Genetic analysis and molecular characterisation of laboratory and field mutants of Botryotinia fuckeliana (Botrytis cinerea) resistant to QoI fungicides

BACKGROUND: QoI fungicides, inhibitors of mitochondrial respiration, are considered to be at high risk of resistance development. In several phytopathogenic fungi, resistance is caused by mutations (most frequently G143A) in the mitochondrial cytochrome b (cytb) gene. The genetic and molecular basis of QoI resistance were investigated in laboratory and field mutants of Botryotinia fuckeliana (de Bary) Whetz. exhibiting in vitro reduced sensitivity to trifloxystrobin. RESULTS: B. fuckeliana mutants highly resistant to trifloxystrobin were obtained in the laboratory by spontaneous mutations in wild‐type strains, or from naturally infected plants on a medium amended with 1–3 mg L^?1 trifloxystrobin and 2 mM salicylhydroxamic acid, an inhibitor of alternative oxidase. No point mutations were detected, either in the complete nucleotide sequences of the cytb gene or in those of the aox and Rieske protein genes of laboratory mutants, whereas all field mutants carried the G143A mutation in the mitochondrial cytb gene. QoI resistance was always maternally inherited in ascospore progeny of sexual crosses of field mutants with sensitive reference strains. CONCLUSIONS: The G143A mutation in cytb gene is confirmed to be responsible for field resistance to QoIs in B. fuckeliana. Maternal inheritance of resistance to QoIs in progeny of sexual crosses confirmed that it is caused by extranuclear genetic determinants. In laboratory mutants the heteroplasmic state of mutated mitochondria could likely hamper the G143A detection, otherwise other gene(s) underlying different mechanisms of resistance could be involved. Copyright © 2012 Society of Chemical Industry     

Resistance to benzimidazole can be caused by changes in β-tubulin isoforms

The summary reports work that indicates that resistance in Rhynchosporium secalis to benzimidazole fungicides could result from substitution of the normal wild-type benA β-tubulin gene by other β-tubulin isoforms. © 1999 Society of Chemical Industry     

Effets de divers herbicides sur la croissance mycélienne de souches de Botrytis cinerea pers et de Penicillium expansum Link, sensibles ou résistantes à certains fongicides

Effects of various herbicides on mycelial growth of strains of Botrytis cinerea. Pers. and Pénicillium expansum Link, sensitive or resistant to certain fungicides Of seventy herbicides tested, thirty-eight were slightly toxic to B. cinerea (CI50, concentration giving 50% inhibition of the speed of mycelial growth, exceeding or equal to 100g of herbicide/ml of nutrient solution) and, of those, ten had no effect even at 1000 g/ml. The most active products (CI50 less than 10 g/ml) were Chlorthiamid, dichlobénil, nitrofen, propyzamide and phenolic derivatives (DNOC, ioxynil, PCP). In most cases P. expansum was less sensitive than B. cinerea except to endothal, propachlor, prynachlor and certain substituted ureas. Strains resistant to the benzimidazole fungicides (carbendazim, thiabendazole etc.) show increased sensitivity to certain carbamade herbicides (barban, chlorbufam etc.), this indicates the existence of a negative cross resistance between these groups of antimitotic pesticides. Strains resistant to the cyclic-imide fungicides (iprodionc, procymidone, vinclozolin) and to various aromatic compounds (biphenyl. chloroneb, dicloran etc.) may also be resistant to bipyridilium, dinitroanilinc and diphenylethcr herbicides, to chlorthamid. dichlobénil and oxadiazon. This cross resistance between pesticides with different biochemical modes of action has yet to be explained.     

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     

A Reliable Method for Testing the Sensitivity of Botryotinia fuckeliana to Anilinopyrimidines In Vitro

Cyprodinil is a representative of the new class of broad-spectrum anilinopyrimidine fungicides. The effect of cyprodinil on mycelial growth of Botryotinia fuckeliana on solid agar medium depends on the composition of the medium and on the age of the mycelium to be used for the bioassay. An in-vitro method was developed to study the sensitivity distribution to cyprodinil in two wild-type populations of B. fuckeliana from fruits of strawberry with grey mould. To validate the applicability of the method, sensitivities to cyprodinil, mepanipyrim and pyrimethanil were monitored in populations of B. fuckeliana from grapes with grey mould from different vineyards, including one trial vineyard where reduced performance of cyprodinil had been encountered. The monitoring procedure was based on the inhibition of the mycelial growth on a synthetic medium containing L -asparagine (asp-agar) amended with the active ingredients. The mycelium was grown on asp-agar discs, starting from a spore suspension, for 17 h prior to the beginning of the test. This procedure proved to be efficient. The two wild-type populations from different sampling sites showed similar distributions of the sensitivity to cyprodinil. Some isolates from the trial site with reduced performance of anilinopyrimidines showed reduced sensitivities to cyprodinil, mepanipyrim and pyrimethanil, demonstrating cross-resistance between these anilinopyrimidines.     

Transfer of the β‐tubulin gene of Botrytis cinerea with resistance to carbendazim into Fusarium graminearum

BACKGROUND: Resistance to carbendazim and other benzimidazole fungicides in Botrytis cinerea (Pers. ex Fr.) and most other fungi is usually conferred by mutation(s) in a single chromosomal β‐tubulin gene, often with several allelic mutations. In Fusarium graminearum Schwade, however, carbendazim resistance is not associated with a mutation in the corresponding β‐tubulin gene. RESULTS: The β‐tubulin gene conferring carbendazim resistance in B. cinerea was cloned and connected with two homologous arms of the β‐tubulin gene of F. graminearum by using a double‐joint polymerase chain reaction (PCR). This fragment was transferred into F. graminearum via homologous double crossover at the site where the β‐tubulin gene of F. graminearum is normally located (the β‐tubulin gene of F. graminearum had been deleted). The transformants were confirmed and tested for their sensitivity to carbendazim. CONCLUSION: The β‐tubulin gene conferring carbendazim resistance in B. cinerea could not express this resistance in F. graminearum, as transformants were still very sensitive to carbendazim. Copyright © 2010 Society of Chemical Industry     

Detection and characterization of benzimidazole resistance of <Emphasis Type="Italic">Botrytis cinerea</Emphasis> in greenhouse vegetables

From 2003 to 2006, a total of 426 single-conidial isolates of B. cinerea collected from greenhouse vegetables in China were characterized for resistance to benzimidazole fungicides and diethofencarb according to inhibition of mycelial growth. Rapid development of double-resistance to benzimidazoles and diethofencarb was observed. Three types of benzimidazole-resistant isolates, Ben R1, Ben R2 and Ben R3 were detected. A new phenotype, Ben R3, which showed low level of resistance to benzimidazole fungicides and resistance to diethofencarb, was detected with frequencies of 6.8%, 10.0%, 13.2% and 12.4% from 2003 to 2006, respectively. Further studies indicated that Ben R3 was caused by a point mutation from GAG in sensitive(S) isolates to GTG at codon 198 in the β-tubulin gene, predicted to cause a change from glutamic acid to valine. Ben R3 isolates had comparable growth, sporulation and pathogenicity ability as isolates of other phenotypes but were more sensitive at lower temperatures.     

New Cases of Negative Cross-resistance between Fungicides,Including Sterol Biosynthesis Inhibitors

A survey of fungicide resistance in Mycosphaerella graminicola and Tapesia acuformis, two major pathogens of winter wheat in France, respectively responsible for speckled leaf blotch and eyespot, led to the characterization of two types of resistant strains to sterol 14α-demethylation inhibitors (DMIs). Most of the strains of M. graminicola collected in France in 1997–1998 were resistant to all DMIs, and only in a few strains was the resistance to several triazoles associated with increased susceptibility to pyrimidine derivatives (i.e., fenarimol, nuarimol) and triflumizole. On the other hand, in T. acuformis the most prevalent strains were those which exhibited negative-cross resistance between DMIs. In both fungi such a phenomenon could be related to changes in cytochrome P450 sterol 14α-demethylase, the target site of these fungicides. For Botryotinia fuckeliana, the causal agent of grey mould, the extensive monitoring conducted in French vineyards before the marketing of fenhexamid revealed the presence of highly resistant strains to this promising botryticide (only in tests involving mycelial growth measurements). Negative cross-resistance to edifenphos and several sterol biosynthesis inhibitors, such as prochloraz and fenpropimorph, was observed in fenhexamid resistant strains. Synergism of the antifungal action of fenhexamid by cytochrome P450 inhibitors, such as the DMI fungicides, was only recorded in fenhexamid resistant strains. These data and those previously obtained with edifenphos resistant strains of Magnaporthe grisea (rice blast pathogen) suggest that in fenhexamid resistant strains of B. fuckeliana the same cytochrome P450 monooxygenase could be involved in detoxification of fenhexamid and activation of edifenphos. Received 6 September 1999/ Accepted in revised form 13 September 1999     

Characterization of the mechanism of action of the fungicide fenpicoxamid and its metabolite UK‐2A

BACKGROUND

Fenpicoxamid is a new fungicide for control of Zymoseptoria tritici, and is a derivative of the natural product UK‐2A. Its mode of action and target site interactions have been investigated.

RESULTS

UK‐2A strongly inhibited cytochrome c reductase, whereas fenpicoxamid was much less active, consistent with UK‐2A being the fungicidally active species generated from fenpicoxamid by metabolism. Both compounds caused rapid loss of mitochondrial membrane potential in Z. tritici spores. In Saccharomyces cerevisiae, amino acid substitutions N31K, G37C and L198F at the Qi quinone binding site of cytochrome b reduced sensitivity to fenpicoxamid, UK‐2A and antimycin A. Activity of fenpicoxamid was not reduced by the G143A exchange responsible for strobilurin resistance. A docking pose for UK‐2A at the Qi site overlaid that of antimycin A. Activity towards Botrytis cinerea was potentiated by salicylhydroxamic acid, showing an ability of alternative respiration to mitigate activity. Fungitoxicity assays against Z. tritici field isolates showed no cross‐resistance to strobilurin, azole or benzimidazole fungicides.

CONCLUSION

Fenpicoxamid is a Qi inhibitor fungicide that provides a new mode of action for Z. tritici control. Mutational and modeling studies suggest that the active species UK‐2A binds at the Qi site in a similar, but not identical, fashion to antimycin A. © 2017 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.     

Studies on the Inherent Resistance Risk to Fenhexamid in Botrytis cinerea

After chemical mutagenesis with N-methyl-N-nitrosoguanidine (MNNG) two phenotypes that were highly or moderately resistant to fenhexamid, were isolated from a wild-type strain of Botrytis cinerea, at a mutation frequency of 0.9 × 10^–5. Resistance factors, based on EC⁵⁰ values, were 460–570 and 10–15, respectively. The mutation(s) for resistance to fenhexamid did not affect the sensitivity of mutant strains to the benzimidazole benomyl, the phenylpyridinamine fluazinam, the anilinopyrimidine cyprodinil, the guanidine iminoctadine or to the sterol-biosynthesis-inhibiting fungicides fenarimol, fenpropimorph and tridemorph. On the contrary, an increased sensitivity (EC⁵⁰ ratios of 0.2–0.6) of fenhexamid-resistant strains to the phenylpyrrole fludioxonil and the dicarboximide iprodione was observed. Study of fitness parameters of fenhexamid-resistant isolates of both phenotypic classes showed that these mutation(s) had no effect on mycelial growth and sensitivity to high osmolarity, but they did affect one or more of some other characteristics, such as sporulation, conidial germination and sclerotia production. In tests on cucumber seedlings under greenhouse conditions, all highly fenhexamid-resistant isolates tested presented decreased infection ability compared with the wild-type. Preventive applications of a commercial formulation of fenhexamid, Teldor 50 WP, were effective against lesion development on cotyledons by the wild-type, but ineffective, even in high concentrations, against disease caused by the fenhexamid-resistant isolates. The risk of resistance problems arising during commercial use of fenhexamid is discussed.     

Effect of Phenylpyrrole-resistance Mutations on Ecological Fitness of <Emphasis Type="Italic">Botrytis cinerea</Emphasis> and their Genetical Basis in <Emphasis Type="Italic">Ustilago maydis</Emphasis>

Mutants of Botrytis cinerea and Ustilago maydis highly resistant to fludioxonil were isolated at a high frequency, after nitrosoguanidine or UV mutagenesis, respectively, and selection on media containing fludioxonil. Tests on the response of mutant strains to high osmotic pressure resulted in the identification of two fludioxonil-resistant phenotypes (FLD_osm/s and FLD_osm/r), regarding the sensitivity to high osmolarity. Approximately 95% of fludioxonil-resistant mutants were found to be more sensitive to high osmotic pressure than the wild-type parent strains. Genetic analysis of phenylpyrrole-resistance in the phytopathogenic basidiomycete U. maydis, showed that fludioxonil-resistance was coded by three unlinked chromosomal loci (U/fld-1, U/fld-2 and U/fld-3), from which only the U/fld-1 mutation coded an osmotic sensitivity similar to that of the wild-types. Cross-resistance studies with fungicides from other chemical groups showed that the mutations for resistance to phenylpyrroles affect the sensitivity of mutant strains to the aromatic hydrocarbon and dicarboximide fungicides, but not to the benzimidazoles, anilinopyrimidines, phenylpyridinamines, hydroxyanilides or the sterol biosynthesis inhibiting fungicides. A study of fitness parameters in the wild-type and fludioxonil-resistant mutants of B. cinerea, showed that all osmotic sensitive (B/FLD_osm/s) isolates had significant reductions in the characteristics determining saprophytic fitness such as mycelial growth, sporulation, conidial germination and sclerotial production. Contrary to that, with the exception of mycelial growth, the fitness parameters were unaffected or only slightly affected in most of the osmotic resistant (B/FLD_osm/r) isolates. Tests on cucumber seedlings showed that the osmotic-sensitive strains were significantly less pathogenic compared with the wild-type and B/FLD_osm/r strains of B. cinerea. Preventative applications of the commercial products Saphire 50 WP (fludioxonil) and Rovral 50 WP (iprodione) were effective against lesion development on cotyledons by the wild-type and the mutant strains of B. cinerea that were resistant to the anilinopyrimidine cyprodinil (B/CPL-27) and to the hydroxyanilide fenhexamid (B/FNH-21), but ineffective, even at high concentrations, against disease caused by the fludioxonil-resistant isolates (B/FLD) and a mutant strain resistant to the dicarboximide iprodione (B/IPR-1). Experiments on the stability of the fludioxonil-resistant phenotype showed a reduction of resistance, mainly in osmotic-sensitive isolates, when the mutants were grown on inhibitor-free medium. A rapid recovery of the high resistance was observed after mutants were returned to the selection medium. Studies on the competitive ability of mutant isolates against the wild-type parent strain of B. cinerea, by applications of a mixed conidial population, showed that, in vitro, all mutants were less competitive than the wild-type strain. However, the competitive ability of osmotic-resistant mutants was higher than the osmotic-sensitive ones. Furthermore, competition tests, in planta, showed a significant reduction of the frequency of both phenylpyrrole-resistant phenotypes, with a respective increase in the population of the wild-type strain of the pathogen.     

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.     

The Pyrethrins and Related Compounds. Part XLI. Structure–Activity Relationships in Non-ester Pyrethroids against Resistant Strains of Housefly (Musca domestica L.)

A series of pyrethroids, related to NRDC 200 and etofenprox (MTI500) in which the central region is represented by a non-ester link, have been tested against one susceptible and two resistant strains (kdr and super-kdr) of houseflies (Musca domestica L.). A range of structural variations in the central region have been examined. Resistance factors mostly fell within narrow ranges for both resistant strains i.e. 10–50-fold resistance against kdr and 50–150-fold against super-kdr; thus no correlation of resistance with structural features was detectable for this region. Other changes examined were the substituent on the phenyl ring in the ‘acid’ component and the bridging group in the ‘alcohol’ component where small variations in response were observed. Examination of the effect of varying the ‘alcohol’ side chain was limited by lack of active analogues.     

Benzimidazole resistance in Pseudocercosporella herpotrichoides—monitoring programme in Denmark1

Surveys in Denmark show decreasing effectiveness of benzimidazole fungicides against Pseudocercosporella herpotrichoides since 1980, and a high% of fields with resistant isolates of the fungus. Farmers have only escaped serious losses because of good weather at harvest. New recommendations are made for fungicide use.     

Using allele-specific oligonucleotide probes to characterize benzimidazole resistance in Rhynchosporium secalis

Benzimidazole fungicides are important mixture components in strategies to combat fungicide resistance in Rhynchosporium secalis Davis. To monitor the performance of these strategies, a rapid, accurate assay has been developed to detect point mutations in the β-tubulin gene which confers resistance of benzimidazoles. The β-tubulin gene of a benzimidazole-resistant strain of R. secalis has been cloned and sequenced. Except for the difference in the position of one of its six introns, this gene showed a strong homology with other β-tubulin genes from filamentous fungi. Resistance was related to a point mutation in codon 198 which caused a glutamic acid to glycine change in resistant field strains, but glutamic acid to lysine in a laboratory mutant. A DNA fragment surrounding codon 198 was amplified directly from diseased lesions using a ‘nested’ set of PCR primers. Combining PCR amplificiation of a target DNA sequence with hybridization of Allele-Specific Oligonucleotide probes (ASO_s, 15-mers) allowed accurate detection of benzimidazole resistance. Only two probes, one sensitive and one resistant, were sufficient to monitor current field populations. Detection was achieved using either ³²P-labelled probe, or non-radioactively using a biotin-labelled probe coupled to streptavidin/alkaline phosphatase. This rapid method using ASOS can detect benzimidazole resistance within 48 h compared with 6–8 weeks by conventional assay procedures.     

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.     

Multiple resistance of Botrytis cinerea from kiwifruit to SDHIs,QoIs and fungicides of other chemical groups

BACKGROUND: Botrytis cinerea Pers.: Fr. is a high‐risk pathogen for fungicide resistance development that has caused resistance problems on many crops throughout the world. This study investigated the fungicide sensitivity profile of isolates from kiwifruits originating from three Greek locations with different fungicide use histories. Sensitivity was measured by in vitro fungitoxicity tests on artificial nutrient media. RESULTS: Seventy‐six single‐spore isolates were tested for sensitivity to the SDHI fungicide boscalid, the QoI pyraclostrobin, the anilinopyrimidine cyprodinil, the hydroxyanilide fenhexamid, the phenylpyrrole fludioxonil, the dicarboxamide iprodione and the benzimidazole carbendazim. All isolates from Thessaloniki showed resistance to both boscalid and pyraclostrobin, while in the other two locations the fungal population was sensitive to these two fungicides. Sensitive isolates showed EC₅₀ values to boscalid and pyraclostrobin ranging from 0.9 to 5.2 and from 0.04 to 0.14 mg L^?1 respectively, while the resistant isolates showed EC₅₀ values higher than 50 mg L^?1 for boscalid and from 16 to > 50 mg L^?1 for pyraclostrobin. All QoI‐resistant isolates carried the G143A mutation in cytb. Sensitivity determinations to the remaining fungicides revealed in total eight resistance phenotypes. No isolates were resistant to the fungicides fenhexamid and fludioxonil. CONCLUSION: This is the first report of B. cinerea field isolates with resistance to both boscalid and pyraclostrobin, and it strongly suggests that there may be a major problem in controlling this important pathogen on kiwifruit. Copyright © 2010 Society of Chemical Industry     

Further studies in increased sensitivity to N-phenylanilines in benzimidazole-resistant strains of Botrytis cinerea and Venturia nashicola

The ring-substituted N-phenylanilines, N-(3-chlorophenyl)aniline (MC-1) and N-(3,5-dichlorophenyl)aniline (MC-2) were tested for their antifungal activity against Botrytis cinerea Pers. ex Fr. and Venturia nashicola Tanaka et Yamamoto. In both fungi, increased sensitivity to MC-1 and MC-2 was clearly observed in ‘highly carbendazim-resistant, diethofencarb-sensitive’ (HR, S) phenotypes. Sensitivity was low in ‘carbendazim-sensitive, diethofencarb-resistant’ (S, R) and ‘intermediately carbendazim-resistant, diethofencarb-resistant’ (IR, R) strains. On cucumber cotyledons, other strains of B. cinerea, possessing the phenotype ‘highly carbendazim-resistant, diethofencarb-resistant’ (HR, R) were not controlled by either MC-1 or MC-2. Response to MC-2 was also examined using random ascospore progenies from V. nashicola crosses. In these progenies, high-level carbendazim resistance and MC-2 sensitivity always segregated together. Sensitivity to MC-2 is controlled by a single gene which is either identical to or very closely linked to one conferring high-level resistance to carbendazim.     

Fungicide control of mushroom cobweb disease caused by Cladobotryum strains with different benzimidazole resistance profiles

The benzimidazole fungicides thiabendazole and carbendazim, and the imidazole fungicide prochloraz-Mn, were tested for their efficacy in controlling cobweb disease of mushrooms caused by two Cladobotryum isolates. Isolate 202A was benzimidazole-sensitive in vitro and cobweb growth on the casing was well controlled by both benzimidazole fungicides in cropping experiments. Carbendazim also controlled the development of spotting symptoms much more effectively than thiabendazole. A second isolate (192B1) was benzimidazole-resistant and was highly resistant to thiabendazole in vitro but it showed some sensitivity to carbendazim in vitro at moderate to high concentrations. Despite this, carbendazim did not control disease symptoms in cropping experiments, confirming that isolate 192B1 is cross-resistant to other benzimidazole fungicides. Both isolates showed some sensitivity to prochloraz-Mn in vitro. This fungicide gave between 45% and 65% control of cobweb growth on the casing caused by either 202A or 192B1 but gave no control of spotting symptoms. Reducing the fungicide application volume did not give enhanced disease control. The emergence of benzimidazole resistance reduces the value of benzimidazoles in the control of mushroom pathogens. However, the lack of effective alternatives means they continue to have utility in cases where pathogens are still sensitive but this requires regular monitoring of pathogen populations for resistance.