Effect of chlorothalonil on resistance of Botrytis cinerea to dicarboximides in cucumber glasshouses1

Failures to control grey mould by dicarboximides on winter-grown cucumbers in glasshouses in Israel were attributed to the development of resistance to these fungicides in populations of Botrytis cinerea. A survey of 18 glasshouses in the winter of 1983-84 revealed that resistance is widespread where dicarboximides have been used extensively. During the summer, resistant B. cinerea populations often shift back to sensitivity; however, following few dicarboximide sprays in the subsequent winter, resistant strains reappear, increase rapidly and become dominant in the pathogen population. In two out of three glasshouse experiments, combination of chlorothalonil and dicarboximides in spray programmes slowed down the increase of resistant strains, whereas in a third experiment such a programme had no effect. The usefulness of chlorothalonil in fungicide-alternation programme to suppress dicarboximide resistance is dicussed in relation to the fitness properties of established versus newly-appearing resistant mutants.     

Use of Trichoderma harzianum in combination or alternation with fungicides to control cucumber grey mould (Botrytis cinerea) under commercial greenhouse conditions

A preparation of Trichoderma harzianum was sprayed on cucumber plants in greenhouses in order to control fruit and stem grey mould. Up to 90% control was achieved by the biocontrol agent (0·5–1·0 g/l) which in most experiments under commercial conditions was as effective as the dicarboximide fungicides iprodione or vinclozolin (0·5 g/l each) alone or alternated with diethofencarb + carbendazim (0·25 g/l each). However, in one experiment disease incidence in Trichoderma -treated plots did not differ significantly from the control. A mixture of T. harzianum with a dicarboximide fungicide resulted in up to 96% control of grey mould. In this case control was always significant ( P =0·05) but improvement of control compared with each treatment alone was not significant ( P =0·05). The alternation of sprays with the biocontrol preparation and with a dicarboximide fungicide was tested in three out of the five experiments and was found to be effective, thus enabling a reduction in the use of chemical sprays. Populations of T. harzianum were on a level of 3 × 10⁵-8 × 10⁵ c.f.u. per leaf and ten times lower on one fruit. They remained high after the second and third sprays. Conditions favouring the ability of T. harzianum to control grey mould were temperatures above 20°C and relative humidity between 80 and 97%.     

Studies on resistance to dichlofluanid and other fungicides in Botrytis cinerea

Amongst 489 mass isolates of Botrytis cinerea obtained from protected crops in Humberside during 1989, 67.3% were resistant to benomyl and 45.6% to vinclozolin. Two isolates resistant to dichlofluanid were obtained with EC₅₀ values for mycelial growth on agar of 21.45 and 19.78 μg dichlofluanid/ml, respectively, compared with values of 1.55-2.56 μg/ml for sensitive isolates. EC₅₀ values for germ tube emergence and length were much lower than for mycelial growth. In population studies carried out on tomatoes in a polythene tunnel, resistance to dichlofluanid and to vinclozolin declined over a 12-week period in the absence of fungicides. Field isolates of B. cinerea resistant to dichlofluanid were also resistant to vinclozolin. but strains resistant to dichlofluanid and sensitive to vinclozolin were obtained during population studies. Examination of a number of features of dichlofluanid-resistant strains revealed a degree of lack of fitness as compared with sensitive strains. There is no indication that resistance to dichlofluanid poses a great threat to the control of grey mould in Humberside at present.     

Resistance to diethofencarb (NPC) in benomyl-resistant field isolates of Botrytis cinerea

Based on the 'negative cross-resistance' phenomenon between N -phenylcarbamates and benzimidazole fungicides, the fungicidal mixture of carbendazim + diethofencarb (MBC + NPC) was introduced, to control Botrytis cinerea phenotypes which are either sensitive to benzimidazoles and resistant to NPC (wild-type: Ben^S NPC^R). or resistant to benzimidazoles and sensitive to NPC(Ben^R NPC^S). At one out of four sites where the MBC + NPC mixture was used in commercial cucumber greenhouses, grey mould control failed and a new phenotype of B. cinerea was found. The new phenotype was resistant to benzimidazoles, as was 100% of the population screened in the four sites, but retained resistance to NPC. Accordingly, the new phenotype was designated Ben^R NPC^R. It was pathogenic on cucumber seedlings.     

Failure to detect highly resistant strains in dicarboximide resistant field populations of Botrytis cinerea1

Field isolates of Botrytis cinerea with moderate levels of resistance to dicarboximide fungicides (ED50 1.0–4.9 μg ml^?1) and to dicloran were obtained from glasshouses where vinclozolin and iprodione failed to control grey mould. From sensitive and moderatcly-resistant cultures, laboratory isolates were selected on dicarboximide-amended medium, which were highly resistant to these fungicides (ED50 125->3000 μg ml^?1). Conidia of all the resistant isolates germinated well on media amended with 100 μg ml^?1 of the dicarboximides vinclozolin, iprodione, procymidone and myclozolin and with 5 μg ml^?1 of metomeclan. However, the spores of the moderately resistant isolates did not germinate on 100 μg ml^?1 metomeclan while the spores of the highly resistant isolates germinated well. Using media with 100 μg ml^?1 of metomeclan to distinguish between the two phenotypes, no highly resistant strain was detected among 312 resistant samples from five cucumber glasshouses with a high frequency of moderately resistant strains. From air-borne inoculum of five glasshouses with 100% resistant populations, 1604 colonies were recovered on vinclozolin-amended (100 μg ml^?1) medium and none on metomeclan-amended (100 μg ml^?1) medium. It is concluded that strains of B. cinerea highly resistant to dicarboximides are absent from field populations.     

Strains of Botrytis cinerea resistant to dicarboximide and benzimidazole fungicides in New Zealand vineyards

In a survey of New Zealand vineyards at harvest 1985, isolates of Botrytis cinerea resistant to benzimidazole and to dicarboximide fungicides were common. The mean frequency of resistance in the major vine-growing districts ranged from 8 to 41% for benzimidazoles, and from 51 to 59% for dicarboximides. All benzimidazole-resistant isolates showed high levels of resistance (EC50 greater than 100 mg/l carbendazim based on radial growth response), and all dicarboximide-resistant isolates showed low levels of resistance. Two subgroups of dicarboximide-resistant isolates were recognized, distinguished in the first instance by their osmotic response. Low-level resistant isolates, which formed a dense margin on osmotically amended medium, exhibited an EC50 for mycelial growth on iprodione of c. 3-2 mg/l; ultra-low-level resistant isolates, which formed a fibrillose margin on osmotically amended medium identical to that of sensitive isolates, exhibited an EC50 of c. 1-3 mg/1. In agar culture, radial growth rate, and conidial and sclerotial production of both subgroups were similar to those of sensitive isolates. Virulence (lesion size) and conidial production on grape berries were highest in sensitive isolates, intermediate in ultra-low-level dicarboximide-resistant isolates, and lowest in low-level dicarboximide-resistant isolates. Evidence is presented indicating that ultra-low-level dicarboximide-resistant strains have progressively replaced low-level dicarboximide-resistant strains in the vineyard population. The presence of dicarboximide-resistant strains was linked with a partial loss of fungicide efficacy.     

Incidence of benomyl and iprodione resistance in isolates of Botrytis cinerea in tomato crops in England and Wales in 1986

In 1986, mass-mycelial isolates of Botrytis cinerea from 67 tomato crops in England and Wales were examined for benomyl and iprodione resistance. Of the 706 isolates obtained, 62.7% were resistant to benomyl at 2μg/ml and 43.2% were resistant to iprodione at 2 μg/ml. Iprodione resistance persisted in the absence of a dicarboximide spray programme. The incidence of benomyl resistance has not decreased since the last survey in 1984 in spite of a considerable reduction in the use of benzimidazole fungicides. There were no clear indications that the use of dichlofluanid influenced the incidence of benomyl or iprodione resistance. Disease control was poorer in crops with a higher incidence of iprodione resistance.     

Limitations in the exploitation of N-phenylcarbamates and N-phenylformamidoximes to control benzimidazole-resistant Venturia nashicola on Japanese pear

The efficacy of N -phenylcarbamates and N -phenylformamidoximes against benzimidazole-resistant strains of Venturia nashicola was tested experimentally in Japanese pear orchards. Both compounds gave effective control when applied in an orchard where highly benzimidazole-resistant strains were predominant. However, unsatisfactory control was observed in an orchard where the fungal population was dominated by fungal strains with intermediate or weak benzimidazole resistance. Strains resistant to N -phenylcarbamates and N -phenylformamidoximes were widely distributed in Japanese pear orchards, suggesting that effective use of these compounds to control benzimidazole-resistant strains of V. nashicola would not be practicable. Increased sensitivity to N -phenylformamidoximes in highly benzimidazole-resistant isolates was shown to be controlled by a single chromosomal gene, but progenies that were highly resistant to both a benzimidazole fungicide and a N -phenylformamidoximes appeared in crosses between parents resistant to each fungicide alone. Similar strains were also found in pear orchards, and this'double resistance'was shown to be heritable.     

Distribution and fitness of isolates of Botrytis cinerea with multiple fungicide resistance in Spanish greenhouses

Forty-nine greenhouses of vegetable crops were surveyed in southeast Spain at the beginning of the disease season in December 1992 to estimate frequencies of resistance to benzimidazoles, dicarboximides and N -phenylcarbamates (NPC) in B. cinerea . Out of 261 isolates collected, 28% were sensitive to both benzimidazoles and dicarboximides, 15% were benzimidazole-resistant and dicarboximide-sensitive, 8% were benzimidazole-sensitive and dicarboximide-resistant and 46% were benzimidazole- and dicarboximide-resistant. Resistance to benzimidazole, dicarboximide and N -phenylcarbamate was determined by measuring the ability of each isolate to grow in the presence of carbendazim, procymidone and diethofencarb fungicides respectively. Carbendazim- or procymidone- resistant isolates were found in all surveyed greenhouses. Three isolates were found with resistance to carbendazim, procymidone and diethofencarb collected in two adjacent greenhouses that were sprayed with the carbendazim and diethofencarb mixture. All other isolates were sensitive to the mixture because they were either sensitive to carbendazim and resistant to diethofencarb or vice versa. Fitness of 31 isolates of B. cinerea was determined in vivo by measuring their sporulation and lesion growth rate on leaf disks. No fitness costs were associated with resistance to iprodione (dicarboximide) and benomyl (benzimidazole). Isolates with EC₅₀ values higher than 101 mg/L for benomyl and 1.6 mg/L for iprodione were considered to be field resistant (they caused visible lesions on cucumber leaf disks treated with each fungicide).     

Screening of <Emphasis Type="Italic">Botrytis cinerea</Emphasis> isolates from vineyards in Israel for resistance to fungicides

Plots in two vineyards in the Golan Heights, Israel were treated with six botryticides during three growing seasons with 3 applications per season. Applications of fenhexamid, pyrimethanil and cyprodinil + fludioxonil were effective, resulting in 52–65% and 53–63% mean reduction in grey mould incidence and severity, respectively. Carbendazim, fluazinam and iprodione were ineffective or slightly effective. Five hundred and sixteen B. cinerea isolates were collected from infected berries or trapped from the air in the vineyards, and profiles of sensitivity to benomyl, fenhexamid, fluazinam, fludioxonil, iprodione and pyrimethanil were established for each of the isolates based on a mycelial growth test. Seventy-four percent of the isolates were sensitive to the six tested fungicides, and the other 26% of the isolates were classified into 10 phenotypes characterized by resistance to one or more fungicides. Resistant isolates showed fitness parameters similar or reduced in comparison to sensitive isolates. Resistance to benzimidazoles and to dicarboximides was the most frequent (up to 25%) and apparently pre-existed in the populations tested. Increased frequency of benzimidazole resistance, but not dicarboximide resistance, was observed following the 3 years of applications of the fungicides. High level resistance to pyrimethanil was present at a frequency of about 2% in both vineyards in the first 2 years of the sampling survey and reached 10% in the third year at Site 2. A few isolates were resistant to fenhexamid or fludioxonil (0.8 or 0.2%, respectively). No strong resistance to fluazinam was detected, although numerous, less sensitive isolates, presumably possessing multi-drug resistance traits, were recovered at higher frequency from the plots treated with fluazinam than from the untreated plots.     

Emergence and frequency of highly benzimidazole-resistant Colletotrichum gloeosporioides, pathogen of Japanese pear anthracnose, after discontinued use of benzimidazole

From July to October 1999, two Japanese pear varieties, Housui and Niitaka, were unexpectedly infected with Colletotrichum gloeosporioides causing anthracnose in Saga Prefecture of Japan. More than 90?% of the anthracnose fungus strains collected in 1999 were highly benzimidazole-resistant and distributed at high frequencies across pear-producing areas. The control of anthracnose by benzimidazole fungicides could not be confirmed in orchards where the isolation frequency of resistant strains was high. The frequency of resistant strains did not significantly decline, even 2?C5?years after the last use of benzimidazole fungicides.     

Persistence of dicarboximide-fungicide resistance in populations ofbotrytis cinerea in a warm,dry temperate agroclimate

Failures to control gray mold with vinclozolin and iprodione in cucumber greenhouses in Israel, during the winter and spring of 1981, were attributed to the development of resistance to dicarboximide fungicides in populations ofBotrytis cinerea Pers. In seven out of eight greenhouses where resistance had been found in 1981, most or all of theB. cinerea population was resistant also in January 1982. This demonstrates the capability of resistant strains to survive during the warm and dry Mediterranean summer, and to provide competitive inoculum for crop infection in the subsequent winter.     

Structure—activity relationships of pyridylcarbamates active against both benzimidazole-sensitive and -resistant isolates of Botrytis cinerea

A series of pyridylcarbamates showed high potency against cucumber gray mould (Botrytis cinerea Pers.). The most potent compound, propargyl-N-(6-ethyl-5-iodo-2-pyridyl)carbamate was effective against an isolate sensitive to benzimidazole and dicarboximide fungicides as well as against an isolate resistant to both types of compound. QSAR analyses and molecular modelling studies were carried out to investigate the structural requirements for highly active compounds and the structural feature of the binding site of each strain. Significantly different QSAR equations were obtained only for substituents at the 6-position of the pyridine ring. An ethyl-sized pocket or an ethyl terminal recognition was suggested in the case of the sensitive or resistant isolate respectively. These results could explain the phenomenon of negatively correlated cross-resistance between benzimidazoles and N-phenylcarbamate fungicides. Substituent effects at the 5- or 2-position were governed by steric factors. Substituent effects at the 3-position were explained by steric hindrance or by conformational effects. The propargyl-substituted compound above was the most desirable one from the viewpoint of QSAR.     

梨黑斑病菌抗药性检测及其对啶酰菌胺的敏感性基线

黑斑病是梨的主要病害之一,近年来不少地区反映多菌灵等传统常用杀菌剂对其防治效果已出现下降。作者从浙江、江苏和安徽3省分离了252株梨黑斑病菌Alternaria kikuchiana,采用菌丝生长速率法检测了其抗药性发生情况。结果发现:所检测的黑斑病菌群体(n=252)对苯并咪唑类杀菌剂多菌灵的抗性频率为57.1%,且全部为高水平抗性(HR);对二甲酰亚胺类杀菌剂异菌脲的抗性频率为46.8%,全部为低水平抗性(LR);对甾醇脱甲基抑制剂类杀菌剂苯醚甲环唑的抗性为低水平(LR)及中等水平(MR),抗性频率均为28.6%;表明梨黑斑病菌对常用杀菌剂已产生较为严重的抗性。供试252株梨黑斑病菌对琥珀酸脱氢酶抑制剂啶酰菌胺的EC50值分布在0.12~3.85μg/m L之间,平均EC50值为(1.21±0.12)μg/m L,且其分布呈近似正态的单峰曲线。研究表明,啶酰菌胺可作为潜在的梨黑斑病防治替代药剂,其平均EC50值(1.21±0.12)μg/m L可作为梨黑斑病菌对啶酰菌胺的敏感性基线。     

Properties of dicarboximide-resistant strains of Botrytis cinerea

Dicarboximide-resistant strains of Botrytis cinerea were isolated from natural substrates and also produced in the laboratory. All these strains exhibited a similar degree of resistance to the dicarboximide fungicides iprodione and vinclozolin, and this resistance persisted in the absence of the fungicides. The natural frequency of resistance to both chemicals was approximately three in 10⁷ conidia but could be enhanced by up to 1000 times after a single exposure to sub-lethal concentrations of either chemical. Mycelium of the resistant strains was able to infect fruit and vegetables to a similar extent as that of sensitive strains, although infection of carrot roots was markedly less aggressive. The resistant strains were separable into two groups according to their growth habit on culture media. Both groups were relatively slow growing and showed a marked lack of sporulation compared with most sensitive strains. This lack of sporulation may account for the apparent failure of resistant strains to increase rapidly in strawberry plantations that had received dicarboximide sprays in successive seasons.     

灰葡萄孢抗二甲酰亚胺类杀菌剂研究进展

综述了灰葡萄孢对二甲酰亚胺类杀菌剂抗性的产生及现状,抗性菌株的生物学特性、生理生化特性以及抗药性机制,并就灰葡萄孢对二甲酰亚胺类杀菌剂抗性研究进行了展望。     

Multiple fungicide resistance to benzimidazoles, dicarboximides and diethofencarb in field isolates of Botrytis cinerea in Israel

During the period January-March 1989, 15 greenhouses at 12 sites in Israel were surveyed for the presence of fungicide-resistant strains of Botrytis cinerea , using a fungicide-amended Botrytis -selective medium. Resistance to benzimidazoles (Ben^R) and to dicarboximides (Dic^R) was frequent in most sites. Resistance to carbendazim + diethofencarb (Ben^R NPC^R) was found in all eight sites in which a mixture of these fungicides had been used against grey mould, but not in other sites. A new phenotype of multiple fungicide resistance was found among these isolates. The new phenotype, designated Ben^R Dic^R NPC^R, combines the three previously described characteristics of resistance to benzimidazole, dicarboximide and N -phenylcarbamate fungicides. It was found only in cucumber greenhouses that had been sprayed with the fungicide mixture carbendazim + diethofencarb against grey mould. Isolates of this phenotype were pathogenic in artificial inoculation of cucumber cotyledons treated with carbendazim, iprodione or carbendazim + diethofencarb.     

An hypothesis to account for the behaviour of dicarboximide-resistant strains of Botrytis cinerea in vineyards

Ten trials were established in the 1986 vintage year on vineyards in three vine-growing districts of New Zealand, to examine changes in the frequency of resistance to dicarboximide fungicides of the Botrytis cinerea population under different fungicide programmes. The vineyards chosen encompassed sites with pre-season resistance frequencies ranging from 0 to 97%. Under a non-dicarboximide programme, using dichlofluanid and chlorothalonil, resistance frequency had decreased at harvest irrespective of the initial frequency. In programmes incorporating dicarboximides (chlozolinate, iprodione, procymidone, vinclozolin), the resistance frequency tended to increase if pre-season resistance frequency was low, and decrease if it was high. The hypothesis is presented that for a given dicarboximide programme, resistance frequency tended to one value, the balance value, where selection for resistant strains due to fungicide application balanced selection against the resistant strains due to their low fitness. This value was estimated for the 1986 vintage year to be 38% for a programme where application of dicarboximides was restricted to the period from the veraison (sugar rise), and 64% where dicarboximides were applied from the pre-bunch closure phase. Actual values observed at harvest 1987, after these same programmes had been applied for two consecutive years, were 39% (S.E. 8-5%) and 68% (S.E. 8-8%), respectively.     

Nested PCR‐RFLP is a high‐speed method to detect fungicide‐resistant Botrytis cinerea at an early growth stage of grapes

BACKGROUND: Grey mould caused by the fungus Botrytis cinerea Pers. ex Fr. is one of the major diseases in grapes. The use of fungicides is a simple strategy to protect grapes against B. cinerea disease. However, phenotypes exhibiting resistance to fungicides have been detected in B. cinerea populations. The variation of fungicide‐resistant B. cinerea isolates renders B. cinerea disease control difficult in grapevine fields. RESULTS: The authors have developed a nested polymerase chain reaction–restriction fragment length polymorphism (PCR‐RFLP) method to detect fungicide‐resistant B. cinerea isolates at an early growth stage of grapes in grapevine fields. The nested PCR‐RFLP method was carried out to detect benzimidazole‐, phenylcarbamate‐ and/or dicarboximide‐resistant B. cinerea isolates from grape berries and leaves at Eichorn–Lorenz growth stage 25 to 29. This method successfully detected fungicide‐resistant B. cinerea isolates at an early growth stage of grapes. In addition, only 8 h was required from tissue sampling to phenotyping of fungicide resistance of the isolates. CONCLUSION: It is proposed that the early diagnosis of fungicide‐resistant B. cinerea isolates would contribute to further improvement of integrated pest management against B. cinerea in grapevine fields, and that the nested PCR‐RFLP method is a high‐speed, sensitive and reliable tool for this purpose. Copyright © 2008 Society of Chemical Industry     

Population dynamics of dicarboximide-resistant strains of Botrytis cinerea on grapevine in New Zealand

Changes in the proportion of dicarboximide-resistant strains of Botrytis cinerea were monitored in two vineyard trials conducted during the period from October 1985 to November 1987. Dicarboximide resistance frequency in samples taken prior to flowering from wood (dead cane-ends, dead tendrils, old bunch remains) was high (66%) in the first trial, and low (12%) in the second trial. In treatments where resistance frequency was high on wood in spring, it decreased during the phase of disease establishment in new bunches irrespective of whether or not dicarboximide fungicides were applied. In the absence of dicarboximide applications, resistance frequency showed little change during the phase of disease development in bunches. The resistance frequency increased, however, if dicarboximide fungicides were applied, the extent of increase depending on the number and timing of applications and on disease severity at the time of application. Where resistance frequency was high on the wood at harvest, it decreased over the following winter but increased again in spring even though no dicarboximides had been applied. The best disease control in both trials was obtained with a programme in which dicarboximide fungicides were only applied during the period from veraison until harvest.