III! II IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII 0441917 L.!IBHARY Michigan State University This is to certify that the thesis entitled LEUCOSTOMA SPECIES IN MICHIGAN PEACH ORCHARDS presented by Dechun Wang has been accepted towards fulfillment of the requirements for Master of Science degree in Horticulture Plant Breeding & Genetics; v/v/Vl’ly :7 N?” e: (”c “’ Major professor '0 ’09“ r / 1 Date fl” “.1 \ 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution _ _- PLACE ll RETURN BOXtonmavothb Whom minced. TOAVOID FlNESntunonorbdonddodm. DATE DUE DATE DUE DATE DUE MSUIOMWMQHOWIW LEUCOSTOMA SPECIES IN MICHIGAN PEACH ORCHARDS By Dechun Wang A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Horticulture Plant Breeding and Genetics 1994 ABSTRACT LEUCOSTOMA SPECIES IN MICHIGAN PEACH ORCHARDS By Dechun Wang Perennial canker is the most important disease limiting peach production in the northern U.S. and Canada. The pathogens involved, Leucostoma spp., were characterized in Michigan peach orchards to: 1) identify which Leucostoma subgroups are most prevalent on the peach cultivars Loring and Redhaven; 2) determine if the canker infections on trunks and twigs are caused by similar Leucostoma profiles; and, 3) examine the number of maternal lineages present in one orchard. 403 isolates of Leucostoma were isolated from cankers on Redhaven and Loring cultivars in 3 southwest Michigan orchards. Based on colony margins and small nuclear rDNA size polymorphisms, 89% of the isolates were identified as L. persoom'i group 1, 10% were L. cincta, and 1% were L. persoom'i group 2. Significant differences in pathogen profiles of branch cankers were found between Redhaven and Loring. Significant differences were also found in the pathogen profiles of cankers on trunks and twigs. 95% of 1,020 pairs tested among isolates of L. persoom'i group 1 were vegetatively incompatible. A minimum of 11 and a maximum of 17 maternal lines were identified among 69 isolates of L. persoonii group 1 from one orchard based upon mtDNA RFLPs. The frequency of incompatibility and the number of maternal lines indicate that wide genetic variations exist within the Leucosroma populations in Michigan peach orchards. ACKNOWLEDGEMENTS I wish to thank my major professor, Dr. Amy Iezzoni, for her financial and educational support, guidance, and editorial assistance. I am grateful to my advisor and committee member, Dr. Gerry Adams, for his guidance, technical advice, and editorial assistance. I appreciated his permission to use his lab to conduct the whole project. I would like to express my appreciation to my committee member, Dr. Jim Hancock, for his guidance and patience. I would like to thank Dr. John W. Taylor and Dr. David Jacobson for providing mitochondrial DNA clones and for the use of Dr. Jacobson’s lab facilities. Special thanks to my wife Cuihua for her constant support, patience, and love. iii TABLE OF CONTENTS LIST OF TABLES .................................... LIST OF FIGURES .................................... INTRODUCTION AND LITERATURE REVIEW ................. List of references .................................. MATERIALS AND METHODS ............................ Canker sampling .................................. Leucostoma isolation ................................ Leucostoma identification ............................. Examination of vegetative compatibility .................... Subgrouping of the most prevalent group of isolates ............ Prasmid preparation of mitochondrial DNA clones ............. DNA isolation ................................... Amplification of DNA fragments using PCR ................. Southern Blotting analysis using Genius nonradioactive system ...... Separation of mitochondrial DNA from chromosomal DNA ........ Data analysis .................................... RESULTS The most common pathogens of Leucostoma canker in Michigan . . . . Pathogen profiles of cankers on Redhaven and Loring cultivars ..... Pathogen profiles of cankers on tree trunks and branches ......... Vegetative compatibility among isolates of L. persoonii group 1 ..... Target DNA hybridized by the two mitochondrial DNA probes ..... Subgrouping of L. persoonii group 1 ...................... DISCUSSION ........................................ LIST OF REFERENCES ................................. iv 9 9 11 11 12 12 14 14 15 17 18 19 19 23 23 27 27 35 39 APPENDICES A. Table 7. Percentage of cankers caused by Leucostoma persoonii group 1, group 2, and L. cincta isolates from trunks and branches of Redhaven and Loring from 3 Michigan orchards ....... B. Output of ANOVA analysis using MSTAT-C software ........... Bl. B2. B3. B4. B5. Output of ANOVA analysis using MSTAT-C software. Cultivars, canker sites, and pathogens were considered as experimental factors and analyzed together as described below ............................ Output of ANOVA analysis using MSTAT-C software. Arcsine transformed percentage data of L. persoonii group 1 on branches of Redhaven and Loring were analyzed as described below .................... Output of ANOVA analysis using MSTAT-C software. Arcsine transformed percentage data of L. cincta on branches of Redhaven and Loring were analyzed as described below .......................... Output of ANOVA analysis using MSTAT-C software. Arcsine transformed percentage data of L. persoonii group 1 on cultivars and canker sites were analyzed as described below .......................... Output of ANOVA analysis using MSTAT-C software. Arcsine transformed percentage data of L. cincta on cultivars and canker sites were analyzed as described below ............................ Page 42 43 43 44 45 46 47 LIST OF TABLES Table Page 1. Number and percentage of cankers caused by each of the pathogens isolated from 3 Michigan orchards ................. 22 2. Percentage of cankers on branches of Redhaven and Loring caused pathogens. The data is summed over 3 orchards ...................................... 22 3. Percentage of cankers on branches and trunks caused by differen pathogens. The data is summed over 3 orchards and two cultivars .................................. 24 4. Percentage of cankers on tree trunks of Redhaven and Loring caused by different pathogens. The data is summed over 3 orchards ............................. 24 5. Percentage of vegetatively incompatible pairs among 182 isolates of L. persoonii group 1 ...................... 25 6. Number and percentage of isolates belonging to each subgroup of L. persoonii group 1 based upon mitochondrial DNA polymorphism ....................... 31 7. Percentage of cankers caused by Leucosmma persoom'i group 1, group 2, and L. cincm isolates from trunks and branches of Redhaven and Loring from 3 Michigan orchards ....... 42 vi LIST OF FIGURES Figure 1. Flow chart of Leucosroma investigation .................... 2. Colony characteristics representative of the three Leucostoma subgroups; a) L. persoonii group 1 with a lobate colony margin, b) L. persoonii group 2, c) L. cincta, with uniform radial colony margins .............. 3. Electrophoretic separation of PCR products of nuclear small rDNA (primers NS21/NS22) on 1% agarose gel stained with ethidium bromide and viewed under ultraviolet light. An insertion of about 450bp is present in the isolates of L. cincta (lanes 2, 3, 5, 13, 14) as compared to L. persoom'i (lanes 4, 6-12, 15, 16). Lane 1 is a lkb lambda DNA molecular weight marker ......................................... 4. Pairing of isolates of L. persoonii group 1 on clarified oatmeal agar medium. Barrage zones formed between vegetatively incompatible isolates ................... 5. L. persoom'i DNA hybridized to Neurospora crassa mitochondrial DNA probe B3. A. Ethidium bromide stained gel viewed under ultraviolet light; B. Autoradiograph after hybridization of probe B3. 1 and 2, L. persoonii; 3, yeast chromosome DNA size standard, Saccharomyces cerevisiae, strain YNN295 ............ 6. L. persoom’i DNA hybridized to Fusarium oxysporum mitochondrial DNA probe A13. A. Ethidium bromide stained gel viewed under ultraviolet light; B. Autoradiograph after hybridization of probe A13. 1 and 3, L. persoonii; 2, yeast chromosome DNA size standard, Saccharomyces cerevisiae, strain YNN295 ............ vii Figure 7. Autoradiographs of Southern blotting using mitochondrial DNA as probes. A. HpaII digested genomic DNA hybridized with labeled B3, lanes 1-8, L. persoonii group 1; B. HaelII digested genomic DNA hybridized with labeled B3, lanes 1-6, L. persoonii group 1; lane 7, DNA size standard; C. HaeIII digested genomic DNA hybridized with labeled A13, lanes 1-8, L. persoonii group 1 ................................ 32 Banding patterns of subgroups of L. persoonii group 1 ........... 33 Unique banding patterns of subgroups of L. persoom'i group 1 ............................... 34 viii INTRODUCTION AND LITERATURE REVIEW Leucosroma canker is one of the most important diseases limiting peach [Prunus persica (L.) Batsch] production in the northern United States and southern Canada (Biggs, 1986; Dhanvantari, 1978; Hildebrand, 1947; Kern, 1955; Layne, 1984). The disease is also called perennial canker, Cytospora canker, and Valsa canker. It limits peach production in Michigan, West Virginia, New Jersey, Pennsylvania, New York, Illinois, Colorado, Idaho, Ontario, and British Columbia (Biggs, 1989). A recent survey of the disease in 93 orchards in Ontario revealed that 98% of the 2,000 trees examined were infected and 90% of the trees had cankers on scaffold limbs (James and Davidson, 1971). Based on the survey, it was estimated that a loss of $68,000 per hectare could result from the disease alone (Biggs, 1989). Leucosmma canker is characterized by extensive perennial cankers on limbs and branches (Biggs, 1986). The pathogens invade peach trees through wounds or dead tissue. Winter injury, a recurring problem of peach in Michigan, is one of the most common infection sites (Dhanvantari, 1978; Tekauz and Patrick, 1974; Willison, 1937). Cankers are first apparent in the spring at nodes, fruit pedicels, and oriental fruit moth injury sites at twig terminals (Dhanvantari, 1978). If left untreated, the cankers from the infected twigs rapidly invade scaffold limbs and large branches (Biggs, 1989). Extensive cankers result in dying of branches, girdling of trunks and scaffold limbs, and ultimately 2 a short orchard life for the infected trees (Biggs, 1986; Dhanvantari, 1978). Upon close examination, infections of small twigs appear as sunken, discolored areas near winter- killed buds or leaf scars. Cankers on trunks, branch crotches, scaffold limbs and older branches begin with excessive exudation of amber-colored gum. As cankers age, the gum becomes dark brown to black, the infected bark dries out, and cracks open, exposing blackened tissue beneath elliptical cankers along the length of the stem (Biggs, 1989). Histopathological investigations reveal that the disease causes xylem dysfunction which result in deficiencies of Ca, Al, B, Mg, Mn, P, and Zn in foliage distal to even nongirdling cankers (Hampson and Sinclair, 1973) and in noticeable reduction in water transport (Chang et (11., 1991). The causal organisms of this disease are two Closely related species of fungi, Leucostoma cincra (Pers. & Fr.) Hohn and Leucostoma persoonii (Nits.) Hohn. Either of the two species could be the prevalent pathogen in a particular growing region. L. cincta is the more common canker pathogen in some regions such as Ontario (Dhanvantari, 1982), while in some other regions such as Colorado (Luepschen, 1981), Utah (T reshow and Scholes, 1958; Treshow, Richards, and Scholes, 1959), New York (Hildebrand, 1947), and Michigan (Kern, 1955), L. persoom'i is the prevalent pathogen. Variation between isolates of the same species was reported (Adams et al., 1989; Konicek and Helton, 1962; Surve-Iyer, 1992; Wysong and Dickens, 1962). Based on isozyme polymorphisms, Surve—Iyer (1992) found that there were two distinct groups of L. persoonii (group 1 and group 2) and two distinct groups of L. cincta among the isolates from Prunus spp. hosts in Michigan, and another group of L. Cincra among the 3 isolates from Malus hosts which also caused peach canker. A L. persoonii isolate from California was found to differ from both of the two L. persoonii groups found in Michigan. Virulence of the pathogens was reported to vary among species and isolates of the same species. L. persoonii was reported by some investigators to be of low virulence on peach as compared with L. cincta (Helton and Konicek, 1961; Willison, 1937). Some other investigators found L. persoom'i was more virulent in warm weather and L. when: more virulent in cool weather (Hildebrand, 1947; Wensley, 1964). In Michigan, a wide range in virulence was found among isolates of L. persoom'i and L. cincta (Adams et al., 1989), and Group 1 of L. persoom'i was found to be the most virulent and Group 6 of L. cincta the least virulent on peach among the 6 groups defined by Surve-Iyer (1992). Leucostoma canker is difficult to control. Once a tree is infected, the disease can not be controlled with fungicides (Helton and Rohrbach, 1967). Current strategies for control of the disease are largely based on avoiding the factors that predispose the trees to this disease. A combination of cultural practices, including removal of infected branches and prevention of insect, rodent, and winter injuries, may reduce the incidence of the disease (Biggs, 1989) but may not always be practical. The best long-term strategy is to develop cultivars with disease resistance (Biggs 1989). Resistance to the disease, although not high or immune, has been found in the peach germplasm (Chang and Iezzoni, 1989; Chang et al. 1989; Luepschen, 1981; Scorza and Pusey, 1984). Cultivars Yennoh and Reliance were reported to be among the least susceptible cultivars, Redhaven among the intermediate, while Loring and Elberta 4 were among the most susceptible cultivars (Chang and Iezzoni, 1989; Chang et al. 1989; Luepschen, 1981; Scorza and Pusey, 1984). Heritability of the disease resistance was reported to be as high as 0.72 (Chang and Iezzoni, 1991) suggesting that it should be possible to develop resistant cultivars. One of the constraints to the development of resistant cultivars is the lack of knowledge regarding pathogen variation (Biggs, 1989). Prior to extensive breeding efforts for host resistance to Leucostoma canker, the Leucostoma species present in orchards must be characterized. Differences in pathogen variation based upon infection site and host cultivar should also be considered. Genetic variation in the pathogen can be estimated by determining the frequency of inherited markers in Leucostoma. Vegetative compatibility groupings can serve as markers (Adams et al., 1990), as can isozyme and DNA polymorphisms (Surve-lyer, 1992). However, isozyme polymorphisms are not commonly encountered in L. persoonii in a local region (Surve- Iyer, 1992). Polymorphisms in mitochondrial DNA (mtDNA) are more common (Taylor et al., 1986; Brooks er al.,1990). Mitochondrial DNA in fungi evolves approximately 5-8 times faster than nuclear DNA (Bruns and Szaro, 1992). Length mutations are the most prevalent source of variability in mtDNA of Ascomycetes like Leucostoma (Taylor et al., 1986; Sanders er al., 1977). Within isolates of Neurospora crassa the total size of mtDNA varies by 25% due to length mutations. Additionally, biases appear in the location of mtDNA mutations in fungi and specific regions are more susceptible to mutation, particularly the 8kb region encompassing the COXB-COXl and a 10kb region of the Oli2-Olil-COX2 genes (Taylor at al., 1986). When an identifiable region of 5 mtDNA consistently shows a high level of mutation, specific probes can be chosen to hybridize to mtDNA fragments that encompass conserved regions with highly variable flanking regions. Length mutations in mtDNA has been used successfully to study variability among isolates within biological species (Taylor er al. , 1986; Brooks et al., 1990) and permit determination of maternal inheritance among individuals in a local population. The objectives of this study are to: 1) identify which Leucostoma species and subgroups are most prevalent on the peach cultivars Loring and Redhaven in Michigan orchards; 2) determine if the canker infections on trunks and twigs of Redhaven and Loring are caused by similar Leucosroma profiles; and, 3) examine the genetic variability and the number of maternal lineages present in one orchard. LIST OF REFERENCES Adams, G.C., S.A. Hammar, and A. Iezzoni. 1989. Optimum sample size for detecting virulence differences in Leucosmma isolates from peach. Plant Dis. 73:754-759. Biggs, A.R. 1986. Comparative anatomy and host response of two peach cultivars inoculated with Leucostoma cincra and Leucostoma persoonii. Phytopathology 76:905-912. Biggs, A.R. 1989. Integrated approach to controlling Leucostoma canker of peach in Ontario. Plant Dis. 73:869-874. Brooks, S., T. D’Souza, and GO Adams. 1990. Relationship of three distinct populations of Fusarium graminearum examined by allozyme and ribosomal DNA polymorphisms. Phytopathology 80:963. Bruns, TD, and T.M. Szaro. 1992. Rate and mode differences between nuclear and mitochondrial small-subunit rRNA genes in mushrooms. Mol. Biol. Evol. 9:836- 855. Chang, L.S. and A.F. Iezzoni. 1989. Excised-shoot assay for tolerance of peach to Leucosroma persoom'i. HortScience 24: 101 1-1012. Chang, L.S., A.F. Iezzoni, G.C. Adams, and GS. Howell. 1989. Leucostoma persoonii tolerance and cold hardiness among diverse peach genotypes. J. Amer. Soc. Hort. Sci. 114:482-485. Chang, L.S., A.F. Iezzoni, G.C. Adams, and F.W. Ewers. 1991. Hydraulic conductance in susceptible versus tolerant peach seedlings infected with Leucostoma persoonii. J. Amer. Soc. Hort. Sci. 1162831-834. Chang, LS. and A.F. Iezzoni. 1991. Heritability of Leucostoma persoom'i canker resistance among diverse peach genotypes. HortScience 26:60-62. Dhanvantari, B.N. 1978. Cold predisposition of dormant peach twigs to nodal cankers caused by Leucosroma spp. Phytopathology 68:1779-1783. Dhanvantari, B.N. 1982. Relative importance of Leucosmma cincm and L. persoonii in perennial canker of peach. Can. J. Plant Pathol. 4:221-225 Hampson, M.C., and W.A. Sinclair. 1973. Xylem disfunction in peach caused by Cytospora leucosmma. Phytopathology 63:676-681. 7 Helton, A.W., and DE. Konicek. 1961. Effect of selected Cyrospora isolates from stone fruits on certain stone fruit variaties. Phytopathology 51:152-157. Helton, A.W., and K.G. Rohrbach. 1967. Chemotherapy of Cymspora canker disease in peach trees. Phytopathology 57:442-446. Hildebrand, EC. 1947. Perennial canker and tree canker complex in New York, with methods of control. Cornell Univ. Agr. Sta. Mem. 276:3-61. James, W.C., and TR. Davidson. 1971. Survey of peach canker in the Niagara peninsula during 1969 and 1970. Can. Plant Dis. Surv. 51:148-153. Kern, H. 1955. Taxonomic studies in the genus Leucosroma. Pap. Mich. Acad. Sci. Arts and Letters 4029-22. Konicek, DE, and A.W. Helton. 1962. An optimum environment for the culturing of Cytospora isolates from stone fruits. lV. Hydrogen-ion concentration. Mycopath. Mycol. Appl. 16:243-248. Layne, R.E.C. 1984. Breeding peaches in North America for cold hardiness and perennial canker (Leucosroma spp.) resistance - review and outlook. Fruit Var. J. 38:130-136. Luepschen, NS. 1981. Criteria for determining peach variety suceptibility to cytospora canker. Fruit Var. J. 35:139-140. Sanders, J.P.M., C. Heyting, M.P. Verbeet, F.C. Melijlink, and P. Borst. 1977. The organization of genes in yeast mitochondrial DNA. 111. Comparisom of the physical maps of the mitochondrial DNAs from three wild-type Saccharomyces strains. Mol. Gen. Genet. 157:239-261. Scorza, R., and L. Pusey. 1984. A wound-freezing inoculation technique for evaluating resistance to Cytospora leucostoma in young peach trees. Phytopathology 74:569- 572. Surve-Iyer, RS. 1992. Phenetic analysis of Leucosroma from Prunus and Malus. PhD dissertation. Michigan State University. 112pp. Taylor, J .M., B.D. Smolich, and G. May. 1986. Evolution and mitochondrial DNA in Neurospora. crassa. Evolution 40:716-739. Tekauz, A., and Z.A. Patrick. 1974. The role of twig infections on incidence of perennial canker of peach. Phytopathology 64:683-688. 8 Treshow, M., B.L. Richards, and J .F. Scholes. 1959. The relation of temperature to pathogenicity of Cytospora rubescens (abs.). Phytopathology 49:114. Treshow, M., and J .F. Scholes. 1958. The taxonomy of some species of Cytospora found in Utah. Utah Acad. Proc. 35:49-51. Wensley, RN. 1964. Occurrence and pathogenicity of Valsa species and other fungi associated with peach canker in southern Ontario. Can. J. Bot. 42:841-857. Willison, RS 1937. Peach canker investigations 111. Further notes on incidence, contribution factors, and related phenomena. Can. J. Res. 15:324-339. Wysong, D.S., and LE. Dickens. 1962. Variation in virulence of Valsa leucostoma. Plant Dis. Rep. 46:274-276. 9 MATERIALS AND METHODS The flow chart in Figure 1 outlines the research approach followed in the investigations discussed below. Canker sampling Diseased tissues were collected from cankers on trunks and large scaffolds, and cankers on smaller canopy branches of each tree (approximately 12 years old) of two varieties, Redhaven and Loring, grown in three orchards, GO, BA and KB, located in southwest Michigan. The sampled Redhaven and Loring plots were adjacent to each other in each orchard. Thirty six trees of each variety were sampled from each orchard in a block of six consecutive rows (with six consecutive trees in each row) right next to the plot of the other variety. A total of 432 cankers were sampled. limosmma isolation Diseased tissue excised from the margins of cankers was surface sterilized by soaking in 5% sodium hypochlorite for 3 minutes and then blotting dry between sterile paper towels. The tissue was pushed into the flesh of firm apples (’Granny smith’) and the pathogenic fungi allowed to grow at room temperature (20-24°C) for 7 - 10 days. Small pieces of apple flesh from the margins of each diseased lesion were then placed on agar-solidified Leonian’s medium (1.2 g KHzPO4, 0.6 g MgSO4, 6.25 g maltose, 6.25 g malt extract in 1 L of distilled H20) (Leonian, 1923) in separate Petri dishes under sterile conditions. The fungi were cultured in the Petri dishes at room temperature for 7 - 10 days and then stored at 4°C. 10 Sampling of 432 cankers from Redhaven and Lorin in 3 orchards (GO. BA. K [9060510013 Isolation in apple Leucastama identification via colony margin L peace/III group i 4 Unidentified Sorting by orchards l identification via rD NA size polymorphism 11E (10 ? Vegetative compatibility grouping L cincta L persoani/ Subgrouping via Group 2 mtD NA RFLPS Figure 1. Flow chart of Leucastoma investigation ll Leucosroma identification The isolates were phenotypically grouped according to the characters of colony margins of the isolates on agar-solidified Leonian’s medium. L. persoom'i group 1 (PGl) had lobate colony margins which clearly distinguished this group from others (Surve- Iyer, 1992). Based on this criterion, all isolates of Leucostoma were grouped into two preliminary groups, "L.p group 1" and "unidentified". Isolates belonging to L. cincra were distinguished from other isolates in the "unidentified" group based on the characteristic presence of a unique insertion in the ribosomal DNA (rDNA). A specific fragment of the nuclear rDNA was amplified by polymerase chain reaction (PCR) (see methods below) using NS21 (5’- GAATAATAGAATAGGACG-3’) and NS22 (5’-AA'ITAAGCAGACAAACTC-3’) as a pair of primers (Gargas and Taylor, 1992). The nuclear small rDNA of L. cincta isolated from Prunus has an insert of about 450 base pairs in the fragment amplified using these two primers while the nuclear small rDNA of L. persoonii does not have the insert (Surve-lyer, 1992). The relative molecular weights of the PCR products following electrophoretic separation were used directly to distinguish L. cincta from L. persoom’i isolates in the "unidentified" group. The group identity of the rest of the "unidentified" L. persoonii isolates was determined phenotypically by colony color, pycnidial size, and temperature maxima, as described by Surve-Iyer (1992). Examination of vegetative compatibility The most prevalent phenetic group among the Leucosroma isolates was further 12 examined for the number of vegetative compatibility groups among isolates. The isolates were grown on agar-solidified Leonian’s medium for 4 days at room temperature. Plugs (4 mm in diameter) from colony margins of the culture were paired on clarified oatmeal agar medium (Adams et al.,1990). Plugs were placed 1.5 cm apart from each other in 150 x 15 mm Petri plates such that 61 plugs were on each plate. The plates were then incubated in the dark at room temperature (20-24°C) for 2-3 weeks. Compatibility or incompatibility between a pair of isolates were determined based on the absence or presence of a barrage zone between the pair (Adams et al., 1990). Subgrouping of the most prevalent group of Leucosmma isolates The most prevalent phenetic group of the Leucostoma isolates was further grouped using two mitochondrial DNA probes, B3 (Taylor and Smolich, 1985) and A13 (Jacobson and Gordon, 1990). B3 is a 8.8 kb EcoRI restriction fragment of Neurospora crassa 74A mtDNA and was provided by Taylor and Smolich (1985). A13 is a 5.3 kb Pstl mtDNA restriction fragment of Fusarium oxysporum f. sp. melonlis strain P-2 and was provided by Jacobson and Gordon (1990). The genomic DNA of each isolate was digested with two restriction endonucleases, Hpall and Haelll. The digested DNA was analyzed by Southern Blotting (Southern, 1975). isolates were grouped according to the banding patterns revealed by the two mitochondrial DNA probes. Plasmid preparation of mitochondrial DNA clones Recombinant plasmids pBR325 with B3 fragment and pUCl 19 with A13 fragment were isolated from overnight cultures of E. coli strains 113101 and MV1139 (Taylor and 13 Smolich, 1985; Jacobson and Gordon, 1990), respectively, using WizardTM Minipreps (Promega, Madison, WI). The E. coli strains were cultured in LB medium (Maniatis et al., 1982) containing 60pg/ml Ampicillin (for strain MV1139) or 60ug/m1 Ampicillin and 50ug/ml tetracycline (for strain HBlOl) overnight at 37°C with shaking (250 rpm). The cells were harvested from 3ml of culture by centrifugation at 13,000g for 2 minutes and resuspended in 200,111 of cell resuspension solution (50mM Tris-HCl, pH7.5, lOmM EDTA, lOOpg/ml RNase A) in a 1.5m1 eppendorf tube. 200u1 of cell lysis solution (0.2M NaOH, 1% SDS) was added to the tube and mixed by inverting the tube several times. 200,111 of neutralization solution (1.32M Potassium acetate, pH 7.5) was then added to the tube and centrifuged at 13,000g for 5 minutes. The clear supernatant was transferred to a new 1.5m] eppendorf tube. lml of the WizardTM Minipreps DNA purification resin (Promega, Madison, WI) was added to the supernatant and mixed by inverting the tube several times. The resin/DNA mixture was transferred to a syringe barrel attached with a WizardTM Minicolumn (Promega, Madison, WI) and pulled through the Minicolumn by applying a vacuum. The Minicolumn was washed by drawing 2ml of column wash solution (83mM NaCl, 8.3mM Tris-HCl, pH7.5, 2mM EDTA, 55% ethanol) through the Minicolumn with vacuum and then transferred to a 1.5ml eppendorf tube and centrifuged at 13,000g for 20 seconds to remove any residual column wash solution. The Minicolumn was then transferred to a new 1.5m] eppendorf tube and 50m of preheated (65°C) TE buffer (10mM Tris-HCl, pH 7.5, lmM EDTA) was applied to the Minicolumn. The plasmid DNA was eluted from the Minicolumn by centrifugation at 13,000g for 20 seconds and collected in the attached eppendorf tube and stored in the l4 eppendorf tube at -20°C untill use. DNA isolation The isolates were cultured in 100 ml of liquid Leonian’s medium at room temperature for 10-14 days. The mycelia were then harvested by vacuum filtration through miracloth (Calbiochem Corp, San Diego, CA), lyophilized and stored at -20°C. DNA was extracted from lyophilized hyphae by the method of Lee et al. (1988). Lyophilized mycelia was ground in a 1.5 ml eppendorf tube with 750 p1 of lysis buffer (50 mM Tris-HCl, 50 mM EDTA, 3% SDS, 1% 2-mercaptoethanol) and incubated at 65°C for 1 hour. 700 pl of phenolzchloroform (1:1) was added and centrifuged at 12,000g for 10 minutes. The aqueous phase was removed to a new 1.5 m1 eppendorf and another 700 pl of phenolzchloroform (1: l) was added and centrifuged at 12,000g for 10 minutes. The aqueous phase was collected and mixed with 700 pl of chloroform2isoamyl alcohol (24:1) in a new eppendorf tube and the mixture was centrifuged at 12,000g for 5 minutes. The top phase was removed to a new tube and DNA was precipitated with 0.1 volume of 3 M NaOAC (pH 8.0) and 600 #1 ice cold isopropanol. The DNA pellet was washed with 1 ml of 70% ethanol and dried overnight at room temperature. The extracted DNA was stored in TE buffer at -20°C. Amplification of DNA fragments using PCR The DNA fragment in the nuclear small rDNA used for the identification of L. Cincta was amplified by polymerase chain reaction. The PCR reactions were set up with Amplitaq DNA polymerase (Perkin-Elmer Corp., Norwalk, CT) in 25111 volume using 15 buffer conditions recommended by the manufacturer. The primer pair used was NS21 and N822. Thirty PCR cycles were performed on a Perkin-Elmer-Cetus thermocycler with the following parameters, 94°C denaturation for 1 minute, 48°C annealing for 1 minute, 72°C extension for 1 minute plus 4 seconds extension after each cycle, and with a final extension at 72°C for 10 minutes. 8 pl of each PCR products were separated according to molecular weight by electrophoresis on agarose minigels (1% Nusieve + 2% Seakem agarose in 1X TAE, FMC Bioproducts, Rockland, ME) along with 1 kb DNA weight marker (Boehringer Mannheim Biochemicals, Indianapolis, IN) and viewed under ultraviolet light. Southern Blotting anaflsis using Genius nonradioactive system The most prevalent phenetic group of the Leucosmma isolates was further analyzed with Southern hybridization. 1-3 pg of genomic DNA of each isolate was digested with 10 units of restriction enzyme (Hpall or Haelll) at 37°C for 2 hours. The products of digestion were separated by electrophoresis in 0.9% agarose gels in TAE buffer with Digoxigenin-labeled DNA weight marker lIl (Boehringer Mannheim Biochemicals, Indianapolis, IN) as a standard. The electrophoresis was carried out at 80 volt for 7 hours at 4°C. Prior to Southern transfer, the gel containing the separated DNA fragments was submerged, in order, in the following solutions: 0.25 M HCl (5 minutes), 0.5 M NaOH plus 1.5 M NaCl (45 minutes), and 1.0 M Tris-HCl (pH 8.0) plus 1.5 M NaCl (45 minutes). The DNA was then transferred from the gel to Boehringer Mannheim’s positively charged Nylon membranes (Boehringer Mannheim, Indianapolis, IN) by capillary blotting overnight, using 10X SSC (1.5 M NaCl, 0.15 M sodium citrate; 16 pH 7.0) as transfer buffer. The DNA was fixed to the membrane by UV. crosslinking for 1 minute at 120,000 microjoules in a UV. crosslinker (Stratagen model 2400, La Jolla, CA). The membrane was incubated in prehybridization solution (5X SSC, 2.0% blocking reagent for nucleic acid hybridization, 0.1% N-lauroylsarcosine, 0.02% sodium dodecyl sulfate, 50% formamide) at 42°C for 2 hours and then incubated in hybridization solution (prehybridization solution plus 10-20 ng/ml digoxigenin-labeled probe) at 42°C overnight. After hybridization, the membrane was washed twice (5 minutes each) in 2X wash solution (2X SSC, 0.1% SDS) at room temperature and twice (15 minutes each) in 0.5X wash solution (0.5X SSC, 0.1% SDS) at 42°C. After wash, the membrane was immediately treated for the detection of hybridization. The detection procedure was the same as described in the User’s Guide of the Genius System (Boehringer Mannheim, Indianapolis, IN) with slight modification. The membrane was briefly (1 minute) soaked in filtered Genius buffer 1 (100 mM Tris-HCl, 150 mM NaCl; pH 7.5) and then soaked in Genius buffer 2 (2% blocking reagent for nucleic acid hybridization dissolved in Genius buffer 1) for 45 minutes. The membrane was then incubated in anti-digoxigenin- alkaline phosphatase solution (0.1 unit/ ml polyclonal sheep anti-digoxigenin Fab- fragments in Genius buffer 2) for 30 minutes and washed twice (15 minutes each) in filtered Genius buffer 1. The washed membrane was equilibrated in Genius buffer 3 (100 mM Tris-HCl, 100 mM NaCl, 50 mM MgC12; pH 9.5) for two minutes and then placed between the two sheets of a plastic page protector. While the membrane was still wet, Lumi-Phos 530 (Boehringer Mannheim, Indianapolis, IN), in the amount of 0.2 ml/ 100 cm2 of membrane, was quickly dropped and evenly spread on the membrane. 17 The membrane was immediately exposed to X-ray film (Kodak XAR) for 1-2 hours at room temperature to detect hybridization. The probes used in the hybridization were labeled with digoxigenin using the Genius 1 DNA labeling kit (Boehringer Mannheim, Indianapolis, IN). A random primed labeling reaction in the volume of 40 pl was set up according to the instructions in the Genius System User’s Guide. The labeling reaction was carried out at 37°C overnight. The yield of the labeling reaction was estimated according to the instructions in the User’s Guide. Separation of mitochondrial DNA from chromosomal DNA In order to examine whether the two mitochondrial DNA probes used in the Southern analysis hybridize only to mitochondrial DNA of Leucostoma, intact mitochondrial DNA of L. persoonii was separated from intact chromosomes in 8% agarose gels by pulse field electrophoresis (Skelly and Malexzka, 1989). Electrophoresis, using the CHEF-DRII system (BIO—RAD Laboratory, Melville, NY), was for 18 hours at 10°C at 200 volt with a pulse of 60 seconds in 0.5X TBE. Intact chromosomes were distinquished from cytoplasmic DNA by the relative distance of migration in the agarose gel which is correlated to the sizes of the molecules. The intact mitochondrial and chromosomal DNA sample was prepared as follows. 23 500ml-Erlenmeyer flasks containing 50ml of 1.3X Leonian’s medium were inoculated with 3ml of L. persoom‘i conidia (107 conidia/ ml) and incubated on a reciprocal shaker (300rpm) for 36 hours at room temperature (20-24°C). The hyphae were harvested on miracloth and rinsed with sterile H20 and cold (4°C) 0.45mM EDTA. The hyphae were then scrapped into 5ml 18 of protoplasting medium (0.1 M NaPO4 buffer, pH 5.8; 1 M mannitol; 0.1% NovoZym 234, Novo BioLabs, Danbury, Conn.; 0.05% chitinase from Serratia, Sigma Chemical Company, St. Louis, MO; and 1% low-melt agarose) in a 50 x 15 mm Petri plate and incubated overnight at 33°C with an overlay of 10ml of EDTA-mannitol solution (0.45 M EDTA, pH 9.0; 1 M mannitol; 9 mM Cysteine-HCI) and then overnight at 50°C with an overlay of 10ml of proteinase-SL8 solution (0.2% proteinase XIV, Sigma Chemical Company, St. Louis, MO; 1% N-lauroylsarcosine sodium salt; 0.45 M EDTA, pH 9.0; 10 mM Tris-HCI) (McCluskey er al., 1990). Agarose plugs containing the treated hyphae were desalted and equilibrated by soaking for 4 hours at 4°C in 0.5X TBE buffer, pH 8, then directly loaded into the wells of 1% agarose gel prepared for CHEF DRII pulse field electrophoresis system and sealed in with warm 1% agarose. The L. persoom'i samples were electrophoresed alongside yeast chromosome DNA size standards of Saccharomyces cerevisiae, strain YNN295, and Schizosaccharomyces pombe, strain 972 h- (Bio-Rad Laboratories, Melville, NY). Data analysis Percentage data were arcsine transformed, i.e. arcsine of the square root of the value of percentage data, and analyzed as split-plot design with cultivars as main plot factor and canker sites and pathogens as subplot factors. The data were analyzed using MSTAT-C computer software (Freed er al., 1988). 19 RESULTS 403 isolates of Leucosmma were isolated from the 432 cankers sampled. The 403 isolates were phenotypically placed in 2 groups (L. pers'oonii group 1 and "unidentified") according to the characteristics of the colony margins. L. persoonii group 1 has lobate colony margins and therefore could be easily distinquished from the other Leucostoma isolates (Figure 2). The unidentified isolates were determined to be L. persoonii group 2 or L. cincta based upon rDNA size differences. The nuclear small rDNA of L. cincta from Prunus has an insertion of approximately 450 bp between the annealing sites of the 2 primers NSZI/NS22, while the nuclear small rDNA of L. persoonii does not have the insertion (Figure 3). The most common pathogens of Leucostoma canker in Michigan Loring and Redhaven trees in the 3 southwest Michigan peach orchards were found to be infected by 3 Leucostoma canker fungi (Table l). L. persoonii group 1 was the most common causal organism isolated from the cankers sampled. Of the 403 cankers sampled from three orchards, 88.6% were caused by L. persoonii group 1. L. cincta was the second most common pathogen causing 10.2% of the cankers. L. persoonii group 2 was the least common pathogen causing only 1.2% of the cankers sampled. Pathogen profiles of cankers on Redhaven and Loring cultivars Table 2 presents the percentage of cankers on the branches of the two cultivars caused by each pathogen. Significant differences between cultivars were observed in the 20 percentage of cankers caused by L. persoom'i group 1 and L. cincra. L. persoom'i group 1 was responsible for significantly more cankers in Redhaven versus Loring. L. persoonii group 2 was only isolated from cankers on Loring. Figure 2. Colony characteristics representative of the three Leucosroma subgroups; a) L. persoonii group 1 with a lobate colony margin, b) L. persoom'i group 2, and c) L. cincta, with uniform radial colony margins. 1018bp— 505119— Figure 3. 21 12 3 4 5 8 T 8 910111213141516 Electrophoretic separation of PCR products of nuclear small rDNA (primers NS21/N822) on 1% agarose gel stained with ethidium bromide and viewed under ultraviolet light. An insertion of about 450bp is present in the isolates of L. cincra (lanes 2, 3, 5, 13, 14) as compared to L. permonii ( lanes 4, 6-12, 15, 16). Lane 1 is a lkb lambda DNA molecular weight marker. 22 Table 1. Number and percentage of cankers caused by each of the pathogens isolated from 3 Michigan orchards. Pathogenl No. of cankers caused % of the total cankers sampled L. persoom'i group 1 357 88.6 a2 L. persoom'i group 2 5 1.2 c L. cincta 41 10.2 b Total 403 100 ‘Pathogens were grouped based upon characteristics of colony margins and rDNA polymorphism. 2Different letters indicate the means are significantly different at P < 0.01 (Appendix B1). Table 2. Percentage of cankers on branches of Redhaven and Loring caused by different pathogens. The data is summed over 3 orchards. Percent cankers for each causal pathogenl Cultivar L. person/iii group 1 L. cincta L. persoonii group 2 Redhaven 88.77 a2 11.23 b2 0 Loring 76.33 b 20.77 a 2.9 ‘Pathogens were grouped based upon characteristics of colony margin and rDNA polymorphism. 2Different letters indicate the means in columns are significantly different at P3005 (Appendix B2, Appendix B3). 23 Pathogen profiles of cankers on tree trunks and branches Cankers on tree trunks and branches were found to be caused by different pathogen profiles (Table 3). A significantly larger percent of cankers on tree trunks were caused by L. persoonii group 1, than were cankers on branches. On the other hand, a significantly larger proportion of cankers on branches were caused by L. cincta, than were cankers on tree trunks. Significant differences were found between the two cultivars, Redhaven and Loring, in the pathogen profile of cankers on branches, as shown in Table 2. However, if only cankers on tree trunks were compared, no significant difference was found between the two cultivars, except that no isolates of L. persoonii group 2 were found on Redhaven (Table 4). Vegetative compatibility among isolates of L. person/iii group 1 One hundred eighty two isolates of L. persoonii group 1 from cankers of adjacent trees were paired on agar solidified medium to examine vegetative compatibility. Of a total of 1,020 pairs examined, 95 % of the pairs were incompatible with each other (Table 5, Figure 4). Such a high incompatibility among isolates made it impractical to divide L. persoonii group 1 into vegetative compatibility subgroups. 24 Table 3. Percentage of cankers on branches and trunks caused by different pathogens. The data is summed over 3 orchards and 2 cultivars.‘ Percent cankers for each causal pathogen2 Canker location L. persoonii group 1 L. cincta L. persoonii group 2 Branch 82.55 b3 16.00 a4 1.15 Trunk 94.50 a 4.50 b 0.17 ‘Leucosroma was isolated from 403 cankers. 2Pathogens were grouped based upon characteristics of colony margin and rDNA polymorphism. 3Different letters indicate the means in column are significantly different at the P<0.01 (Appendix B4). 4Different letters indicate the means in column are significantly different at the P<0.05 (Appendix B5). Table 4. Percentage of cankers on tree trunks of Redhaven and Loring caused by different pathogens. The data is summed over 3 orchards.‘ Percent cankers for each causal pathogen2 Cultivar L. persoonii group 1 L. cincra L. persoonii group 2 Redhaven 94.9 5. 1 0 Loring 94.1 3.9 2.0 ‘Leucosmma was isolated from 403 cankers. 2Pathogens were grouped based upon characteristics of colony margin and rDNA polymorphism. 25 Table 5. Percentage of vegetatively incompatible pairs among 182 isolates of L. persoonii Group 1 Test‘ Total No. of pairs No. & % of incompatible pairs No. % 1 69 62 90 2 88 88 100 3 88 87 99 4 88 81 92 5 107 99 93 6 88 88 100 7 107 104 97 8 81 73 90 9 107 99 93 10 62 60 97 11 84 78 93 12 51 48 94 Total 1020 967 95 ‘The number represent the order in time of different pairing tests. 26 Figure 4. Pairing of isolates of L. persoonii group 1 on clarified oatmeal agar medium. Barrage zones formed between vegetatively incompatible isolates. 27 Target DNA hybridized by the two mitochondrial DNA probes In order to investigate whether the two mitochondrial DNA probes, B3 and A13, which were used in subgrouping of L. persoonii group 1, also hybridized to nuclear DNA, hyphae in agarose were protoplasted and lysed in agaro and intact chromosomes were separated from intact mitochondrial DNA by pulse field electrophoresis. Following electrophoresis, two dense bands of DNA were evident on the ethidium bromide-stained agarose gels. One band of chromosomal DNA occurred that was greater than 1.6 megabase, which is in the common range of chromosome length (1.3 to 6.2 megabase) for ascomycete fungi (Mills and McCluskey, 1990). The other band of DNA, the putative mitochondrial DNA, occurred at less than 0.6 megabases. The separated DNA was probed with the two mitochondrial DNA probes. Neither probe hybridized to the intact chromosomal DNA nor elswhere on the gel, except to the DNA smaller than 0.6 megabases (Figure 5 and Figure 6). Subgrouping of L. permonii group 1 Sixty nine isolates of L. person/iii group 1 from one orchard, BA orchard, were analyzed by Southern hybridization with the two mitochondrial DNA probes. Considerable mitochondrial DNA variation was found among the isolates (Figure 7). Eleven subgroups, defined as 2 or more isolates having the same banding pattern, were identified within this group isolated from BA orchard. The banding patterns of the 11 subgroups is shown in Figure 8. The number and percentage of isolates belonging to each subgroup is shown in Table 6. Two major subgroups, 1 and 2a, comprised 55% of the 69 isolates investigated. Subgroup 3a, 3b, 4b, 4c, 5a were only isolated from 28 cankers on cultivar Redhaven and subgroup 2b and 6 were only isolated from cankers on Loring (Table 6). In addition to the 11 subgroups, 6 single isolates, each with a unique banding pattern (Figure 9), were found on cultivar Redhaven. 29 A l 2 3 — Sampievnl intact chromosomes _ 1,61]ka (Moplastic DNA — 565k!) — 225kb B i 2 3 — Sampiewel Intact chromosomes _ 1.600kb Oytoplastic DNA D .' y _ 565 kb — 225 kb Figure 5. L. persoonii DNA hybridized to Neurospora crassa mitochondrial DNA probe B3. A. Ethidium bromide stained gel viewed under ultraviolet light; B. Autoradiograph after hybridization of probe B3. 1 and 2, L. parsoonii; 3, yeast chromosome DNA size standard, Saccharomyces cerevisiae, strain YNN295. Sample__ well 1.600 H)- 565 kb— 225 kb— Figure 6. L. persoonii DNA hybridized to Fusarium oxysporum mitochondrial DNA probe A13. A. Ethidium bromide stained gel viewed under ultraviolet light; B. Autograph after hybridization of probe A13. 1 and 3, L. persoonii; 2, yeast chromosome DNA size standard, Saccharomyces cerevisiae, strain YNN295. 30 Sample well 1.600 kb 565 kb 225 kb 31 Table 6. Number and percentage of isolates belonging to each subgroup of L. persoom'i group 1 based upon mitochondrial DNA polymorphism‘ Host Subgroups Redhaven Lorin g Total Percent 1 1 1 15 26 37.7 2a 5 7 12 17.4 26 0 4 4 5.8 3a 4 0 4 5.8 36 2 0 2 2.9 4a 1 2 3 4.4 4b 2 0 2 2.9 4c 3 0 3 4.4 5a 3 0 3 4.4 5b 1 1 2 2.9 6 0 2 2 2.9 Other2 6 0 6 8.7 ‘The isolates were Tsolated from cankers on two cultivars at the BA orchard. 2Each isolate had its unique banding pattern which only appeared once in the 69 isolates examined. Figure 7. 32 Autoradiographs of Southern blotting using mitochondrial DNA as probes. A. Hpall digested genomic DNA hybridized with labeled B3, lanes 1-8, L. persoom'i group 1; B. HaeIII digested genomic DNA hybridized with labeled B3, lanes 1-6, L. persoom'i group 1; lane 7, DNA size standard; C. HaeIII digested genomic DNA hybridized with labeled A13, lanes 1-8, L. persoom'i group 1. 33 Subgroups DNA size PB?” 8 1 2a 2b 3a 3b 4a 4b 41: 5:: Sb 5 marker ............. zym _2123kb 5. 5 _::__:::::_-4-i7 — 4.27kh BBiHaelll — 3.531(1) — — - _ 2.03kb — 1.91m: — 1.51m: 5. 5kb —4.27kb BBinall "" "' "" _ —3.53kb _ 2.0311: — 1.90 kb — 21.23 kb A131Haelll _ _ _ _ _ _ Egg — _ - . --------------------- — 4.2? kb '— 3.53 Id) Figure 8. Banding patterns of subgroups of Ape/$001217 group 1 34 Isolates DNAsne Probe! A B C D E F Enzvme .............................................................................................................. T2153. _ 21.23 k0 — — 5.151(1) — — — — — 4.9m: BSiHaelll _ — 3.53kb — — — — _ 2.03kb — 1.90 kb — 1.5010: — — 1.30kb —4.27kb BBinall "' = — — —3.53kh _ 2 03 kb — 1 90 kb — 21.23 kb AlSiHaelIl ‘ - — - — - -2159 —4.27kb — 3.53 kh ............................................................... Figure 9. Unique banding patterns of L peace/Iii gI'OUp l 35 DISCUSSION L. persoonii group 1 is the predominant subgroup of Leucosroma in Michigan peach orchards. L. cincra has been reported to be a primary pathogen in some northern extremes of the peach production area (Dhanvantari, 1982; Northover, 1976; Tekauz and Patrick, 1974; Willison, 1936), however, it is second to L. persoonii group 1 in importance in Michigan. Differences in the isolation frequencies of L. persmmii and L. cincta from different canker sites on peach trees were reported by Dhanvantari (1982). In his study, L. cincta predominated in the isolations from node cankers while L. persoonii predominated in the isolations from perennial and fruit pedicel cankers. In our study, differences were also observed in the isolation frequencies of L. persoonii group 1 and L. cincra. from the two different canker sites, tree trunk and branches. Although L. persoonii group 1 predominated in the isolations from both canker sites, it was significantly less frequent in the isolations from branch cankers than from trunk cankers. On the other hand, L. cine-ta was significantly more frequent in the isolations from branch cankers than from trunk cankers (Table 3). The epidemiological factors that result in these differences still need to be determined. Possible factors may include availability and relative virulence of different Leucosrm-na subgroups at the time when the different canker sites are susceptible to the disease. L. cincta has been reported to be more virulent in cool weather and L. person/Iii more virulent in warm weather (Dhanvantari, 1969; Hildebrand, 1947; Wensley, 1964). Leaf scars and winter injuries predispose small branches to infection in late fall and early spring when L. cincta could 36 be relatively more virulent. Any injuries to the scaffolds and tree trunks during summer such as sunburns, mechanical injuries caused by field equipment and harvesting might predispose the trees to the disease when L. persoonii inoculum is most abundant (Luepschen and Rohrbach, 1969) and more virulent. Significant differences in the profiles of branch canker pathogens on the cultivars Redhaven versus Loring are observed in this study. The difference might be related to the cultivar difference in canker tolerance. Redhaven was reported to be relatively tolerant to canker (Dhanvantari, 1982) and Loring very susceptible to the disease (Chang et al., 1989). Pathogens of low virulence may have a better chance to invade and establish cankers on a more susceptible cultivar than on a tolerant cultivar and this appeared to be the case in our study. Field tests showed that L. persoonii group 1 was more virulent than L. cincta, and L. cincra isolated from Prunus spp. was more virulent than L. persoonii group 2 in Michigan on peach (Surve-Iyer, 1992). L. cincta caused a significantly larger percentage of cankers on the branches of Loring than on the branches of Redhaven in our investigation. L. persoonii group 2 was only isolated from cankers on Loring. The commonly observed vegetative incompatibility among isolates from cankers of adjacent trees in this study is consistent with the findings by Adams et al. (1990). Each incompatible reaction indicates that the two isolates are different individuals. The pairings herein demonstrate that many individuals are parasitizing the trees in a small orchard area. This supports the suggestion that clonal spread might be less frequent than spread by sexual recombinants, and thus ascospores are important propagules in the 37 dissemination of L. persoom’i. The frequency of incompatible reactions and the number of maternal lines are a demonstration of the amount of genetic variation among individuals of L. persoonii in the orchards. However, isozyme polymorphisms are not commonly encountered in L. persoonii in a local region (Surve-Iyer, 1992). The DNA restriction fragment length polymorphisms (RFLPs) of mitochondrial DNA were used to identify maternal lines among L. persoonii group 1 from one split- cultivar block containing 72 trees. As shown in Figure 5 and Figure 6, the two probes used hybridized only to the putative mitochondrial DNA (DNA smaller than 600kb). The size of the intact DNA molecules detected by the two probes suggested their mitochondrial identity. And the probes themselves are restriction fragments of mitochondrial DNA from related species. Maternal inheritance has been demonstrated to be the principal mode of mitochondrial DNA inheritance in fungal species (Mannella et al., 1979; May and Taylor, 1989; Milgroom and Lipari, 1993; Mitchell and Mitchell, 1952; Taylor, 1986b). Each grouping of isolates of L. persoonii group 1 with distinct RFLP patterns of mitochondrial DNA in this study could be considered as a maternal line, of which all the isolates had a common maternal ancestor. At least 11 maternal lines of L. persoonii group 1 were found in the BA orchard. Lines 3a, 3b, 4b, 4c, and 5a were only isolated from cultivar Redhaven and lines 2b and 6 were only isolated from Loring. Whether differences in host specificity, virulence or local dissemination exist among the maternal lines needs to be further investigated. Significant variation in mitochondrial DNA of Leucosnma was revealed by RFLPs in this study. A total of 17 different RFLP patterns were detected with only three 38 probe-enzyme combinations among 69 isolates of L. persoonii group 1 from a 72 tree block. Intraspecific variation of mitochondrial DNA was reported for fungal species Armillaria mellea. (Smith and Anderson, 1986), Cochliobolas hererostrophus (Garber and Yoda, 1984), Cryphonecn‘ia parasitica (Milgroom and Lipari, 1993), Fusarium oxysporum (Taylor, 1986b), and Neurospora crassa (Collins and Lambowitz, 1983; Taylor, 1986a). In Neurospora species, length mutations were found to be the most important source of variability in mitochondrial DNA (Collins and Lambowitz, 1983). Nucleotide substitutions are also common in Neurospora mitochondrial DNA (Taylor, 1986a). Recombinations of mitochondrial DNA during sexual reproduction have been reported for some fungal species (Baptista-Ferreria (2101., 1983; Borst and Grivell, 1978; Economou 6101., 1987). It would be interesting to determine which sources contributed to the variability in Leucosmma mitochondrial DNA. LIST OF REFERENCES Adams, G.C., S.A. Hammar, and T. Proffer. 1990. Vegetative compatibility in Leucostoma persoonii. Phytopathology 80:287-291. Baptisa-Ferreria, J .L.C., A. Economou, L.A. Casselton. 1983. Mitochondrial genetics of Caprinus: recombination of mitochondrial genomes. Curr. Genet. 7:405-407. Borst, P., and LA. Grivell. 1978. The mitochondrial genome of yeast. Cell 15:705-723. Chang, L.S., A.F. Iezzoni, G.C. Adams, and GS Howell. 1989. Leucostoma persoonii tolerance and cold hardiness among diverse peach genotypes. J. Amer. Soc. Hort. Sci. 114:482-485. Collins, R.A., and A.M. Lambowitz. 1983. Structural variations and optional introns in the mitochondrial DNAs of Neurospora strains isolated from nature. Plasmid 9:53-70. Dhanvantari, B.N. 1969. Comparative physiology and pathogenicity of Leucostoma spp. on peach. Phytopathology 59:1023. Dhanvantari, B.N. 1982. Relative importance of Leucostoma cincra and L. persoonii in perennial canker of peach. Can. J. Plant Pathol. 4:221-225. Economou, A., V. Lees, P.J. Pukkila, M.E. Zolan, L.A. Casselton. 1987. Biased inheritance of optional insertions following mitochondrial genome recombination in the basidiomycete fungus Coprinus cinereus. Curr. Genet. 11:513-519. Freed, R., S.P. Eisensmith, S. Goetz, D. Reicosky, V.W. Smail, and P. Wolberg. 1988. MSTAT-C a software program for design, management, and analysis of agronomic research experiments. Michigan State University, East Lansing, MI. Garber, RC, and O.C. Yoder. 1984. Mitochondrial DNA of filamentous ascomycete Cock/inbolus hererostrophus. Curr. Genet. 82621-628. Gargas, A., and J.W. Taylor. 1992. Polymerase chain reaction (PCR) primers for amplifying and sequencing nuclear 18$ rDNA from lichenized fungi. Mycologia 84:589-592. Hildebrand, E.G. 1947. Perennial canker and three canker complex in New York, with methods of control. Cornell Univ. Agr. Sta. Mem. 276:3-61. Jacobson, D.J., and TR. Gordon. 1990. Variability of mitochondrial DNA as an 39 40 indicator of relationships between populations of Fusarium oxysporum f. sp. melom's. Mycol. Res. 94:734-744. Lee, S.B., M.G. Milgroom, and J.W. Taylor. 1988. A rapid, high yield mini-prep method for isolation of total genomic DNA from fungi. Fungal Gen. Newsl. 35:23-24. Leonian, L.H. 1923. The physiology of perithecial and pycnidial formation in Valsa leucostoma. Phytopathology 13:257-272. Luepschen, NS, and K.G. Rohrbach. 1969. Cytospora canker of peach trees: Spore availability and wound susceptibility. Plant Dis. Rep. 53:869-872. Maniatis, T.E., E.F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, p 545. Mannella, C.A., T.H. Pittenger, and A.M. Lambowitz. 1979. Transmission of mitochondrial deoxyribonucleic acid in Neurospora crassa sexual crosses. J. Bacteriol. 137: 1449-1451. May, G., and J.W. Taylor. 1989. Independent transfer of mitochondrial plasmids in Neurospora crassa. Nature 339:320-322. McCluskey, K., B.W. Russell, and D. Mills. 1990. Electrophoretic karyotyping without the need for generating protoplasts. Curr. Genet. 18:385-386. Milgroom, M.G., S.E. Lipari. 1993. Maternal inheritance and diversity of mitochondrial DNA in the Chestnut Blight fungus, Cryphonectria parasirica. Phytopathology 83:563-567. Mills, D., and K. McCluskey. 1990. Electrophoretic karyotypes of fungi: The new Cytology. Mol. Plant-Microbe Interact. 32351-357. Mitchell, M.B., and HR. Mitchell. 1952. A case of "maternal" inheritance in Neurospora crassa. Proc. Natl. Acad. Sci. USA 38:442-449. Northover, J. 1976. Protection of peach shoots against species of Leucosroma with benomyl and captafol. Phytopathology 66: 1 125-1128. Skelly, P.J., and R. Malexzka. 1989. Isolation of mitochondrial DNA using pulsed field gel electrophoresis. Nucleic Acids Res. 17:7537. Smith, M., and JB. Anderson. 1986. variation in mitochondrial DNAs of the North 41 American biological species of Armillaria. Mycol. Soc. Amer. Newslett. 37:43. Southern, EM. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98:503-517. Surve-Iyer, RS. 1992. Phenetic analysis of Leucosroma from Prunus and Malus. PhD dissertation 112 pp. Michigan State University, East Lansing, MI. Taylor, J.W., and 8D. Smolich. 1985. Molecular cloning and physical mapping of Neurospora crassa 74-OR23-1A mitochondrial genome. Current Genetics 9:597— 603. Taylor, J.W. 1986a. Mitochondrial DNA and evolution in Neurospora species. Fungal Genet. Newslett. 33:14. Taylor, J.W. 1986b. Topical review: fungal evolutionary biology and mitochondrial DNA. Exp. Mycol. 10:259-269. Tekauz, A., and Z.A. Patrick. 1974. The role of twig infections on incidence of perennial canker of peach. Phytopathology 64:683-688. Wensley, RN. 1964. Occurrence and pathogenicity of Valsa species and other fungi associated with peach canker in southern Ontario. Can. J. Bot. 42:841-857. Willison, RS. 1936. Peach canker investigations 11. Infection studies. Can. J. Res. 14:27-44. APPENDICES 42 Appendix A Table 7. Percentage of cankers caused by Leucostoma persoonii group 1, group 2, and L. cincta isolates from trunks and branches of Redhaven and Loring from 3 Michigan orchards. Canker No. (%) of L.persoonii NO- (%) 0f Orchard Cultivar Location Group 1 Group2 L. cincta GO Redhaven Trunk 30 (90.9) 0 (0.0) 3 (9.1) Branch 28 (87.5) 0 (0.0) 4 (12.5) Loring Trunk 34 (94.4) 0 (0.0) 2 (5.6) Branch 23 (67.6) 2 (5.9) 9 (26.5) BA Redhaven Trunk 30 (93.8) 0 (0.0) 2 (6.2) Branch 27 (87.1) 0 (0.0) 4 (12.9) Loring Trunk 29 (93.5) 1 (3.3) 1 (3.3) Branch 25 (78.1) 0 (0.0) 7 (21.9) KB Redhaven Trunk 34 (100.0) 0 (0.0) 0 (0.0) Branch 33 (91.7) 0 (0.0) 3 (8.3) Loring Trunk 34 (94.4) 1 (2.8) l (2.8) Branch 30 (83.3) 1 (2.8) 5 (13.9) 43 Appendix B Output of ANOVA analysis using MSTAT-C software. Percentage data were arcsine transformed as described in Materials and Methods before analysis. Appendix Bl Output of ANOVA analysis using MSTAT-C software. Cultivars, canker sites, and pathogens were considered as experimental factors and analyzed together as described below. Function: FACTOR Experiment Model Number 11: Randomized Complete Block Design for Factor A, with Factors B and C as Split Plots on A Factorial ANOVA for the factors: Replication (orchard) with values from 1 to 3 Factor A (cultivar) with values from 1 to 2 Factor B (canker site) with values from 1 to 2 Factor C (pathogen) with values from 1 to 3 Variable: arcsine transformed percentage data ANALYSIS OF VARIANCE TABLE Degrees of Sum of Mean F Source Freedom Squares Square Value Prob Replication 2 1.96 0.98 1.00 0.50 Factor A 1 24.14 24.14 24.64 0.04 Error 2 1.96 0.98 Factor B l 1.00 1.00 0.03 AB 1 1.00 1.00 0.03 Factor C 2 31197.93 15598.97 479.23 0.00 AC 2 306.90 153.45 4.71 0.02 BC 2 871.17 435.59 13.38 0.00 ABC 2 62.17 31.09 0.96 Error 20 651.00 32.55 44 Appendix B2 Output of ANOVA analysis using MSTAT-C software. Arcsine transformed percentage data of L. persoonii group 1 on branches of Redhaven and Loring were analyzed as described below. Function: ANOVA-2 Two—way Analysis of Variance over variable 1 (orchard) with values from 1 to 3 and over variable 2 (cultivar) with values from 1 to 2. Variable: arcsine transformed percentage data ANALYSIS OF VARIANCE TABLE Degrees of Sum of Mean F Source Freedom Squares Square Value Prob Orchard 2 53.08 26.54 3.34 0.23 Cultivar 1 132.82 132.82 16.74 0.05 Error 2 15.87 7.94 Non-additivity l 9.98 9.98 1.69 Residual l 5.89 5.89 Total 5 201 .77 45 Appendix B3 Output of ANOVA analysis using MSTAT-C software. Arcsine transformed percentage data of L. cincra on branches of Redhaven and Loring are analyzed as described below. Function: ANOVA-2 Two-way Analysis of Variance over variable 1 (orchard) with values from 1 to 3 and over variable 2 (cultivar) with values from 1 to 2. Variable: arcsine transformed percentage data ANALYSIS OF VARIANCE TABLE Degrees of Sum of Mean F Source Freedom Squares Square Value Prob Orchard 2 47.38 23.69 6.94 0.13 Cultivar l 82.73 82.73 24.23 0.04 Error 2 6. 83 3.41 Non-additivity 1 5.16 5.17 3.10 Residual l 1 .66 1 .66 Total 5 136.94 46 Appendix B4 Output of ANOVA analysis using MSTAT-C software. Arcsine transformed percentage data of L. persoonii group 1 on cultivars and canker sites were analyzed as described below. Function: FACTOR Experiment Model Number 9: Randomized Complete Block Design for Factor A, with Factor B a Split Plot on A Factorial ANOVA for the factors: Replication (orchard) with values from 1 to 3 Factor A (cultivar) with values from 1 to 2 Factor B (canker site) with values from 1 to 2 Variable: arcsine transformed percentage data ANALYSIS OF VARIANCE TABLE Degrees of Sum of Mean F Source Freedom Squares Square Value Prob Replication 2 138.28 69.14 5.17 0.16 Factor A 1 122.88 122.88 9.20 0.09 Error 2 26.72 13.36 Factor B 1 421.03 421.03 21.02 0.01 AB 1 27.18 27.18 1.36 0.31 Error 4 80.1 1 20.03 47 Appendix BS Output of ANOVA analysis using MSTAT-C software. Arcsine transformed percentage data of L. cincm on cultivars and canker sites were analyzed as described below. Function: FACTOR Experiment Model Number 9: Randomized Complete Block Design for Factor A, with Factor B a Split Plot on A Factorial ANOVA for the factors: Replication (orchard) with values from 1 to 3 Factor A (cultivar) with values from 1 to 2 Factor B (canker site) with values from 1 to 2 Variable: arcsine transformed percentage data ANALYSIS OF VARIANCE TABLE Degrees of Sum of Mean F Source Freedom Squares Square Value Prob Replication 2 161.58 80.79 8.68 Factor A 1 48.36 48.36 5.19 Error 2 18.63 9.31 Factor B 1 450.07 450.07 30.74 0.01 AB 1 34.92 34.92 2.38 0.20 Error 4 58.57 14.64 "IIIIIIIIIIIIIIIIIIIII