Dicarboximide-resistant Strains Research Articles

Resistance of Botrytis cinerea Isolates from Vegetable Crops to Anilinopyrimidine, Phenylpyrrole, Hydroxyanilide, Benzimidazole, and Dicarboximide Fungicides.

During February 2005, 55 single-spore isolates of Botrytis cinerea were collected at the end of the season from vegetable crops grown in 18 greenhouses on the island of Crete, Greece. They were tested for sensitivity to the anilinopyrimidine fungicides pyrimethanil and cyprodinil, the hydroxyanilide fungicide fenhexamid, the phenylpyrrole fungicide fludioxonil, the dicarboximide fungicide iprodione, and the benzimidazole fungicide carbendazim. Results of the study showed the existence of benzimidazole- and dicarboximide-resistant strains at frequencies of 61.8 and 18%, respectively. Moreover, for first time, the development of resistance to anilinopyrimidine fungicides by B. cinerea was detected in greenhouse vegetable crops on the island of Crete. High resistance frequencies of 49.1 and 57.4% were observed for pyrimethanil and cyprodinil, respectively. In addition, one isolate was found to be resistant to the hydroxyanilide fungicide fenhexamid, while no strains resistant to the phenylpyrrole fungicide were detected. Among the 55 isolates tested, 13 were resistant only to carbendazim, 6 were resistant only to anilinopyrimidines, 3 were resistant to both benzimidazoles and dicarboximides, 17 were resistant to both benzimidazoles and anilinopyrimidines, 6 were resistant to both dicarboximides and anilinopyrimidines, 1 was simultaneously resistant to benzimidazoles, dicarboximides, and anilinopyrimidines, 1 was resistant to both anilinopyrimidines and hydroxyanilides, and 8 were sensitive to all fungicides tested. A strong cross-resistance relationship was found between the two anilinopyrimidine fungicides tested when log transformed EC50 values of the isolates were subjected to a linear regression analysis (r = 0.71). Despite the detection of several phenotypes with simultaneous resistance to chemically unrelated active ingredients, in none of the remaining possible fungicide pairs was there observed any kind of cross-resistance relationship.

Mechanisms of resistance to fungicides in field strains of Botrytis cinerea.

Field strains of Botrytis cinerea Pers ex Fr, the causal agent of grey mould diseases, were collected from French vineyards between 1993 and 2000. Several phenotypes have been characterized according to the inhibitory effects of fungicides towards germ-tube elongation and mycelial growth. Two types of benzimidazole-resistant strains (Ben R1 and Ben R2) could be detected; negative cross-resistance to phenylcarbamates (e.g. diethofencarb) was only found in Ben R1. Benzimidazole resistance was related to point mutations at codon 198 (Ben R1) or 200 (Ben R2) of the beta-tubulin gene. Most dicarboximide-resistant strains were also weakly resistant to aromatic hydrocarbon fungicides (e.g. dicloran) but remained sensitive to phenylpyrroles (e.g. fludioxonil). These resistant field strains (Imi R1) contained a single base pair mutation at position 365 in a two-component histidine kinase gene, probably involved in the fungal osmoregulation. Three anilinopyrimidine-resistant phenotypes have been identified. In the most resistant one (Ani R1), resistance was restricted to anilinopyrimidines, but no differences were observed in the amino-acid sequences of cystathionine beta-lyase (the potential target site of these fungicides) from Ani R1 or wild-type strains. In the two other phenotypes (Ani R2 and Ani R3), resistance extended to various other groups of fungicide, including dicarboximides, phenylpyrroles and sterol biosynthesis inhibitors. This multi-drug resistance was probably determined by over-production of ATP-binding cassette transporters. The hydroxyanilide fenhexamid is a novel botryticide whose primary target site is the 3-keto reductase involved in sterol C-4 demethylations. Apart from the multi-drug-resistant strain Ani R3, three other fenhexamid-resistant phenotypes have been recognized. For two of them (Hyd R1 and Hyd R2) fenhexamid-resistance seemed to result from P450-mediated detoxification. Reduced sensitivity of the target site could be the putative resistance mechanism operating in the third resistant phenotype (Hyd R3). Increased sensitivity to inhibitors of sterol 14 alpha-demethylase recorded in Hyd R1 strains was related to two amino-acid changes at positions 15 and 105 of this enzyme.

Patterns of cross-resistance to fungicides in Botryotinia fuckeliana ( Botrytis cinerea) isolates from French vineyards

Field strains of Botryotinia fuckeliana were isolated from diseased grape berries which were collected at the harvest, between 1993 and 1997, from French vineyards located in Alsace, Armagnac, Bordeaux, Champagne and Loire Valley. According to the inhibitory effects of fungicides towards spore germination, germ-tube elongation and mycelial growth, several phenotypes could be characterized. Thus, two types of benzimidazole (e.g. carbendazim, thiabendazole)-resistant strains were detected; negative cross-resistance towards diethofencarb, diphenylamine and dicloran was found only in one type. Most dicarboximide (e.g. iprodione, procymidone, vinclozolin)-resistant strains were also weakly resistant to aromatic hydrocarbon fungicides (e.g. chloroneb, dicloran, tolclofos-methyl) but remained sensitive to phenylpyrroles (e.g. fenpiclonil, fludioxonil). However, in some other dicarboximide-resistant strains, resistance was observed either as being restricted to dicarboximides or as extending weakly to phenylpyrroles. Towards anilinopyrimidines (e.g. cyprodinil, mepanipyrim, pyrimethanil), three resistant phenotypes have been identified. In the most resistant one, resistance was restricted to anilinopyrimidines, whereas in the others, resistance extended to various other groups of fungicides including dicarboximides, phenylpyrroles, sterol biosynthesis inhibitors (e.g. tolfanate, prochloraz, tebuconazole) and the hydroxyanilide derivative, fenhexamid. According to the pattern of cross-resistance, two phenotypes were characterized within these multifungicide-resistant strains. In vitro tests conducted with fenpropimorph, fenpropidin and spiroxamine, revealed the existence of strains highly susceptible to these sterol Δ 14- reduction inhibitors. This property was correlated with a decreased sensitivity towards fenhexamid.

Spatial and temporal characteristics of dicarboximide‐resistant strains of Monilinia fructicola and brown rot incidence in stone fruit

The spatial and temporal distribution of dicarboximide‐resistant strains of Monilinia fructicola were investigated in six peach and nectarine orchard blocks in 1987–89 using a dispersion index (Lloyd's Patchiness Index, LPI), and spatial, temporal and spatio‐temporal autocorrelation analyses (Moran's Coefficient, I). The LPI values indicated that resistant strains were aggregated in all blocks in all years. Spatial correlations were not significant beyond one quadrat for any spatial proximity pattern in five of six blocks. Thus the spread of resistant strains was mostly restricted to the vicinity of the original focus. An absence of significant temporal correlation between years in five of the six blocks indicated poor persistence of resistant strains at specific locations. Only one significant temporal correlation was detected at one block and this could have arisen by chance. Significant spatio‐temporal correlation was not detected, suggesting that there was no focus expansion or carry‐over of resistant strain inoculum from the previous sampling date. Spatial, temporal and spatio‐temporal autocorrelation analyses were consistent with previously reported laboratory results that resistant strains had not acquired all the necessary characteristics to remain in, or dominate, field populations. The spatial pattern of brown rot incidence was investigated at one block in a separate study in 1988 and 1989. There were no significant spatial correlations for brown rot incidence in 1988 for any of the spatial proximity patterns analysed. In 1989, however, significant correlations indicated ellipsoid aggregates of brown rot orientated along the orchard rows.

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.

Dicarboximide-Resistant Strains of Microdochium Nivale in New Zealand.

Isolates of Microdochium nivale resistant to the dicarboximide iprodione were recovered from six golf courses in the northern half of the North Island, New Zealand. Overall frequency of dicarboximide resistance was 19%. EC50 (iprodione) values, based on radial growth response, were 4.5 mg/L for a sensitive isolate, and from 14.1 to 54 mg/L for dicarboximide-resistant isolates. All dicarboximide-resistant isolates except one were also benzimidazole resistant.

Survival of dicarboximide-resistant strains ofBotrytis cinerea in plant debris during summer in Israel

Survival- ofBotrytis cinerea was monitored during two summer seasons. Mycelium and conidia were found dead on the surface of plant debris within 2 months of incubation, whereas a high level of viability was detected in thallus of the pathogen which was 1–2 mm inside the dry host tissue. Of the 148 samples of infected senescing cucumber female fruits, 8% survived seven warm months; half of these isolates ofB. cinerea were resistant to dicarboximides (5 (µ/ml iprodione). Of the stems of cucumber infected withB. cinerea in winter, 18% yielded the pathogen at the beginning of the following winter; 15% of the surviving isolates were resistant to dicarboximides. Cucumber seedlings artificially infected byB. cinerea did not yield the pathogen longer than 9 weeks after establishment of infection, even when incubated in the shade. Plant debris with symptoms of gray mold were kept in the shade during the summer; at the beginning of winter it was possible to establish infection ofB. cinerea from the dry debris.

Influence of spray schedules on fungicide resistant populations of Botrytis cinerea Pers. on grapevine

Treatments with dicarboximide fungicides provided only 20 to 40% control of grey mould on grape (cv. Moscato d'Asti) in vineyards where benzimidazole and dicarboximide resistant strains ofBotrytis cinerea Pers. constituted about 50% of the fungal population and a high disease pressure existed. The percentage of dicarboximide resistant strains increased after treatment. Fungicide combinations of a benzimidazole with diethofencarb, sprayed twice per season or once alternated with a dicarboximide, provided satisfactory control of grey mould and decreased the percetage of benzimidazole resistant strains. A combination of thiram and procymidone controlled grey mould, but increased dicarboximide resistance in the population.

Deposition techniques for the preparation of thin film nuclear targets: A H F Muggleton,Vacuum,37, 1987, 785–817

Field strains of Botryotinia fuckeliana were isolated from diseased grape berries which were collected at the harvest, between 1993 and 1997, from French vineyards located in Alsace, Armagnac, Bordeaux, Champagne and Loire Valley. According to the inhibitory effects of fungicides towards spore germination, germ-tube elongation and mycelial growth, several phenotypes could be characterized. Thus, two types of benzimidazole (e.g. carbendazim, thiabendazole)-resistant strains were detected; negative cross-resistance towards diethofencarb, diphenylamine and dicloran was found only in one type. Most dicarboximide (e.g. iprodione, procymidone, vinclozolin)-resistant strains were also weakly resistant to aromatic hydrocarbon fungicides (e.g. chloroneb, dicloran, tolclofos-methyl) but remained sensitive to phenylpyrroles (e.g. fenpiclonil, fludioxonil). However, in some other dicarboximide-resistant strains, resistance was observed either as being restricted to dicarboximides or as extending weakly to phenylpyrroles. Towards anilinopyrimidines (e.g. cyprodinil, mepanipyrim, pyrimethanil), three resistant phenotypes have been identified. In the most resistant one, resistance was restricted to anilinopyrimidines, whereas in the others, resistance extended to various other groups of fungicides including dicarboximides, phenylpyrroles, sterol biosynthesis inhibitors (e.g. tolfanate, prochloraz, tebuconazole) and the hydroxyanilide derivative, fenhexamid. According to the pattern of cross-resistance, two phenotypes were characterized within these multifungicide-resistant strains. In vitro tests conducted with fenpropimorph, fenpropidin and spiroxamine, revealed the existence of strains highly susceptible to these sterol Δ14- reduction inhibitors. This property was correlated with a decreased sensitivity towards fenhexamid.

Control of low-level dicarboximide-resistant strains ofBotrytis cinereaby dicarboximide fungicides

Abstract The dicarboximide fungicide iprodione (‘Rovral’) gave a high degree of control of infection by low-level dicarboximide-resistant strains of Botrytis cinerea Pers.: Fr. on detached leaves of French bean (Phaseolus vulgaris L.) when the inoculum was in the form of conidia, but not when a mycelial plug inoculum was used. This difference in response partially explains how low-level dicarboximide-resistant strains can be present in crops where dicarboximide fungicides are in use, without causing obvious losses of disease control.

A survey of resistance to the dicarboximide fungicides inBotrytis cinerea

Abstract Isolates of Botrytis cinerea Pers.: Fr. collected from a range of crops in New Zealand, were tested for resistance to the dicarboximide fungicides by measuring radial growth on agar media amended with iprodione. In addition, each isolate was tested for resistance to the benzimidazole fungicides, by growing on medium amended with carbendazim, and for sensitivity to high osmotic pressure, by growing on medium amended with 0.68 M NaCl. In outdoor crops (grape, kiwifruit, strawberry, boysenberry) dicarboximide-resistant strains were found on only 2 of the 18 properties examined, one a vineyard and the other a strawberry planting. In neither instance was any loss of disease control apparent. No dicarboximide resistance was found on the single glasshouse grape property sampled, but on glasshouse vegetable crops (tomato, French bean, cucumber) dicarboximide resistance was found on all 7 properties. In these vegetable houses resistant strains were often predominant and in some instances there appeared to...

Development in New Zealand of resistance to dicarboximide fungicides inBotrytis cinerea, to acylalinines inPhytophthora infestans, and to guazatine inPenicillium italicum

Abstract Strains of Botrytis cinerea resistant to dicarboximide fungicides were isolated from glasshouse vegetable crops in the Auckland region in 1980. The isolates showed cross-resistance to 4 dicarboximide fungicides (dichlozolinate, iprodione, procymidone, and vinclozolin), to dicloran, and to quintozene, and most were also resistant to benzimidazole fungicides. Growth was reduced, but not stopped, on media containing 10-1000 mg/litre of dicarboximide fungicides. The resistant isolates varied in their ability to invade plant tissue. In the glasshouses sampled, presence of dicarboximideresistant strains of B. cinerea appeared to reduce fungicide efficacy slightly. Strains of Phytophthora infestans resistant to metalaxyl were isolated from poroporo and potato crops where blight control based on the use of this fungicide was no longer effective. These isolates attacked poroporo or potato plants sprayed with 500 mg/litre metalaxyl, and a poroporo isolate also attacked plants sprayed with either ofurace or...