WWW mama ; j Hammer-m mama: j _ manna-sacraasm . .~ _ VENTURIMMEQUAUS _ w ‘ r ,DisSertation fori‘the Degree of Ph. 'D. MtCHIGAN STATE UNIVERSITY “KEITH‘SEMIYGDER ’ _ j11914.3 ' _ ‘ University This is to certify that the thesis entitled TOLERANCE TO DODINE AND INHERITANCE OF AN ASCOSPORE ABORTION FACTOR 'IN VENTURIA INAEQUALIS presented by Keith Sem Yoder has been accepted towards fulfillment of the requirements for .__P.h.._D.._ degree in _P.lan.t_.Ea.thology E i Q . W Q, professor Date__£1_l_LLL_J.9_LlL_ 0-7639 LIBRARY ° Michigan State ‘ . Wmmuml irifi'hav HUAB 3 SflNS' BGUK BlNDERY INC. LIBRARY Bl NDERS ABSTRACT TOLERANCE T0 DODINE AND INHERITANCE OF AN ASCOSPORE ABORTION FACTOR IN VENTURIA INAEQUALIS By Keith Sem Yoder Tolerance to dodine (Cyprex) in Venturia inaequalis, first reported in New York in l969, could have serious consequences on the control of apple scab in Michigan because of similar growing conditions and considerable dodine usage. Monoconidial isolates of v, inaequalis, collected in 1969 from dodine-treated and non—treated situations in Michigan, were compared with New York isolates by growth inhibition zones surrounding dodine-treated paper assay discs on potato-dextrose agar, germination of conidia in water solutions of dodine on glass slides, and growth inhibition by dodine in malt extract broth. Normal isolates were inhibited by a dodine concentration of approximately 1 ug/ml. The least sensitive Michigan isolates tolerated twice the inhibitory dodine concentration of normal isolates and were about one-half as tolerant as the least sensitive New York isolates. The tolerance levels of Michigan isolates tested did not correlate with the degree of control by dodine in orchards from which the isolates were taken. Isolates with different levels of tolerance were crossed in_ vitro and progeny were isolated as ordered tetrads, unordered tetrads, Keith Sem Yoder or random ascospore progeny. In crosses of the most tolerant isolate with normal isolates, the presence of tetratype asci having four dis- tinct levels of tolerance to dodine suggests the additive action of at least two major independent genes conditioning the level of tolerance to dodine. This hypothesis is strengthened by the presence of highly tolerant and highly sensitive recombinant progeny from a cross of two isolates of the two intermediate tolerance levels in tetratype asci. The presence of some progeny slightly more tolerant than either of two parents of normal sensitivity may suggest a recombina- tion of several minor genes affecting the level of tolerance to dodine. In crosses of highly tolerant parents and in a cross of a normal isolate with one of an intermediate tolerance level, no progeny were significantly more tolerant than the most tolerant parent, or more sensitive than the most sensitive parent. Testing of progeny from crosses of a green color mutant with isolates representing intermediate and high levels of tolerance indi- cated that there was no close linkage between the genes for dodine tolerance and the green color gene of a known 2, inaequalis linkage group. An inoculation experiment with isolates of high and low sensitivities showed significant differences in control of infection on trees treated with dodine at 200 ug/ml, but symptoms produced by the two isolates were not uniform. Later experiments with isolates producing uniform symptoms showed that the tolerance levels of the isolates were factors in the control of infeCtion on trees treated with dodine at 4 ug/ml but not at l0 ug/ml dodine. Keith Sem Yoder Evidence suggests that inoculum density, temperature, and nutrient conditions may affect the tolerance levels of isolates. Such factors could explain the poor correlation of the dodine tolerance levels of isolates with the degree of control by dodine in situations where the isolates originated. Because of its possible effect on inheritance of tolerance to dodine, an ascospore abortion factor arising from SR4, the most tolerant isolate, was further investigated. The presence of F1 progeny as tolerant as SR4 but permitting normal development in test crosses indicated that the factors causing ascospore abortion were not the same as the genes conditioning higher levels of tolerance to dodine. The effects of ascospore abortion in progeny of isolate SR4 are similar to some previously reported cases in other fungi. Evidence supports both genetic and environmental influences in the induction of ascospore abortion. The strongest evidence supporting a genetic in- fluence is the recurrence of certain ratios of ascus types of crosses of SR4 X normal isolates in intercrosses of F1 progeny and test crosses of F1 progeny with normal isolates. The high, variable frequency of asci having odd numbers of spores is not readily explained genetically and suggests that cultural conditions may influence ascospore abortion. TOLERANCE TO DODINE AND INHERITANCE OF AN ASCOSPORE ABORTION FACTOR IN VENTURIA INAEQUALIS By Keith Sem Yoder 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 1974 T0 ESTA ii ACKNOWLEDGMENTS I express my sincere gratitude to Dr. E. J. Klos who provided guidance and the opportunity to learn and grow throughout this course of study. I appreciate the advice and effort of my guidance committee, Dr. N. G. Fields, Dr. C. w. Laughlin, and Dr. J. L. Lockwood in thier review of the manuscript. Special thanks are due to Dr. Halina Nowacka and Anna Bielenin for sharing their skills and patience in the isolation and testing of the ordered ascospore tetrads and to the technical assistance of So-Yung Jane Chai, Mary Pontoni, and Kassandra Keever. I am indebted to Dr. D. M. Boone, University of Wisconsin for his helpful suggestions and contribution of Venturia inaequalis color mutants, to Dr. E. B. Williams, Purdue University for offering advice and sharing culture techniques, and to Dr. J. D. Gilpatrick, New York Agricultural Experiment Station for donating infected apple leaves. The love, understanding, and patient assistance of my wife, Esta, have been most important to the completion of this project. The encouragement and forbearance of our parents and families is appreci— ated. The support of the Michigan State Agricultural Experiment Station and American annamid Company is gratefully acknowledged. TABLE OF CONTENTS PART I TOLERANCE T0 DODINE IN VENTURIA INAEQUALIS INTRODUCTION ......................... LITERATURE REVIEW ...................... MATERIALS AND METHODS .................... Isolation from leaves ................... Inoculum production .................... Testing of isolates .................... Crossing of isolates and isolation of ascospores ...... Greenhouse studies . . ................... Analysis of data ...................... RESULTS ........................... Screening of field isolates ................ Crossing of isolates .................... Testing of F1 progeny ................... Crosses of F progeny ................... Significance of ascospore abortion in inheritance of dodine tolerance ................... Non-linkage of dodine tolerance genes and green color gene ..................... Inoculation studies . . . ................. Effect of temperature on germination of conidia in dodine .................... DISCUSSION .......................... Tolerance levels of isolates .. ............... Inheritance of tolerance .................. Mechanisms for tolerance to dodine ............. Fungicide tolerance as a factor in control of apple scab ...................... LITERATURE CITED ....................... iv Page to oooouasuim U1 u N PART II INHERITANCE OF AN ASCOSPORE ABORTION FAgTOR ARISING FROM VENTURIA INAEQUALIS ISOL TE SR4, AN'ISOEATE_HI NT TO DODINE INTRODUCTION ........................ LITERATURE REVIEW ..................... MATERIALS AND METHODS ................... Crossing and isolation of progeny ............ Ascospore counts ..................... Perithecia on leaves ................... Staining of asci ..................... RESULTS .......................... Apparent types of abortion ............... Frequency of abnormal asci in crosses of SR4 with normal isolates .................. Ascospore abortion in crosses of FA progeny of SR4 . . . . Ascospore abortion induced by fiel isolates with normal dodine sensitivity . . ......... Differences in individual perithecia of a single cross I I : Ascospore abortion in crosses involving color mutants Induction of ascospore abortion by New York isolates . . Z : Ascospore abortion in natural perithecia from New York . . . DISCUSSION ..................... . . . . Evidence suggesting genetic influence on ascospore abortion ....................... Evidence suggesting non- genetic influence on ascospore abortion ................... Significance of induction of ascospore abortion by Poray Orchard isolates ............... LITERATURE CITED ...................... APPENDICES Al Inhibition of Venturia inaequalis field isolates in potato-dextrose agar by dodine diffusing from paper assay discs ..................... Page 53 54 56 58 6l A2 A3 A4 A5 A6' A7 A8 A9 Bl 82 B3 Inhibition by dodine of germination and growth of conidia of monoascosporic progeny of Venturia inaequalis cross SR4 X SR7 ............... Inhibition of random progeny of Venturia inaequalis cross O4SR4-l-2 X 04SR4-6-l in potato-dextrose agar by dodine diffusing from paper assay discs Inhibition by dodine of germination and growth of conidia of tetrad progeny of Venturia inaequalis cross SR4 X O4SR4-3-2 ................. Inhibition by dodine of germination of growth of conidia of tetrad progeny of Venturia inaequalis cross C2 X O4SR4-5-2 ................. Inhibition by dodine of germination of growth of conidia of tetrad progeny of Venturia inaequalis cross C2 X O4SR4-7-2 ................. Inhibition of random progeny of Venturia inaequalis cross S4 X C25R4 7—4 in potato-dextrose agar by dodine diffusing from paper assay discs ........ Inhibition of random progeny of Venturia inaequalis cross Sl X 2295-2 (green color mutant) in potato- dextrose agar by dodine diffusing from paper assay discs ...................... Inhibition of random progeny of Venturia inaequalis cross C25R4-7-3 X 2295-2 in potato-dextrose agar by dodine diffusing from paper assay discs ...... Frequency of ascospore abortion in crosses of F1 progeny of 04 X SR4 .................. Frequency of ascospore abortion in crosses of normal isolates with selected isolates similar to SR4 in dodine tolerance ................... Frequency of ascospore abortion in crosses involving color mutants ..................... vi Page 67 69 7O 71 72 73 74 76 77 79 80 Table 10. LIST OF TABLES Page Inhibition of Venturia inaequalis in potato-dextrose agar by dodine diffusing from paper assay discs ..... lO Growth of mycelia of selected Venturia inaequalis isolates after 2 wk incubation at 19 C in malt extract broth ...................... ll Inhibition by dodine of germination and growth of conidia of the ordered monoascosporic cultures of a tetratype ascus of Venturia inaequalis cross 04 X SR4 ........................ 13 Control of infection by Venturia inaequalis isolates 04 (normal dodine sensitivity) and SR4 (high dodine tolerance) on trees treated with dodine (Cyprex 65W) ...................... l9 Control of sporulating lesions of Venturia inaequalis isolates H6 (normal dodine sensitivity) and HGSR4 6-l (high dodine tolerance) on trees treated with dodine (Cyprex 65W) ...................... 20 Effect of inoculum density of Venturia inaequalis isolate 04 (normal dodine sensitivity) on apple scab control by dodine (Cyprex 65W) ............. 21 Per cent germination of conidia of two Venturia inaequalis isolates on PDA and in water at indicated temperatures ............ . ......... 22 Frequency of ascospore abortion in crosses of SR4 with normal isolates ............. . ...... 45 Frequency of ascospore abortion in test crosses of F1 progeny of O4 and SR4 .................. 46 Frequency of ascospore abortion in crosses of SR4 X normal isolates and in test crosses of F1 progeny of SR4. . . . vii Table Page ll. Further reduction of the number of spores per ascus by crosses of aborting color mutants with aborting F1 progeny of SR4 ............ 51 12. Frequency of ascospore abortion in crosses involving New York isolates .............. 52 viii LIST OF FIGURES Figure 1. Inhibition of isolates from ascus tetrads of SR4 X normal crosses by dodine diffusing through potato-dextrose agar from paper assay disc ...... 2. Frequency distribution of dodine inhibition zones of 7l progeny of cross O4SR4-l-2 X 04SR4-6-l, isolates representing the two intermediate tolerance levels in tetratype asci .......... 3. Effect of temperature on dodine inhibition of germination of conidia of two Venturia inaequalis isolates ........... . ........... 4. Ascospore abortion in progeny of Venturia inaequalis isolate, SR4 . . . . . ................ ix Page l4 l5 PART I TOLERANCE TO DODINE IN VENTURIA INAEQUALIS INTRODUCTION With the advent of more highly selective fungicides, fungi- cide resistance by plant pathogens may occur more frequently (7, ll). The tolerance of Venturia inaequalis (Cke.) Wint. to dodine is an example of this problem. Since its introduction in the mid- 1950's this organic fungicide has effectively controlled a narrower range of target organisms than many of the earlier fungicides. Dodine has been remarkably effective as a fruit fungicide for the control of apple scab (y, inaequalis) and cherry leaf spot (Coccomyces hiemalis). In 1969 Szkolnik and Gilpatrick (35) reported the failure of dodine to control apple scab in certain areas of New York State while other standard fungicides gave adequate control. This failure was attributed to fungal resistance acquired over a ten years' exposure to dodine. This report stimulated the present work in Michigan where growing conditions and practices are similar to those of New York State. The purpose of this study was to determine the dodine toler- ance levels of a group of natural isolates of Venturia inaequalis, to test the inheritance of dodine tolerance, and to observe the effects of the dodine tolerance level on control of the apple scab fungus. LITERATURE REVIEW Natural tolerance of v, inaequalis to dodine, first appearing in l969 (35), has been the subject in recent reports (12, 36, 37, 38). This tolerance was shown to be inherited under the control of at least two genes (29, 30, 40). Ultraviolet-induced resistance to dodine in Nectria haematococca var. cucurbitae (15) was controlled by four mutant genes which had an additive effect on the degree of resistance when combined in progeny (16). Natural variations have been detected in sensitivity of v, inaequalis to copper sulfate (25, 32), Paris green (25), captan, dichlone, glyodin, phenyl mercury acetate, and sulfur (32). An ultra- violet mutant grew on a medium containing a concentration of antimycin- A 5000 times greater than that required to inhibit the nonirradiated strain (19). Attempts to develop strains of v, inaequalis resistant to captan (26), thiram, ferbam, ziram or zineb (27) by culturing the fungus in successively higher rates of the toxicants were unsuccessful. A wild type isolate developed adaptive tolerance to dodine after con- tinuous culture on a medium containing dodine, but this tolerance was lost after brief culture on a dodine-free medium (22). The rate of 6 spontaneous mutation to dodine tolerance of about one in 10 conidia was not affected by ultraviolet irradiation (22). Fungal species differ greatly in sensitivity to dodine (2, 6, 23). In Fusarium solani f. sp. phaseoli a terminal decrease in reten- tion of 14C-labelled dodine was attributed to a modification of the dodine compound and subsequent release of a less toxic labelled compound (3). Although a slight terminal decrease in retention was also noted in Y, inaequalis, evidence explaining the loss was lacking. Studies of the mode of action of dodine have suggested that it competes for anionic binding sites (5, 33), alters membrane perme- ability (5, 33), and inactivates certain vital enzyme systems (5). The inhibitory effects of dodine on natural apple scab de- velopment include the prevention of scab infection by inhibition of spore germination (l, l3, l4, 24, 3l), prevention of sporulation of established lesions (l, 14, 24), and a darkening and thickening of the cell walls in the subcuticular stroma (l4). The maintenance of adequate surface residue is imperative to apple scab control with dodine (24). Inhibition of spore germina- tion is dependent upon the amount of dodine available per spore, within the normal range of 1.7—2.6 X 10'6 ug/conidium (23, 24). Residue dis- appearance studies have demonstrated a rapid loss of dodine surface residue during the first week after application, followed by a slower rate during subsequent weeks (10, 24, 34). MATERIALS AND METHODS Isolation from leaves Scab-infected apple leaves were collected in Michigan from unsprayed orchards, orchards with a long history of dodine usage, orchards where scab control with dodine seemed unusually difficult and from wild crabapples. Samples were also collected from the Poray Orchard, Sodus, New York, where the tolerance problem was first dis- covered. Monoconidial cultures were isolated by rubbing detached lesions across the surface of Difco potato-dextrose agar (PDA, pH 5.6) and transferring single germinating conidia to fresh FDA in petri plates. Stock cultures were maintained at 1-5 C in screw cap tubes. Inoculumgproduction Inoculum for various tests was produced on cheesecloth wicks in 8 oz prescription bottles containing 30 ml 4% Difco malt extract broth, according to the methods suggested by Williams (39). Spores were collected by rinsing the nutrient medium from the bottles, adding 30 ml sterile deionized water, shaking the bottles vigorously to loosen the conidia, and straining the conidia through two layers of cheese- cloth. Spore suspensions were standardized turbidimetrically by a modification of Kirkham's method (18). A standard spore suspension contained about 4-6 X 105 conidia/ml when adjusted to an optical density at 0.34-0.41 in a colorimeter at 525 mu with distilled water as a reference. Testing:gf isolates Cultures were tested for dodine tolerance using a disc assay, spore germination, and growth inhibition in liquid culture. The stand- ard spore suspension was magnetically stirred into PDA at 42 C at the rate of 1 ml per 10 ml PDA for the disc assay. Twenty-two ml of the seeded agar was immediately poured into 9 cm plastic petri dishes and allowed to solidify. Two hours after the agar hardened a 12.7 nm assay disc (VWR Scientific, S and S No. 740E) was saturated with 0.163 ml of a 50 or 300 ug/ml dodine concentration and placed on the seeded agar. The plates were incubated at 19 C for one week before determin- ing the zone of inhibition using indirect lighting against a light background. Two or three replicate plates were measured per test with three or four tests for each isolate. For spore germination, 0.05 ml of the desired dodine concen- tration was pipetted onto ceramic-ringed glass slides and allowed to dry. An equal volume of the standard spore suspension was pipetted onto the same area of the slides. In an alternate method dodine was diluted into suspensions of conidia and the mixture was pipetted onto the slides. A minimum of 50 spores per 10x field were counted in each of the three fields of three or four replications after 24 hours incu- bation at 20 C. Inhibition of growth of v, inaequalis was determined in 250 ml Erlenmeyer flasks containing 50 ml 4% malt extract broth with several rates of dodine. The cultures were inoculated with 3 ml of a standard spore suspension and incubated at 19 C for two weeks. The mycelium was filtered, oven-dried at 60 C and weighed. The initial tests were conducted with the Cyprex 65W formu- lation of dodine. Subsequent tests used an equivalent rate of the technical grade of dodine (American Cyanamid Co., Princeton, N. J.) diluted with water from an ethanol stock solution with the appropriate ethanol controls. Crossing of isolates and isolation offascospores Isolates were mated in 9 cm plastic petri dishes according to the methods of Keitt and Langford (17). The medium contained 0.5% malt extract and 2.5% Difco agar amended with apple leaf decoction. Aqueous spore suspensions of two single-spore isolates were mixed in the petri dish. The cooling agar was added and swirled to assure a uniform mixture. The dishes were incubated at 19 C for two weeks and then transferred to 8 C. After six months the ordered or unordered ascospore tetrads were isolated with a micromanipulator or by hand with a glass needle. Random ascospores were isolated from five or six perithecia crushed in water using sterile techniques. Single discharged spores were picked up with a small diameter capillary tube. All spores were germinated and cultured on PDA. Greenhouse studies Greenhouse inoculations were conducted on 2 year-old McIntosh apple trees. Actively growing trees were sprayed with Cyprex 65W ap- plied to the runoff point with a paint sprayer (DeVilbiss Type G03). After drying, the trees were spray-inoculated with spore suspensions of selected isolates containing approximately 2 X 105 conidia/m1. Im- mediately after inoculation the trees were placed in a moist chamber at 18-23 C for 2-4 days. Three weeks after inoculation the lesions on the one or two most heavily infected leaves of each shoot were counted and the data calculated as per cent control. Statistical analysis Significance of disc assays was determined by analysis of variance in a completely randomized design. Spore germination and greenhouse tests were analyzed in completely randomized or random block designs after converting percentages to per cent of control and apply- ing the arcsine transformation as appropriate. RESULTS Screening of field isolates The data in Table l are representative of those found in the disc assay screening of 144 isolates from apple leaves. Cultures were tested at two rates by soaking the assay discs in dodine concentrations of 50 ug/ml and 300 ug/ml. Isolate SR4, from the Poray Orchard where dodine tolerance was first noted, was the most tolerant field isolate collected. 51, from a dodine-treated commercial orchard, was the most tolerant Michigan isolate. Although zones ranging from 1 to 3 cm were noted in the 50 ug/ml test, isolates with average zone diameters of greater than 2.4 cm were considered to be quite normal. The most tolerant isolates came from orchards where dodine was used extensively, but the tolerance level of the isolate was not always correlated with the length of dodine usage or with the apparent degree of control by dodine. $1, the most tolerant Michigan isolate, came from an orchard with excellent commercial control, while normal isolates L2, L4 and V2 were from orchards with scab control problems. Complete data on the survey of field isolates are presented in Appendix 1. Isolates SR4, $1, and 04, representing three significantly different levels of tolerance, were chosen to determine their ability to grow in the presence of dodine. Growth of 04, a normal isolate, was almost completely inhibited by a dodine concentration of 1 ug/ml 10 Table 1. Inhibition of Venturia inaequalis in potato-dextrose agar by dodine diffusing from paper assay discs. Inhibition zone diam (cm)a Isolate Origin 50 ug/ml 300 ug/ml C2 Unsprayed tree, Michigan 3.05 3.75 L4 Commercial orchard, Michigan 3.03 3.60 V2 Commercial orchard, Michigan 3.00 3.67 B4 Unsprayed tree, Ohio 2.83 3.27 G5 Unsprayed tree, New York 2.73 3.53 04 Abandoned orchard, Michigan 2.68 3.63 CR2 Unsprayed crabapple, Michigan 2.60 3.38 54 Commercial orchard, Michigan 2.58 3.38 H6 Unsprayed tree, Maryland 2.58 3.05 L2 Commercial orchard, Michigan 2.40 3.30 51 Commercial orchard, Michigan 2.18 3.18 SR7 Poray Orchard, New York 1.70 2.58 SR4 Poray Orchard, New York 0.83 1.63 LSD .05 0.48 0.34 LSD .01 0.65 0.46 aMean of four tests. Zones were measured after seven days' incubation at 19 C. Concentration of dodine with which assay disc (12.7 mm diam) was saturated. (Table 2). $1, the most tolerant Michigan isolate, grew well at l ug/ml but was inhibited at 2 ug/ml. SR4, the most tolerant New York isolate collected, grew well at 2 ug/ml but was inhibited by 5 ug/ml. 11 Table 2. Growth of mycelia of selected Venturia inaequalis isolates after 2 wk incubation at 19 C in malt extract broth . Dry weight (mg) at indicated dodine concn (us/ml)a Isolate Tolerance Levelb 0 0.2 1.0 2.0 5.0 04 low 42.7 40.0 5.8 NGC NG 81 intermediate 37.3 36.4 32.3 NG NG SR4 high 34.4 34.5 40.0 38.6 NG 3Mean of two replications. bTolerance levels based on inhibition zone diam in 50 and 300 ug/ml disc assays: 04 (2.68 and 3.63 cm); 51 (2.18 and 3.18); SR4 (0.83 and 1.63 cm). cNG = no growth. Crossing_of isolates Initial crosses of the most tolerant isolates with normal isolates were made to determine if the level of dodine tolerance is heritable. Test crosses of F1 progeny with the most tolerant parent and with normal isolates, and intercrosses of F1 progeny were set up to test the two-major-gene hypothesis for inheritance of dodine toler- ance. Finally, two isolates having different levels of tolerance were crossed with 2295-2, a green color mutant from Dr. D. M. Boone, Dept. of Plant Pathology, University of Wisconsin to study possible linkage between genes for tolerance and the green color gene of a known V, inaequalis linkage group. 12 Progress of this study was impeded by an ascospore abortion factor arising from the most tolerant isolate, SR4. 0f the asci pro- duced by crosses of SR4 with normal isolates, only about 2% had eight spores. This greatly impaired the isolation of ascospore tetrads and further evidence was needed regarding this factor before firm con- clusions about inheritance of dodine tolerance could be drawn. These studies will comprise a later portion of this dissertation. Testing_of F1 progeny Progeny of the initial crosses were tested for tolerance by disc assay and germination of conidia in dodine (Table 3). In both types of tests differences in results of two members of the same spore pair were insignificant but differences between members of different pairs were significant. In these tetratype asci one pair of spores gave rise to cul- tures having a tolerance level similar to that of the normal parent (04), one pair was similar to the more tolerant parent (SR4), and two pairs had distinct intermediate levels of tolerance. Tetratype asci characteristic of a two gene interaction (8, 9), were found in crosses of SR4 with several unrelated normal isolates although the intermediate levels were not always significantly different. In a survey of these crosses 12 of 20 asci tested were considered to be tetratypes based on the presence of one pair of cultures having a level of tolerance similar to SR4. Usually the tolerance level of the more sensitive parent was also discernible in one pair of cultures from a tetratype ascus, although this level depended somewhat on the isolate that was Table 3. 13 Inhibition by dodine of germination and growth of conidia of the ordered monoascosporic cultures of a tetratype ascus of Venturia inaequalis cross 04 X SR4. Position Inhibition zone diam (cm)a % inhibition Isolate in ascus 50 ug/ml 300 ug/ml of germinationb 04SR4-1-2 1 1.80 2.75 73.5 ab O4SR4-2-2 2 1.90 2.60 79.4 b 04SR4-3-2 3 1.23 2.33 65.9 a 04SR4-4-2 4 1.13 2.23 63.9 a 04SR4-5-2 5 2.30 3.15 94.7 cd 04SR4-6-2 6 2.27 3.25 94.0 c 04SR4-7-2 7 2.98 3.62 97.9 de 04SR4-8-2 8 2.70 3.30 98.1 e 04 parent 2.68 3.63 ---C SR4 parent 0.83 1.63 --- LSD .05 0.41 0.35 LSD .01 0.56 0.48 aMean of four tests. Inhibition by dodine diffusing from 12.7 mm diam paper assay disc in potato-dextrose agar after seven days' incubation at 19 C. Dodine concentration with which paper assay disc was saturated. bInhibition in l ug/ml dodine in water. Generally 4 ug/ml completely inhibited germination of the most tolerant isolates. Means followed by the same letter are not significantly different (P = .05) accord- ing to Duncan's Multiple Range Test. cParent isolates not tested same as offspring. crossed with SR4. Two of these asci were thought to be nonparental ditypes having only intermediate levels of tolerance and one was a parental ditype ascus. Five of the 20 asci did not clearly fit any 14 of the above tetrad types due to experimental error. Figure l illus- trates dodine tolerance levels demonstrated by types of asci in the disc assay at 50 ug/ml. TE'I’RATYPE h—-’—. -. ~__ . ___. _. .. _ _. . _.__-_.H-___....___-_---—_~. -...- ._~-...._... _._ .__..,_. Figure 1. Inhibition of isolates from ascus tetrads of SR4 X normal crosses by dodine diffusing through potato-dextrose agar from paper assay disc. The two most tolerant isolates, SR4 and SR7 (Table l), were crossed to determine whether recombination of these two tolerance levels would yield progeny with dodine tolerance levels greater than SR4. None of the 39 progeny was more tolerant than SR4 (Appendix A2). Two isolates were slightly more sensitive than SR7, but not as sensi- tive as normal isolates. Apparently there was no recombination of major tolerance genes in this cross. 15 Crosses of F1 progeny Some progeny of a cross of isolates O4SR4-6-l and 04SR4-1-2, representing the two intermediate tolerance levels of tetratype asci, demonstrated inheritance of the tolerance levels of 04 and SR4 in the 50 ug/ml test (Figure 2). This indicated a recombination of two major genes conditioning dodine tolerance. Fewer significant deviations from the tolerance levels of the intermediate parents were seen in the 300 ug/ml test, although eight of the 71 progeny were significantly more tolerant than the more tolerant parent, 04SR4-1-2. A complete tabulation of the data composing Figure 2 is presented in Appendix A3. -‘5 I l I l l 1 ['5 I —300 IFS/ml 1:150 rig/ml >”.10 - p _ C) 2 a 32.05 +- _ IL 0' III I l I , 0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 INHIBITION ZONE DIAMETER IN CM Figure 2. Frequency distribution of dodine inhibition zones of 71 progeny of cross 04SR4-1-2 X 04SR4-6-1, isolates represent- ing the two intermediate tolerance levels in tetratype asci. Assay discs were saturated with indicated dodine concn. Four replications measured after 1 wk incubation at 19 C. LSD .05: 300 ug/ml (0.63 cm); 50 ug/ml (0.47 cm). Zone diam of parents in 50 and 300 ug/ml tests: 04SR4-l-2 (1.80 and 2.68 cm); 04SR4-6-1 (2.38 and 3.20 cm). 16 In a backcross of SR4 with 04SR4-3-2, representing the high- est dodine tolerance level in a tetratype ascus (Table 3), none of 30 progeny were significantly more sensitive than the more sensitive par- ent or significantly more tolerant than the more tolerant parent (Appendix 4). In a test cross of a normal isolate, C2, with 04SR4-5-2, intermediate in tolerance in a tetratype ascus (Table 3), none of 31 isolates were more sensitive than C2 to dodine (Appendix A5). Although seven progeny had slightly smaller zones than 04SR4-5-2 in the 300 ug/ml test, none were significantly more tolerant than this parent at 50 ug/ml. Fourteen of 32 progeny of cross C2 X 04SR4-7-2 were signifi- cantly more tolerant at 50 ug/ml than either of their parents (Appen- dix A6). Seven of the isolates reacted to 300 ug/ml with significantly smaller zones than either of the parents of these progeny, although not significantly smaller than 04, normal parent of 04SR4-7-2 (Table 3). In these three crosses, SR4 X 04SR4-3-2, C2 X 04SR4-5-2, and C2 X 04SR4-7-2, there was no apparent recombination of major toler- ance genes. The presence of some progeny slightly more tolerant than either of the two parents C2 and 04SR4-7-2, but not significantly more tolerant than 04, may indicate the recombination of minor genes affect- ing the level of tolerance to dodine. 17 Significance of ascospore abortion in inheritance of dodine tolerance The ascospore progenies whose test results are reported above were all isolated from crosses with a high frequency of asco- spore abortion. Because the causal nature of this phenomenon is not known, conclusions regarding frequencies of different levels of toler— ance in these crosses must be restricted. Linkage of the ascospore abortion and dodine tolerance factors cannot be excluded on the basis of present evidence. However, all tolerance levels found in tetratype asci were also found among progeny isolated from asci with less than eight spores. Also the discovery of several monoascosporic isolates similar to SR4 in dodine tolerance but not inducing ascospore abortion indicates that the two phenomena are not controlled by the same factor or factors. Ascospore abortion was not a factor in a later cross of a normal isolate, S4, with C2SR4 7-4, an F1 progeny as tolerant as SR4. The frequencies of isolates at different tolerance levels in the 50 ug/ml and the 300 ug/ml tests were similar in 40 progeny of this cross (Appendix A7) and in progeny of 04SR4-1-2 X 04SR4-6-l (Figure 2). This is evidence that ascospore abortion probably did not affect the frequency distribution of dodine tolerance levels in the earlier crosses. Non-linkage of dodine tolerance genes and green color gene Isolates representing two significantly different levels of tolerance were crossed with 2295-2, a green color mutant with normal 18 dodine tolerance, to study possible linkage of the genes for dodine tolerance with the green color gene of an identified V, inaequalis linkage group (4). Distribution of the tolerance levels of 20 wild type and 24 green progeny was not significantly different throughout the tolerance range demonstrated (Appendix A8). The tolerance levels of 27 progeny of 2295-2 and C2$R4-7-3, an F1 progeny similar to SR4 in tolerance, were also distributed throughout the tolerance range of their parents (Appendix A9). This random distribution of tolerance levels of green and wild type progeny suggests free recombination and non-linkage of the genes for dodine tolerance and the green color gene. Inoculation studies In an initial inoculation experiment (Table 4) significantly greater control by 200 ug/ml dodine was achieved on trees inoculated with normal isolate 04 than on trees inoculated with the most tolerant isolate, SR4. However, a qualitative difference in symptoms caused by the two isolates on the unsprayed trees was noted. 04 had normal heavily-sporulating lesions, but SR4 produced lesions which were more chlorotic with sporulation somewhat reduced. Because of the difference in macroscopic symptoms, experi- ments were repeated with tolerant and normal isolates selected for uniform symptoms on the unsprayed trees (Table 5). When total lesions were counted there was no significant difference in per cent control of the two isolates by the same rate of the fungicide at any rate. How- ever, there was significantly less suppression of sporulation of the more tolerant isolate, H65R4 6-1, by dodine 4 ug/ml than of normal isolate H6. 19 Table 4. Control of infection by Venturia inaequalis isolates 04 (normal dodine sensitivity) and SR4 (high dodine tolerance) on trees treated with dodine (Cyprex 65W).a Percent Percent shoots Isolate Dodine(ug/ml) Lesions/leafb controlC with infection 04 unsprayed 12.9 0.0 b 100.0 a 04 200 1.5 88.3 a 37.5 b 04 300 0.2 98.4 a 16.7 bc 04 600 0.0 100.0 a 0.0 cd SR4 unsprayed 7.7 0.0 b 100.0 a SR4 200 5.3 31.2 b 72.2 a SR4 300 0.8 89.6 a 19.4 bc SR4 600 0.1 98.7 a 8.3 cd aCompletely randomized design, three replications. Trees held in moist chamber four days at 19-22 C. b CPercent control = 100 - different (P = .05). 'lesions/leaf, sprayed lesions/leaf, unsprayed Column means followed by the same letter are not significantly X 100 Lesions per single most heavily-infected leaf per shoot. 20 Table 5. Control of sporulating lesions of Venturia inaequalis isolates H6 (normal dodine sensitivity) and H65R4 6-1 (high dodige tolerance) on trees treated with dodine (Cyprex 65W Percent control of b Isolate Dodine ug/ml sporulating lesions H6 4 86.7 ab H6 10 81.9 bc H6 200 96.9 a H6SR4 6-1 , 4 66.8 c H65R4 6-1 10 90.3 ab H6SR4 6-1 200 86.1 ab aRandomized block design, averages of three experiments with five replications each. Trees were held in moist chamber two days at 19- 22 C. Inhibition zone diameters in 50 ug/ml and 300 ug/ml disc H6 (2.58 and 3.05 cm); H6SR4 6-1 (0.3 and 1.4 cm). assays: b Based on two most heavily-infected leaves per shoot. lesions determined by macroscopic symptoms. Sporulating Column means followed by the same letter are not significantly different (P = .05). 21 An experiment was conducted to assess the effect of inoculum density on apple scab control by dodine. After fungicide treatment trees were spray-inoculated with suspensions containing 81,000 and 312,000 conidia/m1 of normal isolate, 04. At dodine concentrations of 100 ug/ml and 300 ug/ml significantly more shoots were infected on trees under the heavier inoculum conditions than under the lighter inoculum conditions (Table 6). Thus a heavy buildup of inoculum caused by negligent control measures early in the growing season Could be an important factor in control by dodine throughout the entire season. Table 6. Effect of inoculum density of Venturia inaequalis isolate 04 (normal dodine sensitivity) on apple scab control by dodine (Cyprex 65W).a Percent shoots with infection at indicated inoculum density Dodine (pg/m1) 81,000 conidia/ml 312,000 conidia/m1 Significance unsprayed 100.0 100.0 N.S. 100 41.1 86.7 P = .01 200 49.0 75.3 N.S. 300 13.7 57.4 P = .05 aCompletely randomized design, three replications. Effect of temperature on_germination of conidia in dodine Germination tests of conidia of two isolates in dodine at controlled temperatures of 10 C, 19 C, and 26 C in water and on PDA showed that temperature had a differential effect on the germination 22 of the controls of the two isolates in water, but not on PDA (Table 7). Although H6SR4 6-1 (highly tolerant) had poorer germination than H6 (normal dodine sensitivity) at all three temperatures, the effect was most noticeable at 10 C and 19 C. Temperature also had a differential effect on percent inhi- bition of germination by dodine on PDA (Figure 3-A). At all tempera- tures H6SR4 6-1 was less inhibited by dodine than H6. H6 was most inhibited by dodine at 10 C and least inhibited at 26 C. H6SR4 6-1 was most inhibited at 19 C and least inhibited at 10 C. Table 7. Per cent germination of conidia of two Venturia inaequalis isolates on PDA and in water at indicatedtemperatures.a 10 C 19 C 26 C H6 H6SR4 641 H6 H6SR4 6-1 H6 H65R4 691 Water 78 44 72 51 8O 68 PDA 81 89 84 82 75 77 aMeans of two three-replicate tests. Dodine inhibition of germination in water (Figure 3-B) was not greatly affected by temperature except at 10 C, where H6 was much more inhibited at lower dodine concentrations than H6SR4 6-1. 0n PDA and in water H6SR4 6-1 was least inhibited by dodine at 10 C. These data cannot be interpreted to say that the more toler- ant isolates generally react to temperature in dodine inhibition in this manner, but they show that temperature may affect the relative 23 B — HGSM 6-1 --H6 4 2: $2 I: E! z: E! .3 2: S? 3 3 35 0 ‘ 10 C 1001 ' at 1 ‘ 043 (L4. Owe 0L5 130 2L0 34) DODINE [lug/ml] Figure 3. Effect of temperature on dodine inhibition of germination of conidia of two Venturia inaequalis isolates. A) On PDA. Both isolates tested at 0.3 and 0.6 ug/ml dodine. B) In water. Isolate H65R4 6-1 tested at 1.0, 2.0, and 3.0 ug/ml dodine. Isolate H6 tested at 0.5, 1.0, and 2.0 ug/ml. 24 tolerance level of an isolate. Lower inhibition by dodine at cooler temperatures could be a significant factor in apple scab development during the earlier part of the season. DISCUSSION Tolerance levels of isolates The relative dodine sensitivity levels of field isolates tested in this study varied somewhat depending on the testing method, but the most tolerant isolates were inhibited by dodine concentrations about five times the concentration inhibitory to the most sensitive isolates. These differences in dodine tolerance levels are comparable to those noted previously (12, 22, 36, 40). Actual differences be- tween sensitivity levels reported here and those reported elsewhere may be due to differences in cultural pH and nutrient conditions or inoculum density. The relative differences in sensitivity to dodine are less than those previously reported for phenyl mercury acetate, similar to those reported for dichlone and sulfur, and about two times the natural variation in sensitivity reported for glyodin, captan, copper sulfate and Paris green (25, 32). The degree of natural variation in sensitivity to dodine by V; inaegualis in this study is similar to, or slightly less than, that expressed by the dodine-resistant mutants in Nectria haematococca var. cucurbitae developed by Kappas and Georgopoulos (16). The variation in tolerance in V; inaequalis shown here is about 1000 times less than that of the antimycin A-resistant ultraviolet mutant developed by Leben et a1 (19). 25 26 Inheritance of tolerance The pattern of inheritance of tolerance to dodine found in this work appears to follow more closely that proposed by Kappas and Georgopoulos for two Nectria mutants (16) than that suggested by Polach for V; inaequalis (30). In the system proposed for Nectria, each of two genes conditioned different levels of tolerance when in- herited separately, and were additive when inherited together. In the [—3 system suggested by Polach (30) a nontolerant isolate would not grow ! at 0.25 ug/ml, one gene would allow growth at 0.25 ug/ml, and a second i;u gene, effective only when the first is in dominant form, allows growth at 0.5 ug/ml dodine. In the present study disc assay of asci of crosses of the most tolerant isolate with normal isolates has shown four distinct levels of tolerance (Table 3). An explanation for this phenomenon is the independent action of two separate genes, each con- ditioning distinct levels of tolerance, having an additive effect when inherited together (8, 9). This has been confirmed by a cross of the two intermediate types to yield recombinant progeny more tolerant or more sensitive than either of the intermediate parents. The differences in reaction of progenies of crosses in this work and those of Polach may be the tolerance levels of the parent isolates. SR4 was the most tolerant of 64 New York isolates in this screening (Appendix Al), including 48 from the Poray Orchard, 1969- 1971, and 16 from the McQueen Orchard, 1969, another area where the tolerance problem was found (36). The tolerance level of the more sensitive parent of a cross also could contribute to a different re- action by the progeny. One difference in the testing techniques 27 employed in the two studies may be that in the disc assay each isolate could respond to the dodine concentration gradient to indicate a dis- tinct level of tolerance not evident when the isolates were screened at set concentrations. Further testing must be conducted to assure that recombinant progeny more tolerant than SR4 cannot arise from crosses of $1 with isolates having tolerance levels similar to SR4. If the gene condi- tioning the tolerance level of S1 is the same as one of the genes in if MW" ‘ i SR4 then there should be no buildup in tolerance from this cross. However, if three separate genes are involved, then recombinant progeny should appear with no tolerance genes or with all three tolerance genes. The reaction of the progeny having three tolerance genes would depend on whether the action of the third gene is additive or whether its action is masked by the other two genes. In a cross of $1 with a highly tolerant progeny of SR4, none of 10 progeny were as tolerant as SR4, nor did they have the normal level of sensitivity which would indicate a recombination of tolerance genes. Mechanisms for tolerance to dodine The mechanisms for the genes for tolerance to dodine are unknown. 0f the previously suggested general mechanisms (7, 11) either decreased permeability or detoxification seems to be the most likely possibility for a mechanism conferring such a comparatively low dif- ference in sensitivity. This is in contrast to fungicides with more specific sites of action to which resistant strains have shown a 100 to 1000-fold decrease in sensitivity (7). A re-investigation of the 28 retention of 14 C by tolerant and sensitive conidia following exposure to 14C-labelled dodine could shed insight on the problem of detoxifi- cation described by Bartz and Mitchell (2, 3). The interaction of response to dodine with natural variations in response to different nutrient (20, 28), pH, and temperature conditions (25) may be impor- tant to an understanding of the problem of tolerance to dodine under field conditions. Fungicide tolerance as a factor in control of apple scab Inheritance of tolerance to dodine in V, inaequalis has been clearly demonstrated. However, one must experimentally assess the importance of the degree of fungal sensitivity to dodine as a factor in apple scab control to place it in proper perspective with other factors such as weather factors, inoculum density, proper coverage and timing and rate of dodine application (21). A natural mutation rate to dodine tolerance as low as 1 in 106 (22) should be high enough to as- sure ii constant population with this level of tolerance in the orchard if the selection pressure of dodine is the major factor inhibiting survival of the fungus. The limited emergence of populations sufficiently tolerant to have notable economic importance in spite of a relatively high mu- tation frequency has been attributed to onerkill" in commercial practice (22). The ability of dodine to prevent sporulation has un- doubtedly limited the population screened for dodine tolerance in na~ ture. Additional factors limiting the success of strains with high 29 levels of dodine tolerance may be reduced pathogenicity or reduced sporulation. Isolate SR4 sporulated well in culture, but sporulation was limited on plants in the greenhouse. The majority of the cases of poor scab control by dodine investigated in Michigan demand an alternate explanation because iso- lates collected from heavily-infected orchards have shown a normal level of tolerance to dodine while the most tolerant Michigan isolates A A" ”3 have come from orchards with good control. Frequently poor control in . _ T _ - i_-__ mid-season can be attributed to an early buildup of inoculum due to late timing of an early application, poor coverage due to improper pruning, or careless spraying. Inoculum density has been shown to be an important factor in apple scab control by dodine (Table 6). Since inhibition of spore germination is dependent upon the amount of dodine available per spore (24), an early season buildup of inoculum means the rate of application must be increased or the spray interval reduced, or both, to maintain the level of residue essential to adequate control. PART I LITERATURE CITED $“3m-nnmy—uur PART I LITERATURE CITED ALBERT, J. J. and F. H. LEWIS. 1962. Effect of repeated appli- cations of dodine and of captan on apple scab foliage lesions. Plant Dis. Reptr. 46:163-167. BARTZ, J. A. and J. E. MITCHELL. 1970. Comparative interaction of n-dodecylguanidine acetate with four plant pathogenic fungi. Phytopathology 60:345-349. BARTZ, J. A. and J. E. MITCHELL. 1970. Evidence for the meta- bolic detoxification of n-dodecylguanidine acetate by ungerminated macroconidia of Fusarium solani f. sp. phaseoli. Phytopathology 60:350-354. BOONE, D. M. and G. W. KEITT. 1956. Venturia inaequalis (Cke.) Wint. VIII. Inheritance of color mutant characters. Am. J. Botany 43:226-233. BROWN, IRWIN F. and HUGH D. SISLER. 1960. Mechanisms of fungi- toxic action of n-dodecylguanidine acetate. Phytopathology 50:830-839. BYRDE, R. J. W., D. R. CLIFFORD and D. WOODCOCK. 1962. Fungi- cidal activity and chemical constitution, XI. The activity of n-alkyl guanidine acetates. Ann. Appl. Biol. 58:457-466. DEKKER, J. 1972. Resistance, p. 156-174. In R. W. Marsh [ed.]. Systematic Fungicides. John Wiley and Sons, New York. ESSER, KARL and RUDOLF KUENEN. 1967. Genetics of fungi. Trans. Erich Steiner. Springer-Verlag Inc. New York. 500 pp. FINCHAM, J. R. S. and P. R. DAY. 1971. Fungal Genetics. 3rd. ed. Blackwell Scientific Publications, Oxford. 402 pp. FREAR, DONALD E. H. and ELIZABETH C. SMITH. 1960. Dodecylguan- idine acetate (dodine) residues on apples. J. Agr. Food Chem. 8:465-466. GEORGOPOULOS, S. G. and C. ZARACOVITIS. 1967. Tolerance of fungi to organic fungicides. Annu. Rev. Phytopathol. 5:109-130. 30 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 31 GILPATRICK, J. D. and D. R. BLOWERS. 1974. Ascospore tolerance to dodine in relation to orchard control of apple scab. Phytopathology 64:649-652. HEUBERGER, J. W. and R. K. JONES. 1962. Apple Scab II. Effect of serial applications of fungicides on leaf lesions on previously unsprayed trees. Plant Dis. Reptr. 46:159-162. JONES, R. K., J. W. HEUBERGER, and J. D. BATES. 1963. Apple Scab III. Effect of serial applications of fungicides on leaf lesions on previously unsprayed trees-inhibition of conidial germination, removal (suppression) of the organism, and subsequent development of late terminal infection. ~_. Plant Dis. Reptr. 47:420-424. ' KAPPAS, A. and S. G. GEORGOPOULOS. 1968. Radiation-induced re- sistance to dodine in Hypomyces. Experientia 24:181-182. L» KAPPAS, A. and S. G. GEORGOPOULOS. 1970. Genetic analysis of dodine resistance in Nectria haematococca (Syn. Hypomyces solani). Genetics 66:617-622. KEITT, G. W. and M. H. LANGFORD. 1941. Venturia inaequalis (Cke.) Winter. 1. A groundwork for genetic studies. Am. J. Botany 28:805-820. KIRKHAM, D. S. 1956. A culture technique for Venturia supp. and a turbidimetric method for the estimation of compara- tive sporulation. Nature 178:550-551. LEBEN, C. D., D. M. BOONE and G. W. KEITT. 1955. Venturia inaegualis IX. Search for mutants resistant to fungicides. Phytopathology 45:467-472. LEBEN, CURT and G. W. KEITT. 1948. Venturia inaequalis (Cke.) Wint. V. The influence of carbon and nitrogen sources and vitamins on growth in vitro. Am. J. Botany 35:337-343. LEWIS, F. H. and K. D. HICKEY. 1972. Fungicide usage on decid- uous fruit trees. Annu. Rev. Phytopathol. 10:399-428. MACNEILL, B. H. and JANICE SCHOOLEY. 1973. The development of tolerance to dodine in Venturia inaequalis. Can. J. Bot. 51:379-382. MILLER, L. P. 1960. Uptake and innate toxicity of dodine (n-dodecylguanidine acetate) to fungus conidia. Phytopath- ology 50:646 (Abstr.). 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 32 MITCHELL, J. E. and J. DUAIN MOORE. 1962. Relation of dodine residue levels and scab development on apple fruit and leaves. Phytopathology 52:572-580. PALMITER, D. H. 1934. Variability in monoconidial cultures of Venturia inaequalis. Phytopathology 24:22-47. PARRY, K. E. and R. K. S. WOOD. 1959. The adaptation of fungi to fungicides: adaptation to captan. Ann. Appl. Biol. 47:1-9. PARRY, K. E. and R. K. S. WOOD. 1959. The adaptation of fungi to fungicides: adaptation to thiram, ziram, ferbam, nabam, and zineb. Ann. Appl. Biol. 47:10-16. PELLETIER, REAL L. and G. W. KEITT. 1954. Venturia inaequalis (Cke.) Wint. VI. Amino acids as sources of nitrogen. Am. J. Botany 41:362-371. POLACH, F. J. 1972. Reaction of Venturia inae ualis ascospore progeny to dodine. Phytopathology 62:782-783 (Abstr.). POLACH, F. J. 1973. Genetic control of dodine tolerance in Venturia inaequalis. Phytopathology 63:1189-1190. POWELL, DWIGHT. 1960. The inhibitory effects of certain fungi- cide formulations to apple scab conidia. Plant Dis. Reptr. 44:176-179. ROSS, R. G. and S. A. HAMLIN. 1961. Variation in the sensitivity of isolates of Venturia inaequalis (Cke.) Wint. to fungicides. Can. J. Plant Sci. 41:499-502. SOMERS, E. and R. J. PRING. 1966. Uptake and binding of dodine acetate by fungal spores. Ann. Appl. Biol. 58:457-466. STELLAR, W. A., K. KLOTSAS, E. J. KUCHAR, and M. V. NORRIS. 1960. Colorimetric estimation of dodecylguanidine acetate residues. J. Agr. Food Chem. 8:460-464. SZKOLNIK, M. and J. D. GILPATRICK. 1969. Apparent resistance of Venturia inaequalis to dodine in New York apple orchards. Plant Dis. Reptr. 53:861-864. - SZKOLNIK, M. and J. D. GILPATRICK. 1970. Tolerance of the apple scab fungus to dodine (Cyprex). Proc. New York State Hort. Soc. 115:246-249. SZKOLNIK, M. and J. D. GILPATRICK. 1971. Investigations on apple scab fungus tolerance to the dodine fungicide in 1970. Proc. New York State Hort. Soc. 116:34-37. 38. 39. 40. 33 SZKOLNIK, MICHAEL and J. D. GILPATRICK. 1973. Tolerance of Venturia inaequalis to dodine in relation to the history of dodine usage in apple orchards. Plant Dis. Reptr. 57:817- 821. WILLIAMS, E. B. 1969. Personal communication, p. 161. In John Tuite. Plant Pathological Methods Fungi and Bacteria. Burgess Publishing Company, Minneapolis. YODER, K. S. and E. J. KLOS. 1972. Evidence for inheritance of tolerance to dodine in Venturia inaequalis. Phytopathology 62:799 (Abstr.). PART II INHERITANCE OF AN ASCOSPORE ABORTION FACTOR ARISING FROM VENTURIA INAEQUALIS ISOLATE SR4, AN ISOLATE HIGHLY TOLERANT TO DODINE INTRODUCTION The intriguing discovery of the induction of ascospore abortion in Venturia inaequalis by SR4, the isolate most tolerant to dodine, demanded further investigation. The simultaneous occurrence of both phenomena in a single isolate suggested a common genetic cause. Conclusions regarding inheritance of the dodine tolerance level of this isolate could have been erroneous without further know- ledge of the relationship between the two factors because aborted meiotic products could not be examined for dodine tolerance. Further crossing of isolates and testing of their progeny revealed that the two factors did not always occur together in the offspring. Some F1 progeny were found to be as tolerant to dodine as SR4, but produced only normal asci in crosses with other isolates not inducing abortion. Some progeny were normal with respect to dodine tolerance, but produced abnormal asci. These two points are conclu- sive evidence that the two factors do not have a common genetic cause. From the present data we cannot exclude the possibility that both factors may be caused by linked genes. This paper reports observations on the magnitude of ascospore abortion induced by isolate SR4 and by progeny of this isolate, and compares the morphological characteristics of this phenomenon with pre- viously reported cases. The possible basis of the phenomenon and its effect on the organism's natural survival are discussed. 35 LITERATURE REVIEW The ascospore abortion phenomenon has been recognized in the literature. Dodge (11) pointed out that in the Ascomycetes one usually observes gene expression in the organisms' haploid structures, and that the asci with their fusion nuclei are the only cells of structures in the entire life cycle in which one could demonstrate simple mendelian dominance. In an X-rayed line of Neurospora tetrasperma a recessive lethal factor prevented ascospore delimitation when inherited in the homozygous condition (9, 10). Eight-sporedness appeared to be dominant in a cross of a four-spared N, tetrasperma with an eight-spared N, sitophila because all the resulting asci had eight spores (8). The first baCkcross of F.l progeny with the eight-spored parent usually gave asci with eight spores, but a backcross with the four-spored parent gave asci with spore numbers ranging from one to eight, mostly three to six (11). "Aborta", a variant strain of Hypomyces Ipomoeae (7), induced the formation of only four rather than eight spores when crossed with normal isolates. This ascospore abortion was apparently due to the inability of nuclei carrying the aborta factor to delimit spores. Aberrant ascospore segregations occurred as a result of translocation in Neurospora crassa (3, 21). Translocations, inversions, and deletions can be distinguished by the distribution of abnormal 36 37 asci (13, 14). The effects of spindle orientation and dominance modi- fiers on the development of aberrant asci have been demonstrated in Neurospora (23, 25, 29). The "sterile ascus" phenomenon in selfed monoascosporic iso- lates of Cochliobolus heterostrophus (22) was probably controlled by more than one factor because certain backcrosses produced perithecia with varying ratios of sterile to normal asci. If inhibition of ascospore formation were controlled only by a single dominant or a single recessive gene, either all sterile asci or all fertile asci should have resulted. The occurrence of asci containing from one to eight ascospores was also reported in C, satngs (l6). Asci of Coniochaeta malocotricha containing one to eight spores were found to arise by abortion or non-maturation or by the failure of one or more nuclei to be included into the ascospores (26). The most frequent spore numbers in asci with fewer than eight spores were four and five. Development of perithecia, asci and ascospores of Sordaria fimicola was directly related to biotin content of the growth medium (2). The frequency of abnormal asci with one to seven ascospores in- creased as the biotin concentration was decreased. Venturia inaequalis cultures of a mutant white sector, al- though producing abundant perithecia in crosses with normal isolates, induced partial or complete abortion of four of the eight ascospores in each ascus (30). Aborted spores were one- or two-celled, color- less and usually misshapen. Aborted spores which were viable always gave rise to white cultures. 38 A number of nitrogen mustard-induced color mutants of V, inaequalis (5), when crossed with normal isolates, caused partial or complete abortion of the spores carrying the mutant genes. Two white mutants gave rise to 3:1 and 1:3 ratios of normal to defective spores as well as the more common 1:1 ratio when crossed with normal isolates. Conclusive evidence explaining these aberrant segregations was not attained. An investigation of the chromosome number of V, inaequalis encountered abnormalities among crosses of wild-type cultures (6). One cross gave rise to two types of perithecia, one type with all normal asci, and one type with all asci showing abnormal 4:4 segrega- tions. In one perithecium, a single 4:4 ascus was suggested to have resulted from crossing-over in an inversion heterozygote. Two other cytological studies of ascus development in V, inaequalis (l, 17) did not report such abnormalities. MATERIALS AND METHODS Crossing_and isolation of progeny All isolates used in this study were part of the isolate collection for the dodine tolerance study. The techniques for crossing and isolation have been described in Part I. In addition to the medium of Keitt and Langford (18), a modified potato-dextrose agar (5) was also tested. Ascospore counts Spores were counted at 430x after placing several perithecia in water on a microscope slide and applying pressure to the coverslip to break open the perithecia and expose and spread the asci. In making the counts, only those spores with mature coloration were considered normal. Counts were not conducted until spores were fully mature in all perithecia of a cross. Plates with mature perithecia were main- tained at 4 C until counted. Perithecia on leaves Leaves from several orchards in New York were collected during the month of May (l969-1971) by Dr. J. D. Gilpatrick, New York Agricultural Station, Geneva, New York. The leaves were dried and refrigerated until the perithecia were removed for counting. 39 40 Staininggof‘asci Preliminary efforts to stain nuclei in asci were only par- tially successful. Mature spores were quite impermeable to stain and immature spores did not stain well within the ascus. The pressure needed to flatten the asci to give good microscopic resolution fre- quently displaced the contents of the asci. The most successful stains were iron haematoxylin (19, 20) and 0.5% toluidine blue dissolved in water. Both stains gave good differentiation of nuclei in ascogenous hyphae, but were less success- ful for nuclei inside the asci. The methods of O'Donnell, Tai and Beneke were followed for fixation and hydrolysis for iron haematoxylin staining (24). Length of hydrolysis in TN HCl at 65 C varied from two 10 minute periods to two 2 hour periods with no appreciable dif- ference in results. Toluidine blue dissolved in water stained nuclei as clearly without hydrolysis as with hydrolysis, but the asci were more inclined to burst in unhydrolyzed material. Toluidine blue in 70% ethanol (15) failed to give the desired contrast and 0.5% toluidine blue in 50% propionic acid was unsatatisfactory. RESULTS Apparent types of abortion Several types of ascospore abortion may be influencing the number of spores per ascus in different crosses. Because immature asci were difficult to discern from asci which had reached their maxi- mum development without any spore delimitation, asci without spores were not included in the counts. To allow complete spore maturation before counting, perithecia were maintained at a low temperature after initial maturity was reached. This storage may have allowed some disintegration of early maturing spores. However, in most cases, asci containing less than eight spores appeared to be a result of under- development rather than disintegration of spores. Figure 4-A illustrates three developmental types of asci found in these crosses. Ascus 1 appears to be in the process of as- cospore delimitation and may represent an immature form. Ascus 2 apparently failed to delimit more than four spores. The lack of mature coloration in the smaller cell of the terminal spore in this ascus may signify that this cell is inactive and could disintegrate to give rise to a one-celled spore. Ascus 3 reached its maximum development with- out delimiting any spores. Empty asci were found in crosses of iso- lates not inducing ascospore abortion as well as in crosses of isolates inducing extreme ascospore abortion. 41 42 Figure 4. Ascospore abortion in progeny of Venturia inae ualis isolate, SR4. A—F) Normal and aberrant asci. G-J) Abnormal ascospores. A) l, maturing ascus; 2, four-spared ascus, smaller cell of terminal spore is poorly pigmented; 3, aborted ascus. B) Normal eight-spared ascus. 43 Asci containing one to eight spores were found in crosses of SR4 with normal isolates. In most of these asci reduction of spore numbers appeared to be due to improper spore cleavage (Figure 4-A, 4-E, and 4-F) or failure of cleaved spores to mature (4-B, 4-C, and 4-D). Occasionally odd numbers of spores in asci may have resulted from disintegration of one member of a spore pair (Figure 4-E, three- spored ascus). In one four-spared ascus a culture of one of the four spores had a much higher level of tolerance to dodine than the other three. This indicates that members of three spore pairs were present in this four-spored ascus, implying that one member of each of two spore pairs either disintegrated or failed to develop. Very rarely, highly abnormal ascospores occurred in perithecia of various crosses. Figure 4-F shows an ascus containing single-celled spores. It is not apparent if these arose by disintegration of one cell as suggested by Figure 4-A, or by failure of the spore to undergo the final mitotic division essential to give two-celled spores, or if the two cells of a spore separated and rounded as they matured. An abnormally developing globose ascus (Figure 4-0) con- tained structures similar in shape to the smaller cells of normal spores. An immature ascus (normally somewhat globose) is pictured in Figure 4-E. Several other highly abnormal, mature-colored spores (Figure 4-G-J) occurred only rarely in crosses of SR4 or its progeny, but they may represent an unusual modification of factors contributing to the more common types of ascospore abortion. The three cells of the spore 44 in Figure 4-G remained intact when the ascus was broken. The bulboid protuberance from one cell of the spore in Figure 4-H suggests the possibility that one cell may undergo an additional mitotic division to yield a three-celled spore. The misshapen ascospore in Figure 4-J may be undergoing germination. An unusually large spore (Figure 4-1) maintained a fairly normal shape although it was nearly two times the normal size. Another one-celled spore nearly filled an ascus about half the size of a normal eight-spared ascus. Frequency of abnormal asci in crosses of SR4 with normal isolates Crosses of SR4 with normal isolates (not inducing ascospore abortion) produced a high frequency of asci containing one, three, five, or seven spores (Table 8). The ratios of these asci with odd numbers of spores to asci with even spore numbers varied considerably with the parental combination. Combined frequencies of adjacent cate- gories (one- and two-spored asci, three- and four-spared asci, etc.) were fairly consistent regardless of the parental combination. Since abortion due to inheritance of lethal genes should occur in pairs of spores rather than single spores, the variable distribution of asci with odd spore numbers suggests a non-genetic response to some variable cultural condition causing the underdevelopment or disintegration of one member of a spore pair. The higher consistency of frequencies of asci with odd spore numbers combined with asci having even spore num- bers shows that usually spores were aborted in pairs rather than singly and suggests a strong genetic influence in ascospore abortion. 45 Table 8. Frequency of ascospore abortion in crosses of SR4 with normal isolates. Percent asci with indicated number of spores per ascus Total cm“ 1 2 3 4 5 6 7 85‘5“ SR4XT2 2 (14)a 12 14 (43) 29 25 (42) 17 o (1) 1 77 SR4XB4 1 (11) 1o 28 (59) 31 20 (36) 16 1 (4) 3 110 SR4XB6 1 (17) 16 17 (52) 35 4 (29) 25 o (2) 2 114 SR4XH6 5 (27) 22 16 (48) 32 1o (22) 12 o (3) 3 116 SR4XC2 3 (25) 22 22 (49) 27 1o (25) 15 o (1) 1 78 SR4XCR6 o (25) 25 30 (50) 20 13 (25) 13 o (0)0 56 SR4XL1 o (19) 19 9 (38) 29 19 (38) 19 o (5) 5 21 SR4XL5 3 (12) 9 15 (52) 37 24 (36) 12 o (0)0 33 SR4X04 o (14) 14 29 (43) 14 14 (43) 29 o (0)0 7 %of total 2 (19) 17 18 (48) 30 14 (31) 17 o (2)2 612 aFigures in parentheses represent totals of two adjacent categories. Overall, in crosses of SR4 with normal isolates, only two percent of the asci had eight spores. Asci having one or two spores ranged from 11 to 28% (19% of total). Asci with three or four spores ranged from 38 to 59% (48% of total). Those having five or six spores included 31% of the total with values ranging from 23 to 43%. Ascospore abortioniin crosses of F1 progeny of SR4 To study inheritance of the ascospore abortion factors, crosses involving F1 progeny of SR4 were examined for abortion fre- quencies (Table 9). In crosses of groups of normal isolates with mem- bers of the same spore pair, frequencies of ascus types were quite 46 .mmweommumu “amounts o3» mo mpmuo» pcmmmgamg mommspcmcma cw mgmnszzn .muuuemmeo “mum- ucm ~1muemm¢o "mic- can ~1m1emm¢o ”~1N- new Nupuemmeo "autumn mpmcwm m yo mgwma mgoam we mgmnsmz .Fm xvvcmaa< cw cmgcmmmca m_ mommoeu Fmaup>wucw seem moan mo cowpmpaamh .mmpmpomw Pesto: we masocom NON N A. v o 55 Ammv m NN Ammv m N_ ANNV _ saw x N-4- N5 o Ne V 4 NM Neev m. NN Nagy N_ 5 No V N 5mm x N-N-5Nm¢o NON 55 Nva N m NN v N NN Name N N NN V o z x N-N-¢Nm¢o om o_ ANNV NN mp ANNE m_ NN NNNV 8 NF NN_V o z x N-8-5Nm¢o Na 8N Nemv N N N5 V N we NNmV N o_ NONE o z x N-m-¢mm¢o qu _ NNV o 5N NNNV N me Name _F NN NeNV N z x N-5- NNo m. NNV 5. on Noev ON m4 Nva N 5 AN V _ z x N-N-¢Nmeo mm_ N. NNV N. N_ ANNV o_ m_ Npev 8N NN Nva N z x N-N- 84. o NNV P NN Name NN 5N . Amev 5 mp nNNNV N 52 x N-N-5Nm¢o peach mzomc emu mesonm mo amass: mmocu topmomuew sup: Puma accuse; .wmm use we No mammoga pm we mummoco pump cw cowpconm mcoqmoomw mo adamaamcu .m «New» 47 consistent in two combined categories, but varied considerably in the other two. For example, the proportions of asci having three or four spores and those having seven or eight spores were similar in crosses of normal isolates with 04SR4-1-2 and O4SR4-2-2 of one spore pair and with O4SR4-3-2 and O4SR4-4-2 of another spore pair, but in these crosses there was more variation in the combined frequencies of asci having one or two spores and those having five or six spores. Crosses of O4SR4-5-2 with normal isolates and crosses of 04SR4-6-2 with normal isolates were similar in individual combined frequencies of one- or two-spared asci and combined frequencies of seven- or eight-spared asci, but were more variable in the three- or four-spore category and in the five- or six-spore category. This consistency in two classes and inconsistency in two other classes was also noted in crosses with SR4 (Table 9) and in crosses of members of one spore pair with members of another spore pair (Appendix Bl). If the ascospore abortion is entirely under genetic control then reactions in crosses of isolates with the genetically identical members of the same spore pair should be consistent in all categories. The similarity of combined frequencies in only two of the four com- bined categories of ascus types suggests that certain reactions were under more stable control than ones which were more variable. The frequencies of asci in each numerical category were similar in crosses of SR4 X normal isolates, 04SR4-3-2 and -4-2 X normal isolates, O4SR4-l-2 and -2-2 X 04SR4-7-2, and O4SR4-3-2 X O4SR4-6-2. The frequencies of different ascus types in the backcros- ses O4SR4-3-2 and -4-2 X SR4 were also expressed in crosses of 48 04SR4-1-2 and -2-2 with O4SR4-5-2 and -6-2. This similarity of the frequencies of the ascus types in crosses of SR4 X normal isolates and the frequencies in test crosses of F1 progeny definitely indicates an inherited tendency to produce aberrant asci. The ratios of ascus types of 04SR4-l-2 and -2-2 X normal isolates, O4SR4-5-2 and -6-2 X normal isolates and O4SR4-7-2 X normal isolates did not closely ap- proximate ratios found in other crosses, but all showed some degree of ascospore abortion (Table 10)- Several F.I progeny of SR4 having dodine tolerance levels similar to SR4 were testcrossed with normal isolates to determine whether ascospore abortion was induced in crosses with all isolates having this level of dodine tolerance. Among this group of isolates representing nine spore pairs, isolates representing four spore pairs permitted normal development of at least 95% of the asci in combination with one or more normal isolates (Appendix Bl). Thus the genetic fac- tors conditioning the dodine tolerance level of SR4 were not responsi- ble for the ascospore abortion of its progeny. Ascospore abortion induced by field iso- lates With normal dodine sensitivity A clearcut abortion of four spores occurred in crosses of most non-aborting isolates with three field isolates having normal do- dine sensitivity. In crosses with other isolates shown to induce as- cospore abortion, the mean number of spores per ascus was lower than when either of the isolates was crossed with isolates not inducing abortion. 49 .NmNNommuNo ucmumwum No NNNuop acmmmgqme mmmmcpcmcma :N mmgszN .Ncmmoga NN suNz mm NNNNH .emm cup: "mommocu NNNNN>NucN No upon a .NN chcNNN< .mmamNomN NNENoc No masocwm NN N NN V N NN NNNV N NN NNNV NN NN NNNV N N-N- x N-N- NNN N NN V N NN NNNV N NN NNNV NN NN NNNV N N-N- x N-N- N N-N- NN N NN V N NN NNNV NN NN NNNV N NN NNNV N N-N- N N-N- x N-N- N N-N- NNN N NN V N NN NNNV NN NN NNNV N N NNNV N NNN x N-N- N N-N- NNN NN NNNV N N NN V N NN NNNV N N NN V N z x N-N- NNN NN NNNV NN N NNNV N NN NNNV N NN NNNV N z x N-N- N N-N- NNNN N NN V N NN NNNV N NN NNNV N NN NNNV N z x N-N- N N-N- NNN N NN V N NN NNNV NN NN NNNV NN NN NNNV N z x N-N- N N-N- NNNNN NNN N NN V N NN NNNV NN NN NNNV NN NN NNNNV N Nz x NNN “mmuw N N N N N N N N NNNNN NNUNN NNN NmNoNN mo NNNENN uoumuwucw NNN: Nome Nance No acmugmm .Nmm mo Nemmoca Nu No mmmmogu «may cw new NmpNNoNN Nessa: x Nam mo mwmmogo cN :oNuNonm mNonoomN No Nocmacmgm .oN NNNNN 50 Differences in individual perithecia of a single cross In some cases individual perithecia of single cross varied in their degree of ascospore abortion. In seven of ten perithecia examined from crosses of three normal isolates with an aborting iso- late, 91% of 128 asci contained four spores and none contained more than four spores. In three perithecia, 94% of 49 asci contained eight spores and none had fewer than six spores. Individual perithecia in crosses of normal isolates with N-156, a white color mutant producing light-colored perithecia, also varied in degree of abortion. In these cases individual perithecia contained asci having mostly four spores or mostly two spores. Such differences could not be attributed to the parental origin of the perithecium because the differences were found in both light-colored and dark-colored perithecia. Ascospore abortion in crosses involving color mutants A number of Venturia inaequalis color mutants were reported to induce ascospore abortion (5, 30) when crossed with normal or abortion-inducing isolates. Crosses of normal isolates with a white mutant (N-156) and a yellow mutant (N-150) yielded mostly four-spared asci. When N-156 and N-150 were crossed with isolates inducing ascospore abortion, the combination further reduced the mean number of spores per ascus (Table 11) as had the three dodine-sensitive field 51 Table 11. Further reduction of the number of spores per ascus by crosses of aborting color mutants with aborting F1 progeny of'SR4. Mean spores per ascus in perithecia 0f indicated parental combinations Cross normal 04SR4-1-2 O4SR4-3-2 isolates O4SR4-2-2 O4SR4-4-2 normal isolates --- --- 4.0 (281)a 4.3 (998) N-289b 7.9 (27) 3.5 (175) --- --- N-156 3.8 (34) 2.8 (198) 2.8 (58) N-150 4.2 (85) 3.0 (115) 2.4 (53) aFigures in parentheses indicate total asci counted. bN-289 is nearly normal with respect to ascospore abortion. isolates. Pink color mutant, N-289, and green color mutant, 2295-2, reacted quite normally in combination with normal and abortion-inducing isolates (Table 11 and Appendix 83). Induction of ascospore abortion by New York isblates All five Poray Orchard isolates tested were found to induce some degree of ascospore abortion (Table 12). SC8a crossed with C2 gave a ratio similar to that produced by isolates O4SR4-5-2 and -6-2 when crossed with C2 (Table 10). The degree of abortion produced in CZ X SC8b was comparable to that occurring in crosses 0f N-150 X O4SR4-3-2 and -4-2 (Appendix B3). The ascus ratio in cross C2 X SClZc was similar to that induced by the white color mutant N-156 in crosses with normal isolates (Appendix 83). SR7 in combination with SR4 re- sulted in more ascospore abortion than C2 crossed with SR4. 52 Table 12. Frequency of ascospore abortion in crosses involving New York isolates. Percent asci with indicated number of spores per ascus Total Cross . 1 2 3 4 5 6 7 8 asci c2axs114 3 (25)b 22 22 (49) 27 10 (25) 15 0 (1) 1 78 02 x SC8a o (15) 15 0 (40) 40 0 (6) 6 2 (39) 37 176 02 x SC8b 11 (75) 64 4 (25) 21 0 (0) 0 0(0) 0 28 c2 xsc12c 4 (16) 12 0 (84) 84 0 (0) 0 0(0) 0 80 SR4 x SR7 19 (42) 23 19 (58) 39 0 (0) 0 0(0) 0 26 aNormal isolate. bFigures in parentheses represent totals of adjacent categories. Ascospore abortion in natural perithecia from New York Examination of a few leaves collected from the Poray Orchard in 1970 showed that three of ten perithecia contained asci with asco- spore numbers ranging from four to eight. The development of seven perithecia had been interrupted at a stage prior to the development of ascospores. It was difficult to determine whether the ascospore abortion was a result of the drying or some other factor, but the effect appeared similar to that occurring in the controlled crosses. On these leaves there were also perithecia with all normal asci. DISCUSSION Because most of these data originated from crosses made to study inheritance of tolerance to dodine rather than inheritance of the ascospore abortion factor, certain vital pieces of evidence are lacking. Despite the preliminary status of the work, some lines of evidence can be drawn to support hypotheses of both genetic and non- genetic influences on the ascospore abortion phenomenon. Crucial to a discussion of the possible causes of this phenomenon is the evalu- ation of the descriptive and quantitative data supporting these hypotheses. Evidence suggesting a genetic influence on ascospore abortion Three lines of evidence suggest that the ascospore abortion occurring in progeny of isolate SR4 is under genetic control: the recurrence of similar frequencies of abortion in crosses of SR4 and in test crosses of F1 progeny of SR4; the tendency for ascospores to be aborted as pairs rather than singly; and an additive abortive effect in crosses combining the abortion-inducing SR4 progeny and the aborting color mutants. Several previously demonstrated genetic bases for ascospore abortion could be active mechanisms in the abortion of SR4 progeny. Dimock concluded that the aborta factor inactivated or deleted a single 53 54 gene or gene-complex necessary for delimitation (7). Those nuclei inheriting the aborta factor were unable to delimit spores. Genetic studies with the color mutants of V, inaeqoalis (4, 5, 30) have shown an abortive effect on the spore pairs inheriting the mutant factor. While mutant genes usually segregated with a 1:1 ratio of normal to aborted spores, 3:1 and 1:3 ratios also occurred. Thus several patterns of inheritance of a single gene resulted in two, four, or six spores per ascus. Several mechanisms of chromosome segmental interchange have been shown to yield asci containing four or eight spores (l3, 14, 21). Such mechanisms also may prevent the development of any spores. The frequency of empty asci could help to distinguish the type of inter- change, but in this and other studies (5) empty asci were quite diffi- cult to distinguish from immature asci. Combinations of lethal and modifying genes, segmental inter- changes, or a lethal gene and a segmental interchange could conceivably yield asci with spore numbers ranging from zero to eight. Such mechanisms would cause abortion of spore pairs unless the factors would be active only under certain marginal cultural conditions. The ratios of types of abnormal asci could reflect the linkage of genes or modifying factors or the distance of segmental interchanges from the centromere. Evidence suggestingVa non-genetic influence on ascospore abortion Although most of the ascospore abortion of SR4 progeny ap- pears to be under some form of genetic control, some of the effects reported here are not readily explained by genetic mechanisms. 55 The relatively high, variable frequency of asci containing odd numbers of spores may suggest the activity of some variable non- genetic factor such as a suboptimal cultural condition which could prevail over the genetic control of a spore's development. These asci could result from abortion of a spore after its delimitation, de- generation of a nucleus, or the cooperation of two or more nuclei in the delimitation of a single spore. In crosses of normal isolates the presence of asci which have reached their most advanced stage of development without delimit- ing any spores is comparable to effects of suboptimal nutrient conditions in other fungi. In Sordaria biotin-starved cultures produced this type of ascus more frequently (2). The nutrient medium affected the degree of ascospore abortion in Neurospora (10, 12). Thiamine was essential to ascospore formation in V, inaequalis (28). In certain crosses some perithecia contained mostly abnormal asci while other perithecia contained mostly normal asci. Such differ- ences could reflect local differences in nutrient status of the crossing medium or a differential rate of development (5, 11). Further cytological, cultural and genetic studies should re- solve which effects are caused by genetic or non-genetic factors. Cytological studies would locate nuclei and determine the developmental stage at which abortion occurs. Cultural studies should assess the mini- mal requirements of normal and abortion-inducing isolates on a defined medium (27, 28) and with natural overwintering. Genetic investigations could confirm chromosome aberration by testing linkage with genetic markers. 56 Significance of induction of ascospore abortion by Poray Orchard isolates The induction of ascospore abortion by all Poray Orchard iso- lates must have some relationship with the dodine tolerance situation in this orchard, although the two factors are not caused by the same genes. Several possible relationships exist. Possibly mutations for dodine tolerance and for ascospore abortion were induced simultaneously by some mutagenic factor prior to 1969. Low rates of dodine application would have selected for the dodine- tolerant strain during its first growing season, allowing a buildup of the abortion-inducing population as well. It does n0t seem likely that the population of the self-limiting, abortion-inducing strains would in- crease over several growing seasons unless the genes for tolerance and the ascospore abortion factors are closely linked. Close linkage does not seem likely because of the number of highly tolerant progeny inducing very little ascospore abortion. Several of these isolates arose from partially aborted asci and this selection may have represented a dilution of abortive factors, but the partially aborted asci composed the greatest proportion of the asci in these crosses so there is only a slim chance of close linkage between the abortive factors and the genes for tolerance. A second possible relationship could be that ascospore abortion does not occur in nature, but the factors causing abortion may be closely linked to genes for dodine tolerance. Preliminary evidence from a survey of leaves from the Poray Orchard suggests a natural occurrence of the phenomenon, but this abortion could have been caused by drying of the leaves during ascospore development. It may be significant that the two 57 cytological studies of ascus development of V, inaequalis not reporting ascospore abortion were conducted on perithecia produced in nature (1, 17), while the study reporting ascospore abortion examined perithecia produced in culture (6). Perhaps this again reflects cultural conditions which are slightly suboptimal for ascospore production. If ascospore abortion does not occur in nature in strains such as SR4, then the strain would not be self-limiting and its frequency in the population would in- crease readily with the frequency of the tolerant strains under the selection pressure of dodine. A final possibility could be that dodine induces minor muta- tions causing ascospore abortion. If this situation exists, long contin- ual usage of dodine would stimulate an increase in the abortion-inducing population while also selecting for dodine-tolerant strains. The factors causing the two phenomena would not have to be linked genetically and could easily be separated jn_vitro in F1 progeny. PART II LITERATURE CITED 10. 11. 12. PART II LITERATURE CITED BACKUS, E. J. and G. W. KEITT. 1940. Some nuclear phenomena in Venturia inaequalis. Bull. Torrey Bot. Club. 67:765-770. BARNETT, H. L. and V. G. LILLY. 1947. The effects of biotin upon the formation and development of perithecia, asci, and asco- spores by Sordaria fimicola Ces. and DeNot. Am. J. Bot. 34: l96-203. BARRY, E. G. 1967. Chromosome aberrations in Neurospora, and the correlation of chromosomes and linkage groups. Genetics. 55:21-32. BOONE, D. M. 1971. Genetics of Venturia inaequalis. Ann. Rev. Phytopathol. 9:297-318. BOONE, D. M. and G. W. KEITT. 1956. Venturia inaequalis (Cke.) Wint. VIII. Inheritance of color mutant characters. Am. J. Bot. 43:226-233. DAY, P. R., D. M. BOONE and G. W. KEITT. 1956. Venturia inaequalis (Cke.) Wint. XI. The chromosome number. Am. J. Bot. 43: 835-838. DIMOCK, A. W. 1937. Hybridization studies on a zinc-induced vari- ant of Hypomyces ipomoeae. Mycologia 29:273-285. DODGE, B. O. 1928. Spore formation in asci with fewer than eight spores. Mycologia. 20:18-21. DODGE, B. O. 1934. A lethal for ascus abortion in Neurospora. Mycologia. 26:360-376. DODGE, B. O. 1935. A recessive factor lethal for ascospore formation in Neurospora. Bull. Torrey Bot. Club 62:117-128. DODGE, B. O. 1936. Interspecific hybrids involving factors for ascus abortion in Neurospora. Am. J. Bot. 23:555-561. DODGE, B. O. 1939. A new dominant lethal in Neurospora: the E locus in N, tetrasperma. J. Hered. 30:467-474. 58 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 59 ESSER, KARL and RUDOLF KUENEN. 1967. Genetics of fungi. Trans. Erick Steiner. Springer-Verlag Inc. New York. 500 pp. FINCHAM, J. R. S. and P. R. DAY. 1971. Fungal Genetics, 3rd. ed. Blackwell Scientific Publications, Oxford. 402 pp. FURTADO, J. S. 1970. Alcoholic toluidine blue: a rapid method for staining nuclei in unfixed mycetozoa and fungi. Mycologia. 62:406-407. HRUSHOVETZ, S. B. 1956. Cytological studies of ascus development in Cochliobolus sativus. Can. J. Bot. 34:641-651. JULIEN, J. B. 1958. Cytological studies of Venturia inaequalis. Can. J. Bot. 36:607-613. KEITT, G. W. and M. H. LANGFORD. 1941. Venturia inaequalis (Cke.) Wint. I. A groundwork for genetic studies. Am. J. Bot. 28: 805-820. LU, B. C. 1967. The course of meiosis and centriole behaviour during the ascus development of the ascomycete Gelasinospora calospora. Chromosoma (Ber1.) 22:210-226. LU, B. C. and N. B. RAJU. 1970. Meiosis in Coprinus. II. Chromo- some pairing and the lampbrush diplotene stage of meiotic prophase. Chromosoma 29:305-316. MCCLINTOCK, B. 1945. Neurospora 1. Preliminary observations on the chromosomes of Neurospora crassa. Am. J. Bot. 32:671-678. NELSON, R. R. 1959. Genetics of Cochliobolus heterostrophus. II. Genetic factors inhibiting ascospore formation. MyCOTOgia 51:24-30. NOVAK, D. R. and A. M. SRB. 1971. Genetic alterations of ascus development in Neurospora tetrasperma. Genetics 67:189-199. O'DONNELL, K. L., W. TAI, and E. S. BENEKE. 1974. Nuclear be- havior and spindle pole bodies during ascosporogenesis in Peziza guelepidotia. J. Gen. Microbiol. 81:303-314. PINCHEIRA, G. and A. M. SRB. 1969. Genetic variation in the orientation of nuclear spindles during the development of asci in Neurospora. Am. J. Bot. 56:846-852. ROGERS, J. D. and L. F. GRAND. 1971. Coniochaeta malacotricha: anomalous asci and the conidial state. Can. J. Bot. 49: 2239-2240. 27. 28. 29. 30. 60 ROSS, R. G. and S. A. HAMLIN. 1962. Production of perithecia of Venturia inaequalis (Cke.) Wint. on sterile apple leaf discs. Can. J. Bot. 40:629-635. ROSS, R. G. and S. A. HAMLIN. 1965. Influence of nutrients on perithecial production of Venturia inaequalis (Cke.) Wint. Can. J. Bot. 43:959-65. RUSSELL, P. J. and A. M. SRB. 1972. Dominance modifiers in Neurospora crassa: phenocopy selection and influence on certain ascus mutants. Genetics 71:189-199. SHAY, J. R. and G. W. KEITT. 1945. The inheritance of certain mutant characters in Venturia inaequalis. J. Agric. Res. 70:31-40. APPENDICES 61 NNNNNNNNNN N NN.N NN.N NN N NN.N NN.N NN N NN. NN.N NN N NN.N NN.N NN N NN. NN.N NNNNNNNNNNNN .NNNNNNNNN .>.z .N>N=NN NNNN .NNNN NN N NN.N NN.N NNNNNENNNNNN .NNNNNN N2 .N2 .NNNNNNN NNNN NNNN .NNN NN N NN.N NN.N NNNNNENNNNNN .xNNNNN N2 .N2 .NNNNNNN NNNN NNNN .NNN NN N NN.N NN.N NNNN N NN.N NN.N . NNN N NN.N NN.N NNNNNENNNNNN .NNLNNN .Nz .NNNNNNN NNNN NNNN .NNN NNN N NN.N NN.N NNN N NN.N NN.N NNN N NN.N NN.N =NNNN NNNNNNN= NNN N NN.N NN.N NNNNNNNNN .NNNNNNNNN .Nz .NNNNNNN NNNN NNNN .NNN NNN N NN.N NN.N NN N NN.N NN.N NN N NN.N NN.N NN N NN.N NN.N NNNNNNNNNNNN .NNNNNNNNN .Nz .NNNNNNN NNNN NNNN .NNN NN N NN.N NN.N NN N NN.N NN.N NN N NN.N NN.N NNNNNENNNNNN .NNNNNN .Nz .NNNN>NNNNN NNNN .NNNN NN N NN.N NN.N NN N NN.N NN.N NN N NN.N NN.N NN m mm.m oo.m women a:_NcmmN umeNNNcs .go .mcwmuconNNmm mmmN .poo Nm N NN.N NN.N NNN N NN.N NN.N NNNNNNNNN NNN .NNNNNNENN .Nz .SNNNN NNNN Ni :2 NNNNNNN NENNN NNN NENNN Nm NNNENNNNN NNNNNNNN NNNNNNNNNN NNNNNNN -NNNmm uAEoV ENNN mcou a mgmo wauwcouocoz .Numwu NNNNN NNNNN seem NewmsNNNn mcpvou Na Nome mmochmnnoumaoa cw NNNNNoNN uNoNN NNNnaummeN NNN2u=m> No :oNNNnN;:N N< x~ozw¢m< 62 N NN.N ON.N Nemgoco NNNNN .> .z .Nznom oNNN .NNmm NNNum N mN. NN.N umum m mm.N om.N Nmum N NN.N mm.N Newcogo NNNNN .> .z .mauom onmN .NNmm Nmom m oo.m NN.N . Now m om.N NN.N NNNucmEEou .Nz .NNNNNm NNNN .m:< Now N NN.N mm.N Nm m NN.N No.N mm N NN.N NN.N NNNusmeeou .Nz .NNNNNm NNNN .uuo Nm m NN.N ON.N mm N om.m mo.N NmpcmchmeN .meNau .> .z .N>m:mu mmNN .uNmm Na N oN.m wo.m mo N NN.N NN.N No N NN.N ow.N No N ON.m mN.N NNNNNNNN: .Nz .meNNcNN NNNN mmNN .uuo No N om.N om.N NNNoNoEEoo .Nz .NENNN NNNN .Nuo Nz N om.m oo.m N2 N NN.N NN.N m: m NN.N NN.N N: m NN.N No.m NNNugossou .Nz .3NN zNN NNNN .Nuo N: N om.m mo.m N4 N 0N.m ow.N m4 N om.m ON.N NNNuemesou .Nz .Nuoewm NNNN .Nuo N4 N mo.m mm.N a: N om.N om.m N: N om.m 0N.N m: N Nm.m NN.N N: N 0N.m oN.N umzmgamca .uz .mNNN>Nu:NNw mmmN .Nuo N: NcoNNNo E a: com Ns\on om NcmEmeNN :oNuNuoN nepomNNoo muNNoNN -NNNmm N MAEuV ENNN mcoN N N mama NNNuNcouocoz NNNNNNNNNN N.NNNNV .N< NNNNNNNN 63 N oo.m oo.m m N om.m mm. N N 0N.m mw.N NmNoLmsEou .Nz .ZNN 3mm NmmN .xmz N NNNu m ON.m om.N m m no.m om.N N m mm.m mN.N o N mm.m mo.N m m um.m mn.N N N ou.m oo.m m m 0N.m mm.~ N m om.m om.N NmNoNoEEou .Nz .zma 3mm NNmN .xmz N NNQ NNNNNNN N mm.m mm.N N3 N om.m mm.N NemsumcowN NmNuLwEEou .Nz .wuogmm momN .Noo N3 N om.m ON.N N> N om.N om.N m> N nw.m oo.m N> N oo.m om.N NmNoLwEEoo .Nz .muogmm momN .poo N> N um.N 0N.N ouNoncaw NmpcmENLmnxm .Nz .mcmmcm4 ummm momN .uoo Nzh N NN.m mm.m ouNuchzm Nmucmewsmaxm .Nz .chmcm4 ummm mmmN .Nuo Nah m om.m NN.m mvwormczm Nmucmsmgmaxu .Nz .mcwmcw4 “mum momN .Nuo Nb N mm.~ os.N Nam N mo.N mm.o ucmsugo ance; .> .z .mauom mmmN .uamm Nam N um.N mm.N UNNum N om.N MN.N aNNum NcoNuNu Nexmn com NENNN om ,NemEuNmNN :oNuNuoN mcmuumNNoo muNNoNN -NNNmm NeuV ENNN m=o~ a once NNNNNcouocoz U NNNNNNNNNN N.NcouV .N< NNNNNNNN 64 N om.N om.N mum- N ON.N om.N mum- N oN.N oo.N ugmzugo huge; .> .z .mzuom onmN .xuz mum- N oN.N om.N muN- N ON.N om.N mum- N om.N mm.N muN- N mN.N ON.N muN- N oN.N mm.N Num- N mm.N om.N NuN- N om.N mN.N Nuo- N ON.N oo.N Num- N mm.N om.N N-N- N mN.N mm.N Num- N ON.N mm.N N-N- N mm.N mm.N N-N- N oN.N mN.N Nnm- N mo.N mo.N Numu N oo.N mm.N N-o- N oN.N mN.N N-m- N om.N mo.N NuN- N mm.N ON.N Num- N om.N om.N NuN- N 0N.N om.N ugmsugo huge; .> .z .mauom onN .zmz NuN- QNN N mm.m oN.N m N mm.m oo.m N N om.m om.N o N 0N.m om.N m N mN.m om.N N mcoNumu Ns\mn com Ns\mn om ucmsummgN coNucuoq mumuumNNou mcmgumN -NNamm NEUV EmNu mean 3 upon U =o_aPnP;=H A.u=ouv .N< xwucman< 65 N om.N mN.N NuN- N mm.N mm.N N-N- N mN.N om.N Num N ON.N mm.N NuN N om.N om.N Nuo N om.N om. Nnm N om.N om.N NuN N om.N mm.N Nnm N mN.N ow.N NuN N mm.N mw.N ugmzugo cmmsouz .> .z .omom gpgoz momN .Nmz NuN as N om.N oo.N Num- N mN.N mm.N NuN- N mm.N mo.N N.@. N mN.N mN.N Nnm- N ON.N oN.N NnN- N ON.N ON.N Num- N mN.N mm.N NnN- N om.N om.N NnN- N oN.N oo.N Nuw- N mo.N mm.N NuN- N om.N ON.N Num- N mm.N ON.N Num- N ON.N mm.N NuN- N mN.N oo.N Num- N mo.m 0N.N NnN- N mo.m ON.N ugmgugo xmgoa .> .z .mauom NNmN .xmz NuN- NNN N ON.N om.N m-w- mcoNumu Ns\ma com Newmnom ucmsucmLN :oNumuo4 mvmuumNNou mumgpmN -Nqum uNswg EmNc mcoN a mpmo :oNuNnNch A.u=ouv ._< x_n=mgq< 66 .umpmgsumm mm: umNu xwmmm EmNu as N.NN susz zuNz chuou Na :oNumgpcmucou .u mN um coNgmnaucN .mzmu cm>mm gmumm umgzmmms mmcoNu .xumNLm> zgmochuz= Eogw :mxmp mgmz mmumNomN NNm .uwNNNumam mmmch .mmuNuchaw NmpcmeNLmaxm cpNz umzugam go xmsazu guNz umxmgam .vmzmgamcz smsuNm wgmz mvgmgugo NmucmENgmaxw sogm umamNN mmumNomN .Ewgmoga Nagam gmszmg w No “gag mm xmxazu um>Nmumg NmNugmsEou mm umumNN mmpmNomNn .pcmspmwgu comm EoLN mpmNomN meNN ms» Low NNco umpmNN ago pcmEammgp use coNpmuoN .muwom N mN.N mm.N Nuw- N mm.N oo.N NuN- N oN.N mo.N N-m- N oN.N om.N Num- N oo.N om.N NuN- N mm.N mN.N Num- mcoNpmu Ns\mn com NE\mn om npcwsummgN :oNNmqu mvmuumNNou mvmgumN -NNamm uaauv EmNu chN mgmo :oNuNnNch 353 .2 5:32 APPENDIX A2 Inhibition by dodine of germination and growth of conidia of mono- ascosporic progeny of Venturia inaequa1is cross SR4 X SR7 Inhibition zone diam (cm)a % germinationb Iso1ate 50 ug/m1 300 ug/m1 1 ug/m] Eight-spared asci SR4SR7-1-1 1.48 2.35 30.2 -2-1 1.35 2.43 37.4 -3-1 2.25 3.13 8.7 -4-1 2 05 2.90 12.2 -5-1 1 53 2.40 25.9 -6-1 1 33 2 35 31.4 -7-1 --- --- 14.3 -8-1 1.90 2.73 13.7 -1-2 1.55 2.58 23.1 -2-2 1.50 2.50 23.1 -3-2 1.88 2.88 10.7 -4-2 1.75 2.60 11.7 -5-2 1.70 2.53 22.1 -6-2 1.68 2.55 21.5 -7-2 1.35 2.20 28.2 -8-2 1.33 2.33 . 28.2 Six-spared asci -1-1 2.05 3.05 35.0 -2-1 1.83 2.60 33.4 -3-1 1.53 2.38 41.7 -4-1 1.58 2.70 43.8 -5-1 1.75 2.80 19.1 -6-1 1.95 2.95 12.5 -1-2 1.38 2.33 38.1 -2-2 1.98 2.85 35.1 -3-2 1.58 2.55 36.2 -4-2 1.58 2.48 36.6 -5-2 1.48 2.55 37.4 -6-2 1.43 2.30 34.6 67 Appendix A2. (Cont.) 68 Inhibition zone diam (cm)a % germination b Iso1ate 50 ug/mI 300 pg/m1 1 pg/m1 Four-spared asci -1-1 2.03 2.83 30.7 -2-1 1.80 2.80 35.6 -3-1 1.90 2.83 17.2 -4-1 1.53 2.33 20.0 -1-2 1.53 2.48 35.9 -2-2 1.50 2.38 36.5 -3-2 2.10 2.95 17.2 -4-2 1.98 2.78 16.9 Two-spared asci -1-1 1.45 2.65 32.3 -2-1 1.78 3.00 28.4 -1-2 1.40 2.65 32.1 -2-2 1.45 2.53 36.0 LSD .05 0.40 0.40 LSD .01 0.52 0.52 aMeans of four rep1ications. Zones measured after seven days' Dodine concentrations with which 12.7 mm Inhibition zone diam in 50 pg/m1 incubation at 19 C. assay disc was saturated. and 300 ug/m1 tests: 2.58 cm). bPercent of germination of contro1 conidia in water. SR4 (0.83 and 1.63 cm); SR7 (1.70 and APPENDIX A3 Inhibition of random progeny of Venturia inaegua1is cross 04SR4-1-2 X 04SR4-6-1 in potato-dextrose agar by dodine diffusing from paper assay discs. Inhibition Inhibition zone diam (cm)a zone diam (cm) Iso1ate 50 ug/m1 300 ug/mi Iso1ate 50 pg/mT* 300 ug/mi -1 0.80 1.95 -40 1.70 2.45 -2 0.97 2.02 -41 2.15 2.90 -3 1.90 2.52 -42 3.00 3.57 -4 2.10 2.77 -43 2.85 3.30 -5 0.67 1.65 -44 2.60 3.05 -6 2.17 2.65 -45 1.65 2.40 -7 2.55 3.32 -46 2.40 3.22 -8 2.50 3.25 -47 2.30 2.90 -9 2.22 3.00 -48 2.02 2.87 -10 2.70 3.35 -49 2.37 3.02 -11 2.12 2.95 -50 1.30 1.97 -12 2.22 3.15 -51 1.85 2.62 -13 1.70 2.62 -52 1.65 2.40 -14 2.52 3.22 -53 2.18 2.90 -15 2.37 3.07 -54 2.50 3.15 -16 1.57 2.40 -55 2.42 3.12 -17 2.37 3.17 -56 1.25 2.07 -18 2.02 2.77 -57 1.75 2.67 -19 2.37 2.95 -58 1.30 2.30 -20 1.77 2.45 -59 3.07 3.62 -22 1.92 2.82 -60 2.15 2.90 -24 1.97 2.72 -62 1.95 2.95 -25 0.42 1.65 -63 0.87 1.95 -26 2.35 3.15 -64 2.52 3.30 -27 2.17 2.90 -65 2.35 3.15 -28 1.90 2.70 -66 1.85 2.77 -29 2.10 2.77 -67 1.90 2.70 -30 2.70 3.20 -68 1.97 2.77 -31 1.70 2.47 -69 1.82 2.55 -32 0.62 1.77 -70 3.02 3.60 -33 1.70 2.50 -73 1.55 2.67 -34 1.12 1.92 -74 2.30 2.80 -35 2.82 3.52 -75 2.27 3.05 -36 2.47 3.10 -76 1.20 2.10 -37 2.40 3.05 -38 2.27 2.92 LSD .05 0.47 0.63 -39 2.35 3.17 LSD .01 0.62 0.83 aMeans of four rep1ications. Zones measured after seven days' incuba- tion at 19 C. Dodine concentrations with which 12.7 mm assay disc was saturated. Inhibition zone diam in 50 ug/m1 and 300 ug/m1 tests: 04SR4-1-2 (1.80 and 2.68 cm), 04SR4-6-1 (2.38 and 3.20 cm). 69 APPENDIX A4 Inhibition by dodine of germination and growth of conidia of tetrad progeny of Venturia inaequa1is cross SR4 X 04SR4-3-2. Inhibition zone diam (cm)a % germinationb Iso1ate 50 ug/m1 300 ug/m1 dodine 1 ug/m1 1-1 .50 1.70 28.1 2-1 .73 1.68 33.8 3-1 .70 1.73 46.5 4-1 .48 1.63 61.6 5-1 1.00 1.88 58.6 6-1 .70 1.75 46.2 7-1 1.20 1.85 53.3 8-1 .63 1.73 66.8 -2-3 1.40 2.13 29.8 3-3 .43 1.43 49.9 4-3 .45 1.63 63.0 -5-3 .90 1.78 73.5 6-3 .70 1.68 74.9 7-3 1.20 1.65 72.2 8-3 .55 1.63 40.5 1-4 1.23 1.98 38.2 2-4 .83 1.65 65.1 3-4 .88 1.75 60.1 4-4 1.15 1.93 44.2 5-4 .63 1.60 60.5 6-4 1.28 2.00 25.3 7-4 .68 1.70 49.4 8-4 .53 1.65 65.9 -1-6 .45 1.63 60.9 -3-6 .75 1.70 69.7 4-6 .60 1.65 63.9 5-6 .93 1.80 61.9 6-6 1.23 2.00 34.3 7-6 .73 1.73 30.3 8-6 .68 1.65 49.6 LSD .05 0.40 0.20 LSD .01 0.53 0.26 aMeans of four rep1ications. Zones measured after seven days' incuba- tion at 19 C. Concentrations of dodine with which paper assay disc was saturated. Inhibition zone diam in 50 ug/m1 and 300 ug/m1 tests: SR4 (0.83 and 1.63 cm); 04SR4-3-2 (1.23 and 2.33 cm). b o o o o 0 Percent of germination of contro1 conidia in water. 70 APPENDIX A5 Inhibition by dodine of germination and growth of conidia of tetrad progeny of Venturia inaequa1is cross C2 X 04SR4-5-2. Inhibition zone diam (cm)a % germination b Iso1ate 50 ug/m1 300 ug/m177 dodine 1 ug/m1 -1-3 2.32 2.80 13.3 eg-3 2.80 3.20 6.3 -3-3 2.70 3.23 2.4 -4-3 2.80 3.30 9.9 -5-3 2.10 2.63 23.1 -6-3 2.22 2.73 15.7 -7-3 2.42 2.78 10.8 ~8-3 2.57 2.70 4.4 -1-14 2.72 3.13 4.0 -2-14 2.72 3.13 2.3 -3-14 2.24 2.85 8.6 -4-14 2.62 2.98 7.8 -5—14 2.38 2.90 7.6 -6-14 2.44 2.88 9.4 -7-14 2.36 2.80 12.5 -8-14 2.28 2.78 23.6 -1-15 2.20 2.75 11.2 -2-15 2.37 2.83 12.3 -3-15 2.75 3.18 1.2 -4-15 2.23 2.73 14.9 -5-15 2.30 2.78 19.4 -6-15 2.63 3.03 3.3 -7-15 2.62 3.05 2.9 -8-15 2.50 2.98 1.8 -1-18 2.25 2.88 19.8 -2-18 2.45 2.98 21.3 -3-18 2.32 2.90 18.0 -4-18 2.32 2.83 13.2 -5-18 2.50 2.95 8.5 -6-18 2.33 2.80 7.0 -7-18 2.85 3.23 10.6 -8-18 2.55 3.10 5.0 LSD .05 0.34 0.34 LSD .01 0.45 0.45 aMeans of four rep1ications. Zones measured after seven days' incuba- tion at 19 C. Concentrations of dodine with which paper assay disc was saturated. Inhibition zone diam in 50 ug/m1 and 300 ug/m1 tests: C2 (3.05 and 3.75 cm); 04SR4-5-2 (2.30 and 3.15 cm). bPercent of germination of contro1 conidia in water. 71 APPENDIX A6 Inhibition by dodine of germination and growth of conidia of tetrad progeny of Venturia inaequa1is cross C2 X 04SR4-7-2. Inhibition zone diam (cm)a % germination b Iso1ate 50 pg/m1 300 ug/m1 dodine 1 ug/m1 -1-2 2.45 3.03 2.5 -2-2 2.63 3.15 3.4 -3-2 2.68 3.05 3.3 -4-2 2.58 3.03 0.9 -5-2 2.50 2.93 4.9 -6-2 2.80 3.33 1.0 -7-2 2.88 3.40 0.1 -8-2 2.43 2.85 0.2 -1-5 3.03 3.40 3.6 -2-5 2.78 3.25 2.9 -3-5 2.85 3.33 7.1 -4-5 2.33 2.85 10.9 -5-5 2.60 2.98 1.2 -6-5 2.55 2.98 4.4 -7-5 2.43 2.90 15.4 -8-5 2.20 2.70 2.0 -1-7 2.40 2.83 15.1 -2-7 2.50 2.90 7.4 -3-7 2.75 3.08 1.6 -4-7 2.45 3.03 1.8 -5-7 2.78 2.93 0.7 -6-7 2.53 3.03 0.3 -7-7 2.45 2.90 4.5 -8-7 2.35 2.85 5.8 -1-11 2.60 3.10 1.6 -2-11 2.40 2.80 3.9 -3-11 2.50 3.03 18.0 -4-11 2.57 3.05 5.8 ~5-11 2.55 3.08 11.5 -6-11 2.37 2.80 6.8 -7-11 3.02 3.45 4.8 -8-11 2.83 3.20 8.7 LSD .05 0.44 0.66 LSD .01 0.58 0.87 aMeans of four rep1ications. Zones measured after seven days' incuba- tion at 19 C. Concentrations of dodine with which paper assay disc was saturated. Inhibition zone diam in 50 pg/m1 and 300 ug/m] tests: C2 (3.05 and 3.75 cm); 04SR4—7-2 (2.98 and 3.63 cm). bPercent of germination of contro1 conidia in water. 72 APPENDIX A7 Inhibition of random progeny of Venturia inaequa1is cross S4 X C2$R4 7-4 in potato-dextrose agar by dodine diffusing from paper assay discs. Inhibition zone diam (cm)a Iso1ate 50 ug/m1 300 pg/m1 ~1 2.3 2.9 -3 1.9 2.5 -5 2.2 2.7 -6 2.3 2.9 -7 2.4 2.9 -8 2.2 2.8 -9 1.2 2.1 ~10 1.0 2.0 ~11 2.0 2.6 ~12 2.4 2.8 ~13 2.3 2.7 ~15 2.0 2.7 ~16 1.6 2.4 ~17 1.4 2.3 ~18 2.1 2.8 ~19 1.5 2.3 ~21 2.0 2.7 ~27 2.2 2.8 -28 1.9 2.3 ~31 1.6 2.4 ~32 1.8 2.5 ~33 2.0 2.5 ~35 1.6 2.1 ~37 2.5 2.8 ~38 1.4 1.9 ~39 1.4 2.0 ~40 2.2 2.7 ~41 1.3 2.1 ~42 1.6 2.1 ~44 2.3 2.9 ~46 2.1 2.7 ~47 2.0 2.6 ~50 2.2 2.8 ~51 2.1 2.7 ~52 1.9 2.7 ~54 2.4 2.9 ~55 1.1 1.9 ~56 1.9 2.6 ~57 2.0 2.8 ~58 1.8 2.3 aA singie rep1ication. Zones measured after seven days' incubation at 19 C. Dodine concentrations with which 12.7 mm assay disc was saturated. Inhibition zone diam in 50 ug/m1 and 300 ug/m1 tests: S4 (2.58 and 3.58 cm); C2$R4 7-4 (1.10 and 2.15 cm). 73 APPENDIX A8 Inhibition of random progeny of Venturia inaequa1is cross S1 X 2295-2 (green co1or mutant) in potato-dextrose agar by dodine diffusing from paper assay discs. Inhibition zone diam (cm)a Iso1ate 50 ug/m1 300 pg/m1 Co1or ~1 2.3 3.0 wi1d type ~2 2.6 3.2 wi1d type -3 2.2 2.8 wi1d type -4 1.8 2.6 green ~5 1.8 2.5 wi1d type ~6 1.9 2.8 green ~7 2.6 3.2 green -8 2.7 3.2 green -9 2.4 2.9 green ~10 2.5 3.0 green ~11 2.1 2.7 green ~12 2.3 2.9 green ~13 2.1 2.8 wi1d type ~15 2.0 2.7 green ~16 2.6 3.3 wi1d type ~17 2.9 3.4 wi1d type ~18 2.5 3.1 wi1d type ~20 2.2 2.8 wi1d type ~24 2.1 2.7 green ~26 1.8 2.6 green ~27 2.0 2.8 wi1d type ~29 2.1 2.9 green ~31 2.2 2.8 wi1d type ~32 2.0 2.6 green ~33 2.7 3.2 wi1d type ~34 2.0 2.6 wi1d type ~36 1.9 2.5 wi1d type ~37 2.2 2.7 green ~38 2.0 2.7 green 74 75 Appendix A8. (Cont.) Inhibition zone diamjcm)? Iso1ate 50 ug/m1 300 ug/m1 Co1or ~42 1.8 2.5 green ~43 2.5 2.9 wi1d type ~44 2.8 3.2 green ~45 2.2 2.8 wi1d type ~46 2.4 3.0 wi1d type ~47 2.1 2.8 wi1d type ~48 2.2 2.7 green ~49 2.2 2.8 wi1d type ~50 2.5 3.1 wi1d type ~51 1.9 2.4 green ~52 2.1 2.7 green ~53 2.1 2.7 green ~54 2.0 2.6 green ~55 2.5 3.0 green ~56 2.5 3.1 wi1d type aResu1ts of one or two rep1ications. Zones measured after seven days' incubation at 19 C. Dodine concentrations with which 12.7 mm assay disc was saturated. Inhibition zone diam in 50 ug/m1 and 300 ug/m1 tests: 31 (2.18 and 3.18 cm); 2295-2 (3.00 and 3.57 cm). APPENDIX A9 Inhibition of random progeny of Venturia inae ua1is cross C2$R4~7~3 X 3295-2 in potato-dextrose agar by dodine diffusing from paper assay 1SCS. Inhibition zone diam (cm)a Iso1ate 50 ug/m1 300 ug/mI Color -1 2.5 3.0 green ~2 2.2 2.8 wi1d type ~4 2.2 2.5 wi1d type ~5 2.5 2.9 wi1d type ~7 1.8 2.3 wi1d type ~8 2.2 2.6 wi1d type ~10 2.2 2.7 wi1d type ~12 2.5 3.0 wi1d type ~13 2.9 3.5 green ~15 1.3 2.0 wild type ~20 1.6 2.2 wi1d type ~22 2.1 2.7 green ~24 1.5 2.1 wi1d type ~27 2.6 3.2 wi1d type ~28 1.4 2.0 green ~30 2.2 2.7 wi1d type ~31 1.2 2.0 wi1d type ~34 1.8 2.6 wi1d type ~35 2.4 2.9 wi1d type ~38 2.6 3.1 green ~39 2.1 2.6 wi1d type ~46 2.4 2.9 wi1d type ~48 3.1 3.4 wi1d type ~49 1.8 2.5 green ~53 1.5 2.1 green ~54 2.3 2.8 brown aResu1ts of one or two rep1ications. Zones measured after seven days' incubation at 19 C. Dodine concentrations with which 12.7 mm assay disc was saturated. Inhibition zone diam in 50 pg/m1 and 300 ug/m1 tests: C2SR4-7-3 (1.20 and 1.90 cm); 2295-2 (3.00 and 3.57 cm). 76 APPENDIX B1 Frequency of ascospore abortion in crosses of F1 progeny of 04 X SR4. Percent asci with indicated number of spores per ascus Cross Tota1 1 2 3 4 5 6 7 £3 asci 04SR4~12 X C4 3 (24)a 21 7 (42) 35 15 (34) 19 0 ( 0) (I 74 x 51 1 (56) 45 (22) 22 0 (22) 22 0 ( 0) 0 9 X TH1 0 ( 3) 3 13 (46) 33 11 (49) 38 2 ( 2) 2 63 04SR4~22 X C4 1 (34) 33 30 (45) 15 6 (19) 13 1 ( 2) 1 90 X S1 0 (33) 33 34 (34) 0 0 (33) 33 0 ( 0) 0 3 X TH1 5 (17) 12 16 (33) 17 19 (50) 31 0 ( 0) 0 42 04SR4~32 X C4 1 ( 4) 3 8 (44) 36 15 (50) 35 0 ( 2) 2 124 X 04 0 ( 8) 8 4 (46) 42 10 (44) 34 2 ( 2) 0 134 X S1 0 ( 9) 9 12 (60) 48 14 (31) 17 0 ( 0) 0 58 X 54 0 (23) 23 21 (46) 25 10 (31) 21 0 ( 0) 0 48 X T01 2 ( 9) 7 5 (49) 44 4 (39) 35 1 ( 3) 2 89 X TH1 0 ( 1) 1 2 (63) 61 (36) 28 0 ( 0) 0 174 X SR4 2 ( 6) 4 17 (44) 27 15 (46) 31 4 ( 4) 0 48 04SR4~42 X C4 3 (24) 21 11 (50) 39 3 (26) 23 0 ( 0) 0 74 X 04 0 (28) 28 2 (52) 50 10 (18) 8 0 ( 2) 2 40 X 51 1 (40) 39 (49) 43 (11) 6 0 ( 0) 0 67 X 54 0 (27) 27 16 (55) 39 (18) 16 0 ( 0) 0 49 X T01 2 (15) 13 14 (56) 42 10 (26) 16 0 ( 3) 3 125 X TH1 0 ( 5) 5 6 (69) 63 21 (26) 5 0 ( 0) (J 19 X SR4 1 (13) 12 5 (33) 28 9 (53) 44 0 ( 1) 1 102 77 78 Appendix 81. (Cont.) Percent asci with indicated Cross number of spores per ascus Tota1 1 2 3 4 5 6 7* £3 asci 04SR4~52 X C2 0 (10) 10 7 (52) 45 2 ( 4) 2 8 (34) 26 97 04SR4~62 X C2 0 (12) 12 6 (28) 22 13 (28) 15 22 (32) 10 60 04SR4-72 X C2 0 ( 7) 7 2 (34) 32 2 ( 7) 5 8 (52) 44 108 04SR4~12 X ~62 0 ( 3) 3 3 (26) 23 10 (68) 58 0 ( 3) . 3 31 ~22 X ~52 7 (33) 26 10 (29) 19 21 (38) 17 0 ( 0) 0 42 ~12 X ~72 4 (30) 26 19 (24) 15 9 (36) 27 0 ( 0) 0 53 ~22 X ~72 2 (11) 9 6 (55) 49 8 (31) 23 2 ( 3) 1 105 ~32 X ~62 6 (18) 12 19 (63) 44 0 (19) 19 0 ( 0) 0 16 aFigures in parentheses represent tota1s of adjacent categories. APPENDIX 82 Frequency of ascospore abortion in crosses of norma1 iso1ates with se1ected iso1ates simiIar to SR4 in dodine to1erance. Percent asci with indicated Cross number of spores per ascus Tota1 1 2 3 4 5 6 7 £3 asc1 C2$R4-35 x 04 0 ma 1 o (7) 7 1 (6) 5 o (86) 86 92 X 51 0 0) (I 0 ( 0) 0 0 ( 0) 0 (J ( 0)100 11 X S4 0 0) 0 0 ( 2) 2 0 ( 4) 4 0 (94) 94 136 X TH1 0 E1; 1 0 E I) 1 1 S 7; 6 1) $91; 91 75 X T01 0 2 2 0 23 23 0 2 2 0 73 73 52 % of tota1 0 (1) 1 0 ( 6) 6 1 ( 5) 4 0 (88) 88 366 CZSR4-37 X C4 0 0) 0 0 ( 1) 1 0 ( 3) 3 0 (96) 96 114 X 04 0 0 0 2 ( 2) 0 0 ( 3) 3 0 95 95 40 X S1 0 2 2 0 E0) 0 2 10; 8 7 88 88 87 X 54 0 1 1 0 0 0 0 3 3 0 96 96114 S T01 0 (0) 0 0 (20) 20 3 20) 17 3 (60) 57 66 ‘1. of tota1 0 (1) 1 0 ( 3) 3 1 ( 7) 6 2 (89) 87 410 C2$R4~48 X C4 0 $0; 0 0 1) 1 0 ( 4 4 0 (95) 95 97 X 51 0 0 0 0 2; 2 2 24 22 9 74 65 46 X S4 0 (0) 0 0 0 0 0 2 0 98 98 66 X 101 0 (1) 0 0 ( 8) 8 2 10 1 (79 78 160 % of tota1 0 (1) 1 0 ( 4) 4 1 (10) 9 1 (85) 84 369 C2SR4 74 X C4 0 (0) 0 0 ( 1) 1 0 ( 0) 0 0 (99) 99 184 X 04 0 $0; 0 0 (11) 11 0 g 0; 0 0 (84; 84 44 X S4 0 0 0 0 ( 0) 0 0 0 0 0(100 100 34 X TH1 0 (4) 4 4 ( 8) 4 0 ( 0) 0 0 (88) 88 26 % of tota1 0 (0) 0 0 ( 3) 3 0 ( 1) 1 0 (96) 96 288 aFigures in parentheses represent tota1s of adjacent categories. 79 APPENDIX B3 Frequency of ascospore abortion in crosses invo1ving co1or mutants. Percent asci with indicated Cross number of spores per ascus Tota1 1* 2 3 4 5 6 7 8 asci N~156 X 04 o (12)312 o (88) 88 o (0) 1 1 (0) o 34 2295-2 0 (10) 10 0 (90) 90 0 ( 0) 0 0 ( 0; 0 41 C2SR4-35 1 ( 2) 1 0 (95) 95 0 ( 3) 3 0 E 0 0 129 C2SR4-37 0 ( 53 5 1 (95) 94 0 ( 0) 0 0 01 0 80 C2$R4~48 0 ( 0 0 0 (100) 100 0 ( 0) 0 0 E 0; 0 42 C2$R4 74 0 (18; 18 0 (78) 78 2 (2) 0 2 2 0 55 04SR4~12 3 (52 49 6 (45) 39 0 ( 3) 3 0 ( 0) 0 120 04SR4~22 6 (70) 64 4 (30) 26 0 ( 0) 0 0 ( 0) 0 78 04SR4~32 0 (50) 50 10 (50 40 0 E 0; O 0 E 0; 0 52 04SR4~42 0 50) 50 25 50 25 0 0 0 0 0 0 4 N~150 X 04 0 (10) 10 6 (68) 62 5 (20) 15 0 (2) 2 85 C2SR4-35 0 ( 6) 6 6 (88) 82 0 ( 6; 6 0 E 0) 0 17 C2SR4-37 0 ( 0) 0 1 (100) 99 0 ( D 0 0 0) 0 114 C2$R4 74 0 (58) 58 0 (42) 42 0 0 0 0 0 0 12 04SR4~12 10 (43 33 9 (52; 43 3 5 2 0 0 0 58 04SR4~22 2 249 47 2 51 49 0 0 0 0 0 0 57 04SR4~32 4 84 80 0 (16) 16 0 (0) 0 0 0 0 44 04SR4~42 0 (44) 44 11 (56) 45 0 (0) 0 0 0) 0 9 N~289 X C2$R4~48 0 ( 0) 0 0 ( 0) 0 0 ( 7) 7 0 (93) 93 27 04SR4~12 2 (39; 37 5 E40; 35 4 (21; 17 0 (0; 0 101 04SR4~22 3 41 38 9 40 31 5 (19 14 0 0 0 74 2295-2 X C2SR4-51 0 (10) 10 4 (47) 43 10 (43) 33 0 (0) 0 219 C2$R4-61 1 ( 9) 8 8 (43) 35 11 (45) 34 '1 3) 2 127 04SR4~62 0 ( 4) 4 0 ( 6) 6 0 ( 0) 0 0 (90)90 53 aFigures in parentheses represent tota1s of adjacent categories. 80 Typed and Printed in the U.S.A. Professional Thesis Preparation Cliff and Paula Haughey , 144 Maplewood Drive '7 East Lansing. Michigan 48823 " Telephone (517) 337-1527 HHIHIIIIIIHIIIU 111[111111111111111111211111 3123