JIlllllllllllllllllllllllllllllllll L 3 1293 01106 4742 LIBRARY Michigan State University This is to certify that the thesis entitled Virulence and Hypovirulence in Leucostoma spp. presented by Sue Ann Hammar has been accepted towards fulfillment of the requirements for ”.5. degree in Botany 8: Plant Pathology Major professor Date ”3] 19, 1988 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution ___.__-__. ___l ,_ _ 7 , 7 7 MSU LIBRARIES m -w..- RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. tum u 9 s...)- 2 5‘ .~ . WMWD‘ W99. 32mm A“ all-11131 to State university Michigan mpmufulfillmtoffinrquim forfindagreeof Wtofhtmymflflmtl’atmlogy 1988 552- are; WWWINWSPP. SueAnnHaumar An isolate of Leucostana M with low virulence, amornal mrphology, and lacking conidia was found to contain double-stranded RNA (dsRNA) as determined by polyacrylanide gel electrophoresis. Specific m segments were lost in partially cured subcultures resulting in increased but not normal virulence or sporulation. Protoplast derived dsRNA-free subcultures hai normal virulence and sporulation. A mind viral infection was indicated. The optiml experimental design for evaluatirg isolate virulence in the orchard, based on sampling variances, vasto imculateonebranchpertreeinG—Qtreesforeadi isolate. Criteria for differentiating L_. _c_i_ngt_a_ fran In. Ersoonii by cultural damteristics was evaluated. Vegetative canpetibility (vc) groupings were determined for two orchards by pairing isolates derived francenleers. Isolateswithinanorchardwereinmmsmnvcgruzps. Isolates from several cankers within me tree and ammg closely spaced trees usually differed in vc grouping. The epidemiology and sexuality of .12. my. is discussed- Tomyparents For teachingmethevalue ofaneducation ii WW I wish to thank my major professor, Dr. Gerry Adams, for his sipport, guidance, and friendship. I am grateful to my cannittee members, Dr. Denis Fulbright for the use of his facilities, personnel, and technical advice. and Dr. Allan Jones for the use of his orchard and his editorial assistance. Special thanks to my husband Stephen for his support, patience, and love which were essential to my finishing this project. Last but not least. thanks to Free and Jasmine for their perpetual entlmsiasm and devotion vim times were rough. iii TABIEGFOONIEN'I‘S LIST OF TABLES . .................. . LISTOFFIGURES . . . .......... GENERALINTRODIXS‘I‘ION.... Literature cited ......... . ............. SMION 1 MASSWIATIONOFDOUBLE-S'IRANDEDRNAWITHWVIWLENCEAND IDSSOFSPORUIATIONINANISOMTEOFWPERSOONIIFRQWPEACH ABSTRACTW ............... . ........ MATERIALSANDMEH-ms. .. ..... . Virulencetests........... .......... . IsolationofdsRNA... ............... . Bennyl-resistant laments . . . . . . ......... . . 'l‘r-ansferofdsRNA........... .. . mm... 000000000000 O 0000000000 O Isolates...... ..... . ....... . . .. Virulencetests ScreeningisolatesfordsRNA . ...... Curing of isolate 14.“ . . ......... . ........ Tremissimexperinents. ..... ........ DISCUSSION... ........... . .............. LITERAIIRECITEDHHWW ..... . ..... SECTIONII VESETATIVE mmm IN mm PERSDONII m 0 I O O O I O O O O O O O O O O O O O O O O O O O O O blata O O O O O O O I O I I I O O I I O O O ..... Determination of vegetative mtibility . . iv 10 12 12 12 13 13 15 16 16 16 16 17 17 21 25 29 32 34 34 35 RESULTS ...... . ..... ............. Sexuality onLErsoonii . .................. Distributionofvcgrcups....... ......... DISCUSSION ..... WCITED ...... .. SECTIONIII wmmvmmmmmmmm WMRBISTANCETOCYTOSPORACANKER mmmmnms ....... . RESULTS ...... ...... DISCIJSSIQI............. ..... ImmWMOOOO......COOOOOOOOIOO... 49 53 57 65 69 72 LISTOFTABLES Table Section I 1. Virulmce of isolates of Ieucoetana spp. on ten trees of GarretBeautypeachrankedindecreasirgorderbycemeer length and the association of double—stranded RNA with each isolate.... 2. Assay of virula'rce of isolates of Leucostana grsoonii on mle mt O O O I O O I I O O I O '0 O O O O O ..... 3. Canparison of virulence of [ancestors pgrsoonii wild-type isolates, 14.4A, and subcultures of 14.“ m branches (1? m diam.) onpeachtrees . .................... SE'I'IONIII 1. Isolates of limestone spp.: Origin. colony characteristics, author designated species, and source or reference ....... 2. Analysis of variance table for optimum inoculation procedure ofteucostanaisolatesinpeach . . . . . . . . ........ 3a. Virulence of isolates of narcos___3:__ana spp. on 10 trees of GarnetBeautypeachranledinorderofdecreasingcanker length . ................ . . ........ 38.Randcmized0anpleteBlockDesign..... ...... .. 4. Choice of experimental design for the met efficient determination of the relative virulence of the mthogen or resistance of a cultivar requiring the least labor and cost (observations of inoculated branches and trees) and providing the most accurate experimental malts (greatest reduction in ample variance). A) Total number of observatiom (or inoculations) required with differart imculation models (experimtal designs). 8) Sampling variances expected for the differentdesignsinA. V1 59 64 LISTOFFIGURES Figure 1. SECTIWI Pl'ictograph of an ethidium bromide s’ceired 5% polyacrylamide gelshcwingspecified segments ofdsRNAextractedfran Immos_____t__ana spp. a) isolate 14. 4A, b) partially cured 14. 4A, andc)isoalte9.2. . ......... Characteristic cultural morphology of 30-day-old dsRNA infected andwredstrainsof Leucostanagrsoonii onLMAa) strain 14.4A with light pigmtation, less compact mycelium, and no pycnidia, b) "partially cured" strain with darker pigmentation, capact myceliun, and pycnidial initials, and c) "cured" strain with sporulating pycnidia, and lobate colony nargins characteristic of most isolates of _r_._. m frail canleers mpeach. ...... . . . ..... SECTIONII Caliperison of the clarity of reaction lines (barrage cares) formed at the line of ccmtact batman 15-day—old incomatible colmies of Ieucostan pgrsoonii. Pairings nade on a) oatmeal agar and b) clarified oatmeal agar. . . ........ .MromofpeechtreesinorchardA.Mmbersidentifythe vegetative metibility groups of isolates of Ieucostana Mischtedfmacankerineachtree ....... . Spatial arrangement of trees and vegetative canpatibility groups of 65 isolates of leucostana mi; isolated from 42 trees inorchardB. .......... . .......... Colony characteristics of a) Leucostara cincta with a mifornally radial coldly margin and large (1-3 m) pymidia and b) _L__. m; with a lomte colony margin and snail (<1 m) wail—die. . .............. vii 2O 23 38 41 42 WWW m WIN Wtospora miner of peach is of considerable economic importance and is the limiting production factor for this crop in most northern climate peach growing areas (9,14,25,29). This disease is also known as peach grmosis, valsa canker, perermial canker, die-back of peach, and peach canker (30,46). The same organises that cause Cytospora canker on peach also cause an important disease on apple, cherry, apricot, prune, and plum (19,20). The disease is often destructive in young orchards where it curses prenature death of the trees. Older infected trees gradually lose productivity and longevity is decreased (36,44). Sympt. of Cytospora canker on peach include dieback of twigs and branches, girdling and wilting of branches and major limbs, perennial cankers on the trunk, scaffold limbs, and branches, and premature leaf senescence and defoliation (9,18,40). Ganlners are first apparent in the spring at nodes and fruit pedicels (14,36). Typical canker symptoms are collapsed cortical tissue, bark necrosis, gunning, pycnidia, and a zonate margin (6). The causal agents of the disease are Ianostana 9__inc_g (Pers.: Fr.) Hoel'm. (mph: W 93:93 (Sacc.) Hoetm.) and rennet-u grsoonii (Nits.) Hoel'm. [anamorphs W leucostala (Pers.) Hoel'n]. The fimgi cursing altospora canker on peach were originally placed in the genus gals; in 1849 based on the oblique position and circinate pattern of anerging perithecia in the determinant struna. As early as 1917 Kestrel separated species of E2 sensu late into E193 sensu stricto and Leucostana (3,26). Ieucostara is differentiated from y_a_l§g sarsu‘ stricto by the presence of a distinctive dark conceptacle of capact furgal tissue delimina‘um the stranta around the fruiting bodies. The unnorphs that developed with a similar carcsptacle were segregated from m and placed in W (3.26.37). The amorph and tsliomorph of the species that came Cytospora canker of peach form the distinctive conceptacle. megsrmsreucostanaisintheDiaportheae. Thepycnidia, andvd‘ren present, perithscia, develop in a strain. Perithscia are oblique with few to mmsrous beaks converging and enmpent thrmgh the disc (3,29). Stromatic tissues are prosarchymtous or pseudcparsnchymatous, forming determinant white or grayish brown sctostrcmatic discs. The str'omaca are delimited by a dark camptacls. The cmidia and ascospores are hyalins. cylindroid to al lantoid, and one-eel led. Asci are short-stalked and ellipsoid and have an apical ring mostly visible as two strongly refractive bodies. The asci often becane free fran their attactnent and mymittheperitheciainaslimsmatrix. Thscaiidiaarsproducsdina simple, irregularly chambered pycnidial cavity which is formed either in the center of a perithscial strum or alone in a strana. The pycnidiun usuallyopensbymecsntral pore throughwhichccnidiasamde in long threads or cirri. Kern (29) recognized two species associated with Cytosporacankerofpeachbassdmdiffsrencss incolorandstapeof the strmnticdiscandascosporssizs. mhmmstranaticdisc is whitish or white and lenticular in slaps with ascosporss 8-14 x 1.5-3 u. mammdiscisgrayishbmtodarkbrmandinshapewith ascospores8-20x2—4u. nemidcessandshapeofthsconcepteclearxi ascosporesizerangsall servetodslimit speciesinthsgenus Isucostana (3,29). thforturntely L._._ mi; and ; Limits; are not readily distinguishable by cultural characteristics or anamorph morphology ad the tsleanorph is scarce or ephemeral. For these reasons plant pathologists are seldan able to positively identify the patlmgsns ad thus the pathology literature because difficult to interpret. Inkezic at al. (32) have suggested that the tm species are synonymous. both being derivable frail ascospors progeny of a single psrithscium. Hotvever. other raearchers recognize them as distinct, occuring in the geograrhic locals or m the tree (15.ad I... Spielman. personal cannmication) . The first mention of the parasitic nature of Lsucostana spp. on peach in the American literature was made in 1900 by Stewart at al. (38). Aderhold's reference in 1903 to attack of frost-weakened trees was one of the first recognitions of the importance of predispositional factors in host susceptibility to the disease (1). In 1916, Walton and Babcock (43) found that infections could be initiated by inoculating Ieucostamspp. ontreetrunks. largsrbraiches. adtwigsofpsach trees. In 1918. Mommbin (33) discovered that V_. leucostoma was able to invade healthy tissues ad form canksrs when inoculated onto dead or dyim tissue. Defago (1935) caducted an extensive investigation of Valseae parasitic on declining stone fruit trees (13). He concluded that tin degree of infection was governed by both virulence of the isolate ad host vigor. William (46.47) obtained macimum necrosis with inoculations made at leaf fall. Simmer inoculations were canparatively inocuous under certain conditims. William also reported on the characteristics ad roles played by the two species of y_a_l§§ responsible for peach calmer. Lazcostma spp. are mud-parasites (20.46.48) ad are most likely to cause infection diet: the trees are dormant (7.14). The fungus enters mds caused by low temperature winter injury to peach flower buds. twigs. ad bark (14.21.22.40). pruning (25.47). stmsceld (10). ad fruit moth ad borer injuries (10.42). than the fungus enters the wound it coloninsthsbarkadcenbial tissuesardxylem tissue inadvancs of visible canker margins (9.47). The develomt of the disease is associated with manerous stress factors including moisture stress (6). nutrient stress (sg. potassiun stress). ad high clay content in the soil (5.6). Once in the xylem the fungus may plug vessels or locally destroy the canbiun ad inpeds water flow causing wiltim ad defoliation (2.9.18). canker expansion is greatest Just prior to bud break mils host activity is slight ad temperatures are increasing ad approaching optimal for fungal growth (25.28.46). These pathogais are able to inhibit or overcans host- mediated nonspecific responses such as callus fonnatim. peridsm differentiation. and lignificatim in the bark and the xylem (8). Attempts to cartrol cytospora canker of peach using fungicides ins bear ineffective due to the deep penetration of the fungus in the host (23.35) ad the couplsxity of predisposing ad interacting factors (41). Prunim ad other cultural practices are often inadequate for satisfactory control (44) ad are often time canning. labor intensive. ad costly (41). Oytospora canker might be caitrolled biologically. Work minducedresistancestggests thsvirularcs ofthspathogencenbe radioed if the peach trees are inoculated previously with avirulent pathogera (10.24.27). The disease might also be controlled using hypovirulenca. Hypovirulence is cytoplasmical 1y tramissibls and results in attsrmatim of virulence of the pathogen (42). The most thorough studies an hypovirulence are those on the plant pathogen We mince) [Murr.] Barr. (sardothia mince (Murr.)). the causal agent of chestnut blight. Hypovirulence of g; Eitica has been correlated with the presence of double-straded RNA (dean!) mlecules (12.42). Hypovirulent strains of 9; mince were firstdiscoveredinmiropevheretheymrereducingthe impact of virulait straim of the pathogen (17). Grants ad Sauret (17) mutated that these hypovirulent strains were daninant when mixed in a tree with normal virulaxt isolates. This daninance was later fourd to be due to transmission of dsRNA molecules from hypovirulent to virulent isolates (42). 310109in control of chestnut blight has scoured naturally in Italy (34) ad in Michigan (16) ad the disease is being ccntrolled in Praise by artificial spread of similar strains (17). In all of these cases. the presence of hypcvirulent strains of 9; mince is correlated with the survival of the chestrrut trees in spite of infection (16.17). The purpose of this research vac to explore the pwsibilities of using hypovirulanoe as a means of biologically control ling Cytospora canlmr of peach. To detect hypovirulent isolates of Lancstana. a reliable virulence test was developed to screen for isolates with reduced virulence. Secadly. dsRNA has found associated with the loss of virulence in an isolate of 1:.‘ m; . 'mirdly. the vegetative ccupatibility system for k grsconii was amined to determine if the spread of hypovirulenoe would be possible within the fungal population. “These tests also gave us insight into sane aspects of the biology of the fungus ad epidaniolcgy of disease spread. 10. 11. 12. 13. LITERATURE CITED Anonymous. 1904. Aderhold. R. Uber das Kirschbaum—sterben am Rhein. seine Ursachen und seine Behadlung. Arb. K. Geandheitsamtes Biol. 3. No. 4: 309-363. 1903.Abstr. In Expt. Sta. Record 15:270. Banks. T. J. and A. W. Heltcn. 1974. gytosmra -induced changes in st- of Prams Ersica. Phytopathology 64: 899-901. Barr. M. E. 1978. me Diaporthales in North Merica. Mycol. Mem. No. 7. Cramer Pub.. Lehre. Germany. pp 146-147. Bertrad. P. F. ad H. English. 1976. Relwe and dispersal of ccnidia ad ascospores of Vales leucostcma. Phytopatholcgy 66:987-991. Bertrand. P. F.. H. English and R. M. Carlson. 1976. Relation of soil physical ad fertility properties to the occurence of Cytospora canker in Fraich prme orchards. Phytopathology 66:1321-1324. Bertrand. P. F.. H. English. K. Uriu and F. J. Schick. 1976. Late season water deficits and development of Wtospora canker in French prune. Phytopathology 66:1318—1320. Bertrad. P. F. ad H. miglish. 1976. Virulence ad seasonal activity of m leucostana ad Q; cincta in Praich prune trees in California. Plant Dis. Rep. 60: 106-110. 81008. A. R. 1984. Bourdry zone formation in peach bar): in response to wards ad m leucostana infection. Ca). J. Bot. 62:2814— 2821. Bims. A. R.. 1986. Ganparative anatany ad host response of two peach cultivars inoculated with Leuccstana cincta ad Leucostana mg. Phytopathology 76:905-912. Braun. J. W. ad A. W. Helton. 1971. Induced resistance to gytosmra in Prunus EEC—9&3.- Phytopatholcgy 61:685-687. Chiarapgn. L. 1960. Distribution ad a mode of spread of Cytospora canker in an orchard of the President Plum variety in California. Plant Dis. Rep. 44 (8):612-616. Day. P. R.. J. A. Dcdds. J. E. Elliston. R. A. Jaynes. and s. L. Ariagnosmkis. 1977. Double-straded MIA in Erdothia witica. Phytopathology 67 :1393-1396. nefago. G. 1935. (at certain Valseae von I-Icel'mel parasitic on dying- off stare fruit trees) Thesis. Boole Polytechnique Federals Zurich. 111p. (Abstr.. Rev. Appl. Mycol. 15 447. 1936). 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. Dhanvantari. B. N. 1978. Cold predisposition of dormant peach twigs to nodal cankers by Iaicostana spp. Phytopatholcgy 68:1779-1783. Dhanvantari. B. N. 1982. Relative importance of Leuccstcm cincta ad a perm in peramial canker of peach in southwatern Ohio. Can. J. Plant Path. 4(3):221-225. Fulbright. D. W.. W. H. Weidlich. K. 2. Haufler. C. S. Thomas. and C. P. Paul. 1983. Chestnut blight ad recovering Amrican chestnut trees in Michigan. Can. J. Bot. 61:3164—3171. Grazte. J.. S. Berthelay-Sauret. 1978. Biological control of chestnut blight in France. Proc. Am. Chestmit 3pm.. Morgantown. W. Va. pp 30-34. Hampson. M. 0.. and W. A. Sinclair. 1973. Xylem disfunction in peach caused by m leuccstus. Phytopathology 63:676- $1. Helton. A. W. and J. S. Moisey. 1955. Qytosmra damage in Idaho prune orchards. Plant Dis. Rep. 39:931-943. Heltcn. A. W. ad D. E. Kcnicek. 1961. Effects of selected m isolates frcm stone fruits on certain stone fruit varieties. Phytopathology 51:152-157. Beltm. A. W. 1961. Low temperature injury as a contributing factor in m invasion of plum trees. Plant Dis. Rep. 45:591- 597. Helton. A. W. 1961. Influence of location on invasion of dry-ice— killed tissues on Italian prune trees by naturally disseminated m fungi. Plait Dis. Rep. 45:918-920. Helton. A. W. and K. G. Rohrbach. 1967. Chemotherapy of Qytosmra canker disease in peach trees. Phytopatholcgy 57:442-446. Helton. A. W. and J. W. Braun. 1970. Relation of number of m infecticms on Prunus donatica to rate of expansion of individual cancers. Phytopathology 60:1700-1701. Hildebrad. E. G. 1947. Peramial canker ad the canker couple): in New York. with methods of cmtrol. Cornell Univ. Agr. Sta. Man. 276. 66m- Hoehnel. F. von. 1917. System der Diaportheen. Ber. Deut. Bot. Gas. 35:631—638. Hubert. 3. ad A. W. Helton. 1967. A trazslocated resistance phenanenon in Prams 3:333:12 irduced by initial infection with m cincta. Phytopathology 57:1094-1098. Jones. A. 0. ad N. S. Luepschen. 1971. Seasonal development of Cytospora cater on peach in Colorado. Plant Dis. Rep. 55:314-317. 29. 30. 31. 32. 36. 37. 41. 42. 43. Kern. H. 1955. Tammie studies in the genus Leucostara. Pap. Mich. Acad. Sci. Arts ad letters 40:9—22. Luepschen N. S.. K. G. Rohrbach. A. C. Jones. and L. E. Dickens. 1975. Susceptibility of peach cultivars to Cytospora canker under Colorado orcl'ard corditions. Hort. Scieme 10(1):76-77. Luepschax. N. S. 1981. Criteria for determining peach variety susceptibiliy to 01tospora canker. Fruit Var. J. 35:137-140. Lukezic. F. C.. J. E. Devay and H. English. 1965. Comparative physiology ad pathogenicity of Leucostoma pm; ad Rhodosticta mgr-cine. Phytopathology 55:511-518. McCubbin. W. A. 1918. Peach canker. Canada Dept. Agr. Bul. 37:1-20. . Mittenperghen. L. 1978. The presmt status of chestnut blight in Italy. Proc. Am. Chestnut Symp.. Morgantown. W. Va. pp 34-37. Palmiter. D. H.. ad K. D. Hickey. 1970. Relative resistance of 26 peach cultivars to bacterial spot ad Valsa canker. Plant Dis. Rep. 54 (5):395-399. Royse. D. J. and S. M. Rice 1978. Detection of Cytospora species in twig elements of peach ad its relation to the incidence of paramdal canker. Phytopatholcgy 68:663-667. Spielman. L. J. 1985. Anonograph of Valsa a: hardcods in North America. Can J. Bot. 63:1355-1378. . Stewart. F. C.. F. M. Rolfe. ard F. H. Hall. 1900. A fruit disease survey of western New York in 1900. N.Y. Agr. Exp. Sta. Bul. 191 : 291-331 . Sutton. B. C. 1980. The Coelonycetes. Counmwealth Mycol Inst.. New. airrey. Bylad. pp 579-580. Tekauz. A. ad 2. A. Patrick. 1974. The role of twig infections on the incidmce of perenial canker of peach. Phytopatholcgy 64:683- 688. Traquair. J. A. 1984. Potential for biological control of m canker m stare fruits. Stone Fruit tree Decline Workshop Proc. Oct. 30— Nov. 1. Appalacian Fruit Res. Sta.. Kearneysville. W. Va. VanAlfen. N. R.. R. A. Jaynes. S. L. Anagnostakis. and P. R. Day. 1975. Chestmtt blight: Biological control by transnissible hypovirulenoe in Erdothia gagitica. Science 189:890-891. Walton. R. C. ad D. C. Babcock. 1916. The parasitism of Valsa leucostana. Phytopathology 6:112-113. 4‘. 46. 47. waver. G. M. 1963. A relationship betweai the rate of leaf abscission ad perennial canker in peach varieties. Can. J. Plant Sci. 43:365-369. Wensley. R. N. 1964. Occurerde ad pathogenicity of Valsa (may species ad other fungi associated with peach canker in Southern Ontario. Can. J. Bot. 42:841-857. Willison. R. S. 1936. Peach canker investigations II. Infection studies. Can. J. of Res. 14:27-44. Willison. R. S. 1937. Peach canker investigations III. Further notes on incidence. contribiting factors. ard related pham Car. J. Res. 15:324-339. Wysong. D. S.. and L. E. Dickens. 1962. Variation in virulence of Valsa leuccstcna. Plant Dis. Rep. 46:274-276. SMION I ‘lhe Association of Double-Stranded RNA with Low Virulence and Loss of Sporulation in an Isolate of Leuccstana pgrsconii fran Peach nematimofme-etramdMthhImVirulanoead InsofSporulatiminarIaolateofLeuoostc-amflmeeech An isolate of m m; (14.4A) with low virulence. abnormal culture morphology. ad an inability to produce pycnidia when grumonculturemediawas fourd to ccntainninesegments of double straded RNA (dsRNA) when nucleic acid extracts fron mycelium were mined by polyacrylanide gel electrophoresis. Sane dsRNA segments were eliminated when hyptnl tips of 14.4A were removed ad subcultured. The formatim ad regeneratim of protoplasts ad the reisolation of 14.4A frail susceptible plant tissue also led to the loss of dsRNA. Specific dam seguarbs were more easily eliminated than others. Partially cured strains were more virulent than 14.4A in apple fruit virulaice assays but comparable in virulence to the uncured 14.4A in peach tree assays. Curedstrains. inmichdsRNAvasnot detectable. mm through protoplast regeneration ad selection for conidia production. The dsRNA- free cultm'es grew similar to isolates considered mrmal ad dunonstrated increased virulence on peach trees what ccupared with fully virulait isolates. Four detectable segmaxts of dsRNA were transferred by hyphal autos-sis frcn 14.4A to a genetically marked strain. True results irdicate tint 14.4A may be infected with tm distinct dsRNA viruses. Cytospora caxker of peach is cased by Leucostm cincta (Pers.:Fr.) 10 11 Hoehn. [W W m (Sacc.) Hoehn.) ad Leucostoma persoonii (Nits.) Hoehn. [anmorph= W leucostona (Pers.) than] Symptans of cytospora canker include cambial tissue necrosis ad perennial cankers which are occasionally acccnpanied by copious gun sedation. Infected branches wilt ad die back fron girdling ad plugging of the xylan vessels (4.18.20). Fungicides have not been effective in ccntrcllim the disease. The isolate of k M med in this study is dramatically reduced in virulence. has an almormal culture morphology ad contains double—straded RNA (dsRNA) ad virus-like particles. Although the vast nnjority of mycoviruses apparently have no deleterious effect on their host (17). sane mycoviruses adversely affect the virulence of their host. This suggests tint sane plant pathogenic fungi might be control led with viral infectian (23). Biological cmtrol of Cytospora canker is an appealing concept. Reduced virulence or hypovirulence in some pathogenic fungi is associated with the presence of double-straded MIA (dsRNA) ad has been associated with biological control of plant diseases (8.9.29). Hypcvirulence has best been described in the plant pathogen Mi; mince [Murr.] Barr. (BErdothia mitica (Murr.)) (2.29). which causes a peramial canker disme similar to Cytospora canker. Thepurposeofthisstudywastodetemirn ifthemenceofdsm could be directly correlated with the loss of virulence ad other m1 characteristics of L_._ m. This was acconplished by eliminating the dsRNA fran the fungus ad determining whether or not virulence was regained. In addition. we attemted transmission of the 12 dsRNA to non-infected strains. me All) umms Isolates Isolates used in virulence tests were recovered from cankers on peach trees grow: in Michigan. West Virginia. california. Pennsylvazia. ad North carolina (Tables 1-2). Cultures were unintained on Leonian's Malt Agar (LMA) (24) ad stored on peach bark agar (25) at 4 C with annual subculturing. Isolates were identified as L; m; or L_._ ganta; Wed on colony color. size of pycnidia. whether or not the colony margin was lobate or uniforually radial (31) ad by the presence or absence of growth at 37 C (21). The morphology of each isolate was also compared to the morphology of single ascospore isolates of L._ m. Virulence Last; The virulence of isolates of Leucostoma spp. was evaluated on 8-yr- old peach trees [Pmig grsica (L.) Batsch 'Garnet Beauty']. ad on apple fruit [_v_1_a_l._\:g danestica L. 'Golden Delicious']. Peach trees were inoculated in October of 1985 fol lowing the method of Scorn ad Pusey (26). 'mo-yr-old peach braxches measuring 17 m in diameter were wounded to the xylem with an empty. had-held stapling gun. The wourd area was sprayedfor 5 secwithacamnercial aerosol freezingproduct (100% dichlorofluorcmetrnne. Chemtrmics. Inc.. Hauppauge. NY) ad irnculated witha 5 mmycelial pltigtakenfruntrnmarginofas-day-oldculture on LMA. Inoculatian were wrapped with parafilm to prevent dessication. Caiker lengths were measured the fol lowing May after stripping off the bark to reveal the length of the necrotic area distal to the inoculation point. 13 'l‘he virulence test using apple fruit was performed by removing a 9 nm dianeter x 4 nm deep plug of tissue with a sterile cork borer. Mycelial pltm were placed mycelium side down in contact with the wcurded apple tissue (15). The site of inoculation was covered with tape ad the apples were placed at roan tanperature in open plastic bags. The width of each lesion was unsecured after 10 days. The presence of Leuoostana spp. in inoculated tissues was verified by reisolaticn fron the lesions. Wood pieces were surface sterilized by soaking in a 10% solution of a cannercial laurdry bleach (5.253 NaOCl) for 2-3 min and blotted dry with sterile paper towels. Apple tissue was surface sterilized by swabbing the lesion margin with 95Xethalol. Tissuewasaccisedfrcmtlnnnrginof the lesicnsad embedded in either LMA or potato dextrose agar (Difco. PDA) ad incubated at noon temperature. Isolation 9; ggrg DsRNA was attracted using modifications (16) of the procedures of Day at a}, (9) ad of Dodis (10). Nucleic acid samples were layered on 596 polyacrylamide slab gels ad electrophoresed at 40 mA for 12 hr. Molecular weight estimates of dam were calculated fran standard curves plottirg electrophoretic mobility verses the log of the mlecular weights usim coelectrophoresed stardards of reovirus serotype 3 (27). C_. mince (em) (16). ard Bipglaris Egg (acct 32450). 93.1.13! Lila!“ Five procedures ware used to obtain subcultures of isolate 14.4A with coldly mormology canidered typical for L_. grsocnii isolates. First. myoelial plugs of 14.4A were transferred to LMA media awarded with "\ 14 either 0.25 to 50 m of cyclohexamide (Sigma Chemical Co.. St. Louis. Mo) per milliliter (15) or 25 to 50 ug ribavirin (Sigma Chemical Co.. St. Louis. Mo) per milliliter. After 2 wk. agar plugs were transferred frunthemarginsof thecoloniestofreshPDA. Secad. 100hyphal tips were taken tron four 2-day-old colonies of 14.4A grown on LMA. After 2 daysgrowth. ahyphal tipwastransferredfraneachresultingcolomrad the process was continued ten consecutive times at 2-day intervals. The resulting 100 colaiies ware grown for 4 wk (the length of time for typical cultures to sporulate) before evaluation. Third. reisolations werenadefrcmthemarginofanexpadinglesioninapplefruit inoculated with 14.4A. Fourth. 15. 4-day old cultures of 14.4A were transferred to LMA. ad the plates were incubated in the dark at 33. 36. ad 38 C for 2 wk. Mycelial transfers of axy living colonies were made to LMA ad the plates ware incubated at room tanperature with anbient laboratory lighting. Fifth. protoplasts were isolated ad regenerated. To obtain protoplasts. mycelia were grown 36 hr in 500 ml flasks cantaining 100 ml of a calplete broth medium without glucose (9). Myceliawere collectedwithabuchner funnel andincubatedahat 330 in a solutim curtaining 1 ml of 1 M sodium phosphate buffer at pH 5.8 ad 9 ml of 1 M mdtol amaded with a filter sterilized 1 ml solution cmtaining 10 m of Novozyme 234 (Novo Laboratory. Inc.. Wilton. 0N) ad 1 m chitinase fran Serratia marcescens (Signs). The protoplasts were separated fron large hyptnl fragments by filtering the solution through four layers of sterile cheesecloth. then through a 15 In mesh nylon filter (Tetko. Inc.. Elmsford. NY) ad collecting the filtrate in a centrifuge tube. Protoplasts were centrifuged at 2000 run for5min.rea:spendedin1Munnnitol. recentrifuged. resuspardedin2 15 ml of 1 M marmitol ad layered onto 4 ml of 1 M sucrose. The solution was centrifuged at 1000 rpn for 5 min. The protoplasts. located in an opaque interface between the two solutions (19). were removed ad added to 2 ml of 1 M mannitol. The layering procedure was repeated until a highly purified protoplast preparation was obtained. The final protoplast pellet was diluted in 1 M mannitol to 102 protoplasts per milliliter; 1 ml was pipetted onto 1 M mannitol in couplets medium agar (9). In 48-72 h after plating. regenerated protoplasts were irdividual ly transferred to LMA. In each of 20 experiments. 50-100 protoplasts shaving hyphal initiation ware irdividual ly transferred. With each of the five procedures. subcultures exhibiting a more normal cultural morphology or showing sporulation were tested for dsRNA by polyacrylamide gel electrophoresis. Mil-resistant mtants Balcmyl-resistant mutants were isolated by collecting pycnidia from 4-wk-old colonies of dsRNA-free 14.4A subcultures ad grinding than in sterile water with a mortar ad pestle. The slurry was passed twice througha5umfilter so that only the conidia inwater rennined. Oneml of a solution of 106 conidia per milliliter was pipetted onto LMA amended with 4 ug bermyl per milliliter. The conidia were irradiated for 1.5 min with a 254 an ultraviolet light (uv) from a UVSL-58 Mineralight lamp with an output of 1.25 x 104 ergs can-2 sec-1. This exposure killed approximately 908 of the conidia. Irradiated plates were stored in the dark until conidia germinated ad formed colonies. Six bamyl-resistant mutants were tested to verify lack of dsRNA prior to transmissim experiments. 16 _____Transfer at 28319. To transfer dsRNA from isolate 14.4A to benanyl-resistant (BenR) isolates, mycelial plugs of 14.411 ad a 3is isolate were placed side by side in a petri dish containing LMA or Difoo oatmeal agar. Plates were allowed to incubate until the hyphae of the two isolates made contact. Phys were removed from various areas on the plate ad subcultured on LMA amended with 4 ug bermyl per milliliter. Isolates that contained dsMadgrewonthebamyl emerdedmediawerewsmedtotavebeen converted by isolate 14.4A. Isolates Isolate 14.4A, designated as g; arsoonii, had an abnormal culture morphology includim lysing hymal tips ad did not produce pycnidia or caddie in culture or inoculated stalls. All other isolates tested had a manual or expected morphology of Leucostana spp. (31) ard produced spores in culture. Virulaice 353s; Inoculations performed on apple fruit irdicated that 14.4A was the least virulent of the isolates tested (Table 2). In ordard tests, 14.4A was me of the least virulalt as evidamd by the stall cankers it cured in relation to most other isolates tested (Table 1). It's ranking in Table 1 is higher tl'm expected due to the contamination of 14.411 inoculation sites with wild type strains. while most of the isolates tested had variable virulence fran one test to another (Table 1 ad 2; isolate 14.1 ad 8.2), isolate 14.4A was consistently very low in all virulence ”says (unplblished data). l7 _s_c_re_e_ru_m _____isolates :91: 23.119; The only field isolates in which dsRNA was detected by electrophoresis ware 14.4A ad 9.2 (Table 1 ad 2). Nine segments of dsRNA were visible in nucleic acid extracts fran 14.4A subjected to polyacrylamide gel electrophoresis (Fig 1). Segments B, 0, ad D were consistently associated with 14.4A. Segments A ad F were usually present bit stained nuch fainter with ethidium branide than segments B, 0 ad D after polyacrylamide gel electrophoresis. Segments E, G, H, ad I appeared inconsistently. Segments B, 0, ad D ranged in size from a molecular weight 0‘ 2°90 1: 106 to 2.05 x 106. The segments found in 14.411 were identified as dsRNA based on their resistalce to RNase at high ionic stralgth (0.3 M NaCl), sanitivity to mass at low ionic strength (1120), and resistance to DNase tramps. Isolate 9.2 cmsistaltly yielded three distinct dsRNA segments in every gel observed (Fig 1). Egg; 9; isolate M All partially cured isolates were irdistinguishable fran one another regardless of the curing method from which they were derived. The partially cured isolates were darker in pigment tran 14.4A, the hyphae waremreconpactintextureadm longer lysedat thetips, ardi'onned fruitim bodies which lacked conidia (Fig 2). 0f the one-twdred partially cured strains tested, all retained segments A ad P (Fig 1) but lost segments B, C, D, E, G, 11, ad I. The partially cured strains were consistaltly more virulent than 14.4A in apple fruit assays (Table 2), however, virulence still could not be considered normal since virulalce in peach tree assays was not significantly greater than isolate 14.4A (Table 3). 18 Table 1. Virulence of isolates of Iaucostoma spp. on 10 trees of Garnet Beautypeachranhed indecreasingorderbycaniaer lengthadthe association of double-straded RNA with each isolate State of Isolate Canker lalgth (cm)a mb originc MI MR-l 11.00 Ad - m 8.2 9.98 A8 - CA F—46 9.83 A80 - CA Ctr-5 8.40 BGD - MI 010.8 7.78 sons - MI 119.5 7.64 BCDEF - WV 0-3-1 7.50 CDEEI - WV C-S-20 7.24 DEFG - MI 6-3 7.08 DEE'GH - MI F-4 6.59 DEFGHI - CA Ma—4 6.49 DEFGHIJ - WV C—MI-5 6.48 DEFGHIJ - PA P—i 6.06 DEFGHIJK - W C—jm—18 5.94 EFGHIJK - MI 10.9 5.46 EFGHIJK - no 9.2 5.34 FGHIJK + m 14.1 5.11 m - CA P-45 4.92 GHIJK - m 14,“;9 4.81 HIJK 4- MI 117.13 4.79 HIJK - MI 36.15 4.75 HIJK - MI 119.11 4.71 IJK - MI 11.11 4.18 JK - CA I-80 4.02 K - caltrol 0.5 L a/Inoculations ware nade by placing myceliun embedded in LMA into words made an had-held s 1 ol owed reeze jury. sitesbgf indcumgtim were wratélpeduign $1 11111. Sevlgl Jmonths 1{later triage tarkwm removedadcanlner lengths (distal franthepoint of inoculation) were measured. Meals are based (11 ten replications (10 trees, 1 branch/tree). b/-,no dsRNA present; +,dsRNA present as determined by gel electrophoresis c/Initials of state where isolates were recovered d/Means fol lowed by the same letters are not significantly different (P-.05) e/Higherrantingduetothecontaminatimofthreeoftheten replications. The mean for the seven mtaninated reps =- 1.98. 19 Table 2. Assay of virulence of isolates of Legggsggmg pgggggnii on apple fruit State of Isolate Lesion DsRNAP originc width (cm)a NC 14.1 8.33 Ad - MI T-5 7.77 AB - NC 9.2 6.80 BC + MI 10.8 6.67 BC - MI 011.7 6.58 BC - MI R-l 6.53 BC - NC 8.2 6.48 C - MI RD-l 6.08 C - MI M-S 5.72 C - NC 14.4A 1.52 D + control 0.5 E a/ Inoculations were made by placing mycelial plugs into wounds made by removing apple tissue with a cork borer. The site of inoculation was covered with tape and the apples were placed at room temperature in open plastic bags. Lesions were measured after 10 days. Each value is the mean of ten replications. b/-,no dsRNA present; +,dsRNA present as determined by gel electrophoresis c/Initials of state where isolates were recovered d/Means not followed by same letters are significantly different by the least significant difference test (LSD), P-0.05 20 Figure 1. Photograph of a1 ethidium branide stained 596 polyacrylamide gel showing specified segments of dsRNA extracted from Ieucostana spp. a) isolate 14.4A, b) partially cured 14.4A, ad c) isolate 9.2. 21 Partially cured subcultures of 14.4A were readily obtained by subculturirg hyphal tips, reisolation of 14.4A fran apple fruit or protoplast regeneration. The percentage of subcultures with the partially cured phenotype (method, 14.4A phenMype: partially cured phemtype) were: hyphal tipping, 980:20= 9896; apple fruit, 38:12= 7696; protoplast regenerates, 96:# 9696; treatments of 25 ug/ml Ribavirin. 2:25- 7.42;; ad cyclohetamide treatment, 0:0= 0%. Higher concentrations of Ribavirin did not yield a greater percentage of partially cured strains. Unlike the other field isolates tested, 14.4A did not grow on PDA containing concentrations of 0.25 to 5.0 ug/ml cyclohexamide. Isolates with the partially cured morphology were also readily obtained fran older cultures that had been frequently subcultured onto laboratory media. Isolate 14.4A grew only at 33 0 ad none of the subcultures were cured of dsRNA. The najority of the colonies regenerated fran protoplasts of 14.4A exhibited the herding pattern ard cultural phenotype of partially cured strains: however, three protoplast colonies (103, 105, ad 107) were blackish grey in pigmentation with a more lobate colony margin than 14.4A or the partially cured strains (Fig 2). The protoplast regenerated cultures formed pycnidia ad conidia in culture, resembled field isolates in colony morphology, ad exhibited virulalce equivalent to field isolates in peach tree virulence assays (Table 3). No dsRNA was detected in these isolates in 15 attanpts by electrophoresis. Transmission miments The 381“ nnltalts were derived fran the dsRNA cured isolates 105 and 107. Since 105 ad 107 originated fran protoplasts of 14.4A, hyphal 22 Table 3. Comparison of virulence of Leugosggma pegsgggii wild-type isolates, 14.4A, and subcultures of 14.4A on branches (17 mm diam.) on peach trees Mean canker Isolate length (cm)8 DsRNAb Cured 14.4A-105 4.67 Ac - Cured 14.4A-107 4.48 AB - Cured 14.4A-103 4.33 AB - Wild-type MTS 4.11 AB - Wild-type MR-l 3.65 AB - Benomyl resistant cured 14.4A 3.80 AB - Partially cured 14.4A 1.13 C + 14.4A 0.72 C + a/Inoculations were made by placing mycelium embedded in LMA into wounds made by an empty hand-held stapling gun followed by freeze injury. The sites of inoculation were wrapped in parafilm. Seven months later the bark was removed and canker lengths were measured. Means are based on ten replications (10 trees, 1 branch/tree). b/-,no dsRNA present; +,dsRNA present as determined by gel electrophoresis c/Means followed by same letters are not significantly different by the least significant difference test (LSD), P-0.05. 23 Figure 2. Characteristic cultural morphology of 30—day—old dsRNA infected ard cured strains of Leucostana Ersoonii on LMA a) strain 14.4A with light pigmentaticn, less canpact mycelium, ad no pyulidia, b) "partially cured" strain with darker pigmentation, canpact mycelium, ad pycnidial initials, ad c) "cured" strain with sporulating pycnidia, ad lobate colony nargins characteristic of most isolates of I._._ argon—iii from cancers on peach. 24 fusion should occur readily betweena BenR marked mutant ad 14,411,, The BenR mutants had no detectable levels of dsRNA when evaluated by gel electrophoresis ad were irdistinguishable in morphology ad virulence flan 105 ad 107 prior to exposure to uv light. The hyrhae of the two inoculum plugs appeared to intermingle freely when the fusion plates were preinoculated with 14.4A two days prior to inoculation with the mutant. If the fusion plate was not preinoculated with 14.4A, the untant overgrew the plate ad restricted 14.4A growth. No sectors with altered morphology were omerved at the interface between the two isolates (1). Tralsmission by hyphal alastanosis of dsRNA fran 14.4A to a BenR mutant was successful, although the efficiency of tramissim was very low. DsRNA segments A, B, 0, ad B were found in the converted mtant after gel electrophoresis but they stained faint irdicating a low titer. The converted mutant was obtained after tranferrim a partial of mycelia: frun the m of interaction betweal the paired strains to a plate of LMA with 4 ug bamyl/ml. After incubation for 3 win the culture produced a hyphal sector. Subcultures frmthissectorcaitaineddsRNAadgrawmIMAaumadedwithbamyl. To verify tlat the sector was not a spontaneous mtant of 14.4A, 80 plates of [MA amended with 4 ug/ml benanyl were each inoculated with four mycelial plugs of 14.411. No colony growth or hyphal sectors were produced after 2 umths incubation. The infected mutant was morphologically distinct frcn the original mutant ard tron 14.4A. The coldly characteristics of the dsRNA containing balanyl resistant mtant have been altered frun that of the dsRNA-free mtant. The mycelium was less dense ad tad a brown pigmentation instead of the blackish grey color of the original reliant. The strain retained the ability to form 25 pycnidia. In preliminary virulence tests in apple fruit the infected mtalt is less virulent than the dsRNA-free mutant (P =- 0.05). “a! Most mycoviruses are aswnptanatic or latent in their hosts (17). This does not appear to be the case with _IL._ grsoonii isolate 14.4A. We have found a correlation between the diseased state of 14.4A ad the prwence of dsRNA. This is illustrated by charge in growth, sporulation, ad increased virulence when dsRNA is systenatical ly eliminated fran 14.4A. Although it is difficult to completely cure 14.4A, some segments of damn can be readily eliminated. when the partially cured phenotype of 14.4Aisobtained, dsRNAsegmentsB, C, adDare lost. Thesethree sewn-its were always lost in calcert. This suggests an interdependence ofall threesegments, ordeperdenceofoneormresegmentsmalother. In canperisal to the easily eliminated B, C, D segnents of dsRNA fourd in 14.4A, semits A ad F of the partially cured strains were difficult to eliminate. 'me phenotypic charges that accomany the loss of the B, C, D segments are very characteristic ad constant, incltdirg the absence of lysirg tips, mre canpact hyphal growth, increased virulence, ad fomtial of sterile pycnidial initials. These apparalt fruit bodies reveal only .. conidiogalous layer in electron microscopy (28). The menotypic charges correlated with loss of segnents A ad F are also constalt ad include : ofintertwinedhypheeadnofertile an even greater increase in virulence, fornation of conidia, increased hyphal pignentaticn ad a more lobate (more normal) colony on PDA. This sunsets that 14.4A might have a mixed infection of at least two different dsRNA viruses. 26 Mixedviral infection isoomrinfurgiadhasbeenreportedingg mitica (11,12) ad 9; ggami__x_l__is (6). DsRNA segments A ad F appear to be quite stable in the furgus which is still reduced in virulence ad calidiation when tme segments are present. These partially cured strains are less debilitated to hypinl growth than the conbined infection seen in 14.4A. Isolate 14.4A curtains the dsRNA of the less debilitatirg infection seal in the partially cured strains as well as nary additionl dsRNA seglents B, C, D, E, G, H, ad I. The virus-like particles (VIP's) of 14.4A are not present in the prtially cured strains (Snyder _e_t_ 9L, unpublished). The additiornl dsRNA segnarts may bethegenoneoftheVLP's. 'mepartiallycuredstrainsnnycontaina virus tint has lost the capacity to encepsidate the dsRNA. The unalcapsidated virus apparently replicates its game effectively in the rapidly dividirg hyphal tip cells raking it difficult to eliminate. In cortrast, tin virus tint form VIP's may not marble the particles fast enough to keep pace with the growth of hyphal tip cells. Further studies would be required to determine the degree of relatedness, if aly, of the two «bilitatixg waits. So far, virus-like particles inve not been associated with tramissible cytoplasmic factors tint diminish vigor in cultural growth and host patiggenicity in plant pathogenic furgi (22). In C_. Mitica the dsRNA is either free in the cytoplasm or associated with membranous vesicles (14). No VIP's inve bear isolated or detected in the dsRNA infected isolate of B; £1333, which has been reported involved in Rhizoctcnia decline (8). VIP's inve been fourd infi_._gami_x_i_ig var. tritici, but they were not generally a cause for the reduction in pathogenicity (7). 27 The concentration of VLP's in the cytoplasm of 14.4A is unusually high in several thin sections observed with transmission electron microscopy (28) The particles appear side-by—side, filling the cytoplasn between organel ice. The presence of the VLP's is associated with lysis of hyphal tips. It is therefore not surprising tint infected hyphae are less capable of causing disease. Assessing the role of the VLP's in reducing virulance is difficult. No VLP's can be detected in the partially cured strains, and the strains do not appear as debilitated as 14.4A. In laboratory tests using apple fruit, the isolates with no detectable VIP's are consismntly more virulent. In peach trees, braver, both 14.4A ad partially cured isolates were very low in virulence. Thus, elimination of detectable particles and associated dsRNA segnents still results in lav virulence and no sporulation. The dsRNA segments renninim in the partially cured strains apparently are more ilportant tinntheVIP's indetenninimvirulenceanthehost. In 9; gallium, where damn is cytoplasmically borne, the transmission of the dsRNA by hyphal anstanosis from hypovirulent to vegetatively cmgntible virulent isolates occurs readily (3). Additianl ly, trananissian of dsRNA sanetinns occurs batman isolates that are not vegetatively canpatible (2,29). It is surprising that detecting dsRNA transfer between 14.4A ad a cured 14.4A BanR mutant amearstobera'ebacansethisisconsideradaselfingresponseandmt fusian between two different isolates. (he explanation is tint the lysing hyflnal tips of 14.4A might hinder hypinl anstanosis with anotiner isolate. Hypnl tip lysis may unite isolate 14.4A too debilitated to use for transmission of dsRNA. The discovery of isolate 14.4A is an irdicatian tint hypovirulent 28 strains exists in natural Leucostann populations. The partially cured strains may be better for potential biologin cmtrol of Cytospora canker. These strains still inve very low virulence but lack the lysis of inyfinl tips. Thus, hypinl anstandsis ad cytoplasmic transfer of the dsRNA may occur more readily with the partially cured strains. These strains do not sporulate, invever, ad rntural spread in the orcinrd may prove difficult. Isolate 9.2 was the only other isolate in these studies found to contain dam The dsRNA did not affect tine virulence or morphology of isolate 9.2. The mere presence of dsRNA in 12._._ m; does not necessarily indicate tint an isolate will be hypovirulent. Isolate 9.2 was found to carry dsRNA molecules representing different sizes than those dsRNA molecules in 14.4A. This indicates tint specific molecules my canfer specific Mtypic changes in the host fnmgus. This my be diatothe lackof specificgenesanthedsmmtint intarferawith fungal host metabolism the tolerance of the fungal host to the virus infection, or attemntion of the virus. Even in the g agitica system, a few strains inve bean found vinere dsRNA does not correlate with hypovirulence in the infected strains. Time strains are nearly as pathogenic as the virulent isolate (13,30). 10. 11. 12. 29 mm Arngnostakis, S. I... ad Day, P. 1979. Hypovirulennce conversion in Endothia wince. Phytopathology 69:1226-1229. Anagnostakis, S. I... 1978. The American chestnut: New hope for a fallen giant. Conn. Agric. Exp. Stn. Bull. 777, New Haven, CT. 9 m. Anagnostakis, S. I.., and Waggoner, P. E. 1981. Hypovirulence, vegetative incanpatibility, ad growth of ankers of chestrmt bligint. Phytomthology 71:1198-1202. Banko, T. J., and Helton, A. W. 1974. Cytospora induced changes in stems of Prunus pgrsica. Phytopathology 64:899-901. Buck, K. W. 1979. Replication of double-straded RNA mycovirusas. in: Virmes an Plaanids in Fungi, P. A. Lenka, ed., Marcel Dekker, Inc., New York. pp 94-151. Buck, K. W., Almond, M. R., McFadden, J. J. P., Romance, M., and Ravlinson, C. J. 1981. Properties of thirteen viruses ad virus variants obtained fran eight isolates of the wheat take-all mucus, W M var. tritici. J. Gen. Virol. 53:235—245. Buck, K. W. 1986. Viruses of the meat take-all fungus, W M var- ____tr1tici- In Fungal Virologv- an overview in Fungal Virology, CRC Press, Bonn inton, Fla. pp 221- 236. ‘ Castanho, 3., Butler, E. 3., and Shepherd, R. J. 1978. The association of double-straded RNA with Rhizoctonia decline. Phytopthology 68:1515—1519. Day, P. R.. Dodds, J. A., Elliston, J. 3., Jaynes. R. A., ad Anagnostakis, S. L. 1977. Double-stranded WA in Endothia mgitica. Phytopathology 67:1393-1396. Dodis, J. A. 1970. Revised estimates of the molecular weights of dsRNA segments in hypovirulennt strainns of Endothia witha. Phytopathology 70:1217-1220. Elliston, J. B. 1985. Preliminary evidennce for tm debilitating cytoplasmic wants in a strain of Endothia pgr_a_§1tica frann western Micinigan. Phytopathology 75:170-173. Elliston, J. E. 1985. Further evidence for two cytoplasnic hypovirulence agents in a strain of Erdothia Eagitica fran vastern Michigan. Phytopathology 75:1405-1413. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 30 Elliston, J. E. 1985. Characteristics of dsRNA-free and dsRNA- conntaining strains of Endothia witica in relation to hypovirulance. Phytopathology 75:151-158. Ellzey, J. T., Hammons, T. I... and Cooper, M. O. 1986. An ultrastructural study of unanbrane—bounded particles witinin hypovirula'nt strainns of Erdothia W) wince. Mycologia 78(2):313-315. Fulbright, D. W. 1984. Effect of eliminnting dsRNA in hypovirulant Endothia pga_sitica. Phytopathology 74:722-724. Fulbright, D. W., Weidlich, W. R.. Haufler, K. 2., Thomas, C. 3., ad Pail, C. P. 1983. Chestnut blight ad recovering American chestnut trees in Michigan. Can. J. Bot. 61(12) 3164-3171. Ghabrial, S. A. 1980. Effects of fungal viruses on tineir hosts. Annu. Rev. Pinytopatinol. 18:441-461. Mampson, M. C., and Sinclair, W. A. 1973. Leucostonna canker of peach: symptms in relation to vater supply. Plant Dis. Rep. 58(11) :971-973. flashiba, T. , ad Yanada, M. 1982. For-nation ad purification of protoplasts frmn muzoctania solani. Phytopathology 72:849-853. Halton, A. W., and Randall, H. 1975. Canbial gnmlnsis in Prunnus danestica infected with m cincta. Plant Dis. Rep. 59:340- 344. Hildebrad, E. M. 1947. Pa'annialpeachcankeradthecanker couple): in New York, with methods of control. Cornell Exp. Sta. Man. 276. pp 5-66. Hollinas, M. 1978. Mycoviruses- virnnes tint infect fungi. Advances in Virus Research 22:2-53. Hollirgs, M. 1982. Mycovirusas ad plant patinology. Plant Dis. 66:1106-1112. Damian, L. H. 1923. The physiology of peritinecial ad pycnidial formation in Valsa leucostcnna. Phytopatinology 13:257-272. Royse, D. J., and Rise, 8. M. 1978. The influence of fungi isolated from peach twigs an pathoganicity of Mr; cinncta. Phytopathology 68:603-607. Scam, R., ad Pusey, P. L. 1984. A wound-freezing inoculatian technique for evaluating resistance to m lauoostcma in young peach trees. Phytopathology 74:569-572. Shatkin, A. J., Sipe, J. D., and Loh, P. 1968. Separation of ten reovirus game segnants by polyacrylanide gel electrophoresis. J. Virology 2:986-991 . 31 28. Snyder, B. A., Adams, 6. C., and Fulbright, D. W. 1986. Association of a virus-like particle with a diseased isolate of L_. cincta the causal agant of Wtospora canker of peach. (Abstr.) Phytopathology 76:1126. 29. Van Alfan, N. K. 1982. Biology and potential for disease conntrol of hypovirulence of Endothia mince. Annm. Rev. Pinytopathol. 20:349-362. 30. Van Alfen, N.K. 1986. Hypovirulence of Erdothia W) flitica ad Rinizoctonia solani In M Virolggy, Buck, K. W. Ed., CRC Press, Boca Raton, Fla., 285 pp. Chap. 4. 31. Willison, R. S. 1936. Peach canker investigtions II. Infection studies. Can. J. Res. 14:27-44. SMION II Vegetative Canpatibility in Leucostana pgrsoonii Vegtative Cuntibility in neucostm m Isolates of Ieucostmn mi; were paired on various media to determinn a suitable median for differentiating vegetative canpatibility (vc) groupings. Vegetative incanpatibility was evident only on oatmeal marasdark linnsadpycnidiaformingalongtin linnof mycelial contact betwean expanding colonies. Ascospore colonies derived from a single paritinciun segregated into several vc groups indicating that the fungus outcrosses and is most likely heterotinllic with several alleles cantrolling vegetative culpability. Isolates of g mi; fran cankersanpeachtneeswithinasingleorcinndclusteredintommnrous vcgroups. Isolatesfronseveral caninemwithinonn treeadanong closely spend trees usually differed in vc grouping. Spatial clustering of vcgroupsintinorcinrdwerenotaggregated. Conidiafranasingle pycnidiunnwere inanvc group. Tin frequancyadspatial Wt of vc groups in an orcinrd indicates tint ascospores (or air-blown conidia) could play a major role in disaanination and in initiatian of nan innfectians of peach. Cytosporacaniuerofpaachisanimportantdiseaseaffectingtin lagavity ad productivity of peach trees [Prams pgrsica (L.) Batsch] inmst peachgrmingregians of tinUnited States. Peach trees are susceptible to tvvo fungal species, Ieucostana semi (Nits.) Hoehn. 32 33 [W W leucostann (Pers.) Hoehn.) ad Ieucostcnn m (Pers.:Fr.) Hoehn. [annnorpiF Waggon-a m]. Synptons of the disease may include perennnial cankers on the laterals, scaffold branncines, ad the trunkof thetree, adtwigadbranchdieback (17). The corresponding reduction in tree vigor may result in tree death (22). Currantly, tinre are no effective chemical or cultural controls for peach canker. A potantial biological conntrol for Cytospora canker is through the nse of hypovirulance. Hypovirulant strains of fungi cantain cytoplasnical ly transmissible determinants winich reduce the pathoganicity of virulant wild-type strains (23). Double-stadad RNA (dsRNA) has bean assumed to be responsible for tin hypovirulane ad natural recovery phanman of cinatnut blight caused by W witica (Murr.) Barr (11). IowvirulanceinsbaanassociatedwithtinpresanceofdsRNAinan isolate of L_. pgrsoanii (13). The potantial for biological control of altosporacanioer of paachusing hypovirulancennybedepadanton transnissian of the dsRNA to virulant Iancostcnn isolates through hyfinal anstannosis. Separating isolates of Q; m; into vegetatively emptible (vc) groups mid aid studies of transmissian of dsRNA betwean tvn isolates. Identifying the freqnuncy of differant vc groups innnturevnuldassist inascartaining tin potantial spread of the dsRNA within tin populatian of the pathogan. Thestndyofti'nfrequancyof vcgroupsinanascocarp, onnatree, annnarbytrees, orthronunoutanorcinndorgeographical araamight provide valuable information on tin sexuality of tin pathogan ad on the epidemiologyof tindisease.Foranmple, onevcgroup inanascocarp 34 would indicate a holnotinllic (inmomictic) sexuality winreas several groups would be indicative of outcrossing or interothallian (dimixis). If dimictic, the frequancy of vc groups in a tree ad nnearby trees might provide evidance indicating wintinr disease is spreading primarily thronghinfectianbyoanidiaorasoospones. ‘I'nefrequancyof vcgroups within an orcinrd or a geografinical area might provide an estinntion of tin frequency of sexual raconbinntion in tint region. The objective of this research was to develop methods to first amine vegetative comatibility in I._._ m; ad tinan to look at populations of L; Egg—n11 within certain peach orcinrds. The sexuality of the fungus is revealed and tin implications of the frequency of vc groupsisdiscussadinreferance toboththetrasnnissionofdsRNA mediated hypovirulance and tin epidaniology of Cytospora canker of peach. Isolates TinisolatesnsedinthisstudywereobtainadfrancankersonBto 9-yr-old peach trees in two orcinrds in Clarksville, Michigan. Twenty fourtreasfrantmmvnresampledinanorcinrd(orcinrdA)ad42 treasfrmfivaromwaresaupladinasecondorchardmrcinrdB). Onn canker was sampled from each tree and multiple cannkers were sampled frann eigint trees. Encisadpiecas (2x2an) of barkandwood franthe nnnrgins of canker-s were sterilized by soaking in a solution of 0.5% sodium inypocinlorite for 3 min and than blotting dry betwaan sterile paper towels. 'nn excised pieces vars anbadded in petri dishes cantaining Ieonian's malt agar (LMA)(16) ad incubated at roan 35 tangerature (20-24 C) for 4-6 days. Isolations were unintainned ad stored on LMA. Species determinntions of the Clarksville isolates were made med on colony color, morphology, pycnnidial size, ad nnximnmn tanperature for growth (13). For ascospore isolations, peach branch segments containing sexual fruiting bodies were collected by Tyre Proffer in a Michigan State University orcinrd ad by Allan Biggs in orcinrds in Ontario, Canada. Peritincia were dissected fran strunn on surface disinfected stans with a sterile scalpel. Peritinecia were pushed ad rol led over solidified water agar to remove conidia fran the surface of the fruiting bodies. After slicing openn the peritincia, ascospores were dispersed in 1 ml of sterile distilled water with a innd-operated tissue inancgantor. The slurry was filtered through a double layer of 15 um nylon mesh filter (Tetko, Inc., Elmsford, NY) ad than diluted to 102 ascospores per milliliter. (kn-tenth ml of tin ascospore solution wes streaked across tin madiunn surface on each petri plate containing LMA. Plates were incubated 24-36 hr at roan tanperature. Single germinated ascospores were isolated with the aid of a dissecting microscope and sterile, drain glass nnadles, ad were transferred to slants containing LMA. Single conidia were isolated fran a pyanidium in tin sane mannnnr. Determinatian _o_f_ m tibili Culturasweregraenfor 4daysonLMAunder fluorescent ligints (Ganral Electric 20w cool-white lame) at 20-24 C. Plngs (4 nun dianeter) frcnn colony nnrgins of the cultures were paired on various media to determine tin best medium for detecting tin mycelial intaractian sans (barrages) characteristic of vegetative incanpatibility reactions (2,21). Plugs were placed 1 on apart fran each 36 otiner in 100 x 15 run petri plates such tint 21 plugs were on each plate (19). Each isolate was paired with itself and with each of the other isolates. The pairing plates were inncubated in the dark at room tangerature for 10-20 days ad than, based on the absance or presence of barrage zones (21) between isolates, rated as canpatible or incompatible respectively. Tin media tested were Difoo potato dextrose agar (PDA) (Difco Laboratories, Detroit, MI), acidified PDA, PDA plus 1% activated charcoal (neutralized activated cinrooal, Sigma Chanical Co., St. Iouis), 2% water agar, Endothia canplete agar (9), Leonian's malt agar . (LMA), Difoo corn meal agar, Difco oatmeal agar, oatmeal agar (10), Clarified oatnneal agar, CV agar, V-8 juice agar (18), 1.25% malt extract agar, ad Neurospora synthetic crossing medium (25). Clarified oatmeal agr was nnde as follows: 75 g of oatnnal in 1 L of water was autoclaved for5min., bladedinawnringblader for5min, adpouredthrough two layers of cinese cloth. The resulting liquid was centrifuged at 11,000 rpn for 30 min and 200 ml of the supernntant was diluted to 1 L with water, 1 ml of a vitamin stock solution (22) ad 20 g agar were added ad tin medium was autoclaved for 30 min. CV agar consisted of 10 g mltwe, 1.2 9 10121304, 0.6 g M930.1,"5'I20, 1 ml of vitamin stock solution (1), 1 ml trace elannnt stock solution (1), ad 20 g agar per liter. Each canpatibility test was conducted at least twice ad each treaMnt plate in a test was replicated twice. All subequent studies were oaducted as above on clarified oatmeal agar. was wtnn pairing _L; m isolates on media the most frequent problem ancountered was tin formation of an irregularly lobate colony margin ad 37 avoidance betwean colonies. Avoidance was mannifested in several ways: tinhypine frompaired isolates wouldgrowawayfraneachother in opposite directions, the hyphae of the two isolates would meet ad grow around each otiner with nno nnargence, or the colonies would cease growth before hypine made contact. 9; grsoanii grew sporadically or not at all an2%weteragarwhichwesusedas thepreconditioningstepfor vc pairing in Leucostm Lo_nnz_e;;l_ (19). Preconditioning was not necessary with E. grsoanii. Isolates grew with uniformly radial margins, merged, ad exhibited no immediate antagonism only on the media containing oatmeal. In 7-16 days sane adjacant colonies formed dark brown or black linnes (barrage zones) at points of contact (Pig. 1). In 30-35 days pycnidia formed along the barrage linne. Such pairings were considered vegetatively incompatible. Pairings were considered vegetatively canpatible whan merging isolates found nno barrage zonn. Growth ad interactions on clarified oatmeal agar were caparable to oatmeal agar. The clarified medium was preferred for testing canpatibility because the clarity allowed easier recognition of tin barrage reaction (Fig. 1). Oatmeal agar ad clarified oatmeal agar were pregnned fresh because Difco oatmeal agar gave inconsistent results. Results on oatmeal containing media were variable if media were poured too deeply (>45 ml in 100 x 15 petri plates) or when plates were not irncubatadintindark. Lightcasedtinhyphaeof Ewito pignant, and nnsk barrage zones. m .1. L. M All canidial isolates derived fran a givan pycnnidium were vegetatively compatible ad did nnot fora barrage zones when paired. 38 L______.<_,__ ,_,¥ ._, Figure 1. Canparison of the clarity of reaction lines (barrage zones) formed at the line of contact between 15-day—old incompatible colonies of Ieucostcnn Ersoanii. Pairings made an (A) oatmeal agar ad (B) clarified oatmeal agar. 39 Whereas, single ascospore isolates from a givan perithecium oftan exhibited barrage zones. Numerous (greater than six) vc groups were prasant in a peritincium. This indicates tint alleles at several loci cantrol vegetative canpatibility ad tint outbreeding has occurred. Thus, _L; m; is most likely a interotinllic (dimictic) pyramycete. Production of the peritincinm is necessary to confirm senonlity. This, however, has nnot yet bean achieved. Distribution g _vg m In orcinrd A, 13 vs groups were idantified among 24 isolates sapled frcn24treesintworows. Groupsladzcontainndfour isolates each, adgrwpsaad4oontainndthreeisolateseech. Insaneinstances adjacent trees were infected with strains from the same vc group int more frequent were adjacant trees with cankers caused by isolates in differant vc groups (Fig. 2). In orchard B, 23 vs groups were idantified mag 65 isolates frun cankerson42traesinfiverows. Several vcgroupsoccurredwith relative frequency in the orchard: Vc groups 9, 12, 23, 10, ad 11 were recovered true 10, 9, 6, 5, ad 4 trees respectively. Otinr vc groups were represanted by a single isolate (groups 1, 3, 5-8, 14, 15, 17-22). Sptial distributian of vc groups in orchard B did not appear aggregated or clustered around foci (Fig. 3., eg., note vc groups 9, 11, and 12). InorcinardB, isolatestakanfrantweormorecankerswithinatree generally differed in vc grouping (Fig. 3). In one tree (Fig. 3., row 1 tree 12) five isolates, each from a differant canker, belonged to differant vc groups, winerees, in a second tree (row 5 tree 6), isolates fran six cankers all belonged to tin same vc group. Isolates frun four offivecankersineacinoftwetraes(row4tree4adrow5tree14) were differant vc grouping. The ranining four trees that were sampled contained two to tinree cankers each, and all cankers within a tree were found to be caused by isolates fran differant vc groups. OrcinrdAadorchardBwereadjacent, separatedbyaone lane road. Three isolates in orcinrd A were vegetatively compatible with four isolates inorchardB, however, nno other vcgroupwespresent inboth orcinrds. 41 Orchard A Tree DONOGbUM-b .5 O duh-5d“ «004.0» a O I®®®6®®®®®I®I I@I®G IE; I I l I l I l0®®l®l®®®®®0l I: a 0 5.) . Figurez. Tweroweot'peachtreesinorchardA. Numbers identifythe vegetative oanpetibility groups of isolates of Ianostana grsoonii isolatedfrunakaer ineach tree. 42 Orchard B 5. _ _®@®m®_®@@_ _.@_ 4_@@_®o_o@_o®®_ _@_ ma. _ _ _ _ _ _ .0. _@®©@_@ g____@.@@_o®@o__@ 1.@_§_ _ _®_Om_@._ .I a; a. 4. .o a. 1. a. .u nu 1. a; so 4. .3 cu so.» Ieuoostoua grsoonii isolated fran 42 trees in Figure 3. Spatial armament of trees and vegetative oomatibility grams of 65 isolates of orchards. 43 W]!!! The difficulties encountered when pairing isolates on media other than oatmeal agar were avoidance, irregular radial growth, and growth inhibition. We initially hypothesized that these growth patterns might be due to vitamin deficiencies in the media. However, pairings on media oontainirg all cannon vitanins (including choline, myo-inositol, thiamine, and biotin which are reported to be growth requirenents of sane isolates of L; pgrsoonii (17)) did not significantly alter the growth characteristics. We also hypothesized that avoidance and growth inhibition might be due to excretion of toxic metabolites in the media during growth. However, the addition of activated charcoal to the media to absorb waste products did not improve growth. Further experiments will be required to achieve a: muiemtanding of the unique characteristics of oatmeal nedia in inducing myoelial reactions. Baeedcnthernmerof vcgroupsfoundinthisstudy, thereappears to be high diversity in the L_. grsoonii population, even within an orchard. Isolates within one tree and between adjacent trees characteristically differ in vc grouping. The results indicate that a reappraisal of theprevailingviewthatrainspreadconidiaarethe effective infective propagules is warranted (7,26). The great number of vc groups identifiable in close proximity to one another in the orchard slugest a hypottesis that the primary propagules of infection might be ascospores. However, if cmidiaarespread longdisoencesbywind, a preponderance of vc groups similarly might result. Conidia are generally believed to spread infection because masses of conidia are present eanadingfranverymmerouspymidiammst wankers. Also, theconidia are infective when trees are wound inoculated with pure smpensions of conidia in water (13,20). The alternative infective propagule, the ascospore, is rareorat least theascocarpisgenerallyrarelyseenby investigators and, when found, occurs sparsely in comparison to pycnidia. It is our interpretation that the preponderance of vc groups in an orchard is most likely due to ascospore dissemination of g; mum furtrerresearchisneededmpropaguledisseminatimto clarify the roles of conidia and ascospores. The lack of occurrence of cannon vc groups in isolates fran adjacent trees does not support prevailing views on the primary importance of infected nursery seedlings serving as foci of infection in newly established orchards (24). Conidia forming on an infected seedling adjacent to disease-free plants should spread the disease to eventually produce an aggregaticn of trees with cankers caused by isolates of a canon vc group. Such a clustered pattern of vc groups within an orchard was not seen in our studies. Therefore, the initial infective propagules might more likely be air blown ascospore or conidia dissaninated from other orchards. The orchard in study, however, Ind been established for 9 years. We have planted new orchards to nmitor spread of infection from nursery seedlings. Bertrand and niglish (7) amested conidia would be the major or only inoculum in orchards where standard practices of pruning out diseased branctes are followed bemuse perithecia are formed on branches 2 years after branch death. In Michigan we found the perfect state on livim scaffold branches. Both the frequency and site of canker formation in Michigan thus precludes effective pruning. Time, ascospores might be important propagules evaiinwell pinnedorchards. 'Ihe collections of the sexual spores from perithecia on conkers and 45 the pairing of the spores from a perithecitmx on oatmeal agar uncovered the many vc groups segregating during recombination. The results confirm the outcrossim behavior of I._._ grsoonii and reveal tint multiple loci cmtrol vc canpatibility. Apparently, E pgrsoonii is heterothallic but an alterrntive possibility is that, like 9_._ mitica, it may be a hanothallic fungus tint has the capacity to out breed (2), and readily does so. Leonian (16) mentioned the fornntion of a sexual state in culture by an isolate of k m; thus indicating the species might be hamtinllic. However, fructification in culture has not been confinedbysuwequautresearcinrsandhasnotbeenrepeatableinour laboratory. i It is hypothesized tint a natural method of virus transmission in fungi is through hyphal arnstanosis'and heterokaryon formation (14). Vegetative conpatibility or the ability of fungal mycelia to fuse and establish cytoplasnic contact is important in determining the spread of cytoplasmic virus-like infecticns in a furml population (8). Hyphal arnstcmosis is controlled by vegetative canpatibility (vc) genes (2,4,8). It is aggested tint incmpatibility is a cellular defense against genetic infection arfi might serve to protect mycelia fran invasion of viruses did other suppressive cytoplaanic determinants follmim hypinl arnstanosis (8). Vegetative inconpatibility any reduce the effectivaxess of viral transfer and biological cmtrol of diseases in hypovirulmt system (2,4,6). For instance, biological control of cinstnut blight in Europe has been successful (11) , however, there is no evidmce tint hypovirulence is spreading wurably in the United States (6). Isolates of Q_._ witica (a hanotinllic Pyrenanycete) from Wareinasinglevcgroupandinmropeonlya fewgroups exist 46 (12). In the United States, 35 vc groups have been reported in Ccn'lnecticut alone (5). line proliferation of vc groups is the probable factor limiting establishment of biological control in Connecticut, in contrast to Europe where there is little diversity (6). Incanpatibility is a disadvantage for the spread of dsRNA mociated hypovirulence in a hamtinllic fungus. It is encouraging, however, tint sale vegetatively inoamatible pairings of ; wince allow the transfer of hypovirulaace determinants (3). This pram termed hypovirulence conversion compatibility is less stringent than vegetative compatibility (2,6). The influence of vcgrozpsmthespreadofdsRNAandassociated virus particles can mly be hypothesized at this stage of our mderstardim of hypovirulence in _I._._ M; However, it is probable tint the nultiplicity of vc groups will slov the natural spread of hypovirulence (3). _I_.._ mi; any differ fran Q: mince in being heterotinllic. Heterothallism might favor trumission if dsRNA or virions can be transmitted during mating . The virulane reducing factors in our studies of _I._._ grsoaxii (13) are dsRNA and virus-like particles (VLP's). The spread of VIP's, evidently constructed with a capsid in order to survive and infect outside the cytoplasm, might have a mechaniun to overcome the barrier to hyphal anastanosis and plmgany imposed by the vc or semi cmpatibility system. _L_.p_e_zs_;ec_n;:_l_i_ and its virulence reducing dsRNA and VIP's might afford a good model system for research exploring the factors controlling transmission of hypovirulence, dsRNA, and virions in a frequently outcrossing (probably heterothallic) ascauycete with mltiple loci controlling vc coupatibility. 10 . 11. 12. 13. 14. 47 mom Adams, 6., Johnson, N. Leslie, J. P., and Hart, P. 1987. Heterokaryons of Gibberel la zeae formed fol lowing hyphal anastonosis or protoplast fusions. Experimental Mycology 11:339-353. Arngnostakis, S. L. 1977. Vegetative incanpatibility in Endothia parasitica. Experimental Mycology 1:306—316. Arngnostakis, S. L., and Day, P. R. 1979. Hypovirulence conversion in Endothia mince. Phytopathology 69:1226—1229. Atngnostakis, S. L., and Wagganer, P. E. 1981. Hypovirulence, vegetative incalpatiblity, and growth of cankers of chestnut blight. Phytopathology 71:1198-1202. Anagnostakis, S. L. 1983. Conversion to curative morphology in Eindothia flitica and its restriction by vegetative canpatibility. Mycologia 75(5):?77-780. takis, S. L., and Kranz, J. 1986. Diversity of vegetative Anagnos conpatibility groups of W pgagitica in Connecticut and Europe. Plant Dis. 70:536-538. Bertrand, P. P., and English, H. 1976. Release and dispersal of conidia and ascospores of Valsa leucostana. Phytopathology 66:987-991. caten, C. B. 1972. Vegetative incalpatibility and cytoplasmic infection in fungi. J. Gen. Microbiol. 72:221-229. Day, P. R.. Dodds, J. A., Elliston, J. E., Jaynes, R. A., and takis, S. L. 197?. Double-stranded RNA in Endothia Arngnos pagitica. Phytopathology 67:1393-1396. Goodiru, G. V., and Lucas, 6. B. 1959. Factors influencing sporangial fornntim end zoospore activity in W with; var. Niootianae. Phytopathology 49:277-281. Grente, J., and Bertiely-Sauret, S. 1978. Research carried out inFranceintodiseasesofthechestnut tree. InProceedingsof the American Chestnut Synposium, West Virginia University Books, Morgantown, West Virginia. pp. 88—92. Grente, J. 1981. Les variants hypovirulents de l'nndothia wince et la lutte biologique contra le chancre du cintaignier. Ph.D. thesis. Universite de Bretagne Occidentale, Brest. France. 195p. runner, S. 1988. Virulence ani hypovirulence in reucostana Spp. MS 'mssis. Michigan State University, East Lansing, MI. Hollinoa, M. 1978. Mycoviruses- viruses tint infect fungi. Advances in virus research 22:2—53. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 48 Kern, H. Taxomnic studies in the germs Leucostana. Michigan Academy of Science, Arts annd Letters 40:9-22. Leonian, L. H. 1923. The physiology of perithecial and pycnidial ' fornntion in Valsa leucostm. Phytopatinology 13:257-272. Lukezic, F. L., DeVay, J. R.. and English, H. 1965. Comparative physiology and pathogenicity of Leucostann grsoonii and Rinodosticta quiz-cine Phytopatinology 55:511-518. Mircetich, S. M., and Matineron, M. E. 1976. Phytophthora root and crown rot. Phytopathology 66:549-558. Proffer, T. J., annd Hart, J. H. Vegetative compatibility groups in W kunzei. Phytopathology 78:256-260. Scorca, R.. and Pusey, P. L. 1984. A wound-freezing inoculation technique for evaluating resistance to W leucostanna in young peach trees. Phytopatinology 74:569-572. Sonoda, R. M., Ogawa, J. M., Essar, T.E., and Manji, B. T. 1982. Mycelial interaction zones among single ascospore isolates of Monilia fructicola. Mycologia 74(4):681-683. Tekauz, A., and Patrick, Z. A. 1974. The role of twig infections an the incidence of perennial canker of peach. Phytopathology 64:683-688. Van Alfen, N. R.. Jaynes, R. A., Anagnostakis, S. L., and Day, P. R. 1975. Chestnut blight: biological cantrol by transmissible hypovirulence in Bndothia wince. Science 189:890—891. Wensley, R. N.. 1964. Occurrence and pathogenicity of Valsa (Mm) species and otiner fungi associated with peach canker in Soutinern Ontario. Can J. Bot. 42:841-857. Westergaard, M., and Mitchell, H. K. 1949. Ne__u_rosm_r§ B. A synthetic medium favoring sexual reproduction. Am. J. Bot. 34:573-577. William, R. S. 1936. Peach canker investigations II. Infection studies. Can. J. Res. 14:27-44. SECl'Iw III Evaluating Virulence in Leucostann for Screening Peacln Cenlnpla- for Resistance to Cytospora center MmtirgVimleneianorScreeningachGemlaafor WmcytosporaCaker m A wide regs in virulence was founnd ameg 24 isolates of m M14. and Ieuoostana Linc}; after inoculation of each isolate on one brannchper tree intenpeachtrees (MMflJ Batsch cv. 'Garnet Bauty'). The most reliable metinod for consistently obtaining ‘ successful infection of peach with Iaucostoma spp. was the canbinnation of wounding to xylem depth, injuring to surrounding tissues with freezing, inoculating, and wrapping the wound in parafilnn. Ann optinnl experimental designn for detecting differences in virulence among isolates of Leucostolna spp., and differences in tolerance of peach to Cytospora miner, wasdevelopedbasedonmeasurementof variancein susceptibility of innoculated trees annd brannchss. Inoculating more than one branch per tree did nnot conntribute to reducing experimental error tut increasing the number of trees innoculated (one branch/tree) to greater than four trees significantly reduced the statistical variannce. Inoculatirg six to nine trees, one branch/tree, gave high precisian in the detection of differences in virulence or tolerance winile utilizing few trees and reducing labor. Criteria for differentiating L; 9_inc_ta_ and imamcultmewaseminedandasystembasedonthecolany margin, size of pycnnidia, maximum tenperature for growth, and colony color was founnd to be suitable. 49 50 CytosporacanfloerofpeachcensedberncostanaM(Fr.)Hoem [Inperfect state, W 9.1% (Sacc.) Hoehn.) and m. M (Nits.) Hoehn. [imperfect state, W leuccstoma (Pers.) Hoehnn] is a destructive disease of peacln in Michigan (19). The disme is often the limiting factor in peach production as well as a deterrent to replanting and establishment of new orcinrds (24). Cytospora canker is also an inportant disease of prune, apricot, cherry. and plum (8,17). The disease is characterized by premature leaf semcennce, twig annd branch dieback, and ertensive perennial caniners on the trunk, brancines, and scaffold limbs (16,20,35). Cytospora canker is difficult to control. Once a tree is infected. the disease can not be control led using fungicides (18) and pruning out the infectionn is nnot always practicle. All of the currently gram peach cultivars are sneceptible to 01tospora canker (6,10). Tin nest advantageous nnsans of controlling Wtospora canker of peach would be the introdnction of horticultural ly acceptable disease resistant cultivars. Breeding prograns have been initiated to develop canker resistant varieties (24,36) but no gernplasm with suitable disease tolerance has been identified (10.24.28). Recently. an effort ins been initiated in the Department of Horticulture at Michigan State University to identify genetic resistance to Cytospora canker in a brood-based population of m @3129 (L.) Eamon. Essential needs of the breeding progran innclude the means for consistant neasurennent of genotypic variation in host response to inoculation with Ieucostana and measurenent of the relative virulene of the isolates used in screening the germplasm for resistance to Cytospora canker. To effectively determine genotype variation in susceptibility it is 51 desirable toknowapprondmatelyhonnnnybranches inatreeorhowmany trees of one genotype need to be inoculated (ecperinnental size) to determine real differential responses. Additionnlly, such informatian is needed to deternnine the relative virulence of the fungal isolates used in the screening. Others inve reported difference in peach varietal resistance to cankers ad differences in isolate virulence (3,17,36,38). However. variationn due to sampling error (canker size on innoculated brancins of a tree) and replicatian error (canker size on different inoculated trees of one genotype) was nnot reported. Generally, four or fewer trees of one cultivar were inoculated at numerous loci or branches to determine cultivar or isolate responses (3.17.29.36.38). One concern ininnoculatingseveral locionafewtrees is that it has been denunstrated tint differences in host vigor (7) and in enviranmental stresses an hosts (3,26) affect susceptibility of individual trees to Cytospora miner. For exanple, variatian in the clay content of soils within one orchard can account for 88% of the variation in susceptibility to Cytospora canker among prune trees (1). Also, trees in an orchard are likely to be individual 1y subjected to different severities of postinrvest moisture stresses, scale injury. and nematode infections which influence relative susceptibility to Wtospora canker (2.13.29). Thus, variationn along trees of one genotype might nnsk differences between cultivars or fungal isolates if inadequate replications are tested. Annotiner concern is tint branches of different dianeter or wood age might similarily increase variation and hinder . accurate determinatian of cultivar response or isolate virulence. A reliable method for testing differences in virulence among isolates of Ieuoostann might provide information on disease resistance by 52 influencing the experimental designs for peach breeding programs. It could also be inportant for clarifying the relative inportance of host and patiogen variatian. Additianally, it is important to determine the relative virulence of isolates of L_. Ersoonii and £1; g_in£t_a_ since they have been reported to differ greatly. In one study (38) two of ten isolates of L; M on peach were avirulent and in a separate study (17) an of four isolates of L_., m were avirulent. when seventy-six isolates (mostly L. mg were inoculated on french prune, two isolates produced cannkers greater than twice the size formed by 703 Of the remaining isolates (3). k M has been reported to be of low patiogenicity canpared to L_._ g_i_ng__ta_ on peach (19.37). however. in other researchgmgwasmorevimlent inwarmweather (mean teperature greater than 16 C) and L_ginngta; more virulent in cool weather (1.19.36). The objective of this stndy was to determine the relative virulence ofannnoerof isolates ofLLErsoaniiadLngLctgmamnifom source of germplasm. The contributions to variation in the experimental results are calculated for the error in senplirg ad the error in replications. Fran this data we develop reconnnadations for approximating the optiInnl experiment size ad design for detecting a desired percentage difference in viruleoe among isolates of the pathogen while mummy swarm of plants and labor. Fran this information we ertrapolate the experimental size ad design required to detect a desired amounnt of cultivar resistance (expressed as percentage reduction in canker size canpared to a susceptible cultivar) with a selected level of assurance tint the detected resistanoe is gennine. The evaluatians of peach cultivars for resistance to Cytospora canker 53 reported in the literature are discussed in relation to experimental design. Criteria for characterizing isolates of Leucostana spp. from peacharediscussedandmetiodsof inoculatingthetreesareeamined. Twenty-four isolates were obtained fran Michigan. North Carolina. and West Virginia. Cultures were grown on Leonian's malt agar (LMA)(25) for 5 days at 25 0 prior to use in inoculation experiments. Designation of isolates as _L.._ pings; or L; p_e_rsoonii wm based on a canparison of their cultural characteristics after 30 days growth at 25 C an LMA to descriptions by Hildebrand (19) and William (37). Characteristics tint were emined included (i) whether the colony margin was lobate with restricted growth. or uniformily radial growing to fill the culture dish, (ii) whether pycnidia were snnnll (<1 nun) or large (1-3 m). (iii) the colony color, ad (iv) the ability to grow at 37 C. In acditian. the cultural cinracteristies of the 24 isolates were canpered to the cultural characteristics of single ascospore isolates of L; grsoonii derived from perithecia. Variations in the inoculation metiod were tested to determine the optimun procedure for obtaining consistant infectionn. In this test the trunk of 62 2—year-old trees were mecinnicallywonnded with a hand held stapling gunn fol lowed eitiner with or without the aerosol freeze treatment. Thewanndswere thenwramed ineither parafilm or cloth tape (which fell off after 2 weeks.) The freeze spray and wrap treatments were tested in all canbinntions ad replicated three tinnes. me would freeze inoculation metiod of Scorza and Pusey (32) was wed in the isolate virulence study. The inoculation site was first cleaned 54 withgauzesoekedinQSX E'I'OH. Thetreeswerewcundeddowntoxylem depth using an enpty hand held stapling gun. The resulting 12 x 2 nun wound was frozen by spraying with a cannercial aerosol cryogen (100% Dichlorofluorcnnetl'ene, Cheutrcnics, Inc., Hauppauge, NY) for five seca'ds at a distance of approximately 15 cm. Inoculum consisted of a 5 m diameter mycelial ping. 'lhe inoculun was placed on the wounded bark and wrapped in perafilm to prevent dessicaticn. Control trees were wounded in an identical manner except a plug of INA was substituted for the mycelial png. Tm vigorous 8-yr-old peach trees (3; pgrsica cv. 'Garnet Beauty') were each inoculated with nine isolates of Q; gi_n§_t_a_ ad 15 isolates of k m; in October 1985 (isolate virulence plot). Each of the 24 isolates and a control were placed on each tree and there were ten replications. Each inoculation was nade on a separate 2-yr-oldbranchatapointmthebranchneasuring17mindiuneter. Wwereremovedandcenker lengthwasmeasured (lengthcf necrotic area distal to inoculation point) at the time of leaf elongation during the spring fol lawing inoculatian. Statistical analysis of data incltded analysis of variance (AIDV) of randunized camlete block design with trees as blocks (replicates). Isolates were ranked according to average canker length using the least significant difference (LSD) at the P- 0.05 level. An isolate was inoculated into a single tree cm 20 seprate branches to test for within tree variation (branch variance plot). Branches were measm'edwitha caliper and aportion of thebranchwith 2-yr-oldwecd ad neamring 17 m in diameter was inoculated. Isolate NC 9.2 contained double-stranded RNA (dsRNA) and was inclLded to evaluate whether wounded and inoculated brancles were successfully colonized by the inoculum or 55 invaded by wild strains. (endeared branches that had been innoculated tl'e previous fall with isolate NC 9.2 were removed fran tl'e ten trees and brought into the laboratory. Pieces of tissue were removed along the side of the necrotic margin of the canker and soaked in a 0.525% hypochlorite solution for 3 min. The tissue was blotted dry with sterile paper towels and tran'eferred to LMA. 'me ten resulting cultures were tested for the presence of dsRNA using a modified double cellulose column attraction procedure and electrophoresing on a 5% polyacrylamide slab gel (11,14). the following uethods were used to develop an experimental design providing the most efficient determination of the relative virulence of a pathogen or the relative resistannce of a peach cultivar. Tre enperinnental design requiring tne least labor and providing the greatest reduction in sapling and enperimentel errors was determined using a method frun Sahel ed Rohlf (33). Re senple variannce (MSS) fran tre mm of data of tne branch variance plot and tie mean square error (MSE) from tl'e Amv of data of the virulence plot were used to determine the minimal experiment size and optiml design for reducing canponent variannce. This was achieved by the construction of two tables, one of tie total number of observations or inoculations required for various experimtal designs (Table 3A), and a seced table (Table SB) of the sapling variannce expected for each of tie experimental designs in Table 3A. The values in Table 38 are calculated using the fol lowing fornnilae (33). 56 Sailingermams mplicatianerrcr= MSE-IVSS (no. of branches inoculated per tree) Smlirg variance- sapling error replication error + (no. of trees 1: (no. of branches (no. of trees inoculated) inoculated/ tree) inoculated) 'neenperinental designis ctmenfrcm'rableaAanithtl'emmber of trees to be inoculated. when limiting the size of the experiment to a certainmmerof treeeanestinatiencanbemadeof treabilityofthe emerinent to detect differences in isolate viruleoe or cultivar resistance. Differences in isolate viruleoe or cultivar resistance (exprmed as a percent difference in canker legth following inoculation with a fungal isolate) are detectable at a closen nagnitude with a chosen assuranoe that the detected differeoes are true. Using MSE: as an estinate of the variance, croosirg tie assurance level desired at 90% (0.90) and the level of significanoe to be used in the actual experiment at P- 0.05. the following formula and a table of t values are used to eatinate detectable differeoes (34): 57 2 Percent detectable difference was) (to - t1) in isolate virulence or = cultivar resistance (center legth) number of trees inoculated per isolate or per cultivar t0 = t table (two tailed) value for P: 0.05 using degrees of freedan associated with DEE. t1 = t table (two tailed) value for P = 2(1 - 0.90) = 0.20. [0.90 is tle assurance level ctoeen] using degrees of freedan associated with BBB. mus Mong the 24 isolates tested, 15 were identified as I; pgrsoonii and nine as Engine—ta. All isolates of 5m had a lobate, restricted colony margin, an olivaceous or darker colonny color after 30 days growth en LMA, and snall (<1 nun) pycnidia (Fig 1). Camerisons with cultures derived frann single ascospores fran an ascnis identified as _l_‘.._,_ pe_rs_o_gn.i;_i aided our identification of L_. grsoenii by cultural characteristics but the cecal state of L.._ §___inc_t_a_ was not available. The eight L._ 2413933 isolates were nore variable (Table 1). Six of tie isolates hai characteristic, nrnifornally radial nargins, large pycnidia (1-3 an) often having a cotteny appearance, and en olivaceous to buff colony color (Fig 1). The other two isolates red unniformal ly radial margins but lacled the characteristic pycnidia. 'ne Penn isolate (Table 1) had all (<1 m) pycnidia and isolate H6 had no observable pycnidia after 30 days. These two isolates were identified as E». gi_nnc_t_§ based on colcnny color, nergin, and inability to grow at 37 C. Isolates designated as I; ci_nn_c_ta;failed togrowat 37 thereas tlosedesignatedaskpgrsoonii 58 Figure 1. Ooleny characteristics of a) L_. cincta with a unniformal 1y radial coleny nargin and large (1—3 nun) pycnidia and b) E; M with a lobate colony nargin and all (<1 m) pycnnidia. TABLE 1. Isolates of m spp.: Origin, colony characteristics, author designated species, and source or reference Isolate Origin. Colony colorb eonow° Pycnidi 4 Growth Designated. Source or Margin Siss st 37 C Species Reference $8.2 1c Olivscoous buff P I. - c (12) $9.2 1c Olivscsous P I. - c (1.2) ”14.2 no Olivscsous I. S + p (12) End 1C Grssnish olivscsous P I. - c (12) C-J-l W Grssnish olivscsous I. S + p anon: 58385: C-HZI-S WV Olivscsous I. S + p C.I.. Hilson C-Jn_15 W Olivsosous I. S + p C.I.. Hilson c-s-zo WV Issbsllins I. 8 + p C.I.. Hilson 310.9 HI Grosnish olivscsous I. 3 + p 8.A. Hun-r ..11 HI Iran grey I. 8 + p 8.A. Banner 39.5 MI Grssnish olivscsous I. 3 + p 8.A. ulnar 85.15 MI Olivsosous I. S + p S.A. Banner 87.13 MI Pusoous black I. s + p 3.A. lunar 0-3 III I. lbuss grey Y I. + c S.A. Ban-r lfl-l HI Olivscsous I. S + p SA. H-sr F-t HI Duff 1’ S - c SA. finer P-l PA key olivscsous I I. + 1: LA. finder 11.11 MI Orsanish olivsosous I. s + p 8.A. l-sr 10.8 HI Olimoous I. 3 + p 3.A. Bun-r Cl-S CA Ground-h olivsosous P I. - c 3.“. 03m Hs-Q CA Olivsosous I. 8 + p J.H. “an I-BO CA Olivsosous P I. ID 0 J.H. 03m P-is CA Olivsosous P I. - c 001 (3) P45 HI Honey I D - c F-QO (3) sl Initials of state where isolsto originated. All mopt 1’45 and P-QG were fraposch In] Color key used: Runner's Colour dart (31) o/ P- am. uniforlnally redial margin. I.- lobsto margin dl 3- .sll (<1 - di-stsr) midis, I.- lsrss (1-3 III di-stor) pycnidis .’ P' M- P m fl Auriosn 13p. Culture Collection 60 grew at 37 C or greater temperatures. The best inoculation method for the virulence screen was determined in a preliminary test. The largest and most consistent cankers were obtained using Soorza's method (32). 'me most effective treatment consisted of the freeze spray in combination with the parafilm wrap (Table 2). Infection was not as consistent when the freeze spray was eliminated or when the mud was wrapped with tape instead of parafilm. Therefore, we used the freeze spray in combination with the parafilm wrap for all subsequent inoculation experimts. In the virulence screen all inoculations resulted in wanker formation emept the controls. The results show a wide range in virulence of the isolates. The virulaioe of isolates [based on mean m length of ten replications (trees)] are listed in order of decreesiry virulence (Table 3A). Isolate I-ao tron Chlifomia is the least virulent with a mean canker length of 4.02 an, vhile Riley, a Michign isolate. is the most virulent with a mean cancer length of 11.0 on. 'me mographic origin of an isolate was not a factor in its virulence. For instance, the four most virulent isolates were isolated tron Michigan, North Gerolina, and California, while the four least virulant isolates were isolated in Michigan and Gelifoznia. The relative virulence of Lam ompared to L_. grsoonii has not appreciably different and _1.; $1223; isolates were dispersed evenly anong the 1:._._ PM. isolates in the virulence ranking (Table 3A). Individual trees of a cannon cultivar (scion germplasn) varied significantly in their reepmseetothepatrngenwmthevariatimbetmentreeevasramved by the two-way AIDV the different Leuoostana isolates mre revealed to rave significant differences in virulence (Table 33). Branches within a 61 Table 2. Analysis of variance table for optimum inoculation procedurea of nggggtgmg isolates in peach Degrees of Mean F value freedom square Isolateb 2 25.154 12.58** Freeze spray alone 1 .302 .15 nsc Isolate x freeze spray 2 .863 .43 ns parafilm wrap alone 1 .667 .33 ns Isolate x parafilm wrap 2 .174 .09 ns freeze spray x parafilm wrap 1 5.523 2.76** Isolate x freeze spray x parafilm wrap 2 9.730 4.87** Error 24 2.000 a/2-yr-old trees were mechanically wounded with a stapling gun followed either with or without an aerosol freeze treatment. The wounds were wrapped in tape (which fell off and exposed the wound within 2 wk) or parafilm. b/Two isolates were inoculated three times for each treatment. The two isolates were significantly different in their virulence. c/ns - not significant 62 Table 3A. Virulence of isolates of Leucostcsna spp. on 10 trees of Garnet Beauty peach ranked in order of decreasing canker length Origin Isolate Species Average canFr designation length (cm) MIa R—l g; Ersoonii 11.00 AC NC 8.2 3.1.; cincta 9.98 A8 CA F—46 _I_._._ cincta 9.83 ABC CA CL-5 I: cincta 8.40 BCD MI 10.8 E Ersoonii 7.78 BCDE MI 119.5 E. M 7.64 BCDEF WV C-j-l I: grsoonii 7.50 CDEF WV C-S-20 I; pgrsoonii 7.24 DEFG MI G-3 LL cincta 7 .08 DEFGH MI F—4 L; cincta 6.95 DEFGHI CA Ma-4 E Ersoonii 6.49 DEFGHIJ WV C—MI-5 L_._ Ersocnii 6.48 DEFGHIJ PA P—1 in Ersoonii 6.06 DEFGHIJK WV C-jm-18 Q; Ersoonii 5.94 EFGHIJK MI H10,9 L_._ pgrsoonii 5.46 EE'GHIJK m 9.2 _L_._ cincta 5.34 FGHIJK It: 14.1 g; cincta 5.11 GIIIJK CA F—45 I; cincta 4.92 GHIJK It: 14.4A L2. pgrsoonii 4.81 I-IIJ'K MI H7.13 _IL Ersoonii 4.79 HIJK MI 36.15 _I_._._ m 4.75 EUR MI 39.11 g m; 4.71 IJK MI 11.11 h Ersoonii 4.18 JK CA I-80 L._._ cincta 4.02 K Control 0.5 L a/ Initials of state where strains originated b/Length of worker distal to the inoculation point c/Means followed by the sane letters are not significantly different by the least significant difference test (LSD) (P=0.05) B. Randanized Canplete Block Design Degrees of than F significant freedom square at P less than: Total 228 Peach tree (reps) 9 53.015 .000 Isolates 23 36.033 .000 Error 196 10.116 63 single tree did not vary significantly in susceptibility. The standard deviation (8) for branches within a single tree was .453 whereas the standard deviation between trees was 3.18. This is strong indication that there is considerably more variation between trees than within a tree. Cultures reisolated fran branches on ten trees inoculated with strain m 9.2 exhibited the typical dsRNA baniing pattern of NC 9.2 in all imtances. misprovidedassurancethatcankerswerecamedbythe strains used in the tests. The AmV of the data from the virulence plot and the branch variance plot permit the calculations in Table 4A & B. Enemination of Table 4A 6: Breveal trat increasingthemndaerofbranchesinoculatedpertreehas an insignificant effect on reducing the sample variance. Increasing the number of trees inoculated (one branch per tree) greatly reduces the sunple variance. However, increasing the mmbar of trees inoculated aboveninegivesminorreductions invariance ttatperhapsarenotworth the increased labor or plant material. Fran our data, the experimtal design of choice for the most efficient determinatim of the relative virulence of an isolate of the pathogen or the relative resistance of a peach cultivar would be to inoculate one branch par tree and use between six ani nine trees to screen a cultivar or a furgal isolate. Replicatim inoculations on ten trees detects differences as low as a 4.63 in virulence (canker length) at P = 0.05 and with an assurance of 90% that the detectable difference is true. This can be interpreted as m ability to detect a 4.638 difference in resistance to canker in peach cultivars when ten trees per cultivar are inoculated, one branch per We A. Total observations (inoculations) 3 . Number of trees Inoculated g 2 1 3 0 0 12 15 “ c '- 1 1 3 6 9 12 15 £3 2 2 0 12 10 24 30 .63 3 3 9 18 27 38 45 _g 4 4 12 24 30 40 00 33 5 5 15 30 45 00 75 E 8 1O 10 30 60 90 120 150 25 2O 2O 60 120 180 240 300 8. Sampling variances 1.- . Number of trees inoculated 5; 1 3 0 0 12 15 ‘2 1 10.12 3.37 1.00 1.12 0.04 0.07 S 2 0.00 3.23 1.01 1.00 0.01 0.05 ~53 3 0.54 3.10 1.50 1.07 0.00 0.04 .3 4 0.40 3.15 1.50 1.00 0.70 0.03 3'5 5 0.42 3.14 1.57 1.05 0.70 0.03 5310 0.3 3.11 1.55 1.04 0.70 0.02 35 20 0.3 3.10 1.55 1.03 0.77 0.4 Table 4. Choice of experimental design for the most efficient determination of the relative virulaace of the pathogai or resistance of a cultivar requiring the least labor and cost (observations of inoculated branches and trees) and providing the most accurate experimental results (greatest reduction in sample variance). A) Total mmber of observations (or inoculations) required with diffemt inoculation models (experimtal designs). B) Sampling variances acpacted for the different designs in A. 65 13mm! The aralysis of the plot data reveal the relative importance of sampling error and replication error. calculations based on the data are used to estimate the optiml experimental size and design for screening isolates of Leucostm spp. to determine their relative virulence. This experimental design should minimize the sources of experimental variance that would interfere with the precision of screenirg peach gern'plasm for resistance to Cytospora canker. Variance due to inoculation of different branches in a tree is essmtially of no consequence. Thus, inoculatirg morethanonebranchper tree givesno increasedprecision in differentiating isolate virulence or varietal resistance. Variance due to inoculation of several trees (one branch per tree) of the same gernplaan is the major source of experimental error. Increasing the nunber of plants inoculated greatly increased the precision of the screeningtests. Theimculatimofmretimfmn'treesisneceesaryto detect the true response of the trees of one germplasn to the virulence of a tested isolate of the mthogen. Under our experimental conditions, the experimental size and design for optimal precision using the least labor and amount of trees of (me gemlaan will require inoculation of mebrarnhpertreeforsixtoninetrees (Table4AandB). Webelieve tint the sources of experimental error unier these conditions should be similar in other locales. In our plots there are no recognizable environuental factors that might account for the varimxce seen among individlal trees of the suns germplasm. Apparently, there is no significant variation batman branches of uniform dianeter and age on a peach tree in regards to response to pathogenesis. Assuning that the identified sources of acperimental error in our 66 tests are similar to those of past tests, it becomes difficult to interpret the results of experiments where numerous branches or loci were inoculated on four or fewer trees in screening tests. It is notemrthy tint two sttdies evaluating resistance to Cytospora canker in peach trees (27,32) utilizing numerous replicatiorn fourd only weak or insimificant disease resistance. This is clear evidence that the existing level of Leucostana resistance in North American germlasm is quite low. The morphologial aid pathological characteristics of isolates med inabreedingprogramneedtobe thoroughlydescribed prior toscreening of germplasn so tint species identification will be universally interpretable. Identification of the pathogen to species is not easily achievedwithCytosporacanker. Cytosporacanker iscausedbytwo species that are caniderad to be distinct in the north east aid north central states. although Lulazic _e_t_ a_.'l_. (29) inve cpestioned the validity of separating the two species. The inportant cinracteristics for identification are those of the sexual fniiting bodies (teleomorphs) which do not form under laboratory corditions. Because of the relative scarcity of the teleanorph of the two species in nature, §Lc__:lnc_ta; and L._ pm; are ccnmonly idmtified by cultural characteristics (19,36). Researchers generally inve utilized the cultural cinracteristica described by Williscn (37) as criteria for differentiating L.._ gan_t_a_ from _I_.._ M. In this study we found the culture pintogrann aid optimum growth tmerature studies by Hildebrard (19) to also be useful for distinguishing betwem the two species. Colcny color vas the most variable cinracteristic aid was the least inportant for cinracterization. Hildebrard fourd tint the maxilmm growth 67 temperature forangEwnsaocmnreasforEMitwnsSQC. Only isolates designated as g m; (based on lobate colony margin, small pycnidia, andcolony coloronLMA) wereable togrowat 37 C in our studies. Tine, differences in cultural cinracteristies were further corroborated with differences in maximum tenperature allowing growth. Growth at 37 C is apparently a relatively reliable character for distinguishing g m from k my Differentiating species by cultural characteristics, however, was dependant an growth on LMA at stardard tenperature and light conditions because differences in isolates desigrnted as 1:._._ gm ard E; M; were often indistinct when grown on other undia. Kern (23) suggested tint the typical spaciwnofhgifledEMmight represent ectrennsard tint intermediate forms were possible. Many of the isolates tint we have examined were intermediate in pycnidial size, colony color, colony margin, or temperature maximm. No cinracteristic was singly reliable in distingiishing cultures of the two species, however, the multiple cinracteristics used in this study are suggested as useable until isozyme patterns or more precise criteria bacanne available. Our data show significant variation in virulence among isolates of _I_.._ 91.15.; and 9; Mi. Vb have not fourd significant numbers of avirulent isolates as reported in otiner studies (17,38). However, the evident difference in virulence emphasize the importene of obtaining a measurenant of the relative virulence of an isolate prior to utilizing it for screening gennplasm. We did not observe the virulence differences betmenLLLingtge'dggrsoonii as reported intine literature (19,37). Hildebrand (19) noted that g; g_inc__tg always censed larger cenkers tinnkmgwdnen treeswere inoculated during the cooler 68 nnnnths of the year. Our tests showed an even distributionn in virulence between the two species. The cooler temperatures of Michigan winter and early spring did not favor the growth of £2 pilots; over L_. m; Similarily, Dinanvantari (9) found tint in trees artificially inoculated in autumn the two species of Leucostana were equally virulent. The geographic origin of the isolates tested was not correlated with the level of virulence. Peachtreesintheorcinrdaremst susceptible to infectionintine early spring following dornnnncy (4.5.22). Thus, trees should be irnoculated in late fall or winter. Differences in methods of inoculating peach trees with Leucoatcnna spp. cann affect the reliability of resistance evaluations. Others have reported that the combination of injury to xylen depth, freeze injury to surrounding tissues, and wrapping the mind in parafilm is most reliable in inducing infection beyondtheinjuredarea(32). Theimportanceof freezeinjuryandthe superiority of parafilm wrap to cloth tape for providing a nnore optimunn envirennent for infection is confirmed in our tests. 10. ll. 12. 69 mom Bertrand, P. P., B. English and R. M. Carlson. 1976. Relation of soil physical and fertility properties to the occurence of Cytospora canker in French prune orcinrds. Pinytopatinology 66:1321-1324. Bertrand, P. P., H. English, K. Uriu and F. J. Schick. 1976. Late season water deficits and developnent of Cytospora canker in French prune. Phytopatinology 66:1318—1320. Bertrand, P. F. and H. English. 1976. Virulence and seasonnl activityofm leicostanaardggcinctainli‘renchpnmetrees in California. Plant Dis. Rep. 60:106-110. Bertrand, P. F. and ii. English. 1976. Release and dispersal of T cmidia and ascospores of Valsa leucostann. Pinytopatinology 66:987- 991. Bier, J. E. 1964. The relation of some bark factors to canker susceptibility. Phytopathology 54:250—252. Biggs, 11.12.. 1986. 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After harvesting the mycelial pad was pressed between layers of absorbent paper to remove excess liquid. The fungal tissue (0.5-6.0 g)wesfrozeninliquidnitrogenandgrmnxiinachillednortarand pestle with glass beads (490 um). The resulting fine pader was added to ceitrifuge tubes eld camined with 10 ml STE buffer (0.05 M Tris, 0.1 M NaCl, and 0.001 M EDTA, pH 7.0) containing 0.196 beitonite, 1.5 ml 10% sodium dodecyl sulfate, and 15 ml of redistilled phenol saturated with STE hiffer. Followirg centrifmation at 8,000 rm for 15 minutes, the Imperaqueomphasewnsremovedeidadiedtoflx ethanol fora final ceaceitratim of 15% ettumol. This was passed thromh cellulose columns (Ratman CF-ii) and washed with 15% ethanol-STE to remove all the species of nucleic acids (ribosanal RNA, transfer RNA, and DNA) except dsFNA end viroid material. The dam was eluted from the cellulose with STE and collected. Etl'unol was adied to make a final concentration of 1596 ethanol-STE. The colum procedure was repeated and the solution was stored overnight at -20C. The precipitate that formed was collected by centrifugation aid then resuspended in electrophoresis buffer (0.04 M Tris, 0.02 M sodium acetate, 0.001 M EDTA, pN 7.8). The sanples were layered on 5% polyacrylamide slab gels (17.5 cm wide x 16 on long x 1.5 m thick) and electrophoresed at 40 mA for 12 hr. The gels were placed in a 500 ml ethidium branide solution (0.05 ug/ml) for 30 minutes followed W astaining for 15 min in 320. Banding patterns were recorded 72 73 by placing gels on an ultraviolet transilluminator and photographing through a No. 4 yellow filter with Polaroid type 55 film. Some gels were treated with deoxyribonuclease (DNase) and with ribonuclease (RNase) to renove DNA or ssRNA. Gels were placed in a solution of RNase A (Signa) at a concentration of 50 ug/ml in a 0.3 M NaCl solution for 30 minutes. The gel was stained‘with.ethidiumlbromide, observed, and.then.placed in a RNase and water solution to renove all RNA's. If any bands retained after the RNase inxwater treatment, they were treated‘wdth.10 ug/ml DNase»I (Sigma) in 5 mMImagnesium.chloride and stained.againwwith ethidium.bmomdde to determine if the bands were DNA. Bands digested.by ribonucleasehinwwater'but not by RNase in.NaC1 were assumed to be dsRNA (13) HICH mmW