Comparative studies on genetic variability and fungicide resistance in fuckeliana (de Bary) Whetzel against vinclozolin and the phenylpyrrole CGA 173506

Inauguraldissertation zur Erlangung der Würde eines Doktors der Philosophie vorgelegt der Philosophisch- Naturwissenschaftlichen Fakultät der Universität Basel

von

Urs Werner Hilber aus Degersheim- Magdenau SG

Basel, 1992

n BASLER - SCHNELLDRUCK : ~ BERNHARD SCHLATTMANN ~@Jm:.t-;(.!:...JURASTAA.SSE 5-5 TElefON 081 35 37 37 40 5 3 8ASEL

Comparative studies on genetic variability and fungicide resistance in (de Bary) Whetzel against vinclozolin and the phenylpyrrole CGA 173506

Inauguraldissertation zur Erlangung der Würde eines Doktors der Philosophie vorgelegt der Philosophisch- Naturwissenschaftlichen Fakultät der Universität Basel

von

Urs Werner Hilber aus Degersheim- Magdenau SG

Basel, 1992 Genehmigt von der Philosophisch- Naturwissenschaftlichen Fakultät auf Antrag der Herren Professoren Dr. F.J. Schwinn und Dr. T. Boiler

Basel, den 28. April 1992

Prof. Dr. J.-M. Le Tensorer (Dekan) Dedicated to MajaBodmer Contents

Preface 1 Disease cyde 2 Structure formulae 3 General Introduction 4

1. Field study on the population dynamics of Botryotinia fuckeliana (de Bary) Whetzel resistant to dicarboximides 8 1.1 lntroduction 8 1.2 Materialsand Methods 8 1.3 Results 10 1.4 Discussion 12

2. Studies on the base~line sensitivity of Botryotinia fuckeliana (de Bary) Whetzel against CGA 173506 15 1.1 Introduction 15 1.2 Materialsand Methods 15 1.3 Results 18 1.4 Discussion 22

3. Stu.dies on the occurrence of laboratory resistance to vindozolin and CGA 173506 in Botryotinia fuckeliana (de Bary) Whetzel and its influence on fitness parameters 24 1.1 Introduction 24 1.2 Materialsand Methods 25 1.3 Results 31 1.4 Discussion 47

4. Studies on the inheritance of resistance to CGA 173506 and vinclo~ zolin and related fitness parameters in Botryotinia fuckeliana (de Bary) Whetzel 51 1.1 Introduction 51 1.2 Materialsand Methods 51 1.3 Results 54 1.4 Discussion 58

General Discussion 61 Summary 65 Zusammenfassung 67 Bibliography 69 Curriculum vitae 78 Preface

The following studies were realized at the Swiss Federal Research Station at Wädenswil from 1989 to 1992. I like to thank all who helped me with their contributions to achieve the results presented in my PhD thesis. I am grateful to my teachers Prof. Dr. F.J. Schwinn and PD Dr. H. Schüepp who gave me the exceed­ ingly interesting topic and helped me at al1 times through their valuable scientific discussions. I am very grateful to PD Dr. H. Schüepp for promoting me at the Research Station and giving me the chance to cooperate with our guest scientists, Yufen Zhang from China and Prof. Dr. Robert N. Goodman from USA. I further thank Prof. Dr. Th. Holler for his mandate as a referee. Plant protection and fungicide resistance are topics of high socioeconomic interests. The present pro­ ject was most interesting as the constellation of a cooperation of academia (University of Basle), national au­ thorities (Swiss Federal Research Station) and industry (Ciba-Geigy Ltd.) gave promise of a weil balanced approach. In this context I express my thanks to Ciba-Geigy Ltd., Agricultural Division, for financial support and to its representatives Dr. Th. Staub, Dr. P. Urech, Dr. D. Sozzi and Dr. C. Nuninger for helpful discus­ sion of the project, and to the Director of the Swiss Federal Research Station at Wädenswil, Dr. W. Müller and his staff of administration for their hospitality. I thank, W. Siegfried and Dr. E. Bosshard who were excellent teachers in the field and our court photographer, K. Vogelsanger, for technical support. For making the SEM available to me and for technical assistance, I thank Prof. Dr. H. Hohl and U. Jauch from the University of Zürich. I also appreciate the help from my close friend Dr. M

1

'Disease cyc{e of 'Botryotinia fuc,kgliana {ae 'Bary) Wfietze{

multinuclear conidiaA

ascus and liberated ascospores single ascospore cultures

multinuclear ascospores in ascus"

ana, fud

sclerotia and apothecia of Botryotinia fuckeliana inoperculate ascus with 8 ascospores

apothecium, detail of disc centre0

develop multinuclea.r genn tubes,._

anastomoses, exchange of nuclei,._

conidiophore culture on pea agar medium norph of Botryotinia ;eliana ()

• sderotium0

microconidia,._

,._HO Giemsa stain 0 SEM photographs technical assistance: U. Jauch

ment of s!.3lked apothecia0 apothecia arising from sclerotium0

Structure formulae of compounds used in these studies

Methylbenzimidazolcarbamate fungicides

CO-NH-C4H9 1

©(N>-NH-COOCH3 rir H benomyl carbendazim

Dicarboximide fungicides

0 Cl 0 0

Cl 0 vinclozolin procyrnidone

0 0 l _,CH3 / - N-C-NH-CH 0 \J ~ 'CH, Cl 0 iprodione

Phenylpyrrole fungicides

0 CN xo 0 0 CN 0 1/ ~ N 1/ ~ 1 N H fenpiclonil CGA 173506 h

3 General Introduction

Tue genus Botrytis (ßcrtpu~"' a bunch of grapes) was culties of obtaining apothecia (Grindle, 1983). Even established by Micheli in 1729. lt was validated by though the procedure still remains laborious and time Persoon in 1801 and revalidated by Nocca and Bai- consuming, apothecia can now be readily induced in bis in 1821 and by Fries in 1832 (Gregory, 1949, Coley- vitro and the sexually produced ascospores are ac­ Smith, 1980). Although Fries described Botrytis ein- cessible for genetic analysis. erea under the rules of botanical nomenclature, Per- soon's description of B. cinerea is generally referred B. fuckeliana can infect an extremely wide range of to (Groves and Loveland, 1953). host plants including field and glasshouse vegetables, In 1866, de Bary reported for the first time Pe­ fruits, omamentals, bulb and com producing mono­ ziza Juckeliana to be the teleomorph of B. cinerea. Three cotyledons and forest tree seedlings. In addition this years later Fuckel established the new genus Sclero­ can cause considerable damage to plants as tinia with fuckeliana the second spedes well as plant products during Storage and transpor­ mentioned (Gregory 1949). The connection of B. cin­ tation (Coley-Smith, 1980, Jones and Sutton, 1984, erea and P. fuckeliana, or S. juckeliana, respectively, Rosenberger, 1990, Bulit and Dubos, 1988, Snowdon, was generally accepted until the beginning of this 1990, 1991). century but was then questioned because de Bary's Grey mould is the most widespread and im­ specimens and descriptions were lost. The situation portant disease of grapes, occurring wherever the was resolved, when in 1949, Gregory discovered de crop is grown (Snowdon, 1990). Quality as well as Bary's type specimens in the British Museum of Natu­ quantity of grapes can be drastically reduced by Bot­ ral History in London. He found a slide with sec­ ryotinia bunch rot Modified chemical composition tions of apothecia labelled P. fuckelüma in de Bary's in grape berries, conversion of simple sugars (glu­ handwriting. Whetzel established the genus Botryot­ cose and fructose) to glycerol and gluconic acid as inia and described the new combination Botryotinia weil as the production of enzymes catalyzing the oxi­ fuckeliana. Details on the history of the description dation of phenolic cornpounds are due to the infec­ of B. fuckeliana are summerized by Gregory (1949). tion of B. fuckeliana; this may lead to serious qualita­ tive damage in vine production (Bulit and Dubos, Groves and Drayton (1939) for the first time reported 1988). Vines produced frorn rotten grapes have off the production of apothecia of B. fuckeliana under flavors and are sensitive to oxidation and bacterial semi-controlled conditions. Besides this report, there contamination which makes them unsuitable for ag­ are only a few other reports published concerning ing. Even though in most cases B. Juckeliana causes the occurrence of B. juckeliana in vitro (Groves and severe damage it can be beneficial when occurring Loveland, 1953, Lorenz and Eichhorn, 1983) or in the shortly before harvest, causing a condition termed field (Kublitzkaya and Rjabtzeva, 1968, 1970, Polach noble rot. Due to damage of the cuticle by B. fuckeli­ and Abawi, 1974, 1975). Faretra and Antonacci (1987), ana , water evaporates from the grape berries. As a Faretra and Pollastro (1988) and Faretra et al. (1988) result, the concentration of sugar rises which leads investigated the production of apothecia of B. fuckeli­ to heavy sweet vines, as the Tokays of Hungary, the ana under fully controlled environmental conditions. Sauternes of France and Trockenbeerenauslese of They developed a reliable method for the produc­ Gennany. tion of apothecia in vitro which was also success­ fully used in the present study. The genetics of B. Chemical control of plant diseases and occurrence of juckeliana were hardly investigated due to the diffi- resistance problems are of a special socioeconomic

4 importance (Schwinn, 1982, Köller and Scheinpflug, does therefore not develop as quickly as in the pre­ 1987). The first highlights in fungicide development viously mentioned cases; for example, powdery mil­ were the introductions of Bordeaux mixture (1855) dew of small grain cereals is only slowly shifting to­ and organomercurials (1913) (Eckert, 1988). These wards reduced sensitivity (Schwinn and Morton, fungicides were non selective and provided environ­ 1990). However, in both cases, of one step or multi­ mental problems due to the input and accumulation step resistance the genetic potential for overcoming of heavy metals in the ground. In the Eastern part of the fungicide toxicity is present and the resistant Switzerland, between World War I and II, the input biotype is nurtured by a selective environment that of copper was up to 80kg per ha and year (Räz et al., favors its multiplication over that of other compo­ 1987) whereas it is today, due to modern chemical nents of the population (Georgopoulos, 1987, Eckert, control measures, 3 to 4kg per ha and year (Anony­ 1988). With major gene control of resistance, the sen­ mous, 1991). Until the early 60's the inorganic com­ sitivity distribution of the target population is dis~ pounds were partly replaced by synthetic, protec­ continuous and the response to selection is qualita­ tive, multisite inhibitors (Eckert, 1988), mainly dithio­ tive. In polygenic resistance the overlapping of geno­ carbamates and phthalimides. Although these com­ typic classes results in a continuous, unimodal fre­ pounds were widely and exclusively used, the de­ quency distribution. The observed change is a grad­ velopment of resistance was not encountered (Köller ual, quantitative shifting of the whole distribution and Scheinpflug, 1987). Pram the late 60's onwards, toward reduced sensitivity (Georgopoulos, 1988). starting with the introduction of the methyl-benz­ imidazolecarbamate fungicides (see p. 3), selective Before the introduction of methyl-benz­ single- site inhibitors came an the market. These and imidazolecarbamates, no highly effective means of other systernic, highly selective fungicides like the control were available for grey mould. Substantial phenylamides, the SBI's (Sterol ßiosynthesis Inhib­ improvement was obtained by their introduction in itors) as weil as the non- systemic dicarboximides the late 60s and again by the introduction of the dicar­ show a very positive ecological and toxicological be­ boximides (see p. 3) in the early 70s. In both cases, havior. Their high selectivity, however, contains the however, few years after wide and exclusive practi­ risk of facile development of resistance (see chapter cal application of these chemicals, buildup of resis­ 3). This is demonstrated by the accelerated increase tance was encountered. (Schüepp and Lauber, 1977, in the number of resistant fungal genera since their Holz, 1979). The buildup of resistance to methyl­ market introduction. In most cases the development benzimidazolecarbamates in B. Juckeliana in Swiss of resistance can be explained by the selection of vineyards provided a classic example of how such spontaneous mutants in the pathogen population that resistance appeared. and persisted (Staub, 1991). The have a decreased biochemical target affinity. Most stability of resistance in B. juckeliana was shown by cases of rapid development of practical resistance Schüepp and Küng (1981) who compared the fre­ could be ascribed to mutants possessing a single ma­ quency of resistant B. Juckeliana isolates from Swit­ jor gene for resistance (Eckert, 1988). In one step re­ zerland, collected in 1974 when the use of methyl­ sistance, mutation of a single major gene is all that is benzimidazolecarbamate fungicides was discontin­ required for the fungus to acquire the highest resis­ ued, with that of resistant strains collected in 1978. tance possible (Georgopoulos, 1988). In the case of Even though the selection pressure was absent for SBI's, however, resistance is of polygenic nature, i.e., four years the proportion of resistantstrains persisted it results from the additive effects of several minor between 50 to 60%. In contrast, in the case of the genes for resistance (Köller and Scheinpflug, 1987, dicarboximides the frequency of resistant strains de­ Georgopoulos, 1988). Practical resistance in the field creased after stopping treatments (Schüepp and

5 Siegfried, 1983, Martinetti, 1986). They did, how- alleles for resistance towards a novel fungkide can ever, not disappear totally (Löcher et al., 1987). easily be selected when selection pressure exerted by Under these circumstances the phenylpyrrole this novel fungicide is applied. Leroux et al. (1977), compound CGA 173506, presented by Gehmann et Schüepp and Küng (1978) and Pommer and Lorenz al. (1990), is a highly welcome novel active ingredi- (1982) reported the ease of selecting dicarboximide ent to control B. fuckeliana. Ta ensure its long-term laboratory resistant strains of B. fuckeliana without use in practice, the early evaluation of the risk of re­ using UV irradiation or other mutagenic agents. sistance, and the development of a suitable antire- The occurrence of laboratory resistant strains sistance strategy are of prime importance. The latter does, however, not necessarily imply the buildup of may consist of altemating spray programs or pre­ resistance in the field (Dekker, 1982a). The relative packed mixtures (DeWaard, 1988) whereby today fitness of resistant strains compared to sensitive mixtures are prevailing (Schwinn and Morton, 1990, strains in the field is of paramount importance for S!aub, 1991). the development of resistant populations {Dekker, Tue use of dicarboximides as companion part- 1982a). Acquisition of resistance is often linked with ners in mixtures with CGA 173506 depends on their loss or decrease of fitness (Dekker, 1982a, Brent et resistance risk. In this context the population dynam­ al., 1990). For the assessment of a resistance risk it is ics of B. Juckeliana field resistant to dicarboximides thus essential to study the fitness of resistant and was studied in a vineyard. The influence of one to sensitive strains. Although in the laboratory only a two dicarboximide applications per season as well limited number of fitn.ess parameters {e.g., growth as the effect of one year free of applications was in­ rate, sporulation, osmotolerance, competitive ability, vestigated in a population of B. fuckeliana on the low pathogenicity and virulence) can be studied, whkh susceptible grape-vine variety Blauburgunder, clone reflects a limited view of the much more complex Mariafelder; for details see chapter 1. situation in the field, studies on the fitness of resis- tant strains (compared to sensitive strains) provide Hansen and Smith (1932) and Menzinger (1965) de- an important tool in the evaluation of the resistance scribed the formation of anastomoses and the ex- risk. In the absence of selection pressure, strains of changeof nuclei between different hyphae in B. jucke- B. fuckeliana that are resistant to methyl-benz- fiana. Menzinger (1965) proved that numerous mono- imidazolecarbamates typically are as fit as sensitive conidial isolates of Botryotinia were heterokaryons strains which leads to a stable proportion of resistant and, only in one case, did he succeed in producing strains in the field. However, strains that are resis­ morphologically recognizable heterokaryons from tant to dicarboximides, show a decreased fitness in­ homokaryons. dicated by a decreasing proportion of resistant strains Beyond the genetic variability which is, in com- in the field in the absence of selection pressure. In mon for all living organisms, caused by mutation this study the effect of the acquisition of laboratory and sexual reproduction B. fuckeliana shows addi- resistance to CGA 173506 in B. fuckeliana an fitness tional genetic flexibility caused by the selection of parameters was studied with regard to the signifi­ different alleles within the heterokaryon, a charac- cance for the buildup of a resistant population in the teristic feature of multinucleate (heterokaryotic) or- field (chapters 3 and 4). ganisms. Due to the spontaneous occurrence of muta- Good fungicide resistance monitoring (= testing for tions towards fungkide resistance and the possible sensitivity of target organisms in field populations) occurrence of nuclei carrying alleles coding for sen- ls a comerstone of the management of fungicide re­ sitivity or for resistance in the same heterokaryon, sistance (Brent, 1988). As the detection of resistant

6 strains is based on the cornparison with data obtained the pathogen was compared in vitro and base-line from sensitive isolates, the evaluation of base-line sensitivity conceming both active ingredients of two sensitivity (= the genuine 5ensitivity of untreated wild wild type populations was evaluated. In chapter 3 type isolates) is crucial (Georgopoulos, 1982, Geor­ the potential of the fungus to develop laboratory re­ gopoulos and Dekker, 1982, Brent, 1988 and Brent et sistance against CGA 173506 and against vinclozolin al. 1990). Isolates of B. fuckelüma from strawberries was studied. Morphological characters and fitness that never received any fungicidal treabnents in the parameters of selected mutants were compared. The field were collected at two locations. In the second genetic basis of resistance against CGA 173506 and chapter of this investigation the base- line sensitivity against vinclozolin was studied by using sexual of CGA 173506 was established for these isolates. progenies of B. fuckeliana (chapter 4). Inheritance of fitness parameters affected by the acquisition of re­ Because the sexual stage of B. fuckeliana rarely oc­ sistance to vinclozolin or CGA 173506 was studied curs, genetics of fungicide resistance have not been by tetrad analysis (chapter 4). investigated fully in this pathogen. However, recent work by Faretra and Pollastro (1991), who managed to obtain sexual reproduction under laboratory con­ ditions, has clarified the genetic basis of resistance against vinclozolin and methyl-benzimidazolecar­ bamates. Resistance to both fungiddes is each en­ coded by a single polymorphic major gene. Faretra and Pollastro (1991) further recognized three classes of alleles at each locus. Based on the methods of Faretra and Pollastro (1988) the inheritance pattems of resistance against CGA 173506 and against vin­ clozolin and of related fihless parameters in B. Jucke­ Unna were investigated (chapter 4). In contrast to Faretra and Pollastro (1991) who were using random spare analysis, the genetic basis of resistance and pleiotropic effects on fitness characters were studied using dassical tetrad analysis.

In summary, the goal of the present investigations was to study genetic variability in B. f11ckeliana by comparing fungicide resistance to vinclozolin and the phenylpyrrole CGA 173506 and to assess the resis­ tance risk of the novel compound CGA 173506. In a field study (chapter 1), the occurrence of vinclozolin field resistance under the influence of restticted use of dicarboximides in a weakly susceptible grape va~ riety was studied. Population dynamics of B. fuckeli­ ana resistant to dicarboximides were evaluated dur­ ing 3 successive seasons from 1989 to 1991. In chap­ ter 2 the effect of CGA 173506 and of vinclozolin an

7 1. Field study on the population dynamics of Botryotinia fuckeliana (de Bary) Whetzel resistant to dicarboximides

1.1 Introduction

Iprodione and vinclozolin were first introduced in tists started to study the population dynam.ics of B. Switzerland in 1976 for large field trials and were juckeliana resistant to dicarboxirnides (Leroux and later registered in 1977 (Schüepp, 1977). Due to good Clerjeau, 1985, Martinetti, 1986, Löcher et al. 1987, performance they were intensively used to control B. Pak et al. 1990). They mainly investigated seasonal fuckeliana (grey mould); 4 to 5 treatments per season changes in the frequency of resistant strains of B. were allowed in grape·vines (Schüepp, 1977, Beetz fuckeliana in the population on susceptible grape va­ and Löcher, 1979). Leroux et al. (1977) and Schüepp rieties in intensively treated vineyards. The influence and Küng (1978) could easily select highly resistant of reduced selection pressure on the development strains of B. fuckeliana in the laboratory. During the of the resistant population has not been investi­ intensive monitoring program which comrnenced in gated to date. Delp (1980) and Staub and Sozzi (1984) 1976, the first low level field resistant strains were assumed that a reduction of the number of treat­ found in 1979, three years after the first use of the ments would prevent or at least delay the buildup dicarboximides in Switzerland. Holz (1979) reported of resistance. In this context, the changes in sensitiv­ for the first time lass of efficacy of the dicarboximides ity of B. juckeliana to dicarboximides were moni­ in the Mosel valley in Germany. In the vineyards, tored over three years under practical conditions in monitored by Schüepp and Siegfried (1983), the per­ the vineyard in Walenstadt, Switzerland, where centage of resistant strains increased drastically from dicarboximides were applied at the most 2 times per about 40% in 1979 to almost 100% in 1982 but ade­ season. During this investigation only one treatrnent creased efficacy only was found from 1982 onwards. at early bunch dosure was applied in the first year Whereas Schüepp and Siegfried (1983) no longer and no treatment was applied in the second year. In advised the use of dicarboximides in Swiss vineyards the third year one third of the vineyard remained where field resistance occurred, other authors rec­ untreated whereas the other two thirds were treated ommended a limitation to two, in exceptional cases twice with vinclozolin. three, applications per season (Gouot, 1988, Löcher Due to the low susceptibility of the grape va­ et al. 1985, Löcher et al. 1987, Löcher, 1988, Wade, riety Blauburgunder, clone Mariafelder, the disease 1988). In contrast to the resistance to methylbenz­ pressure in the vineyard in Walenstadt, Switzerland, imidazoles which was stablely maintained in the has been low, Dicarboximides were therefore only population in the absence of a selection pressure of sprayed 7 times since the beginning of the introduc­ the fungicide, the resistance to dicarboximides was tion of this group of fungicides (Table 1.1) and be­ not stable. Although the resistant population did not fore these studies were started. disappear completely, even after several years with­ 1.2 Materials and Methods out a selection pressure (Martinetli, 1986, Löcher et al. 1987), the proportion of resistant strains in the The field trials were carried out in the upper plane pathogen population decreased (Wade, 1988). of the vineyard in Walenstadt which consists of 39 rows with up to 30 plants of Blauburgunder clone After the occurrence of field resistance, many seien- Mariafelder per row (Fig. 1.1). This plot totally re-

8 ceived 10 treatments with dicarboximides from 1983 The detection Hmit of resistant strains in a fungicide to 1991 (Table 1.1,2). During the vegetation periods resistance monitoring program directly depends on of 1989 to 1991 varied treatments of dicarboximides the number of samples (isolates), e.g., the minimum were applied (Table 1.2). Iprodione or vinclozolin, detection limit with ten isolates is 10% (1 resistant respectively, (Maag, AG; 1kg a.i. per ha) were isolate out of 10) whereas it is 5% with 20 isolates (1 sprayed with a high pressure (2MPa) main sprayer resistant isolate out of 20) and 1% with 100 isolates connected to a tube with tv.ro nozzles of 1.2mm width (1 resistant isolate out of 100) (Leroux and Oetjeau, using a water rate of 1500 to 2000L/ha. In 1989 1985). For dicarbo:ximide resistance monitoring a de­ iprodione was applied at early bunch closure to the tection limit of 5%, that is a sample size of 20 isolates entire plot. During 1990 no dicarboximides were used was feasible. at all. In 1991 rows 5 to 17 were treated with vin­ Freshly sporulating isolates of B. juckeliana (19 clozolin twice, at early bunch closure and at verai­ to 25) were randornly collected within the rows 6 son, with an air blast sprayer using 400L/ha whereas and 26 from leaves, pea sized berries, and mature rows 18 to 43 remained untreated. The proportion of berries, respectively. Conidia were transferred from isolates of B. fuckeliana, resistant to dicarboximides sporulating colonies to malt agar (12g agar-agar, was monitored at early bunch closing, prior to treat­ Merck and 15g malt extract, Difco) amended with ment, and before harvest. Control of pests and patho­ 100mg streptomycinsulfat/L and 100mg oxytetracy­ gens other than B. fuckeliana was carried out follow­ clin/L. After an incubation period of 3 days at 20'C ing the Swiss plant protection recommendations for in the dark, the colonies were transferred to pea agar vine growing (Anonymous, 1991). (15g agar-agar, Merck, 168g green peas and 5g sac-

Fig. 1.1: Vineyard in Walenstadt, Switzerland

plant number plant rows N 0 t 5 s

0

Y '-~1:.;o(..._,...6.:;1sL __o:__,;25:_ _ _:30::_ _ __::35:_ _ _:40:..., Färber grape variety 10m grapes row Blauburgunder, clone Mariafelder number

9 charose per Iiter, pH=6) and incubated. at 2o·c under resistant at a level of 1mg vindozolin/L. Isolates from fluorescent and black light to enhance sporulation. leaves being resistant at a level of 3mg vinclozolin/L Spore suspensions of 105conidia/ml were prepared were not detectable, whereas 5% of the isolates from from intensively sporulating cultures. SOµl of the sus­ pea sized berries were resistant at thls level. After pensions were added onto malt agar discs of a di­ the treatments the abundance of dicarboximide field ameter of 10mm. The discs were incubated for 17h at resistant strains was high; all the isolates taken from 2o·c in the dark and then placed upside down onto mature berries after treatment were resistant at a level malt agar plates amended with 1, 3 and, in 1990 and of 1mg vindozolin/L and 16% also were resistant at 1991, with 10mg vinclozolin/L. Mycelial growth was a level of 3mg vindozolin/L. There was no signifi~ assessed by measuring the mean colony diameter af­ cant difference in the per cent of resistant isolates ter an incubation period of 3d at 20"C in the dark. from leaves and pea sized berries, neither for a level Mycelial growth on agar amended with fungicide of 1 nor of 3mg vinclozolin/L. The difference in the was compared to mycelial growth on fungicide free proportion of resistant isolates from leaves, pea sized agar (check). Isolates which were able to grow on berries and mature berries was highly significant agar amended with 1, 3 or 10mg vinclozolin/L more (Contingency table analysis, p=-0.0001). than 50% of the diameter of the control were classi­ In 1990 no dicarboximides were applied at all. fied as resistantat a Jevel of 1, 3 or 10mg vinclozolin/ None of the isolates from leaves, pea sized and ma­ L, respectively. All experiments were replicated three­ ture berries was resistant at a level of 3mg vin­ fold. clozolin/L (Fig. 1.2B). On malt agar amended with 1mg vinclozolin/L 21% of the isolates from leaves, 1.3 Results 20% of the isolates from pea sized berries and 5°/,, of Table 1.2 shows the dates of dicarboximide applica­ the isolates from mature berries were resistant, the tions and dates of isolations of B. fuckeliana from differences were not significant (Fig. 1.2B). From this 1989 to 1991. Before the dicarboximide treatments it was conduded that the increase in the frequency the abundance of dicarboximide field resistant strains of strains that were resistant at a level of 1 and 3mg was low; as shown in figure 1.2A in 1989 prior to the vinclozolin/L in 1989 was due to the one application field treatment with iprodione 24% of the isolates of iprodione before bunch closure. from grape leaves and 25% of pea sized berries were

Table 1.1: Applications of dicarboximides from 1983 to 1988 in the vineyard in Walenstadt

year numberof dateof fungicide aeElications aE:elication 1983 0 1984 1 21. August iprodione 1985 1 19. August procymidone 1986 2 22. July procymidone 21. August iprodione 1987 2 5. August iprodione !. September iprodione 1988 1 28. July iprodione

10 Fig. 1.2: Percent of field isolates growing ~ 50% of control an agar amended with 1 and 3 mg vinclozolin/L

ffill 1mg vinclozolin/L 1111 3mg vindozolin/L % n=19 b i::: 100 i::: ~ 90 0 -~ 80 ]""' "'lJ> 70 p...... 60 g.,§ 50 - 0 40 ~ ~ 30 n=25 n=24 ~ cl 20 < -~ 10 0

00 100 .,,§ 90 80 "'u '"00 70 "'g..§""-~ 'Cl 60 50 0 ~ ~ .0 40 0~ 30 "'~:-a 13 20 10 "'··- 0 0

0 N -"' 10 0,'! " 0 9 " ~ 80 ].S 70 "" iß 60 ""-0"' -~ 50 g -~ 40 ~ 0 ~ ,e 30 ':"": .§ 20 n=20 n=19 u 'Cl 10 0 0 § -00 00 i::: ·; 100 n=20 0 ~ ~ z: a;I Cl> 90 .;::!"' "'~ 'tj> 80 - 0 ~ 70 "" ~ "' 60 """' s '"~ 0 "i:: ::1 50 i ~ i':l 40 ~ ] V 30 8; 2 -6 ~u§... s 20 n=20 Q > .0 10 n=19 0

isolates from isolates from isolates from leaves pea sized berries mature berries

11 In 1991 B. juckeliana was isolated from rows 6 (contingency table analysis p= 0.0001) in the treated and 26 (Fig. 1.1). Row 6 was treated before bunch row 6 (Fig. 1.2C). All the isolates from mature ber­ closure and at veraison whereas row 26 remained ries of row 6 were resistant at a level of 1mg vin­ untreated (Table 1.2). Figures 1.2C and D show that clozolin/L and 3mg vinclozolin/L. none of the isolates taken from leaves and pea sized From 1989 to 1991 isolates that were resistant berries of row 6 were resistant at a level of 3mg vin­ at the level of 10mg vinclozolin/L were not found, clozolin/L whereas 10% isolated from leaves and 5% their frequency was thus lower than 5%. isolated from pea sized berries from row 26 did show resistance. The differences between leaves and pea 1.4 Discussion sized berries and between row 6 and 26 prior to treat­ Although the buildup of populations of B. fuckeliana ment were not significant, neither for the level of 1 resistant to dicarboximides occurred soon after the nor of 3mg vinclozolin/L. 15% of the isolates from intensive use of these fungicides (Holz, 1979, Beever mature berries of the untreated row 26 were resis­ and Brien, 1983, Schüepp and Siegfried, 1983, tant at a level of 1 and of 3mg vinclozolin/L (Fig. Northover and Matteoni, 1986) the occurrence of re­ 1.2C). There was no significant increase in the per sistant strains was not always positively correlated cent of resistant strains between leaves, pea sized with loss of efficacy (Schüepp et al. 1982, Pommer berries and mature berries in the untreated row 26 and Lorenz, 1982, Northover, 1988). Beever et al. (Fig. 1.2C), whereas there was a significant increase (1991) suggested the frequency of resistance genes to

Table 1.2: Monitoring from 1989 to 1991 in Walenstadt; evaluation of seasonal shifts in dicarboximide sensitivity prior and after treatments with dicarboximides

date of sampJed part date of treatment application sampling of plant stage 6.7.89 leaves 6.7.89 young berries 16.8.89. veraison 28.9.89 mature berries 12.7.90 leaves 12.7.90 young berries 15.10.90 mature berries 26.6.91 leaves 18.7.91 young berries 24.7. 91 bunch closure 20.8. 91 veraison 26.9.91" mature berriesb 26.9.91 · mahne berriesc

a all rows treated with Rovral (iprodione) b from row 6, treated with Ronilan (vinclozolin) c from row 26, untreated

12 be the result of the interplay between selection for The present study, however, even showed that in these genes determined by fungicide application, and the restrictively treated vineyard in Walenstadt, dur­ selection against these genes due to their effects on ing 1989 one single application of iprodione and dur­ fitness. Resensibilisation of a resistant population to ing 1991 two applications of vindozolin, were also a high extent due to reduced fitness in resistant strains sufficient for the resistant population to reach a level is still not fully understood. Results from laboratory of 100%. In agreement with Löcher et al. (1985), our experiments did not always refled the situation in results from harvest 1989 and early bunch closure the field as it will also be demonstrated in chapter 3. 1990 indicate a decrease in resistance frequency from Under laboratory conditions only a limited number 100% to at rnost 21 % for strains of a level of 1mg of fitness parameters can be investigated whereas in vindozolin/L (Fig. 1.2). In the absence of a selection nature the number of parameters affecting fitness is pressure in 1990 and in the untreated rows also in enormous. This might be a possible explanation for 1991 resistant strains at the level of 1mg vinclozolin/ the partly contradictory results from field and labo­ L did not disappear. ratory studies. Delp (1980) and Staub and Sozzi (1984) suggested Northover (1988) showed in one vineyard a decrease that the development of fungicide resistance can be of the resistance frequency from September to July prevented or retarded by limiting the number of treat· whereas in three other vineyards he found no sig­ ments in order to reduce the selection pressure and nificant difference in the resistance frequency in Sep­ by combining or alternating active ingredients with tember and in July. Löcher et al. (1987) concluded different modes of action. The present investigation from a declining proportion of resistant strains from in the vineyard at Walenstadt clearly demonstrated harvest to the first application of dicarboximides to that the restriction in the number of applications of treat as late as possible in the growing season. They dicarboximides to at most two treatments per sea­ showed two properly timed treatments to be as ef­ son, from the very beginning of the commercial in­ fective as four treatments. Leroux and Clerjeau (19&5) troduction of the dicarboximides as weil as the use reported similarities in the population dynamics be­ of less susceptible grape varieties as Blauburgunder, tween vineyards in the Champagne and the Alsace clone Mariafelder did not prevent the buildup of a where they found high resistance frequencies at har­ resistant population. These results complemented vest and in the following spring. They found thus a those of Löcher et al. (1985), Leroux and Clerjeau different behavior in the Loire Valley where they (1985), Martinetti (1986) and Pak et al. (1990) who in­ observed a decrease in resistance frequency during vestigated the seasonal fluctuations in resistance fre­ this period, which they attributed to a dilution effect quency in vineyards that were intensively treated of sensitive inoculum from surrounding vineyards. with dicarboximides for several years. Even though Pak et al. (1990), however, by conduding from their the population of B. fuckeliana was small, due to the own results, attributed these findings of Leroux and reduced susceptibility of the chosen grape variety Clerjeau (1985) to dynamics within the vineyards and the selection pressure by only one or two fungi· themselves. dde applications was Jow, the population dynamics In the intensively treated vineyard in Wä• observed during the three years field study were com­ denswil, Martinetti (1986) applied 4 treabnents of parable to those described by the aforementioned au­ vinclozolin which resulted in 100% resistant strains. thors.

13 1hls study showed that within the small popu­ lation of B. juckeliana in the vineyard at Walenstadtr a resistant subpopulation did build up despite cau­ tious use of dicarboxitnides. In this context, the novel phenylpyrrole fungicide CGA 173506, might pro­ vide an important, additional tool in the chemical control of the pathogen.

14 2. Studies on the base-line sensitivity of Botryotinia fuckeliana (de Bary) Whetzel against CGA 173506

2.1 Introduction

Gehmann et al. (1990) was first to report the use of prior to the use of a new fungicide, i.e., the base-line the pheny1pyrrole fungicide, CGA 173506; common sensitivity. The aim of the following investigation name n.n. trade mark Saphire®. In contrast to fen­ was to determine such hase-line sensitivity data of B. pidonil (CGA 142705) of the same chemical dass Juckeliana conceming CGA 173506. In comparison which is used for seed treatment (Nevill et al., 1988), with the results reported by Martinetti (1986) and CGA 173506 has been developed as a foliar fungi­ Leroux (1991), working with fenpiclonil and a dicar­ dde for grapes, stone fruit, vegetab1es, rice, field crops boximide fungicide, the effects of CGA 173506 and and turf and as a seed treatment for cereal and non­ vinclozolin on the mycelial growth and on the ger­ cereal crops. As a foliar fungicide it provides a high mination process of conidia were compared. inherent activity against species of the genera: Bot­ ryotinia, Monilinia, Sclerotinia, Rhizoctonia and Alter­ 2.2 Materials and Methods naria. In the laboratory, Gehmann et al. (1990) did Isolation of B.fuckeliana not find cross resistance to methyl-benzimidazole­ Isolates from naturally infected strawberries of dif­ carbamates, dicarboximides or guanidines when they ferent varieties including Tenira were collected from investigated field resistant strains of B. fuckeliana. fields in Oedischwend, Wädenswil (Fig. 2.1) andin Martinetti (1986) and Leroux (1991) reported Fondli, Dietikon (Fig. 2.2). In none of the fields had parallels in the effect of fenpiclonil and the dicarbox­ any fungicides been applied. The field in Oedi­ imide fungicides an B. fuckeliana in vitro. Leroux schwend consisted of three rows, with a total length (1991) concluded frorn his results, that the mode of of 70m each. The rows were numbered from I to III. action of the phenylpyrrole fungicides and of the Each row was divided in three sectors A, B and C dkarboximides is the same. However, the mode of each consisting of 56 plants. Eight strawberry action of the phenylpyrrole fungicides has not been samples, infected with B. fuckeliana, were taken per darified yet, and conceming the mode of action of row and sector. TI1ese samples were numbered from the dicarboximide fungicides, numerous contradic­ 1 to 8. Each isolate could thus later be localized by tory results have been described in the literature the row number, the sector number and the sample (Corbett et al., 1984, Endlich and Lyr, 1987, Ruiz number of the strawberry from which it was iso­ Bmge, 1988). lated. Tue field in Fondli consisted of 5 rows (I to V) which were divided in three sectors (X, Y and Z). During the development of novel fungicides, infor- From 20 plants per row and sector, two infected mation on the potential of the target organisms to strawberry samples were taken. The nomenclature develop resistance under both laboratory and field of the isolates of the samples from Fondli was equiva­ conditions is required (Georgopoulos and Dekker, lent to the nomendature for isolates from Oedi- 1982, Löcher and Lorenz, 1991). Georgopoulos (1982), schwend. Georgopoulos and Dekker (1982), Brent (1988) and Brent et al. (1990) emphasized the importance of de- Strawberries which were latently infected with grey termining natural sensitivity of field populations mould were incubated with a wet cotton plug at 2o•c

15 Hg.2.1: Strawberry field: Oedischwend, Wädenswil row numberof sector number strawberry s< N strawberry plant labe!"--.._ \ ~lant number example for labelling: B18 / row /

,..... N t"'l I -e~ (§0~ f)w --.tE)-.o®l51 -®"'®II"\ \Oe 00 II (row number) -' N c<'>'

A B C (sector labe!)

20 isolates (Oand@) were stored in liquid nitrogen 10 isolates (@}) were selected for further experiments

Fig. 2.2: Strawberry field: Fondli, Dietikon row sector number strawberry s labe! \ ?lant number example for labell~ 12 numberof berry plant ~ N /straw ~ row @ (Z)' @ (z> !' 2 I @) 2 @. 2 CD (z> II (row number) CD @ @ 2 @ (z> III CD (z> 1 @ CD 2 N CD 2 @ 2 1 ~ V 19.6.1989 11m

z y X (sector label)

20 isolates (0 and@) were stored in liquid nitrogen 10 isolates (@) were selected for further experiments

16 for several days to enhance sporulation. Conidia warmed up at room temperature for 20 to 30 min­ were transferred to malt agar (12g agar-agar, Merck, utes. The agar discs were then aseptically transferred 15 malt extract, Merck) amended with 100mg strep- to either malt agar plates or to test tubes containing tomycin/L and 100mg oxitetracydin/L. Colonies pea agar. growing on agar amended with antibiotics were transferred onto pea agar (160g frozen green peas, Fig. 2.3: Dia gram of ternperature development of the cryochamber and the probe during a Coop, 20g agar-agar, Merck and 5g saccharose, Merck controlled freezing process in IL distilled water, pH = 6) and incubated for 7d at 2o·c in 12h dark, 12h fluorescent light (Osram L 20W /25S) and black light (Philips TLD 18W /08). supercooling of probe 20 i reheating of probe 0 Cryopreservation of B. fuckeliana in liquid nih'o­ 1-' -20 gen .E ru -40 ~ The method used for cryopreservation of young my­ .a -60 I ~ cold shock celium from B. fuckeliana is adopted from Dahmen ru~ -80 probe et al. (1983) and from Hohl and Iselin (1987). Spore S"" -100 2 cryochamber suspensions of isolates from Oedischwend, Wä• -120 denswil were adjusted to 105conidia/ml. Malt agar -140 discs of a diameter of 10mm were inoculated with 0102030405060 50µ1 of the spore suspension and then incubated for minutes 17h at 20°C in the dark. Three to five discs contain- ing young mycelium of 17h were put into 2ml plas- tic cryotubes (NUNC, Kamstrup, Denmark); skim milk (17% skim milk, 20%, glycerol, Difco, Merck in a Fungicides and media mixture 1:1) was added as a cryoprotectant to each Vindozolin and CGA 173506, both technical grades, cryoh.Ibe up to the mark at 1.8ml. Ten cryotubes for were provided by Ciba-Geigy Ltd„ From stock solu- each of 20 strains of B. fuckeliana from Oedischwend, tions, prepared by dissolving 300mg and 100mg vlll- Wädenswil (encircled in Fig. 2.1) and from Fondli, clozolin or CGA 173506, respectively, by ultra sound Dietikon (encircled in Fig. 2.2) were cryopreservated for 5 minutes at room temperature in 100ml ethanol in liquid nitrogen. For controlled freezing a program­ abs. further stock solutions of 0.3mg to 30mg vin- mable liquid nitrogen freezing unit (Planer Program~ clozolin and of 0.01 to 30mg CGA 173506/lOOml etha- mable Temperah.Ire Controller PTC 303, Sunbuny nol abs. were obtained by 10 fold dilutions. The stock on Thames, England) was used. Fig. 2.3 shows the solutions were stored at 0°C and were reprepared at development of temperature in the cryochamber and least every third month. in the probe during the controlled cryopreservation process. Reheating of the supercooled probes during For agar plate assays, media amended with fungi- the freezing process were minimized by using the cides were prepared adding 10ml of stock solutions freezing program described for Phytophthora spe· of vinclozolin or CGA 173506, respectively, to auto­ cies by Hohl and Iselin (1987). lt provided an aver- claved (120°C, 101,2kPa, 20 minutes), and cooled aged cooling rate of 1.5'C/min. For thawing, the (50°C) malt agar (12g agar·agar and 15g malt ex­ cryotubes were removed from the storage tank and tract/L, Merck). For final concentrations of 100mg 17 vinclozolin/L, 100mg of the active ingredient were agar plate test by placing three discs containing the dissolved in 10ml ethanol abs. and then added to the mycelium upside-down on malt agar (diam. &m) medium. amended with 0, 0.03, 0.1, 0.3, 1, 3 and 10mg vin- clozolin/Lor 0,001, 0.003, 0.01, 0.03, 0.1, 0.3, and 1mg Sensitivity to dicarboximide fungicides CGA 173506/L, respectively. Myce1ial growth was To ensure that all isolates (encircled in Fig. 2.1 and assessed by measuring a mean colony diameter after Fig. 2.2) were sensitive wild types, their reaction on an incubation period of three days at 20°C in the dkarboximides was determined roughly by placing dark. The diameter of the inoculation disc (10mm) 10mm mycelial discs (17h old mycelium) on malt was subtracted from each value to give the true value agar amended with 0, 0.3, 1 and 3mg vinclozolin/L. that was compared to the control plates containing Colony diameters were assessed after an incubation 10ml ethanol abs. but no fungicide. For the germina­ period of 3 days at 20°C in the dark. Colonies on tion test 50µ1 of a suspension of 105 conidia/ml were media amended with vinclozolin that grew more than placed on malt agar discs amended with 0, 0.1, 0.3, half of the diameter of the controls containing no 1, 3 and 10 mg vinclozolin/L or 0.01, 0.03, 0.1, 0.3 fungicide were dassified as resistant at the appropri­ and 1 mg CGA 173506/L, respectively. Germination ate level. rates of conidia that have developed germ tubes which were at least as long as the conidia, were as­ Dose-response relationship of vinclozolin and CGA sessed after an incubation period of 13h at 20°C in 173506 the dark. Percentage of inhibition of growth and ger­ Ten vinclozolin sensitive strains, from both locations rnination, respectively, on agar amended with fungi­ each, were chosen from the twenty strains which were cides were determined by reference to the controls. cryo-preservated in Jiquid nitrogen (shaded cirdes Values of EC50 were determined graphically. in Fig. 2.1 and Fig. 2.2). The dose-response relation­ 2.3 Results ship of both ingredients was assessed for the germi­ nation process of conidia and for mycelial growth of Sensitivity to dicarboximide fungicides young mycelium. The base-line sensitivity concern­ Of the twenty isolates from Oedischwend, Wädens~ ing both ingredients was determined for the popula­ wil all but two were sensitive to vinclozolin. Their tions of Oedischwend and Fondli. The reaction of growth rate was less than half of the control on agar the young mycelium (17h old) was detennined in an amended with 0.3mg vinclozolin/L. Strains Al5 and

Table 2.1; Number of isolates growing less than 50% of the control on agar amended with 03, 1 and 3mg vindozolin/L (* AIS and BI5, 0 XIV1, XV2 and ZVl)

Number of isolates with ECSO values for mycelial growth

origin of < 0.3mg vinc./L 0.3-lmgvinc./L 1-3mg vinc./L > 3mg vinc./L isolates Oedischwend, Wädenswil 18 0 2• 0 Fondli, Dietikon 17 30 0 0

18 Fig.2.4: Inhibition of conidia gennination: Dosage-response relationship of CGA 173506 and of vinclozolin in 10 strains from Oedischwend, Wädenswil and from Fondli, Dietikon, respectively

CGA 173506 vindozolin

100 100

..; C C ! 80 80 -&• 160 60 "'e !' 40 40 a ~ ,.g :::1 0 20 20 ~!!!~ -:;; {I-~ 0 0 ]~: -20 -20 .01 .1 1 .1 1 10 100 100

C .g 80 80 •C ,g ·, 60 60 C w 0 40 40 ~ ...' C a 0 20 8 ~ 20 - ,:: ;5 IB o :i'! 0 -;; ~.. 5 0 .ß a # -20 -20 .01 .1 1 .1 1 10 concentration in mg/L concentration in mg/L

Fig. 2.5: Inhibition of mycelial growth: Dosage-response relationship of CGA 173506 and of vindozolin in 10 strains from Oedischwend, Wädenswil and from Fondli, Dietikon, respectively

CGA 173506 vinclozolin

.s 100 100 ~ i w 80 80 I 60 60 e' 1... 40 40 §= ] 20 20 ~ "'. ' ~ 0 ..!!l• :g• .5 0 .~~ * -20 -20 .001 .01 .1 1 .01 .1 1 10 = 100 100 'ew 80 80 1! 60 60 ~ e C e' ,2 ... 40 40 C a 20 20 _jl C i ,, 0 0 l~ .5 ~ -~ ·-" -20 -20 " .001 .01 .1 1 .01 .1 1 10 concentration in mg/L conccnlration in mg/L

19 Pig.2.6: Distribution of EC50 values of ten wild type strains from Oedischwend and from Fondli, respectively; minimum and maximum values are indicated for each combination

CGA 173506 vinclozolin

0.2 0.19 3.0 j 0.18 Ö O \...Q 0 0.1 • • • r;;ö .o 0 • o.077 0.1 •o o i i 0.072 0 .. 00-j-----"'=--- 1.0+------

0.025 0.30 0.27

0.020 0.016 i 0.25 • 0.013 6 • !21 0.015 \,,_ • ei •• ,.,. 0 • 0.20 0.010 o oö ;oo 0.002~• A O 0 0 0.19 0.005 \. u • • • 0.15 0 0 Ü ö, 0.0042 0.000+------0.10+------==---'--0.12 wild type strains 1 to 10 wild type strains 1 to 10 Legend: 0 isolates frorn • isolates from Oedischwend FondH

Table 2.2: Response of B. Juckeliana to vinclozolin and to CGA 173506, respectively, in a spare germination test and a mycelial growth test

ECSO"· spare germination test mycelial growth test vinclozolin CGA 173506 vindozolin CGA 173506 Oedischwend mean (stdev) 1.9 (0.04) 0.15 (0.047) 0.17 (0.03) 0.0092 (0.0037) range 1.8-2.0 0.072-0.19 0.12-0.21 0.0027-0.016 Fondli mean (stdev) 1.9 (0.08) 0.14 (0.033) 0.22 (0.02) 0.0084 (0.0031) range 1.7-2.0 0.077-0.18 0.19-0.27 0.0042-0.013

*values are means (stdev} in mg/L of 10 strains

20 BI5, were therefore classified as vinclozolin field re­ As shown in Figures 2.4 and 2.5 dose-response curves sistant, growing more than 50% on 1mg but less than are similar in shape for vinclozolin and for CGA 50% of the control on agar amended with 3mg vin­ 173506. Both ingredients show a strong inhibitory clozolin/L (Table 2.1). All the twenty strains isolated effect on mycelial growth but CGA 173506, however, from strawberries from Fondli, Dietikon were sensi­ has shown a higher inherent activity than vinclozolin. tive to vindozolin, their growth rate being less than For the spore germination test, the minimal inhib­ half of the control on agar amended with 1mg vin­ itory concentration (MIC) was 3mg/L for vindozolin dozolin/L. Of the twenty strains, seventeen had a and 0.3mg/L for CGA 173506 (Fig. 2.4), respectively, growth rate less than 50% of the control on agar whereas in the mycelial growth test it was lmg/L amended with 0.3mg vinclozolin/L whereas strains for vindozolin and 0.lmg/L for CGA 173506 (Fig. XIVl, XV2 and ZVl, grew more than half of the con­ 2.5). trol on 0.3 but less than half on agar amended with In the germination test the mean ECS0 values lmgvinclozolin/L (Table 2.1). of the two populations of Oedischwend, Wädenswil and Fondli, Dietikon, both represented by 10 strains, Dose-response relationships did not differ significantly (t-test, p>0.05). However, On agar amended with increasing amounts of CGA in the mycelial growth tests on agar amended with 173506 or vindozolin, respectively, response of B. either vinclozolin or CGA 173506 the mean EC50 val­ Juckeliana to both fungicides was determined in a ues of the two populations differed significantly (t­ mycelial growth test and an all-or-none response was test, p <0.05) for vinclozolin but not for CGA 173506 indirectly determined in a spore germination test. (Table 2.2 and Fig. 2.6). Although the statistically sig-

Table 2.3: List of ECS0 values of sensitive wild type isolates of B. fuckeliana evaluated by various authors

ECS0 values in mg/L conidia gennination mycelial growth author 0.8 0.1-0.3 (0.45) Albert, 1979 12 0.17 Pappas and Fischer, 1979 1.4 0.1 Pappas, 1982 0-1 Lorenzini, 1983 0.1-0.2 Katan, 1982, 1985 0.15 Leroux and Besselat 1984, 1985 5.3-16 0.11-0.36 Davis and Dennis, 1981 0.12 Wang et al., 1986 0.86 0.19 Beever et al., 1989 0.45-0.84 0.04-0.22 Rewal et al., 1991 1.7-2.0 0.12-0.27 Hilber, 1992 (see table 2.2)

21 nificant difference conceming mycelial growth on several pesticides were applied to the young plants agar amended with vinclozolin (Fig. 2.6. and Table before they were sold. For the cv. Tenira the treatM 2.2) was not explicable, for further studies the strains ments induded vinclozolin. Strawberries cv. Tenira from Oedischwend, Wädenswil, which showed the were only sold to the farmer in Oedischwend, WäM Iower mean ECS0 value were used. denswil, where the two field resistant isolates were detected, but not to the fanner from Fondli, Dietikon, Base--line sensitlvity of CGA 173506 and vinclozolin where no strains were found with a decreased senM Fig. 2.6 shows the distributions of the ECS0 values of sitivity to vinclozolin. By applying only a few treat­ vindozolin and CGA 173506 evaluated for 10 strains ments of dicarboximide fungicides in the field the from Oedischwend, Wädenswil and for 10 strains selection for resistant strains is very rapid as shown from Fondli, Dietikon. Minimum and maximum valM in the previous chapter for the vineyard in Walen­ ues are indicated by arrows. In the two populations stadt, Switzerland. The minute, but statistically sig­ Oedischwend and Fondli, the base-line sensiti.vity for nificant, differences between the mean ECS0 values vindozolin ranked between 1.7 and 2.0mg/L and for of vinclozolin for mycelial growth in the two popuM CGA 173506 from 0.072 to 0.19mg/L, respectively, lations Oedischwend, Wädenswil and Fondli, DieM for the germination of sensitive wild type conidia. In tikon are of no practical relevance. However, because the mycelial growth assay the base-line sensitivity of the treatments with vinclozolin during the proM ranked between 0.12 and 0.27mg/L for vinclozolin duction of young plants, strains of B. fuckeliana that and behveen 0.0027 and 0.016mg/L for CGA 173506, were resistant to dicarboximides in the field survived respectively (Table 2.2). during the season and could be detected. This indi­ cated the presence of isolates with nuclei canying 2.4 Discussion the gene for resistance to dicarboximides. Although, The strawberries from Oedischwend, Wädenswil and the proportion of isolates carrying nudei with the from Fondli, Dietikon were cultivated without appliM resistance gene is very small in population of OedisM cation of fungicides and were sold as "pick your chwend, Wädenswil, the influence of the resistance own" on the field. Grey mould was indirectly meM gene cannot be completely excluded. chanically controUed by the consumers who not only The ECS0 value is a general purpose value picked healthy fruits for consumption but also had (Brent, 1988) which, commonly, is graphically deterM to remove macroscopically visible infected fruits from mined by plotting percentage inhibition of mycelial the plants. Although no dicarboximides at all had growth against the logarithm of fungicide concen~ been applied in Oedischwend, Wädenswil, from tration (Grindle, 1983, Lorenzini, 1983, Beever et al., twenty tested samples, two were found to be vin­ 1989, Köller and Wubben, 1989). The precision of its clozolin field resistant. Due to this unexpectedly high determination, however, depends an the number and frequency of field resistant strains, 40 samples were the range of concentrations that are used to assess tested additionally. None of these showed a decreased the dose·response relationship. Due to practical rea· sensitivity to dicarboximides indicating the resistance sons the number of concentrations used to evaluate frequency tobe much lower than it was first anticiM doseMresponse curves is limited. Probit analysis does pated. often not satisfy mathematically as frequently too fe1,v Both farmers bought part of their young strawM values are available for a reliable regression analy­ berry plants from Häberli AG, Switzerland where sis. Even though ECS0 values are generally used to

22 characterize dose-response relationships the afore­ mentioned reservations have to be considered. To assess the base-line sensitivity of CGA 173506 and of vinclozolin in B. fuckeliana EC50 val­ ues of 10 strains from each of two populations were graphically determined from dose-response curves. In table 2.3 the results for vinclozolin are compared with data given in the literature. Buchenauer (1976), Albert (1979), Pappas and Fisher (1979), Pappas (1982), Davis and Dennis (1981), Beever (1989) and Rewal et al. (1991) found the spore gennination proc­ ess much less affected by vinclozolin than the my­ celial growth. As indicated in Fig. 2.4 and 25 in table 2.2 the same effect could be demonstrated for the phenylpyrrole compound CGA 173506. Conceming mycelial growth the range of ECSO values of vin­ clozolin from sensitive wild type strains indicated in Fig. 2.6 agrees with the data given in the literature (Table 2.3). However, Buchenauer (1976) found slightly higher values whereas Rewal et al. (1991) found strains with slightly lower ECSO values. Re­ garding the germination test, the range of EC50 val­ ues given in the literature for vinclozolin is much broader (fable 2.3). As some conidia produce a non vital germ tube, the evaluation of the conidia genni­ nation test is difficult which is reflected in the broad range of EC50 values of vinclozolin determined by different authors.

23 3. Stu.dies on the occurrence of laboratory resistance against vinclozolin and CGA 173506 inBotryotiniafuckeliana (de Bary) Whetzel and its influence on fitness parameters

3.1 Introduction

Most fungi have a short generation time and pro­ Gisi and Csech (1988a,b) and Brent etal. (1990) duce a high number of propagules, among which, who refer to the aforementioned authors, recently assum.ing a frequency of mutations towards resis­ published resistance risk evatuation schemes for new tance from 1Q--4 to 10-10 (Dekker, 1987, Gisi and Csech, candidate chernicals to be used in disease control. 1988a) resistant progenies are likely to occur. There­ The overall resistance risk is split into inherent risk fore the occurrence of resistant strains in a field popu­ and management risks. The inherent risk is compris­ lation is the rule rather than the exception (Gisi and ing parameters conceming the effect of the fungicide Csech, 1988a). Brent et al. (1990) assumed the resis­ on the pathogen with respect to its biology and to tance alleles in a natural population to occur below the environmental conditions whereas the manage­ an expected frequency of lQ-6 due to the often re­ ment risk includes the factors related to the practical duced fitness of the resistant pathogens. As fungi­ use of the fungicide (Staub and Sozzi, 1984, Gisi and cide resistance is a major problem in plant disease Csech, 1988a, b). Although emergence of strains that control (Dekker, 1987), for the development of new are resistant to single site inhibitors (e.g., dicarbox­ products it is essential to study, in an early stage, the imides, phenylamides, methyl-benzimidazolecar­ potential of the target organisms to develop resis­ bamates and SBI's) in the Iaboratory is common, this tance against the novel active ingredient (Dekker, does not necessarily imply the buildup of a resistant 1982b, Schwinn, 1982, Gisi and Csech, 1988a,b, Brent population in the field (Dekker, 1982a, 1987). Never­ et al., 1990, Schwinn and Morton, 1990). To assess theless the occurrence of laboratory resistant strains the resistance risk it is tlms crucial to know the in­ indicates the risk of resistance development and thus herent genetic variation in sensitivity towards the should not be underestimated (Brent et al. 1990). The fungicide in the pathogen population (Gisi and Csech, question whether the fungidde resistance risk in the 1988b, Brent et al., 1990). Due to genetic flexibility field can be estimated from laboratory and green­ (see p. 6) overall variability can be drastically in­ house tests is discussed by Dekker (1982a). For the creased in fungi carrying two or more genetically assessment of the inherent risk of a new product it is different nuclei in the same cytoplasm forming a essential to compare fitness parameters of sensitive heterokaryon. Anastomosis furthermore allows the wild type strains with strains showing a reduced sen­ free exchange of nuclei within the fungal popula­ sitivity to the new active ingredient. tion. If only few nuclei that carry a gene for fungi­ Based on their preliminary results from stud­ cide resistance are present in a fungal population the ies on the occurrence of resistance in B. J11ckeliana to exposure to the appropriate fungicide will conceiva­ fenpidonil and vinclozolin or iprodione, respectively, bly increase the proportion of nudei which carry the Martinetti (1986) and Leroux (1991) assumed that the gene encoding for resistance (Georgopoulos, 1987). inherent resistance risks of dicarboximides and B. fuckeliana, due to its biology, is therefore a patho­ phenylpyrroles are similar in this fungos. In the pres­ gen "at risk" carrying a high potential to develop ent studies the occurrence of CGA 173506 laboratory fungicide resistance to new chemical compounds. resistant strallls of B. fuckeliana was studied with

24 regard to changes in relative fitness of resistant respectively, when the surface of the potato dextrose strains. Inherent resistance risk parameters were stud­ agar in the test tubes was completely covered with ied and compared for vinclozolin and CGA 173506. sporulating mycelium (Tab1e 3.1). Irregular sporula­ tion (agar only partly covered with sporulating my­ 3.2 Materials and Methods celium) was described as medium (Table 3.1). Tue Strains colours of the colonies shaded off in pale grey, grey, The occurrence of laboratory resistance was studied dark grey and brown grey. Production of sderotia in 10 sensitive wild type strains from Oedischwend, was qssessed as either yes or no. Wädenswil. These strains were characterized quali- tatively by assessing growth, sporulation, colour and Growth rates production of sderotia an potato dextrose agar (39g Spore suspensions of 105conidia/ml were prepared potato dextrose agar /L, Merck) as weil as quantita- from sporulating cultures on potato dextrose agar. tively by measuring daily increase in colony diame- Suspensions (50µ1) were added to malt agar discs ter on malt agar and virulence on . (diam. 10mm). The discs were incubated at 20°C for 17h in the dark and then either used in a pathogen­ Visual assessment of growth, sporulation, colour icity assay (see p. 26) or placed upside-down in the and production of sclerotia on potato dextrose agar centre of malt agar plates (diam. 15cm) to assess my­ Colonies that grew for two weeks at 20°C under fluo- celial growth. The mean colony diameter of three rescent light in test tubes which contained potato replicates were measured during 6 days. Minimal dextrose agar were visually assessed. Growth and and maximal daily increase in diameter and mean sporulation were described as regular and as strong, daily increase over 5 days were calculated.

Fig.3.1: Adaptation on sublethal concentrations of vindozolin and CGA 173506; scheme of subcultures on agar amended with 0.005 to 0.01mg CGA 173506/L or 0.15 to 0.3mg vinclozolin/L

sensitive wild type strains stored in liquid nitrogen AIIB, Alll4, Bl7, Bll2, BIIl4, CIIS ·-··· ····· ······· ··········· ········ CGA 173506 vindozolin control I control II subculture 1 0.005mg/L 0.15mg/L Omg/L

subculture 2 O.OOtmg/L 0.15mg/L Omg/Lt •t subculture 3 O.Jmg/L 0.3mg/L Omg/L• subculture 4 O.Oltg/L 0.3:tg/L ig/L subculture 5 O.Olmg/L• 0.3mg/L• Omg/L pe,agar petagar peat agar ~ ... t / comparison of EC50 values

25 Pathogenicity assay them, however, proved to be sensitive. The pathogenicity assay was adopted from Schüepp "Runaways" were produced on agar contain­ and Küng (1978). cv. Golden Delicious were ing fungicides in the concentrations: 1mg vindozolin/ divided in half, surface sterilized with ethanol 70% L, 3mg vinclozolin/L, 0.3mg CGA 173506/L or 1mg and circularly cut-injured with a cork borer at three CGA 173506/L, respectively. As parental strains the places on each half. The circularly cut parts were aforementioned 10 wild type strains from Oedis­ removed and replaced by mycelial discs. As a con­ chwend, Wädenswil were used. Agar discs were in­ trol treatment, the removed parts of the cutide were oculated with 50µ1 from a Suspension of 106 conidia/ replaced by sterile malt agar discs. Diameters of the ml. After an incubation period of 17h at 20°C in the rot lesions were assessed after an incubation period dark, three of these mycelial discs were placed up­ of 3 to 4 days at 20°C in the dark. Each wild type side--down into each of 10 agar plates per concentra­ strain was tested in 6 replkates. tion and parental wild type strain. The plates were further incubated at 20°C in the dark. Tue appear­ Adaptation test ance of "runaways" was checked every second day. In an adaptation test 6 sensitive wild type strains For further characterizalion "runaways" were trans­ were subcultured on malt agar amended with 0.005 ferred to labeled test tubes containing potato dex­ or 0.01mg CGA 173506 and 0.15 or 0.3mg vinclozolin/ trose agar. The labels were consisting of 3 parts: the L, respectively (Fig. 3.1). By choosing these concen­ name of the parental strain from which they were trations near the EC50 values of the sensitive strains, derived (A~C/I-III/1-8), the fungicide concentration their mycelial growth was only slightly inhibited. on which they were produced (0.323*, 123*, lV** or

Spontaneously arising sectors did not occur under 3Y,•*; * 23 := CGA 173506, **V""' vinclozolin) and the these conditions. After the 5th subculture the strains plate on which they occurred (1-10). were transferred to fungicide free pea agar. The spores produced on pea agar were used for a my­ Characterization of "runaways" celial growth test on agar amended with 0.03 to 3mg Morphological classification vindozolin/L or 0.001 to 0.3mg CGA 173506/L, re­ A total of 107 "nmaways" that developed spontane­ spectively; the dose-response curves of the strains ously from the sensitive wild type strains were mor~ subcultured on agar amended with either CGA phologically classified. Fig. 3.2 shows the 5 morpho­ 173506 or vinclozolin and on non amended agar, re-­ logical classes in which the "runaways" were cate­ spectively, were assessed and compared to the curves gorized. of the original strains that were not subcultured. Fast growing "runaways" of types IA, !B, !C and ID spontaneously arose from the mycelial disc. Production of "runaways" with CGA 173506 and They either grew as circular colonies (IA and lß) or vinclozolin as sectors (I.C and Jl2). Type LA differed from lß: in Strains spontaneously developing from sensitive wild the form of the edge of the colony. In IB. the edge type strains on agar amended with lethal or strongly frayed out, whereas it was regular in IA. Type IC. inhibiting concentrations of CGA 173506 or vin­ contrasted with type ID. by developing tvvo or more clozolin, respectively, were termed "runaways". sectors. Most of them showed a reduced sensitivity to CGA In contrast to the fast growing "runaways" of 173606 and to vinclozolin, and surprisingly some of types IA- ID the "runaways" of types IlID and IIIE

26 arose from strongly inhibited growing mycelium. riphecy of 4d old cultures or by incubating discs for Type IIlJ2 developed sectors, comparable to the sec- 17h at 20°C on which 50µ1 of a suspension of 105 tors found in 112, whereas IIIE only fonned small conidia/ml were placed. Tue mycelial discs were sectors that were limited in vitality. placed upside-down on the agar plates. After an in- cubation period of 3d at 20°C in the dark, the mean Tolerance of vinclozolin and CGA 173506 colony diameter of tllree replicates per plate was as­ Mycelial growth on agar amended with 1, 3 and sessed. The inhibition of mycelial growth compared 100mg vinclozolin/L and 0.3, 1 and 3mg CGA to the control was calculated. "Runaways" which 173506/L, respectively, was compared to the mycelial grew more than 50%, of the control were referred to growth on fungicide free medium. Mycelial discs as laboratory resistant at the appropriate level. were prepared by either cutting them from the pe-

Fig.3.2: Classification of ''runaways"; the binary classification consisted of growing types (I and III) and morphologic types (A, B, C and D)

Type!A TypeIB circular colony growth circular colony growth

fast colony growth fast colony growth

regular edge

malt agar disc

TypeIC TypeID

sectorial growth with two sectorial growth with or more origins one origin

fast colony growth fast colony growth 10mm

Type Il!D Type IIIE fast sectorial growth from strongly inhibited colony limited sectorial growth from strongly inhibited colony

strongly inhibited colony growth strongly inhibited colony growth

27 Dose-response relationships and pathogenicity Conidia of the sensitive strain CII5, the low assay laboratory resistant strain Cil5 IV 10 and the highly Of a total of 107, 34 "runaways" were chosen to as­ laboratory resistant strain Cil5 0.323 2 were used to sess dose-response relationships of vinclozolin and develop an accurate and rapid technique to measure CGA 173506 in a mycelial growth test. Malt agar the lengths of fungal germ tubes by image analysis. discs (diam. 10mm) were inoculated with 50µ1 of a One drop of a suspension, adjusted to 5xlO'spores/ suspension of lOSconidia/ml and incubated for 17h ml, was dispensed onto 10mm diameter agar discs. at 20°c in the dark. Tue discs were placed upside­ The discs were incubated for ten hours at 20°C in the down on agar amended with 0.1 to 100 mg vin­ dark. Afterwards a drop of methylene blue in lactic clozolin/L or 0.001 to 10 mg CGA 173506/L, respec­ acid (0.16%) was added on each disc to stain the tively. Inhibition of colony growth compared to the germ tubes and to stop further growth. The discs controls on fungicide free agar was calculated. Val­ were then stored for 12h at 0°C in the dark. During ues of EC50 were determined graphically. this time the excess stain solution was absorbed by Virulence on apple was assessed in a patho­ the agar disc. Seventeen germinated spores were genicity assay as described for the sensitive wild type measured per treahnent. Tue experiment was re­ strains. peated 3 times. The image analyzing system ASBA ® (Wild and Measuring fungal germ tubes by using an image Leitz Ltd., Switzerland) detected 256 different shades analyzing system of gray from 0= black to 255= white (Anonymous, Vimlence on apple and sensitivity to vinclozolin and 1988). The intensively blue stained germ tubes con­ CGA 173506 were assessed for strains CII5, Cll5 IV trasted clearly with the agar disc and could be easily 10 and CII5 0.323 2 in an apple pathogenicity test deteded. The germ tubes were examined by light and in a mycelial growth test, respectively. microscopy (Leitz Diaplan) at 250x magnification. The

Fig.3.3, A genninated conidiumof B.fuckeliana on maltagar is stained with methylene blue, the white line within the germ tube represents the skeleton of one pixel (picture element) in width. Fig. 3.3y) is the magnification of the area indicated within dotted lines in Fig. 3.3x).

x) y) A 40µm lOµm

s ,-.... ___ ,,_,,_ i ! l' B 1 j B ! 1_,_,,,.,,_, __ ,_,'

28 image was recorded by a video camera (Kappa CF6), Nuclear staining (HCI Giemsa staining and HCI digitized by the graphic tablet (HlPAD™ digitizer, Boroviczeny staining) Model EDT 11 AA P, Houston instruments, Austin The number of nuclei per of sensitive wild Texas) and analyzed by the processor (Z8002). type strains was compared with that of the corre­ The softw-are program allowed. an image tobe sponding laboratory resistant conidia. reduced in length and width stepwise. For the caku­ A modification of the commonly used HCI lation of the total length, the reducing processes were Giemsa staining method (Menzinger, 1965, Sing, 1983, repeated until the image was reduced to a skeleton Akutsu et al., 1983, Lorenz and Eichhorn, 1983, Fare­ of one pixel (picture element) in width (Fig. 3.3). With tra and Antonacci, 1987, Shirane et al., 1988 and each step of the reducing process the genn tube im­ Shirane et al., 1989) was used for the nuclear stain­ age was thus reduced from all sides by two pixels ing of B. juckeliana. For nuclear staining three drops per step. The reduction at the tips therefore amounted of a suspension of 106 conidia/ml were air dried on to the number of reducing steps times two pixels. a glass slide. For fixation of the conidia the slide was The reduction in width was two pixels per step at submerged in ethanol glacial acetic add (3:1) for 10 both sides of the germ tube, which amounted to the minutes. The conidia were then washed. for 5 min­ number of reducing steps times 4 pixels. When the utes each in ethanol 35% and distilled water before skeleton of one pixel in width was accomplished, no they were hydrolyzed for 7 minutes in HCI 10M at further reducing of the skeleton occurred. The actual room temperature. To remove the excess add, the Iength in µm (1) of the germ tube was calculated by slides were quickly washed in distilled water and using the following formula: 1 :c:: f(na + s): where f = rinsed in phosphate buffer (P-buffer KH PO./ 2 translation factor: pixels to µm, n = number of tips Na2HP04; pH=. 6.9) for 1 minute. Finally the conidia (at A and at B shown in Fig. 3.3x), a= the number of were stained for 30 minutes in the staining solution reducing steps times two pixels and s= length of (80ml P-buffer, 20ml Giemsa staining solution skeleton in pixels. The translation factor (f) was the (Flucka)). The staining procedure was terminated by quotient of a measurement in µm of a defined, rinsing the slides in P-buffer to remove excess stain. straight distance measured with the micrometer and A cover glass was mounted and the nuclei were of the measurement in pixels of the same distance counted using a microscope Ortholux II (Wild and measured with ASBA®. Leitz) at magnification 1250x using oil immersion. In order to verify the stained organelles to be Conidial dimensions and number of nuclei per co­ the nuclei, a second DNA binding dye was used. In nidium the HCl Boroviczeny staining method lg Toluidin­

Conidia of laboratory resistant strains were compared , blue, 0.5g Safranin, lg K,.HP0.1 0.5g KH2PÜ4 and to those of their sensitive wild type parental strains 200ml Hp were mixed and filled with methanol up with regard to dimensions. 50µ1 of a suspension of to lL (Reiss, 1965). The staining procedure was the 105 conidia/ml were placed on malt agar discs. Co­ same for the HCI Giemsa staining as for the HCI nidia were stained with rnethylene blue in lactic acid Boroviczeny staining. The specimens were stained as described for germ tubes previously. Vectorial for 20 seconds to 20 minutes in the undiluted stain lengths and widths of a total of 100 conidia were solution. By the HO Boroviczeny staining the same measured interactively by using the image analyz­ organelles as by the HCI Giemsa staining were ing system ASBA ®. For statistical analysis the meas­ stained; from this result it was concluded the organ­ urements of all 3 discs were pooled. elles were nuclei. 29 Competition test Stabillty of resistance The competitive ability of 8 laboratory resistant Stability of laboratory resistance was assessed after strains of B. fuckeliana in mixture with conidia from successive transfers either from apple over pea agar the corresponding sensitive wild type strains was to apple, or from pea agar to pea agar only. Myce­ investigated by assessing the proportion of resistant lium of 40 laboratory resistant and sensitive strains conidia in the generations following mixed inocula­ was subcultured from pea agar 10 times in irregular tion. A mixture of a dicarboximide field resistant intervals during 6 months. In the test including apples (CGA 173506 sensitive) strain (G 6.15 1.6) and a sen­ 24 laboratory resistant and sensitive strains were sitive (CGA 173506 and vinclozolin) strain (G 6.15 transferred from apples to apples via pea agar three 1.8), both derived from single ascospores of the same times. Dose-response relationships were assessed as ascus were additionally included in the experiment. described previously. EC50 values of vinclozolin and Equal portions of suspensions of U}' laboratory re­ of CGA 173506 that were detennined before and af­ sistant and sensitive conidia/ml each were mixed. ter the transfers over pea agar and over apple, re­ From every combination 100µ1 of the mixture were spectively, were compared. plated on each of 8 malt agar plates containing 0.3mg CGA 173506/L, 3 or 10mg vinclozolin/L, respec­ Fitness of laboratory resistant strains as compared tively, while 50µ1 were plated on each of 8 fungicide to their vinclozolin field resistant parental strains free plates. Two test tubes containing pea agar were Five vindozolin field resistant strains of B. Juckeliana inoculated with 50µ1 of the same mixhtre. Colonies were isolated from freshly sporulating grapes in a growing on fungicide free agar were counted after 2 vineyard in Walenstadt before harvest 1991 (chapter days whereas colonies growing on agar amended 1). For each strain dose-response curves were deter­ with fungicide were counted after 3 days. Percent­ mined for vinclozolin and for CGA 173506 in a my­ age of resistant conidia was calculated (r=cc100(m/2n); celial growth test. Simultaneously response to in­ r= % resistant conidia, n= number of counts on fun­ creased osmotic pressure and pathogenicity on apple, gicide free medium, m= number of Counts on me­ cv. Golden Delicious, were assessed. To test osmo­ dium amended with fungicide). From sporulating tolerance, mycelial discs containing 17h old myce­ cultures on pea agar of the second generation sus­ lium were placed upside-down on malt agar plates pensions of 103 conidia/ml were prepared. Propor­ amended with 1, 3, 5, 7, 10, 15 and 30% glucose/L. tions of resistant conidia were evaluated as described. Mean diameters of three replicates per plate were above and 2 fresh test tubes containing pea agar were assessed. Mycelial growth on medium supplemented inoculated. The experiment was run for four genera­ with glucose was compared to the growth of the con­ tions. Agar plates were incubated at 20°C in the dark; trol on non supplemented medium. "Runaways" of to induce sporulation the test tubes were incubated the vinclozolin field resistant strains were produced under white light and black light in a 12h rhythm. on agar amended with 10mg CGA 173506/L. Tue The ability of sensitive, laboratory resistant and "runaways" were transferred to pea agar;

Visual assessment of growth, sporulation, colour All strains were pathogenic on apple. They and production of sclerotia on potato dextrose agar caused rot lesions which showed mean diameters As shown in table 3.1 all sensitive wild type strains that ranked arnong 17 and 30mm after three days. grew regularly on potato dextrose agar medium and Controls which were inoculated with sterile malt agar showed medium to strong sporulation. Slight phe­ discs did not rot. The virulence differed significantly notypic variations were visible in the colour of the within the populations as shown in Fig. 3.4. colonies which were pale grey, grey, dark grey or brown-grey, respectively. Four sensitive wild type Adaptation test strains produced sclerotia on potato dextrose agar In a training experiment the occurrence of a shift in whereas no sclerotia formation was observed in the sensitivity to vinclozolin or CGA 173506, respectively, other six strains. during successive subculturing on media amended with sublethal doses of both fungicides was investi­ Growth rates and pathogenicity assay gated. Although the mycelial growth of strains Bil2 The mean daily increase in colony diarneter an an and AII8 was strongly inhibited by 0.3mg vin­ average over 5 days did not differ significantly among clozolin/L the formation of "runaways" was not ob­ 10 strains from Oedischwend, Wädenswil (Kruskal served. All strains subcultured on agar amended Wallis test p>0,05). As shown in table 3.2 the values with either vinclozolin or CGA 173506 maintained of mean daily increase, on an average over 5 days, their sensitivity towards both fungicides. As shown ranked from 16 to 20mm/ day, whereas daily in­ in Fig. 3.5 in this experiment the phenotypic vari­ creases ranked from 10 to 32mm/day. ations with respect to the sensitivity towards vin-

Table 3.1: Visual assessment of characteristics of sensitive wild type strains growing on potato dextrose agarmedium

morphological characteristics on PDA medium

strains growth sporulation color production of sclerotia

Al4 regular strong grey-brown yes Al7 regular strong pale grey yes AIIB regular medium darkgrey yes Alll4 regu1ar strong pale grey no Bl7 regular strong grey no B II 1 regular medium pale grey no B 112 regular medium darkgrey yes B lll 4 regular strong pale grey no Cl4 regular strong pale grey no Cil5 regular medium darkgrey no

31 Table 3.2: Mean daily increase assessed on an average over 5 days and minimal and maximal daily increase in colony diameter

daily increase in colony diameter in mm

strains average over 5 days a stdev min. max.

Al4 18 5 11 24 Al7 16 3 10 20 AIIS 20 5.6 15 32 Aßl4 19 2.3 15 23 Bl7 19 5 13 25 Bill 16 3.4 11 21 Bll2 19 4.1 12 24 Blll4 16 3.5 12 21 CI4 17 3 12 20 CII5 19 3 15 23

a increase in colony diameter was measured in three replicates from the 2nd to the 6th day

Fig. 3.4: Mean virulence of 10 sensitive wild type strains from Oedischwend, Wädenswil, assessed in a pathogenicity assay on apples cv. Golden Delicious. Numbers on top of the bars indicate to which strains significant differences occurred (Anova, p< 0.05).

35

30 25 .s 20 15

10

5

0

wild type strains

32 Adaptation test vinclozolin CGA 173506 100 100 80 80

0 60 60 00 "".5 40 40 - § < "' 20 20 ßj5 ·- 0 0 .. -20 -20 100 100 80 80 60 60 ...*5 40 40 Fig. 3.5: = " 20 Adaptation test: comparison of <$ 5 ·- 0 dose-response curves of 6 sen· ß~ -20 -20 sitive wild type strains being . ·- stored without subculturingor 100 100 being subcultured for 5 times 80 80 onagar amended with0.005 to 60 60 0.01mg CGA 173506/L, 0.15 to *.5 40 40 ~ § 0.3mgvinclozolin/Loronnon "' "' 20 amended agar, respectively. 0 11. ·- -20 -20 Legend: 100 80 80 0 subcultures on 60 60 fungicide free media *.5 40 40 ~ C - 0 20 20 "'E ·-"' D no subcultures "rc :9 0 0 ti :!l -20 -20 100 100 80 80 0 subcultures on agar 60 amended with 0.15 and 60 -o:t< .E 0.3mg vinclozolin/L * 40 40 =""'.,,- 0 20 20 .E :ö • subcultures on agar 0 0 amended with 0.005 and .§ ~ -20 -20 0.01mg CGA 173506/L . ·- 100 100 80 80 60 60 C 40 IQ·~* 40 U·.;::- 0 20 20 ·rcE ·-:9 0 ;, -§ 0 . ·- -20 -20 mg/L.01 .1 1 10 mg/L.001 .01 .1 1

33 clozolin and CGA 173506 were minute. Dose-re- ence (contingency table analysis p=0.75) in the num­ sponse curves from the cultures which were subcul- ber of "runaways" that arose on either vinclozolin or tured for 5 times on fungicide amended media did CGA 173506 amended agar was found when the neither differ from the controls that were subcultured "runaways" of type IIIE (55) which showed abnor­ on fungicide free media nor from the dose-response mal, restricted growth were excluded. curves from the original strains that were not sub- cultured. Characterization of "runaways" Morphological classification and tolerance of vin­ Production oi "runaways" with CGA 173506 and clozolin and CGA 173506 vinclozolin Fig. 3.6 describes the distribution of the "runaways" As shown in table 3.3, 233 "runaways" were pro- with respect to their morphological type and their duced within 14d. The number of "runaways" pro- sensitivily to vindozolin and to CGA 173506. The duced on agar amended with 0.3 mg CGA 173506/L 107 "runaways" could all be ascribed to three pat­ ranked from 1 to 17, whereas it ranked from O to 7 tems: on agar amended with 1mg CGA 173506/L. On agar Pattern 1: EC50 vinclozolin < lmg/L, EC50 CGA amended with 1mg vinclozolin/L 1 to 28 and on 173506 < 0.3mg/L agar amended with 3mg vinclozolin/L 2 to 20 "run- Pattern 2: ECSO vindozolin > lmg/L, ECSO CGA aways" occurred. 173506 > 0.3mg/L and Pattern 3: ECSO vinclozolin > lmg/L, ECSO CGA Induding aJl types of "runaways" into account, sig- 173506 < 0.3mg/L. nificantly less "runaways" developed on agar amended with CGA 173506 (87), than on agar Strains showing ECSO values for CGA 173506 > amended with vinclozolin (146) (contingency table 0.3mg/L and < lmg/L for vindozolin could not be analysis p=0.0001). However, no significant differ- found (Fig. 3.6).

Table 3.3: Number of "runaways" occurring within 14d on agar amended with lethal or strongly inhibiting concentrations of CGA 173506 or vinclozolin, respectively

number of "runaways" a parental CGA 173506 vinclozolin strains 0.3 mg/L lmg/L lmg/L 3mg/L AI4 1 3 1 2 AI7 8 7 2 16 AIIS 1 4 1 18 (9) AIII4 3 1 1 4 Bl7 4 4 28 (27) 6 BIil 5 0 6 (3) 8 (3) Bil2 3 1 2 4 Bill4 11 2 2 10 (6) CI4 5 3 2 (2) 20 (1) CIIS 17 4 3 10 (4)

a including "runaways" of type IIIE numbers in() indicate "runaways" ofType IIIE

34 The ECS0 values of the majority of the "run- tory resistant strains (VHR) which showed ECSO val­ aways" were either > 100mg/L for vinclozolin and > ues > 30mg/L (Fig. 3.7). The border between sensi­ lOmg/L for CGA 173506 (upper end of pattem 2) or tive and low resistant strains was arbitrarily set at < 1 for vinclozolin and < 0.3 for CGA 173506 (pat- the ECS0 value of lmg/L. The dose-response curves tem 1). Of 40 "runaways", morphologically charac- of CGA 173506 apparently reflected a continuous terized by the types IA or IB, respectively, 37 were spectrum, however, the results of genetic analysis in ascribed to pattern 2. From 17 "runaways" of type chapter 4 will show that a classification in different IlIE, all but 1 were assigned to pattem 1. Nine "run- sensitivity dasses can be made. The resistance level aways" of type IC were ascribed to pattem 2 whereas for vinclozolin and for CGA 173506 was identical for 3 strains were attributed to pattem 1. "Runaways" all but 3 strains (Table 3.4) which indicates that vin­ of type 1D and IIID behaved intennediate. dozolin/CGA 173506 resistances might be coupled. In further experiments, these 3 strains, however, were Dose-response relationships not stable (data not shown), Dose- response relationships of vinclozolin and CGA 173506 were assessed for 7 strains of pattern 1, 19 Low laboratory resistant strains and field resistant strains of pattem 2 and 8 strains of pattem 3. Dose- strains apparently showed the same resistance pat­ response curves for both fungicides are given in Fig. tem for vinclozolin. Table 3.4, however, shows that 3.7. Conceming vindozolin two groups of curves in contrast to the vinclozolin field resistant strains in were differentiated. The first group induded sensi- the low laboratory resistant strains decreased sensi­ tive (VS) and low laboratory resistant strains (VLR), tivity to vinclozolin was linked to decreased sensi­ whereas the second group comprised highly labora- tivity to CGA 173506.

Fig. 3.6: Distribution of "runaways" with respect to rnorphological type and sensitivity to vindozolin and CGA 173506

numberof ''runaways''

morphological types range of EC50 (vinclozolin)/ range of ECSO (CGA 173506)

35 Table3k ECSOvalues of vinclozolin and CGA 173506 of strains shown in Fig. 3.7

adopted resistance pattems EC50 values in mg/L (mycelial grow.th) "runaways" CGA 173506 vindozolin CGA 173506 vinclozolin BI71V 1 CS vs 0.0057 0.26 Bl73V3 CS vs 0.00855 0.26 Biil lV 3 CS vs 0.00655 0.19 BII141V 2 CS vs 0.00805 0.37 BIII4 3V 7 CS vs 0.0206 0.62 BIII4 0.323 3 CS vs 0.00595 0.21 Cll53V 1 CS vs 0.0156 0.335 Al4123 6 CHR VHR 0.685 > 100 All81V 7 CHR VHR 0.385 > 100 All8 3V 1 CHR VHR 2.05 > 100 All81234 CHR VHR 2.9 > 100 AIII4123 9 CHR VHR 0.92 > 100 Bl71Z3 10 CHR VHR 05 > 100 BIil 0.323 6 CLR VHR 0.25* > 100 BII21V 6 CHR VHR 6.3 > 100 BII20.3231 CHR VHR 2.56 > 100 BII2 123 7 CHR VHR 5.3 > 100 BIIl4 3V 6 CHR VHR 0.75 > 100 CII5 3V 7 CHR VHR 0.38 > 100 CII51231 CLR VLR 0.26 7.6 CII51Z38 CHR VHR 0.92 > 100 All 8 0.323 7 CHR VHR > 10 > 100 AIIl43V2 CHR VHR >10 > 100 Alll4 0.323 1(4) CHR VHR > 10 > 100 BI71Z31 CHR VHR > 10 > 100 Cl412310 CHR VHR > 10 > 100 Al71V 8 CS VLR 0.0225 3.1s- All83V6 CLR VLR 0.03 2 BI71V 2 es VLR 0.0245 1.35** BIII41V9 CLR VLR 0.0945 6.3 BIIl4 3V10 CLR VLR 0.078 6.3 BIII4 0.323 5 CLR VLR 0.075 6.75 CII53V 6 CLR VLR 0.23 7.25 CII50.3Z3 5 CLR VLR 0.245 7.15

• ECSO value of CGA 173506 was most likely estimated too low ** ECSO value of CGA 173506 was most likely estimated too high

CS CGA 173506 sensitive strains (ECSO < 0.03mg/L) CLR CGA 173506 low resistant strains {0.03mg/L < ECSO < 0.3mg/L) CHR CGA 173506 highly resistant strains (ECSO > 0.3mg/L) vs vinclozolin sensitive strains (EC50 < lmg/L) VLR vindozolin low resistant strains (lmg/L < EC50 < 30mg/L) VHR vindozolinhighly resistant strains (ECSO > 30mg/L)

36 Fig.3.7: Dose-response curves of vinclozolin and CGA 173506 of 34 selected "runaways"

100 "'.s 90 ;; 80 e~ 70 00 60 50 ~u 40 §' 30 0 20 - 10 :~ 0 '9 -10 ] -20 .01 .1 1 10 100 mg vinclozolin/L

100 "'.s 90 -" 80 -~ 70 e00 60 'iil 50 40 l 30 20 -0 C 10 .9 0 ~ -10 .s -20 .001 .01 .1 1 10 mg CGA 173506/L

Pathogenicity assay The mean virulence on apple and the EC50 values of strains AI7 1V 8, Bl7 lV 2 and BII2 lV 6 the Iabora­ vindozolin and CGA 173506 of 34 laboratory resis- tory resistant strains generally showed a decreased tant and sensitive strains are shown in Fig. 3.8. EC50 virulence. They caused lesions with diameters Iess values of sensitive, low and highly laboratory resis- than 20mm. As remarked in table 3.4 strains AI7 IV tant strains range within the indicated threshold bar- 8 and Bl7 1V 2 are presumably sensitive strains be­ ders. After an incubation period of 5 days at 20°C cause EC50 values of vindozolin were found to be sensitive strains caused lesions with diameters that < lmg/L in further tests (data not shown). ranged between 22 and 45mm. With the exception of

37 Fig. 3.8: Pathogenicity assay of 34 selected "runaways" on apple cv. Golden Delicious

..., ..., --- 100 t ttttttttttttttttt --- bO 50 =i 1f E 10 aEi ~ 1 0~ 40 ~~ - < 1 "~- ·>~ü u 30 0 0 ~ --0 0 .1 lß [j 20 ~ 0 0 lt ii .01 "s· CO • • 10 i:)i:) • • • § .001 """0. 0 .-, <':'l 00 Nr:<:1 ;:; ~;:; ;:;~ t:. ~::: i:o -.t<..... St.i:o "'"'"'- "'

strains arranged according to increasing ECSO values

Legend:

t = ECSO vinclozolin > 100mg/L o EC50 values ofvinclozolin mg/L t = EC50 CGA 173506 > 10mg/L • ECSO values of CGA 173506 mg/L range: -c'.'././_:_::,':z::_: : vindozolin low resistant strains ~ stdev of mean range ~~ : CGA 173506 low resistant strains LJ mean lesion diameter in mm (3 replicates)

Measuring fungal germ tubes by using an image ute differences in germ tube lengths can be detected. analyzing system In the mycelial growth test strain CII5 was sen­ Germ tube measurement is often used to study the sitive to both active ingredients, whereas strain 015 effect of fungicides on the eady development of fungi. 1V 10 was low laboratory resistant and strain 015 Typically a micrometer has been used to measure 0.323 2 was highly laboratory resistant to both fun­ vectorial lengths of the germ tubes. The micrometer gicides (Fig. 3.9). In the pathogenicity assay the labo­ method is feasible only for measuring straight germ ratory resistant strains were less virulent than their tubes. A new technique using the image analyzing sensitive parental strain (Fig. 3.9). system ASBA ~ was developed for accurate and rapid Germ tube lengths of seventeen germinated measurement of fungal germ tubes, independent of spores were measured per treatment. The experiment their shape. Since an image analyzing system allows was repeated 3 times. Fig. 3.10 shows the means of inclusion of the curvature in the measurement, min- the three experiments and their standard deviations.

38 Fig.3.9: Dose-responserelationship of vinclozolinand CGA 173506 assessed ina mycelialgrowth test(Aand B) and virulence on apple cv. Golden Delidous (C) of strains Cll5, CII51V 10 and Cil5 0.3Z3 2.

-.,_ .s 100 A B 50 C ;:; 90 ~ Legend: ~ 80 .s 40 e 70 strain 11" • } "" 60 30 s DIS ~ 50 40 strain J 20 ~} ~ 30 Cll51V 10 0 .9 - 20 "• .9" 10 -" 10 strain ;§ 0 tmJ cns 0323 2 .s-" -10 0 .01 .1 1 10 100 .001 .01 .1 1 10 s LR HR mg vinclozolin/L rngCGA 173506/L strains

Fig. 3.10: Germ tube lengths (bars) and inhibition of germ tube lengths (curves) of a sensitive, a low and a highly labora tory resistant strain on agar arnended with increasing amounts of vinclozolin or CGA 173506, respectively

sensitive low laboratory resistant highly Jaboratory resistant strain CIIS strain CIIS 1V 10 strain CJIS 0.323 2 200 180 160 140 120 100 80 60 40 20 0 O"""! ("'),...; ("')o o 8 O~t>1.-tMoos Oci ,...;M,...; 0 O ,... M,... mg vinclozolin/L mg vinclozolin/L mg vinclozolin/L 200 180 160 140 120 100 80 60 40 20 0 0,...;t")"""!C"1,...;Mo C:C:oo ,...; 0 0 rng CGA 173506/L mg CGA 173506/L mg CGA 173506/L

39 Bars represent the germ tube lengths in µm while nificantly in the number of nuclei per conidium. Two curves show the inhibition of germ tube lengths in Jaboratory resistant strains, both derived from the percentage. Vindozolin inhibited germ tube growth sensitive wild type strain B17 differed significantly of the sensitive strain 015 completely at concentra­ in lengths, widths and in the number of nuclei per tions of 3mg/L. Germ tube growth of the medium cell (Mann Whitney U, p< 0.05). resistant strain, CII5 lV 10, was significantly reduced (Fisher's PLSD test, p<0.05) at concentrations of Fig. 3. 11: Notched box plots, description of 5 per­ 10mg/L but not at concentrations of 0.1 or 0.3mg/L. centi]e points of a frequency distribution There was no statistically significant inhibition of germ tube growth of the highly resistant strain, 015 0 extreme values 0.323 2, at concentrations up to 100mg/L vinclozolin. 0 90th percentile Germ tube growth of the sensitive strain 015 was 25th percentile completely inhibited by 0.1mg CGA 173506/L; on median* agar amended with 0.03mg CGA 173506/L the inhi- 25th percentile bition was 66%. Strain CIIS 1V 10 was significantly 10th percentile (Fisher's PLSD test, p<0.05) inhibited at concentra- 0 0 extreme values tions above 0.01 mg CGA 173506/L. lt was com- with 95% confidence pletely inhibited by 3mg CGA 173506/L. The germ * bands about the median tube growth of the highly resistant strain CTIS 0.3Z3 2 was significantly reduced at the concentrations 0.1, 3 and 10mg CGA 173506/L, at concentrations below Com.petition test 0.lmg/L as weil as at 0.3 and lmg/L the reduction The laboratory resistant strains tested were not as did, however, not differ significantly from the con- competitive as their sensitive parental strains. As trol. shown in Fig. 3.13 in the first generation the propor- tion of laboratory resistant conidia decreased. From Conidial dimensions and number of nuclei per co­ the second to the fourth generation the proportion of nidium. resistant conidia became approximately stabilized at Fig. 3.12 shows the frequency distributions of lengths a low level of resistant conidia. Although the com­ and widths of conidia and the number of nuclei per petitive ability of laboratory resistant strains was low conidium. Results are shown in notched box plots compared to the sensitive parental strains, they did which display five percentile points (Fig. 3.11). not disappear totally. In contrast to the laboratory As shown in Fig. 3.12 no clear trends for dif­ resistant strains the dicarboximide field resistant ferences between laboratory resistant strains and their strain (G 6.15 1.6) was more competitive than its sen­ sensitive parental strains were found. Of 5 resistant sitive competitor (G 6.15 1.8) from the same ascus st:rains, derived from the sensitive wild type strain (Fig. 3.13). Iltls mixture, however, became contami­ AUB, 3 strains differed significantly in lengths, nated after the 3rd generation and the dynamics in whereas all 5 differed significantly in widths. There the population could not be followed further. was, however, no difference in the number of nuclei The strains included in the competition test per conidium. Concerning strain Affi4, 2 of 3 labora­ were further used to study the influence of the ac­ tory resistant strains differed significantly in Jengths quisition of laboratory resistance on other fitness and one differed in width. One strain differed sig- parameters. Fig. 3.14 shows the mean daily increase 40 Fig. 3.12: Notched box plots of the frequency distribution of lengths, widths and number of nudei per conidium oflaboratory resistant strains and their sensitive parental wild type strains. Shadowed box plots indicate significant differences among the parental strains and their laboratoryresistantstrains (t-test, p< 0.05) lengths of conidia widths of conidia (n = 100) (n = 100) 16 • 16 • • • • ! 1 • 14 . : . • 14 • 1 • 1 . : • '• . • • • §_ 12 • • §_ 12 • • .s • i .5 ' 10 10 • • • • t ] 1 i 1 • •1 • • • • 1 8 • 1 • • '·!!':• • • • • • • • • • • 6 • • • :HI • 1 • 4-',-,-r,-,-,-r,-,--r-,r,-,-

parental strains (bold) followed by parental strains (hold) followed by their laboratory resistant strains their laboratory resistant strains

number of nuclei per conidia (n = 100) 14

-~ 12 0 ] 0 8 10 0 0 0 0 ~ • 0 0 • 0 • 0 0 0 0 0 0 i B 0 • 0 0 • 0 0 0 0 0 0 0 0 6 • -0 4 $$$$ 2 i 0 0 0 0 0 0 0 $~+~~+0 0 0 0 *• 0 0 0

parental strains (bold) followed by their laboratory resistant strains

41 in colony diameter over 3 days. 5 from 8 laboratory same ascus. Fig. 3.14 shows the mean lesion diame­ resistant strains showed a significantly lower increase ters assessed on Golden Delicious. Although the dif­ in colony diameter than their sensitive parental strains ferences in the mean lesion diameters were signifi­ (Mann Whitney U test, p<0.05). The field resistant cant between. the field resistant strain and the sensi­ strain grew as fast as the sensitive strain from the tive strain from the same ascus and between the Iabo­ same ascus. ratory resistant strain CIIS 0.323 2 and its sensitive In the pathogenicity assay all the resistant parental strain 015, these resistant strains showed a strains were significantly less virulent than their sen­ higher virulence than the other laboratory resistant sitive parental strains or the sensitive strain from the strains.

Fig.3.13, Changes in the proportion of resistant and sensitive conidia during transfer experiments. Resistant and sensitive strains were mixed 1:2.

Legend:

Ü AIIB/All80.3Z3 7 O Cl4/041Z310 • Cil5/Cll5 0.3Z3 2 0 Aill4/ Alll4 3V 2 D Cil5/CII5 lV 10 • Cll5/CII5 1231 /:;. B17 /Bl71Z31 A Cil5/Cll5 3V 7 • G 6.15 (1.8)/G 6.15 (1.6) bold letters: sensitive wild type strains plain letters: laboratory resistant strains italic letters: dicarboximide field resistant strain

100 vinclozolin 90 80 70 60 50 40 30 20 10 0 100 CGA 173506 90 80 70 60 50 40 30 20

1~ L_.,.___ -..';::==::=*=~----....:;;;:___ GO Gl G2 G3 G4

generations

42 Fig. 3.14: Mean daily increase in colony growth and virulence on apple of strains used in the competition experiment (parental strains: black bars; laboratory resistant strains: shaded bars).

:€ 20 e 40 .§_ I 18 35 "16 :lj 30 ~ 14 " .!l 12 -"'• 25 "C " 210 "'~ 20 .9 8 ~ 8 ;; 15 .s 6 " 10 4 .,,-~ 5 2 0 0 " 0

parental strains (bold) followed by parental strains (bold) followed by their laboratory resistant strains or their laboratory resistant strains or by the field resistant strain (italic) by the field resistant strain (italic) in alphabetic order in alphabetic order

Stability of resistance 10, BIT! 0.323 !, BIT2 lV 6, Bil2123 7, BIIl4 3V 6, CI4 The EC50 values for vindozolin of 10 sensitive wild 123 10, CII5 0.323 2 and CII5 3V 7), all maintained type strains were significantly higher (t-test, p< 0.05) their resistance to both fungicides during 10 subcul­ after 10 subcultures on pea agar. However, the resis­ tures on pea agar. From 9 highly laboratory resistant tance pattem of strains AI4 to CIIS (Table 35) to strains, 6 remained unchanged (AUS 3V 1, AIIl4 0.323 vinclozolin and to CGA 173506 did not change after 1(4), AIII4 3V 2, BII2 lV 6, Cl4 123 10 and CIT5 3V 7) 10 subcultures on pea agar (Table 3.5). conceming their resistance level, whereas two strains Six strains (Bl71V 1, Bl7 3V 3, Bill lV 3, Bffi4 (AIII4 1Z3 9 and CII5 123 8) became sensitive to 0.323 3, Bffi4 lV 2, CIIS 3V 1) that were derived both fungicides when transferred on apple three from sensitive wild type strains on agar amended times; one strain (AII8 1V 7) further tolerated high with various amounts of CGA 173506 or vinclozolin, concentrations of vinclozolin but lost the ability to respectively, but did not show decreased sensitivity grow on agar containing high concentrations of CGA to either active ingredient were transferred on apples. 173506 (Table 3.5). Five of these six strains were likewise subcultured Stability of resistance was tested for five low on pea agar medium. All strains kept their sensitiv­ laboratory resistant strains (BIII4 0.323 5, BIII4 1V 9, ity during the subculturing and the transfers on B1114 3V 10, CIIS 123 1 and CTI5 3V 6) by transfer­ apples (Table 3.5). ring them on apple and for 6 low laboratory resis­ Of 16 highly laboratory resistant strains (AI4 tant strains (BIII4 0.323 5, BIII4 lV 9, B1114 3V 10, 123 6, Ail8 0.323 7, AII8 lV 7, AII8 123 4, AIIl4 Cll5 0.323 5, CII5 lV 10 and CTIS 3V 6) by sub-cul­ 0.3231(4), AIII4123 9, AIIl4 3V 2, Bl7123 1, Bl7 123 turing them on pea agar. From the five strains trans-

43 Tab1e 3.5: EC50 values in mg/L (mycelial growth test)

before 1st transfer after 3rd transfer on apple after 10th passage on

strains vinclozolin CGA 173506 vinclozolin CGA173506 vinclowlin CGA 173506 AI4 0.19 0.012 0.52 0.0017 AI7 0.15 0.0027 0.37 0.0028 AII8 0.18 0.0076 0.82 0.0091 AIII4 0.20 0.0079 0.57 0.0067 BI7 0.21 0.0066 0.47 0.0066 Bill 0.13 0.0030 0.23 0.0026 Bll2 0.19 0.012 0.55 0.0087 BID4 0.20 0.0083 0.55 0.011 04 0.14 0.0070 0.22 0.0034 CIJ5 0.22 0.016 0.67 0.020 AI4 123 6 >100 0.69 >100 1.2 Al71V 8 3.2 0.023 0.53 0.0046 AII8 0.3237 >100 >10 >100 >10 AII81V 7 >100 0.39 >100 0.24 >100 0.83 AIIB !234 >100 2.9 >100 >10 All8 3V 1 >100 2.1 >100 1.5 AII8 3V 6 2.0 0.020 2.2 0.024 3.7 0.044 AIII4 0.323 1(4) >100 >10 >100 >10 >100 >10 AIII4123 9 >100 0.92 0.52 0.0064 >100 1.9 Alll4 3V 2 >100 >10 >100 >10 >100 >10 ßl71V 1 0.26 0.0057 0.48 0.0044 ßl71V 2 1.4 0.025 6.3 0.027 2.7 0.025 B171231 >100 >10 >100 >10 BI712310 >100 0.50 >100 0.95 BI73V 3 0.26 0.0086 0.44 0.0059 0.63 0.0075 Blll 0.323 6 >100 0.25 >100 0.11 0.0038 BIil IV 3 0.19 0.0066 0.47 0.0028 0.22 0.0026 BII2 0.3231 >100 2.6 75.0 >10 B112 lV 6 >100 6.3 33.8 3 >100 >10 B112 123 7 >100 5.3 >100 >10 Bill4 0.323 3 0.21 0.0060 0.55 0.0078 0.64 0.016 BIII4 0.323 5 6.8 0.075 0.56 0.013 >100 0.16 BIII41V2 0.37 0.0081 0.57 0.0078 0.65 0.015 BIII4 IV 9 6.3 0.095 5.1 0.083 6.4 0.14 Blll4 3V 10 6.3 0.078 7.3 0.061 7.4 0.16 Bill4 3V 6 >100 0.75 >100 2.2 B1114 3V 7 0.62 0.021 1.4 0.027 Cl412310 >100 >10 >100 >10 61.0 >10 Cll5 0.323 2 >100 >10 >100 >10 Cll5 0.323 5 7.2 0.25 >100 0.40 CII51V 10 5.6 0.17 87.0 0.27 Cll5123 l 7.6 0.26 >100 0.27 Cll5123 8 >100 0.92 0.58 0.0046 CII5 3V 1 0.34 0.015 0.55 0.010 0.60 0.021 Cll53V6 7.3 0.23 4.9 0.028 23.0 0.29 CIIS 3V 7 >100 0.38 >100 0.44 >100 0.63

44 Table 3.5 (page 44): 173506 (BIIl 0.32.3 6) were compared after subcultur­ ECS0 values of sensitive and vinclozolin and CGA ing on pea agar or on apples, respectively, with the 173506 laboratory resistant strains prior and after original ECSO values. After 3 transfers an apple the subculturing on apples or on pea agar, respectively. strain which was highly laboratory resistant to vin~ Classification of resistance pattems (based on results clozolin and low laboratory resistant to CGA 173506 given in chapter 4): (BIil 0.323 6) maintained its atypical resistance pat­ CGA 173506 sensitive (CS) (EC50 < 0.03 mg CGA tem. One of the 3 vinclozolin low laboratory resis­ 173506/L), CGA 173506 low resistant (CLR) (0.03 mg tant, CGA 173506 sensitive strains became sensitive CGA 173506 < ECSO < 0.3 mg CGA 173506/L), CGA to both active ingredients (AI7 lV 8), whereas two 173506 highly resistant (CHR) ECSO > (0.3 rng CGA maintained their low laboratory resistance to vin­ 173506/L) dozolin after three transfers on apple (Bl7 1V 2 and vinclozolin sensitive (VS) (ECSO < 1 mg vinclozolin/ BIIl4 3V 7). During the subculturing on pea agar, L), vinclozolin low resistant (VLR) (1 mg vinclozolin one strain kept its uncharacteristic resistance pattern < ECSO < 30 mg vinclozolin/L), vinclozolin highly (Bit 1V 2) while in another strain (Ail8 3V 6) the resistant (VHR) EC50 > (30 mg vinclozolin/L) sensitivity to CGA 173506 decreased to the level of low laboratory resistance and one strain was not tested (Table 3.5). ferred on apple 2 maintained their resistance (B1114 lV 9 and BIIT4 3V 10), whereas 1 strain became sen- Fitness of laboratory resistant strains as compared sitive (BIII4 0.3Z3 5) and 2 strains behaved interme- to their vinclozolin field resistant parental strains diate (CIIS 123 1 and CIIS 3V 6). Three of 6 strains Fig. 3.15. A and B show the dose-response curves for subcultured on pea agar maintained their low labo- vinclozolin and CGA 173506, respectively, of field ratory resistance (BIII4 lV 9, BIII4 3V 10 and CIIS 3V resistant strains and of laboratory resistant strains, 6), while two strains kept their low laboratory resis- derived from these field resistant strains. Tue field tance to CGA 173506 but increased their resistance resistant strains showed low resistance to vindozolin to vinclozolin (B1114 0.3Z3 5 and CTIS lV 10) and 1 (0.3mgvinclozolin/L < EC50 < 30 mgvinclozolin/L) strain (CIIS 0.323 5) became highly resistant to both while they were sensitive to CGA 173506 (ECS0 < active ingredients (Table 3.5). 0.03mg CGA 173506/L) (Fig. 3.15A and B left charts). As it was expected for the multinucleate, Tue laboratory resistant strains, however, were highly heterokaryotic B. fuckeliana, intermediate forms oc- resistant to vinclozolin (Fig. 3.15 A right chart) and curred. lt has thus to be considered that the thresh- they showed, in contrast to the field resistant strains, old levels separating sensitive, low and highly labo- a decreased sensitivity to CGA 173506. Six out of ratory resistant strains were defined arbitrarily and seven strains were highly resistant to CGA 173506 the transitions between the resistance patterns are whereas one strain showed low resistance (Fig. 3.15B smooth. Values of EC50 for four intermediate strains right chart). (Al7 lV 8, AII8 3V 6, BI7 lV 2 and BIil 0.323 6), On media amended with up to 30% glucose, three of which low laboratory resistant to vinclozolin the field resistant strains were inhibited by less than but sensitive to CGA 173506 (AI7 1V 8, AUB 3V 6 50%, compared to the control which grew on glu­ and BI7 lV 2) and one highly laboratory resistant to cose free media. Mycelial growth increased on agar vinclozolin and low laboratory resistant to CGA amended with low concentrations of glucose (Fig.

45 Fig. 3.15 A-D: Dose~response curves of vinclozolin and CGA 173506, osmotolerance and virulence of vinclozoHn and CGA 173506 laboratory resistant st:rains and their vinclozolin field resistant parental strains

Legend: Legend: 0 WT6.l l:,. WT6.8 0 WT6.l 10Z3 • WT 6.6(2) 10Z3 0 WT6.3 • WT6.12 0 WT6.310Z3 • WT6.810Z3 D WT6.6 D WT6.3100V • WT 6.12 1023 l:,. WT 6.6(1) 10Z3 :.§ 100 w ~ 80 ~ .g ,;, 60 c.·-0 " . ~ .s 40 _§ ~~ 20 .g e j 0 „ 0 < u .s -20 .01 .1 1 10 100 .01 .1 1 10 100 mg vinclozolin/L mg vinclozolin/L

<'l 100 ~ "'~ 80 w- ·<§c:, ,;, 60 5ru .s 40 w " c ';< .Q ~ 20 •w. w .g i: ~ 0 „ 0 "' "' u .s -20 .001 .01 .1 1 10 .001 .01 .1 1 10 mg CGA 173506/L mg CGA 173506/L "'" ""• & c O 100 0 u 80 o-" 0 :c bO 60 ,;, ~-~·- "' 40 i~ .s 20 ·- 'O "'--a w 0 8 c -20 ""w 1 „ 8 Um "'.s -40 -60 0 1 3 5 7 10 15 30 01 3 5 7 10 15 30 concentration of glucose in% concentration of glucose in% ,._ 8 ••~ 0 8 20 .c-·o• 0 .s :~o 2" 15 1/,,o ; 10 0 w 'O "' :g -i'Lü0 .8" 5 „ c J!l• 0 0 0 00 ~~ -q~ _,; '°_,; _,; ...,~ ·N e<:> "?f3 «:if3 ~f:3 - ::i "' '°o '° 0 f,< ~8...... ~o..... ~o...... ~o...... ~\Co ...... :: ~ ~ ~ i ~- ~- -C -s:!..

46 3.15C left chart). In contrast to the response of vin­ (1977) considered resistant strains to be already exis­ clozolin field resistant strains the mycelial growth of tent before the introduction of a new chemical. Re-­ laboratory resistant strains decreased drastically when sults of experiments with CGA 173506 supported this the osmotic pressure was increased (Fig. 3.15C right hypothesis. From ten sensitive wild type strains of B. chart). Five laboratory resistant strains were com­ fuckeliana laboratory resistant strains could be se­ pletely inhibited and one strain was inhibited by more lected with the same ease on media amended with than 80% on agar amended with 30% glucose (Fig. 1etha1 concentrations of CGA 173506 as it was previ­ 3.15C right chart). Strain WT 6.3 100V showed mor­ ously demonstrated for dicarboximide fungiddes. In phologically abnormal colony growth on media contrast to Lorenz and Eichhorn (1982) who reported amended with glucose; its response to high osmotic dicarboxirnide resistance to be a classical case of adap­ pressure could therefore not be investigated. tive (= physiological) resistance, Hisada et al. (1979), In the pathogenicity assay all the laboratory Davis and Dennis (1981) and Ruiz Borge (1988) con­ resistant strains were less virulent than their sensi­ cluded from their results the resistance to be tive parental field resistant strains. constitutional (= genetically fixed). In an adaptation The Iaboratory resistant strains differed from test including CGA 173506 and vinclozolin there was their vinclozolin field resistant parental strains in no change in sensitivity neither to the phenylpyrrole sensitivity to vinclozolin and to CGA 173506 as well nor to the dicarboximide fungicide after 5 transfers as in osmotolerance and virulence. In contrast to the on agar amended with sublethal concentrations of Iaboratory resistant strains their field resistant pa­ both active ingredients, which indicated that resis­ rental strains tolerated higher concentrations of glu­ tance to CGA 173506 as we11 as to vinclozolin was cose in the medium and were more virulent on apple. not adaptive. The acquisition of laboratory resistance was thus re­ In the literature, the occurrence of laboratory lated to a lass of fitness in all cases. resistant strains has been described as sectorial growth. 1his is, however, an over-simplification as 3.4 Discussion the "runaways" do not necessarily have to emerge Tue ease of selecting laboratory resistant strains with­ in the classical form of a sector. Schüepp and Küng out treatments of UV irradiation or other rnutagenic (1978) showed that fast growing sectors either devel­ agents was described by various authors for dicar­ oped frorn slowly growing colonies or from totally boximide fungicides (Leroux et al., 1977, Schüepp inhibited colonies, respectively. In the binary classi­ and Küng, 1978, Hisada et al. 1979, Maraite et al. fication system used here, these two types corre­ 1980, Schüepp et al., 1982, Martinetti, 1986 and Wang sponded to growing type I and m, respectively. Al­ et al. 1986). They found fast growing sectors which though the results were not clear cut, it was obvious developed from sensitive strains on agar amended that "runaways" of type IA and IB mainly were with lethal or strongly inhibiting concentrations of highly resistant to vinclozolin and CGA 173506, re­ the fungicides. Hisada et al. (1979) and Martinetti spectively, while "runaways" of type IIlE and IC (1986) also found colon.ies emerging frorn conidia on rnostly did not show a reduced sensitivity to either agar amended with either procymidone or vinclozolin of the two active ingredients. Even though it was not in high concentrations. From their experience with possible to derive their resistance pattem from the methyl-benzimidazolecarbamates and dicarbox­ morphology of the "runaways" in all cases, it was, imides, respectively, Bolton (1976) and Leroux et al. however, surprising that there was a clear evidence

47 for the connection of high laboratory resistance to Under laboratory conditions resistant strains with a vinclozolin and CGA 173506, respectively, and the reduced fitness could survive whereas under field morphological appearance of type IA and IB. conditions they became suppressed. Vinclozolin field Dicarboximide field resistant B. fuckeliana resistant strains were all of low resistance. As these strains differ from laboratory resistant strains with strains could easily be detected in the field they were respect to their sensitivity to CGA 173506. While the competitive to a certain extent. In the laboratory their former are sensitive to CGA 173506 (Gehmann et al., high fitness was confirmed by their osmotolerance 1990) the laboratory resistant strains show a reduced and their comparably high virulence on apple. Vin­ sensitivity also towards CGA 173506. Similarly Marti­ clozolin and CGA 173506 double resistant strains netti (1986) found no cross resistance to the phen­ were never found in the field but they were readily ylpyrrole fungicide fenpiclonil in vinclozolin field re­ induced under laboratory conditions. sistant strains but she found reduced sensitivity to Katan (1985) and Wang et al. (1986) produced both active ingredients when working with labora­ laboratory resistant strains from vindozolin field re­ tory induced mutants. The close association of dicar­ sistant strains. The present studies showed that labo­ boximide and phenylpyrrole resistance in laboratory ratory resistant strains that occurred from vinclozolin resistant strains might be interpreted as cross resis­ field resistant strains were highly resistant to vin­ tance between the two groups of fungicides, how­ clozolin and to CGA 173506 but lost their fitness and ever, the high CGA 173506 sensitivity of dicarbox­ thus were not able to survive in nature. These results imide field resistant isolates is contradictory to this provided an explanation for the Jack of double resis­ hypothesis. Faretra and Pollastro (1991) showed in tant strains in nature but not for the absence ofhighly sexual crosses the segregation of vinclozolin field re­ vinclozolin resistant or CGA 173506 resistant strains. sistance ("" low resistance) and laboratory resistance lt was, however, assumed that the fitness of highly (= high resistance) in the ratio one to one, which vinclozolin resistant strains was seriously affected, indicates that low resistant and highly resistant phe­ which leads to a suppression of these strains under notypes are caused by mutations in the same gene. field conditions whereas strains that were resistant Therefore it can be concluded that dicarboximide to only CGA 173506 were lethal as they could not laboratory and dicarboximide field resistance are due even be found under the favorable conditions in the to different alleles of the same gene. laboratory. While Leroux (1991) assumed the same mode The comparison of mycelial growth rates of of action for vinclozolin and fenpiclonil Martinetti vinclozolin and CGA 173506 laboratory resistant (1986) proposed the existence of two different modes strains and their sensitive parental strains reflected of action for fenpiclonil and vindozolin and two dif­ the contradictory results given in the literature. Pom­ ferent modes of resistance for field and laboratory mer and Lorenz (1985) and Ruiz Borge (1988) only resistance to vinclozolin which was later disproved found slight differences in mycelial growth between by Faretra and Po11astro (1991). To elucidate the con­ sensitive and dicarboximide resistant strains whereas nection of modes of action and modes or resistance Davis and Dennis (1981) found growth rates of dicar­ in phenylpyrroles and dicarboximides more data on boximide laboratory and field resistant strains to be the genetic basis of resistance to these active com­ inferior to sensitive strains. Wang et al. (1986) found pounds are needed. minute differences in colony growth between dicar­ boximide field resistant, low laboratory resistant and

48 sensitive strains but they found considerable differ­ culture if all the nuclei are genetically identical. In ences in growth when comparing highly laboratory accordance with the results presented here for vin­ resistant strains to sensitive strains. clozolin and CGA 173506 laboratory induced resis­ The infective ability of B. fuckeliana is appar­ tant strains, Lorenz and Eichhorn (1982), who inves­ ently dependent on the host. Davis and Dennis (1981) tigated vinclozolin field resistant strahls, did not find found no difference in the infective ability of dicar­ differences in nuclear numbers among resistant and boximide resistant strains when tested on strawber­ sensitive strains. However, Hansen and Smith (1932), ries or tomato leaves. Corresponding results were Lauber (1973) and Shirane et al. (1988), in contrast to reported by Wang et al. (1986) for cotyledons of cu­ Menzinger (1965), found a positive correlation be­ cumbers and from Lorenz and Eichhorn (1978) for tween conidial dimension and number of nuclei, and leaves of Vicia faba. On the contrary, laboratory re­ Phillips et al. (1987) reported a positive correlation sistant strains were less virulent than sensitive strains of increased glucose concentration in the growth when tested on carrots (Davis and Dennis, 1981). Ler­ medium and increase in volume, nuclear number and oux et al. (1977) working with cucumbers and aggressiveness of the spores produced an the me­ Schüepp and Küng (1978) workingwith apples found dium. some laboratory resistant strains to be as virulent as Menzinger (1965) underlined the importance sensitive strains but, in accordance to Pommer and of the heterokaryotic state of B. fuckeliana and the Lorenz (1985), they too found laboratory resistant formation of anastomoses with regard to the strains that lost their pathogenicity. Tue presentstud­ enormous variability in this fungus. This high vari­ ies on the infective ability of 34 strains which were ability is of practical relevance as it allows the rapid either sensitive or low to highly laboratory resistant development of fungidde resistance leading to seri­ to CGA 173505 and vinclozolin confirmed a gener­ ous problems in chemical control (Lorenz, 1983). ally decreased virulence of laboratory resistant However, various factors influence the fast buildup strains. Vinclozolin laboratory resistant strains have of a resistant population. Resistant strains have to be been reported tobe poor competitors with sensitive as competitive and as virulent as sensitive strains types and have frequently shown a tendency to de­ and the resistance must be stable. Fot strains resis­ cline in or disappear from rnixed populations (Hi­ tant to methyl-benzimidazolecarbamate fungiddes sada et al., 1979, Gullino and Garibaldi, 1981, Pom­ both assumptions are true (Bolton, 1976, Schüepp, mer and Lorenz, 1985, Wang and Coley-Smith, 1986, 1981, Pommer and Lorenz, 1982). In the dicarbox­ Ruiz-Borge, 1988). In a competition test in absence imides the situation is different, in practice popula­ of selection pressure CGA 173506 laboratory resis­ tions of B. fuckeliana may show extreme fluctuati.ons tant strains behaved as described above for vin­ in sensitivity from year to year (Schwinn and Mor­ clozolin resistant strains. ton, 1990). Under laboratory conditions, however, laboratory resistance to vinclozolin and to CGA In mycelial ce11s of B. fuckeliana Menzinger (1965) 173506 was generaUy stable although in some cases found 3 to 120 nuclei whereas in conidia he found 3 resistance was lost after transfers in the absence of a to 15 nuclei. Hansen and Smith (1932) suggested that selection pressure. For vinclozolin these results were the basic unit of an individual is the nucleus and not confirmed by Davis and Dennis (1981), Lorenzini the cell; a multinucleate spore then represents a col­ (1983), Wang and Coley-Smith (1986) and Ruiz-Borge ony which will only give rise to a genetically pure (1988).

49 Heterokaryosis provides a mechanism to main­ tain nuclei carrying different genetic infonnation, e.g., for resistance or sensitivity, respectively, in the same ce11 or hyphal strand (Summers et al., 1984). In B. Juckeliana the (genetically different) nuclei can be ex­ changed between hyphal strands from different my­ celia through anastomoses and within hyphal strands of the same mycelium through the septa between the hyphal cells. From crosses of diploid organisms the interplay of parental alleles leading to a uniform Fl generation is weil known. Similarly it can be speculated about an interplay between different al­ leles from different haploid nuclei in multinucleate cells leading to a functional polyploidy. Selection pressure, e.g., exerted by a fungicide, may lead to the se1ection of cells (or hyphal strands) with a high proportion of nuclei that carry the suitable resis­ tance gene. Esuruoso and Wood (1971) hypothesized that a sufficient number of nuclei carrying the gene for resistance must be present in hyphal strands or in spores of resistant fungal strains. They, however, proposed that spores and cells with too many nuclei which carry the infonnation for resistance might lose their viability when the resistance gene has a nega­ tive pleiotropic effect on fitness. Prolonged and ex­ dusive use of a fungicide group under high disease pressure can eventually lead to field resistance re­ gardless of reduced fitness of resistant mutants (Staub and Sozzi, 1984). It can be hypothesized that in these heterokaryotic strains mutant and wild type alleles form an equilibrium in which the expression of re­ sistance is caused by gene products of the mutant allele whereas the fitness of the fungus is influenced by the gene products from the wild type allele.

50 4. Study on the inheritance of resistance to CGA 173506 and vinclozolin and related fitness parameters in Botryotinia fuckeliana (de Bary) Whetzel

4.1 Introduction

Faretra and Antonacci (1987) and Faretra and Pollas­ genetic flexibility, which is a characteristic feature of tro (1988) developed a reliable method to produce heterokaryotic, asexually reproducing, field isolates the sexual stage of B. fuckeliana. Faretra and Pollastro of B. fuckeliana (Pommer and Lorenz, 1982). The in­ (1991) were able to elucidate the genetics of resis­ stability and variability within progeny of individ­ tance to methylbenzimidazoles and dicarboximides, ual heterokaryotic isolates might be caused by the with special consideration of the effects of resistance selection and reassortment of genetically different genes on phenotypic characters relevant to crop pro­ nudei duringhyphal growth (Grindle, 1979). tection. They analyzed random samples of 300 asco­ In the present investigations the osmotic sen­ spores from each apothedum and thus could distin­ sitivity and the pathogenicity of sexual progenies guish sensitive and resistant ascospores according to from sensitive strains, from strains carrying a labora­ Leroux and Gredt (1981). Random spare analysis, tory-induced resistance to CGA 173506 and to vin­ however, did not allow distinguishment between clozolin, respectively, and from vinclozolin field re-­ progeny of different levels of resistance. In contrast sistant strains were compared. to Faretra and Pollastro (1991) in the present investi­ gations classical tetrad analysis was performed; 8 as­ 4.2 Materials and Methods cospores of single asci were cultivated and their col­ Isolates were collected from strawberries and grapes ony growth on agar was assessed by using a wide at three locations in the Northeastern part of Swit­ range of fungicide concentrations. The inheritance of zerland (chapter 1, 2). Isolates resistant to vinclozolin resistance to CGA 173506 and to vinclozolin was stud­ in the field were collected from a vineyard that had ied and the genetic basis of resistance against both been treated with dicarboximides for about 15 years active ingredients was compared. (chapter 1). Laboratory induced resistant strains, which were resistant to vinclozolin and to CGA Grindle (1983) showed the vinclozolin resistance gene 173506, were derived from sensitive parental strains Yin-1 in Neurosporn crassa Shear & Dodge to be alle-­ from strawberry fields which had never received any lic with the gene for osmotolerance Qi-1. Beever (1983) fungicidal treatments. They were obtained by sub­ showed that all of 150 isolates, with one exception, culturing from sectors spontaneously arising from that were resistant to dicarboximide in the field to be the sensitive isolate incubated on malt agar amended abnonnally sensitive to high osmotic pressure. He with 1mg vinclozolin/L, 3mg vinclozolin/L, 0.3mg raised the hypothesis that strains showing medium CGA 173506/L or 1mg CGA 173506/L, respectiveiy to high resistance to dicarboximides are always ab­ (chapter 3). nonnally sensitive to osmotic pressure and Ums will, because of their reduced fitness, occur infrequently Mycelium, sclerotia, microconidia and macroconidia in the field. The high number of nuclei per cell from sensitive strains, from dicarboximide resistant (Grindle, 1979, Lorenz and Eichhorn, 1982), the field strains and from CGA 173506 and dicarbox- heterokaryosis (Davis, 1966) and the frequent forma- imide laboratory resistant strains were produced on tion of anastomoses (Lorenz, 1983) Jead to the high malt agar (Oxoid No. 3). Sclerotia which deveioped 51 for 4 weeks at 15°C and for another 4 weeks at 0°c germination, they were transferred to new malt agar in the dark were picked with forceps from the agar plates. These plates served as mother plates from and placed in distilled sterile water in test tubes. The which the single ascospore isolates were mass trans­ sderotia were then spermatized by adding a suspen­ ferred to pea agar. After 5 to 10 days sporulating sion of microconidia, macroconidia and mycelial frag­ cultures were stored at4°C in the dark. ments which were scraped from those plates from which the sclerotia were picked before {detailed pro­ Spore suspensions of lOSconidia/ml were prepared cedure see Faretra and Pollastro (1988)). Apothecia from sporulating cultures and 50µ1 were added to were formed approximately 2 months after the sper­ agar discs of 10mm diameter. The agar discs were matizing process. In the sexual crosses the microco­ incubated at 20'C in the dark for 17h. They were nidia which are uninucleate and apparently have only then placed upside-down on fungicide amended agar a sexual function (Grindle, 1979, Whetzel, 1929,1945) and incubated at 20'C in the dark for 3 days. Vin- were used as male parental partners whereas sde­ clozolin was used in six concentrations for sensitive rotia were used as the fernale partners. The role of and field resistant strains, ranging from 0.03mg/L to rnacroconidia and mycelial fragments in the suspen­ 10mg/L and in eight concentrations for laboratmy­ sion that is used for spermatizing the sclerotia has resistant strains, ranging from 0.03mg/L to lOOmg/ not been investigated yet. Uninucleate microconidia L. CGA 173506 was used in six concentrations for which are produced from a homokaryotic strain of sensitive and field resistant strains, ranging from B. fuckeliana (which is very rare in nature) are all 0.001mg/L to 0.3mg/L and in nine concentrations genetically identical whereas microconidia from a for laboratory-resistant strains, ranging from heterokaryotic strairl (which is the typical case in 0.(Xllmg/L to 10mg/L. The mean diameters of colo- asexually produced strains) are genetically different. nies of 3 replkates were assessed and the percentage Because of the frequent formation of anastomosis, of inhibition of growth was calculated. between two different hyphae, the definition of an individual is difficult in this fungus. Therefore the To assess osmotolerance, rneasured as glucose toler- use of microconidia from one (heterokaryotic) strain ance, malt agar discs, 10mm diameter, were inocu­ or from a couple of strains is in this case of minor lated with 50µ1 of a spore Suspension of B. fuckeliana importance. containing 105 conidia/ml and incubated for 17h at 20'C in the dark. The malt agar discs were placed Apothecia were crushed in 200µ1 of sterile distilled upside-down on the glucose (sorbitol) agar medium water ona cavity slide. Approximately 50µ1 of a single (malt agar amended with 1, 3, 5, 7, 10, 15 and 30% crushed apothecium in water were placed in the cen- glucose (or sorbitol)). The colony diameter of three ter of a malt agar plate. The plates with the frag- replicates was measured after 3 days and inhlbition ments of the crushed apothecium, the liberated asco- of growth was cakulated. spores and the entire asci were exposed in a sterile cabinet to evaporate the excessive water. Asci either The pathogenicity assay described by Schüepp and hurst and released their ascospores or ascospores Küng (1978) was slightly modified to assess the viru­ were liberated by breaking the ascus apart using a lence of the single ascospore cultures (for details micromanipulator equipped with two fine glass about the apple pathogenicity test see chapter 3.2, needles. Tue 8 ascospores were separate

52 Table 4.1: Characterization of strains used for the production of apothecia

Sclerotia of single strains were spermatized with mixtur€s of microconidia, macroconidia and mycelial fragments from various strains.

Sclerotia Origin Resistance Pattern Spennatizing Partner of Sclerotia Partner" Partnerst>

Oe4 strawberry, Wädenswilc vs CS group I G6.7 grape, Walenstadic vs CS group n G6.9 grape, Walenstadt" vs CS group n WBIIS grape leaf, Walenstadic vs CS group III B 26.21 grape leaf, Walenstadt" VLR CS group IV G6.ll grape, Walenstadt" VLR CS group V G6.15 grape, Walenstadf VLR es group V A1183V6 selected for resistance on 3mg vinclozolin/L d VLR CLR group VI A118 3V 1 selected for resistance on 3mg vinclozolin/L d VHR CHR group vn AIII43V 2 selected for resistance on 3mg vinclozolin/L d VHR CHR group VII A1181Z34 selected for resistance on 1mg CGA 173506/L d VHR CHR group VII

• Resistance pattern of sclerotia partner VS vinclozolin sensitive (ECS0 < lmg/L) CS CGA 173506 sensitive (ECS0 < 0.03mg/L) VLR vindozolin, low resistance (lmg/L < EC50 CLR CGA 173506, low resistance (0.03mg/L < EC50 <30mg/L) <0.3mg/L) VHR vindozolin, high resistance (ECS0 > 30mg/L) CHR CGA 173506, high resistance (ECSO > 0.3mg/L)

b spermatizing partners group I 3 strains from strawberries, Wädenswil group II 4 strains from grape leaves, Walenstadt group III 12 strains from grapes and strawberries; various origins group IV 3 strains from grape leaves, Walenstadt group V 4 strains from grapes, Walenstadt group VI 40 strains from strawberries, Wädenswil and Dietikon group VII 7 strains from strawberries, Wädenswi1 and Dietikon

The strains of group I, VI and VII were all sensitive whereas those of group II, III, IV and V contained sensitive as well as strains with low resistance level to vinclozolin. None of these strains showed decreased sensitivity to CGA 173506. c isolates from naturally infected fruits or leaves d strains selected in the laboratory from isolates of strawberries from Wädenswil

53 4.3 Results

Table 4.1 shows the combinations of sclerotia part­ as to CGA 173506 occurred by spermatizing strain ners and spermatizing partners which yielded in AIIS 123 4 being highly laboratory resistant to vlll­ apothecia with ripe ascospores. The progeny of these clozolin and CGA 173506 with severa] sensitive ascospores was used to study the genetic variability strains (Fig. 4. lB). Pattern three reveals a genotype with regard to fungicide resistance as well as viru­ that is low resistant to both active ingredients. The lence on Golden Delicious and osmotolerance. Sensi­ ascospores tested originate from strain Ail8 3V 6 tive strains, vinclozolin field resistant strains and which was low resistant to vinclozolin and to CGA strains of different levels of laboratory resistance to 173506 and was spermatized with the same mixture vinclozolin and CGA 173506 were selected as sde­ of sensitive strains that was used to spennatize scle· rotia partners. For the spermatizing step, strains were rotia of strahl AII8 123 4 (Fig. 4.lC). grouped. In contrast to the results in Fig. 4.1 that were obtained from experiments with laboratory resistant Fig. 4.1 shows the reactions of progeny of eight spores strains, Fig. 4.2 shows representative data from 8 from a single ascus to vindozolin and CGA 173506. single ascospores of a single ascus of experiments For vinclozolin, the classification of Faretra and Pol­ with field resistant strains. Resistance pattem 4 (Fig. lastro (1991) was slightly modified: ECSO of sensitive 4.2) shows single ascospore cultures that were ob­ strains 30mg/L whereas ECSO values of strains with low vinclozolin field resistant strain. G6.15 was sperma­ resistance levels ranged from lmg/L to 30mg/L. tized with microconidia from three sensitive strains With regard to CGA 173506 strains were classified and from one dicarboximide field resistant strain. as sensitive strains with an EC50 < 0.03mg/L, as low Conceming vinclozolin the single ascospore cultures resistant with an EC50 ranging from 0.03mg/L to which originated from combinations of field resis­ 0.3mg/L and as highly resistant strains with an ECSO tant strains and sensitive strains reacted in the same > 0.3mg/L. way as those produced from laboratory resistant Tue 120 single ascospore cultures tested, can strains and sensitive strains. However, no resistance all be assigned to four resistance patterns to vin­ to CGA 173506 was found in progenies of strains clozolin and CGA 173506. Fig. 4.1 and Fig. 4.2 show which were field resistant to vinclozolin. data of representative asci for each of the four pat­ terns. Conceming both ingredients, strains attributed Results from tests on media supplemented with sor­ to the first pattern were sensitive, strains ascribed to bitol, which has the same osrnotic effect as glucose the second pattem were split into sensitive and highly but cannot be metabolized by B. fuckeliana did not resistant in the ratio one to one, whereas pattem three differ from results obtained from tests on media sup­ shows sensitive and low resistant strains in the ratio plemented with glucose (data not shown). one to one. Single ascospore cultures which are sen· Conceming osmotolerance and pathogenicity sitive to both ingredients resulted from the combina­ on apple, identical results were obtained from 48 tion of sderotia of the sensitive strain Oe4 with a single ascospore cultures of pattem 1 from 6 asci of mixture of three sensitive spermatizing strains (Fig. different apothecia. Fig. 4.3A shows the data of a 4.lA). An equal splitting of single ascospore cultures representative set of 8 ascospore isolates of a single in sensitive and highly resistant to vinclozolin as well ascus. These 8 strains showed a significant increase

54 Fig.4.1: Pattern of genotype reaction to vinclozolin and CGA 173506 of progeny of eight ascospores from single asci

CGA 173506 vinclozolin ,fj ,:: 100 100 ~ ] 80 80 i 60 60 0 40 40 § ..,, 20 20

j 0 0 * -20 -t--r---r-r-.-,,-.----...---, -20, -t---r---r-r-.-,-.----,---, .001 .01 .1 1 10 .01 .1 1 10 100 ,fj ,:: 100 100 ~ „-~ 80 80 l 60 60 O 40 40 § "°' 20 20 j 0 0 ""0 -20 -,-,---,--,--,--,-,-.-, -20 +-.---.---..--,--,----,----, .001 .01 .1 1 10 .01 .1 1 10 100

1100 100 ~ "ia 80 80 l 60 60 Q 40 40 § "°' 20 20 j 0 ""0 -20 -i--,--,-.--,---,----,-,--, .001 .01 .1 1 10 .1 1 10 100

concentration in mg/L concentration in mg/L Legend: -.. -, ...... resistant progeny ---sensitive progeny

55 Fig.4.2: Pattern of genotype reaction to vindozolin and CGA 173506 of progeny of eight ascospores from single asci of dicarboximide field resistant sderotia of strain G. 6.15

CGA173506 vinclozolin .fi 100 100 0 ~ ~ "00 0 80 80 "' -"' "'8 "'[ 60 60 "'u s -00 ~ 0 40 40 s.,,o,... § 20 20 J:I -~ 2()'° ..0"' ~E, i 0 0 0~ J:,"' C.oo 0~ -20 -20 .001 .01 .1 1 10 .01 .1 1 10 100 concentration in mg/L concentration in mg/L

in colony diameter compared to the control on malt motic pressure. However, compared to the four sen­ agar amended with up to 10% glucose (Mests, sitive isolates the four vinclozolin low resistant iso­ p<0.05). Assays on Golden Delicious showed patho­ lates showed a small, but significant increase (t-tests, genicity of all strains. Indeed, in a one factor analysis p<0.05) in osmotic sensitivity on agar amended with of variance, mean lesion diameters on apple differed 3 to 10% glucose. Of the cultures that were obtained significantly between different single ascospore cul­ from two moreasci of pattem 4 from different apothe­ tures of the same ascus (F-test, p=0.0001). However, cia the single ascospore cultures showed the same these differences are minute compared to the differ­ virulence on apple as the 8 cultures which were de­ ences in lesion size between resistant and sensitive rived from the ascus shown in Fig. 4.3B. The differ­ strains (Fig 4.3C,D). ences in osmotolerance were however minute and Tue results shown in Fig. 4.3B were obtained thus obscured by other phenotypic variation of these from an ascus of pattern 4. The 8 ascospore cultures isolates (data not shown). were all pathogenk on Golden Delicious. The mean Seven asci of pattem 3 were obtained from 6 virulence of the four vinclozolin field resistant proge­ different apotheda. The characteristics of the 8 single nies and the four sensitive progenies, respectively, ascospore cultures from a single ascus shown in Fig. was not significantly different (t-test, p=0.13). The 4.3C are representative for all the 7 asci that were four field resistant isolates were tolerant to high os- tested, conceming increased glucose concentrations

56 Fig.4.3: Pathogenicity assay on apple cv. Golden Delicious and sensitivity to high osmotic pressure (measured as glucose tolerance of 8 spores of single asci

• } sensitive single ascospore cultures III resistant single ascospore cultures (s) (r) J

pathogenicity assay on sensitivity to osmotic pressure of 8 Golden Delicious ascospore cultures from a single ascus

35 100 E ~ s 30 w 80 0 .5 s', 60 l 25 Ei C l'l" 40 o m- 20 0 -·- w - 20 "'J; s 15 C l s Cw •w ., .9 0 10 e-t "C -20 0.. ·;; C -~ 5 ~t -40 < w ·- 8 - -" 0 # "" -60 0 1 3 5 7 10 15 30 m C C "mj'l - -w... E ~ E 30 w 80 -"-w "w .5 s', 60 "' ;,::: 25 Ei • w l'l" 40 0.- w 20 -0 -" C 20 "' E E 15 Jr, s Cw ·-X ., :8 0 C: C 10 ....ßO-€

"Ct'.) < m U C ~--;:; .... .-~ ' - 20 l s C -0 ..0 s 15 C wbO...,.:; "'m ., .9 0 o.c 10 " ooß "C ~ ,5 -20 .c 5 C ~ "'·- R -~ ·- 0 -40 Cl m - -" 0 """ -60 * 0 1 3 5 7 10 15 30 8 ascospore cultures from a single ascus concentration of glucose in % 57 and the virulence on apples. As shown in Fig. 4.3C clarified so far only for the methylbenzimidazoles response to high osmotic pressure and pathogenicity (Davidse, 1988, Sisler, 1988). Studies on the genetics of the 8 single ascospore cultures derived from a of resistance by Faretra and Pollastro (1991) showed single ascus split in the ratio one to one. All the strains that field isolates and laboratory mutants of B. fucke­ that showed decreased sensitivity to either vinclozolin liana can be classified into three phenotypes regard­ or CGA 173506 showed increased sensitivity to high ing their response to benomyl or to vinclozolin, re­ osmotic pressure and significantly decreased viru­ spectively: sensitivity, low resistance and high resis­ lence or lack of pathogenicity an apple (t-test, tance. The polymorphic major gene conferring resis­ pc::cO.()(H)l). On agar amended with 1 to 30% glucose tance or sensitivity to the dicarboximide vinclozolin the four strains which showed a decreased sensitiv­ has been designed as Qafl, that to methylbenz­ ity to both active ingredients differed significantly imidazoles as Mw;l. Alleles coding for sensitivity, from the sensitive strains in the inhibition of my­ low resistance and high resistance have been assigned celial growth (Mest, p<0.05). The four strains that lla.flS, QaflLR and .QaflHR for vinclozolin and were sensitive to vinclowlin and to CGA 173506 were M.lu;lS, M.h,cl LR and M..l;ti:lHR for methylbenz­ pathogenic on Golden Delicious and they also toler­ imidazoles (Faretra and Pollastro, 1991). In the pres­ ated high osmotic pressure. ent investigation, genetic studies using classical tet­ Fig. 4.3D shows data of 8 single ascospore cul­ rad analysis showed the same patterns of sensitivity tures which derived from a single ascus of pattem 2, or resistance, respectively, to phenylpyrrole as dem­ that represented the progenies of 4 asci from 4 dif­ onstrated by Faretra and Pollastro (1991) for dicar­ ferent apothecia. Pathogenidty on Golden Delicious boximides and methylbenzimidazoles by random and tolerance to high osmotic pressure split in the spare analysis. From this it was concluded that resis­ ratio 1:1. Resistant strains showed a significantly de­ tance to CGA 173506 was also located in a single creased virulence (Mest, P"" 0.001) or even lack of gene. Tue major resistance gene responding to CGA pathogenicity. On media amended with increasing 173506 is suggested to be designated as gene ~1 concentrations of glucose the sensitive strains be­ following Yoder et al. (1986). lt was further hypothe­ haved as described and explained in Fig. 4.3A, sized that three alleles (ea:1s, ,ea:lLR and ~IHR) whereas the resistant strains behaved as described are coding for sensitivity, low resistance and high and explained in Fig. 4.3C. resistance, respectively. This hypothesis, however, remains tobe verified in future tests. 4.4 Discussion Hornokaryotic dicarboximide resistant sexual For the control of B. fuckeliana in grapes methyl- progenies from crossings of heterokaryotic dicarbox- benzimidazoles were withdrawn from the market in imide resistant field strains with heterokaryotic sen- most vine growing areas because of the high genetic sitive strains are weakly resistant to vinclozolin and stability of the resistance and the high füness of re- they are sensitive to CGA 173506 (Fig. 4.2). Homokar- sistant strains in the field (Schüepp and Küng, 1981, yotic CGA 173506/ vinclozolin laboratory resistant Saracchi and Lorenzini, 1985). In contrast, in the case sexual progenies from crossings of heterokaryotic of the dicarboxirnides the generally lower fitness of CGA 173506/ vinclozolin 1aboratory resistant strains resistant field strains still allows use in certain areas with heterokaryotic sensitive strains, show decreased (Beever et al., 1991, Pak et al., 1990). The mode of sensitivity to vinclozolin and to CGA 173506 (Fig. action of the aforementioned botryticides has been 4.1). In contrast to the situation with methyl-benz-

58 imidazolecarbamates and dicarboximides, where by an allele of Da.fl that only causes reduced sen­ Faretra and Pollastro (1991) demonstrated the inde­ sitivity to vinclozolin but neither has an effect an pendent segregation of the two major genes, the re­ the sensitivity to CGA 173506 nor has a pleiotropic sults reported here show cosegregati.on of P,)a:l and effect on osmotolerance. ]2afl (Fig. 4.1). This may lead to the following hy­ With respect to the use of the novel phenylpyrrole in potheses: the field a single locus for resistance to vindozolin 1. The genetic basis of field resJStance in B. fuckeli­ and to CGA 173506 is obviously more critical than ana is different for vinclozolin and for CGA two different Iod. As the development of field resis­ 173506. However, it is identical in Jaboratory re­ tance cannot be excluded, shifts in sensitivity of B. sistance for both active ingredients. This hypothe­ Juckeliana to the new fungicide in the field have to sis provides an explanation for the lack of cross be carefully monitored. Past experiences with the resistance between CGA 173506 and vinclozolin dicarboximides and this particular pathogen, as wen in the field and for the presence of cross resis­ as these results, clear1y indicate the need for a suit­ tance between the phenylpyrrole and dicarbox­ able anti-resistance strategy based an which the imides in laboratory resistant strains. It is, how­ phenylpyrrole fungicide should be introduced into ever, contradicted by the results reported by the market. Further studies on the resistance risk of Faretra and Pollastro (1991) who demonstrated CGA 173506 must focus on testing the above men­ dicarboximide field resistance (12aflLR) and tioned hypotheses. Sexual progenies from crosses of dicarboximide laboratory resistance (QaflHR) to sensitive strains with CGA 173506/ vindozolin labo­ be caused by mutations in the same gene. They ratory resistant strains that were selected from dicar­ suggested that DaflLR and D.aflHR are allelic. boximide resistant field strains or crosses between 2. l2afl is different from ~1 and both genes are vinclozolin resistant field strains and CGA 173506/ dosely linked. This hypothesis explains the lack vinclozolin laboratory resistant strains should be of cross resistance in the field but it is contra- analyzed. Random spare analysis is, due to the pos­ dicted by the fact that independent segregation sibility of testing a much bigger sample of ascospores, of ~1 and l2afl was never found. 111e closer to prefer over classic tetrad analysis in this case where the linkage of the hvo genes the higher is the a close linkage is expected and recornbinants are rare. number of parental ditypes. The number of ana- lyzed tetrads might then, however, have been Mutants of N. crassa Shear & Dodge and of Snccharo­ too small to find the rarely occurring recombi- myces cerevisiae Meyen ex Hansen which were hyper­ nant ditypes (= all the 8 sexual progenies within sensitive to high osmotic pressure were character­ a single ascus are recombinants) and the tetra- ized by Mays (1969) and Singhand Shermann (1978). types (= 4 of the 8 the sexual progenies within a Mays (1969) and Se1itrennikoff et al. (1981) specu­ single ascus are recombinants and 4 of them are lated that the products of the corresponding genes identical with their parental strains). are involved in cell wall synthesis, i.e., in the supply 3. Qafl is identical with ~1 and the genetic basis of precursors for the cell wall synthesizing enzymes of field and laboratory resistance is the same. In (Mays, 1969). This hypothesis was supported by the this case Jack of independent segregation is ob- results of Livingston (1968, 1969) who found in os­ vious. Tue Jack of cross resistance in dicarbox- motically sensitive mutants of N. crassa some differ­ imide resistant field strains can then be explained ences in the overall cell wall composition as well as

59 in the cell wall gross appearance. A structural change a11y found an increase in osmotic sensitivity of field in the membrane which affects cell wall synthesis resistant isolates when they compared them with may have occurred. Slight conformational disturbance sensitive strains. They found, however, the differ­ of the cell wall synthesizing enzymes, or a change in ences to be so small that they were easily obscured the transport capabilities might cause such distur- by other variations in the phenotypes. The virulence bance in cell wall metabolism and therefore lead to of low level dicarboximide field resistant strains was abnormal osmotic se.nsitivity. not reduced compared to the sensitive strains (Fig. 4.3B). Cosegregation of resistance to vinclozolin and CGA 173506, sensitivity to high osmotic pressure and pa- The finding of a connection between osmotic sensi­ thogenicity suggest that sensitivity to high osmotic tivity and resistance against dicarboximides which pressure and pathogenicity are pleiotropic effects of was reported by various authors working with N. the resistance gene(s). Beever (1983), working with crassa and B. fuckeliana (Grindle and Temple, 1982, 20 laboratory-induced dicarboximide resistant, asex- Beever, 1983, Beever and Brien, 1983, Grindle, 1983, ual strains of B. fuckelirma found 3 strains which Endlich and Lyr, 1987, Ellis et al., 1991) was also were weakly resistant and did not show an increase found in this investigation on B. fuckeliana resistant in osmotic sensitivity. In contrast to Beever {1983) in to CGA 173506. This leads to the question whether the present investigations, all strains derived from changes in ce11 wall synthesis and membrane per­ single ascospore isolates of single asci, without ex- meability, which probably are involved in the in­ ceptions, carried laboratory-induced resistance to crease of sensitivity to high osmotic pressure, are dicarboximides and to CGA 173506 and showed an the cause of decreased sensitivity to dicarboximides increase in sensitivity to high osmotic pressure and a and phenylpyrroles, respecti.vely. Tue understand­ decrease of virulence, independent of the level of ing of the biochemical basis of osmotic sensitivity in resistance. The aforementioned genetic flexibility of B. fuckeliana rnight help to elucidate the biochemical B. fackeliana might provide an explanation for the rnode of action of dicarboximides and phenylpyrroles. contradictory results. Mutations of only one of the nuclear components of a heterokaryon would be masked by the prevailing a!Iele in the other compo- nents. The phenotype depends on the kind and pro~ portion of the nuclei present in the heterokaryon (Esuruoso and Wood, 1971, Grindle, 1979). Beever and Brien (1983) also tested 150 low level dicarboximide field resistant isolates of the pa­ thogen. With only one excepti.on, all were abnormally sensitive to high osmotic pressure. Sexual progenies from three crossings of low level dicarboxirnide field resistant strains with sensitive strains showed small, but significant differences in sensitivity to high os­ motic pressure in one case. These results are in ac­ cordance with Faretra and Pollastro (1991) who usu-

60 General Discussion

B. fuckeliana is a genetically highly variable organ­ Siegfried, 1983). ism with a worldwide distribution. Belonging to the In the study presented here B. fuckeliana was most ubiquitous facultative plant pathogens it causes chosen as a suitable model organism to investigate damage of great economic importance in grapes, genetic variability and development of fungicide re­ small fruits, vegetables, bulbous monocotyledons, sistance for various reasons: its genuine genetic vari­ forest tree seedlings and glasshouse crops Oarvis, ability is vast, it is a fast growing, facultative patho­ 1977, Domschet al., 1980, Coley-Smith, 1980, Mathys, gen that can be easily cultivated an artifidal media, 1982). Tue fact that B. fuckeliana is capable of attack­ wild type isolates are abundant on a wide variety of ing many diverse crops at almost any stage in their hast plants, sexual progenies are accessible for ge­ growth andin Storage, and of affecting all plant parts, netic studies, occurrence of resistance and damage including the roots, cotyledons, leaves, stems, flow­ caused by this organism is of economical importance. ers and fruits makes it an extremely difficult fungus First reports on similarities in the mode of resistance to control (Maude, 1980). between the new chemical group of phenylpyrroles The response of fungal populations to fungi­ (Gehmann et al. 1990) and the chemically unrelated cides used for their control, and hence the risk of group of dicarboximides (Martinetti, 1986, Leroux:, disease control failure due to resistance depends on 1991) were the starting point for the present investi­ the biochemical mode of action of the active ingredi­ gations. ent, on the extent of genetic variability available in the pathogen (Georgopoulos and Skylakakis, 1986) Repeated use of fungicides with the same mode of and the use pattem of the product. In B. juckeliana action leads to a shift of sensitivity of the target fun­ the genetic flexibility is enormous due to its gus population (Delp, 1988). Alongwith other meas­ heterokaryotic state (Hansen and Smith, 1932, ures restricted spray program, i. e., achieved by the Hansen, 1942, Menzinger, 1965, Lauber, 1971, Grindle, use of less susceptible grape varieties that require 1979). By the formation of anastomoses, genetically less treatments has been advised to prevent or to de­ different nuclei obtained by spontaneous rnutation, lay the buildup of resistance in the field (Staub and can be freely exchanged in the fungal population. Sozzi, 1984, Staub, 1991, Schwinn and Morton, 1990). Due to this peculiarity in its biology B. Juckeliana is Tue vineyard in Walenstadt provided an excellent able, to a high degree, to adapt to changes in envi­ possibility to study the population dynamics of B. ronmental conditions rapidly. For example labora­ Juckelinna under such conditions (chapter 1). In a tory resistant strains develop within a few days in three year field trial the pathogen population was vitro, when high selection pressure is exerted by the challenged by only one dicarboximide application in antifungal compounds fenpidonil or vinclozolin (Ler­ the first year, in the second year no dicarboximides oux: et al., 1977, Schüepp and Küng, 1978, Martinetti, were applied and in the third year one third of the 1986). In the field, high selection pressure exerted by population was challenged by two dicarboximide the intensive and exclusive use of methyl-benz­ applications, whereas the remaining two thirds were imidazolecarbamates and dicarboximides resulted in left untreated. However, one or two applications of the development of field resistance and in lass of ef­ dicarboximides in the first and in the third season, ficacy within a few years (Holz, 1979, Schüepp and respectively, resulted in the increase of the resistant Küng, 1981, Schüepp et al., 1982, Schüepp and population to a level of 100%. The proportion of re-

61 sistant strains decreased from harvest in the first year respectively, the biological phenomenon of the spon· to spring in the second year and remained unchanged taneous emergence of "runaways", that, so far was during the second year and also during the third claimed to be unique in dicarboximide fungicides year when no dicarboximides were applied. Surpris­ (Leroux et al., 1977, Schüepp and Küng, 1978), was ingly, the population in the vineyard in Walenstadt identically found in the phenylpyrrole. showed the same behavior as was reported by Le­ Most "runaways" that spontaneously occurred roux and Oerjeau (1985), Martinetti (1986), Pak et al. on artificial media amended with strongly inhibiting (1990) and Beever et al. (1991) for populations on or lethal concentrations of CGA 173506 or vinclozolin, susceptible grape varieties that were heavily treated. respective]y, generally proved to be resistant, how­ These results clearly indicate that, in this case, the ever, in some cases they were neither altered in sen­ reduction of the selection pressure achieved by re­ sitivity to dicarboximides nor to CGA 173506. The stricted dicarboximide applications due to the use of assessment of dose-response relationships concem­ a less susceptible grape variety could not prevent ing CGA 173506 and vinclozolin in 34 "runaways" the buildup of a resistant population in the field. revealed resistance levels from high1y sensitive to Martinetti (1986) and Leroux (1991} studied the highly resistant (chapter 3). In contrast to the results ability of B. fuckeliana to develop cross-resistance obtained from tetrad analysis of (genetically ho· against dicarboximide fungicides and the phen­ mogenous) sexual progenies (chapter 4) that clearly ylpyrrole fenpiclonil. Their speculations on a com­ indicated the existence of 3 distinct resistance pat· mon mode of action of dicarboximides and phen­ tems for both active ingredients the classification of ylpyrroles were, however, contradictory (see chap­ resistance levels of "runaways" was difficult. Grindle ter 3). The phenylpyrrole CGA 173506 provided a (1979) discussed the hypothesis that the phenotype most suitable tool to study the potential of the fun­ in a heterokaryon depends on the kinds and propor­ gus to develop resistance and to study the occur­ tions of nuclei present. He indicating that mutations rence of cross resistance to the chemically not related in a minority of the nuclear component of a hetero­ dicarboximides. Studies on the variability of sensi­ karyon could be masked by the majority of normal tivity to CGA 173506 and to vinclozolin, respectively, alleles in the other components. As in B. fuckeliana showed an only small variation in the base-line sen­ anastomoses allow the exchange of nuclei, conclud­ sitivity in both active ingredients. CGA 173506 proved ing from the results from Grindle (1979) it can be to be more active than vinclozolin; by both com­ speculated that due to the random combination of pounds spare germination was less inhibited than sensitive and resistant nuclei theoretically, any resis­ mycelial growth. Dose- response curves were similar tance level could be reached in asexual progenies. in shape. They showed a steep slope in the linear Although Parmeter et aL (1963) in their review on part of the J -shaped curves. Significance and practi­ heterokaryosis and variability in plant pathogenic cal importance of the assessment of the base-line sen­ fungi emphasized the need of further evaluation of sitivity is explained in detail in chapter 2. While the the concept of heterokaryosis, unfortunately the in~ variations in sensitivity of wild type strains was small, terplay and the state of activity of resistant and sen­ pretending genetic homogeneity, under the selection sitive nuclei in heterokaryons was not studied so far pressure of CGA 173506 or vinclozolin, respectively, and therefore discussions of possible effects on the the genetic flexibility of B. fuckeliana became obvi­ resistance level are still mainly hypothetical. ous. In cornparative studies on the occurrence of labo­ While Leroux et al. (1977) and Schüepp and ratory resistance against CGA 173506 or vinclozolin, Küng (1978) found a rapid development of dicarbox- 62 imide laboratory resistant strains in vitro, the selec­ resistance to benzimidazole and dicarboximide fun­ tion of resistant strains was much slower under field gicides in B. Juckeliana. The present shldies confirm conditions. First reports about dicarboximide field the results of Faretra and Pollastro (1991) for vin­ resistant strains in vineyards were encountered after dozolin by using the technique of dassical tetrad three years of practical use of these fungiddes (Holz, analysis. In addition (chapter 4) laboratory resis­ 1979, Schüepp et al., 1982). The occurrence of dicar­ tance to CGA 173506 was found to l:>e linked with re­ boximide resistant strains in the field was, however, sistance to vinclozolin. not correlated with lass of efficacy at that time (Schüepp et al. 1982, Pommer and Lorenz, 1982, Table 5.1 shows in a combination square all inde­ Löcher et al., 1985). When the resistance frequencies pendent combinations of the six alleles (UaflS, reached nearly 100%, in Swiss vineyards the lass of I2a.{1LR, I2a.{1HR, ric,:1s, ric,:1LR, l';a:lHR) which are efficacy of the dkarboximides was also found coding for sensitivity, low and high resistance to vin­ (Schüepp and Siegfried, 1983). Whereas in the labo­ clozolin or to CGA 173506, respectively. From the ratory, growing conditions are optimized and selec­ nine expected phenotypes only four were repeat­ tion pressure is only exerted by the fungicide in the edly found in the present investigation (shaded medium, under field conditions the selection pres­ squares). Three hypotheses an the genetic basis of sure is much more complex and a disadvantage in the connection between resistance to dicarboximides fitness by the acquisition of resistance genes often and to phenylpyrroles were discussed in chapter 4. leads to a retardation of the occurrence of resistant Genette studies were extended to two fitness strains {Brent et al., 1990). Comparative studies on parameters, osmotolerance and pathogenicity, that the fitness of strains laboratory resistant to vinclozolin were described to be altered in dicarboximide and CGA 173506, showed a general decrease. While (Beever, 1983, Schüepp and Küng, 1978) and CGA in chapter 3 a close correlation of the acquisition of 173506 (chapter 3) resistant strains (details in chap­ laboratory resistance to both active ingredients and ter 4).Vinclozolin/ CGA 173506 double resistant the lass of virulence, osmotolerance and competitive strains showed decreased osmotolerance and reduced ability was demonstrated, dicarboximide field resis­ pathogenicity an apple when compared to the sensi­ tant strains were sensitive to CGA 173506 and showed tive strains from the same ascus. In contrast to the no lass of fitness. The present studies not only re­ laboratory resistant strains, dicarboximide field re­ vealed a clear difference in the fitness between field sistant strains did not show any or only a minute resistant strains and laboratory resistant strains but decrease in osmotolerance and pathogenicity on also in the level of resistance. apple. Table 5.1 shows that all strains which are re- sistant to CGA 173506, independent of the level of Until recently genetics have only been marginally resistance, showed an increased osmotic sensitivity studied in B. fuckeliana as the production of apothe- too. In contrast dicarboximide field resistant strains cia was difficult and few authors reported produc- (which are sensitive to CGA 173506) and one strain tion of apothecia (Groves and Drayton, 1939, Groves which was reported from Faretra (pers. comm.} tobe and Loveland, 1953, Lorenz and Eichhorn, 1983, Fare- highly resistant to vindozolin and sensitive to CGA tra and Antonacci, 1987, Faretra et al., 1988, Faretra 173506, were normally tolerant to high osmotic pres- and Pollastro, 1988). As mentioned before {chapter sure. 4) Faretra and Pollastro (1991) for the first time pro- Absence of strains highly resistant to dicar­ vided data an the analysis of the genetic basis of boximides in the field and Jack of strains resistant to

63 CGA 173506 but sensitive to vinclozolin, in vitro as clozolin either are lethal under field conditions or weil as in vivo, lead to the following speculations: 1. cause a loss of fitness that has not yet been over~ the acquisition of resistance to CGA 173506 also im­ come. A new tenn is needed to suitably describe this plies resistance to vinclozolin (and increased osmotic connection of resistance to dicarboximides and sensitivity) but not vice versa and 2. acquisition of phenylpyrroles in B. juckeliana as neither the defini­ high resistance to vinclozolin in the field and resis­ tions of cross resistance nor of multiple resistance tance to CGA 173506 linked with resistance to vin- given by Dekker and Delp, (1985) match in this case.

TabJe 5.1: Combination square: independent combination of 6 alleles coding for sensitivity, low and high resistance to dicarboximides and phenylpyrroles. Only the phenotypes described in the shaded squares could be stablely maintained in the present investigation.

' ',reaction to ' '~inclozolin sensitive low resistant highly resistant ' (=S! (=LR! lmg/L < (=HR) ' ECSO <1ing/L reaction to ' ', EC50 < 30mg/L EC50 > 30mg/L CGA 173506 ' ' SHR sensitive (=S) only 11ab. res. strain (Faretra, EC50 < 0.03mg/L pers. comm.)

low resistant LR5 LRHR (=LR) 0.03mg/L < EC50 < 0.3mg/L notfound notfound or unstable •

highly resistant HRS HRLR (=HR) EC50 > 30mg/L notfound notfound

e hypersensitive to increased osmotk pressure measured as glucose tolerance

64 Summary

In the present investigations genetic variability in B. not to vinclozolin were never found. Acquired labo­ fu.ckeliana was studied with regard to fungicide re­ ratory resistance was generally stable after several sistance to vinclozolin and the phenylpyrrole CGA transfers on apples and on pea agar, respectively, 173506. however, some strains lost their resistance. Investi­ Population dynamics of B. fuckeliana resistant gations on the morphological appearance of "run­ to dicarboximides were studied in a vineyard at aways" and the level of resistance revealed a corre­ Walenstadt, Switzerland on the grape variety Blau­ lation of morpho-type and resistance level which was burgunder, clone Mariafelder, which is weakly sus­ however not clear cut in all cases. Conidial dimen­ ceptible to grey mould and therefore dicarboximides sions and number of nuclei of vinclozolin and CGA were rarely used. In a three year field trial, one and 173506 laboratory resistant strains did not differ. two applications of dicarboximides in the first and However, differences in fitness were significant Ac­ in the thlrd season, respectively, resulted in the in­ quisition of Jaboratory resistance to CGA 173506 and crease of the resistant population to a Jevel of 100%. vinclozolin was correlated with loss of virulence, The proportion of resistant strains decreased from osmotolerance and competitive ability. Dicarboximide harvest in the first year to at most 20% in the spring field resistant strains were as virulent as sensitive of the second year and further decreased but did not strains and their osmotolerance was not altered. Viru­ disappear during the second year nor during the third lence and osmotolerance, however, decreased when year when no dicarboximides were applied. Re­ they additionally acquired laboratory resistance to stricted applications of dicarboximides did not pre­ vinclozolin and CGA 173506. vent the buildup of a resistant population. 1his popu­ Homokaryotic sexual progenies of hetero­ lation behaved like populations that were selected karyotic sensitive strains or heterokaryotic strains under a heavy selection pressure. having acquired resistance in the field to dicarbox­ Variability in the sensitivity to CGA 173506 and imides, or in the laboratory to dicarboximictes and vindozolin of B. juckeliana isolated from strawber­ CGA 173506, respectively, were produced using field ries from fields that never ever received any fungi­ isolates from naturally infected strawberries and cidal treatments was investigated. Spore gennination grapes. Resistance to CGA 173506 was inherited by was less inhibited than mycelial growth by both ac­ one major gene which was termed as gene ~l. Tet­ tive ingredients; EC50 values ranged from 1.7-2.0mg rad analysis of sexual progenies confirmed the re­ vindozolin/L and 0.072-0.19mg CGA 173506/L for sults from Faretra and Pollastro (1991) who found, spare germination and from 0.12--0.27mg vindozolin/ by random spare analysis, resistance to vinclozolin L and 0.0027-0.016mg CGA 173506 for mycelial to be inherited by three alleles (I2af1S, Daf_lLR and growth. 12af1HR) of the major gene Dafl. Based on analogy Resistance to CGA 173506 and to vinclozolin between the results in dicarboximides and phen­ was genetically fixed that is, it was not caused by ylpyrroles, it is suggested that resistance to CGA physiological adaptation. "Runaways" (strains spon­ 173506 is inherited by three alleles (ea:1S (sensitive), taneously arising an fungidde amended agar) gen­ ~lLR (low resistant) and f'AlHR (highly resistant)) erally proved to be resistant to vinclozolin and to of the major gene I'.}a:l. In tetrad analyses eu:1 and CGA 173506, however some of them were sensitive. Dafl did not segregate independently. Dicarboximide Strains with a reduced sensitivity to CGA 173506 but field resistant strains were sensitive against CGA

65 173506, whereas laboratory resistant strains showed. double resistance against vinclozolin and CGA 173506. Three hypotheses considering dose linkage of ß:1:1 and Qafl or identity of the two genes are discussed. Osmotolerance and pathogenicity of sexually produced (homokaryotic) strains was assessed. Strains which were resistant to dicarboximides and to CGA 173506 were less virulent and showed a de­ creased osmotolerance, irrespective of the Ievel of resistance. Dicarboximide field resistant strains, how­ ever, while as virulent as sensitive strains, did not show any or only minute differences in osmotoler­ ance when compared. to the sensitive strallls from the same ascus.

66 Zusammenfassung

In der vorliegenden Arbeit wurde die genetische Va­ clozolin/l und 0.0027-0.016mg CGA 173506/1. riabilität von B. fuckeliana im Zusammenhang mit Sowohl die Resistenz gegen Vinclozolin wie der Enstehung von Fungizidresistenz gegenüber Vin­ auch gegen CGA 173506 sind konstitutionell (gene­ dozolin und dem Phenylpyrrol CGA 173506 unter- tisch fixiert). "Ausreisser" (auf Fungizidmedium sucht. spontan wachsende Isolate) waren resistent Populationsdynamische Vorgänge in einer gegenüber CGA 173506 und Vinclozolin; es konnten Dicarboximid-resistenten Population wurden in aber auch Stämme gefunden werden, deren Sensiti­ einem dafür besonders geeigneten Rehberg in Walen- vität gegenüber diesen Wirkstoffen nicht vermindert stadt, Schweiz, untersucht. Die dort angebaute war. Stämme, die eine reduzierte Sensitivität Traubensorte Blauburgunder, Klon Mariafelder, weist gegenüber CGA 173506, nicht aber gegenüber Vin· gegenüber der Graufäule eine reduzierte Anfälligkeit clozolin aufwiesen, wurden nicht gefunden. Labor­ auf, weshalb Dicarboximidfungizide nur wenige Male resistenz gegenüber CGA 173506 und Vinclozolin war eingesetzt wurden. In einem dreijährigen Feldver· im allgemeinen stabil, obwohl einige Stämme ihre such führten ein bis zwei Dicarbox.imidspritzungen Resistenz nach mehrmaliger Passage über Äpfel oder im ersten und im dritten Versuchsjahr zu einem An· über Erbsenagar verloren. In der Mehrzahl der un· stieg der resistenten Population bis auf 100%. Der tersuchten Fälle bestand ein Zusammenhang zwi- Prozentsatz resistenter Stämme nahm von der Ernte sehen Erscheinungsform von "Ausreissern" und des ersten Versuchsjahrs bis zum Frühjahr des deren Resistenzniveau. Bezüglich Längen- und zweiten Versuchsjahrs bis auf maximal 20% ab. Im Breitenmessungen sowie Kernzahl konnten laborre· Verlauf des zweiten und dritten Versuchsjahres ging sistente Stämme nicht von sensiblen unterschieden er weiter zurück, sofern keine Dicarboximidbehand- werden. Erworbene Laborresistenz war jedoch mit lungen vorgenommen wurden. Resistente Stämme einem Verlust an Virulenz, Osmotoleranz und wurden jedoch nicht vollständig verdrängt. Eine re- Konkurrenzfähigkeit korreliert Dicarboximid·feldre- duzierte Anzahl Dicarboximidbehandlungen verhin· sistente Stämme zeigten keinen Unterschied in ihrer derte den Aufbau einer resistenten Population nicht. Virulenz und ihrer Osmotoleranz im Vergleich zu Diese Population verhielt sich hinsichtlich popu· sensiblen Stämmen. Beide Eigenschaften waren lationsdynamischer Vorgänge gleich wie resistente jedoch stark vennindert, wenn feldresistente Stämme Populationen, die unter hohem Selektionsdruck zusätzlich Laborresistenz gegenüber CGA 173506 und entstanden sind. Vinclozolin erworben hatten. Die natürliche Variabilität in der Sensitivität Homokaryotische Stämme wurden aus gegenüber CGA 173506 und Vinclozolin von B. Kreuzungen von heterokaryotischen sensiblen mit fuckeliana- Erdbeerisolaten aus nie mit Fungiziden be- heterokaryotischen Dicarboximid-feldresistenten oder handelten Parzellen wurde untersucht. Beide Dicarboximid- und CGA 173506- laborresistenten Fungizide hemmten die Sporenkeimung weniger als Stämmen gewonnen. Die heterokaryotischen Frei- das Myzelwachstum, wobei CGA 173506 die höhere landisolate stammten von Erdbeeren und Trauben. Wirkung zeigte als Vinclozolin. ECSO Werte für Nach einer Ruheperiode bei erniedrigter Tempera· Sporen·keimung lagen zwischen 1.7- 2.0mg Vin- tur wurden reife Sklerotien einzelner Stämme mit dozolin/L und 0.072-0.19mg CGA 173506/1, für einer Suspension von Mikrokonidien, Makrokonidien Hemmung des Myzels zwischen 0.12-0.27mg Vin· und Myzelfragmenten verschiedener Stämme sper-

67 matisiert. Resultate aus Kreuzungsexperimenten zeigten, dass die Laborresistenz gegen CGA 173506 durch ein Strukturgen vererbt wird, welches als ea:1 bezeichnet wurde. Die Tetradenanalyse bestätigte die Resultate von Faretra und Pollastro (1991}, die aufgrund von Massensporenanalysen schlossen, dass die Resistenz gegen Oicarboximidfungizide durch das Hauptgen !2af1, welches in 3 Allelen (QaflS, IaflLR und ]2af1HR) vorkommt, vererbt wird. In Analogie zu den Resultaten, die bei der Untersuchtung der Vererbung der Dicarboximidresistenz gefunden wur­ den, wird angenommen, dass das Phenylpyrrolre­ sistenzgen ebenfalls in drei verschiedene Allelen (~lS (sensibel), l:.}alLR (mittelresistent} and ~lHR (hochresistent)) vorkommt. In Tetradenana­ lysen konnte keine unabhängige Segregation zwischen ~1 und 12afl nachgewiesen werden. Dicarboximid- feldresistente Stämme zeigen keine verminderte Sensibilität gegenüber CGA 173506. Laborresistente Stämme sind dagegen in allen Fällen sowohl gegen CGA 173506 als auch gegen Vinclozolin resistent. Drei Modelle welche ein enge Kopplung der Gene ~1 und Dafl oder die Identität der bei­ den Gene berücksichtigen werden diskutiert. Osmotoleranz und Pathogenität der sexuellen (homokaryotischen) Nachkommen wurde untersucht. Stämme die eine Laborresistenz gegenüber Dicarbox­ imiden und CGA 173506 aufwiesen, zeigten sowohl eine verminderte Virulenz wie auch eine reduzierte Osmotoleranz. Sexuell erzeugte Nachkommen Dicar­ boximid-feldresistenter Stämme zeigten jedoch weder in der Virulenz noch in der Osmotoleranz eine deut­ liche Veränderung im Vergleich mit sensiblen Stämmen aus dem gleichen Ascus.

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77 Curriculum. vitae

29.3.1963 Born in Basel Son of Hilber- Müller, Anne- Marie, Eugenia and Hilber, Walter, Emil, 4402 Frenkendorf, BL

1970- 1979 Primary School and Progyrnnasium Frenkendorf 1979- 1982 Gymnasium Llestal, Matura C

1983- 1989 University of Basel Studies in botany, zoology and chemistry

1986 Traineeship at Ciba-Geigy Ltd. Basel, Insect control department (Dr. G. Voss and Dr. T. Schläpfer) Development of a laboratory test to assess the effect of selected, slowly killing insecticides on Myws persicae

1987 Traineeship at the Glasshouse Crops Research Institute at Littlehampton (U.K.), Insect pathology department (Dr. D. Burgess and Dr. A. Gillespie} Studies on culturing methods of Beauveria bassiana, a fungus used to bio­ control Nilparvata /ugens

1987- 1988 Grant of the Jubilee- Foundation of the Basellandschaftliche Kantolnalbank for studies in agriculture and plant pathology at the ETH at Zürich

1988- 1992 Assistance in research projects and collaboration in advisory service at the Swiss Federal Research Station at Wädenswil

1988- 1989 University of Basel and Swiss Federal Research Station Diploma thesis in plant pathology on the infection biology of Gymnosporangium .fuscum (Supervisors: Prof. Dr. F.J. Schwinn, University of ßasel and PD Dr. H. Schüepp, Swiss Federal Research Station}

1989- 1992 PhD thesis at the University of Basel and the Swiss Federal Research Station at Wädenswil, in collaboration with Ciba-Geigy Ltd. (Disease control) (Supervisors: Prof. Dr. F.J. Schwinn, Prof. Dr. T. Boller, University of Basel and PD Dr. H. Schüepp, Swiss Federal Research Station)

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