OGiCAL lAE OLDECAL AND CYTOL N GNOMONlA FRAGAR KLEBAHN MORPH STUDES i Thesis hat the Degreo of M. S. MKHIGAN STATE COLLEGE Sung Huang 19139 mnmmmmmu 31293 10696 3485 This is to certify that the thesis entitled Morphological and Sytological Studies inGnomonia framariae Klebahn. presented by dung Huang has been accepted towards fulfillment of the requirements for the Lab, degree in I‘JIORPHOLOGICAL AIID CYTOLOGICAL STUDIES IN GNOMONIA FRAGARIAE MEN 33' Sung gang A THESIS Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Botany and Plant Pathologr 1949 :[Htibts CONTENTS ACKNOTVJTLEml'ENT 0.00.00.00.00.00.00.00.000.000......l I. IImODUCTION .OCOOOOOOOOOOOOOOOOOOOO00.......0 2 II. REVIEW OF LITFJRATU‘RE 0.00.00.00.00...000...... 3 III. EXTERIMENTAL METHODS A. Media Used B. 1. corn meal agar 0000000000000000000000... 8 2. Maljt extract agar ...................... 8 3. Malt extract and corn meal agar ........ 9 4. Strawberry runners ..................... 9 Methods in Studying Spore Germination and Fruiting Body Initials.................1O Staining methods 1. Staining Paraffin sections .............11 2. I‘Iaceration methOd cocoo~oooooooooooooooell OBSERVATIONS AND EXPERIMENTAL.RESULTS A. B. C. D. E. F. G. Vegetative Hyphae .........................l4 Pycnidia and Pycnospores ..................14 Development of Pycnidia ...................16 Pycnospore Germination ....................17 Perithecia. Asci and.Ascospores ...........18 PerithecialDevelopment 1. The production of perithecia ...........2O 2. Perithecial initials ...................21 Cytology and Development of Asci ..........2l (“r-"3's r: 3'.“ (T) J'& .‘l‘f'j". ,5. )1 H. Germination of Ascospores ..................24 V. DISCUSSION A. Pycnidial Development ......................25 B. Sex Organs and Perithecial Development .....26 C. Crozier Development and Ascus Formation ....28 VI. SUMMARY.AND CONCLUSIONS .......................SO BIBLIOGRAPHY OOOOOOOOOCOOOOOOOOOOO0.0.0.0...0......32 - 1 .. ACKNOWIEDGMENT The writer is indebted to Dr. C. J. Alexopoulos for his helpful suggestions and invaluable guidance throughout the whole course of her experiments and in the preparation of the manuscript; to Dr. G. B. Wilson for his help in the cytological studies; to Dr. E. F. Woodcock for instruction in histological work. and to the Librarians of Michigan State College and the University of Michigan for arranging the loan of Klebahn's “Haupt-und Nebenfruchtformen der Ascomyceten' for reference. This work was done while the writer was on a scholar- ship granted by the Graduate School of Michigan State Colle- ge, which is hereby acknowledged with grateful thanks. The writer also wishes to thank the United States State Depart- ment for extending financial aid which enabled her to con- tinue her studies in the United States at a time when it was impossible to receive mail from her home in Shanghai, China. -2- CHAPTER I INTRODUCTION Among various isolations from diseased Robinson straw- berries collected in Lawrence, Michigan, a few proved of particular interest. Like most of the isolations, they pro- duced pycnidia with conddiophores and spores resembling those of Dendr0phoma obscurans, but they developed round, black perithecia with long beaks, typical of Gnomonia £13- m a fungus hitherto unreported from the Western Hemi- sphere. The purpose of this thesis is to study spore germina- tion, pycnidial and perithecial formation, and the cytology of ascus development. Because of time limitation, some phases of the work have not been completed, but the results obtained thus far are of sufficient interest to warrant this paper. Three isolates designated as DB1, DL2 and A1 were used in this study. DB1 was isolated from a berry, as a singlewpycnospore; mg was isolated from a leaf, also as a single pycnospore; Al was derived from a single ascospore isolated from a perithecium of DB1. Later D131 lost? its power to produce perithecia; and consequently most of the work was done with DI.2 animal. Dendrophoma obscurans cul- tures were used occasionally for the comparision of the two. - 5 - CHAPTER II REVIEW OF LITERATURE Gnomonig framiae was first described by Klebahn in 1918 (:57). He noticed the fungus in 1908, while searching for strawberry diseases in Hamburg, Germany. {According to Klebahn's description, the fungus produces black round per- ithecia which are sunkwin the tissues with their long beaks protruding, 'me asci are elongated and spindle-shaped, and the ascus wall is very much thickened at the gets forming a narrow canal. The spores are spindle-shaped, colorless, two-celled. Germination of ascospores takes place easily on agar. Germ tubes are produced after twenty-four hours from one or both cells. The mcelium consists of dense, thick walled, brown colored hyphae, and the aerial melium is grayish. Perithecia measure 250;: to 400}! in diameter; the beaks are approximately 1.5mm. long; the asci are 50-80}: x 7-10u; the spores are 2.5-3.5p X 13-19p. Klebahn does not mention pycnidia. He probably did not notice them on the host,and in culture the agar used was apparently not favorable for pycnidial formation. In 1930 G. and M. Arnaud studied strawberry diseases in France (7). On the surfaces'of some fruits they found small, light brown protuberances (pycnidia), which oozed spores easily when moist. Beneath the fruit tissue, they - 4 - also found young globose perithecia associated with the py- cnidia. Artificial cultivation of the fungus on quaker-oat agar, produced pycnidia only. When transfered to plum agar, the fungus produced perithecia with oncto four beaks. According to Arnaud, the pycnidla are light brown and sunken. The conidiophores are needle-shaped and bear at their tips cylindrical, oblong spores with two oil guttules one at each end. The spores are 6-7); long and about 2}). wide. Perithecia in culture are at first light brown, form one to four beaks, then turn black, carbonaceous. They are globose, 300 to 500,: in diameter, with one to four beaks, 500-1000u in length and 35-50}: in width. A great many eight spored, 33}: long asci are produced. Ascospores are oblong, fusiform, colorless and unicellular, with five to six oil guttules. Some spores are constricted toward the middle and two-celled. They measure 1143.5}; I 2-2.5u. Arnaud identified the perfect stage as Gnomonia liege- ;igg Klebahn var. Fructicola G. and M. Arnaud. Inspite of the fact that he recognizes the pycnidial nature of the im- perfect stage, he classifies it as Gloeosporium fragariae Nob" probably influenced by the fact that most known species of .nomonia have an acervulus as an imperfect stage. In 1941 Wormald and Montgomery (56, 5'?) found a Leaf -5- Blotch.fungus of strawberry leaves in Kent, England. The most important characteristics of the fungus are the small biguttulate spores measuring about 6 x 2p, borne on long branched conidiophores, and the brownish, waxy, pycnidia. The fungus is associated with large yellowish brown blotches bordered.by a dark line on the strawberry leaves. They referred this fungus to Phyllosticta gggndimaculannguback and Krieger, then decided to change to Zzthia fragarias Lei- bach'because of the fructifications which are brownish (pale yellow in culture), not black as in a typical Phyllosticta, and the consistency of the fruiting bodiesdtfiéEZaxy, not coriaceous or carbonaceous. Perithecia were found in 1944 (58) both in culture and on infected strawberry leaves. They are black spherical bodies with long necks. The asco- spores are small, with.four oil drops and some of them are two-celled. Theyassigned the perfect stage to Gnomonia, but did not designate the species. Their description of the perfect and the imperfect stages close resemble those of Gnomonia fragariae as given.by.Arnsud and Arnaud (7). Alexnpoulos and Cation (1) while investigating various strawberry rots in Lawrence, Michigan in 1947, found a very large percentage (76.3%) of the diseased fruits examined, bearing the pycnidia of a fungus they believed to be Egg- drophoma obscurans. Pure cultures were obtained by tran5~ lferring pycnospores or diseased tissue from fruits, calyces, and peduncles of strawberries to agar. Such cultures produ- ced fruiting bodies identical with.those produced by isola- tions from.diseased leaves with typical symptoms of Dendro- phoma leaf blight. Perithecia were discovered on agar and diseased parts of strawberries associated with the pycnidia. Plantings of ascospores and pycnidiospores both produced perithecia and pycnidia. This proved the relationship be- tween the perfect and the imperfect stages of the fungus. Different media were tried and it was found that no peri- thecia were produced on potato-dextrose, oat, nutrient, or maltose agar (with.two exceptions). In corn meal agar tubes, perithecia were crowded together forming a black band at the base of the slant. Alexcpoulos and Cation identified the perfect stage as a Gnomcnia and pointed out the similarity between the peri- thecia of their fungus and those of wermald and Mbntgomery found in England. They did not mention.Klebahn's fungus in their paper. The isolates used in the present investigation were isolated by these workers and turned over to the writer for further study. A comparison of the fungus at hand with the descriptions of Gnomonia fragariae as given by Klebahn and especially by Aynaud, leave little doubt that the Michigan -7- fungus is the same species as the European one. The rela- tionship of Gnomonia fragariae to Dendrophoma obscurang, however, needs further investigation. No other literature on Gnomonia fragariae appears to exist. -8- CHAPTER III EXPERIMENTAL METHODS A- W £529- 1. Corn meal agar. It is made by dissolving 19 grams of Difco corn meal agar into 1000 m1. of distilled water. The agar is brought to a boil, filtered through cotton, and ster- ilized in the autoclave at 15 pounds for 20 minutes. This medium is very clear, and consequently good for studying the germination of spores and the develop- ment of pycnidia. Perithecia are produced from three weeks to two months with difficulty. Therefore it was not used for studying perithecial development. 20 Malt eXtI‘aCt 8331'. In order to produce the perithecial stage more rapi- dly, malt extract agar was tried. It was prepared by mixing the following ingredients: Bacto nutrient agar............. 23 gm. Malt extract .. 10 gm. Dextrose ....................... 15 gm. Distilled water ...... 1000 ml. -9- This medium is good for mycelial growth, but is not good for studying fbrmation of fruiting bodies because as soon as the fungus grows, the medium turns black. 3. Malt extract and corn meal agar. 2-5% malt extract was added to Difco corn meal agar and was prepared in the usual manner. It is found that 2% malt extract is as efficient as 5% for perithecial production, therefore 2% was used thereafter. The fungus grows quite well on this medium and forms perithecia in about two weeks. Pycnidia are developed considerably late; so thatXwas found to be the best med- ium for studying perithecial development. 4. Strawberry runners. Since the fungus is found on strawberries in nature, parts of the plant that can be infected should be a good medium for cultivation. Strawberry runners were cut and put into test tubes with moistened cotton at the bottom. The test tubes were then plugged with cotton and steri- lized in the autoclave at 15 pounds for 20 minutes. Spo- res or mycelium on a bit of agar were inoculated on the runner B a After two days, white mycelium could be seen withthe naked eye. Two weeks later yellow pycnidia were found -10.. pushing through the epidermis of runners inoculated with A1. Perithecia were recognized by their long, black beaks within one month after inoculation with DL2 and A10 Transfers from runners to the plates. seem to yield colonies with new strength. This method was used for reviving the fungus. B. Methods 1.3; Studying §pore Germination and Fruitng Body Initials. A large cover glass was placed in a Petri dish and sterilized in the oven at about 150°}? for four hours. For studying spore germination and pycnidial formation, corn meal agar was poured on the cover glass so as to form a layer somewhat less than 1mm. in thickness. For perithe- cial formation, 2% malt extract was added to corn meal agar. Spore suspensions were made by touching the spore exu- dates with a sterile needle and transferring into a drop of sterile distilled water. A small loopful of the suspension was then transferred onto the agar over the central part of the cover glass. At the same time a glass ring 40 mm. in diameter, was sealed on a Petri dish with vasiine, and a little water was ~poured in the ring. The free edge of the ‘ ring was sterilized by wiping with 75% alcohol. Then the - 11 - cover glass with agar was cut out of the Petri dish and inverted on the ring forming a van Tieghem cell. Spores were examined for germination every four hours. In studying the development of pycnidia and perithecia, a bit of agar with mycelium.was transfered instead of spore suspension. {After the fungus grew about 20 mm.from.the inoculum, a Van Tieghem cell was made in the manner descri- bed. .A daily search was made under microscope for perithe- cial initials. Any structure suspected of being a perithe- cial initial stage was checked for further development. C. Staining methods. 1. Staining paraffin sections. Bits of agar with various stages of perithecia were cut from the plates and fixed in chrome-acetic acid. lficrotome sections were made from.3u to 7 P in thickness, and it is found that 5P is the most satisfactory. Heid- enhain's iron-alum.hematoxylin and triple stains were tried. For studying the structures of the perithecium, that is, the wall, the beak, the cavity and the arranges ment of asci, both stains are satisfactory. But for the cytology of asci, this technique was found unsatisfactory. 2. maceration method. This method is described (48) as follows: - 12 - 1/ Fix in 3:1 absolute aleohol-propionic acid (45%) solution for 48 hours. 2/ Carry through various concentrations of alcohols to water which is slightly acidified with a few drops of propionie acid. 3/ Transfer to 4% iron alum for ten.minutes. 4/‘Wash with acidified water overnight with several changes. 5/'Stain with propionocarmine. In following these directions, step 2 was once omit- ted by accident, and the results were found to be super- ior to those obtained.by following the above method. The procedure which was finally adopted as a result may be outlined as follows: 1/'Ki11 in absolute ethyl alcohol-propionic acid solution for 48 hours. 2/ 4% iron-alum 10 minutes. a/‘Wash in running water for four hours. 4/ Stain on slide with propionocarmine. The stain was made according to Sass's acetocarmine - 13 - formula. (48), by substituting propionic acid for the acetic acid. The slide is made as follows: l/'Hacerate a perithecium.in a drop of the stain on a. Slide. 2/ Drop cover glass over mount and tap gently. 3/ Pass slide quickly over an alcohol lamp flame Ieveral times. 4/ Drain off excess stain, and seal the edges of the cover glass with glycerince Jelly. 5/ The slidesshould be aged. They show good results usually after two weeks or longer. - 14 - CHAPTER IV OBSERVATIONS AND EXPERIMENTAL RESULTS A. Vegetative H! phae. 'me , The hyphae appear hyaline underAmicroscope and white to the naked eye. There are two types of hyphae; one is thin, the other broad ( Fig. 1 )_. The thin hyphae measure from.3.5u to 5.25u in diameter. These are the young and actively growing hyphae which enter in the formation of fructifications. The cytoplasmic con- tent is dense and granular and exhibits small oil drops. Sometimes the hyphae are highly vacuolated. It has not been possible to stain nuclei with the technique employed. Septa are very few and are far apart, but in a few cases close septations do occur. The broader hyphae vary from 8.75? to 10.5u in dimme- ter. They represent the older hyphae. No fruiting bodies have been found in association with this kind of hyphae. The cytoplasm is restricted to a very thin layer. There are large oil drops and vacuoles. The septa are quite close to each other. Aerial mycelium is produced when the culture is old. B. gycnidia and gycnospores. - 15 - Pycnidia are developed beneath the epidermis of various parts of strawberries. As they grow, the epidermis breaks open in a stellate manner, and the short beaks of the pyc- nidia protrude. In the presence of a considerable amount of moisture, the pycnidium.swells and the spores ooze out in droplets of water or in long gelatinous cirrhi through the ostiole ( Fig. 3 ). In nature the pycnidia are yellowish.brown with slight- ly darker beaks, but in culture they are pale yellow or white. The wall of the pycnidium is made of pseudoparenchy- ma cells about two to three cells in.th1aknell. The con- sistency of the wall varies with.moisture; it may be car- bonous or waxy. Pycnidia are circular in surface view, ranging from 135p.to 500p.in diameter, and are somewhat flattened in side view (Fig. 3 ). The height of the pycnidia without the beaks varies from.105u to 285p. The beaks are quite short, about 75p.to lOQp in length and 7Qp to 95p in width. Lining the inner wall of the pycnidium are numerous branched conidiophores ( Fig. 4). There may be two, three or four branches, the most common ones being two-branched. Pycnospores are borne at the tips of the conidiophores. After successive culturing,the fungus lost its power to form.conidiophores, and only a mass of spores within the thin walled pycnidium could be found. Meet of the spores are bacilloid, with round ends ( Fig. 6b ). Few of them are ellipsoidal ( Fig. 6c) and some are intermediate; that is, one end is round and the other is pointed (Fig. 6a). There are two to three guttules or oil drops in the spores, usually one at each end. They are very conspicuous and are often as large in diameter as the width of the spore. The size of the spores varies from 1075‘2p I 5.25-7.311- C. Development g£_§ycnidia. Kemptom.(36) mentioned two types of pycnidial develop- ment. mf the pycnidium initiates from the interlacing and anastomosing of hyphal branches from.different mycelial threads, its development is of the symphogenous type. If the pycnidium arises from the proliferation of one hyphal cell or from.branches of the same hypha,its development is of the simple meristogenous or compound meristogenous type respectively. Pycnidia of most fungi develop according to one of the two types. Mercer ('42) in his studies of Ms; richardiae found both types of development in that species. This seems to be also true of Gnomonia fragariae. The symphogenous type or pycnidial development is t- 1? - found in strain.A1. Pycnidial initials can be found under the microscope four days after inoculation. The first evi- dence of a pycnidial initial is the growing of several hyph~ as toward a common point ( Fig. 7 ). The tips of the hyphae then bend and intertwine ( Fig. 8 ). They intersect each other and form.the pseudoparenchymatous wall of the pycni~ dium ( Fig. 9, 10 ). mature pycnidia ( Fig. 2, 3 ) are found about two weeks after inoculation. The compound meristogenous type is found in strain.DL2. Instead of several hyphae intertwining, there is usually one hypha branching ( Fig. 11 ) and forming knots ( Fig. 12). These then proliferate into a.young pycnidium. The young pycnidium.is distinguished from.the young per- ithecium.in that the former is more or less irregular in shape and the latter is spherical and more compact ( Fig. 24) even in an early stage of development. The origin and develOpment of conidiophores and spores have not been studied. D. gzcnospore Germination. Hanging drops of corn meal agar were used for the stu- dy of pycnospore germination. No more than two or three per cent of the spores germinate within twenty-four hours. Forty eight hours after sowing, more spores are found to be germinating and the germ tubes of some are already forming branches. No further germination has been observed after forty eight hours. Total germination reaches about 15%. At the first stage of germination, the spore swells, usually enlarging in width rather than in length. A little protuberance now buds out from the spore ( Fig. 5a ) and in some cases one of the guttules moves to the edge, at the initiation of the bud ( Fig. 5e ). Germination usually occurs on the side of a pycnospore, near the center ( Fig. 5a ) or to one side ( Fig. 5B ). Germ tubes have been found pushing out from the ends of spores ( Fig. 5f ) , but this type of germination is very rare. Figures 5a to 5d show different stages in spore germination. Branches are formed very soon» after the germ tubes have elongated, but the sep- ta are formed considerably later. Profusely branched my- celium can be seen within the third day of germination. E. Perithecia, Asci and Ascospores. In nature perithecia are recognized by their black, long, bristle like beaks which protrude from the strawberry tissues in which the perithecial bodies are deeply buried. Normally each perithecium has one beak. In culture, however, from one to four beaks are usually produced. Sometimes groups fl of six to ten beaks were found on a single perithecium, -‘19 - but such perithecia, when crushed, cdntained no ascospores. Figure 48 shows some of the various types of perithecia that have been observed. The wall of the perithecium is shining and leathery, and consists of four to seven layers of pseudoparenchymatous cells ( Fig. 47 ). The wall is black when examined with the naked eye or hand lens, but The dark brown when observed undeericroscope. Within the perithecium.are numerous eight-spared asci. They are characterized by the thick apices provided with a canal ( Fig. 53, 54 ). The ascus wall disintegrated'before the ascospores are released from.the perithecium. Therefore when a very mature peritheciun is crushed, there are no asci, but a mass of ascospores held together in groups of eight. The ascospores are hyaline, spindle-shaped, and two- celled. When the spores are young, they are one-celled with six to seven oil droplets ( Fig. 49, 55 ). ‘As they become mature, a septum develops, dividing the spore into two cells. The two cells are slightly unequal, each of them usually contains three oil droplets ( Fig. 50, 54). Average measurements are as follows: Perithecia 250- 550? in diameter, with beaks 550-1500p in length and 45- 60? in.width; asci 55-85u>XZ7-llp; ascospores 7-lgp X.2- 3P0 F. Perithecial Development. 1. The production of perithecia. various methods have been tried in efforts to pro- mote the production of perithecia in different fungi. The effects of thiamin, biotin, and different sources of sugars have been studied by many workers (11, 12, 29, so, 51, 32, as, 39 ). The influence of chemical stimulation and extracts from other fungi are also em! phasized (9, 54, 40, 54 ). Furthermore, ultra-violet irradiation was found to be very useffil in some fungi (49, 50). Gnomonia fragariae will form perithecia on corn meal agar from two weeks to two months irregularly. To study the initial stages of the development is quite a problem; when too much agar is poured on the plate, it is impossible to see the structures with the aid of the microscope, and when a thin layer of agar is poured, it will dry out before the initials are formed. With 2-5% malt extract in the corn meal agar, plxxi of hyphae were found within four days,vblack young perithecia were obtain- ed within seven days and long-beaked.mature perithecia occurred about two weeks from the date of inoculation. This result was quite satisfactory, therefore other methods were not tried for the production of perithecia. - 21 - 2. ferithecial initials. Perithecial initials could not be determined with certainty. There were several interesting branches and coils growing out the hyphae ( Fig. 13 - 21 ) about the third day of inoculation. They were suspected to be the perithecial initials, but further development could not be traced. Some of the advanced stages ( Fig. 23, 24 ) were found, but their origin was unknown. No spermagonia have been found during the develop- ment of perithecia. The branches shown in figures 15, 20, 21 may represent antheridia. Since fertilization has not been studied, the above statement is subject to further investigation. o. mum and W 215. A__..sci- When a young perithecium with an already differentiated beak about 20? long ( Fig. 43 ), is crushed and stained, a group of ascogenous cells ( Fig. 25 ) come out. Mbst of them.are binucleate, few are uninucleate. Occasionally croziers are found among them. When the beak reaches its full length, but with the ostiole not yet open ( Fig. 44), various stages of the asci can be found within the peri- thecium. Croziers are initiated by the binucleate ascogenous - 22 - cells. A hook-like structure is produced from the ascoge- nous cell ( Fig. 26 ). One of the two nuclei moves to the hook and the other remains in the cell ( Fig. 27 ). Then those two nuclei divide simultaneously giving four nuclei ( Fig. 28 ). Septa are developed so as to separate the four nuclei into three cells; one in the hook, two in the penul- timate cell and one in the basal cell ( Fig. 29 ). nuclear fusion takes place in the penultimate cell ( Fig. 30, 31 ) immediately after the septa are laid down. No division figures have been.observed in croziers. The ascus originates from the elongation of the penul- timate cell after nuclear fusion. Figure 52 which shows the long big fused nucleolus with chromosomes, probably re- presents the early prophase I. .At metaphase I chromosomes are arranged on the equato- rial plate (Fig. 35 ). The propiono-carmine does not have affinity for spindle fibers, so those structures are not shown. The nuclear vacuole at this stage is spindle shaped. At anaphase I the chromosomes are reduced very much in size and the homologues are separated into two groups ( Fig. 34 ). The individual chromosomes are very indistgkt in telophase I (Fig. 55). Prophase II is apparently of very short duration, for - 23 - this stage was missing in all preparations. Vishaped chromosomes ( Fig. 36 ) which.are the sister chromatids that attach to each other by means of centromeres, are the characteristics of the metaphase II. At anaphase II the W sister chromatids become separated to opposite poles (Fig. 57 ). Chromosomes of those two stages are fairly distinct so the basic haploid chromosome number is determined as eight. The two nuclei at that time may lie side by side (Fig. 36 J, or separate ( Fig. 37 ), each of them contain- ing sixteen chromatids. A very interesting figure was observed in metaphase III. One of the asci had two nuclei with chromosomes arranged on equatorial Plates, and the other two nuclei with chromosomes arranged in rings ( Fig. 58 J. The ring arrangement is typi- cal of meiotic division. If this is the case, a.braohymeio- sis as proposed by Gwynne4Vaughan and‘Williamson (27) for Ascobolus magnificus ma£riaking place here. The other fig- ures of metaphase III, anaphase III (Fig. 59) and telophase III (Fig. 40 ) are normal mitotic divisions. The chromosome numbers here are eight in each nucleus. The octonucleate stage of asci with large nucleoli have been found ( Fig. 42 ). While the walls of the ascospores are formed ( Fig. 41 ), numerous oil droplets present, inter- fere with the staining of the nuclei. At this time the wall of the ascus begins to thicken at the apex and the canal is formed. When the spores are mature, they are two-celled. Re— peated attempts to stain their nyclei with propiono-carmine, have failed. H. Germination g£.Ascospores. The ascosporls as described before are two-celled. The two cells are unequal in size. The germ tube usual- ly come out from the larger cell of the ascospore (Fig. 52a, b, c, d, f ). Germ tubes emerging from.both cells (Fig. 52e) or from the tips of the smaller cells (Fig. 52g) have been found, but such cases are very rare. Ascospores usually germinate within twenty four to forty eight hours. - 25 - CHAPTER V DISCUSSION A. chnidial Development. hempton (36) who made extensivabtudies of pycnidial deveIOpment in several of the Sphaeropsidales, classified the types of development into three groups: 1. the simple meristogenous type; that is, development of a pycnidium by the proliferation of one hypha, 2. the compound meristoge- nous type in which a pycnidium is developed from the inter- twining of branches from the same hypha; and 3. the sympho- genous type in which the interlacing of different hyphae takes place. He found that some fungi such as Phoma her- barum,_§eptoria helianthi, and Sphaeronemella fragariae developed their pycnidia entirely by the first of these methods, that the pycnidia of Sphaeropsisvmalorium, and of Hendersonia opuntiae according to the second.method, and that of Diplodia manilliana, Sphaerella nigerristigma and gicinnobolus cesatii developed their pycnidia according to the third method. On the other hand he also noted that a number of fungi may employ two or even all three methods in develop- ing their pycnidia. Among them are: Phoma destructive, Spharopsis citricola, Sgptoria polygonum etc. In Gnomonia fraeariae, the pycnidia have been found to develop both by the compound meristogenous and the symphogenous methods. The first of these was found to prévail in strain.A1, and - 25 - the second in strain DL2. In its pycnidial development Q. fragariae resembles most Fhoma richardigg found by Hercer (42) to form.its pycnidia according to both the meristo- genous and the symphogenous types of development. B. Sex.0rgan§ and.Perithecial Development. Nichols (44) found two kinds of perithecial initials in Pyrenomycetes. The first type of initials are developed from.swollen cells in the mycelium. A single cell divides to form a solid sphere of pseudoparenchymatous tissue, the asci arising from the differentiation of interior cells of this tissue. The second type of initials are curved bran- ches or several coiled structures which develop into peri- thecia. Arnold (8) who studied.gporonia leporia Nieaal founi:¥he perithecia originate as single twollen cells, each of which contains one nucleus. Such types of cells are not found in Gnomonia fragariae. In melanospora zamiae (18), Neurospora tetraspermaDodge (l7), Ceratostomalla fimbriata (5, 10, 28 ), and Ceratostomglla nuitignngigg”(4), etc. peri- thecia are found to develop from either recurved branches or coiled structures. Such kind of structures have been found in Gnomonia fragariae but actual perithecial develop- ment h35fi not been traced directly to them. Antheridia were found in Ceratostomalla fimbriata (10), Ceratostomglla multiannulata and Mycosphaerella melonis - 27 - (17). Elliott even saw a single nucleus from the antheri- dium.pass through the trichogyne int&:%ogonium.in Cerato- stomglla fimbriata. Gwynne-vaughan and Broadhead (27) stuiéd the same fungus, about ten.years after, but failed to find antheridia. .Although.Andrus (4) found antheridia in.ggratgptomgllgLmplfiiannulata, he could not trace their function. Most of the workers (14, 18, 19, 4:5, 45, 52) did not find antheridia. In Gnomonia f;agariae, the presence of antheridia is uncertain. Figures 15, 20 and 21 show structures which somewhat resemble antheridia, but their function is unknown. Spermagonia were found in Gnomonia erythrostoma (l4) and Polystigma rubrum.(13), but were re- garded as functionless. Trichogynes were found in Gnomonia fizzghgggggm§.(14) and Polystigma rubrum (13), but the con! nection with the ascogonium.was uncertain. Brooks consi- dered them.as respiratory and Blackman and wellsford said that they were vegetative in function. Trichogynes have not been found in Gnomonia fragariae. Blackman and'Wellsford (13) considered the ascogenous hyphae to develop later from purely vegetative cells within the perithecium. NUclear fusion.takes place in the ascus andisometimes in the ascogenous hyphae. Caley (16) reach- ed the same conclusion in Nectris galligena. MbIntosh (41) considered the hyphal outgrowth from ascogonia of Nectria mammoidea to be abortive structures. Cookson (l9) believed -28- the central cord of Melanospora zanfiae Corda to become dif- ferentiated into fertile and sterile elements. The frag- mentation of the fertile portion gives rise to ascogenous cells. The crozier type of cell division among the asco- genous cells give origin to the asci. This is the case in Gnomonia fpagariae. C. Crozier Development and Ascus Formation. Croziers were first found by Dangeard (21), as hook- 1ike structures arising from the ascogenous hyphae. Asci developed from the penultimate cells of the crozier. Some- times the binucleate ascogenous cells give rise to asci directly without the formation of crozier, such as in the case of Gnomonia erythrostoma (14) Qghigbolus gramini; (35)etc. Backus and Keitt studied venturia inaegualis (10), and found the primary ascus nucleus to be large and to show prominent chromatin beads. Three successive nuclear divi- sions in the ascus result in the formation of eight nuclei. The number of chromosomes is questionable, the haploid num- ber being gamewhere between four and six. The method of formation of the eight spores in the ascus seems to be more or less uniform.in most cases, but the number of chromosomes and the process of meiosis are difficult to determine and are therefore uncertain. Colson found that the haploid - 29 - number of chromosomes is six and that reduction takes place in the first ascal division in Neurospora tetrasperma (18). In Glomerella (53) the number of chromosomes was determined as four in some of the pseudoparenchyma cells surrounding the ascogenous hyphae. In Gnomonia fragariae, the haploid number of chromosomes is eight, but it was not possible to determine with certainty where reduction takes place. 1. 2. 3. 4. 5. 6. 7. 8. - 30 - CHAPTER VI SUMMARY AND CONCLUSIONS The mycelium.of Gnomonia gragariae consists of two types of hyphae. the thin type and the broad type. Fruiting bodies are found to be derived from the thin type hyphae. Pycnidial formation is both symphogenous and compound meristogenous. The pycnospores germinate within twenty four to forty eight hours. Germ tubes may originate at any part of the spore, but usually emerge from the sides. Initial stages of perithecial formation could not be determined with certainty. It is believed that the recurved or coiled structures are the most probable initial stages. Croziers are formed from the binucleate ascogenous cells. Asci are developed from the penultimate cells of cro- ziers. after nuclear fusion. Three successive divisions of the nucleus in an ascus form eight spores. Each spore forms a septum.at matu- rity, so as to become two-celled. Germ tubes of germinating ascospores are usually found on the side of the large cell. Sometimes both cells germinate. 9. A comparison is made between Gnomonia fragariae and other Pyrenomycetes. - 32 - BIBLIOGRAPHY l. Alexopoulos, Const. 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F. and J. C. Walker. 1949. Mbrphology and 18. 19. 20. 21. 22. 25. 24. variability of the cucurbit black rot fungus. Jour. of Agr. Research, Z§:81-102. Colson, B. 1934. The cytology and morphology of Neurospora tetrasperma Dodge. Ann. Bot., 58:212. Cookson, I. 1928. The structure and development of the perithecium in melanospora zamiae, Corda. Ann. 3017. , $3255-2690 Cutter, an M. Jr. 1946b. Smear methods for the study of chromosomes in.Ascomycetes. Stain Tech., 21: 129-131. Dangeard, P. A. 1894. La reproduction sexuelle des Ascomycétes. Botaniste, 5.30. De Bary, A. 1887. Comparative morphology and biology of the fungi, mycetozoa and bacteria. Engl. transl. Dodge, B. 0. 1923. Origins of the central and ostio- late cavities in pycnidia of certain fungus para- sites of fruits. Jour. Agr. Res., 23:745-759. 1937. The perithecial cavity formation - 35 - in a Leptosphaeria opuntia. Nycologia, 22:707-716. 25. Elliott, J. A. 1925. A cytological study of Ceratos- tomella fimbriata. thtopath., 15:417. 26. Gaflmann, E. A. and C. w. Dodge. 1928. Comparative morphology of Fungi. 701 pp. MCGraw-Hill Book Co. Inc., New York. 27. Gwynne-vaughan, H. C. I. and.Broadhead, Q. E. 1936. Contributions to the study of Ceratostomella fim- briatao Ann. 3013., EQ:747-7590 28. and H. S. Williamson. 1932. The cytology and development of Ascobolus magnifi- cus. Ann. Bot.,4§:653-670. 29. Hawker, L. E. 1939. The influence of various sources of carbon on the formation of perithecia by Melano- gpora destruens Shear in the presence of accessory growth factors. Ann. Bot. N. 8., 3:455-468. 30. 1936. The effect of certain accessory growth substances on the sporulation of melanospora destruens and of some other fungi. Ann. Bot., 12 699-7180 31. 1939. The nature of the accessory growth factors iniiuencing growth and fruiting of 32. 34. 35. 36. 37. 39. - 55 - melanospora destruens Shear and of some other fungi. Ann. Bot... gem-eve. Hawker, L. E. 1939. Effect of growth substances on growth and fruiting of Melanospora destruens. Na- ture, 142:1038. Harris, H. A. 1935. morphological studies of Septoria lycopersici. Phytopath., 25:790-799. Heald, F. D. and Venus W. Pool. 1909. The influence of chemical stimulation upon the production of peri- thecia by melanospora pampeana Speg. Neb. Agr. Exp. Sta. Rept.. 235130-132. Jones, S. G. 1906. The development of the perithecium of gphiobolus grmminis Sacc. Ann. Bot., 49:607-629. Kempton, F. E. 1919. Origin and development of the pycnidium. Bot. Gaz., 68:233-254. Klebahn, H. 1918. Haupt-und Bebenfruchtformen der Askomyzeten. Ester Teil, Leipzig, 285-288. Leonian, L. H. and Virgil G. Lilly. 1945. The compa- rative value of different test organisms in the microbiological assay of B vitamins. west Virginia Agr. Exp. Sta. 3111. NO. 5190 Lilly, V. G. and H. L. Barnett. 1947. The influence 40. 41. 42. 43. 44. 45. 46. 47. - 57 - of pH and certain growth factors on mycelial growth and perithecial formation by Sordaria fimicola. Amer. Jour. of Bot., 22:131-137. EeCormick, Florence A. 1925. Perithecia of Thielavia basicola Zopf. in culture and the stimulation of their production by extracts from other fungi. Conn. Agr. Exp. Sta. Bul., 269:539-544. MCIntosh, A. E. S. 1927. Perithecial development in Nectria mammoidea. Brit. Assn. Adv. Sci.,2§:387-388. Mercer, W. B- 1913. On the morphology and development of Phoma richardiae, n. sp., mycology Centr., 2:244- 253. Miles, L. E. 1921. Leaf spots of the elm. Bot. Gaz., 11:161-196. Nichols, M. A. 1896. The morphology and development of certain Pyrenomycetous fungi. Bot. Gaz., 22:301. Pomerleau, Rene. 1937. Recherches sur le Gnomonia ulmea (schw.) Thum. (Biologie-Ecologie-Cytologie) Naturalists Canadien, Nov.l937-Oct. 1938. Rumbold, Caroline T. 1941. Ascospores of Ceratostomella ips and Ceratostomella montium. 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Effects of fungous extracts upon the initiation and growth of the perithecia of Ven- turia inaegualis (cke.) Wint. in pure culture. - 39 - Phytopath. , 11:835. 55. weir, F. A. and.F. T. Wolf. 1947. The Fungi. V01. I. viii+438 pp. John Wiley & Sons, Inc., Hew'York. 56. wormald, H. and H. B. S. Mbntgomery. 1941. Leaf blotch of strawberries. Gard. Chro. 3d Ser., 110:180. 57. 1941. Strawberry leaf blotch. E. Malling Res. Sta. Rpt. 1941, p.44. 58. Wbrmald, H. 1944. Strawberry leaf blotch fungus. Gard. Chl'Oo 3d Ser., 1163160-1610 PLATE I Fig. 1. ---- vegetative hyphae. showing thin and broad types; X 1300. Fig. 2. ---- Mature pycnidium: X 270. Fig. 3. ---- Spores ooze through ostiole of the pycnidium in long gelatinous cirrhi; X.120. . '4! l .... r »' e “' V. 4. "‘ """T‘Ct-‘i. .. .-....l _ ‘7r “‘9: I. "‘1 Q .. -.r . 1. V, .‘ua! Pm. PLATE II Fig. 4. ---- Branched conidiophores; X 1300. Fig. 5. ---- Germination of pycnospores; X 1800. a-d. ---- Different stages of pycnospore germination from the side of the pycnospore. e. ------ The guttule initiates the germ tube. f. ------ Germ tubes emerging from the tip of the pycnospores. g. ------ Germ tube emerging from the side of the pycnospores near the tip. Fig. 6. ---- Different pycnospores: a. - ----- chnospore with one pointed end and one round end. b. ------ chnospore with round enis. c. ------ chnospore with pointed ends. Plate ll PLATE III Figs. 7 - 10.----Symphogenous type of pycnidial develop- Fig. F180 Fig. Fig. Figs Fig. Fig. 7. 8. 9. ment: ----Hyphae growing toward a common point; X 950. ----Hyphae intertwine; X5950- ----Hyphae intersect; X.1500. 10. ---Young pycnidium with pseudoparenchymatouS‘ wall 3 X 1500. 11-12. ------ -Compound meristogenous type. 11. ---Branching of hypha; X 1800. 12 ---Hyphae forming knots; X 1500. Haul" PLATE IV Figs. 13-22 ---- Probable perithecial initials; branches coming from.other hypha shown in figs. 15, 20 and 21 may represent antheridia; X 1800. Figs. 23-24 --- Advanced stages in perithecial develop- ment; X 950. Place u Fig. 25. Fig. 26. Fig. 27. Fig. 28. Fig. 30. PLATE V ---- A group of ascogenous cells: X 1800. ---- Early crozier formation; X 2100. ---- One nucleus moves to the hook; X 2100. ---- Conjugate division of two nuclei:.X 2100. ---- Formation of septa; X 2100. Figs. 29, 31.-Fusion of nuclei in the Penultimate cell; Fig. 320 Fig. 33- - Fig. 340 Fig. 550 Fig. 36. Fig. 37. Fig. 38. Fig. 39. Fig. 40. Fig. 41. Fig. 42. X 2100. ---- Early prophase I; X 2100. --- Metaphase I; X 2100. --—- Anaphase I: X 2100. ---- Telophase I;.X 2100. -—-- metaphase II; X 2100. ---- Anaphase II; X 2100. —--- Metaphase 111; x 2100. ---- Anaphase III;.X 2100. ---- Telophase III;.X 2100. ---- Ascus with eight young ascospores; X 2100' ---- Eight nucleated stage; X 2100. nah-""1 ‘ ~“... ‘73. -'. -.}'.~'.'. 7c~‘\'.' .a >. ‘ ‘_'1_-fJ,\'L' " _. . . L'z - 2"". . \ "51.-’1. ' _. (h. Fig. Fig. F18. Fig. Fig. Fig. 43. 44. 45. 46. 47. 48. PLATE‘VI Young perithecium; X 80. Beak of perithecium.with ostiole closed; X 290. Mature perithecium with two beaks; X 72. Mature perithecium with one beak; X 72. Longitudinal section of mature perithecium; X 120. Different types of perithecia; X 21. I’lllld' 1" . _ ‘ ~ , - ~‘..\.‘*,’~” .' C. hg'l‘y“.." Fig. 49. Fig. 50. Fig. 51. Fig. 52. PLATE VTI Young ascospores;.X 1800. Nature ascospores; X 1800. Mature ascospores when stained with pro- piono-carmine; X 1800. Ascospore germination; X 1500. a,b,c,d and f ----germ tubes emerging from the large cell. e. - -------------- germ tube emerging from both cells. g. -------------- -germ.tube emerging from the tip of the small cell. Fig. 53. ---- Ascus with young ascospores; X 3000. Fig. 54. ---- Ascus with.mature ascospores; X 3000. Plate V II - 'ROOM use ONLY. [M iii-LIBRARY LOAN Ar: (2‘4- ’53 MY 2854 d ‘- - m—‘ti—I -'-a——- ...-..fl-u- “- —.-<—-I'- *- u— g- - MICHIGAN STRTE UNIV. LIBRQRIE III III!” lllll III III Lillll