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RSITY LIBRARIES WM\|\\\\\\\\\\\\\\|WW\ \ us \i \ This is to certify that the dissertation entitled Phenetic Analysis of leucostoma from Prunus and Malus presented by Rupa Surve-Iyer has been accepted towards fulfillment of the requirements for Ph.D. degree inBotany & Plant Pathology WWfi’ MSU is an Affirmative Action/Equal Opportunity Institution 0 i2771 LIBRARY Peiichlggen Si‘iate Ufii‘bfiii’ffiiiy PLACE IN RETURN BOX to remove thic checkout from your record. TO AVOID FINES return on or before data due. DATE DUE DATE DUE DATE DUE ‘ .491}- — JLJFTJ — ii? i usu Is An Affirmative ActiorVEoual Opportunity institution cwmt _._.._._—._ _ PHENETIC ANALYSIS OF LEUCOSTOHA FROM PRUNUS AND HALUS BY Rupa S. Surve-Iyer A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 1992 ABSTRACT PHENETIC ANALYSIS OF LEUCOSTOMA FROM MALUS AND PRUNUS SPP. by Rupa S. Surve-Iyer Isozyme patterns, morphology, virulence on peach and variation in rDNA were used to compare isolates of Leucostoma cincta and L. persoonii collected from Malus and Prunus spp. Leucostoma persoonii isolates formed three distinct phenetic groupings based on isozyme polymorphisms. The three groups were distinct from all phenetic groupings within L. cincta. Based on isozyme patterns of L. cincta, three phenetic groupings exist, a distinct group on Malus and two related groups on Prunus. Far more diversity was evident among isolates of L. cincta from different hosts than among isolates of L. persoonii from different hosts. Five of the six phenetic groups, two of L. persoonii and three of L. cincta were compared for virulence on 3-yr-old multistemmed peach seedlings. All five groups were virulent on inoculated peach including PG6, an L. cincta group found on Malus in nature. L. persoonii isolates in PGl were most virulent while L. cincta isolates in PGS were the least virulent. In culture, isolates of the two taxa were differentiated on the basis of their pycnidial size and color and colony margin. Isolates of all five phenetic groups grew at 27°C, but only isolates of phenetic groups PGl and P62 of L. persoonii showed growth at 330C. The phenetic groups were further compared by fragment length polymorphisms based on nuclear ribosomal genes. Digestion of the ITS-LrDNA with 40 restriction enzymes followed by electrophoresis revealed 9 enzymes that distinguished the isolates of L. persoonii. Several of the L. persoonii groups, defined by ITS-LrDNA phenotypes corresponded to those detected in isozyme studies and others corresponded to host and/or geographic distributions. Less variation in ITS- LrDNA was evident in L. cincta populations in contrast to isozyme studies. Most of the RFLP variation in the ITS-LrDNA was within the internal transcribed spacer rather than in the nuclear large rDNA. PCR amplification of the nuclear small rDNA revealed an insertion in L. cincta isolates from Prunus spp. that is absent in L. cincta isolates from Malus and in L. persoonii isolates. ACKNOWLEDGEMENTS I gratefully acknowledge the guidance and enthusiastic support offered by my major Professor Dr. G. C. Adams. His expertise in fungal taxonomy was absolutely essential in formulating and analyzing this project. I thank my committee Drs. A. Iezzoni, A. Jones, S. Somerville and S. Tonsor for their time and effort over my thesis. I wish to thank C. Sneller (Horticulture), R. Hagen and S. Williams (Entomology), T. D’souza (Microbiology) and M. Case (Botany) for their help with the technical aspects of the project. I would also like to thank my colleagues in the Adams laboratory for their input during the years in his lab. This project would not have evolved so far without the help of T. Proffer who provided me with most of the L. cincta isolates and Dr. J. Taylor (U. California, Berkley) for giving me the opportunity to work in his laboratory to complete the RFLP project. I wish to thank my parents Sadanand and Smita Surve and my brother Girish for their support and encouragment. I also wish to thank my uncle Vijay Kane for his encouragement and support. Finally, I cannot thank my husband Shridhar enough for his unfailing support, encouragement and patience, especially during the latter stages, when it seemed almost immpossible to obtain this degree. iv 0 . TABLE OF CONTENTS LIST OF TABLES. . . . . . . . . . . . . . . . . . Vii LIST OF FIGURES. . . . . . . . . . . . . . . . . . viii GENERAL INTRODUCTION 0 O O O O O O O O O O O O O O O 1 Literature cited . . . . . . . . . . . . . . 18 SECTION I ISOZYME DETECTION AND VARITION IN LEUCOSTOMA SPECIES FROM PRUNUS AND MALUS. ABSTRACT. . . . . . . . . . . . . . . . . . . . . . 25 MATERIALS AND METHODS . . . . . . . . . . . . . . . 30 RESULTS . . . . . . . . . . . . . . . . . . . . . . 34 DISCUSSION. . . . . . . . . . . . . . . . . . . . . 36 LITERATURE CITED. . . . . . . . . . . . . . . . . . 44 ' SECTION II RESTRICTION FRAGMENT LENGTH POLYMORPHISIMS IN NULEAR RIBOSOMAL DNA OF LEUCOSTOMA CINCTA AND L. PERSOONII ABSTRACT. . . . . . . . . . . . . . . . . . . . . . 58 MATERIAL AND METHODS. . . . . . . . . . . . . . . . 58 DNA preparation . . . . . . . . . . . . . . . . . 58 Enzymatic amplification of DNA with the use of PCR. . . . . . . . . . . . . . . . . . . . . . . 63 Data analysis . . . . . . . . . . . . . . . . . . 66 RESULTS . . . . . . . . . . . . . . . . . . . . . . 67 DISCUSSION. . . . . . . . . . . . . . . . . . . . . 70 LITERATURE CITED. . . . . . . . . . . . . . . . . . 76 COMPARISON OF FIVE PHENETIC GROUPS OF LEUCOSTOMA USING MORPHOLOGY AND VIRULENCE ON PEACH ABSTRACT. . SECTION III MATERIALS AND METHODS . . . . Cultural characteristics and temperature response. Virulence tests on 3—yr-old peach seedlings RESULTS . . Cultural characteristics and temperature response. Virulence tests on 3-yr-old peach seedlings DISCUSSION. LITERATURE CITED. CONCLUSIONS AND FUTURE PROSPECTS APPENDIX. SECTION IV vi 86 91 91 91 94 94 95 96 101 107 111 LIST OF TABLES Table Page 1. Leucostoma isolates used in isozyme studies. . . 49 2. Enzyme stains tested with Leucostoma . . . . . . 52 3. Alleles scored at eight loci in Leucostoma. . . . 53 4. Summary of genotypic diversity for species and subgroups in Leucostoma. . . . . . . . . . . . . 55 5. Leucostoma isolates used in RFLP studies. . . . . 80 6. Restriction enzymes used in the digestion of PCR products of the ITS-LrDNA* fragment in L. cincta and L. persoonii. . . . . . . . . . . . . . . . . 81 7. High temperature growth tolerance, culture, pycnidial size and mean canker length of Michigan isolates of Leucostoma. . . . . . . . . . . . . 104 8. Summary of Analysis of variance for the five phenetic groups of Leucostoma. . . . . . . . . . 105 9. Virulence of standard isolates of the five phenetic groups of Leucostoma on 3-yr-old multistemmed seedlings ranked in order of decreasing canker length. . . . . . . . . . . . 106 vii LIST OF FIGURES Figure Page 1. Phenogram based on isozyme analysis showing the groupings of isolates of L. cincta and L. persoonii. The phenogram was constructed with the NTSYS program using UPGMA from Simple Matching coefficient values. The numbers in parentheses correspond to isolate numbers (Table 1). . . . . . 56 Most parsimonious tree generated using the BOOT program of PHYLIP. The numbers on branches correspond to the confidence limit on each branch. Those on the end of the tree correspond to the isolate numbers in Table 1. . . . . . . . . . . . 57 Phenogram of Leucostoma persoonii isolates based on the UPGMA cluster analysis of simple matching coefficient. Similarity generated from restriction fragment length polymorphisms in the portion of the nuclear rDNA containing ITSI, ITSZ, 5.8S rDNA and part of the 28S rDNA. . . . . . . . . . . . . . . 82 Map of a portion of rDNA repeat showing the location of oligonucleotide primer sites to amplify rDNA from Leucostoma spp. . . . . . . . . 83 Restriction profiles of PCR product of the fragment containing the two ITS regions, 5.88 rDNA and a portion of the 28S rDNA after digestion with Hinf I. A70 & Flh are isolates of L cincta and NC22.2, T26,6, LCN, 11.3, Cy4, f.sp. mahaleb and LP10 are isolates of L persoonii. Primers used: ITSS/TW14. . . . . . . . . . . . . . . . . . . . . 84 PCR products from enzymatic amplification of nuclear rDNA using primers NSZl/NSZZ . An insertion of about 400bp present in the isolates of L. cincta on Prunus is missing in isolates of L. cincta on Malus and L. persoonii. . . . . . . . 85 viii GENERAL INTRODUCTION AND LITERATURE REVIEW GENERAL INTRODUCTION AND LITERATURE REVIEW Leucostoma canker, which is also called perennial canker, Cytospora canker and Valsa canker, seriously limits peach production in the northern portions of the region favorable for production of temperate fruits (Biggs, 1986; Dhanvantari, 1978; Hildebrand, 1947 & Kern, 1955). The disease is also a problem on plum and prune (Prunus domestica L.), sweet and sour cherry (P. avium L. and P. cerasus L., respectively), apricot (P. armeniaca L.), wild black cherries (P. serotina Ehrh.), ornamental quince (Chaenomeles speciosa (sweet) Nakai), and apple (Malus domestica Borkh.) among other mainly roseaceous hosts (Helton and Moisey, 1955 ; Helton & Konicek 1961, Proffer & Jones, 1989)._ The disease is characterized by extensive perennial cankers on the limbs and branches, which results in branch dieback, progressive weakening of the tree and eventually the infected branch is girdled and killed (Biggs, 1986; Hampson & Sinclair, 1973; Tekauz & Patrick, 1974). Infections of small twigs appear as sunken discolored areas near winter killed buds or leaf scars. Symptoms on the main trunk, branch crotches, scaffold limbs and older branches begin with gum exudation. As cankers age, the gum becomes dark brown to black, the infected bark dries out and cracks open exposing the blackened tissue beneath elliptical cankers along the length of the stem (Biggs, 1989). The disease is especially destructive in young orchards where it causes the premature death of orchard nursery stock. Fungicides have very little effect on the incidence of new infections or on the expansion of established cankers (Helton & Rohrbach, 1967 ; Palmiter & Hickey, 1970). Pruning and other cultural practices have had limited success in controlling leucostoma canker (Weaver, 1963). This disease is caused by two closely related species of fungi, Leucostoma cincta (Pers. : Fr.) Hoehn. [anamorph = Leucocytospora cincta (Sacc.) Hoehn.] and L. persoonii (Nits.) Hoehn. [anamorph = Leucocytospora leucostoma (Pers.) Hoehn.]. Early literature in the nomenclature of the pathogen shows an obvious conflict. The two species were placed in the genus Valsa by some authors and in the genus Leucostoma by others (Von Hoehnel, 1917). In 1867, Nitschke divided the genus Valsa into five sub-genera based on the structure of the stromata and Leucostoma was included as one of these genera. In 1917, Von Hoehnel elevated the subgenus Leucostoma to an independent genus. He used the presence of a darkened conceptacle or marginal zone . ‘ delimiting the stromatic tissue as the distinguishing feature. Leucostoma and its corresponding imperfect state Leucocytospora exhibit the developed conceptacle, while Valsa and its anamorph Eucytospora do not. (Von Hoehnel, 1981 ; Linda Spielman, 1984) A monographic treatment of Leucostoma was completed in 1958 by Urban in Czechoslovakia (Urban, 1958). Eight species were described, two from coniferous hosts, L. kunzei (Fr. ) Munk and L. curreyi (Nit.) Defago; four with a broad host range of deciduous trees, L. nivea (Hoffm. : Fr.) Hoehn., L. massariana (DeNot.) Hoehn. L. aureswaldi (Nit. ) Hoehn. and L. translucens (Ces. & DeNot. ) Hoehn.; and two from Prunus species L. cincta (Fr.) Hoehn. and L. persoonii (Nit. ) Hoehn. A third species on a conifer host, L. sequoiae Bonar (1928) was described in 1928. The species were differentiated primarily by the size of the stroma, number of spores per ascus, location of the pycnidium in relation to the stroma and the ascospore size. In order to recognize the taxa in Leucostoma, a full range of criteria must be available. Those criteria pertaining to the sexual state are crucial. However, sexual states are rarely found in nature and identification of these species by plant pathologists has been based on cultural characteristics described by Willison (1936 & 1937) and Hildebrand (1947). This has led to confusion in the identification of the two taxa due to the presence of a wide range of variation in cultural morphology (Adams, Hammer & Iezzoni, 1990 ). Kern (1955) suggested that typical isolates of L. cincta and L._persoonii might represent extremes and that intermediate forms are possible. Lukezic et a1 (1965) reported that monoascospore isolates from a single ascus of L. persoonii showed a range of cultural characteristics putatively typical to both species. Optimum growth temperature was suggested for differentiating the two species (Hildebrand, 1947) but this criterion is not used by most workers today (Luepschen et a1, 1975 & Luepschen, 1981). Defago (1934) conducted an extensive investigation of the pathogenicity of three Leucostoma spp. and two Valsa spp. on Prunus. He described nine formae speciales of L. persoonii. His concept of formae speciales was based on differences in the'degree of virulence on each of 10 Prunus species rather than on host specificity on a specific Prunus species. The relative virulence of isolates of L. cincta and L. persoonii identified on cultural characteristics have been reported to differ greatly. L. persoonii has been reported to be of low virulence as compared with L. cincta on peach. (Helton & Konicek, 1961 ; Willison, 1937). However, in other research L. persoonii was found to be more virulent in warmer weather and L. cincta more virulent in cool weather (Hildebrand, 1947 ; Wensley, 1964). Proffer and Jones (1989) identified Leucostoma canker caused by L. cincta as a new disease of apples in North America, specifically in Michigan. It is likely that in previous studies of apples L. cincta was either not identified or was identified as L. persoonii (Leonian, 1921). Thus no characteristic capable of reliably distinguishing cultures of these two species is currently available. This problem has significant consequences in current research on breeding peach trees for disease resistance. Breeding is an expensive and labor intensive process and release of new resistant germplasm faces the risk of being exposed to pre-existing races or biological species of the pathogen that might overcome such a resistance. Although identification of a species is based on observable differences in morphology, species and populations may be genetically distinct even when they are not morphologically separable. In recent years, there has been an increasing interest in new approaches 0 to study genetic variation in fungi at several taxonomic levels using biochemical and molecular techniques. Electrophoretic analysis of isozymes has been extensively and successfully used for years to provide rapid and qualitative estimates of the variation within species of higher plants and animals (Gottleib, 1982 ; Hillis & Davis 1986). Enzymes which are coded by different alleles or separate genetic loci frequently possess different electrophoretic mobilities. Such differences are due to variations in the amino acid content of the molecule, which in turn is dependent on the sequence of nucleotides in the DNA. An analysis of isozyme variation by electrophoresis therefore approximates the analysis of gene variation and has been useful in the studies of population genetics. There are at least three major areas in which isozyme analysis has been useful in studies of fungi. These areas include ; 1. classification and delineation of fungal taxa, 2. identification of fungal cultures, and 3. fungal genetics In classification and delineation of fungal taxa, the interpretation of isozyme banding patterns have proven to be very useful in solving taxonomic problems when few morphological characters are available or when the characters are plastic in nature. Bonde et a1; (1984 ) 9 _ used isozyme analysis to differentiate species of Peronosclerospora causing downy mildew of maize. Isozyme patterns of ten fungal cultures representing three species of the genus Peronosclerospora from Texas, southern India, Brazil, Taiwan and the Philippines were compared to aid species identification. Based on isozyme patterns the authors concluded that P. sacchari in Taiwan was the same pathogen as P. philippines in the Philippines. P. sorghi in Thailand was genetically different from P. sorghi in India, Brazil and the United States and was probably misidentified. They suggested that delineation of species needed further re-evaluation, which could be achieved by isozyme analysis of additional isolates of Peronosclerospora. Micales et a1, (1987) used the same technique to prove that Endothia eugeniae, a pathogen of clove, and Cryphonectria cubensis, a pathogen of eucalyptus, were conspecific. The two species were indistinguishable by the analysis of their soluble proteins. In addition they shared alleles at 16 presumed loci as detected by isozyme analysis. Hanson and Wells (1991) characterized three species of Tremella based on their isozyme banding patterns. The three species were monomorphic at twelve enzyme loci but also retained species specific mobilities. Similar studies were performed using isozyme analysis in Colletotrichum spp. ( Bonde, Peterson and Mass, 1991), Ustilago hordei (Hellman and Christ, 1991) and in the Acremonium/Epichloe complex (Leuchtman and Clay, 1990). Interpretation of isozyme bands to compare allelic ratios expressed by fungal isolates has been used to study phylogenetic relationships among organisms. Stasz et a1, (1989) used isozyme polymorphism to determine phylogenetic relationship in the genus Trichoderma by cladistic analysis. They evaluated five morphological species of Trichoderma. Their study determined that morphological species were not characterized by either specific alleles at single loci or by specific patterns of alleles at multiple loci. Oudemans and Coffey (1991) used isozyme analysis to study the systematics of twelve papillate Phytophthora species. Based on their studies they proposed that two species, P. arecae and P. palmivora were synonymous. Three species, P. capsici, P. magekarya and P. citrophthora demonstrated much higher levels of variation in isozyme pattern. They found no evidence to support the existence of distinct varieties in P. parasitica. In a related study on Pythium, Chen et a1. (1991) reported the separation of seven homothallic species of Pythium on the basis of their isozyme banding patterns. In the identification of fungal cultures, isozyme analysis can be used to compare unknown isolates to previously identified cultures in order to determine their identity. Bonde et a1. (1985) reported that 50% of the genetic loci of Tilletia indica were monomorphic. Tooley et a1. (1985) detected 11 of the 24 loci tested to be monomorphic in Phytophthora infestans. It was suggested that these monomorphic loci be used when comparing a known isolate to an unknown. Burdon and Rolfs (1985) examined the effect of sexual and asexual modes of reproduction on the level of diversity of isozyme variation found within respective North American populations of Puccinia graminis f. sp. tritici. In their study they concluded that sexual populations of P. graminis were more diverse than asexual. Jeng et a1, (1987 ) did a comparative study of electrophoretic characteristics of Eurasian and North American races of Ophiostoma ulmi. In the study they demonstrated additional differences between the two races. Their data also indicated that one of the isolates might be a hybrid between the two races. A comparative study of isolates of Septoria on citrus from Australia and United States was performed by the study 10 of their isozyme banding patterns (Bonde et a1, 1991). The authors concluded that the isolates from both the countries belonged to the same species. In their study of isozyme comparison among worldwide sources of three morphologically distinct sources of Phytophthora, Oudemans and Coffey (1991) detected three enzyme loci that were found to be diagnostic of the three species. Studies like the preceding ones become extremely important when some crop materials are quarantined due to danger of entry of exotic plant pathogens. In such instances, isozyme analysis can facilitate identification when only a few morphological characters are available. In fungal genetics , isozyme analysis has been an effective technique for studying the allelic variation in populations. Kerrigan and Ross (1989) used 18 field isolates of Agaricus bisporus to study genetic variation in the wild Agaricus population. Their study revealed the presence of new alleles supporting the belief that cultivars of A. bisporus escape from commercial cultivation and can reproduce under normal circumstances. It also revealed the potential of expanding the gene pool of commercial varieties. High variability was detected in the related species Agaricus bitorquis (Roux and Labarere, 1991). This 11 species has a phenotype comparable to A. bisporus and it was suggested that the high genetic variability compared to that of A. bisporus could provide a greater genetic base for cultivar expansion. Genetic variation was also reported to be high in Crumenulopsis sororia (Ennos and Swales , 1991). Zambino et a1 (1989) studied genetic variation between varieties of Leptographium wagneri. They reported one electrophoretic type to be abundant and broadly distributed within each variety. Gene diversity within each variety was observed to be low but genetic differentiation between varieties was high. In other studies isozymes have been coupled with other markers for a comparative analysis. For example, Newton et a1 (1985) studied variation for isozyme and double-stranded RNA among isolates of Puccinia striiformis. These studies were aimed at using the two groups of molecules as markers for both the nucleus and the cytoplasm and thus allow the variability in each genome to be examined separately. No differences in isozyme banding patterns were observed among 29 diverse wheat-attacking isolates of P. striiformis (WYR). Isolates of the barley attacking form (BYR) of P. hordei and P. recondita showed similar uniformity. In D 12 contrast, there were differences in the dsRNA phenotypes both among the three species and between WYR and BYR. Burdon et a1, (1985) studied isozyme and virulence variation in asexually reproducing populations of Puccinia graminis and P. recondita on wheat. They detected no correlation between isozyme and virulence diversity. In a related study on Uromyces appendiculatus Linde, et a1. (1990) detected greater 1-~.—.._.__‘—— —< <- diversity in virulence patterns as compared to the' isozyme patterns. Mills et a1.(1991) used isozyme and mitochondrial DNA analyses to study intra- and interspecific variation in Phytophthora cryptogea and P. drechsleri. The majority of the isolates were subdivided into ten groups based on numerical analysis of 24 putative enzyme loci. Analysis of mitochondrial DNA restriction fragment length polymorphisms of selected isolates from each enzyme group supported their isozyme data. Vilgalys (1991) reported association between different intersterility groups of Collybia dryophilla with isozyme differences and postulated that fungal speciation evolves primarily by allopatry. Additional techniques would be helpful both in the identification of species in the absence of sexual states well as to detect intra- and interspecific variation. Recently, restriction fragment length 13 polymorphisms (RFLP), which reflect variation among homologous DNA sequences have provided more precise tools for detecting and quantifying genetic variation. RFLPs have been studied in many organisms including humans (Botstein et a1, 1980), plants (Helentjaris et a1, 1985) and animals (Hillis and Davis, 1986). These polymorphisms can be generated by loss or gain of restriction sites resulting from point mutations, or from rearrangement of DNA sequences. McDonald and Martinez (1990) used RFLP markers to measure the amount and distribution of genetic variation in Septoria tritici. Single and multilocus analysis of their RFLP data indicated that a high level of genetic variability was distributed in the population. RFLPs have also been used to study molecular variation in Verticillium (Carder & Barbara, 1991) and in Colletotrichum gloeosporioides infecting Stylosanthes spp in Australia (Braithwaite and Manners, 1990). Specht et a1. (1984) used RFLPs to study strain specific differences in the nuclear ribosomal DNA (rDNA) of the fungus Schizophyllum commune. Restriction mapping of rDNA using four strains of the fungus revealed strain specific variation with repeat lengths of 9.2-9.6Kbp. These authors were also the first to report methylation of rDNA. RFLP markers 14 were used by Anderson et a1. (1987) to compare biological species within the taxon Armillaria mellea. They identified significant variability and most of the variability occurred between rather than within biological species. One of the difficulties in applying RFLPs to taxonomic problems is in selecting a DNA segment that can resolve at an appropriate taxonomic level, showing neither excessive and uninterpretable variation nor homogeneity at the taxonomic rank of interest. In fungi, rDNA is especially useful in taxonomic studies for the following reasons: 1)the rDNA repeat length is within a range that can be examined by RFLP analysis, 2) the rDNA repeat unit contains both slowly evolving regions (the 188, 5.88, and 28S rRNA genes) and more rapidly evolving regions ( the transcribed and non transcribed spacers) so that information from various levels of evolutionary history can be recovered, and 4) the rDNA evolves in a concerted fashion. (Appels & Dvorak, 1982; Bruns, White & Taylor, 1991). The rDNA repeat unit has been widely used to study phylogenetic relationships and population genetics in fungi. Kohn et al. (1988) compared Sclerotinia species by the analysis of the RFLP patterns of nuclear and mitochondrial rDNA. They observed polymorphisms in the 15 rDNA between rather than within species. In addition extensive variation in RFLPs of mitochondrial DNA between species was also observed. Magee et a1. (1987), used RFLPs in the nuclear rDNA to distinguish various isolates of Candida albicans. Six different classes were detected based on variations in the restriction pattern. Similar studies by Walsh et a1. (1990) using a heterologous probe to detect RFLPs in Entomophaga rDNA sequences showed delineation of genera and species. within Entompthorales but were not useful at lower taxonomic levels. William et a1. (1988) compared RFLP patterns in the nuclear rDNA and mitochondrial (mt) DNA of three Agaricus species. Restriction patterns of one species (A. brunescens) were found to be identical, and the A. bitorquis and A. campestris isolates were subdivided into two. Vilgalys and Gonzalez (1990) detected variation both at the inter- and intragroup level of the 15 anastomosis groups of Rhizoctonia solani used in their study. In a related study on restriction analysis of rDNA of binucleate Rhizoctonia spp. Cubeta et a1. (1991) reported the separation of 13 of 21 anastomosis groups of binucleate Rhizoctonia spp. into distinct groups. These groupings were found to be consistent with prior groupings based on hyphal 16 anastomosis. Egger, Danielson and Fortin (1991) analyzed the nuclear and mitochondrial ribosomal RNA genes to elucidate the species concept among the asexual E- strain mycorrhizal fungi and to examine their population structure. They found that most E- strain isolates could be assigned to the two sexual taxa Wilcoxina mikolae and W. rehmii, which have different habitat preferences. Analysis of the mitochondrial DNA revealed that within each species isolates could be differentiated based on host preference. With the advent of enzymatic amplification, copies of selected regions of the rDNA repeat unit can be selectively amplified as non methylated copies from crude DNA preparations, greatly simplifying RFLP analysis of populations (Bruns, White & Taylor, 1991). Vilgalys and Hester (1990) used this technique for genetic identification and mapping of amplified rDNA of Cryptococcus species. Digestion and electrophoresis of the PCR products by using restriction enzymes produced restriction phenotypes that were unique for each strain or species. Hibbet & Vilgalys (1991) studied evolutionary relationships of Lentinus to the Polyporaceae by restriction analysis of enzymatically amplified rDNA. Their results suggested that Lentinus tigrinus was more closely related to the Polyporaceae 17 than to the Tricholomataceae The aim of this project was to study the variability in the two closely related species of Leucostoma, L. cincta and L. persoonii. This research in particular aims at using markers that would be useful in revealing species and subspecific variation. The following three chapters describe the techniques used to achieve this goal. Chapter 1 describes the use of isozyme markers to clarify and delineate the two taxa. Chapter 2 describes the use of restriction fragment length polymorphisms to study the nuclear ribosomal DNA variation between various isolates of Leucostoma. In chapter 3, pycnidium size, temperature optimum and relative virulence among some of the genetically different phenetic groups within L. cincta and L. persoonii are compared. The concluding chapter stresses the importance of the research in providing a basis for understanding the relationship between the plant pathogenic isolates of Leucostoma and makes recommendations for future research. 18 LITERATURE CITED Adams, G. C., Hammar, S. A. and Iezzoni, A. 1989. Optimum sample size for detecting virulence differences in Leucostoma from peach. Plant Disease 73: 754-759 Anderson, J. B., Petsche, D. M. and Smith, M. L. 1987. Restriction fragment length polymorphism in biological species of Armillaria mellea. Mycologia 79:69-76. Apples, R. and Dvorak, J. 1982. Relative rates of divergence of spacer and gene sequences within the rDNA region of species in the Triticae: Implication of maintenance of homogeneity of a repeated gene family. Theoret. Appl. Genet. 63:361-365. Biggs, A. R. 1986. Comparative anatomy and host response of two peach cultivars inoculated with Leucostoma cincta and Leucostoma persoonii. Phytopathology 76: 905-912. Biggs, A. R. 1989.Integrated approach to controlling Leucostoma canker of peach in Ontario. Plant Disease 73: 869-874. Botstein, D., White, R. L., Skolnick, M. and Davis, R. W. 1980. Construction of a genetic map in man using restriction fragment length polymorphisms. Am. J. Hum. Genet. 32:314-331. Bonar, L. 1928. Studies on some California fungi. Mycologia 20:292-300 Bonde, M. R., Peterson, G. L., Dowler, W. M. and May, B. 1984. Isozyme analysis to differentiate species of Peronosclerospora causing downy mildews of maize. Phytopathology 74:1278-1283. Bonde, M. R., Peterson, G. L., Dowler, W. 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Revise Ceskoslovenskych zaspcu rodu Valsa, Leucostoma, a Valsella. Rozpr. Cesk. Akad. Ved. Rada Mat. Prir. Ved. 68: 1-101. Von Hoehnel. 1918. Mycologische Fragmente. Ann. Myc. 16:127-144. Vilgalys, R. and Gonzalez, D. 1990. Ribosomal DNA restriction fragment length polymorphisms in Rhizoctonia solani. Phytopathology 80: 151-158. 24 Vilgalys, R., and Hester, M. 1990. Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species. J. Bacteriol. 172:4238-4246. Vilgalys, R. 1991. Speciation and species concepts in the Collybia dryophilla complex. Mycologia 83: 758-773. Walsh, S. R., Tyrell, D., Humber, R. A. and Silver, J. C. 1990. DNA restriction fragment length polymorphisms in the rDNA repeat unit of Entomophaga. Expt. Mycol. 14, 381-392. Weaver, G. M. 1963. A relationship between the rate of leaf abscission and perennial canker in peach varieties. Can. J. Plant Sci. 43:365-369. Wensley, R. N. 1964. Occurrence of pathogenicity of Valsa species and other fungi associated with peach canker in Southern Ontario. Can. J. Bot. 42:841-857. White, , T. J., Bruns, S., Lee, S. and Taylor. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics, p.315-322. In Innis, N., Gelfand, J., Sninsky and White, T. (ed), PCR protocols: a guide to methods and applications. Academic Press, Inc., New York. Willison, R. S. 1936. Peach canker investigations II. Infection Studies. Can J. Res. 14: 27-44. Willison, R. S. 1937. Peach canker investigations III. Further notes on incidence, contributing factors, and related phenomena. Can. J. Res. 15:324-339. Zambino, P. J. and Harrington, T. C. 1989. Isozyme variation within and among host specialized varieties of Leptographium wagneri. Mycologia 8:122-133. ISOZYME DETECTION AND VARIATION IN LWCOSTOMA FROM PRUNUS AND MALUS. ISOZYME DETECTION AND VARIATION IN LHICOS'TOMA SPECIES FROM PRUNUS AND MALUS ABSTRACT Isozyme analysis was used to study the relationship between two closely related taxa of Leucostoma, L. cincta and L. persoonii. Isolates were obtained from six Prunus spp. and Malus domestica Borkh. in North America. Monoascospore isolates collected from individual perithecia of L. cincta and L. persoonii, tissue isolates and isolates varying in vegetative compatibility groups of L. persoonii were examined. Thirty alleles were resolved at eight putative loci and the two species were found to be distinct at 48% similarity._ In addition, this analysis separated the two species into six phenetic groups. L. cincta was separated into several closely related groups on Prunus spp. and one group on Malus. L. persoonii was separated into three distinct groups which were not correlated to hosts or geographic origins. Isozyme polymorphisms were not found to be associated with vegetative compatibility groups in L. persoonii. Far more diversity was evident among isolates of L. cincta from different hosts than among isolates of L. persoonii from one perithecium from different hosts. 25 26 Identification of a fungal species is based on observable differences in morphology, but species and populations may be genetically distinct even when they are not morphologically separable. Genetic and biochemical techniques such as isozyme analysis have been useful in combination with morphology to identify genetic variability between and among fungal species (Micales, Bonde & Peterson, 1986; Micales & Stipes, 1987; Hanson & Wells, 1991; Chen, Hoy & Schneider, 1991; Stasz et a1, 1989) and to delineate taxa that are morphologically similar in culture (Bonde, Peterson & Mass, 1991). An alternative method to morphology is needed for differentiating the two closely related and important plant pathogens Leucostoma cincta (Fr.: Fr.) Hoehn. and L. persoonii Hoehn. that cause Cytospora canker of fruit trees.Morphological characteristics distinguishing the two species are present only in the teleomorph and the teleomorph is generally not found in nature associated with the disease symptoms. The Leucocytospora anamorphs, Leucocytospora cincta (Sacc.) Hoehn. and L. persoonii Hoehn., that are abundantly present in infected orchards are not separable morphologically. Because the teleomorph generally is not found the practical delimitation of these two pathogens often has 27 been based on cultural characteristics. Current researchers have not been in agreement on the relevant cultural and physiological characteristics of the two species (Adams, Hammar & Proffer, 1990; Surve-Iyer, Adams & Iezzoni, 1992). Cultural characteristics and growth temperature responses described by Willison (1938), Hildebrand (1947) or Kastirr (1984) have been utilized as criteria for differentiating the two species in some studies (Adams, Hammer & Iezzoni, 1989, Proffer & Jones, 1989) but in others the criteria used for identification is not clear (Endert Kirkpatrick, 1986; Helton & Konicek, 1961, Regner & Johnson, 1990). Thus currently, the presence of the sexual fruiting body continues to be required for the definitive identification of these two species until a more thorough study of cultural characteristics of isolates can be correlated with specific sexual states. In this study we have begun the grouping of anamorphic isolates by comparing their similarity in cultural characteristics to those of isolates obtained from identifiable sexual states, assigning the sexual name to corresponding cultures and comparing their isozyme profiles. This approach provides the foundation of the isozyme study and it effectively clarifies our 28 understanding of the revealed genetic diversity. The usefulness of cultural characteristics alone, as criteria in distinguishing the species, is summarized and discussed elsewhere (Surve-Iyer, Adams & Iezzoni,1992). Our objective is to identify and delineate the two taxa in the absence of the sexual state and to identify genetic variation in the Leucocytospora populations by using isozyme markers. Identifying the extent of genetic diversity in the two pathogens is necessary to construct an effective screening procedure in an ongoing peach breeding program selecting for canker resistance (Chang, Iezzoni & Adams, 1991; Chang et a1, 1989). Breeding is an expensive and labor intensive procedure. Release of new resistant germplasm would face risks of being exposed to pre-existing races or biological species of the pathogen that might overcome such resistance if selection for resistance was based on a limited understanding of the genetic diversity of the pathogens. The two Leucostoma species also cause annual or perennial cankers on nectarine,.sweet, sour and black cherry, plum and prune, ornamental quince, apple, chokecherry, amelancheir, and wild roseaceous hosts. The genetic variation among isolates on various hosts has not been examined thoroughly in the past. Whether host 29 specific isolates or races might exist within the populations of the two Leucostoma is not known but in a study in 1934 Defago concluded that some isolates represented formae speciales. His concept of forma speciales was not based on host specificity rather his formae speciales isolates were virulent on all the hosts he tested and were comparatively more virulent on one host. Some believe the relative virulence of the two species differs greatly; L. persoonii has been re- ported to be of low virulence compared to L. cincta on peach (Helton & Konicek, 1961; Willison, 1989). However, others believe L. persoonii is more virulent in warm weather and L. cincta more virulent in cool weather (Hildebrand, 1947; Wensley, 1964). We attempt in this study to use isozymes to analyze the extent of genetic variability in diverse populations from numerous hosts. This approach has been effective in other studies of fungi (Hellman & Christ, 1991 ; Zambino & Harrington, 1989; Leuchtmann & Clay, 1990; Linde, Groth & Roelfs, 1990; Yoon, Gessner & Romano, 1990). Similarly we wish to examine whether specific populations might be traceable to specific geographic origins, as others have accomplished with isozyme analysis (Bonde, Peterson, Emmett & Menge, 1991; Leung & Williams, 1986; Oudemans & 30 Coffey, 1991). The extent of genetic variation revealed in this study and correlation of the diversity to host and geographic origins is discussed. Isolates representative of the genetic diversity revealed herein are examined in virulence studies elsewhere (Surve-Iyer, Adams & Iezzoni, 1992). MATERIAL AND METHODS The host, geographic origin and source of the isolates of Leucocytospora used in this study are given in Table 1. Isolates of L. persoonii from perithecia were collected from Prunus persica (L.) Batsch. and P. serotina Ehrh. and isolates of L. cincta from perithecia were collected on P. armeniaca L., P. domestica L., and Malus domestica Borkh. Leucocytospora isolates that corresponded in cultural characteristics to L. persoonii ascospore cultures were collected from diseased plant tissue from Michigan, North Carolina, California, and Oregon and Ontario, Canada. Similarly asexual isolates corresponding to L. cincta were collected from Michigan. Isolates of L. persoonii from 9 vegetative compatibility groups (Adams, Hammar & Proffer, 1990) were and 7 European formae speciales ( Defago, 1935) were also compared in isozyme analysis. 31 Isolates were maintained on Leonian’s media (1.2 g KH2P04, 0.6 g M9304, 6.25 g maltose, 6.25 g malt extract in 1 L of distilled water) (Leonian, 1923). Five 1 cm plugs of mycelia grown on Leonian's agar medium were added to 100 ml of liquid Leonian’s media in a 500 ml Erlenmeyer flask. The isolates were grown at room temperature in still cultures. Mycelial mats were collected from each flask after 10-14 days incubation by vacuum filtration onto Miracloth and blotted dry on sterile paper towels. The mats were either used immediately or stored at -70°C. Mycelium was ground in liquid nitrogen and 3 ml of extraction buffer (50 mM Tris HCl, pH 7-7.5, 10% glycerol and 0.1% beta mercaptoethanol) and sand in a cold mortar and pestle. The mixture was centrifuged in a microfuge at 12,700 g for 20 min at 4°C, and 4 ul of the supernatant was applied to thin layer cellulose acetate plates ("Titan III" [94mm 76mm], Helena Laboratories, Beaumont, Texas) and electrophoresed at 12-20 mA for 20 min at 4°C. Excess supernatant was stored at -70°C and reused till the enzyme showed signs of degradation, after which it was discarded. Each isolate was tested three times and each gel was run with two standard isolates (11.3 and ATCC 62910). Five buffer systems were used in electrophoresis (Richardson, 1987); buffer 32 A, 10 mM NaZHPO4, 2.5 mM citric acid, pH 6.4; buffer B, 11.6 mM Na2P04, 8.4 mM NaH2P04, pH 7.0; buffer c, 50 mM Tris, 20 mM maleic acid, pH 7.8; buffer D, 15 mM Tris, 10 mM MgC12, 5 mM NazEDTA, pH 7.8; and buffer I, 25 M Tris, 192 mM glycine, pH 8.5). Enzyme stains were from Richardson (1987) and were scaled to approximately 2 ml total volume and applied to plates as overlays mixed 1:1 with 1.5% Bacto agar in distilled water. For each enzyme assay, mobility of the enzyme reaction was noted for each isolate and similar mobilities were compared in adjacent lanes in subsequent gels. Each band was considered as an allele of a specific locus. The most anodally migrating band was designated "a" and the alleles of the same locus were assigned "b", "c", etc., according to their mobility relative to the "a" allele. The genetic basis of the allelic patterns could not be unequivocally determined because it was not possible to induce formation of sexual states. Therefore we assumed the mobility differences or electromorphs were allelic variants within a given locus. Nomenclature for designating enzymes, loci and alleles followed the convention of Hanson & Wells (1991). Enzymes were identified by a short, upper-case abbreviation, e.g., IDH (isocitrate dehydrogenase). Genetic loci were 33 capitalized and italicized, e.g., IDH. When more than one allele was present an alphabet identifier was added: IDH-a or IDH-b The data as binary codes was analyzed using two computer assisted programs and their subprograms; SIMQUAL, SAHN, COPH & MXCOMP in the NTSYS Version 1.7 (Rohlf, 1992) and BOOT program in PHYLIP (Felsenstein, 1992). The SIMQUAL program was used to calculate similarity coefficients, Simple Matching Coefficient, which emphasizes both the positive and negative matches and the Jaccard's Coefficient, which emphasizes the positive matches over the negative matches. The similarity coefficients were analyzed with UPGMA (Unweighted pair group analysis using arithmetic means ) and clustered using the SAHN subprogram. To estimate the accuracy of the phenograms produced, each similarity matrix was converted to a matrix of cophenetic values, using COPH in NTSYS. Matrix correlations between the original similarity coefficients and the cophenetic values were assessed by MXCOMP in NTSYS. When the cophenetic correlation was greater than 0.9, the fit of the tree to the similarity coefficient matrix was considered very a good fit. The BOOT program of PHYLIP was used to generate the most parsimonious tree by bootstraping using Wagner’s assumptions with global 34 options. In addition genotypic diversity was calculated for each species or subgroup using the procedure of Selander et a1. (1986). Isolates were assigned to electrophoretic types (ETs) each representing a group of isolates with identical phenotypes for all loci scored. The genotypic measure considers the number of distinct ETs found in a species or subgroup and does not assume random mating. Isozyme phenotypes for the ETs of each isolate are given in Table 3. RESULTS Table 2 lists the enzymes that were either undetected, poorly resolved (bands were either faint or did not stain for all isolates) and well resolved on cellulose acetate gels. Thirty alleles were resolved at eight putative loci among the 56 isolates that stained for the eight enzymes. All the eight enzymes stained for a single locus (Table 4). Significantly greater variability was seen among isolates of L. cincta than among L. persoonii. No variability was detected among the representative isolates of the 9 vegetative compatibility groups. The L. persoonii formae speciales, from Europe showed unique alleles that were different from all North American L. persoonii isolates tested. f 35 Californian L. persoonii from nectarine also showed unique polymorphisms at two loci. Genetic Distances and Identities The phenogram in Fig. 1 provides a visual summary of the pattern of genetic differentiation between the two Leucostoma taxa and among several subgroups. The UPGMA phenogram constructed for the Jaccard's and the Simple Matching coefficients had identical topology however, the Jaccard's phenogram had longer branch lengths at major divisions and showed no (0%) similarity between the two Leucostoma taxa. The phenogram of the Simple Matching coefficient assigned closer relationships to the isolates than Jaccard’s coefficient. The phenogram in Fig. 2 is the most parsimonious Wagner unrooted tree calculated from 1000 bootstrap replications (13 hours of 386-33 MHZ PC-computer time) and shows confidence limits calculated for each branch, represented as the percent of the bootstrap estimates. The two taxa, L cincta and L. persoonii were distinctly different at 48.4% similarity and 99.6% confidence. Isolates of L. cincta were separated into two closely related phenetic groups on Prunus species (77.4% similarity and 47.3% confidence) and one distinct phenetic group on Malus (64.5% similarity and 99.6% confidence). L. persoonii 36 was separated into three distinct groups each sharing 74.2% similarity, one widespread group on Prunus spp. (PGI) (100% confidence), a second on Michigan peach and black cherry (P62) (100% confidence) and a third group of on nectarine in California (PG3) (42.8% confidence). Genotypic diversity in the two taxa was higher in L. cincta at .84%, the L. persoonii at 50% (Table 4). Most of the diversity in L. cincta was within the phenetic group occurring on Prunus spp (66%) for which two alleles occurred in each of the PGI, PGM, IDH, G6PD, ME, MAN, , GDH, and PGD loci (Table 2 & 3). DISCUSSION Isozyme analysis has proved to be an useful technique to study the relationship between the isolates of Leucostoma. Plant pathologists in the past have routinely substituted vague cultural characteristics in absence of sexual states to identify the two species. The results presented here clearly show the extent of differentiation between L. cincta and L. persoonii with respect to the electrophoretic banding patterns for eight different soluble enzymes. The eight could be used as diagnostic loci to identify the two species. The patterns of the stained enzyme loci obtained were clear, easy to interpret and readily obtained for 37 isolates from North America. However, several of the L. persoonii formae speciales isolates from Europe did not produce any bands when stained for a particular enzyme. These enzymes may be produced at extremely low levels or below the level of detection of the staining procedure. In addition, this technique has also demonstrated the separation of these two species into six phenetic groups. The phenogram generated by NTSYS-pc (Rohlf, 1987) using the SIMQUAL sub-program and the UPGMA option (NTSYS) resolved the genetic distances between 56 of the isolates and separated them into six phenetic groups. Isolates of L. persoonii were separated into three .phenetic groups (PG1, PG2 and PG3); those of L. cincta were subdivided into at least three phenetic groups, (PG4, P65, PG6). The L. cincta isolates from Malus appeared to be ecologically host specialized (although found exclusively on Malus this group is found to be virulent on inoculated peach seedlings). Proffer & Jones (1989) were the first to report the presence of L. cincta on Malus in North America. The reasons for the absence of these strains on Prunus in nature remains unknown. In addition to their distinct electrophoretic I' 38 patterns, these isolates also showed differences in cultural characteristics when compared to other L. cincta isolates. They released a reddish brown pigment in culture which was not detected in other isolates. These isolates were also slow growing compared to the rest of the isolates used in this study (Proffer & Jones, 1989). Screening of additional L. cincta isolates on Malus to study their isozyme banding patterns will be required to further elucidate the ecological host specialization of this group Unlike the distinct phenetic group found on Malus, the other two phenetic groups consisting of ascospore progeny of L. cincta on Prunus show substantial amounts of diversity both within and between the phenetic groups. This diversity might be due to sexual outcrossing through meiotic recombination and perhaps these groups are a single diverse group found on Prunus species. In nature L. cincta teleomorphs have been more abundant than L. persoonii teleomorphs. Genetic diversity has been reported as being higher in populations of Crumenulopsis soraria ( Ennos & Swales, 1991) where reproduction of this population is through sexual outcrossing than in asexual populations. Similarly high isozyme diversity has been reported in sexually outcrossing fungi such as Magnaporthe grisea I. 39 (Leung & Williams, 1986), Epichloe (Leuchtmann & Clay, 1989), and natural populations of Agaricus bitorquis (Roux & Labarere, 1991) when compared to related asexual species and populations. However it should be noted that isozyme variability might depend on the number of isolates used and the particular enzymes tested. The first phenetic group (PG1) of L. persoonii was geographically widespread and diverse in host range in nature. PG1 consisted of ascospores, isolates representing five vegetative compatibility groups, and tissue isolates of from four Prunus spp. Isolates within this group had identical banding patterns regardless of their origins. The PGZ isolates of L. persoonii have been found only from a peach seedling planting and two canker isolations from native black cherry in Michigan. Isolates in this group were not from sexual states, and sexual fruiting bodies have not been identified with this group so far. Perhaps this group represents a native infection source and hence it’s presence on seedlings rather than on imported nursery stock. Nursery stock might be often shipped infected (Wensley, 1964). The third group, PG3 occurred on nectarine from California. The PG3 isolates were not similar to isolates from California on plum (PG1), but 40 climatic factors vary greatly between the California locations of the plum and nectarine orchards. Therefore, whether the genetic isolations between the groups is due to geographic origins, climatic origins or host preference is speculative. Additional sampling in PG3 will be necessary before further speculations can be made about this group. Several factors may be responsible for the low genetic diversity seen in the phenetic group 1 of L. persoonii. Lack of isozyme variability could occur because of founder effect ( Ennos & Swales, 1991). Individual populations could have been established from founder isolates and it is possible that the introduced pathogen represented only part of the genetic variability that existed in the parent species, especially if the species is native to Europe or Asia. Absence of isozyme variability could be through genetic drift if the population size was dependent on transatlantic importation. Population bottlenecks also could naturally occur through seasonal environmental factors. Though sexual states have been found in this species and the fungus is reported to be heterothallic (Adams, Hammar & Proffer, 1990), lack of polymorphism could be due to the rarity of the sexual cycle or effective lack of a sexual cycle. It is also possible 41 that all the allelic variability is not detected by electrophoresis and the amount of genetic variability and genetic differentiation between groups may be underestimated (Ayala et a1, 1973; Bonde, Peterson & Dowler, 1988). Low levels of isozyme variability have been reported in other plant pathogenic fungi including some species of Phytophthora (Oudemans & Coffey, 1991), Ustilago hordei (Hellman & Christ, 1991) , Tremella (Hanson & Wells, 1991) and also in highly biotrophic fungi in Puccinia Styriformis (Newton, Caten & Johnson, 1985). There are no comparative studies of genetic variation in fungi having biotrophic, necrotrophic or saprotrophic nutrition to make general assumptions on their genetic variability. These results provide considerable information on the relationship between L. cincta and L. persoonii. Based on isozyme polymorphism we have demonstrated the presence of two closely related sympatric species, L. cincta and L. persoonii. Both species and several phenetic groups are known to occur in the same orchard in our study. The amount of variation at the level of local phenetic groups was high both between the two species and within L. cincta. Such patterns have been documented for Drosophila (Ayala et a1, 1973) and 42 Neurospora (Speith, 1975). According to Ayala (1973) the genetic distances between closely related species depends in part on the speed with which reproductive isolation mechanisms have developed and their efficacy during the speciation process. Polymorphism can occur if temporal variation is coupled with spatial heterogeneity (Gillispie and Langley, 1974). This could be the case in L. persoonii whose origin might be traced to Europe or perhaps Asia, having later being transported to the United States on susceptible hosts. Over time L. persoonii may have become three reproductively isolated groups. The large genetic distances between L. cincta and L. persoonii suggests that reproductive isolation between the two may have been quite rapid. In his review of genetic variation in natural populations, Nevo concluded that genetic polymorphism and heterozygosity are correlated with ecological heterogeneity (Nevo, 1978). Perhaps selection pressure due to adaptation to the many host species in the wild or to nursery stocks derived from selective breeding may have contributed to the above process and to the development of ecologically host specific races, as seen in isolates of L. cincta from Malus. This could also lead to formation of localized populations where peaches have been cultivated in the same orchard for decades. This could stabilize certain geographical populations. Perhaps this has occurred in phenetic group 2 of L. persoonii on black cherry, a tree which is native in mixed deciduous woodlands of Michigan. In inoculation experiments, Defago (1934) recognized forms of L. persoonii uniquely more virulent on one Prunus species than on others although all isolates had been found on P. persica. Though the concept of forma specialis was incorrectly used by Defago we have not detected in Michigan the type of host specialized isolates of L. persoonii he observed (Surve-Iyer, Adams & Iezzoni, 1992). However, the phenomenon of ecological host specialization in L. cincta was detected in our study. 44 Literature Cited Adams, G. C., Hammar, S. A. & Proffer, T. J. (1990). Vegetative compatibility in Leucostoma persoonii. Phytopathology 80: 287-291. Ayala, F. J. (1982). Genetic variations in natural populations: problem of electrophoretically cryptic alleles. Proceedings in National Academy of Sciences. U. S. A 79:50-554. Ayala, F. J., Tracey, M. L., Hedgecock, M. & Richmond, R. C. (1973). Genetic differentiation during speciation process in Drosophila. Evolution 28:576-592. Bertrand, P. F. and English, H. (1976). Effect of selected Cytospora isolates from stone fruits on certain stone fruit varieties. Phytopathology 51: 152-157 Bonde, M. R., Peterson, G. L. & W. M. Dowler. (1988). A comparison of isozymes of Phakospora pachyrhizi from eastern hemisphere and the new world. Phytopathology 78:1491-1494. Bonde, M. R., Peterson, G. L., Emmett, R. W. & Menge, J. A. (1991). Isozyme comparisons of Septoria isolates associated with citrus in Australia and United States. Phytopathology 81:517-521 Bonde, M. R., Peterson, G. L. & Mass, J. L. (1991). Isozyme comparison of Colletotrichum species pathogenic to strawberry. Phytopathology 81:1523-1528. Chang, L. S., Iezzoni, A., Adams, G. C. & Howell, G. S. (1989). Leucostoma persoonii tolerance and cold hardiness among diverse peach genotypes. Journal of American Society for Horticultural Science 114:482-485. Chang, L. S., Iezzoni, A. & Adams, G. C. (1991). 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Specific enzyme methods for cellulose acetate electrophoresis. In Allozyme Electrophoresis. pp. 145- 228 N.S.W. Australia: Academic Press. Rohlf, F. J. (1992). NTSYS-pc: numerical taxonomy system for the IBM pc microcomputer (and compatibles), version 1.40 manual. Setauket, New York, U.S.A.: Applied Biostatistics, Inc. Roux, P. & Labarere, J. (1991). Determination of genes and subunit composition of three isozyme activities in Agaricus bitorquis Mycological Research 95: 851-860. Selander, R. K., Caugant, D. A., Ochman, H., Musser, J. M. Gilmour, M. N. & Whittman, T. S. (1986). Methods of multilocus enzyme electrophoresis for bacterial population genetics and systematics. Applied and Environmental Microbiology. 51:410-413. Speith, P. T. (1975). Population genetics of allozyme variation in Neurospora intermedia. Genetics 80: 785-805. Stasz, T. E., Nixon, K., Harman, G. E. & Weeden, N. F. (1989). Evaluation of phenetic species and phylogenetic relationships in the genus Trichoderma by cladistic analysis of isozyme polymorphisms. Mycologia 81:391-403. Yoon, C. S., Gessner, R. V. & Romano, M. A. (1990). Population genetics and systematics of Morchella esculenta. Mycological Research. 82:227-235. Wensley, R. N. (1964). Occurrence and pathogenicity of Valsa (Cytospora) species and other fungi associated with peach canker in Southern Ontario. Canadian Journal of Botany. 42: 841-857. 48 Willison, R. S. (1938). Peach canker investigations II. Infection studies. Canadian Journal of Research 14: 27-44. Zambino, P. J. & Harrington, T. C. (1989). Isozyme variation within and among host specialized varieties of Leptographium wagneri. Mycologia 81: 122-133. 49 Table l. Ieucostona isolates used in the isozyme studies Species Sexual or isolate asexual nulber 6 SUI-e! code * Origin Host Host Group L. cincta (1) A2 5 Michigan M. donestica Proffer 6 Jones P66 (2) A8 5 Michigan M. donestica Proffer 6 Jones P66 (3) A9 5 Michigan M. domestica Proffer 6 Jones P66 (4) 612 5 Michigan 11. donestica Proffer 6 Jones P66 (5) A43 a Michigan M. douestica Proffer 6 Jones P66 (6) A45 a Michigan M. domestica Proffer 6 Jones P66 (7) A48 a Michigan M. domestica Proffer 6 Jones P66 (8) A46 5 Michigan M. domestica Proffer 6 Jones P66 (9) A79 5 Michigan 14. domestica Proffer 6 Jones 966 (10) A32 a Michigan M. domestica * Proffer 6 Jone P66 (11) SP4 5 Michigan P. donestica Proffer 6 Jones P65 (12) 2P2 s Michigan P. domestica Proffer 6 Jones P65 (13) 4P1 s Michigan P. domestica Proffer 6 Jones P65 (14) 1P1 5 Michigan P. dosestica Proffer 6 Jones P64 (15) 062 a Michigan P. donestica Proffer 6 Jones P64 (16) IP63 a Michigan P. domestica Proffer 6 Jones P64 (17) IP66 a Michigan P. donestica Proffer 6 Jones P64 (18) IP70 a Michigan P. donestica Proffer 6 Jones P64 (19) IP73 a Michigan P. armeniaca Proffer 6 Jones P65 (20) IP40 a Michigan P. arseniaca Proffer 6 Jones P65 (21) IP39 a Michigan P. armeniaca Proffer 6 Jones P64 (22) P111 5 Michigan P. arseniaca Adams P64 (23) IP47 5 Michigan P. armeniaca Adams P65 (24) LP49 5 Michigan P. armeniaca Adams P64 (38) A166 62910 a Ontario P. persica ATCC (Biggs) P64 Flu 5 Michigan P. armeniaca Adams Plg 5 Michigan P. arneniaca Adams Flr 5 Michigan P. armeniaca Adams Flb 5 Michigan P. armeniaca Adams 1‘15 5 Michigan P. armeniaca Adams Flf 5 Michigan P. arneniaca Adams 50 Table 1. contd. L. persoonii (25) 11.3 a Michigan P. persica Adams P61 (26) 7584 5 Ontario P. persica Biggs P61 (27) 0855 5 Ontario P. persica Biggs P61 (28) 7553 3 Ontario P. persica Biggs P61 (29) IP8 s Michigan P. serotina Proffer P61 (30) IP10 5 Michigan P. serotina Proffer P61 (31) LP16 a Michigan P. donestica Proffer P61 (32) LP34 a Michigan P. arseniaca Proffer P61 (33) 128.1 a Michigan P. persica Adam 6 Surve P62 (34) 120.3 a Michigan P. persica Adams 6 Surve P62 (35) I14.7 a Michigan P. persica Adams 6 Surve P62 (36) TM a Michigan P. persica Adams 6 Surve P62 (37) 18.6 a Michigan P. persica Adams 6 Surve P62 (39) Cy2 a Oregon P. aviul Spotts P61 (40) Cy3 a Oregon P. avium Spotts P61 (41) Cy4 a Oregon P. aviul Spotts P61 (42) CyS a Oregon P. avium Spotts P61 (43) 128.1 a Michigan P. persica Adams 6 Surve P62 (44) Lplz a Michigan P. cerasus Proffer P62 (45) TM a California P. persica Michailides P63 ' var. nucercisa (46) LCC a California P. persica Michailides P61 var. nucercisa (47) LCM a California P. persica Michailides P63 var. nucercisa (48) T26.3 a Michigan P. persica Adams 6 Surve P62 (49) 14617 a North Carolina P. persica linden-Kirkpatrick P61 (50) 8649 a North Carolina P. persica Ended-Kirkpatrick P61 (51) T16.7 a Michigan P. persica Adams 6 Surve P62 (52) 1469.2 a North Carolina P. persica Ended-Kirkpatrick P61 (53) NC8.2 a North Carolina P. persica Endert-Kirkpatrick P61 (54) T18.1 a ‘ Michigan P. persica Adams 6 Surve P62 (55) RSTIO a California P. domestica Adams P61 (56) R3T16 a California P. domestica Adams P61 f.sp mahaleb a Switzerland P. persica CBS(264.34) f. sp persica a Switzerland P. persica CBS(266.34) f. sp oecononica a Switzerland P. persica CBS(26S.34) f.sp arneniaca a Switzerland P. persica CBS(260.34) f. sp avian a Switzerland r. DGFSlCd *represents the number an the phenogram in Fig. l 0 '5' represents the sexual state and 'a ' represents asexual state 51 table 1. contd. f. sp cerasi a Switzerland P. persica CBS(262.34) 85115 a Michigan P. persica Adam R615 a Midiigan P. persica Adams 85112 a Michigan P. persica Adams 10.14 a Michigan P. persica Adams 11.12 a Michigan P. persica Adams 58 a Michigan P. persica Adams 81112 a Michigan P. persica Adams 10.1 a Michigan P. persica Adams 11.2 a Michigan P. persica Adams R4114 a Michigan P. persica Adams 8114 a Michigan P. persica Adams R1816 a Michigan P. persica Adams 105 a Michigan P. persica Adams R6114 a Michigan P. persica Adams 85111 a Michigan P. persica Adams 52 Table 2. Enzyme stains tested with Leucostoma Enzyme (EC number) Buffer* Undetected enzymes Aconitate hydratase (EC 4.2.1.3) Alcohol dehydrogenase (EC 1.1.1.1.) Adenylate kinase (EC 2.7.4.3) Aldehyde oxidase (EC 2.7.1.40) Fructose diphosphatase (EC 3.1.3.11) Glyceraldehyde-3-phosphate dehyrogenase (EC 1.2.1.12) Glutathione reductase (EC 1.6.4.2) B- Hydroxybutarate dehydrogenase (1.1.1.30) Lactate dehydrogenase (EC 1.1.1.27) Pyruvate kinase (EC 1.2.3.1) Shikimic dehydrogenase (EC 1.1.1.25) (3043 ‘\‘ U!w(§()UC3 U UJOED ‘ CJ>I>UIO<1 wi4>iw0v U \““‘ Poorly resolved enzymes Acid phosphatase (EC 3.1.3.2) Alkaline phosphatase (EC 3.1.3.1) Diaphorase (EC 1.6.4.3) Glucose dehydrogenase (EC 1.1.1.47) Hexokinase (EC 2.7.1.1) Mannose- phosphate isomerase (EC 5.3.1.8) WEDOJV(3>‘ WIDCJUIDCD well-resolved enzymes Esterase (EC 3.1.1.1) Glutamate dehydrogenase (EC 1.4.1.3) Glucose-phosphate-dehydrogenase (EC 1.1.1.49) Glucose-phosphate-isomerase (EC 5.3.1.9) Isocitrate dehydrogenase(EC 1.1.1.42) Malate Dehydrogenase (EC 1.1.1.37) Mannitol dehydrogenase (EC 2.4.2.1) Phosphogluconate dehydrogenase (EC 2.7.5.1) Phophoglucomutase (EC 2.7.5.1) “ {VCJWCHD'wCDCJV U *Buffer A: 10 mM NaZHPO4 and 2.5 mM Citric acid, pH 6.4 *Buffer B = 11.6 mM NazHPO4 and 8.4 mM NaH2P04, pH 7.0 *Buffer = 50 mM Tris and 20 mM Maleic acid, pH7.8 *Buffer D: 15 mM Tris, 5 mM NaZEDTA, 10 mM MgC12, pH 7.8 *Buffer I: 25 mM Tris and 192 mM Glycine Locus Alleles 53 PGM IDH G6PD MAN GDH PGD ME PGI Table 3. Alleles scored at eight loci in Leucostoma. Isolate number* aaaaaaaaaabbbabbbbbbbbbbccccCCCCdddddb bbbbbbbbbbaaaaaaaaaaabbbCCCCCCCCCCCCCb bbbbbbbbbbaaaaaaaaaaabbbCCCCCCCCCCCCCb aaaaaaaaaabbbabaabbbaaaaCCCCCCCCdddddb bbaaaaaaaabbbabbbabbabbbCCCCCCCCCCCCCb bbbbbbbbbbaaababbbbbabbbCCCCCCCCdddddb aaaaaaaaaaababababbbbbbaccCCCCCCdddddb aaaaaaaaaabbbbbbbbbbbbbaCCCCCCCCdddddb 4.5678 01.. 22222 33 32 33 34 35 36 37 38 01234567890123 9 12345678911111111112222 2 39 4o 41 54 contd. Table 3. Cdd Cdd Cdd Cdd 0.0.0 42 43 44 45 46 47 48 49 Cd Cd Cd Cd Cd 50 51 52 Cd Cd Cd Cd Cd 53 54 55 56 *Isolate number corresponds to that on the phenogram in Fig. l 55 om.o m w an wwcoomhom .q 66.0 m m ma momshm co moocwo .q mm.o m m 0H moan: co moocwo .q em.o 0H m mm muocwu .q huwmuo>wo mam Hood mouoHomfl msouonsm oflmxuocmo mo umnaoz mo HonEdz mo nonfioz \mofloomm .mfioumoosoq a“ mmoouonsm ocm mofioomm mom mofiuwmum>flo oflmxuocom mo xumeesm .6 dance 56 Genetic Similarity 0.48 0.64 0.80 0.96 1.1 2 . If I I I 1‘ _. a g to o to ‘ so ‘2 a, N ° E C) a 23_ 0 O 4 o. o. 0) (a 13 ‘2 .3 I. CL 25— :5. C1 0) (I) ‘3 ‘C ‘2 K ‘° ‘= :2. °‘ E 2 43 C) 9 o 32 ‘” ‘3 ~32 8 m 35 g 8 21. xi Fig. 1. Phenogram based on isozyme analysis showing the groupings of isolates of L. cincta and L. persoonii. The phenogram was constructed with the NTSYS program using the unweighted pair-group method with arithmetic averaging (UPGMA) from Simple Matching Coefficient values. The numbers in parentheses correspond to isolate numbers (Table 1). 57 o-—8 '31] -—d3 67 -—5 123 31'9 -—442 4—6 9943 -:—‘-7 _3 _ ’ 428 —' 2 620 -—n07 +—-1 V c.1000 ‘ L—Q . I 1 1 l 4 2 1 Fig. 2 Most parsimonious tree generated using the BOOT program of PHYLIP. The numbers on the branches correspond to the confidence limit on each branch. Those on the end of the tree correspond to the isolate numbers in Table 1. RESTRICTION FRAGMENT LENGTH POLYMORPHISMS IN THE NUCLEAR RIBOSOMAL DNA OF LNGOfi'OMA CI NCTA AND L . PERSOONI I RESTRICTION FRAGMENT LENGTH POLYMOPTHISMS IN THE NUCLEAR RIBOSOMAL DNA OF LEUCOSTOMA CINCTA.AND L. PERSOONII. ABSTRACT Genetic variation within and between groups of two Leucostoma species was examined by analysis of restriction fragment length polymorphisms (RFLPs) of nuclear ribosomal DNA (rDNA). The small nuclear rDNA and the internal transcribed spacers plus a portion of the adjacent large rDNA (ITS-LrDNA, 1600 base pairs) were separately amplified by polymerase chain reaction (PCR). rDNA pattern variation appeared in digests with 10 of 40 restriction enzymes. Phenetic analysis revealed seven groups among various isolates of L. persoonii. Several ITS-LrDNA groups corresponded to those previously detected in isozyme studies and others corresponded to host and/or geographic distributions. Less variation in ITS-LrDNA was evident in L. cincta groups in contrast to isozyme analysis. Most of the RFLP variation in the ITS-LrDNA was within the internal transcribed spacer rather than in the large rDNA. PCR amplification of the nuclear small rDNA revealed an insertion in L. cincta isolates from Prunus spp. that is absent both in L. cincta isolates from Malus and in L. persoonii isolates. 58 59 Leucostoma persoonii Hoehn. and Leucostoma cincta (Fr.: Fr.) Hoehn. are the pathogens that cause Cytospora canker on cultivated peach, nectarine, apricot, plum, prune, sweet and sour cherry, native black cherry, chokecherry, amelanchier and ornamental quince. The most destructive diseases are those on peach, sweet cherry and plum where the canker is perennial. Taxonomy of the two closely related pathogens has been complicated by the lack of availability of species specific characteristics (Kern, 1955), particularly the frequent absence of the sexual state. Kern’s (1955), criteria in literature for identifying the two taxa in the absence of the sexual state have been perceived as inadequate or create confusion judging from frequent current use of incorrect Species epithets (Surve-Iyer, 1992). Misidentification and a lack of understanding of genetic diversity in the taxa present a cogent problem to breeding programs screening for canker resistance in perennial fruit trees (Adams, Hammer, Iezzoni, 1989). Although identification of a fungal taxon is based on observable differences in morphology, biological species and populations may be genetically distinct even when they are not morphologically separable. Recently Surve-Iyer (1992) used isozyme analysis to separate the two Leucostoma species at eight 60 polymorphic loci. In addition to the separation of these two species, isozyme polymorphisms revealed variation within the two species. Three phenetic groups were detected within L. persoonii and three closely related groups were found within L. cincta. Molecular genetic techniques such as restriction fragment length polymorphisms (RFLPs) can identify genetic variability between and within fungal species. RFLP analysis of a region of DNA sequence that evolves at a tempo roughly corresponding to species divergence could reveal whether isozyme phenetic groups correspond to populations or to biological species.’ RFLPs have been useful genetic markers in organisms such as humans (Botstein et a1, 1980 ), crop plants (Helentjaris et a1 ), and animals (Hillis & Davis, 1986). Such polymorphisms can be generated by the loss or gain of restriction endonuclease sites by point mutations or by rearrangement of DNA sequences. In fungi, RFLPs have been used both to resolve taxonomic groups ( Kohn et a1, 1988; Cubeta et a1, 1991) and in population genetic studies (Hulbert et a1, 1985; McDonald and Martinez, 1990; Michelmore & Hulbert,1987). The ribosomal repeat unit (rDNA) has been used extensively for restriction enzyme studies in fungi. 61 Polymorphisms in fungal rDNAs have been used to demonstrate both species and strain specific differences (Kohn et a1, 1988; Specht, Novotony & Ullrich, 1984). In most eukaryotes, the nuclear rDNA exists as a tandemly repeated array of three rRNA genes (183, 5.88 and 28S) separated by internal transcribed (ITS) and non transcribed spacers (IGS). The various rRNAs and their respective DNA coding regions are known for their value as evolutionary markers, since they contain regions of both high and low sequence variability (Apples & Dvorak, 1982). Among fungi, analysis of this region has been applied to numerous genera including Neurospora (Verma & Dutta, 1987), Candida (Magee, D'Souza & Magee, 1987), Schizophyllum (Spect, Novotony & Ullrich, 1984), Coprinus (Wu, Cassidy & Pukkila, 1983), Armillaria (Anderson, Petsche & Smith, 1987) and Lentinus (Hibbet & Vilgalys, 1991). - With the advent of enzymatic amplification, copies of selected regions of the rDNA repeat unit can be selectively amplified as non methylated copies from crude DNA preparations, greatly simplifying RFLP analysis of populations (Bruns, White & Taylor, 1991). This technique has been used by Vilgalys and Hester (1990) for genetic identification and mapping of Cryptococcus species and by Hibbet and Vilgalys (1991) 62 to study evolutionary relationship of Lentinus to the Polyporaceae. In studies presented here we examined variation in portions of the nuclear rDNA repeat of isolates of L. persoonii and L. cincta by analysis of the restriction endonuclease fragment patterns. The small nuclear rDNA (185), and the internal transcribed spacers plus a portion of the adjacent large rDNA (283), were enzymatically amplified using polymerase chain reaction (PCR) and universal primers for fungal rDNA (White, Arnheim & Erlich, 1989; White et a1, 1990). Forty restriction enzymes were used to study the rDNA variation between and within the two species. The data was analyzed by phenetic methods and the results are diagrammed in an unrooted tree. The results were compared to those obtained by isozyme analysis and the various groupings by the two methods are discussed in this paper. MATERIALS AND METHODS DNA preparation The fungal isolates used in this study are listed in Table 6. Formae speciales cultures of L. persoonii obtained from Centraalbureau voor Schimmecultures, 63 Netherlands (CBS) were also used in this study. The fungi were grown for 10-12 days in 100 ml of Leonian's medium (1.29 KH2P04, 0.6g MgSO4, 6.25g maltose, 6.259 malt extract in 1 L of distilled water). _Mycelia were harvested by vacuum filtration through Miracloth (Calbiochem Corp, San Diego, U. S. A), lyophilized and stored at -20°C until the DNA was extracted. DNA was extracted by the method of Lee & Taylor, (1990). Lyophilized mycelium was ground in a mortar and pestle in 750 ul of lysis buffer and incubated at 600 C for 20 minutes. 700 ul of phenol:chloroform (1:1) was added followed by microcentrifugation for 15 minutes. 700 ul of SEVAG was added to the top aqueous phase, the mixture was centrifuged for 10-15 minutes. The aqueous phase was removed and 10ul of 3M NaoAC pH 8 and 0.54 (600 ul) volume of isopropanol was added. DNA ropes were seen precipitating at this stage. The DNA was washed with 1.5 ml of 70% ethanol and the pellet was allowed to dry in a vacuum oven at 50°C for 15 minutes. The extracted DNA was stored in TE buffer at 40C. Enzymatic amplification of DNA with the use of PCR The rRNA coding regions were amplified from genomic DNA by use of the polymerase chain reaction with oligonucleotides complementary to 5’ and 3’ end of the 64 nuclear coding region (White et al., 1990, Fig. 4). The PCR reactions were set up with Amplitaq DNA polymerase (U.S Biochemicals) in either 50- or 25-ul volumes (White et al., 1990) using buffer conditions recommended by the manufacturer. The following primer pairs were used, ITSS/ TW14 (ITSS: ggaagtaaaagtcgtaacaagg, TW14: gctatcctgagggaaacttc) Ctb6/TW13 (Ctb6: gcatatcaataagcggagg, TW13: ggtccgtgtttcaagacg), Ctb6/TW14 and th13/TW14 ( th13: cgtcttgaaacacggacc) (Fig. 4). Restriction patterns of fragments amplified by primers ITSS/TW14 were compared while the remaining primer pairs were used to locate restriction sites or length mutations. For each amplification, one negative control (excluding the DNA template) was used and all the amplification reactions were performed with a positive displacement pipetmen in a PCR-product-free room. Thirty PCR cycles were performed on an automated thermocycler device (Perkin-Elmer-Cetus), with the °c following parameters, 94°C denaturation (1 min), 48 annealing (1 min), 72°C extension (45 sec + 4 sec/cycle) and with a final extension at 72°C for 10 minutes. The PCR products were checked by running 3-4 ul of each reaction mixture on 3% agarose minigels. Previously, a large insert was discovered in the nuclear small rDNA of L. cincta isolate LP59 located 65 between primers NSZl/NSZZ (N521: gaataatagaataggacg, N822: aattaagcagacaaactc) that was absent in L. persoonii isolate LP8 (Mary Berbee, personal communication). A portion of the nuclear small rDNA between the primer pair NSZl/NSZZ was amplified to examine the presence of an insert in the isolates of L. cincta and L. persoonii. Restriction analysis of PCR products After removal of the mineral oil overlay from the PCR reactions, the amplified products were directly used for restriction analysis. 5 ul of the PCR product was digested in a 20 ul volume containing 13 ul of H20, 2 ul of the 10X buffer and 0.1 ul of the restriction enzyme under incubation conditions supplied by the manufacturer. Forty restriction enzymes (Boehringer Mannheim, U.S.A, New England Biochemicals, U. S. A & Strategene, U. S. A) were used in this study (Table 7). The products of the restriction reactions were separated by electrophoresis in 1%+2% agarose gels in TAE, pH 8.1 (1% Nuseive-+2% Seakem, FMC Bioproducts, U.S.A) using le lambda and 123bp ladders as molecular weight standards (Boehringer Mannheim) and non-digested controls. After staining with ethidium bromide, the gels were photographed over an UV transilluminator. 66 Nine enzymes ( AluI, RsaI, MboI, Tan, BSTNI, MseI, BSTUI, HpaII, and ScaI) were further used to study the intraspecific variation within L. persoonii. To identify the areas of variability in the region of ITS- LrDNA, parts of the 28S rDNA were also amplified. Data analysis Digested rDNAs were run side by side on agarose gels and the restriction patterns were compared. Fragments which migrated the same distance during agarose gel electrophoresis as compared to the lambda ladders were considered to be fragments in common. The digested DNA fragments were compared with each other on the gels, their position and molecular weight recorded. To preclude errors in estimates of genetic divergence fragment patterns for each enzyme were coded as different allelic forms of a given locus (Bruns, White and Taylor, 1991). The data marix was entered into a computer file of the Numerical Taxonomy Multivariate System program, version 1.7 (Rohlf, 1987) and the discrete data were converted to Simple Matching coefficient using the SIMQUAL program. The coefficient emphasizes both the positive and negative matches Cluster analysis was performed using the program SAHN by unweighted pair group method using arithmetic averages 67 (UPGMA). For preliminary studies, one isolate from each phenetic group separated by isozyme analysis ( Surve- Iyer, 1992) was used for restriction digestion. Similarities were deduced from the pairwise comparison of isolates by using their ITS-LrDNA profiles. To detect length mutations, the following procedures were performed: 1. For each gel a standard graph was constructed by plotting the molecular weights of the lambda ladders against the distance of migration of the fragments. For every restriction pattern the molecular weight of each fragment was calculated from these standard graphs and were compared with each other. 2. 8% nondenaturing polyacrylamide gels (12 x 16 cm) were run loaded with uncut PCR amplified fragments between primer pairs ITSl/ITS4 & Ctb6/TW14 to obtain relatively precise length comparisons. RESULTS PCR amplification of the nuclear small rDNA between NSZl/NSZZ revealed the preSence of the insert in all of the 16 isolates of L. cincta from Prunus spp. (Fig. 6) (isolate LP66, 65P3, LP49, LP59, LP62, LP47, 2P3, BSS, ATCC72910, Flh, T33.6, 5p4, 4pl, LP40, 351, 1P1). The insert was absent in all of the 13 isolates of L. cincta from Malus (isolate A15, A32, A9, A70, A8, A52, A48, A2, 68 A77, A45, LP39, 383, B82) and did not occur in any isolate of L. persoonii (Fig. 6). The nuclear small rDNA of L. cincta from Malus and L. persoonii were similar in size. Among the 40 restriction enzymes used, 19 recognized sites in the ITS-LrDNA and 10 enzymes showed polymorphisms. The restriction profile of only one enzyme, Hinf I differentiated the isolates of L. cincta and L. persoonii in ITS-LrDNA region. A 268bp band which was detected in all the isolates of L. persoonii was absent in the isolates of L. cincta (Fig. 5). Low variability was seen between the two taxa, but a high level of variability was detected among the isolates of L. persoonii in the region of ITS-LrDNA. Restriction length polymorphism was detected with nine restriction enzymes (AluI, RsaI, MboI, Tan, BSTNI, MseI, BSTUI, HpaII, and ScaI ). For each of these restriction enzymes, two or three patterns of restriction profiles were observed. These patterns were also shared by isolates of L. cincta from both Malus and Prunus and the formae speciales cultures of L. persoonii from CBS. The phenogram generated by the SIMQUAL program using Simple Matching coefficient (Fig. 3) summarizes the relationship between the isolates of L. persoonii. L. 69 persoonii was separated into seven clusters. The first cluster consisted of two isolates (LP6 and 11.3) from peach. Isolates on cherry formed a second cluster, three of these (LP8, LP9, and LP10) were on black cherry, one (LP13) was on sour cherry (all the four isolates were from Michigan) and one (CHR) from Oregon near the Hood river. Five isolates on peach (T18.1, T26.1, T4.7, T16.1, T7.4) collected from a single orchard in Michigan and one isolate (LCN) on nectarine from California clustered together. Two isolates, one from Michigan (LP21) and one from North Carolina (NC9.7) were clustered together. The fifth cluster consisted of two isolates, one on prune (LP2) and the other on peach (NC9.7). Two North Carolina isolates on peach (NC14.1 and NC8.2) formed a sixth cluster. Two California isolates (R3T16 and R5T10) from plum were grouped together to form the seventh cluster. Length mutations appeared to be evident when the restriction patterns were compared by standard graphs. However, comparison of the restriction profiles of ITS- LrDNA and portions of the 288rDNA did not reveal difference in size prior to digestion. Comparison of restriction profiles between IT84/IT85 and Ctb6/Tw14 showed maximum variability in the region between primers ITSS and ITS4 (Fig. 4) 70 DISCUSSION Analysis of the restriction patterns of two separate regions of the rDNA revealed information on the relationship between the isolates of Leucostoma. We were able to detect enzymes that showed restriction length polymorphisms by using 40 restriction enzymes with four to eight base recognition sequences. Based on RFLPs, the isolates of L. persoonii appear to be composed of several genetically heterogeneous groups. Seven clusters were observed, four clusters were separated on the basis of host adaptation, one cluster consisted of isolates from a single orchard in Michigan and isolates from cherry and nectarine. The remaining two clusters were separated by geographical location. Previous studies (Surve-Iyer, 1992) using isozyme analysis have shown the separation of L. persoonii into three groups. These three groups did not reveal significant variation within themselves. One of these groups was widespread, a second group consisted of specimens isolated from one peach orchard in Michigan and one canker isolation on black cherry, and a third group consisted of two specimens on nectarine from California. 71 RFLPs have revealed greater intraspecific genetic variation than isozyme analysis, perhaps this may be due to due to genetic divergence among L. persoonii caused by geographhical isolation. Intraspecific variation as seen here in the groups of L. persoonii has also been observed using Drosophi1a (Williams et a1, 1987). However in similar studies of rDNA variation in fungi less intraspecific variability was detected (Anderson, Petsche & Smith, 1987; Kohn et al., 1988; Braithwaite, Iwrin & Manners, 1990; Egger, Danielson & Fortin, 1991). Sequence analyis of the nuclear encoded rDNA has also shown higher interspecific than intraspecific variability in Laccaria (Gardes et al., 1990) and Gibberella spp. (Peterson and Logrieco, 1991). Recently, there has been one report by Vilgalys & Gonzalez (1991) in which high variability was detected in the nuclear encoded rDNA repeat unit of Rhizoctonia solani both among and within the different anastomosis groups. McDonald and Martinez (1990) have reported a high level of genetic variation in the local population of Septoria tritici using restriction analysis of the nuclear DNA. This form of variation within the species may provide an important method of investigating the identification, origin and spread of certain isolates. 72 Isozyme analysis (Surve-Iyer, 1992) has proven to be more informative with respect to the separation of the two taxa of Leucostoma than RFLP analysis. Isozyme analysis separated the two species L. cincta and L. persoonii at each and every loci tested. RFLPs of the rDNA did not show distinct separation of L. cincta on Malus (PG6) from the isolates of L. persoonii. L. cincta on Malus is likely to represent a genetically isolated biological species in the L. cincta species complex. It differs in isozyme loci and morphological markers and the lack the insertion in the nuclear small rDNA (Surve-Iyer, 1992; Proffer & Jones, 1989). P66 was virulent on 3-yr-old inoculated peach seedlings (Surve-Iyer, 1992), however, in nature PG6 is absent on Prunus spp. This ecological host specialization of P66 may have led to the genetic isolation of L. cincta on Malus and L. cincta on Prunus spp. This study revealed that an insert in the 18S region of the rDNA was present in all specimens of L. cincta from Prunus but absent in L. cincta on Malus and L. persoonii. Inserts in the nuclear small rDNA have not been detected in plants or animals, however, such inserts have been noted in lichens (De Priest and A. Gargas, personal communications) and Chytrids (B. Bowman, personal communication). Furthermore, the PCR 73 amplification of isolates of L. cincta containing this insert often showed two amplified products rather than one. This phenomenon apparently occurs commonly during amplification of DNA containing large inserts (Bruns, personal communication). The collections of L. cincta are limited (inserts have been detected in 16 isolates and absent in 13 isolates) and examination of more widespread collections are needed to confirm that the insert is unique and indicative of L. cincta on Prunus. Calculations of the molecular weights of digest fragments indicated that length mutations were present in the ITS-LrDNA regions. However comparing smaller uncut PCR amplified products separately for the ITS (ITSS/ITS4) and the LrDNA (Ctb6/Tw14) did not reveal length mutations. Quantitative analysis of RFLP pattern comparison can be prone to error if length mutations are present. This can cause overestimations of divergence when multiple enzymes are used. We believe that the variation we have detected in the ITS-LrDNA regions is due to site changes rather than length mutations. Further support for our argument is that of the forty restriction enzymes used, we detected differences with ten enzymes, but nine other enzymes that cut the ITS- LrDNA ( ClaI, AvaI, DdeI, HaeI, NciI, SstI, MnI, EcoRI 74 and Fnu4H) did not reveal variation among diverse isolates. However there is still a possibility that we may have not detected minor length mutations due to comigration of nonhomologous fragments of identical molecular weights. This study has been useful in identifying variation within L. persoonii. In a study on Rana (Hillis and Davis, 1986), allozyme data provided extensive information on the relationship between closely related groups in a species complex. However their RFLP analysis identified several unresolved or poorly resolved portions of the phylogeny at the intergroup level much like our studies of Leucostoma. It may be early to assign evolutionary relationship in Leucostoma ’ based solely on RFLP banding patterns, however this study might be phylogenetically informative when the molecular basis of the variation is known. Detailed sequence data would be useful in understanding the variation in this region. If the variability detected in the groups of L. persoonii precisely reflects the genetic variability present in this species, than this variation should be taken into consideration when screening for resistant peach cultivars. Comparison of levels of variation from isozyme marker and RFLP markers to levels of variation in the mitochondrial DNA 75 and the rDNA intergenic spacer region combined with anastomosis grouping might provide a basis for detailed population genetic and epidemiological studies of the two taxa that would benefit in selecting resistance to perennial canker in peach varieties. 76 LITERATURE CITED Adams, G. C., Hammer, 8. A. & Iezzoni, A. (1989). 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Strain and species identification by restriction fragment length polymorphisms in ribosomal DNA repeat of Candida species. Journal of Bacteriol 169: 1639- 1643. McDonald, B. A., 6 Martinez, J. P. (1990). Restriction fragment length polymorphisms in Septoria tritici occur at a high frequency. Current Genetics 17: 133-138. McDonald, B. A. & Martinez, J. P. (1990). DNA restriction fragment length polymorphisms among Mycosphaerella graminicola (anamorph: Septoria tritici) isolates collected from a single wheat field. Phytopathology 80:1368-1373. Michelmore, R. W. & Hulbert, S. H. (1987). Molecular markers for genetic analysis of phytopathological fungi. Annual Review of Phytopathology 25:383-404. Peterson, 8. W. & Logrieco. A. (1991). Ribosomal RNA sequence variation among infertile strains of some Gibberella species. Mycologia 83:397-402. Proffer, T. J. and Jone, A. L. 1989. 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A. & Silver, J. C. (1990). DNA restriction fragment length polymorphism in the rDNA repeat unit of Entomophaga. Experimental Mycology 14:381-392. White, T. J., Arnheim, N. & Erlich, H. A. (1989). The polymerase chain reaction. Trends In Genetics 5: 185- 189. White, T. J., Bruns, T. D., Lee, S. B. & Taylor, J. W. (1990). Amplification and direct sequencing of fungal ribosomal genes for phylogenetics, p 315-322. In N. Innis, D. Gelfand, J. Sninsky, and T. White (ed.), PCR protocols: a guide to methods and applications. Academic press, Inc., New York. Williams, S. M., Furnier, G., Fuog, E. & Strobeck, C. (1987). Evolution of ribosomal DNA spacers of Drosophila melanogaster: Different patterns of variation on X and Y chromosomes. Genetics 116: 225- 232. Willison,-R. S. (1938). Peach canker investigations II. Infection studies. Canadian Journal of Research 14: 27-44. Wu, M.M., Cassidy, J.R. & Pukkila, P. J. (1983). Polymorphisms in DNA of Coprinus cinerus. Current Genetics 7:385-392. 80 TableES. Leucostoma isolates used in the RFLP studies. Species & Isolate Origin Host L. persoonii LP6 Michigan Prunus persica 11.3 Michigan Prunus persica LP10 Michigan Prunus serotina LP9 Micigan Prunus serotina LP13 Michigan Prunus serotina LP8 Michigan Prunus serotina CHR Oregon Prunus avium Cy5 Oregon Prunus avium T18.1 Michigan Prunus persica T26.1 Michigan Prunus persica T4.7 Michigan Prunus persica LCN California Prunus persica var. nucercisa T7.4 Michigan Prunus persica T16.1 Michigan Prunus persica LP21 Michigan Prunus domestica NC9.7 North Carolina Prunus persica LP2 Michigan Prunus domestica NC17 North Carolina Prunus persica NC14.1 North Carolina Prunus persica NC8.2 North Carolina Prunus persica R3T16 California Prunus domestica R3T10 California Prunus domestica f.sp mahaleb Switzerland Prunus persica L. cincta LP66 Michigan Prunus domestica ATCC 62910 Ontario Prunus persica 5P4 Michigan Prunus domestica A48 Michigan Malus domestica A45 Michigan Malus domestica A9 Michigan Malus domestica 81 Table 6. Restriction enzymes used in the digestion of PCR products of the ITS-LrDNA* fragment in L. cincta and L. persoonii AluI ClaI Hian PvuII AvaI EaeI HpaII RmaI AvaII DdeI HpaII RsaI BamHI EcoNI KpnI SalI BclI EcoRI MboI ScaI BglII EcoRV MnI SstI BglIII Fnu4H MluI StuI BstEII HaeIII MseI Tan BstNI HhaI NruI XbaI BstUI HindIII PstI XhoI *ITS-LrDNA= ITSl, IT82, 5.8SrDNA and portion of the 288rDNA .0.560 0.640 0.720 82 0-800 0.880 0.960 1.040 I__ L98 Fig. 3 Phenogram of Leucostoma persoonii isolates based on the UPGMA cluster analysis of simple matching coefficient. Similarity generated from restriction fragment length polymorphisms in the portion of the nuclear rDNA containing ITS], I'I‘SZ, 5.88 rDNA and part of the 288 rDNA. l7 iiiii 11.3 ' LP10 LP13 ,LPB .qs T18.1 T26.1 T4.7 T7.4 ’T16.1 L921 'NC9.7 NC17 NC14.1 NC8.2 RSTIO R3T16 83 6% 653.8633 Bob <75.— QEEw 3 new: 686 SEE «once—escewze me .8583 65 manta—m anode.— 8 Leeemwuwe “Hueeewwfieowm use was 2.2222 mama men In eesoLLoL mesa: .uefioa eofiuaaseoew o» Leeann Lessee we“ so eseees E 31.3 as". s. as .3 .83 . as: a... 9. W2 AANVLLeeo Leofioo m.emc>em “new: sex 20100 coma so eoLLeesecfi so meme .2 Cause spaces. _CJ a 2 as s . sea ea 3...: .2 as. r... e a.m meson g . «amuse woe eneewe .S aims. was s o.m Assam s . «amuse sue unease .8 miss” she e m.. 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Lodge mafia u an aeeeuefiou eweee: macho mmwoeam eflmflao mumLomH a o as as? 58.88 ea :28 sees e as easmoum- mo mmum “WM 105 88888 88.8 ... 8 88 8.888 888.8 888.0 088.88 088.88 800.8 880.8 880.08 880.8 880.08 880.8 888.08 880.8 808.80 888.8 880.88 88.8 088.0 08.0 080.8 88.0 808.8 888.8 .080.88 80.8 080.88 ( )\ \L‘) (L) 0’4 f\ 88888 888888 888888 888888 888888 8 8888 8 8888 8 8888 8 8888 8 V 8888 88 880 888 800 888 880 . 3.: n 88 808880 88888880 8880 888 88 88888 88 888888888 88.88888888 88 88888888 88 8888388 u 106 TABLE 5?. Virulence of standard isolates of the five phenetic groups of Leucostoma on 3-yr-old peach multistemmed seedlings ranked in order of decreasing canker length. Standard Leucostoma Phenetic Mean cagker isolates species group length (cm) 11.3 L. persoonii PG1 8.9 a LP59 L. cincta PG5 8.2 ab F1h L. cincta PG4 7.2 DC T28.1 L. persoonii PGZ 6.5 cd A3 L. cincta P66 5.9 d a Length of the canker distal to inoculation point. Means of forty replicates followed by the same letter are not significantly different by the least significant difference test, LSD, P = 0.05. CONCLUSIONS AND PROSPECTIVES FOR FUTURE STUDY CONCLUSIONS AND PROSPECTIVES FOR FUTURE STUDY This research has provided the foundation of an understanding of the variation in two closely related taxa of Leucostoma, L. cincta and L. persoonii. Isozyme analysis has been very useful in separating the two species. Isozymes can be used as a diagnostic tool to identify the two taxonomic species. In addition, isozyme analysis has identified six distinct phenetic groups in Leucostoma, three of which were found within L. persoonii, PGI, PGZ and P63, and three within L.cincta, PG4, PG5 and PG6. A higher level of genetic diversity was detected in the populations of L. cincta than L. persoonii, which may indicate that the primary mode of reproduction in L. cincta is sexual outcrossing. In contrast the low level of diversity seen in the populations of L. persoonii may indicate that the primary mode of it’s reproduction is asexual or that this population may have arrived in North America as a founder population. Virulence ,experiments on three year old» peach seedlings were also indicative of the diversity present in Leucostoma. The virulence experiments showed that L. cincta isolates from Malus could also be virulent on peach. The two species, L. cincta and L. persoonii 107 108 could not be distinguished on the basis of their virulence on peach. Cultural morphology also differentiated the phenetic groups of the two species. In culture L. cincta and L. persoonii could be differentiated by their color and pycnidial size. In addition, phenetic group 2 of L. persoonii was differentiated from phenetic group 1 on the basis of it’s color in culture and the absence of the distinct lobate margin typical of P61 cultures. Phenetic group 6 of L. cincta was readily differentiated from PG4 and PGS by formation of a reddish pigmentation in culture. Studies on restriction fragment length polymorphisms revealed populations within isolates of L. persoonii. Seven populations were revealed, several corresponded to those detected in isozyme studies and others corresponded to hosts and or geographic distributions. RFLPs also revealed two populations within L. cincta. The L. cincta isolates on Prunus spp. were differentiated from L. cincta isolates on Malus and L. persoonii by the presence of an insert in the 188 region of the nuclear rDNA. The new information from this research has opened the way to future approaches to the study of speciation, population biology and molecular evolution in 109 Leucostoma. Several directions in research, rich in prospectives are listed and described here. 1. Anastomosis studies can be used to separate the two taxa in terms of their anastomosis groups and correlate these groupings with isozyme analysis. 2. Sequencing of the ITS region will provide information on the type of variation seen in the rDNA, whether it is due to site changes or length mutation. 3. Additional isolates of L. cincta on Prunus should be screened to verify that the insert in the 18S rDNA region is unique to this group. Sequencing will then aid in the identification of the insert and perhaps reveal information on it’s role in evolution of Leucostoma. 4. Fungal ‘mtDNA has been estimated ‘ to evolve approximately 5-8x faster than nuclear DNA. Variation in the mtDNA could be used to study maternal inheritance among individuals in local pOpulations or geographically distinct groups within the two species. 5. Collection of single ascospores from perithecia of L. cincta isolates from various orchards and studying their isozyme patterns will reveal information on the genetic diversity in the population of L. cincta. Such future experimental possibilities will provide 110 valuable information on the population genetics and phylogenetic studies in Leucostoma. APPENDIX Table 11. Alleles scored in Leucostoma study. Locus ME PGI PGM IDH G6PD MPI EST DIA MDH EST Isolate Alleles NC 9.2 c c c c c - a - ab - NC10.2 c c c c c - a - ab - NC22.2 c c c c c - a - ab - NC4A c c c c c - a - ab - 11.1 c c c c c - a - ab - NC23 c c c c c - a - ab - NC8.2 c c c c c - a - ab - NC17 c c c c c - a - ab - NC49 c c c c c - a - ab - T36.1 d d d d - c - - - - T9.3 d d d d - c - - - - T4.1 d d d d - c - - - - T32.2 d d d d — c - - - — T28.1 d d d d - c - - - - T10.6 d d d d - c- - - - - T26.6 d d d d - c - - - - T18.1 d d d d - c - — - - T16.7 d d d d - c - - - - T3.6 d d d d - c - - - - R5T15 c c c c c - ac 0 ac - 10.14 c c c c c - ac 0 ac c 11.2 c c c c c - ac c ac c 58 c c c c c - ac c ac c R1T12 c c c c c - ac c ac c 10.1 c c c c c - ac 0 ac c 11.12 c c c c c - ac c ac c R4Tl4 C c c c c - ac 0 ac c RIT4 c c c c c - ac 0 ac c RT16 c c c c c - ac c ac c 105 c c c c c - ac 0 ac c R6T14 c c c c c - ac c ac c R4T11 c c c c c - ac 0 ac c 111 311.2 Table 12. Restriction frequent patterns of Leucostola persoonii Isolate Restriction enzyle AluI HboI HseI BstUI $an BstEII HpaII E 11.3 1 0 0 0 1 0 0 1 0 0 0 0 l 1 0 1 0 1 0 0 LP8 0 1 0 0 1 0 0 1 O 0 0 0 1 1 0 1 0 0 0 1 LP10 0 1 0 0 1 O O 1 0 0 0 0 1 1 0 1 0 0 0 1 LP9 0 1 0 0 1 0 0 0 0 1 0 0 1 0 1 1 0 1 0 0 LP13 0 1 0 0 1 0 0 1 0 0 0 0 1 1 0 1 0 0 0 1 T4.7 0 1 0 1 0 0 0 l 0 0 0 0 1 l 0 0 1 0 0 1 T7.4 0 1 0 1 0 0 0 1 0 0 0 0 1 1 0 0 1 0 0 1 CHR 0 1 0 0 1 0 0 l 0 0 0 0 1 1 0 1 0 0 O 1 Cy5 0 1 0 0 1 0 O 1 0 0 0 0 1 1 0 1 0 0 0 1 118.1 0 1 O 1 0 0 0 1 0 0 0 0 1 1 0 0 1 0 0 1 LCN 0 1 0 1 0 0 0 1 0 0 0 0 1 1 0 0 1 0 0 1 LP21 0 1 0 1 0 0 0 1 0 0 0 0 1 1 0 0 1 1 0 0 T26.1 0 1 0 1 0 0 0 1 0 0 0 0 1 1 0 0 1 0 0 l NC9.7 0 1 0 1 0 0 0 1 O 0 0 0 1 0 1 0 1 1 0 0 LP2 1 0 0 1 0 0 0 0 1 0 0 0 1 1 0 1 0 1 0 0 NC17 1 0 0 1 0 0 0 1 0 0 1 0 0 1 0 1 0 1 0 O NC14.1 1 0 0 O 0 1 1 0 0 0 0 l 0 0 l 1 0 1 0 0 NC8.2 1 0 0 0 0 1 1 0 0 0 0 1 O 0 1 0 1 1 0 0 R5T10 1 0 0 1 0 0 1 0 0 0 1 0 0 1 O 1 O l 0 0 T16.1 0 1 0 1 0 0 0 1 0 0 0 0 l 1 0 0 l 0 0 1 LP8 0 1 0 0 1 0 0 1 0 O 0 0 l 1 0 1 0 0 0 1 R3T16 1 0 0 1 0 O 1 0 0 0 1 O 0 1 0 1 O l 0 0 HOOOOOOO OHHHHHHH OOOOHOHOOOHOOHHOOHHOOO HHHHOHOHHHOHI—DOOHHOOHHH "111111011111111111111“