EN ‘11—. GENETIC m HGST-vPARA-SHE mmmmu STUDIES OF THE FUNGAL PATHOGEN {Hmosncu 59mg 031m; mmwammsmva m AUXOTROPHEC MUTATIONS Them: ‘00 We Dagmar: (if M. S. MlCfiiGAN STATE UNIVERSITY Dean William Gabriel 1 9 7 6 ABSTRACT GENETIC AND HOST-PARASITE INTERACTION STUDIES OF THE FUNGAL PATHOGEN PHYLLOSTICTA MAYDIS USING TEMPERATURE SENSITIVE AND AUXOTROPHIC MUTATIONS By Dean William Gabriel The mutagenic agent nitrosoguanidine was used to produce thirty temperature45ensitive, ten auxotrophic, and five morphological mutants of Ehyllosticta maydis Arny and Nelson. The auxotrophic mutants have been characterized as to nutritional requirements, and complementation groups have been determined. The temperature—sensitive mutants fell into three categories: those temperature-sensitive on artificial agar media, those temperature-sensitive on host plants, and those temperature- sensitive on both. These temperature sensitive-mutants provide evidence that E, maygi§_follows the gene-for-gene pattern of interaction on Normal (N) cytoplasm corn. ‘Mutations to increased virulence were also obtained, and together with the temperature-sensitive mutants, provide 'support for the hypothesis that the avirulence gene(s) of the parasite is (are) active in promoting incompatibility, and that the virulence allele(s) arise(s) through loss of function. The apparently multinucleated condition of the conidia, and restrictions on the homothallic capability of the perfect stage, are Dean William Gabriel discovered and discussed in terms of the variability they afford the fungus. GENETIC AND HOST-PARASITE INTERACTION STUDIES OF THE FUNGAL PATHOGEN PHYLLOSTICTA MAYDIS USING TEMPERATURE-SENSITIVE AND AUXOTROPHIC MUTATIONS By Dean William Gabriel A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Interdepartmental Program of Biological Science 1976 To my parents, Evelyn and Bill, my wife, Meri Anne, and my son, Ryan 11' ACKNOWLEDGMENTS I wish to thank Dr. Albert H. Ellingboe for his assistance and guidance both in this research and in the preparation and critical evaluation of this manuscript. I am grateful to have known and benefitted from the teaching and encouragement of the late Dr. William Grady Fields, whose loss I and many of his students still feel. I am indebted to Dr. T. Wayne Porter.for his thoughtful advice, guidance and exemplary commitment to teaching. Thanks are also due Dr. James A. Asher, who helped in the review of this thesis. Financial support for this research was generously provided through Dr. A. H. Ellingboe by the United States Department of Agriculture. LIST OF FIGURES LITERATURE REVIEW MATERIALS AND METHODS TABLE OF CONTENTS LIST OF TABLES ....................... INTRODUCTION ........................ Specific recognition: the gene-forrgene hypothesis . . Specific recognition: toxin mediated parasitism Genetics of pathogenicity of E, maydis ........ Formal genetics .................... Phyllosticta maygis cultures ............. Media ......................... Protocol for ultraviolet light (UV) mutagenesis . . . . Protocol for N-Methyl-N'-Nitro-N-Nitrosoguanidine (NG) mutagenesis ..................... Selection procedure: auxotrophic mutants ....... Selection procedure: temperature-sensitive mutants . . Screening for temperature-sensitivity and for changes in virulence .................... Scoring of the temperature-sensitive mutants on the host plant . . . . ................ Inducement of the perfect stage ............ iv OOOOOOOOOOOOOOOOOOOOOO Page vi vii l3 I4 23 23 23 24 25 26 27 28 29 3O TABLE OF CONTENTS (cont.) Page Complementation analysis ................ 3l Crosses ........................ 31 Cytological studies .................. 32 RESULTS ........................... 33 UV Mutagenesis ..................... 33 NC Mutagenesis ..................... 33 Characterization of auxotrophic mutants, complementation and crossing data .................. 37 Cytological studies .................. 40 Characterization of temperature-sensitive mutants . . . 40 Mutations to increased virulence ............ 49 DISCUSSION ......................... 54 SUMMARY ........................... 63 LITERATURE CITED . .' .............. ' . . ; . . . 64 LIST OF TABLES Table Page l. Numbers of mutations obtained using UV and NG mutagenic treatments ................ 34 2. The ten auxotrOphic and five morphological mutants obtained, their nutritional requirements, and descriptions ................... . 38 3. Growth of presumptive temperature-sensitive mutants at 30° C on complete media ..... . . . . 43 4. Summary list of temperature-sensitive mutants. . . . . 45 5. Selected examples of raw data scores of control plants, wild type innoculated, and temperature— sensitive mutant innoculated plants ...... . . . 46 6. Rated and summarized scores of all t§_mutants. . . . . 50 7. A_priori scoring expectations of temperature- sensitive mutants; list of mutants fitting the expectations . . . . . . . . . ........ . 58 vi Figure la. lb. LIST OF FIGURES The 9 possible parasite/host genotypes involving a single gene pair governing compatibility in diploid hosts and parasites ............ The Quadratic Check . . . .............. Toxin mediated parasitism in three systems ...... A temperature-sensitive gene-for-gene interaction . . Expected variations in paraSite populations over time during the evolution of a gene-for-gene relationship and the establishment of basic facultative parasitic capability .......... Semi- -log survival curves of P. maydis conidia on both minimal and complete media following U. V. light treatments, expressed as percentage survival plotted against the duration of U.V. exposure ................... Complementation of six auxotrOphic mutants on minimal media ................... Photographs of feulgen stained spores of E, maydis, 2,000 X magnification ............... vii Page 711 20 2] 35 39 41 INTRODUCTION Yellow leaf blight (Phyllosticta maydis Arny and Nelson) of corn (Zea mays L.) is known to occur across the northern part of the corn belt of the U.S. and in Ontario, and in 1968 appeared in near epidemic proportions in Michigan. A Phyllosticta leaf blight caused by Phyllosticta zeae was first described in 1930 (45). Isolates of Phyllosticta with larger spores were found to be widespread in the northern corn belt regions in the l960's, and these larger spored isolates were considered to be a new species (2). The economic importance of the species was correlated with the use of Texas male sterile cytoplasm (Tms) in corn. The immediate cause of the E, maydis epidemic is considered to be related to its ability to produce a toxin specific for plants with the Tms cytoplasm (6,7,54). This toxin is not mandatory for parasitism, however, as E, maydis is capable of infecting normal (N) cytoplasm corn in some cases as severely as Tms lines. Studies on resistance and susceptibility of N cytoplasm corn indicate that resistance is dominant (3,4,29,32). E, maygj§_may be a valuable link between pathogens in which toxin production is essential for pathogenicity, and those which produce no toxin. One of the purposes of this study was to obtain temperature sensitive mutants and use them to help analyze the genetics and physiology of the host-parasite interactions. Mutants which are temperature-sensitive both on agar and on host plants should affect spore germination, differentiation of germ tubes, appressoria, and basic life processes. Mutants which are temperature-sensitive on agar media but not on the host should be useful to study transfer of materials from the host to the parasite. Most desireable of all are mutants which are not temperature-Sensitive on agar media, but which are temperature-sensitive on the host, for these mutants affect the parasitic ability of the fungus. This last type should exhibit at least two types of reaction: l) At the restrictive temperature, the fungus would ngt_be parasitic, possibly because of a failure of a developmental step or sequence necessary to an active parasitism, such~ as formation of appressoria, or penetration through host cuticle, or production of a toxin. Temperature-sensitive mutants of this kind could conceivably be used to determine the developmental gene sequences necessary to the life cycle of E, maydis, and especially to resolve whether or not parasitism is an active process, the latter an hypothesis which has wide acceptance but with little supporting evidence. 2) At the restrictive temperature, the fungus woulg_be parasitic, because of‘ a loss of the specific gene-for-gene interaction(s) between resiStance gene(s) of the host and avirulent gene(s) of the parasite. The P6/Sr6 interaction of wheat stem rust is one example of a temperature-sensitive interaction following this pattern (l5). 3. maygj§_is an ascomycete, and therefore haploid. Its sexual stage, recently described, is Mycosphaerella zeae-maydis Mukunya and Boothroyd (31). With the discovery of the sexual stage, coupled to the fact that E, maydis is saprophytic and easily cultured in the laboratory, comes the possibility of genetic analyses of induced mutations. Another purpose of this study was to obtain auxotrophic mutants, and to obtain linkage data not only for the auxotrophic markers, but also to attempt to determine linkage relationships between auxotrophic markers and temperature sensitive mutations, since these mutations are indistinguishable unless they are developmentally (temporally) or biochemically distinct. A final purpose of the study was to determine whether or not A E, mgygi§_would form a heterokaryon, and if so, undergo subsequent parasexual recombination. Heterokaryosis is regarded as a prominent feature of the fungi and of great importance to plant pathogens in particular, since the proportion of different nuclei in a heterokaryon may change in response to selection, and since segregation of dissimilar nuclei in conidia can generate variation (33). Occasionally unlike nuclei may fuse to form a diploid, which then apparently undergo mitotic non-disjunction back to the haploid state through stages of aneuploidy, in a process termed parasexuality (38). Since E, maydis under laboratory conditions produces ascospores (sexual cycle) only sparsely, demonstra- tion of parasexuality or heterokaryosis in this fungus would indicate an important alternate source of variability in this pathogen. LITERATURE REVIEW Specific recognition: the gene-for-gene hypothesis.---0ne of the essential features of man's success in supporting such a large population of his own species has been the large-scale planting and cultivation of crops in monoculture. With our basic philosophy of discarding disease- prone or low yielding plant lines and substituting disease-resistant ones (l5), together with our basic economic philosophy of uncontrolled use of certain preferred lines, we have replaced in each major crop older, lower yielding varieties by a few, closely related, "superior" forms (9a). Selection for uniformity in crops---where once there was diversity--- has encouraged concommitant selection for, and rapid multiplication of, particular parasite races. 1 Plant breeders tend to introduce and thus rely upon relatively few genes for resistance to any one disease at any one time. The effect is to expose the host genes one at a time. Few genetic changes in the parasite are necessary to render the host genes so exposed ineffective. One can follow the appearance and disappearance of physiological races. of fungal pathogens according to the use and disuse of various genes for resistance (34). Johnson has called the phenomenon "man-guided" evolution (25). Careful genetic studies of host and parasite variability has revealed a particular pattern, first articulated by Flor as the gene- for-gene hypothesis (l8,l9,20). A host plant with a given gene will be resistant to its parasite only in the presence of a specifically recognized parasite gene, called then, by definition, a gene for avirulence. The given host gene is then, by definition, a gene for resistance. This interaction has been demonstrated or suggested with at least some of the following parasites: rusts, smuts, bunts, mildews, nematodes, insects, bacteria, and viruses (9b). A simple heuristic interpretation of.this gene-for-gene interaction (see Figure l) is that a host "Resistance" (3) gene "recognizes" a parasite "Avirulence" (A) gene. With the use of a host line [131, the segregation of Al_from al_is undetectable in crosses of the pathogen. Since an A_gene, until the appropriate R_gene is found, is initially associated with virulence, it is probable that the "primary role of the A_gene is to function in support of the parasite's success as an intrinsically viable organism" (36). The converse is only partially true; that is, Flor demonstrated the importance of using a wide variety of parasite isolates because the use of parasite strain al§l_does not allow detection of segregation of Bl_from [1, Unlike A_genes, however, R_genes are thought to increase in frequency in response to pathogenic selection, and as such, more accord with the general expectation that dominant genes afford a swifter selective advantage than recessive ones. (Note that the gene for virulence, a, behaves as a recessive, and is not in accordance with this expectation.) The initial situation of pathogenic encounter is hypothesized to be the compatible AlA2A .../rlr2r ... . The host responds by selection of mutations for a recognized R_gene, giving the incompatible interaction AlA2A .../er2r ... . The primary role of figu§§_la,---The 9 possible parasite/host genotypes involving a single gene pair governing compatibility in diploid hosts and parasites. The terms compatible and incompatible are used quite arbitrarily to grade a disease reaction into two categories: "+" denoting compatibility, and "-" incompatibility. These terms are useful in calling attention to the parasite/host interaction, alleviating the confusion involved in describing a host as resistant or susceptible, or a parasite as avirulent or virulent. The nine possible combinations involving a single gene pair in both host and parasite are shown above right. This figure is often shown in abbreviated form, and called the "Quadratic Check", as below right. Figure lb.---The Quadratic Check. Parasite Genes (:0 ll ["5 u |> u [OJ ll Parasite Genes I” I” DJ > Host Genes RR Rr rr - ' .. + - - + + + + a gene specifying resistance in the presence of specifically recognized A_gene. alternate allele of 3, gene specifying avirulence in the presence of specifically recognized R gene. alternate allele of A, Host Genes R- rr - + + + b Figure l the R_gene, then, is probably not in support of the host's success as an intrinsically viable organism, as argued for the A_gene in the pathogen. Because resistance is often phenotypically expressed several days after intimate contact between host and parasite is established, 3 genes are often thought of as inducible genes which are activated by and respond to the presence of the pathogen. At least one gene, however, which specifies resistance has been demonstrated to be constitutive: the_Vb gene in oats. The dominant Vb gene conditions susceptibility to Helminthosporium victoriae toxin and resistance to Puccinia coronata (49). Reaction of oat protoplasts with the Vb_gene to H, victoriae toxin is immediate (40,51), and indicates that the Vb_gene is constitutive in function. The site of action of the toxin appears to be in the plasma membrane; resistant (yb) cell membranes appear to lack the receptor site (40). Since this same Vb_gene confers resistance to E, coronata, then it is by definition an 3_gene, and has been demonstrated to be constitutive. Other B_genes may be constitutive; it is also possible that R_genes may function in a partially inducible manner, responding' to parasitic invasion with increased levels of activity (15). There may be many host genes for resistance, each specific for a corresponding parasite avirulent gene, and any combination specifying incompatibility is epistatic to all others which would allow parasitism to proceed. For a parasite population to overcome a given resistance factor, it needs to lose or alter its gene for avirulence, which is recognized by the host, and if it was a necessary gene, replace it with a gene of identical function, but one which is now unrecognized. Hence the disappearance of certain physiological races, and the appearance of a few, "select" others. The significance of exposing resistance genes one at a time now becomes apparent. The use of plant stocks which are genetically homogenous except for a few discovered resistance genes facilitates the parasite's change to negate the recognition of the host 3 genes. Reliance on horizontal, or general resistance is not likely to be of much value either, since all non-toxin mediated host-parasite interactions appear to follow the gene-for-gene pattern (14). Specific recognition: toxin mediated_parasitism.---E, maygj§_ gained its prominance as a pathogen, not because of selection to avoid a specific dominant resistance factor, but rather because of its ability to produce a host-specific toxin. In commercial seed production, a great yield advantage may be realized through the use of hybrid seed, which seedsmen successfully promoted in the 1930's (9c). Self fertilization may be prevented either by hand detasseling the anthers, or by the use of a cytoplasmic male sterility factor, transmitted by the female parent to all the progeny. While there are many sources of male sterile cytoplasms, Texas male sterile (Tms) had unusual favor because it caused full sterility (no anthers exerted) in a majority of corn inbred backgrounds. Most other sterility factors cause only partial sterility or full sterility in only a few inbred backgrounds. The widespread use of Tms hybrids provided a man-made, genetically uniform background of another kind, allowing epidemic sized parasitic IO infection by any organism able to exploit it. In this case, by genetically uniform is not meant that each host plant was genetically identical---indeed, the Tms cytoplasm was used with a great many different inbred backgrounds---but rather, that a single recognizable genetic factor was present. This factor appeared to be the Tms factor, for all Tms lines and Tms lines with nuclear fertility restorer (Tms-Rf) genes tested were reported uniformly susceptible, while almost all inbreds without the sterility factor (Normal, or N cytoplasm) were reported uniformly resistant (43). The ability of a fungal pathogen to attack a host containing a single factor (susceptibility dominant) as opposed to inability to attack a host containing a single factor (resistance dominant) is characteristic of toxin mediated parasitism (See Figure 2),which is quite distinct from gene-for—gene interactions (Compare Figure l). The former interactions are thought to be relatively early host- parasite encounters in an evolutionary time scale, and the latter are thought to be the more highly evolved and hence more stable or equili- brated interactions (15). For example, fl, victoriae, mentioned previously, produces a toxin which renders all oat varieties containing the single recognizable genetic factor, the nuclear Vb_gene, susceptible. Specificity is, therefore, for a nuclear gene, the site of action appearing to be the plasma membrane (40). Toxin specificity of P. maydis and Helminthosporium maydis, on the other hand, is not for a nuclear gene as such, but rather for the cytoplasmically inherited sterility factors, such as ll Host Vb Oats Vb Tms Corn N Tms Corn N H. victoriae Race T + - H. maydis Toxin + _ P. maydis Toxin + - H;_victoriae Race 0 - - Parasite .H. maydis No toxin - — P, maydis No toxin - ' Figure 2.---Toxin mediated parasitism in three systems. "+" = compatible reaction, "-" = incompatible reaction. Vb_and vb are alternate alleles of a nuclear gene of oats. Tms and N_refer to Texas male sterile and Normal cyt0p1asms of.corn, respectively. Note that this pattern is the exact reverse of the "Quadratic Check" gene-for-gene pattern of Figure l. 12 as Tms (6,7,8,4l,54). Evidence has been presented which suggests that the specificity in reaction to the toxin produced by H. maydis and P, mayg1s_is for Tms mitochondria (6,7,50). Toxin in low concentrations is highly specific, causing uncoupling of oxidative phosphorylation from the electron transport chain and immediate and irreversible swelling of isolated Tms mitochondria, while having no effect on isolated N cytoplasm mitochondria, even when concentrated a thousand fold (6,7). Nuclear genes which restore fertility (Rf) to plants with Tms cytoplasm give a toxin reaction intermediate between that observed with N and Tms plants and mitochondria. In one case toxin had a greater effect on Tms-Rf mitochondria than on Tms without Rf (50). Several possibilities are proposed to account for the observations: l)Rf genes may be closely linked to other genes conditioning toxin sensitivity; 2) Rf genes may modify the toxin itself; and 3) Rf genes may alter mitochondrial or plasma membranes, or both (50). As mentioned previously, without the Tms or other dominant susceptibility factors present, P, maygj§_exhibits the more usual pattern of resistance being dominant. That is, when one considers N cyt0p1asm corn only, then F1 progeny of crosses of these corn lines always show resistance approximately equal to that of the most resistant parental line. This pattern follows the gene-for-gene interaction (Figure 1), and argues strongly against toxin mediated parasitism (Figure 2) in N cytoplasm corn, where susceptibility in the F1 would at least be equal to that of the most susceptible parent. 13 If further studies bear out the preliminary data, then it becomes apparent that toxin production is useless to E, maydis in promoting parasitism of N cyt0p1asm corn. This conclusion is in conflict with an abstract published in 1972 by Yoder and Mukunya (55), in which they report that culture filtrates of P, mgygj§_contain host-specific toxic metabolite(s) having the same specificity as the pathogen in N cytoplasm corn. If this is the case, then they have discovered a metabolite with different specificities than the Tms specific toxin (7). They have published nothing further about it. Since Tms cytoplasm rendered host lines which carried it susceptible to P, maydis, hybrid seed producers have resorted to N cytoplasm corn (with hand detasseling). An advantage is that N hybrids appear to perform better than Tms hybrids even when disease is not a problem (16,39). Of course, conversion to N cytoplasm only obviates the toxin specificity of E, maydis, and incorporation of genetic sources of resistance in corn hybrids are needed for protection and control. Genetics of pathogenicity of P. maydis.--- Yellow leaf blight was first observed on corn in Ohio in 1965 (29), and by 1967 in Wisconsin (3) and in Ontario (24). It was first described by Scheifele and Nelson (42) in 1969 as a Phyllosticta leaf spot, and the causal organism was assumed to be P, zeae_$tout (45), but nine Inonths later Gates and Mortimer (24) determined that it was not. During 1970 it was referred to as Phleosticta s2, until described as a new species by Arny and Nelson in 1971 (2). 14 In terms of pathogenicity, E, maygj§_is considered weak, due largely to its predilection for young seedlings, older leaves of larger plants, physiologically weakened or stressed plants, or those already parasitized (1,24,46). Several studies have been made of the reactions of various inbreds (3,4,29,32,42,43) and these studies all indicate that even though a considerable amount of resistance may be present in inbred lines, the resistance will be partially or totally lost upon conversion to Tms or Tms-Rf cytoplasm. The results of these studies on N cytoplasm corn indicate that resistance in the F1 is as good as the most resistant parent, or is at least intermediate between the two parental lines. These intermediate reactions are not surprising in view of the apparent lack of appreciation of Flor's work demonstrating the necessity of using single isolates to detect different resistance factors. In only two studies are single isolates used to obtain data on pathogenicity. To cite one example, the rating of W64A in the data of Arny et al (3) varied from 2.5 to 5.0 on a 12 point scale. They used "several isolates" of E, maydis. Formal genetics.--- The sexual stage of P. maydis was first described in 1973 as Mycosphaerella zeae-maydis by Mukunya and Boothroyd (31). Mycosphaerella is in the Order Dothideales, of the Subclass Loculoascomycetidae. Diagnostic characteristics are a bitunicate ascus, with the asci borne in locules, the ascocarps of which are stroma. The locules are thus not perithecia, which IS they resemble, but pseudOperithecia, or pseudothecia. As with many fungi, sexual reproduction in E, maygis_is favored by relatively low, and inhibited by relatively high, temperatures. In Pleospora herbarium, sexual reproduction may be triggered by either U.V. light or low temperatures, and subsequent maturation requires light (27). In Venturia inaqualis, initiation of perithecia requires a temperature of 13° C, while the maturation of the ascospores requires a temperature of 20° C (53). P, mayg1s_develops pseudothecial initials in corn debris over winter, and maturation occurs in the Spring; the optimum temperature is about 20° C, with neither light nor cold treat- I ments necessary. Mukunya and Boothroyd report that all of 72 single ascosproe cultures developed mature pseudothecia; they thus considered E, maygjs_to be homothallic (31). Whether the homothallic mechanism is primary or secondary is unknown. Homothallism is thought to be favored by natural selection due to its lack of dependence on spores or hyphae of compatible mating type to ensure sexual reproduction (34). This advantage may be gained by essentially heterothallic fungi as well, such as those capable of secondary homothallism [Neurospora tetrasperma (52)], those which are weakly heterothallic [Glomerella cingulata (52)], and those which mutate from one mating type to the other [Saccharomyces lactis (23), Chromocrea spinulosa (28)]. Indeed, primary homothallism, in which single spore cultures are never self sterile, and in which mating type is stable, appears to be relatively rare and not well understood (48). 16 The most important aspect of heterothallism to a fungus is the requirement of heterokaryon formation [the mixing of unlike nuclei in a common cytoplasm (33)] and the possibilities of recombination afforded it by meiosis or parasexuality. In those fungi in which meiosis is restricted, rare, or unknown (as with the Imperfect Fungi, most of which are considered to be related to the Ascomycetes), parasexual recombination is assumed to be important in generating variatiOn. Mitotic crossing over occurs in A, nidulans according to the following scheme (17): l) Plasmogamy (heterokaryon formation); 6 2) Karyogamy at a frequency of about 10] to form diploid heterozygous nuclei with sectoring of diploid and haploid nuclei; 3) Mitotic crossing over (at a frequency of about 10'2 per nuclear division, compared to 10 per nuclear division for meiotic crossing over); and 4) Haploidization at the rate of about 10'3 per nuclear division. Mitotic, like meiotic, crossing over is reciprocal, occurs at the four strand stage, and only two of the four strands participate in any one exchange (26). Parasexuality in the Ascomycetes has been demonstrated in Helminthosporium sativum and Glomerella cingulata and indicated in Leptosphaeria maculans (47). In all of the above, heterokaryons were forced using auxotrophic or drug resistant mutations, but linkage relationships of markers were not established. In plant pathogens generally, well marked chromosome arms are lacking (33). In part this is due to the use of multiple isolates presenting many heterogenous genetic backgrounds, unlike the cases of N, crassa, and A, nidulans, 17 where genetic analysis is based entirely on two and on one wild type stocks, respectively. In part this is also due to the lack of development of the shortcuts needed to expedite genetic manipulations with a particular (and almost by definition, peculiar) fungus. Perhaps the most extensive genetic work on a single pathogen in terms of identification of avirulent ("pathogenic") genes, auxotrOphic and chemical resistant markers, and their linkage has been done on Venturia inaequalis (5). Nitrogen mustard methyl-bis (P chloroethyl) amine and U.V. radiation were used to obtain 30 amino acid, 19 vitamin, and 39 nucleic acid auxotrophs, representing 11, 8, and 12 loci, respectively. Only the biotin, inositol, nicotinic acid, and pantothenic acid mutants were pathogenic, and the specificity to different host lines was unaffected by the mutations. The rest were all generally nonpathogenic. Although nutritional mutants were able to grow together in complementary fashion, or syntropically, on minimal media, and even though hyphal anastamoses occurred regularly, heterokaryosis was not demonstrated. V, inaegualis proved to be a more difficult fungus to work with than NeurOSpora, despite their resemblance in many respects to each other. Linkage relationships, for example, demonstrated more centromeres than the observed number of chromosomes. Of particular interest are their mapping studies of the 19 avirulence genes, none of which were found to be closely linked or allelic. This finding is in accordance with the general observation that parasite genes recognized as avirulent are numerous and scattered, while host genes 18 recognized as resistant are few, and in general, form multiple allelic series, or are closely linked (22). The significance of many unlinked A genes in parasites and of the long allelic series for A_genes in the host may be appreciated in light of continuing interpretations of the term "virulence" as necessarily indicating an active process on the part of the pathogen gene so labeled in support of the pathogen. For example, Person (1975) writes, "it appears that the primary function of the virulence gene is to negate the primary function of A? (35). And Boone (1971) writes, "the allele for virulence might somehow be active in repressing the expression of the resistance gene of the host” (5). The problem with these interpretations is that alleles are alternative varieties of the same gene at a given locus. Of those alleles which are known to be functional (due to their specific reaction to A_or A), they might be expected to differ from one another by only a few base pairs, perhaps arising via a missense mutation. (A mutation on the order of a nonsense mutation would be expected to eliminate most of the function of the cistron.) Yet the specificity of those A_alleles is for A genes which map all over the parasite genome. If a gene for virulence repressed the expression of one A_allele, why would it not also repress an alternate allele of the same locus in a diploid, that allele presumably almost identical to the one supposedly repressed? Similarly, if the §_gene were providing something the parasite needed for growth which the host r_gene normally supplies and the_A gene does not, it-would require an incredible flexibility of the A 19 cistron to provide so many different gene products from a set of alleles. A far simpler explanation is that a gain in virulence represents either a loss in function, or a shift to a non-identifiable function. If this is so, then mutations to virulence should be easily obtained, but if virulence is an active allele, then mutations to virulence should be difficult. With A, lini, at least, Flor found the former to be the case (21). Boone's group apparently did not attempt similar studies (5). Further support for the idea that A and A_genes actively interact to produce incompatibility is provided by a temperature sensitive interaction. In stem rust of wheat, the P6 gene of Puccinia graminis conditions incompatibility with the wheat gene Sr6. This interaction is temperature-sensitive; that is, at the permissive temperature (20° C), the P6/Sr6 interaction specifies an incompatible reaction, while at the restrictive temperature (25° C), the P6/Sr6 interaction gives a compatible reaction (See Figure 3). This is not to say that gene-for-gene interactions are the only interactions possible. Some host-parasite combinations exist where no gene-for-gene interaction is demonstrable (15). Since the active, specific interaction appears to be for incompatibility, and since for parasitism to occur, this interaction cannot be present, then the gene-for-gene interaction must develop later in an evolutionary time scale than basic compatibility. The gene—for—gene interaction is thus superimposed on a basic parasitic capability (Figure 4). If this is so, then mutations which affect this basic compatibility ought to be P6 p6 20 20° 25° Sr6 sr6 Sr6 sr6 - + P6 + + + + p6 + + |3> loo figgrg_§,---A temperature-sensitive gene-for-gene interaction. A. The Sr6 gene in the wheat variety Red Egyptian interacts with gene P6 of A, graminis to produce the standard gene-for-gene pattern of interaction at 20° C. The same interaction as in A, except that the temperature has been raised to 25° C. Parasite Population 1 Two independent 21 First Encounter Development of harmony; Regulatory machinery synchronized; Basic compatibility. organisms ¥ 7 Second Gene-for-Gene relationship (Parasite gg_genes) 4‘ Gene-for-Gene effects minimized (Host plant in area of origin) ‘ 7' First Gene-for-Gene Relationship (Host A_gene) Evolutionary time Figure 4.---Expected variations in parasite populations over time during t e evolution of a gene-for-gene relationship and the establishment of basic facultative parasitic capability. According to this scheme, the gene-for-gene interaction is superimposed on a basic ability to overcome constitutive physical barriers to parasitism. [After Ellingboe (15)]. 22 obtainable. In particular, temperature-sensitive mutants should be obtainable that disallow this basic compatibility at the restrictive temperature. If A, maygis follows the gene-for-gene interaction, then temperature-sensitive mutants which disallow incompatibility at the restrictive temperature (like P6/Sr6) should be easily obtained also. 23 MATERIALS AND METHODS Phyllosticta maydis cultures.---All cultures of P. maydis used in this study, unless otherwise noted, are descended from a single ascospore isolate obtained from a culture isolated from the field in 1969. This culture grows and sporulates well on minimal media (below), and both ascospores and conidia germinate on water agar, but mycelial growth is light and no sporulation of any type is observed on the latter. Conidial spores measure 12-16 X 4-6 p, and ascospores are 13-19 X 5-6 u, closely fitting the descriptions of P, maygjs_Arny and Nelson, and M, zeae-maydis Mukunya and Boothroyd. All stock cultures were maintained by serial transfer on complete or minimal media. All sectors which arose in the stock cultures were seperately transferred and appropriately labeled. Mggia,---Minimal, Complete, and Corn Leaf Decoction agar media were prepared according to the following recipes: Minimal Media Complete Media Glucose 10 g Glucose 10 9 Salt solution 62 ml Salt solution 62 ml KNO3 3 g KNO3 3 g KH2P04 0.5 g KH2P04 0.5 g KZHPO4 l g K2HPO4 1 g Noble Special Agar 20 9 Yeast extract 2 9 Water to 1 liter Bacto-Peptone 2 g Agar 20 9 Water to 1 liter 24 Salt Solution Corn Leaf Decoction KH2P04 16 9 Corn leaves 50 9 Na SO 4 9 Water to 1 liter, 2 4 . KCl 8 g combine and autoclave . 20 minutes, then f1lter. MgSO4 7 H20 2 g -Infusion filtrate l L Cac'z ' 9 KZHP04 2 9 Water to 1 liter Agar 20 9 Adjust pH to 6.2 The Minimal Media is patterned after Ustilago Genetics Media; the Complete Media is patterned after Schizophillum Complete Media; and the Corn Leaf Decoction Media is patterned after Hay Infusion Media (44). When compacted colony growth was desired, 5 g of sorbose was substituted for 5 g of the glucose in the medias. . For the screening of auxotrophic mutations, variously supplemented media were used, based upon the Minimal Media recipe, to which the supplements were added. All supplements save casein (used at Zg / liter) were kept in stock solutions. All amino acids and nucleic acids were used at a final concentration of 10'4 M. Vitamins were used at the following concentrations: inositol, 0.4%; ascorbic acid, choline, niacin, pantothenate, 0.2%; thiamine, 0.1%; para-amino benzoic acid (PABA), pyridoxine, riboflavin, 0.05%; and biotin, 0.025%. Protocol for ultraviolet light (UV) mutagenesis.—--Conidia were obtained from one to two week old minimal media plates. Plates were flooded with distilled water, the mycelial mat was rubbed lightly with a bent glass rod dipped into Tween-80, and the resulting spore suspension was passed through sterile triple layered cheesecloth to 25 filter out germinated spores, mycelial fragments and whole pycnidia. Spore concentration was adjusted to 102 spores per ml. A11 U.V. treatments were performed in a Microvoid laminar flow hood. The flourescent light in the hood was turned off and plates subsequently kept as dark as possible to avoid photoreactivation of the treated conidia. Seventy-five ml of spore suspension was poured into a glass petri dish, and a magnetic stirrer provided constant swirling throughout the treatment. A low pressure (90% of output @ 2,537 A) .25 amp. Model V-4l Mineralight (U.V. Products, San Gabriel, Ca.) placed 5 inches from the plate was used as the U.V. source. One ml. alliquots of liquid were withdrawn every 30 seconds for 15 minutes after the start of the treatment, and plated on complete or minimal media. Depth of spore suspension varied from an initial 3-4 mm to a final 2-3 mm. of liquid. Protocol for N-Methyl-N'-Nitro-N-Nitrosoguanidine (NG) Mutagenesis.--- Conidia were obtained as above, except that liquid complete media was used in place of water to wash off the spores. Spore concentration was adjusted to 1.0 - 2.5 x 105 spores / ml. LNG supplied by Aldrich Chemical Co. of Milwaukee was kept for up to a month in a stock solution and used at a final concentration of 100 pg / ml. The spore suspension was constantly agitated during NG treatment. Spores were not washed following treatments; rather,the suspension was diluted by 5 X 103 prior to plating. Treated spores were plated on either minimal or complete media (see selection procedure). 26 Selection procedure: auxotrophic mutants.---Spores treated with either NG or UV were screened by either of two procedures. In the first, the layer plate method, spores were evenly dispersed overi petri plates of minimal media at about 100 spores per plate, allowed to grow for two days, and those which germinated were marked as proto- trophs. A layer of 45° C complete media was then poured over the top of the minimal plates. The colonies which grew after two days were isolated as presumptive auxotrophs for further testing. In the second procedure, the random isolate method, spores were plated at about 100 spores per plate directly onto complete plus sorbose media, allowed to germinate, and then 16 hyphal tips were each transferred onto one plate of complete media. Replica plates of these were then made onto minimal media, and those which grew well on complete and ' not at all on the minimal were isolated as presumptive auxotrOphs. Presumptive auxotrophs from either method were then plated onto minimal plus casein, minimal plus vitamins, and minimal plus nucleic acids. AuxotrOphs which grew on one of these three and not on the other two were kept for further analysis. Auxotrophs which responded to casein were presumably amino acid requirers, and were plated on amino acid "pool plates"--—minimal media supplemented as follows: #l #2 #3 #4 #5 #6 alanine aspartic acid leucine serine tryptgphan #7 isoleucine cystine phenylalanine glycine threonine #8 _gysteine lysine glutamic acid tyrosine arginine #9 histidine methionine proline Aglutamine valine 27 An auxotroph requiring, for example, arginine, would grow on plates #5 and #8, but on no others. Auxotrophs which grew only on the vitamin supplemented plates were plated on vitamin pool plates---minimal supplemented as follows: #1 #2 #3 #4 riboflavin . pyridoxine inositol #5 niacin Apantothenate ascorbic acid #6 PABA choline biotin No auxotroph isolated responded to nucleic acid-supplemented plates, so further testing of this expected type was unnecessary. Selection procedure: temperature—sensitive mutants.---Conidia treated with NG were allowed to germinate, and hyphal tips were trans- ferred 16 per plate onto complete plus sorbose media. After two days the colonies were replica plated onto complete media to be held at 30° C, onto minimal to be held at 22° C, and onto minimal to be held at 30° C. This method thus served to screen for auxotrophs as well as temperature- sensitive (ts) mutations. Growth on complete media at 22° was compared to growth on complete at 30°, and little or no growth at high temperature, with good growth at low, indicated temperature—sensitivity. Temperature- sensitive auxotrophs were tested for auxotrophic requirement. Another, simplified method that has the advantage of considerable savings in time and in petri dishes and agar was also used. In this method, a temperature-upshift experiment, hyphal tips were transferred 16 per plate onto complete media, and allowed to grow at 22° for five days. The extent of the mycelial growth was indicated by circling the perimeter of growth with a marking pen on the bOttom of the plate. 28 This complete media plate was replicated once onto minimal media, and then transferred into an incubator to be held for another five days at 30°. Much growth beyond the circled area indicated insensitivity to temperature, while ts_mutants would not grow at all, or only very little. After marking the ts_mutants, these plates were then placed at room temperature (22°), and checked for lethality. An added advantage of this method is that ts_auxotr0phs are automatically eliminated, since they continue to grow beyond the circled boundary. Each mutant was tested at least three, and sometimes four times; most were tested by both methods. Screening for temperature-sensitivity and for changes in virulence.--- Corn lines W117, W64A, and M3500 in both N and Tms cyt0p1asms were innoculated with wild type (wt) E, maygj§_to obtain normal reaction variability; all mutants were innoculated onto W64A in N cyt0p1asm. Conidial suspensions of each mutant tested were obtained by rubbing a two-week—old mycelial mat with a bent glass rod in about 10 m1 of distilled water to which a drop of Tween 80 was added. Each suspension was atomized onto two sets of corn plants (one set = 4-6 plants in one pot) at the 3—4 leaf stage. Fertilizer was added to the pots to help prevent yellowing of the lower leaves. The innoculated plants were placed into a mist chamber in the greenhouse, which provided a water- saturated atmosphere. Temperature in the mist chamber normally varied from 65° -75° F, except where indicated. Innoculated sets of plants were held in the mist chamber for 48 hours, and were then divided and transferred into two Sherer-Gillette Controlled Environment chambers, 29 one set at 22° C, and the other at 30° C. The chambers varied by i 1° - 2° in temperature, and from 35% - 85% in relative humidity. The chambers were set for a photoperiod of 16 hours on a 24 hour cycle. Notes were taken daily beginning with the first day of transfer and continued for about two weeks. Reaction type was scored on a scale of O for no reaction, F for fleck reaction, 1 for first leaves yellowing, 2 for second leaves yellowing and first leaves browning, and 3 for second leaves almost brown, first leaves completely brown. Controls of uninoculated plants were always present. Scoring of the temperature—sensitive mutants on the host plant.--- Factors such as variations in moist chamber temperature, watering frequency, height of the plants, density of the plants, fertilizer concentration, and variation within the environmental chambers made raw data comparisons between sets innoculated on different dates difficult. An effort to control such factors was made by comparing the experimental group scores to fit and control scores obtained on the same date. The raw data was then reduced to a simplified numerical rating system to make inter-group comparisons more meaningful. A score of 3 by the At_on a particular day was taken as the norm for the + reaction, by which all other mutants were scored, according to their deviation from this norm. Plants which reached a reaction of 3 at the same time or before the US were scored "+", or ”+(the number of days the score of 3 preceeded the At)", respectively. Plants which scored 0 at the beginning or which never reached a 3 score ("-?") 30 were considered as reacting much like an uninoculated control. A flecking reaction which later turned to 0 ("F/O”) was considered one of the most diagnostic of scores, for control plants never show it, and it is characteristic of a t§_mutant. Finally, plants which reached a reaction of 3 later than wt_were scored "~(the number of days the score of 3 succeeded the At)". Inducement of the perfect stgge.---Several methods were used in an attempt to obtain easily manageable pseudothecia. In the first, sterile, senescent corn leaves were placed on water agar, a block of mycelium was transferred to the center of the leaf, and incubated for three weeks at 21° C in darkness. This method gives the best growth, and the most consistent looking pseudothecia, but since these structures lie buried in the leaf, their clean removal is difficult. In the second, corn extract agar was used in place of water agar and corn leaves. This method was poor due to the tendency of the mycelium to grow through, rather than on top of the agar, a phenomenon not observed with minimal or complete media. In the third, and perhaps best method, corn extract agar was covered with a piece of cellulose. Mycelial growth usually remained on top, so pycnidia and pseudothecia were easily and cleanly peeled off the cellulose mat. Growth of mycelium and numbers of fruiting structures were reduced compared to either of the two previous methods, however. With all three methods, plates were held at a constant 21 i 1° C in the dark. 31 Complementation analysis.---Six of the best auxotrophs were gorwn together in all possible pairwise combinations in liquid media On a shaker for one week. The mycelial spheres were blended in a sterile blender cup and centrifuged. The mycelial pellet was resuspended in distilled water, centrifuged again, and the washed pellet divided and spread out on three minimal media plates. The plates were inspected after a week's time to determine if new mycelial growth was observed, and if so, a small piece was transferred to a minimal plate. In those plates where mycelial growth appeared limited to the edge of the innoculum, a 1 cm square block of agar was transferred to minimal media. Control plates were made of all six auxotrophs grown in liquid media on the shaker alone and treated similarly to the pairwise mixtures. Complementation was scored as positive if two auxotrophs were able to grow together on minimal, either syntropically, or by hetero- karyon formation. LaCk of complementation was indicated by failure to grow even from an agar block transfer. To determine whether or not heterokaryons were formed in the complementing groups, colonies grown on minimal plates were allowed to grow for several days until spores were produced. Conidia were harvested and plated on minimal media at about 50 spores per plate. Sixteen hyphal tip isolations were made of each complemting group as well, and these were plated on minimal media. Crosses.---Since only limited mycelial growth of auxotrophs occurred on the corn leaves, crosses of complementing auxotrophs were made on sterile corn leaves. A small chunk of minimal media from the 32 growing edge of two complementing auxotrophs was placed face down on a sterile corn leaf, the corn extract agar, and the corn extract agar covered with cellulose. Cytological studies.---Since no report exists in the literature of the number of nuclei per conidial spore, an attempt was made to stain the nuclei with Feulgen reagent. Glass slides thinly smeared with a small amount of albumen were pressed onto the surfaces of mycelial mats of two-week-old P, mgygis_cultures. The slides were lifted, the albumen allowed to dry, and the adhering spores were then fixed for 30 minutes in Carnoy's solution. The slides were then transferred to l N HCl for another 30 minutes, after which they were immersed in freshly prepared leuco basic fuchsin for 30 minutes. As a final step, the slides were rinsed briefly in 45% acetic acid, blotted dry, and covered with Permount. 33 RESULTS U.V. Mutagenesis.-—-U1traviolet light proved to be very effective in producing lethal mutants and presumptive auxotrophs. 0f the approximately 7,000 spores treated in four experiments, 56 presumptive A auxotrophs (0.8%) were isolated. Only one of these remained stable, unleaky, and characterizable. (See Table l). A plot of survival on both minimal and complete media indicated the optimum treatment range for production of auxotrophic mutants, since the survivors on complete media would be comprised of both auxotrophs and prototrophs, whereas survivors on minimal media would be prototrophic only. The curves (Figure 5) diverge the most at the 50-80% survival levels, indicating an optimum treatment time of 4-6 minutes of U.V. exposure. The survival curves unexpectedly demonstrated multiple hit kinetics, suggesting that more than one nucleus is present in each conidia. N.G. Mutagenesis.---Nitrosoguanidine proved to be an effective mutagenic agent, being more efficacious than U.V. light in producing mutants. 0f some 2,160 isolates removed by the random isolation method, 76 were presumptive auxotrophs and 92 were presumptive ts mutations; thus mutation rates were 3.5% and 4.2%, respectively. (See Table 1). Nine stable, Nine stable, unleaky, characterizable auxotrophs, and 30 stable ts mutations were obtained. 34 m OOLOQ‘NMQ'CDOMv—OON O magmas: mp a_aam= wo Lonesz 0F mm om 00>.N 0 e a 00F 0 0 0 00, F 0 NF 00> _ a 0 00> 0 o m 00> 0 0 0 0 0 F m 00_ F NP a 00_ N N_ m 00> m N. 0 00> 0 0m 0 0mm 0 PF NF 00— N NF n 00_ 0 0 0 00 P 1- om 000.5 P -1 up 000.N 0 11 0_ 000.0 0 -1 m_ 000.N 0 1. 0p 000.> mzaocpoxs< mcowumpzz mp mcowpwpzz umpmmh mwcomm m_nmm0 mwvmz L00< owzaocpox0< >0 Emnszz mo Lmnsaz m>w0050mmca m>wpaszmmgm mo Lungsz yo amneaz op oz mp oz op oz mp oz 0_ oz m_ oz NF oz _— oz 0P oz 0 oz 0 oz N oz 0 oz 0 oz 0 >0 0 >0 0 >0 _ >0 L80:52 00w: acmewgquo 0mummgh .mpcmEummLu uwcmmmuze oz 0:0 .>.0 mcwm: cmcwmuao mcowumuae $0 mcmneazuuu.> mpnwh 35 Figure 5.--- Semi-log survival curves of P. maydis conidia on both minimal and complete media following U.V. light treatments, expressed as percentage survival plotted against the duration of U.V. exposure. 400% 300% 200%} 100% 80% \l 0 3‘2 60% ' ' Complete Media 50% ~ \. ' o .\. 40% Minimal Media . o o \, \ 2w \ Percentage Survival 10%' 0 1 2 3 4 5 6 7 8 9 10 1] Duration of Exposure in Minutes Figure 5 37 Characterization of auxotrophic mutants, complementation and crossing data.--- In Table 2 are listed ten auxotrophic and five morphological mutants obtained, their nutritional requirements, and descriptions. Four arinine, two glutamic acid, one cystine, one lysine, one leucine, and one isoleucine requiring mutants were obtained. Although they were expected, no vitamin or nucleic acid requiring mutants were found. Six of the auxotrophic mutants were tested for complementation, and these results are summarized in Figure 6. Four complementation groups were obtained, the first containing NG-39, a glutamic acid requirer (glu'), the second containing WG-28, an arginine requirer (arg'), the third containing NG-3O and WG-35, lysine (lys') and leucine (leu') requirers, respectively, and the fourth containing WG-36 and UVe53, both arg'. The complementing groups were scored unambiguously except where indicated. Individual auxotrophic controls scored completely negative. No crosses have been obtainable after repeated attempts to find a single pseudothecia amongst all possible combinations of mutants; the At_under the same conditions produced few pseudothecia. A single ascospore was isolated from one of these few pseudothecia, and all attempts to pass this isolate through the perfect stage have failed. This indicates a restriction or limitation on homothallism for this fungus which is not apparent until consecutive passes of single ascospore isolates through the perfect stage are made. Table 2.---The ten auxotrophic and five morphological mutants obtained, their nutritional requirements, and descriptions. a. Name UV-53 'NG-3 NG-3O NG-35 NG-36 NG-38 NG-39 WG-72 TS-9 TS-74 0' Name Sandy Cotton Rust Yellow Band Auxotrophic mutants Requirement Description Arginine 'UE in appearance Cystine At_in appearance Lysine Darker than At, dense growth Leucine Lighter brown than UL: Arginine NE in appearance Arginine .Frequent sectoring ‘ Glutamic acid -Fuzzy surface Glutamic acid wt_in appearance Isoleucine At_in appearance Arginine At in appearance Morphological mutants Description Very sick, flat appearance. White border, with overall tan color. Spores misshapen, guttules irregular or lacking. Also known as TS-lO. Surface of mycelia when fresh grows a fluffy white layer which begins to disintegrate to a moldy appearance when old. Orange in color when fresh, ages to a rich dull orange. A bright yellow color. Produces a very distinct banding pattern. Also known as TS-22. 39 Figure 6.---Complementation of six auxotrophic mutants on minimal minimal media. UV-53 NG-3O NG-35 WG-36 NG-38 NG-39 UV-53 - + + — + + WG—30 - - +_ +_ + NG-35 ’ - +- + + NG-36 _ + , NG-38 . - + NG-39 A - The four complementation groups determined by the above: WG-39, glu' WG-38, arg' NG-30, lys NG-36, arg- NG-35, 1eu' uv-53. arg- 1 2 3 4 "+" = growth, "-" = no growth, "+4" = sparse growth, but more than each of the two cultures growing seperately 4O Attempts to force syntropic growth of two auxotrophs on minimal media have been successful, but repeated attempts to demonstrate heterokaryosis have failed. Cytological studies.---Slides prepared as described in Materials and Methods revealed four to six intensely stained objects per conidia. Since the Feulgen reaction is specific for DNA, this observation was taken as evidence that the objects were in fact nuclei. Photographs of two of the slides are presented in Figure 7. Characterization of temperature-sensitive mutants.---Ninety-two temperature-sensitive mutants were selected initially on the basis of their indicating a temperature sensitivity on complete media. Of these, a total of 26 remained stable. Tests were repeated at least three and in some cases four times; this data and a summary is given in Table 3. None of the mutants were lethal after one week at the restrictive (high) temperature. 0f the 26 stable mutants which are t§_on agar, 9 also proved to be temperature-sensitive on the host plant. Four isolates of the 92 originally selected as agar t§_mutants subsequently lost their temperature sensitivity on the agar media, but tested temperature- sensitive on the host plants. A summary of the 30 t§_mutants is given in Table 4. Only mutants tested for pathogenicity at least twice are included in the results. Original data on all 13 t§_mutants are included in Table 5. Also included are the original data on several controls (fertilized, 41 ' Figure 7.---Photographs of feulgen stained spores of P, maydis, 2,000 X magnification. The intensely stained bodies which appear in these conidia are taken to be nuclei; in these pictures, there are four nuclei per spore. 1w?_- 42 Figure 7 43 Table 3.--Growth of presumptive temperature-sensitive mutants at 30° C on complete media. "+” = growth, "-" = no growth. 44 Table 3 Name 5-1 5-6 6-1 6-1' Summary.' Name 5-1 5-6 6-1 6-1 Summary TS-l + + + + 50 + + + + 2 + + + + 51 +- + + + 3 - - + - 52 + + + + 4 + + + + 53 - - - - 5 + + + + 54 - — + — 6 - - +- - 55 +- + + + 7 + + + + 56 + + + + 8 - - + — 57 + + + + 9 — - + — 58 + + + + 10 - - - - 59 + + + + 11 + + + + 60 + + + + 12 + + + + 61 - - + - 13 + + + + 62 +- + + + 14 + + + + 63 +- + + + 15 + + + + .64 +— + + + 16 + + + + 65 - + + + 17 + + + + 66 — - + - 18 - + + + + 67 - - + - 19 - - - - - 68 +- + + + 20 - - a - 69 +- + + + 21 - +- +- - 70 +- + + + 22 +- + - +- - 71 - - + - 23 +- + + + + 72 +- + + + 24 + + + + 73 + + + + 25 + + + + 74 - - + - 26 + + + + 75 + + + + 27 + + + + 76 - - - 28 + + + + 77 - + - 29 + + + + 78 - - - 30 + + + + 79 + + + 31 + + + + 80 + + + 32 + + + + 81 + + + 33 - — + - 82 - - _ 34 + + + + 83 + + + 35 + + + + 84 + + + 36 + + + + 85 + + + 37 + + + + 86 + + + '38 + + + + 87 + + + 39 + + + + 88 + + + 40 — + — + - 89 + + + 41 + + + + 90 - - - 42 + + + + 91 - - - 43 ~ - - - 92 ‘44 + + + + 45 + + + + 46 + + + + 47 + + + + 48 + + + +. 45 Table 4.---Summary list of temperature-sensitive mutants. On agar 0n the host On both TS-3 TS-4 6 6 TS-6 8* 9* 10* 17 19 20* 21 22* 33 33 33 34 4O 40 4O 43 43 43 49 49 49 50 53* 54 61 61 61 66 66 66 67* 71 71 71 74* . 76 76 76 77 ‘ 78 82 90 91 26 13 9' Totals * Indicates discovery of auxotrophy, after initially scoring prototrophic. Except for TS-9 and TS-74, however, the nutritional requirements of these t§_mutants could not be determined. Table 5.---Selected examples of raw data scores of control plants, wild type innoculated, and temperature—sensitive mutant innoculated plants. "0” = no fleck observed; "F" = flecking observed; "1" = first leaves turning yellow; "2" = second leaves yellowing, first leaves brown; "3" = third leaves yellowing, first and second leaves brown. Innoculated with Control--No fertilizer Control-- fertilizer Acetone Tween-80 Wild type TS-4 TS-6 TS-17 13-33 TS-34 I 01 I NC” hNCDOS NNCN #NN—‘Nm-Jm I OWkaD cam-boo 010100 I I I I l 01 ON 01 D I 014300 I Nm—1 0'! N N -1 0100 I N N01 47 Tab1e 5 Daily scores 48 hrs. 22° 1 5 10 15 001122222222222 001111222222222 000000001111111 000001122222222 000001111222222 000000011112222 000000011112222 122222223 FFF111223 F1112222223 11223 112223 1222223 FF112222223 FF11112223 12222223 11222222222222 FFF111222222222 1112222223 FFFFFFF1111223 FFF111222222222 F001111111111 FFFF11122222223 111222222222 FFFFFFO FFFFFFFF112223 122222223 FF111123 12223 after innoculation 30° 1 5 10 15 00011223 0111111223 0000011111 01111112223 0111223 0122223 0122223 12223 1122223 F112223 123 F1223 122223 F11122223 FF1112223 1111112223 123 FFF122223 122223 FF11223 FF1223 FF111123 F111223 112223 F1122223 F1123 13 12222223 1222223 48 Table 5.---(Cont.) Innoculated with Daily scores 48 hrs. after innoculation 22° 1' 5 10 15 30° 1 5 10 -15 TS-40 . FFFFFO F112223 FFFFFFF112223 1111223 0000001122223 000011223 01111222 000123 TS-43 FFFFFOO FF1111223 FF00111122223 FF11223 0111111222222 122223 "TS-49 F000000000011112 FF11223 F11122222222223 F11122223 F1222223 FFF22223 TS-SO 11222223 123 F1222223 F1223 TS—61 F111111111111223 FF11223 1111111223 F112223 FFFFOOOOOOOOOOOO FFF1123 F1112222223 F11223 TS-66 1111111123 0112223 FFFF011112222222 FFF122223 TS-71 1111111122223 F12223 FF11222222223 F12222223 TS-76 FFFFF11111222 FFF1222222223 0F112222223 0F12223 49 non-fertilized, acetone and Tween-80 sprayed, and untreated plants) as well as the scores of At_on W64A, Wll7N, Wll7T, 5002XN, and 5002XT. Data assembled in this manner from different scoring dates were much less meaningful than data compared to gt_and control scores on a particular date and using a particular batch of plants. For example, on 4-29 it took the gt_only 3 days to achieve a rating of 3 at 30° C on W64A, yet it took the same wt_isolate 9 days to achieve the same rating on 3-6 and 5-26. By the same token, it was noted that an uninoculated control plant scores a 3 at high temperature within 7 days. A numerical rating system was therefore devised to evaluate any given isolate's reaction on any date in comparison to any other isolate. The ratings of all 76 tested isolates, analyzed as explained in Materials and Methods, are presented in Table 6. The temperature in the incubation chamber on two occasions rose during the day to well above 30° C; these are the 5-26 and 6-4 study dates. The 22° column of Table 6 of both these dates represents a temperature downshift, for the exposure to 30° began the day of innoculation, and was not ended until two days later, upon transfer to the incubation chamber. The 30° column then, represents a relatively steady temperature state. This situation is the exact reverse of the six previous sets of data in the table, where the 30° columns represent temperature downshift experiments, while the 22° columns represent steadily held temperatures of 22°. Mutations to increased virulence.---Four mutant isolates were discovered which produced disease symptoms much faster than At: TS-23, TS-26, TS-27, and TS-75 (Table 6). 50 Table 6.---Rated and summarized scores of all t§_mutants. i'NSD" = not sufficient data; "+" = scores equivalent to the At; "-" = no growth; "0" = no flecking observed; "F" = flecking observed; "F/O" flecking observed which later disappeared; "O/F'I = 0 score followed by flecking; "-?" = plant never achieved a score of 3 during the experiment; "+1", "+2", etc., = number of days a score of 3 was achieved in advance of the At; "-l", "-2", etc., = number of days a score of 3 was achieved after the wt, "++" = rating of 3 achieved much faster than wt; mutants so marked are considered to have mutations to increased virulence. 51 000000 00000000500 _P< 00000 00000 000 :0 00003 .00 000 000 .00000 0500 0:0 00 .000000000000 00 0500 000 5000 000000 >P0>000000 0000 00000000500 .00000F000000 00000 0000; 00 00000000 000 000 0 500+ 00000000000 :0 000000200 0 0000000000 00000 020 00000 :0 050—00 .000 5000 00000000500 00 0000000 00 0000000000 050—00 000 000 .00000 00000 —_0 c0 .000000000000 00 0500 000 5000 00000 030 00000 :0 0+0: 003 00000000000 000 000 1 . .1 O 002 + ...- 00 + + ++ ++ 0+ 0+ 0+ 0+ 00 + + 1 1 0 0 0 01 00 1 + 00z 0 0 +0 1. + 002 0 0 0 0 00 1 + + + 0 0 + 01 0+ 1 + + 1 + ,0+ 0 0 0+ 01 + 01 0+ 1 + + 1 + 01 + 01 1 up + + + + + +1 0+ + + + + 0+ + 0p + + + + 0+ 0+ 00 + + 002 0+ 01 00 + + + + 0+ 0+ 00 + + 002 ...- 0+ : ... + + + 0 0\0 + m1 + 0+ 00 1 + + + o o I 0.. m - + + + 0 0\0 + 01 + 01 0 1 + 002 01 01 0 + + + 1 +0: 0+ 0.. + 01 0+ ...- 0 1 + sz _.+ 0+ m + + + 1 + 01 0+ 01 _1 0+ 0 + + 1 1 0 0 0 0+ 0 1 + + + + + 0+ 0+ 0 + + 002 o 0- _-00 + + + + + + + + + + + + + + + + + + + + .Hm + + 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0000000 000 000 .000 000 000 000 000 000 000 000 com 000 000 000 000 000 000 000 000 000 0005800 +010 «0010 0010 0010 0010 010 0010 01m 0E0z N005500 000; :0 00mm :0 0 00000 52 0+. 0+ + + + _+ + + 0 0 + 01 + + + + 0+ 0+ 01 01 QWZ Pl + + + 01 1 0+ 0+ + 1 0+ _+ + 01 + 1 + + 01 001 + 0\0 + + + 0+ DWZ + MI + + + 0+ + + + + + + + + + 1 + 0 + _1 _1 0\0 002 01 0\0 002 01 0+ 1.. l O O + _.l + O\....— Omz PI + 002 F1 01 002 F1 01 002 P1 + + + + + 1 + _1 0+ 01 + + 1 0+ 01 0+ 01 _1 0\0 0 0 0+ 01 0\0 01 + + + + 0+ m+ + N1 P1 N+ + + + 0+ —+ + + + 0+ 01 + + + + 0+ + + + ++ ++ 0+ 0+ + + ++ ++ 0+ 0+ + + + + 0+ + _1 0- 000 000 000 000 000 000 000 000 000 000 000 000 N005500 +010 0010 0010 010 0010 000; :0 010 0700200 00>0: 000:3 0:000 u :Gl: .203000 0: u :1: 0003000 u =+= .0:0E0000x0 0:0 0:0:00 m 00:000000 00 00300000 00000 0 u =0\0=000L00000000 000:3 0:000000 u=o\0=00:000000 00: 002000 0000002 u =0= 00000 0003 00 0:000>0:00 203000 2+..U 0 .0::00 00 00 0:0000:00500 00000000 000 0000050000 00 0000000000 00 :300 :00000 000 0:0 .000>0000000L 000 0:0 .00 .0 0000050: :050; 0:0 00 0000:00000 00011100:0 000; 000 :0 0>0000:00 100000000500 00020 0:0 .00:0 0000 :0 0>0000:0010L:00000500 00000 .000; 0:0 0:0 0000 0000 :0 0>0000:0010L:00L00500 00000111000000x0 0:0000005 0>0000:0010L:00000200 00 00000 00000 00000 0000 00100 00.0100 00100 .00.00 .00.00.00.o—.m1mh .00.0m.00.00.mm.00 .00.00.mm.mp.0100 0:02 00000000 m0c00=z + ++ ++ + 01 Lo 0\0 ++ 00 + ++ 00 + 01 00 0\0 + 01 00 0\0 01 00 0\0 + 0\0 Lo 0 + 0 + 0\0 00 0 ++ 00 + 0 00 + 01 00 0\0 0\0 Lo 0 0- 00 000 0 + .uwmuuummm .mmmuuummm .1JmmW1. 1100m01. 000000200 000000: 000000 000000 0000000x0 00:0000 + 1 + + 00000 + 1 + + 00000 1 + + + 0000 + + 1 + 00 + 1 1 + 00 1 + 1 + 00 0.04.0.0. 00.00.1000 0000 000: 000< 000000010005500 .0000000000x0 0:0 0000000 00:0002 00 0000 ”00:0005 0>0000:0010L:00L00E00 00 0:00000000x0 0000000 000000.0111.0 00000 59 as having excaped the original screening. Significantly, though, none of these were found to be temperature-sensitive on the host.) When applied to the host, the spores may or may not germinate at 30°, depending on whether or not spore germination per_§e_is affected by the mutation; in any case, they will continue to infect, once started, a plant held at high temperature, and will cease to infect, or slow greatly, at low temperature. This type of reaction is similar to the P6/Sr6 system in wheat stem rust, as well as to a type III reaction, except that in this latter type, the spores are not tempera- ture-sensitive on the agar. A type III reaction may be regarded as involving some active interaction of parasitism, but not directly related to basic life processes, as in type I. A type IIIa, which shows inactivation at the high temperature, may be regarded as a mutation affecting the basic ability to parasitize. If the nonpermissive temperature is. assumed to be the high temperature (an assumption warranted on the basis of general experience with t§_mutations in this and other organisms), then some gene or gene prodUct which confers ability to promote primary infection on corn is non-functional at the high temperature. By the same token, a type IIIb reaction, non-functional at the high temperature, indicates the dysfunction of the specific interaction necessary for incompatibility, i.e., the inability to parasitize. Type IIIb thus follows the gene-for-gene interaCtion. This type closely resembles type Ib, except in this case, the parasite gene does not appear to function in support of intrinsic viability of 60 the pathogen. The type IIIb reaction is divided into two subgroups, based upon the similarity or lack of it to the wild type reaction at low tempera- ture. One troubling factor in both the Ib and IIIbl reactions is the difficulty in interpreting their low-temperature reactions, as compared to wild type. If the temperature-sensitivity is truly at the high temperature, why does the low-temperature reaction differ from the wild type? Far more satisfactory (convenient?) is the type of score represented by IIIb2, where low temperature reaction is unaffected by the mutation(s), and high-temperature-reaction shows increased compatibility, due to inactivation of the specific interaction necessary for incompatibility---presumably, an avirulence gene(s). With types IIIbl and lb, multiple mutations which in general slow down the metabolic machinery of the fungus could account for the generally increased incompatibility these classes show compared to wt at low temperature, but separation of these effects from those which just affect gene-for-gene interactions would be more satisfactory. The mutant types were fairly evenly distributed. The seven type 11 mutations, which are temperature sensitive on agar, and still are able to parasitize well, but which show no temperature-sensitivity on the host, should facilitate study of materials transferred from host to parasite. ‘To be sure, four of these have turned out to be auxotrophic, but whatever they require is not provided by any of the nutrients used to supplement the minimal media in the screenings, and remain unknown. The other three, however, do not score as auxotrophic. It is possible 6) that these are the missing Ia mutant types---after all, there is only one IIIa type, and it grows so poorly that it was almost missed---it may be difficult to score a mutant as temperature-sensitive at the high temperature on corn due to the very rapid onset of a 3 score. (Compare how fast a 3 is achieved on uninoculated plants at 30°). Consider the single example of type IIIa, TS-34. Since it is not temperature-sensitive on agar, reaction at 30° proceeded without difficulty through germination (Refer to Table 6). In comparison to control data, the high temperature score of TS-34, even though achieving a score of 3 rapidly, did so less rapidly than the wt,, and only somewhat more rapidly than the uninoculated control. By comparison, the rating at 22° followed that of the wt_closely, with attainment of scores of 2 and 3 simultaneously. Further work, using a slightly lower high-temperature setting (say 28°) may help clarify certain of these reactions. The four mutants to increased virulence are probably a result of the loss of function of an avirulence gene(s) which is not harmful to the pathogen. These mutants may be useful in isolating individual resistance factors in host plants, in the absence of natural isolates able to do so. Mutations have been used with effect to obtain dominant resistance factors in the host (37)., and mutations to virulence in the pathogen such as these should be helpful in identifying and isolating newly discovered, and newly created, resistance factors. They should also help avoid the accidental loss, through inbreeding of naturally variable host plants, resistance factors 62 which would otherwise go undetected---overshadowed by other factors. The ease with which the mutations to increased virulence were obtained supports the hypothesis that a gain in virulence represents a loss of function, and that specificity of gene-for-gene interactions is for incompatibility, not compatibility. If specificity were for compatibility, mutations to increased virulence should have been diffiCult to obtain. In addition, the ease with which t§_mutations to increased virulence at the restrictive temperature were obtained supports the same hypothesis: the loss in function occurs at the restrictive temperature and translates into a gain in virulence. If specificity were for compatibility, then t§_mutations to increased virulence at the restrictive temperature should have been difficult to obtain. 63 SUMMARY Preliminary work on E, maygi§_to determine its suitability for more extensive genetic analyses has been done. Both auxotrophic and temperature-sensitive mutations were obtained with facility when care was taken to overcome the apparently multi-nucleated condition of the conidia. Reports of homothallism must be amended in light of indications that the homothallism, while primary, was restricted. These restrictions made classical genetic analysis difficult, and prevented use of a single asCospore background for analysis in the present study. Parasexual analysis may be more profitable. Temperature-sensitive mutations supported previous indications that E, maydis follows a gene-for-gene pattern of interaction on N cytoplasm corn, where toxin production appeared to be useless. All three expected types of t§_mutations were found: those temperature- sensitive on agar only, those temperature-sensitive on host plants only, and those temperature—sensitive on both. Four mutants to increased virulence were found, and should be useful in screening for new or hidden resistance factors. The ease with which the mutations (temperature-sensitive and non-temperature- sensitive) to increased virulence were obtained supports the hypothesis that a gain in virulence represents a loss in function, and that specificity of gene-for-gene interactions is for incompatibility, not compatibility. LITERATURE CITED 9a. 64 LITURATURE CITED Allinson, D.w. and w.w. Washko. l972. Influence of a disease complex on yield and quality components of silage corn. Agronomy Journal 64: 257-258. Arny, D.C. and R.R. Nelson. l97l. Phyllosticta maydis species nova, the incitant of Yellow Leaf Blight of maize. Phytopathology 61: ll70-ll72. Arny, D.C., G.L. Norf, R.w. Ahrens, and M.F. Lindsey. l970. Yellow Leaf Blight of maize in Wisconsin: its history and the reactions of inbreds and crosses to the inciting fungus (Phyllosticta §E,). Plant Disease Reporter 54: 281-285. Ayers, J.E., R.R. Nelson, Carol Koons, and G.L. Scheifele. l970. Reactions of various maize inbreds and single crosses in normal and male sterile cytoplasm to the Yellow Leanglight organism (Phyllosticta 59,). Plant Disease Reporter 54: 277-280. Boone, D.M. l97l.’ Genetics of Venturia inaequalis. Annual Reviews of Phytopathology 9: 297-3l8. Comstock, J.C., C.A. Martinson, and B.G. Gengenbach. l972. Characteristics of a host specific toxin produced by Phyllosticta maydis. Phytopathology 62: llO7.- (Abstract). Comstock, J.C., C.A. Martinson, and 8.6. Gengenbach. 1973. Host specificity of a toxin from Phyllosticta maydis for Texas Cytoplasmically Male Sterile maize. Phytopathology 63: l357-l36l. Comstock, J.C. and R.P. Scheffer. 1972. Production and relative host specificity of a toxin from Helminthosporium maydis Race T/ ' Plant Disease Reporter 56: 247-25l. Day, Peter R., Genetics of Host Parasite Interaction, N.H. Freeman and Co., San Francisco. 1974. p. 189. 9b. 9c. 10. ll. 12. T3. 14. 15. T6. 17. 18. 19. 20. 21. 22. 23. 65 Ibid., p 96,97. Ibid., p 176. Drake, John W. 1970. The Molecular Basis of Mutation. Holden Day, San Francisco. p.165. Dobzhansky, T., 1950. Mendelian populations and their evolution. American Naturalist 84: 401-418. Dubovoy, Celia. 1976. A class of genes affecting 8 factor regulated development in Schizophyllum commune. Genetics 82: 423-428. Edgar, R.S. 1969. The genome of Bacteriophage T4. The Harvey Lecture Series 63: 263-281. Ellingboe, A.H. 1975. Horizontal resistance: an artifact of experimental procedure? Australian Plant Path. Society Newsletter 4: 44-46. Ellingboe, A.H. Genetics of host-parasite interactions. Physiological Plant Pathology. R: Heitefuss and P. Williams, ed., (in press). Ellingboe, A.H., E.C. Rossman, and G.R. Safir. 1974. Epidemiology and genetics of resistance to Yellow Leaf Blight, Phyllosticta maydis, of corn and the remote detection of Southern Corn Leaf Blight. (Unpublished). Esser, Karl and Rudolf Kuenen. 1967. The Genetics of Fungi, Springer-Verlag, Berlin. p. 92-93. Flor, H.H. 1946. Genetics of pathogenicity in Melampsora lini. J. of Agr. Res. 73: 335-337. Flor, H.H. 1947. Inheretance of reaction to rust in flax. J. of Agr. Res. 74: 241-262. Flor, H.H. 1956. The complementary genic systems in flax and flax rust. Adv. Genet. 8: 29-54. Flor, H.H. 1958. Mutation to wider virulence in Melampsora lini. Phytopathology 48: 297-301. Flor, H.H. 1971. Current status of the gene-for-gene concept. Ann. Rev. Phytopathology 9: 275-296. Herman, A. and H. Roman. 1966. Allele specific determinants of Saccharomyces lactis. Genetics 53: 727-740. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 66 Gates, L.F. and C.G. Mortimer. 1969. Three diseases of corn (gea_mays) new to Ontario: Crazy Top, a Phyllosticta leaf spot, and eyespot. Can. Plant Disease Survey 49: 128-131. Johnson, T. 1961. Man-guided evolution in plant rusts. Science 133: 357-362. Kafer, E. 1961. The processes of spontaneous recombination in vegetative nuclei of A. Nidulans. Genetics 46: 1581-1609. Leach, C.M. 1971. Regulation of perithecium development and maturation in Pleospora herbarum by light and temperature. Brit. Mycol. Soc. Trans. 57: 295-315. Mathieson, M.J. 1952. Ascospore dimorphism and mating type in Chromocrea spinulosa. Annals of Botany, N.S. 16: 449. McFeeley, J.C. 1971. Comparison of isolates causing Yellow Leaf Blight of corn in Ohio. Plant Dis. Reptr. 55: 1064-1068. Morris, Ronald M. 1975. Mitotic mutants of Aspergillus nidulans. Genetical Research 26: 237-254. Mukunya, D. M. and C. W. Boothroyd. 1973. Mycosphaerella zeae-maydis sp, n., the sexual stage of Phyllosticta maydis. Phytopathology 63:529-532. Nelson, R.R., J.E. Ayers, and J.B. Beckett. 1971. Reactions of various corn inbreds in normal and different male sterile cyt0p1asms to the Yellow Leaf Blight organism (Phyllosticta sp,). Plant Dis. Reptr. 55: 401-403. Parmeter, J.R. Jr., W.C. Snyder, and R.E. Reichle. 1963. Heterokaryosis and variability in plant pathogenic fungi. Ann. Rev. Phytopathology 1: 51-76. Person, C. 1967. Genetic aspects of parasitism. Can J. of Botany 45: 1193-1204. Person, C. and T. Ebba. 1975. Genetics of fungal pathogens. Genetics 79: 397-408. Person, C. and G. M. E. Mayo. 1974. Genetic limitations on models of specific interactions between a host and its parasite. Can. J. of Botany 52. 1339- 1347. Person, C., and G. Sidhu. 1971. Genetics of Host-Parasite Interactions, Mutation Breeding for Disease Resistance, International Atomic Energy Agency, Vienna., p 31-38. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 67 Pontecorvo, G. 1956. The parasexual cycle in fungi. Ann. Rev. Microbiology 10: 393-400. Rossman, E. C. and A. H. Ellingboe. 1975. Yellow Leaf Blight (Phyllosticta‘_p. ) resistance. (Unpublished). Samaddar, K.R. and R.P. Scheffer. 1968. Effect of the specific toxin in Helminthosporium victoriae on host cell membranes. Scheffer, R.P., R.R. Nelson and R.B. Pringle. 1964. Toxin production and pathogenicity in Helminthosporium victoriae. Scheifele, G.L. and R.R. Nelson. 1969. The occurrence of Phyllosticta Leaf Spot of corn in Pennsylvania. Plant Dis. Reptr. 53: 186-189. Scheifele, G.L., R.R. Nelson, and Carol Koons. 1969. Male sterility cytoplasm conditioning susceptibility of resistant inbred lines of maize to Yellow Leaf Blight caused by Phyllosticta zeae. Plant Dis. Reptr. 53: 656-659. Stevens, Russell B. Mycology Guidebook, University of Washington Press, 1974, pp 514, 691. Stout, G.L. 1930. New fungi found on the indian corn plant in Illinois. Mycologia 22: 271-287. Sutton, J.C., A. Bootsma, and T.J. Gillespie. 1972. Influence of some cultural practices on Yellow Leaf Blight of maize. Can. Plant Dis. Surv. 52: 89-92. Tinline, R.D. and B.H. MacNeill. 1969. Parasexuality in plant pathogenic fungi. Ann. Rev. Phytopathology 7: 147-170. Ullrich, R.C. and J.R. Raper. 1975. Primary homothallism--- relation to heterothallism in the regulation of sexual morphogenesis in Sistotrema. Genetics 80: 311-321. Wallace, A.T., R.M. Singh, and R.M. Browning. 1967. Induced mutations at specific loci in higher plants III. Mutation response and spectrum of mutations at the Vb locus in Avena byzantina. Induced mutations and their utilization. Erivin-Bour-Gedachtnisvorlesungen IV, Akademie- -Ver1ag, Berlin. pp 47-57. Watrud, L.S., A.L. Hooker, and D.E. Koeppe. 1975. The effects of nuclear restorer genes of Tms cyt0p1asm on host response to Helminthosporium maydis race T. Phytopathology 65: 178-182. 51. 52. 53. 54. 55. 68 Wheller, H. and H.S. Black. 1963. Effects of Helminthosporium victoriae and victorin upon permeability. Am. J. of Botany 50: 686-693. Whitehouse, H.L.K. 1949. Heterothallism and sex in the fungi. Biological Reviews, Cambridge Philosophical Society 24: 411-447. Wilson, E.E. 1928. Studies of the ascigerous stage of Venturia inaequalis (Cke.) Wintergreen in relation to certain factors of the environment. Phytopathology 18: 375-416. Yoder, O.C. 1973. A selective toxin produced by Phyllosticta maydis. Phytopathology 63: 1361-1366. Yoder, O.C. and D.M. Mukunya. 1972. A host-specific toxic metabolite produced by Phyllosticta maydis. Phytopathology 62: 799. (Abstract). ”11111111111111fillllllilllllllfllllm