphasized the fact that only the formation of funct— ional secondary hyphae (or haustoria) Should be taken as a 70 Fig. 16. Acid phosphatase activity on and in the haustoria of E, graminis E, gg. tritici. A) Enzy- matic activity 48 hr after inoculation. B) Enzy- matic activity 72 hr after inoculation. 0) Control. (575x) 72 .asosz SsHo oHesaS co Aaooaos .Sm .S mHSHBmMW xmv zowHHa hoHHom Am .hoHHmn SHHSSosz So AHoHHHHP «mm aw mHSHaonm amv BoSHHa pmoSB A4 .mpmoS oHpHpSoomSmSoS So mHmHHHSH HSSSSS SmocSooom mo SOHpoahom .SH .mHS 73 -Q’ N <[ (D? u I N 0‘ HN \ \O \ \ I O '\ 1~ \ ‘\ \42 L l J l l l l i O O O O O O 0 N (D ID Q‘ to N - BVHdAI-l AHVONOOBS JO 39V1N3083d HOURS AFTER INOCULATION 74 criterion for the establishment of a successful host- parasite relationship. DISCUSSION.--Primary infection in powdery mildew is made up of several component phases (30). These phases are distinguished on a morphological basis and by a diff- erence in response of each phase to various environmental conditions. When optimum conditions for every phase were reached, the parasite population was synchronized; that is, most units of the p0pulation reached each phase within a 4 hr period. Also, the primary infection process was shortened appreciably and the infection efficiency was greatly increased. About 90% infection was obtained in susceptible host-parasite combinations of wheat and barley; this is far higher than any other known host- parasite combination. This host-parasite combination, with a fairly well-defined kinetics, lends itself to quantitative studies of host-parasite interaction. It is possible to predict what portion of the parasite popula- tion is in a particular stage of development at a particular time. After maturation of appressoria, a fine penetration peg emerges from the center of the appressorium into the lumen of the cell (11, 40, 41). Under optimum conditions, penetration starts as early as the 10th hr after inoculation. Corner (11) reported that penetration by powdery mildew fungi begins 24 hr after inoculation at 20 C and 100% RH. There seemed to be a considerable lag in the progress of primary infection as reported by Corner (11). Slow 75 76 progress of infection reported by Corner may be due to sub-optimal environmental conditions for each component phase. The infection pegs undergo a series of morphological and cytological changes between the ll-l8th hr after inocu- lation. The changes result in formation of haustorial bodies which reach their full size 18 hr after inoculation. At this time (18 hr after inoculation), secondary hyphal initials begin to appear on the sruface of the inoculated leaf. There was a correlation between the formation of haustorial bodies inside the epidermal cells and the Simultaneous appearance of secondary hyphal initials on the leaf surface. It is reasonable to assume that secondary hyphal initials are not formed until the haus- toria reach a certain stage of development. In all hosts studied, susceptible as well as resistant, there was a uniform pattern of morphological and cytological develop- ment of haustoria except for minor modifications brought about by the major genes for resistance. Microscopic observations of the development of haustoria in susceptible and resistant hosts gave an in- sight to some mechanisms underlying resistance and susceptibility. The action of these major genes for resis- tance was during the early stages of the initiation of primary infection. The effect of all genes was to exclude a portion of the parasite population from infecting plants carrying the genes. It was shown e.g. that whereas about 77 90% of the parasite units could infect susceptible Man- churia barley, only 5, 30, and 33% of the parasite units could infect Algerian, Kwan, and Psaknon barley respec- tively. It is very unlikely that these genes act during pro-penetration stages. Conclusions regarding infection by mildew do not agree entirely with conclusions of other workers, using other models. Flentje (l6) ascribed failure in host penetration to: 1) lack of infection pegs; 2) mechanical toughness of the cuticle; and 3) poor attachment of the appressoria and consequent failure to take the back pressure of penetration. Graf-Martin (20) attributed resistance of older barley plants to E. ggaminis f. Sp. hordei to the abortion of the infection peg due to the hardness or thickness of the cuticle and epidermis. It is conceivable that one or more of these mechanisms could be involved in mildew resistance, but other poss- ibilities Should not be overlooked. It is quite possible that even after infection takes place, the progress of the pathogen could be halted thus constituting a later line of defense in the host. It was demonstrated that even after a fully compatible host-parasite relationship was estab- lished, the Algerian gene suppressed completely the sporulation of the fungus and, consequently, repeating disease cycles were avoided. The Kwan gene induced a hypersensitive reaction 5 days after inoculation. This 78 resulted in the collapse of host cytoplasm and eventual disintegration of most haustoria. Several of the aforementioned mechanisms might be operative in resistant hosts. It is plausible to assume that highly resistant or immune hosts are very well "buffered" by having several of these "protective" mechanisms as compared to less resistant hosts. It would be conceivable, therefore, that the Algerian gene confers most of these mechanisms of resistance, whereas the Psaknon gene confers a more limited number of these mechanisms. Consequently, whereas the Algerian strain of barley is almost immune to barley mildew, the Psaknon strain is relatively susceptible. In compatible host-parasite combinations, secondary hyphal initials elongate and branch giving rise to secondary and tertiary appressoria which eventually develop into macrosc0pically visible pustules. A portion of the secondary hyphal initials never advanced beyond 2 u in length (i.e. never reached the functional form). On all hosts studied (resistant and susceptible), over 90% of the applied conidia produced secondary hyphal initials under optimum conditions. Even in non-compatible host-parasite combinations (barley mildew on wheat and wheat mildew on barley), secondary hyphal initials were formed. Since secondary hyphal initials can form in the absence of developed haustoria, the initiation of secondary hyphal 79 initials might be the result of a mechanical stimulus. Although this might be true, other factors could be in- volved. There seemed to be some specificity even in the formation of secondary hyphal initials. This was sub- stantiated by the fact that whereas over 90% of the para- site units produced secondary hyphal initials on susceptible hosts, only 69 and 24% of the parasite unit; produced secondary hyphal initials on Manchuria barley and Little Club wheat when inoculated with wheat and barley mildew respectively. In all hosts studied, there was a good correlation between the number of functional secondary hyphae on the surface of the leaf and the number of haustoria in the epidermis of the same leaf. The same relationship did not hold for secondary hyphal initials. This meant that secondary hyphal initials could form regardless of whether or not haustoria were formed. In fact, it was observed that while 95% of the applied conidia produced secondary hyphal initials, only 5% of those same conidia produced functional secondary hyphae and haustoria when the host plant possessed the Algerian gene. Therefore only functional secondary hyphae or haustoria Should be the criterion for the establishment of a successful host-parasite relationship. All attempts by other workers to study biochemical changes in the hosts, in response to infection, have shown 80 differences at the time of flecking or sporulation of the fungus (2, 3, 7, 26, 45). Allen (2) and Bushnell and Allen (7) reported that plants infected with powdery mildew had increased respiration, but these respiratory changes were not detected until 6 days after inoculation. Several isozymes of succinate dehydrogenase and malate dehydrogen- ase were reported in rust-infected bean plant 12 days after inoculation (45). In my study, histochemical tests revealed certain enzymatic changes in the host 2 hr after penetration. This was rather well established with succinate dehydrogenase activity at l2, l4, 16, 18, 24, 48, 72 and 144 hr after inoculation. The detection of succinate dehydrogenase in the appressoria before pene- tration, and around the haustoria (in the host) 2 hr after penetration indicate that either the enzyme moved from the parasite to the host or its synthesis was stimulated in the host by the parasite. Whichever the case may be, this is an indication of a host-parasite interaction early in the establishment of parasitism. Whether this is necessary to parasitism or is merely a Side effect was not determined. The optimum environmental conditions for germination, formation of appressorial initials, and maturation of appressoria were determined with relative case. This was not the case with optimum conditions for the formation of functional secondary hyphae. This is not hard to conceive 81 since all processes conducive to the establishment of infection take place prior to the formation of functionalsecondary hyphae. It is the formation of infection pegs at the lower side of the appressoria, the penetration of host cuticle and epidermis, and the development of haustoria that precede the appearance of functional secondary hyphae on the sur- face of the host. The progress of the parasite could, theoretically, be halted at any one of these steps, and finding the overall optimum conditions for this phase of infection might require finding the optimum conditions for each individual step. It was generally observed that the external structures of the fungus were sensitive to various environmental con- ditions. This was not the case with the haustoria, the only internal structures of the fungus. The development of haustoria was insensitive to light and RH. Insensitivity to light could be partly due to limited photosynthetic activity in epidermal cells. Lack of response to RH could mean that the fungus, being inside the host, has plenty of water. High temperature of 35 C blocked haustorial develop- ment. This temperature is well above the optimum for growth of many fungi. This host-parasite combination is a very useful system for the study of host-parasite interaction. Because of the ectoparasitic nature of the fungus, changes in both the host 82 and the parasite can be studied when in close association. The fungus population can be synchronized, allowing for kinetic studies. SUMMARY The differential response of the various stages of primary infection of powdery mildew (E, graminis) to different environmental conditions suggests that the primary infection is made up of several stages. These are: l) germination, 2) formation of appressorial initials, 3) maturation of appressoria, 4) development of the haustorial body, 5) the formation of secondary hyphal initials, and 6) the formation of functional secondary hyphae. Optimum environmental conditions were established for develOpment of each phase. Under optimum conditions, the parasite population was synchronized and most units of the p0pulation reached each phase within a 4 hr period. The host was penetrated around the 10-12th hr after inocula- tion. The haustorial bodies appeared 16 hr after inoculation and full-sized haustoria were detected 34-36 hr after inoculation. Prior to penetration, succinate dehydrogenase activity was detected only in the parasite. Two hr after penetra— tion, succinate dehydrogenase activity was detected on the surface of the young, develOping haustoria. This indicated that either the enzyme moved from the parasite to the host or its synthesis was stimulated by the parasite in the host. In susceptible hosts and in strains of barley with major genes for resistance, the number of functional 83 84 secondary hyphae on the surface of the host equalled the number of haustoria formed inside the host. 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