I I I I III “in III?“ W... .. . I ‘ I. I. -';I‘ ~ 'II IILI'IIII-f‘I I II III IIIIITII' (I -‘ -IIII?_.: f. Ifli‘ III: “I :széII II.;III:!£III;-II II I I). I; ; I I I 'It',':lf\II IIIII I' " II‘I. Il‘ :III.I PM”. Izul 'II'II - III 43'.” III ‘I I'Ul I I , unfllpa'”: ”4|. I]. ‘ y'III I'IIII‘I'HII' . IIFII !' If”; I;;g:.:I>: 15.»; ;. “III, ‘ , AI. I'II’U‘I'MIIfi’f‘ ‘ I“: I I [III [III {F‘Ifi A ‘E‘IA‘I'IIIW {I’II ”II We It; 1IIIrIII. II: t; 1. 613. m II I " INbYI’II-i.'”lr.-r‘ ’\II‘;I‘;"‘I‘I. II I In“; I. : ”'5‘” 141 'IIII .)\ ."- '73. I.I 3" gIfi I m“ ; 31:137. MRI-II ' w MI W"! o u' ‘v I . ml ‘:~1 '1. 3;: .:.l;§ \..‘._.:-, I ,I'mu.‘ ‘ '12.I 5" I... ' WU- ' .- . :29} '. n'. . . ,. S s‘ 4‘. A ‘ 9' ‘ g. ' .. U~€IIII 4 .'n I ' v ‘I I - I ‘. I I ~ " n L" . I» “é"v-JIIrrt‘Icrrw: .1" MIN. «I (Iv . 4n I I I‘ u .~- ,-_.. ‘7 ,.-._— a?“ ; .. .- r... .. .- 3 .I' ‘ . , . ..‘-. “‘5‘“ ‘I‘I‘I ,MI"‘. 'Mh‘. '5 , I I' . $04": I iII/H/ " ':4 "‘ '7 ' I I' ‘ Rf.” l.‘ >. ‘F - .I,I N 1.. .;~;. £31. ‘ . II-' :I r. '.|' ' 1" .L. Y! .. -5)‘£‘:‘I,‘.IL:{I 'v _IH a. I", \‘ 1m“ III.'{I3'II ,2 9‘“ I, III? (MW/I M A; .‘fi 9 I‘““‘\ a 2'"?! I: " 6 =2 o, q?" a , a .1 r- huwu-II “-1 h w -v— 1"- 1 .-'.' ‘ .‘_("‘. L' o-va-vo— This is to certify that the dissertation entitled GENETICS OF BENOMYL RESISTANCE AND REDUCED SENSITIVITY ‘10 STEROL-INHIBITING FUNGICIDES IN VENTURIA INAEQUALIS (COOKE) HINT. presented by Victor Frederick Stanis has been accepted towards fulfillment of the requirements for Ph.D. Plant Pathology degree in Alan L Jones flprofessor mafia? é /?0Vé/ MS U is an Affirmative Action/Equal Opportunity Institution 0- 12771 MSU RETURNING MATERIALS: Place in book drop to uaamuss remove this checkout from -_. your record. FINES will be charged if book is returned after the date ‘ stamped below. a 3‘?“ 1:13 GENETICS OF BENOMYL RESISTANCE AND REDUCED SENSITIVITY TO STEROL-INHIBITING FUNGICIDES IN VENTURIA INAEQUALIS (COOKE) WINT. By Victor Frederick Stanis A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 1985 Victor Frederick Stanis ABSTRACT GENETICS OF BENOMYL RESISTANCE AND REDUCED SENSITIVITY TO STEROL-INHIBITING FUNGICIDES IN VENTURIA INAEQUALIS (COOKE) WIN‘IT By Victor Frederick Stanis Resistance to benomyl and reduced sensitivity to sterol-inhibiting fungicides were studied in field isolates of Venturia inaegglis. Four levels of benomyl resistance were identified among 59 benomyl-resistant isolates of l. inaegu_alis from Australia, Chile, West Germany, Italy, New Zealand, and five states in the United States. Genetic analysis of 23 resistant isolates corroborates reports that in X. inaegu_§iis different levels. of benomyl resistance are controlled by alleles of a single gene. No linkage was detected between the gene for benomyl resistance and four genetic markers for auxotrophy or three colony color markers. Isolates of 1. inaeggalis exhibiting reduced sensitivity to sterol-inhibiting fungicides were obtained from an orchard in West Germany. With the sterol inhibitors BAS 454 06 F (l-(Lfl-dichlorophenyli-Z-(lH-l,2,lt-trizol-l-yl)), bitertanol, CGA 71818 (l-[2-(2,¢-dichlorophenyI)-pentyll -iH-l,2,4-triazole), DPX H6573 (bis(4-fluorophenyl)methyl(lI-l-l,2,4-triazol-l-yl methylisilane), etaconazole, fenarimol, Ro 15-1297, and triflumizole, minimal inhibitory concen- trations preventing colony formation by individual conidia were a to 8 times higher for these less sensitive isolates than for other similarly tested isolates from West Germany and the United States. Levels of sensitivity did not change Victor Frederick Stanis in culture. Genetic analysis of nine isolates with reduced sensitivity indicates that in 1. inaeggalis reduced sensitivity to sterol-inhibiting fungicides is controlled by a single gene. ACKNOWLEDGEMENTS Sincere appreciation goes to Dr. Alan L. Jones for assistance both in the preparation of this thesis and throughout the course of my work at Michigan State University. ‘ Drs. Karen Klomparens-Baker, Joseph Vargas, and John H. Hart are thanked for reviewing the thesis for its content and literary style. I thank the typist, Alice Ellis, for her attention to detail. Finally, the financial assistance of a Michigan State University Recruitment Scholarship (1981) is gratefully acknowledged. TABLE OF CONTENTS Page LIST OF TABLES ................................................. V iv LIST or FIGURES .......................... ‘ ....................... v GENERAL INTRODUCTION AND LITERATURE REVIEW ............... 1 LITERATURE CITED .............................................. 9 PART I GENETICS OF BENOMYL RESISTANCE IN VENTURAL INAEQUALIS FROM NORTH AND SOUTH AMERICA, EUROPE, AND NEW ZEALAND ABSTRACT ...................................................... 13 INTRODUCTION .................................................. 14 MATERIALS AND METHODS ....................... - ............. ,. . . 15 RESULTS .............................. 22 DISCUSSION ..................................................... 33 LITERATURE CITED .............................................. 36 PART II REDUCED SENSITIVITY TO STEROL-INHIBITING FUNGICIDES IN FIELD ISOLATES OF VENTURIA INAEQUALIS ABSTRACT ...................................................... 39 INTRODUCTION .................................................. 40 MATERIALS AND METHODS ....................................... ‘42 RESULTS ........................................................ 46 DISCUSSION ..................................................... 57 LITERATURE CITED .............................................. 60 iii LIST OF TABLES PART I Variation in levels of benomyl resistance among isolates of Venturia inaequalis subjected to a series of benomyl concentrations in potato-dextrose agar ..................... Segregation for benomyl resistance among ascospore progenies of Ventura inaegu_alis ..................................... Segregation for benomyl resistance and genetic markers among Venturia inaequalis progenies .............................. PART II Minimal fungicide concentrations for preventing colony formation by germinated conidia of Venturia inagggalis with two levels of sensitivity to sterol-inhibiting fungicides. Suggested discriminatory concentrations inhibit colony formation by conidia from sensitive isolates but not by conidia from isolates with reduced sensitivity to sterol-inhibiting fungicides ............................................... Inhibition of mycelial growth surrounding assay discs treated with two concentrations of dodine and placed on agar seeded with sensitive isolates of Venturia inaeggglis or with isolates that exhibited reduced sensitivity to sterol-inhibiting fungicides Segregation of single-ascospore isolates of Venturia MEL“ into less sensitive and sensitive phenotypes according to growth on potato-dextrose agar amended with 1.5 ug/ml fenarimol . . . . Page 16 23 3O #7 48 52 LIST OF FIGURES Figure Page PART I l Progeny of Venturia inaeggalis tested on potato-dextrose agar amended with 5, l, 10 and 500 ug/ml benomyl. Twenty-four progeny per plate were evaluated simultaneously at each concentration using bits of mycelium from single-ascospore colonies. Examples show growth 3 weeks after inoculation with progenies from four crosses; A) sensitive X sensitive, B) high resistance X sensitive, C) low resistance X low resistance, D) high resistance X medium resistance ----------------------- 27 PART II 1 Response of Venturia inaeggalis on potato-dextrose agar amended with O, 0.2, 0.4, and 0.6 ug/ml BAS 454 06 F. Five isolates with reduced sensitivity to BAS 454 06 F and four sensitive isolates were tested simultaneously at each concentration. Three weeks after inoculation with single germinated conidia, isolates with reduced sensitivity but not sensitive isolates showed growth at the two higher concentrations .......................................... 49 2 Effects of dodine, sodium lauryl sulfate (51.5), and carbonyl cyanide 3-chlorophenylhydrazone (CCCP) on the toxicity of fenarimol to Venturia inaeggalis. Plates a-d were seeded with an isolate sensitive to sterol-inhibiting fungicides, while plates e-h were seeded with an isolate exhibiting reduced sensitivity to sterol inhibitors. Vertical strips were treated with fenarimol and horizontal strips a,e with distilled water; b,f with dodine; c,g with 51.5; and d,h with CCCP ........................... 54 GENERAL INTRODUCTION AND LITERATURE REVIEW The first of several fungicides based on the benzimidazole structure was benomyl, introduced into agriculture in 1967. Benomyl became the fungicide of choice for control of a number of plant pathogens. For control of apple scab, caused by Venturia inae_a_u_alis (Cooke) Wint., benomyl admirably filled the void created by declining use of dodine after reports of dodine resistance in a number of apple growing regions (18,26). Perhaps foremost among the favorable features of benomyl was its systemic activity which made possible a degree of after infection control previously unknown for agricultural fungicides. Benomyl also fit well into newly evolving programs of integrated pest management which depend on a diversity of options in fungicide application. . Benomyl was used extensively for scab control with excellent results. The success was, however, short-lived. In Australia, after only three years of use, benomyl was found ineffectual for scab control (33). Similar experiences with the failure of benomyl and other benzimidazoles to control apple scab were reported worldwide: Japan (6), West Germany (28), Michigan (8), New Zealand (27), Poland (16), South Africa (19), Maine (15), and France (17). The pattern was invariably the same -,- three to four years after scab control with benzimidazoles was initiated, X. Wig possessed resistance to these fungicides. Studies of a number of fungi, both laboratory-induced mutants (1,2,5,29,30) and field isolates (7,9,10,13,22,23), have substantiated the view that benzimidazole resistance originates through genetic changes in the fungal cell. Initial investigations into the genetics of benzimidazole resistance were done in the laboratory with non-pathogens. This was a result of the advantages of using fungi which are well-understood and especially amenable to genetic study. Specific inquiries deemed important were: the relative ease with which benzimidazole resistance can be induced, often proposed as predictive of emergence of resistance in the field; the number of genes responsible for resistance; the levels of resistance which result from different genes or their interaciton. When other genetic markers were available, mapping or the placement of genes for resistance in known linkage groups was attempted. Thus, in 1971 Hastie and Georgopoulos (5) obtained nine benomyl-resistant mutants of Amgillus nidulans after UV-irradiation of conidia. Five of the mutants were highly resistant, while the remaining four were less resistant. In terms of response to benomyl relative to the wild-type, this amounted to about a 14-fold increase resistance for the high resistance group and a 3-fold increase for the low resistance group. Two mutants, one from each of the two levels of resistance, were used in crosses with other strains carrying genetic markers. From combined meiotic and mitotic analysis, it was concluded that the two levels of resistance were determined by two unlinked genes, respectively, which were located on known linkage groups relative to other markers. Recombinants having both genes for resistance failed to show additive effects of an increased level of resistance. This work was confirmed by van Tuyl (30) who found the same two genes for high and low resistance in UV-mutants of A. nidulans and additionally discovered yet a third gene conferring low resistance. Each of the three genes conferring benomyl resistance was mapped on a different linkage group. Van Tuyl's findings with regard to possible additive effects of combining more than one gene for resistance in the same isolate were, however, somewhat more complicated than those of Hastie and Georgopoulos. Recombinants containing both genes for low resistance had only a slight increase in resistance over the low resistance level. However, crosses with a negatively cross-resistant mutant, showing resistance to another benzimidazole, thiabendazole, and extra- sensitivity to benomyl, and either of the low resistance isolated produced recombinants having an intermediate resistance to benomyl and a somewhat higher resistance to thiabendazole. Borck and Braymer (2) found different levels of benomyl resistance in UV- induced mutants of Neurospora crassg but clear-cut categories were difficult to discern. Rather, a range in levels of resistance up to almost 100 times the level toxic to the wild-type was found among the 14 mutants tested. Genetic mapping of 15 resistant mutants showed that only a single gene for resistance was ' involved, and it was suggested that modifier genes played a role in determining varying degrees of resistance. From the data presented, neither the existence of multiple alleles, each conferring a different level of resistance, nor the possibility of cytoplasmic influence on the single gene for resistance could be discounted. Tests for benomyl resistance among heterokaryons of resistant and sensitive isolates showed resistance to be dominant and at-a level comparable to that found in the resistant member. The first investigations into the genetics of benzimidazole resistance of a plant pathogen was carried out by Jones and Ehret in 1976 (7). Benomyl- resistant isolates of Venturia inaequalis were obtained from Michigan orchards in which scab control with benomyl had failed. When tested on benomyl-amended media, high and low levels of resistance could be distinguished among these isolates. Although the genetic basis of this observation was not pursued, the monogenic inheritance of benomyl resistance in !. inaequalis was demonstrated by the 1:1 ratio of resistant and sensitive progeny produced in each of four crosses between resistant and sensitive isolates. Only a single high level of resistance was found among nine carbendazim- resistant Israeli isolates of Venturia pm the cause of pear scab (21). From an analysis of crosses involving five of the resistant isolates, it ws concluded that resistance is controlled by a single gene, since crosses between resistant and sensitive isolates yielded resistant and sensitive progeny in a 1:1 ratio, while crosses between different resistant isolates yielded only resistant progeny. Resistant progeny always showed the same level of resistance irrespective of whether one or both parents were resistant. This unifbrmity in response ruled out any contribution by modifying genes or cytoplasmic components. Cross- resistance to thiabendazole was also found. It was shown that the trait of carbendazim resistance was not linked to mating-type, but lacking an array of genetic markers as is possible for such fungi as A. nidulans or N. grasg, the subject was not investigated further. Kiebacher and Hoffmann (ll) analyzed a total of five crosses using 1. M isolates from West Germany; three crosses of benomyl-resistant and sensitive isolates resulted in 1:1 ratios of resistant and sensitive progeny, while a cross of two sensitive isolates produced only sensitive progeny, and a cross of two benomyl-resistant isolates produced only sensitive progeny, and a cross of two benomyl-resistant isolates produced only resistant progeny. These data are in full agreement with the idea of a single gene for benomyl resistance in 1. inggualg,’ and the absence of sensitive progeny from the cross of the two resistant isolates indicates that the same gene for resistance was present in the two parents. To account for the six different levels of benomyl resistance recognized in their earlier studies (12), the authors proposed the operation of modifying genes and/or cytOplasmic factors. Utilizing such traits as colony morphology, mycelial coloration, and sporulation, colonies of benomyl—resistant isolates could not be distinguished from those of sensitive isolates, and the examination of progeny from crosses between resistant and sensitive isolates differing in these respects showed no evidence of linkage. Likewise, as in the aforementioned case of l. M2 (21), it was shown that benomyl resistance in 1. ingualis is not linked to mating-type. Concurrent with the report from West Germany was a similar report from France in which Martin et al. (14) showed 1:1 segregation of resistance and sensitivity in the progeny of each of ten crosses between benomyl-resistant and sensitive French isolates of !. inagualis. Resistance was attributed to the action of a single gene, and although high and low levels of resistance were noted, the study only treated those isolates having high resistance. Following UV-irradiation of a conidial suspension from a benomyl-resistant isolate, a benomyl-resistant white mutant was obtained. In subsequent crosses the traits of white mycelium and benomyl resistance segregated independently showing that they were not linked. The genetic basis underlying different levels of benomyl resistance in l. ingualis was studied by Shabi et al. (22) using benomyl-resistant Israeli isolates of the fungus and later Katan et al. (9) who considered those same isolates along with benomyl-resistant isolates from New York. Recognizing four levels of resistance according to growth-rate and sporulation on benomyl-amended media, the inheritance of different levels of resistance was investigated. This work showed that discrete levels of resistance are heritable according to the rules of Mendelian segregation. Analyzing the progeny of twenty crosses made between resistant and sensitive isolates and among different resistant isolates, it was shown that different levels of resistance could be accounted for by different alleles of a single gene. Neither modifying genes nor cytoplasmic inheritance as suggested by Kiebacher and Hoffmann (13) were necessary in the explanation. The newest group of fungicides of importance are the sterol inhibitors. These chemicals offer promise in the control of numerous diseases. Most remarkable is their effectiveness at very low rates of application, and almost all possess some degree of systemicity which results in curative activity. In field trials, results of the use of sterol inhibitors against 1. 'naeggalis has been favorable (20). As yet, many sterol-inhibiting fungicides are still in the developmental stage, but it is expected that they will be used extensively on apple in the near future. Resistance to sterol-inhibiting fungicides in the field has not yet been confirmed. Adaptation to triforine has been obtained in the laboratory for some triforine-sensitive non-obligate plant pathogens, but tolerance was lost upon further subculturing on triforine-free medium (4). Increased levels of tolerance both in the greenhouse and the field have been reported for Erysiphe graminis f. sp. £1921 to tridomorph (31). While isolates varied both in level of tolerance and pathogenicity, tolerance was in most cases stable in laboratory culture (32). Under laboratory conditions, mutants showing permanent resistance to sterol inhibitors have been obtained for both pathogens and non-pathogens by either mutagenic treatment or by selection from wild-type populations. Triarimol- resistant mutants of Cladosporium cucumerinum and Aspergillus fumagatus were obtained from colonies growing in high concentration of the fungicide (25). Mutagenesis through UV irradiation has yielded triarimol-resistant mutants of _C_. cucumerinum, Verticilium albo-atrum (4), and Ustilago _m_§y_d_is (24). Similarly induced triforine-resistant mutants have been obtained for Q. cucumerinum (4). Nitrosoguanidine-induced mutagenesis has yielded isolates of Penicillium expansum resistant to imazalil (30). In a comparison of rates of spontaneous, UV-, and nitrosoguanidine-induced mutation for Aspergillus nidulans, imazilil resistance was found to occur much more frequently in all three cases than resistance to either benomyl or carbonxin (30). While resistance to sterol inhibitors is rather easily induced in the laboratory, the levels of resistance are generally lower than those obtained for many other fungicides. Van Tuyl (29) isolated imazalil-resistant mutants of Aspergillus nidulans having ED5O values 43 and 133 times greater than wild- types, respectively. As a rule, cross-resistance occurs among sterol-inhibiting fungicides and is interpreted as presumptive evidence for a similarity in the mode of action of these compounds. A number of cases of cross-resistance to sterol inhibitors have been summarized by Fuchs et al. (3). As yet, the only genetic study of resistance to sterol-inhibiting fungicides has been the work of van Tuyl (29,30) with imazalil-resistant mutants of A. nidulans. Among 21 mutants examined genetically, seven originated spontaneously, while eight were UV-induced and six were induced with nitrosoguanidine. All 21 mutants possessed single gene mutations of which 11 were allelic to one locus, four were allelic to a second locus, and six represented another six loci. Two mutants selected for cycloheximide resistance were found to be also imazilil-resistance and represent two additional loci. The total of ten genes for imazalil resistance was allocated to six of the eight linkage groups (chromosomes) of A. nidulans. As further shown in mapping, the genes for imazalil resistance were not clustered, but instead distributed throughout the genome. Different levels of resistance were found among the different genes as well as among their different allelic forms. Individually, single gene mutations produced relatively low levels of resistance with a maximum 10-fold increase in minimal inhibitory concentration over sensitive isolates. However, the combination of genes for resistance resulted in positive interaction. In one recombinant strain with two genes for resistance and a modifier gene, which while not conferring resistance itself enhanced the level of resistance, a lOO-fold increase over sensitive isolates was obtained in the minimal inhibitory concentration. In recent years, stringent governmental regulation and rising costs of development have diminished the availability of new fungicides. If the sterol inhibitors are suitable replacements for dodine and benomyl, as expected, they will be well received. To prevent the loss of yet other fungicides to resistance, it is clearly desirable to gain a greater understanding of the problem; its genetic basis, the concomitant physiological changes in the pathogen, and the epidemiology. Such understanding would aid in devising management strategies for delaying or preventing the development of resistance, and ultimately might contribute to the rational development of new fungicides for which resistance will be less likely. The research presented here contributes to an understanding of fungicide resistance in X. inaequalis. The objectives were to i) investigate the ' genetic basis for the different levels of benomyl resistance in X. inaequalis and ii) to determine if the reduced sensitivity to sterol-inhibiting fungicides in certain field isolates of V. inaequalis extended across a range of sterol-inhibiting fungicides and if this differential response was genetically'controlled. 2. 3. 10. ll. 12. LITERATURE CITED Ben-yephet, Y., Henis, Y., and Dinoor, A. 1974. Genetic studies on tolerance of carboxin and benomyl at the asexual phase of Ustilago__ hordei. Phytopathology 64:51-56. Borck, K., and Braymer, H. D. 1974. The genetic analysis of resistance to benomyl in Neurospora crassa. J. Gen. Micrbiol. 85:51-56. Fuchs, A., de Ruig, S. P., van Tuyl, J. M., and de Vries, F. W. 1977. Resistance to triforine: A nonexistent problem? Neth. J. P1. Path. 83 (Suppl. 1):l89-205. Fuchs, A., and Viets-Verweij, M. 1975. Permanent ' and transient resistance to triarimol and triforine in some phytopathogenic fungi. Meded. Fac. Landbouww. Rijksuniv. Gent. 40:699-706. Hastie, A. C., and Georgopoulos, S. G. 1971. Mutational resistance to fungitoxic benzimidazole derivites in Aspergillus sni_£l__ulans. J. Gen. Micro. 67:371-373. Iida, W. 1975. On the tolerance of plant pathogenic fungi and bacteria to fungicides in Japan. Jap. Pest. Inf. 23:13-16. - Jones, A. L., and Ehret, G. R. 1976. Tolerance to fungicides in Venturia and Monilinia of tree fruits. Proc. Am. Phytopath. Soc. 3:84-90. Jones, A. L., and Walker, R. J. 1976. Tolerance in Venturiaina Inaeggalis sto dodine and benzimidazole fungicides In Michigan. Plant Dis. Reprtr. 60:40- 44. Katan, T., Shabi, E., and Gilpatrick, J. D. 1983. Genetics of resistance to benomyl in Venturia inaggalis isolates from Israel and New York. Phytopathology 73 :600-603. Kiebacher, J. 1981. Genetic aspects of the benzimidazole resistance in a natural pOpulation of Venturia inagualis. Neth. J. P1. Path. 87:248-249. Kiebacher, H., and Hoffmann, G. M. 1976. Benzimidazole-Resistenz bei Venturia inaequalis. Z. Pflanzenkr. Pflanzenschutz. 83:352-358. Kiebacher, J., and Hoffmann, G. M. 1980. Qualitative und quantitive Untersuchungen zur Resistenz von Venturia inagggalis gegen Benzimidazol- Fungizide. Z. Pflanzenkr. Pflanzensch. 87:705-716. l3. 14. 15. 16. l7. l8. 19. 20. 21. 22. 23. 24. 25. 26. 27. 10 Kiebacher, J., and Hoffman, G. M. 1981. Zur Genetik der Benzimidazol- Resistenz bei Venturia inaequalis. Z. Pflanzenkr. Pflanzensch. 88:189-205. Martin, 0., Oliver, J. M., and Les inasse, Y. 1981. Obtention in vitro de peritheces de Venturia inaequalis Cke.) Wint.: application a l'analyse de la resistance au benomyl acquise au verger. Agronomie 1:745-749. McGee, D. C., and Zuck, M. G. 1981. Competition between benomyl- resistant and sensitive strains of Venturia inaequalis on apple seedlings. Phytopathology 71:529-532. Novacka, H., Karolczak, W., and Millikan, D. F. 1977. Tolerance of the apple scab fungus to the benzimidazole fungicides in Poland. Plant Dis. Reptr. 61:346-350. Olivier, J. M. 1979. Observations sur les souches de tavelures du pommier et du poirier resistantes aux benzimidazoles. Ann. Phytopathol. 11:135. Ross, R. G., and Newberry, R. J. 1977. Tolerance of Benturia inaequalis to dodine in Nova Scotia. Can. Plant Dis. Surv. 57:57-60. Schwabe, W. F. S. 1977. Tolerance of Venturia inaequalis to benzimidazole fungicides and dodine in South Africa. Phytophylactica 9:47-“. - Schwinn, R. J., and Urech, P. A. 1981. New approaches for chemical disease control in fruit and hops. Proc. Br. Crop Protect. Conf. Insect. Fungic. 3:819-833. Shabi, E., and Ben-yephet, Y. 1976. Tolerance of Venturia pirina to benzimidazole compounds. Plant Dis. Reprtr. 60:451-454. Shabi, E., and Katan, T. 1979. Genetics, pathogenicity, and stability of carbendazim-resistant isolates of Venturia pirina. Phytopathology 69:267- 269. Shabi, E., Katan, T., and Marton, K. 1983. Inheritance of resistance to benomyl in isolates of Venturia inaequalis from Israel. Plant Pathol. 32:207-212. Sherald, J. L., Ragsdale, N. N., and Sisler, H. D. 1973. Similarities between the systemic fungicides triforine and triarimol. Pestic. Sci. 4:710- 727. Sherald, J. L., and Sisler, H. D. 1975. Antifungal mode of action of triforine. Pestic. Biochem. Physiol. 5:447-488. Szkolnik, M., and Gilpatrick, J. D. 1969. Apparent resistance of Venturia w to dodine in New York apple orchards. Plant Dis. Reptr. 53:861- 864. Tate, K. G., and Samuels, G. J. 1976. Benzimidazole tolerance in Venturia inggualis in New Zealand. Plant Dis. Reprter. 60:706-610. 28. 29. 30. 31. 32. 33. ll Vagt, W. 1975. Die Schorfsituation 1974 und unsere Spritzempfehlungen fur 1975. Mitt. Obstbauversuchsring. Alten Landes 30:76-80. van Tuyl, J. M. 1977. Genetic aspects of resistance to imazilil in Aspergillus nidulans. Neth. J. P1. Path. 83 (Suppl. 1):l69-176. van Tuyl, J. M. 1977. Genetics of fungal resistance to systemic fungicides. Meded. Landbouwhogesch. Wageningen 77:1-136. Walmsley-Woodward, D. J., Laws, F. A., and Whittington, W. J. 1979. Studies on the tolerance of Erysiphe graminis f. sp. hord__e_i to systemic fungicides. Ann. appl. Biol. 92: 199-209. Walmsley-Woodward, D. J., Laws, F. A., and Whittington, W. J. 1979. The characteristics of isolates of Erysiphe graminis f. sp. hordei varying in response to tridemorph and ethirimol. Ann. appl. Biol. 92:211-219. Wicks, T. 1974. Tolerance of thea a9ple scab fungus to benzimidazole fungicides. Plant Dis. Reprtr. 58 .886-8 PART I GENETICS OF BENOMYL RESISTANCE IN VENTURIA INAEQUALIS FROM NORTH AND SOUTH AMERICA, EUROPE, AND NEW ZEALAND 12 ABSTRACT Fifty-nine benomyl-resistant isolates of Venturia inaequalis from Australia, Chile, Germany, Italy, New Zealand, and five states in the United States were compared with sensitive isolates for growth on benomyl-amended media. Isolates which failed to grow on media amended with 0.1 ug/ml benomyl were considered sensitive. Resistant isolates were divided into three phentotypes: low resistance, isolates grew on media amended with l ug/ml but not with 10 ug/ml benomyl; medium resistance, growth at 10 ug/ml but not at 25 ug/ml benomyl; and high resistance, good growth on media amended with up to 500 ug/ml benomyl. Genetic analysis of 23 resistance isolates corroborates recent reports that‘different levels of resistance are determined by alleles of a single gene and supports the conclusion that although benomyl resistance in 1. inaequalis developed independently in several areas of the world, resistance was determined by the same gene in each area. No linkage was detected between the gene for benomyl resistance and four genetic markers for auxotrophy or three colony color markers. 13 INTRODUCTION Excellent control of Venturia inaggualis (Cooke) Wint., the pathogen causing apple scab, was possible when benzimidazole fungicides became available for use on apple (M3133 sylvestris Mill.). However, in Michigan and in many other apple growing regions of the world, benzimidazoles became ineffective for scab control about 3 years after their introduction (3,5,11,13-15,l7-20). An explanation for the failure of benzimidazoles came with the demonstration of heritable resistance in isolates of l. 'nagualis from orchards where these fungicides were ineffective. Investigations in Michigan (4), Germany (10). France (12), and Israel (6,17) have all attributed benomyl resistance in y_. inaefllis to mutations in a single gene. To explain widely differing levels of resistance among different resistant isolates (4,8,13,18), the influence of modifying genes and cytoplasmic factors was suggested (10), but recent work provides evidence for a multi-allelic system in which different alleles of a single gene confer different levels of benomyl resistance (6,17). In this study, 59 benomyl-resistant isolates of V. inaequalis from Australia, Chile, West Germany, Italy, New Zealand, and five states in the United States were tested for their level of resistance. Twenty-three of the resistant isolates were analyzed genetically to determine if resistance is always determined by the same gene regardless of the source of the isolate. Linkage between the gene conferring benomyl resistance and other genetic markers was also investigated, using auxotrophic and colony color mutants. 14 MATERIALS AND METHODS Cultures. The original culture designation and geographical origin of each isolate used in this study is given in Table 1. Several sources provided benomyl- reistant isolates, but the majority were provided by E. I. duPont de Nemours 8: Co., Wilmington, DE 19898. The laboratory-induced mutants used in the linkage study were supplied by D. M. Boone, University of Wisconsin, Madison. To ensure genetic purity, a single conidium or hyphal tip was taken from each original culture and used to start a stock culture. Stock cultures, used in all testing and crosses, were maintained at 18°C and subcultured by transfer of single conidia every 5-7 weeks. Media. Cultures were grown on modified potato-dextrose agar (modified PDA: 40 g potatoes steamed 45 min in 200 ml distilled water and then homogenized, 17 g agar, 5 g dextrose, and sufficient distilled water to make a final volume of l L) (1). To make a substrate suitable for inducing the sexual phase of l. inaequalis, a decoction of overwintered apple leaves was included in the modified PDA as described by Keitt and Langford (7). All benomyl-amended media were prepared from Difco PDA (Difco Laboratories Inc., Detroit, MI 48201). Benomyl (Benlate 5096 WP, E. l. dePont de Nemours dc Co., Wilmington, DE 19898) was added to the media prior to sterilization in the autoclave at 121°C for 20 min. Minimal medium for testing the auxotrophic mutants was prepared with distilled water according to Boone et al. (2) as follows (g/L): KNOB, 3.12; K HPO 0.75; KH2P04’ 0.75; MgSOa'7HZO, 0.5; NaCl, 0.1; 2 4’ CaC12°H20, 0.1; dextrose, 5; trace element solution, 1 ml, agar, 17. The trace 15 16 Table 1. Variation in levels of benomyl resistance among isolates of Venturia inaequalis subjected to a series of benomyl concentrations in potato-dextrose agar Level of Isolate and Origin Resistanceb Source U.S.A., Michigan was»)a s A. L. Jones FS6C-12 (+) S A. L. Jones WRR (-) S A. L. Jones WM (-) S A. L. Jones WL (+) S A. L. Jones SPINKS 79 (-) LR E. I. duPont de Nemours 6t Co. RH-4 (-) HR E. I. duPont de Nemours 6: Co. RH1-11B(-) HR E. I. duPont de Nemours & Co. SHl-9B (-) HR E. I. duPont de Nemours 6: Co. SH2-10B HR E. 1. duPont de Nemours 6: Co. SH2-9B HR E. I. duPont de Nemours 6: Co. KV3C (4) HR A. L. Jones CR2C (-) HR A. L. Jones GAVIN 43 HR E. I. duPont de Nemours 6: Co. VAN DRESSEN 1 HR E. I. dIIPont de Nemours 6: Co. PAJ-l3C-P3 HR E. I. duPont de Nemours dc Co. FS-l7C-P3 HR E. I. duPont de Nemours 6: Co. VAN DRESSEN 2 HR E. I. duPont de Nemours & Co. NU-26 DPX-5 HR E. I. duPont de Nemours cit Co. BG—7C HR E. I. duPont de Nemours 6: Co. 17 Table 1. (cont.) BIGLER HR E. I. duPont de Nemours 6c Co. AB-2C-Bl HR E. I. duPont de Nemours 6: Co. FP-TC 139-4 HR E. I. duPont de Nemours 6: Co. SHl-2B HR E. I. duPont de Nemours 6: Co. U.S.A., Minnesota MINN FRUIT ACRES 7 (-) HR E. I. duPont de Nemours 6: Co. MINN 6-1B HR E. I. duPont de Nemours 6: Co. MINN LAUTZ 4 (-) HR E. I. duPont de Nemours 6: C5. OLD HICKORY 9-2 HR , E. I. duPont de Nemours 6: Co. MINN 5C HR E. I. duPont de Nemours 6c Co. MINN 5-3B HR E. 1. duPont de Nemours 6: Co. LAUTZ 10-5 HR E. 1. duPont de Nemours 6c Co. MINN 8-2B HR E. I. duPont de Nemours & Co. OLD HICKORY 6-3 HR E. I. duPont de Nemours 6: Co. MINN 543 HR ' ' E. I. duPont de Nemours 6: Co. MINN 3 HR E. I. duPont de Nemours 6: Co. U.S.A., New York #42 MINNS 118 (-) LR J. D. Gilpatrick #74 PRESTON 7 (+) LR J. D. Gilpatrick #6 MITCHELL 94-1 (-) LR J. D. Gilpatrick #32 MINNS 86-8 HR J. D. Gilpatrick #60 MINNS 86-8 HR J. D. Gilpatrick VAN DUSER 3 HR E. I. duPont de Nemours ck Co. VERDINE 2 HR E. I. duPont de Nemours 6: Co. U.S.A., Maine V-558 (+) MR M. Zuck Table 1. (cont.) SIS-16 (+) V-560 (+) V-565 (+) MAINE 8 (+) MAINE 6. (+) MAINE 23 (+) U.S.A., North Carolina N. C. STATE 1 298 Italy 15-1 (-) l7-1 (+) 14-1 16-1 11-1 New Zealand 75-78 75-150 (+) 75-71 75-26 Australia RI RIII West Germany GERMANY G3-2 (-) Chile CHILE 24B (+) MR MR MR MR MR MR HR HR HR HR HR LR HR HR HR HR HR HR MR M. Zuck M. Zuck M. Zuck E. I. duPont de Nemours 6: Co. E. I. duPont de Nemours 6: Co. E. I. duPont de Nemours & Co. T. B. Sutton E. I. duPont de Nemours at Co. E. I. duPont de Nemours 6: Co. E. I. duPont de Nemoursdt Co. E. 1. duPont de Nemours 6: Co. E. I. duPont de Nemours 8: Co. G. J. Samuels G. J. Samuels G. J. Samuels G. J. Samuels E. I. duPont de Nemours 6: Co. E. I. duPont de Nemours 6: Co. E. I. duPont de Nemours 6: Co. E. I. duPont de Nemours 6: Co. 19 Table 1. (cont.) Unknown origin 5 HR . E. I. duPont de Nemours 6c Co. Laboratory-induced mutantsC U-2340 PYRIMIDINE—3 (-) S D. M. Boone U-3010 PURINE-8 (-) S D. M. Boone U-2599 NICOTINIC ACID-l S D. M. Boone U-3069 ARGININE-3 (-) S D. M. Boone U-Z570 BIOTIN-I S D. M. Boone I906 PYRIMIDINE-G S D. M. Boone U-l907 PYRIMIDINE-Z S D. M. Boone 1261 HISTIDINE-l S D. M. Boone U-l905 CHOLINE (-) S D. M. Boone U-2129 PURINE-3 S D. M. Boone N-l56 WHITE-2 (-) S D. M. Boone N-l BROWN-l S D. M. Boone N-282 BROWN S D. M. Boone N-l5l PALE S D. M. Boone N-10 GREEN (+) S D. M. Boone N-222 YELLOW (+) S D. M. Boone aMating-type of isolates used in crosses is included in parentheses. bstensitive to benomyl at 0.1 ug/ml; LR = low resistance, growth at l ug/ml but not at 10 ug/ml; MR = medium resistance, growth at 10 ug/ml but not at 25 ug/ml; HR = high resistance, growth at 500 ug/ml. cFirst 10 mutants listed are auxotrophs and remaining 6 mutants are color mutants. 20 element solution was prepared as follows (mg/400 ml): ZnSOQ°7HZO, 58.4; CuSOa'SH o, 31.6; MnSO '4H 4 20, 16.2; HBO 3 3, 11.4; MoO 2 7.0; Fe(C6HSO7)°3HZO, 214.2. 3’ Benomyl resistance and auxotrophy. To determine levels of benomyl resistance, all isolates were subjected to a series of benomyl concentrations in PDA ranging from 0.1-1200 ug/ml benomyl. Two methods were used. In the first method, a 2- to 3-week-old colony was excised from its agar substrate, macerated in 4 ml of sterile distilled water with a 7 ml tissue grinder, and 0.5 ml aliquots of the homogenate were spread over the surface of benomyl-amended and unamended PDA in 60 X 15 mm petri plates. Growth was evaluated after 3 weeks and rated relative to the unamended control. In the second method, bits of mycelium (<1 mm in diameter) were taken from the mid-radium area of 2- to 3-week-old colonies and inoculated onto benomyl-amended and unamended PDA plates. Twenty-four isolates per petri plate were tested simultaneously at each concentration. Growth was evaluated after 3 weeks. Resistant isolates were retested 3-5 times to confirm that the level of resistance was stable from test to test. Using bits of mycelium as inoculum, all isolates were tested for growth on minimal medium to confirm the presence of nutritional deficiencies in the auxotrophic mutants and the prototrophy of field isolates. gm. Matings were made according to the procedure of Keitt and Langford (7) except colony homogenates rather than spore suspensions were used as inoculum. Individual 2- to 3-week-old colonies were removed from their agar substrates and macerated in 2 ml sterile distilled water. To make the crosses, a 0.5 ml aliquot of homogenate of each member of the cross was added to a standard 100 X 15 mm petri plate, 30 ml of warm (42°C) modified PDA with apple leaf decoction was added, and the homogenate and agar were mixed by swirling the plate. Inoculated petri plates were incubated 7-10 days at 20°C in 21 an incubator until mycelial growth was evident on the surface of the agar, then sealed with Parafilm (American Can Co., Greenwich, CT 06830), inverted, and held at 8°C for 5-6 months as required for pseudothecia formation and ascospore maturation. All isolates used in crosses were also selfed to confirm self- incompatibility. Testing of progeny. From each fertile pairing, 12-20 pseudothecia were removed from the agar substrate with the aid of a needle and a dissecting microscope and crushed in 1 ml of sterile distilled water using a 7 ml tissue grinder. Portions of the resultant ascospore suspension were plated on 296 water agar and incubated at about 21°C for 24-48 hours. Randomly chosen germinated ascospores were transferred to individual culture tubes containing modified PDA. After 2-3 weeks growth, the single-ascospore colonies were tested on PDA with 0, l, 10, and 500 ug/ml benomyl, using bits of mycelium for inoculum as described earlier. Progenies from crosses with auxotrophic mutants were also tested on minimal medium to determine if they were prototrophic or auxotrophic. RESULTS Levels of benomyl resistance. Testing of isolates on benomyl-amended PDA resulted in the identification of four readily distinguishable groups based on the concentration of benomyl required to inhibit the growth of each isolate (Table 1). Isolates were considered sensitive (S) if they failed to grow on media amended with 0.1 ug/ml benomyl. This group included five isolates from Michigan and the 16 laboratory-induced mutants. Among the 59 resistant isolates, five grew on media amended with 1 ug/ml but not with 10 ug/ml benomyl, eight grew at 10 ug/ml but not at 25 ug/ml benomyl, and 46 showed good growth on media amended with up to 500 ug/ml benomyl. The three phenotypes were designated as having low resistance (LR), medium resistance (MS) and high resistance (HR), respectively. Analysis of crosses. None of the isolates produced pseudothecia when selfed, confirming their self-incompatibility. All 212 ascospores from three crosses among five sensitive isolates were sensitive to 1 ug/ml benomyl (Table 2, Fig. 1A). To determine whether resistance was controlled by more than one gene, 4922 ascospores were examined from 25 crosses of 21 different resistant isolates with sensitive isolates. In all but one cross, resistant and sensitive progeny were found in a 1:1 ratio, and in each cross the level of resistance of the resistant progeny and the resistant parent always corresponded regardless of the level of resistance involved (Table 2, Fig. 1B). This indicates that in each of the LR, MR, and HR isolates tested, resistance was determined by a single Mendelian gene. In the cross KV3C X WRR (HR X S) segregation deviated from 22 23 35 w: NE SN .5. x 88 >z<2mmu 25 a. a: :N «.5 x T: 8... ea c: on .5 x 7: who 3 «2 RN 5 x a mmmu< Esme 22$ 2; «2 e: an 5 x Ta 2 .n ”2 2. ea ~55 x main :4 3 8 e2 .5 x 27:3 :5 e: N: NAN 5 x 27:3 35 mm. a: «a 5 x .12 mxm: S S .5 x .5 8 ON «.5 x 5 e: e: 55 x 2.0%... m x m AA u a 3 m a.— 52 a: .33. «.3880 mo:_m> NX 2:30:25 A65 883 Demo...“ ans—smug mtaucu> mo 3:8on oceanoomm wcoEm 005330.. 380:3 8m Sfiemoemom .N 03m... 24 RN onm mat—cm x UNMU. mm x a: N: R s. as 5 x 73 350:: we 2 .N .3 R m: ”:5 x R 22.8% is $5 .3 NA em 5 x R 9.25 mxma 35 2 9 we 55 x .3 52.2 S .o as 2 a 55 x e ~22: Rd 3 R we ”15 x Sn; 2 .o as 2 a 55 x SA; :3 E R SN 55 x 2.5 R; n:. e: «N 55 x new new; :5 E e: SN 55 x 3m m.=:U mxas B; m: L as :N 55 x .55 and a: 5 ea 5 x s N55 22;. was 8. s: E 5 x 026 3.... 3 e2 RN .5 x 026 2 .m e: «a «a .5 x on: mnK— mm and new dd? X Un>¥ 25 :6 n: o: n- Owl—<2 X UNmU e S . e. 8 2.5. x 026 8R 3 m: RR ”R; x 020 Ree RR. .2 RR 3R 3:5 x 02.0 25 RR. 5 . RR 5.. 350 x 028 a: x «2 RR RR . Ta 320:: 2 x R 20.5.5.“ .5. e2 . e2 R 20.55.. 5.. x «R 92:3 5 x a.— RS .RL: s N55 22:2 x mix». 3 me 325 x RAU >z<2mmo Ra RR R 8mu< :3: 22:2 x 3-5m RRR RRR mafia x 72 SR SR 545 x 27:2 . :R 5 mafia x .12 3 3 BAR x 026 2: 8. 092 x «-3 >z<2amu RR «2 025. x 026 onw OMN 72 X UNMU 26 .Ge a sane E 8...; esteem... 60:333.. 32 X 8537.9. €3.02: u d.— X mi... .5235..." X 8:333. 53... u m X a: .5533 X ozxmcom u m X m :04 63505.3 Banana 3 wcmueooom con—seem 0.5 memmEUm . :.N :4 3.0 ca.— 36 3.— wk me mm nu me he mm mm mm mm m: an n2 ma RN— 3— mm mo— Tea 4.51052 w§ X new MAEU Tea 4.51—0.52 w§ X omnt> a: mZZ—E Na; X .395 Tea JANICE—S 0* X nwnt> m.— X ~22 R 20.5me an‘ X UNMU Tea AflIUH—E o§ X Um>v_ m..— X a: Figure l. 27 Progeny of Venturia inaeggalis tested on potato dextrose agar amended with O, l, 10 and 500 ug/ml benomyl. Twenty-four progeny per plate were evaluated simultaneously at each concentration using bits of mycelium from single-ascospore colonies. Examples show growth 3 weeks after inoculation with progenies from four crosses; A) sensitive X sensitive, B) high resistance X sensitive, C) low resistance X low resistance, D) high resistance X medium resistance. «\- vO'O' 28 h'.| . ‘1'. x I I l't'..~\ I ... ...\H ' I . . ‘ J; Vol‘.‘ I i ,0 O ' f K ,ng,’7 . . ‘ g. I“ m.“ ‘ I. - \\ l 1 l ' l V l / ' ,0... 4.x , l ' ' $1 71 ,u.. 9 . .;.;Ut 't'/: .1; x... O C......x1 " .F— 11.9- 0000 "V .—( “— ,. 4:001,” 0006“” H— \ ~— \\ :.. :l// ..Q I/ \ ‘ ._ ‘\\ ~ _ 1, u 35 ‘ 1&9; +“w' ...'\;'. U. 3 -. . 9.. “o ..' ‘0. ‘ to out". xi: 29 a resistant:sensitive ratio of 1:1. However, no deviation from expected ratios was observed in other crosses involving KVBC. ' From 23 crosses between 21 different resistant isolates, 3702 ascospores were examined to determine whether the mutations for benomyl resistance occurred at the same locus in all isolates. No sensitive progeny were detected in any cross between resistant parents (Table 2). When the parents had the same level of resistance, all of the progeny had a corresponding level of resistance (Fig. 1C), and when the parents had different levels of resistance, the same two levels were found in the progeny in a 1:1 ratio (Table 2, Fig. 1D). The absence of any sensitive recombinant or any recombinant showing higher levels of resistance than the parents indicates allelism at a single locus. To investigate possible linkage of the gene for benomyl reistance with other genes, each of the 10 auxotrophic and six color mutants were crossed with each of the HR isolates of opposite mating type, KV3C and CRZC. Fertile pairings were obtained with seven mutants, but no indication of linkage between any genetic markers and the gene conferring benomyl resistance was found. Each of the crosses of resistant isolates with the auxotrophic mutants U- 23l$0 PYRIMIDINE-3, U-30lO PURINE-8, and U-l905 CHOLINE yielded the four phenotypes in approximately a ratio of l:l:l:l, indicating an independent assortment of the markers (Table 3). Segregation of progeny of the cross with U-3069 ARGININE-3 deviated from a l:l:l:l ratio, but the recovery of similar numbers of progeny in the parental and recombinant classes indicates that the b_eg_ and a_r_g-3 markers recombined freely. With crosses involving the color mutants N-lO GREEN and N-222 YELLOW, progeny of parental and recombinant phenotypes were recovered in a ratio of l:l:l:l (Table 3). In a cross with N-156 WHITE-2, whitish pseudothecia were produced in abundance, but they were often void of asci or contained mostly 3O D + muwcm $.N: mm con + ”S numZ—Z_Um< mwonua X Um>¥ on con ozo mm + + 3 + 9.0 :6— 9. con + RN MZSOIU 32-: X Un>¥ 2 con mica @N + + o— + wing 9.2 cm con + 3 mumzaam Scan: X Um>v_ mm con «LE NM + + me + mica mmfi 3 con + an. mum—Z—o=2_~;m oanmu: X Un>¥ *A“ w _u _ a C .3832 non—50:25 cone—E 339.0 mo:_m> NX omflocozq an coflmwoamom .33. .333 acumen. 320on £3335 3.3:»; 95:3 9.0th otocow o5 3557.8 382.2. com cotmwocwom .n 038. 31 n: a 85 u a a 33> 8625. wnJm Cain m—.N S 2 no me mm mm en ow on em 3 :2. can con can con con :2. N-,? N-,; o.» o> cw cw muwcm : oaN :nw mum—:13 wnfinz X Um>v_ BOAAM> NNNnZ X UNmU mem0 072 X UNmU 32 four-spored asci. All ascospores were pale and thin-walled, and the majority failed to germinate. The two white colony phenotypic classes were absent, indicating that the 1v_h_-2 mutation was lethal to those progeny which inherited it. Progeny of the other two phenotypic classes were recovered in similar numbers indicating free recombination. DISCUSS ION When 59 benomyl-resistant isolates of \_l_. inaggualis from Australia, Chile, Germany, Italy, New Zealand, and five states in the United States were screened for variation in the level of benomyl resistance, three phenotypes were distinguished. The three phenotypes correspond well to those described by Katan et al. (6) in a study of l. in§_egualis from Israel and New York, except HR isolates were not subdivided into an additional very highly resistant phenotype based on growth rate. The predominance of HR isolates among the isolates screened for resistance may be a consequence of the technique used in collecting the primary isolates and therefore not accurately reflect the distribution or prevalence of the LR and MR strains in natural populations. For example, media containing 5 or 25 ug/ml benomyl were commonly used to monitor for resistant strains (4,21). At 25 ug/ml benomyl the LR and MR strains would not be detected and at 5 ug/ml benomyl the LR strains would not be detected. Fifty-one crosses involving 23 different resistant isolates were analyzed. The results show that discrete levels of resistance are inherited according to Mendelian rules. In crosses between resistant and sensitive isolates or between isolates having different levels of resistance, only parental phenotypes in a ratio of 1:1 were recovered among the progeny. In several crosses using the same resistant isolate with a different sensitive partner, the expression of the gene for resistance was not altered by the genetic background of the sensitive parent. Crosses between resistant isolates having the same level of' resistance produced 33 34 only resistant progeny of the same phenotype. It is concluded that the three levels of benomyl resistance are determined by three alleles of a single Mendelian gene. Benzimidazole-resistant and sensitive isolates of X. inaegualis are indistinguishable from each other in culture except on media amended with benzimidazole fungicides. Kiebacher and Hoffmann (9,10) attempted to correlate traits such as colony morphology, mycelial coloration, sporulation, and mating-type with resistance, but the examination of progeny from crosses between resistant and sensitive isolates differing in these traits provided no evidence for their linkage with benomyl resistance. Martin et al. (12) isolated a benomyl-resistant white mutant following UV-irradiation of a benomyl-resistant isolate. Genetic analysis of this mutant showed that the traits of white mycelium and benomyl resistance segregated independently. Similarly, Shabi et a1. (17) found no evidence of linkage of the gene for benomyl resistance and a gene for green colony color. Sixteen laboratory-induced mutants were utilized in an attempt to localize the gene for benomyl resistance relative to other genetic markers. Failure to obtain fertile pairings with nine of the mutants is attributed to physiological disorders of these mutants, since the HR isolates with which they were crossed were suitable partners in many other matings. Analysis of . progenies of crosses between resistant isolates with four auxotrophic and three colony color mutants, did not reveal linkage between any of the genetic markers and the gene for benomyl resistance. Progenies of crosses with U-3069 ARGININE-3 and N-156 WHITE-2 failed to show the 1:1:1:1 phenotypic ratios, but parental and recombinant phenotypes were found in similar numbers indicating free recombination between markers. The two aberrant phenotypic ratios can be related to the abortion of ascospores carrying the mutant alleles. 35 Ascospore abortion with mutant N-156 WHITE-2 has been reported previously (22). Genetic analysis of a large number of isolates from a wide geographical range fully corroborate the recent finding (6,17) that different levels of benomyl resistance in X. inaequalis are controlled by different alleles of the same gene and are not the result of interactions between different genes for resistance. In a comparison of two y, 'naeggalis isolates, one showing low benomyl resistance and the second with an isolate showing high benomyl resistance, Shabi and Gilpatrick (16) reported that the two isolates did not differ in pathogenicity and that both caused typical scab lesions on benomyl-sprayed apple seedlings. Consequently, in contributing to the failure of benzimidazole fungicides to control apple scab, the level of resistance may be less important than the fitness of the particular resistant strain involved. l. 5. 6. 10. ll. 12. LITERATURE CITED Boone, D. M., and Keitt, G. W. 1956. Venturia inaequalis (Cke.) Wint. VIII. Inheritance of color mutant characters. Am. J. Bot. l0:226-233. Boone, D. M., Stauffer, J. P., Stahmann, M. A., and Keitt, G. W. 1956. Venturia inaegialis (Cke.) Wint. VII. Induction of mutants for studies on genetics, nutrition, and pathogenicity. Am. J. Bot. 433199-204, Iida, W. 1975. On the tolerance of plant pathogenic fungi and bacteria to fungicides in Japan. Jap. Pest. Inf. 23:13-16. Jones, A. L., and Ehret, G. R. 1976. Tolerance to fungicides in Venturia and Monilinia of tree fruits. Proc. Am. Phytopathol. Soc. 3:84-89. Jones, A. L., and Walker, R. J. 1976. Tolerance of Venturia inaegalis to dodine and benzimidazole fungicides in Michigan. Plant Dis. Rep. 60:40-04. Katan, T., Shabi, E., and Gilpatrick, J. D. 1983. Genetics of resistance to benomyl in Venturia inaequalis from Israel and New York. Phytopathology 73:600-603. Keitt, G. W., and Langford, M. H. 1941. yinturia inaequalis (Cke.) Wint. I. A groundwork for genetic studies. Am. J. Bot. 28:805-820. Kiebacher, J., and Hoffmann, G. M. 1980. Qualitative and quantitative Untersuchungen zur Resistenz von Venturia inaequalis gegen Benzimidazol- Fungizide. Z. Pflanzenkr. Pflanzenschutz 87:705-716. Kiebacher, J., and Hoffman, G. W. 1981. Zur Entwicklung des Perfektstadiums von Venturia inaequalis in vitro. Z. Pflanzenkr Pflanzenschutz 88:1-8. Kiebacher, J., and Hoffman, G. M. 1981. Zur Genetik der Benzimidazol- Resistenz bei Venturia ingualis. Z. Pflanzenkr. Pflanzenschutz 88:189- 205. McGee, D. C., and Zuck, M. G. 1981. Competition between benomyl- resistant and sensitive strains of Venturia inaggalis on apple seedlings. Phytopathology 71:529-532. Martin, D., Olivier, J. M., and Lespinasse, Y. 1981. Obtention in vitro de peritheces de Venturia inaequalis (Cke.) Wint.: application a l'analyse de la resistance au benomyl acquise au verger. Agronomie 1:745-749. 36. 13. I4. I5. l6. 17. I8. 19. 20. 21. 22. 37 Novacka, H., Karolczak, W., and Millikan, D. F. 1977. Tolerance of the apple scab fungus to the benzimidazole fungicides in Poland. Plant Dis. Rep. 61:346-350. Olivier, J. M. 1979. Observations sur les souches de tavelures du pommier et du poirier resistantes aux benzimidazoles. Ann. Phytopathol. 11:135. Schwabe, W. F. S. 1977. Tolerance of Venturia inaequalis to benzimidazole fungicides and dodine in South Africa. PhytOphylactica 9:“7‘5“. ' Shabi, E. and Gilpatrick, J. D. 1981. Competition between benomyl- resistant and benomyl-sensitive strains of Venturia inaggalis on apple seedlings treated with benomyl and captan. (Abstr.) Neth. J. Plant Pathol. Shabi, E., Katan, T., and Marton, K. 1983. Inheritance of resistance to benomyl in isolates of Venturia inaequalis from Israel. Plant Pathol. 32:207-211. Tate, K. G., and Samuels, G. J. 1976. Benzimidazole tolerance in Venturia ingualis in New Zealand. Plant Dis. Rep. 60:706-710. Vagt, W. 197 5. Die Schorfsituation 1974 and unsere Spritzempfehlungen fur 1975. Mitt. Obstbauversuchsring. Alten Landes 30:76-80. Wicks, T. 19714. Tolerance of the apple scab fungus to benzimidazole fungicides. Plant Dis. Reptr. 58:886-889. Yoder, K. S. 1978. Methods for monitoring tolerance to benomyl in Venturia inaequalg,‘ Monilinia spp. Cercospora spp., and selected powdery mildew fungi. Pages 18-20 in: Methods for Evaluating Plant Fungicides, Nematicides, and Bactericides. E. I. Zehr (ed.). American Phytopathological Society, St. Paul, MN. 141 pp. Yoder, K. S., Klos, E. J., Nowacka, H., and Bielenin, A. 1982. Inheritance of an ascospore abortion factor in Venturia inagualis. Can. J. Bot. 60:2105-2111. PART II REDUCED SENSITIVITY TO STEROL-INHIBITING FUNGICIDES IN FIELD ISOLATES OF VENTURIA INAEQUALIS 38 ABSTRACT . For eight sterol-inhibiting fungicides, minimal inhibitory concentrations for preventing colony formation by individual conidia were a to 8 times higher for Venturia ingualis isolates from one West German orchard than for isolates from a second West German orchard and from orchards in the United States. Reduced sensitivity was exhibited to BAS (45¢ 06 F (I-(2,4-dichlorophenyl(-2-(1H- l,2,4-triazol-1-yl)), bitertanol, CGA 71818 (l-I 2-(2,#-dichloropheny1)-penty1] - 1H-l,2,lt-triazole), DPX H6573 (bis (1+-fluorophenyl)methyl-(1H-1,2,4-triazol-1-yl- methyl)silane), etaconazole, fenarimol, Ro l5-1297, and triflumizole. Isolates with reduced sensitivity to sterol inhibitors did not show increased sensitivity to dodine. Dodine, sodium lauryl sulfate, and the respiratory inhibitor carbonyl cyanide 3-chlorophenylhydrazone did not ‘potentiate fenarimol toxicity toward either sensitive isolates or isolates with reduced sensitivity. Genetic analysis of nine isolates with reduced sensitivity, involving 3261 ascospores from 15 crosses, indicates that reduced sensitivity is determined by a single Mendelian gene. 39 INTRODUCTION Several sterol-inhibiting fungicides are now available for agricultural use, and a number of others are being developed. These fungicides are a chemically diverse group of compounds which specifically inhibit ergosterol biosynthesis (12,16). They are effective at very low concentrations against a broad spectrum of higher fungi, and almost all exhibit some degree of systemicity. Sterol- inhibiting fungicides have proven useful in the control of several important apple diseases (13). With excellent postinfection activity, these compounds offer much needed alternatives to dodine and to the benzimidazole fungicides whose use has declined following the development of resistance in Venturia inagualis (Cooke) Wint., the cause of apple scab. Due to their favorable characteristics, sterol- inhibiting fungicides will probably be used extensively on apple in the near future. The specific mode of action common to the sterol inhibitors suggests the potential for problems of resistance under conditions of intense field use (3-5). Under laboratory conditions, mutants resistant to sterol-inhibiting fungicides are readily induced and cross-resistance often occurs (1,6,8,ll&,15). Fenarimol resistance in the non-pathogen Aspggillus nidulans (Eidam) Wint. was reported to be negatively correlated with dodine resistance (7). If this same relationship were to occur with pathogens in the field, there would be the possibility of using mixtures of alternative applications of dodine to combat the problem of resistance. 40 41 Reduced sensitivity to tridemorph has been reported in strains of Erysiphe graminis f. sp. h_o_rfle;i_ collected from the greenhouse and field (18), but as yet, there is no report of reduced sensitivity to sterol-inhibiting fungicides in natural strains of X. inaequalis. Recently, isolates of y_. inaequalis were obtained from the Federal Republic of Germany which formed sporulating colonies on media amended with sterol-inhibiting fungicides, while isolates from other sources failed to grow at similar fungicide concentrations. This study was conducted to determine if the differential response of these isolates was stable in culture and extended across a range of sterol-inhibiting fungicides. Also, it was determined whether this reduced sensitivity to the sterol-inhibiting fungicides was inherited by Mendelian rules. MATERIALS AND METHODS Cultures. Twelve monoconidial isolates of V. inaequalis, taken from separate leaf lesions, were obtained from the Federal Republic of Germany. Nine of the isolates, designated Bl...B9, were collected from one apple orchard, and three isolates, designated W10...W12, were from a second orchard. Sterol- inhibiting fungicides had been used experimentally in both orchards. Among four monoconidial isolates from Michigan, WB and WRR were opposite mating types and GAVIN 29 and GAVIN 32 were dodine-resistant. The Michigan isolates were collected from orchards where no sterol-inhibiting fungicides had been used. Isolates were grown at 18°C on modified potato-dextrose agar (modified PDA: 40 g potatoes steamed 45 min in 200 ml distilled water and then homogenized, 17 g agar, 5 g dextrose, and distilled water to make a final volume of l L) (2). At 4-6 wk intervals single conidia were subcultured. Fungicides and chemicals. The fungicides used in this study were: l-(2,4- dichlorophenyl)-2-(lH-l,2,4-triazol-1-yl) (BAS 454 06 F 2596 EC) from BASF Wyandotte Corp., Parsippany, NJ 07054; bitertanol (Baycor 2596 WP) from Bayer AG, Leverkusen, Federal Republic of Germany; l-[2-(2,4-dichlorophenyl)- pentyl]-lH-l,2,4-triazole (CGA 71818 1096 WP) and etaconzaole (Vangard 1096 WP) from Ciba-Geigy Corp., Greensboro, NC 27409; dodine (10096 technical grade) from American Cyanamid Co., Princeton, NJ 08540; bis(4-fluorophenyl)- methyl(1H-l,2-4-triazol-l-ylmethyl)silane (DPX H6573 4096 EC) from E. I. duPont de Nemours & Co., Inc., Wilmington, DE 19898; fenarimol (Rubigan 12.596 42 43 EC) from Eli Lilly and Co., Greenfield, IN 46140; Ro 15-1297 (4896 EC) from Maag Agrochemicals, Vero Beach, FL 32960; triflumizole (A-815 3096 WP) from Uniroyal, Naugatuck, CT 06770; and triforine (Funginex 18.296 EC) from EM Laboratories, Hawthorne, NY 10532. Sodium lauryl sulfate (SLS) and carabonyl cyanide 3-chlorophenylhydrazone (CCCP) were purchased from Sigma Chemical Co., St. Louis, MO 63178. Minimal inhibitory concentrations. Minimal fungicide concentrations needed to inhibit colony formation were determined by plating conidia on 296 water agar and after 12 to 24 hours transferring individual germinated conidia to 100 X 15 mm petri plates containing PDA (Difco Laboratories Inc., Detroit, MI 48201) amended with a series of fungicide concentrations. Concentrations ranged from 0 to 5 ug/ml for all fungicides except triforine which was in the range of 20 to 44 ug/ml. Using 9596 ethanol to assist in dissolution, fungicides were added to molten PDA (42°C) after sterilization. The final ethanol concentration never exceeded 196 in either treatment or control. Up to 12 isolates were tested simultaneously in each petri plate (Fig. l). Colony growth was evaluated after 3 weeks incubation at 20-22°C. Dodine sensitivity. Response to dodine was tested using two methods. In the first method, conidia were germinated on 296 water agar and transferred individually to dodine amended PDA.) Dodine concentrations ranging from 0 to 1.0 ug/ml in 0.1 ug/ml increments were prepared by adding dodine in ethanolic solution to molten PDA (42°C) after sterilization. The concentration of ethanol never exceeded 196 in either treatment or control. Dodine resistant isolates GAVIN 29 and GAVIN 32 were included for comparison. Colony growth was evaluated after 3 weeks. In the second method, isolates were compared for inhibition of growth around assay discs treated with 0, 50, and 300 ug/ml dodine. Sterile 13 mm assay discs (Schleicher and Schuell, Inc., Keene, NH 03431) were 44 saturated with 100 ul of distilled water or dodine solution and allowed to dry. For each isolate tested, a 2- to 3-week-old colony was excised from its agar substrate and homogenized in 4 ml of sterile distilled water using a 7 ml tissue grinder. Aliquots (0.5 ml) of homogenate were used to seed the surface of PDA in each of three 100 X 15 mm petri plates, and a treated disc was placed in the center of each plate with light pressure to ensure contact with the agar. Diameters of zones of inhibition were measured after 25 days incubation at 20- 22°C. Interaction of fenarimol and other chemicals. To investigate the effect of certain chemicals on fenarimol toxicity, the crossed paper strip method was used (7). Filter paper strips (1 cm wide) were dipped into a methanolic solution of 1487 ug/ml fenarimol and then allowed to dry. Similarly, strips were prepared with 14,300 ug/ml dodine, 57,600 ug/ml SLS, and 307 ug/ml CCCP. Strips were transferred to the surface of PDA plates previously seeded with 0.5 m1 aliquots of colony homogenate and incubated 3 days at 20-22 'C. In each plate, a fenarimol treated strip was crossed with one of the other chemically treated strips. After an additional 14 days incubation, zones of inhibition about the strips were measured near the edge of the petri plate and near the center where the strips crossed. (_25_os_se§_. Isolates were crossed using the procedure of Keitt and Langford (9), except colony homogenates rather than spore suspensions were used as inoculum. Individual 2- to 3-week-old colonies were removed from their agar substrates and homogenized in 2 m1 of sterile distilled water using a 7 ml tissue grinder. A 0.5 ml aliquot of homogenate of each member of the cross was added to a 100 x 15 mm petri plate, 30 ml of warm (42°C) modified PDA with apple leaf decoction (9) was added, and the homogenate and agar were mixed by swirling the plate. The plates were incubated 7-10 days at 18°C until mycelial 45 growth was evident on the surface of the agar, then sealed with Parafilm (American Can Co., Greenwich, CT 06830), inverted, and held at 8°C for 5-6 months as required for pseudothecia formation and ascospore maturation. All isolates used in crosses were also selfed to confirm self-incompatibility. Testing oLprogenies. For each fertile pairing, 12-20 pseudothecia were removed from the agar substrate with the aid of a needle and a dissecting microscope and crushed in 1 ml of sterile distilled water. Portions of the resultant ascospore suspension were plated on 296 water agar and incubated at 20-22°C for 12-24 hours. Randomly chosen germinated ascospores were transferred to individual culture tubes containing modified PDA. After 2-3 weeks growth, bits of mycelium ( < 1 mm in diameter) were taken from the mid- radius area of the colonies and inoculated onto PDA and PDA amended with 1.5 ug/ml fenarimol. Growth was evaluated after 3 weeks incubation at 20-22°C. Selected single-ascospore colonies were also tested for sensitivity to other sterol-inhibiting fungicides, for sensitivity to dodine, and for their response to fenarimol in the presence of dodine, SLS, and CCCP. RESULTS Minimal inhibitory concentrations. Although colony size of all isolates tested diminished with increasing fungicide concentration, isolates Bl...B9 grew at higher concentrations of each sterol inhibitor except triforine than did isolates W10, W11, W12, WB, WRR, GAVIN 29, and GAVIN 32. Single-ascospore isolates derived from crosses between sensitive and less sensitive isolates behaved either like their sensitive or less sensitive parent. Within the less sensitive and sensitive groups, the response of difference members to different concentrations of each sterol inhibitor except triforine was uniform (Fig. 1, Table 2). In an amendment series with 32, 36, 40, and 44 ug/ml triforine, fewer and fewer colonies were produced, but differences in growth between less sensitive and sensitive isolates were inconsistent in four repetitions of the test. Concentrations for differentiating those isolates with reduced sensitivity were selected for each sterol inhibitor except triforine (Fig. 1, Table 1). At these concentrations, sensitive isolates fail to grow, while less sensitive isolates form colonies. Except on Ro 15-1297 amended media, sporulation of colonies was observed in 3-4 weeks. Response to dodi_n£. When germinated conidia were transferred to dodine amended PDA, growth of isolates withireduced sensitivity to sterol inhibitors did not differ from the growth of sensitive isolates. Both groups showed the same diminution in colony size with increasing dodine concentration and cessation of growth at 0.2-0.3 ug/ml dodine. The dodine-resistant isolates, GAVIN 29 and GAVIN 32, grew at 0.5 and 0.7 ug/ml dodine but not 1 ug/ml. 47 Table 1. Minimal fungicide concentrations for preventing colony formation by germinated conidia of Venturia inaequalis with two levels of sensitivity to sterol- inhibiting fungicides. Suggested discriminatory concentrations inhibit colony formation by conidia from sensitive isolates but not by conidia from isolates with reduced sensitivity to sterol-inhibiting fungicides Minimal inhibitory concentration Suggested ( 11 g] m1)a discriminatory Sensitive Isolates with concentration Fungicides isolates reduced sensitivity (ug/ml) BAS 454 06 F 0.4 2.0 0.9-1.0 bitertanol 1.0 5.0 0.5-1.0 CGA 71818 0.2 0.8 ' 0.3-0.4 etaconazole 0 . l 0 . 8 0 . 2 DPX H6573 0.1 0.6 0.2 fenarimol 0.4 3.0 0.5-1.0 Ro 15-1297 0.1 0.4 0.2 triflumizole 0.2 l. 0 0. 3-0.4 triforine > 32.0 > 32.0 —-- 3Data are the results of numerous tests with field isolates that exhibited reduced sensitivity to these fungicides and with sensitive field isolates, as well as with single-ascospores isolates derived from crosses between sensitive and less sensitive strains. 48 Table 2. Inhibition of mycelial growth surrounding assay discs treated with two concentrations of dodine and placed on agar seeded with sensitive isolates of Venturia inaeggalis or with isolates that exhibited reduced sensitivity to sterol- inhibiting fungicides Inhibition zone (mm)a 50 ug/ ml dodine 300 11g] m1 dodine Sensitive Less sensitive Sensitive Less sensitive Cross isolates isolates isolates isolates B4 X WRR 21.0 (14.6) 17.4 (13.1) 31.8 (14.4) 28.6 (13.8) BS X WB 18.2 (11.8) 15.0 (11.0) 32.0 (11.0) 23.6 (13.3) B6 X WRR 18.6 (12.5) 15.0 (11.9) 29.2 (13.8) 24.2 (14.8) B7 X WRR 14.4 (11.7) 14.4 (11.5) 23.2 (15.8) 23.0 (12.0) B8 X WRR 17.4 (14.0) 15.2 (13.0) 27.2 (14.5) 25.4 (12.9) 39 x WRR 22.0 (12.9) 18.0 (13.3) 36.8 (17.3) 27.3 (13.3) aMean diameters and standard deviations are given for zones of inhibition of five sensitive isolates and five less sensitive isolates from each cross. Values include 13 mm diameter of assay disc. Figure l. 49 Response of Venturia inaequalis tested on potato dextrose agar amended with 0, 0.2, 0.4, and 0.6 @g/ml BAS 454 06 F. Five isolates with reduced sensitivity to BAS 454 06 F and four sensitive isolates were tested simultaneously at each concentration. Three weeks after inoculation with single germinated conidia, isolates with reduced sensitivity but not sensitive isolates showed growth at the two higher concentrations. 51 In tests with assay discs, no zones of inhibition developed around discs treated with water. Around discs treated with solutions containing dodine, growth was inhibited. At both concentrations of dodine, zones of inhibition were slightly less for the isolates with reduced sensitivity to the sterol inhibitors than for sensitive isolates (Table 2). However, according to the t test, differences in mean diameters of zones of inhibition for the two groups were not significantly different at E = 0.05. 1 Effect of chemicals on fenarimol toxicity. Tests were conducted with sensitive isolates, less sensitive isolates, and single-ascospore isolates derived from crosses between sensitive and less sensitive isolates. Zones of inhibition were observed around all strips except the water treated controls (Fig. 2). Around fenarimol treated strips, zones of inhibition were less distinct but wider than zones around dodine, SLS, and CCCP treated strips. Less sensitive isolates were inhibited less than sensitive isolates around fenarimol treated strips. Around dodine, SLS, and CCCP treated strips, no significant differences in the relative widths of zones of inhibition for sensitive and less sensitive isolates were observed. The three test chemicals did not alter the toxicity of fenarimol toward either sensitive or less sensitive isolates. Analysis of crosses. None of the isolates produced, pseudothecia when selfed, thereby confirming their self-incompatibility. Single-ascospore isolates derived from fertile pairings were readily separated into two groups based on their growth on PDA containing 1.5 ug/ml fenarimol. At this concentration, sensitive isolates failed to grow, while less sensitive isolates formed colonies. When crosses were made between sensitive isolates, isolates W10, W11, W12 were fertile with WRR, indicating that WB, W10, W11 and W12 were of the same mating-type (Table 3). All 361 single-ascospore isolates from the three crosses among four sensitive isolates were sensitive. 52 Table 3. Segregation of single-ascospore isolates of Venturia inaequalis into less sensitive and sensitive phenotypes according to growth on potato-dextrose agar amended with 1.5 ug/ml fenarimol Progeny tested (no.) Phenotype ' Less X2 Values Cross Total sensitive Sensitive (1:1)a Sensitive isolates X Sensitive isolates W10 X WRR 90 0 90 W11 X WRR 99 0 99 W12 X WRR 172 0 172 Totals 361 0 361 Less, sensitive isolates X Sensitive isolates Bl X WB 252 142 110 4.06 BZ X WB 228 97 131 5.07 B3 x was 96 a3 53 1.0:: B4 X WRR 250 116 134 1.30 B5 X WB 96 68 28 16.70 B6 X WRR 230 97 133 5.63 B7 X WRR 224 124 100 2.57 B8 X WRR 244 103 141 5.92 B9 X WRR 247 136 111 2.53 Totals 1867 918 949 0 .12 53 Table 3. (cont.) Less sensitive isolates X Less sensitive isolates Bl X BB 229 229 0 Bl X B4 246 246 0 BZ X B3 240 240 B2 X B4 - 246 246 0 B3 X B5 248 248 0 B4 X B5 185 185 0 Totals 1394 1394 0 3The expected Chi-square value at the 196 level of significance is 6.63. Figure 2. 54 Effects of dodine, sodium lauryl sulfate (SLS), and carbonyl cyanide 3-chlorophenylhydrazone (CCCP) on the toxicity of fenarimol to Venturia inaequalis. Plates a-d were seeded with an isolate sensitive to sterol-inhibiting fungicides, while plates e-h were seeded with an isolate exhibiting reduced sensitivity to sterol inhibitors. Vertical strips were treated with fenarimol and horizontal strips a,e with distilled water; b,f with dodine; c,g with SLS; and d,h with CCCP. Inhibition zones around fenarimol treated strips were characterized by a lack of sporulation rather than a complete inhibition of growth. The lack of sporulation is not visible in the photographs although it was clearly visible in the original plates. 55 56 To determine whether reduced sensitivity was controlled by more than one gene, 1867 single-ascospore isolates were tested from nine crosses between sensitive and less sensitive isolates (Table 3). Isolates Bl, B2, and B5 were compatible with WB, and isolates B3, B4, B6, B7, B8, and B9 were compatible with WRR. In all but one cross, sensitive progeny were found in a 1:1 ratio with less sensitive progeny. The only ratio deviating from the 1:1 ratio was that of cross B5 x WB. To determine whether the mutations for reduced sensitive to sterol- inhibiting fungicides occurred at the same locus, crosses between less sensitive isolates were examined (Table 3). A total of 1394 single-ascospore isolates were tested from six compatible pairings resulting from all possible pairings of isolates Bl...B5. No sensitive progeny were detected. From the analysis of crosses between sensitive and less sensitive isolates and between the less sensitive isolates it was concluded that in the isolates studied, reduced sensitivity to the sterol-inhibition. fungicides was determined by a single Mendelian gene. The gene for reduced sensitivity was not linked to mating type. DISCUSSION The concentration of sterol-inhibiting fungicides necessary to inhibit the growth of the West German 1. 'naegualis isolates Bl...B9 ranged from 4 to 8 times that necessary to inhibit the growth of V. inaggalis isolates from a second West German orchard and from orchards in the United States. This differential response in sensitivity was not lost or changed in culture. The level of variation between less sensitive and sensitive isolates was comparatively small, and in this respect much like the difference between dodine-resistant and sensitive V. inaegyglis (11) and unlike some benomyl-resistant l. inaeggalis which grow at several hundred times the benomyl concentration which inhibits sensitive isolates (10). The variation in the sensitivity of !. inaequalis to the sterol inhibiting fungicides indicates a potential for the selection of less sensitive strains under orchard conditions. Using the suggested discriminatory concentrations given in Table I, it should be possible to monitor conidial populations for shifts in sensitivity for establishing the incidence of strains with reduced sensitivity. The inconsistent behavior of _V_. inagggalis on triforine amended media was manifested in two ways. Firstly, while the other eight sterol inhibitors prevented colony formation at concentrations of 5 ug/ml or less, concentrations in excess of 32 ug/ml were needed to prevent colony formation with triforine. Secondly, sensitive isolates could not be differentiated from less sensitive isolates in an amendment series. Despite evidence that triforine produces the accumulation of the same sterol intermediates as a number of other sterol inhibitors including fenarimol (l7) and reports of cross-resistance between 57 58 triforine and other sterol inhibitors (8, l5, 16), isolates Bl...B9 for some unexplained reason did not exhibit a reduced sensitivity to triforine. De Waard and van Nistelrooy (7) reported that two laboratory-induced fenarimol-resistant mutants of Aspergillus nidulans were slightly more sensitive to dodine than a wild-type fenarimol-sensitive strain of the fungus. In our studies, strains of X. 'naggualis with reduced sensitivity to fenarimol and sensitive strains were equally sensitive to_ dodine, and no correlation between reduced sensitivity to sterol inhibitors and increased dodine sensitivity was observed. This suggests that with V. 'naegualis dodine may not provide any particular benefit in combating population shifts toward reduced sensitivity to sterol inhibitors. Also, dodine, SLS, and CCCP potentiated the toxicity of fenarimol toward both sensitive wild-type and fenarimol-resistant mutants of A. nidulans (7). However, in our studies no similar potentiation of fenarimol toxicity was observed toward sensitive isolates or toward isolates with reduced sensitivity to fenarimol. The differences between the results with V. inaequalis and _A_. nidulans may be explained in part} by the fact that reduced sensitivity in _V_. 'naeggalis was controlled by a single gene, while a multigenic system existed for A. nidulans (17). It is still too early to make unequivocal statements concerning the significance of reduced sensitivity to sterol inhibitors in V. inaeggalis. As has been pointed out (4), an outbreak of disease depends on the overall fitness of a pathogen. However, the isolates with reduced sensitivity were obtained from infected apple leaves and thus were pathogenic. Since reduced sensitivity was controlled by a single gene, the potential for a buildup of these strains in the presence of prolonged exposure to sterol-inhibiting fungicides is greater than if several genes were required (3-5). In culture, these isolates grew and sporulated well, which suggests they may have sufficient fitness for survival among 59 populations containing sensitive strains. Based on current data, it seems likely that if sterol-inhibiting fungicides are used exclusively in apple scab control programs, there will be a shift in the sensitivity of the pathogen population. A possible (method for proventing this shift in sensitivity is to use field rates for these fungicides that are sufficiently high to control both sensitive and less sensitive strains. lo. 11. 12. LITERATURE CITED Barug, D., and Kerkenaar, A. 1979. Cross-resistance of UV-induced mutants of Ustilago maydis to various fungicides which interfere with ergosterol biosynthesis. Meded. Fac. Landbouwet. Rijksuniv. Gent. 44:421- 427. Boone, D. M., and Keitt, G. W. 1956. Venturia inaequalis (Cke.) Wint. VIII. Inheritance of color mutant characters. Am. J. Botany 43:226-233. Dekker, J. 1976. Acquired resistance to fungicides. Ann. Rev. Phytopathol. 14:405-428. Dekker, J. 1976. Prospects for the use of systemic fungicides in view of the resistance problem. Proc. Am. Phytopathol. Soc. 3:60-66. Delp, C. J. 1980. Coping with resistance to plant disease. Plant Dis. 64:652-657. de Waard, M. A., and Gieskes, S. A. 1977. Characterization of fenarimol- resistant mutants of Aspergillus nidulans. Neth.'J. Pl. Path. 83 (Suppl. 1):l77-188. de Ward, M. A., and van Nistelrooy, J. G. M. 1982. Antagonistic and synergistic activities of various chemicals on the toxicity of fenarimol to Aspergillus nidulans. Pestic. Sci. 13:279-286. Fuchs, A., de Ruig, S. P., van Tuyl, J. M., and de Vries, F. W. 1977. Resistance to triforine: a nonexistent problem. Neth. J. P1. Pathol. 83 (Suppl. 1):189-205. Keitt, G. W., and Langford, M. H. 1941. Venturia inagglis (Cke.) Wint. I. A groundwork for genetic studies. Am. J. Etany 28:805-820. Keibacher, J., and Hoffman, G. M. 1980. Qualitative and quantitative Untersuchungen zur Resistenz von Venturia inagggalis gegen Benzimidazol- Fungizide. Z. Pflanzenkr. Pflanzenschutz 87:705-716. Polach, F. J. 1973. Genetic control of dodine tolerance in Venturia inaggualis. Phytopathology 63:1 189-1190. Ragsdale, N. N. 1977. Inhibitors of lipid synthesis. Pages 333-363 in: Antifungal Compounds, Vol. II. M. R. Siegel and H. D. Sisler (eds.) Marcel Deld