Negative cross-resistance to N-phenylanilines in benzimidazole- resistant strains of Botrytis cinerea,Venturia nashicola and Venturia inaequalis

A new series of compounds with high contra-selective activity against benomyl-resistant fungal strains was found among ring-substituted N-phenyl-anilines. Hydrophobic substitution in one of the benzene rings, together with the secondary amine character of the molecule, are important factors for high fungitoxicity. The sensitivities of benomyl-resistant isolates to some representatives of the N-phenylanilines equal or even surpass their sensitivity to diethofencarb or MDPC. The negative cross-resistance with benzimdazole fungicides is valid for strains of Botrytis cinerea, Venturia nashicola and Venturia inaequalis. The strains of B. cinerea with double insensitivity to benzimidazoles and phenyl-carbamates are insensitive also to N-phenylanilines. However, the similarly double-insensitive strain of V. nashicola was found to be just as sensitive to the N-phenylanilines tested as the strain resistant only to benzimidazoles. The latter result revealed an important bonus compared with diethofencarb. Preventive application of one of the most active representatives of the N-phenylanilines to young cucumber plants was effective against infection with benomyl-resistant isolates of B. cinerea. Moderate apical translocation after root-dipping was also observed.     

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.     

Mechanism of selectivity to fenpropimorph in Fusarium genus and Pseudocercosporella herpotrichoides

Algorithms developed for use in the study of artificial intelligence also show promise in such quite different fields as the physical and life sciences. These methods may have advantages in speed, scope, or both, over more conventional scientific methods, and expand the range of algorithmic techniques open to the scientist. The mechanism of one of these methods, the genetic algorithm, which is already finding application in a number of areas in science, is outlined.     

Metabolism of fungicidal cyanooximes, cymoxanil and analogues in various strains of Botrytis cinerea

BACKGROUND: The metabolism of cymoxanil [1‐(2‐cyano‐2‐methoxyiminoacetyl)‐3‐ethylurea] and fungicidal cyanooxime analogues was monitored on three phenotypes of Botrytis cinerea Pers. ex Fr. differing in their sensitivity towards cymoxanil. For this purpose, labelled [2‐¹⁴C]cymoxanil was added either to the culture medium of these strains or to its cell‐free extract. RESULTS: In the culture medium of the most sensitive strain, four main metabolites were detected. Three were isolated and identified. Cymoxanil was quickly metabolised by at least three concurrent enzymatic pathways: (i) cyclisation leading, after hydrolysis, to ethylparabanic acid, (ii) reduction giving demethoxylated cymoxanil, (iii) hydrolysis followed by reduction and then acetylation leading to N‐acetylcyanoglycine. In the cell‐free extract of the same strain, only the first and the second of these enzymatic reactions occurred. By comparing the metabolic profile of the most sensitive strain with that of the less sensitive ones, it was shown that the decrease in sensitivity to cymoxanil correlates with a reduced acetylcyanoglycine formation. Among all metabolites, only N‐acetylcyanoglycine is active against the most sensitive strain. Moreover, in a culture of this strain, two other fungicidal cyanooximes were also metabolised into this metabolite. CONCLUSION: The formation of N‐acetylcyanoglycine may play an important role in the fungitoxicity of cymoxanil and cyanooxime derivatives. Copyright © 2008 Society of Chemical Industry     

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.     

Genotyping of benzimidazole-resistant and dicarboximide-resistant mutations in Botrytis cinerea using real-time polymerase chain reaction assays

Botrytis cinerea, an economically important gray mold pathogen, frequently exhibits multiple fungicide resistance. A fluorescence resonance energy transfer-based real-time polymerase chain reaction assay has been developed to detect benzimidazole- and dicarboximide-resistant mutations. Three benzimidazole-resistant mutations-(198)Glu to Ala (E198A), F200Y, and E198K-in beta-tubulin BenA were detected using a single set of fluorescence-labeled sensor and anchor probes by melting curve analysis. Similarly, three dicarboximide-resistant mutations-I365S, V368F plus Q369H, and Q369P-in the histidine kinase BcOS1 were successfully distinguished. Unassigned melting profiles in BenA genotyping assay resulted in the identification of a new benzimidazole-resistant BenA E198V mutation. This mutation conferred resistance to carbendazim as do E198A and E198K mutations. The isolates with BenA E198V mutation showed a negative cross-resistance to diethofencarb, but to a lesser extent than the E198A mutants. A survey of 210 B. cinerea field isolates revealed that most of benzimidazole-resistant isolates possessed the E198V or E198A mutation in the BenA gene, and the I365S mutation in the BcOS1 gene was also frequently observed in Japanese isolates. However, benzimidazole-resistant isolates with BenA F200Y or E198K mutations, which confer the diethofencarb-insensitive phenotype, were rare. Our BenA and BcOS1 genotyping is a rapid and reliable method that is suitable for monitoring the fungicide-resistant field population.     

Distribution of benzimidazole-resistant strains of the onion gray-mold neck rot pathogens,Botrytis aclada and Botrytis allii,in Hokkaido,Japan

Botrytis aclada and Botrytis allii, associated with onion gray-mold neck rot and isolated in Hokkaido, were tested for sensitivity to benzimidazole. Of the B. aclada strains, 59% were highly resistant and the remaining 41% were sensitive; all strains of B. allii were sensitive. Resistant strains were widespread in Hokkaido. We analyzed the sequences of the β-tubulin gene of resistant strains and detected the replacement of glutamic acid (GAG) by lysine (AAG) at codon 198. This is the first report of benzimidazole resistance in B. aclada. This study revealed a difference in fungicide sensitivity between the two Botrytis species.

     

Stability and fitness of anilinopyrimidine-resistant strains of Botrytis cinerea

The fitness of anilinopyrimidine-resistant isolates of Botrytis cinerea compared with that of sensitive isolates, collected from vegetable crops in Greece during 2005, was investigated. Stability of resistance to anilinopyrimidine fungicides was determined after consecutive transfers of the fungal isolates on fungicide-free potato dextrose agar for 16 culture cycles or on fungicide-untreated cucumber seedlings for eight disease cycles. Results showed that after the consecutive transfers of the isolates either in vitro or in vivo sensitivity to cyprodinil was not changed significantly compared to the initial sensitivity in all the isolates tested, suggesting a stable genetically controlled trait. Fitness parameters measured were mycelial growth, spore production in vitro, osmotic sensitivity, virulence, spore production in vivo, percentage of spore germination, and competitive ability of the resistant isolates in four pairs with sensitive isolates both on artificial nutrient medium or on cucumber seedling plants. The measurements of the fitness components in individual isolates showed high variability within both sensitivity groups in all, except virulence, fitness components tested. As a group, resistant isolates showed significantly lower (P < 0.05) mycelial growth and virulence, while they were more osmotically sensitive than the sensitive isolates. In addition the resistant isolates showed higher (P < 0.05) spore production in vivo but there was no difference (P > 0.05) between the two sensitivity groups in spore production in vitro and in the percentage of spore germination. However, the correlation to test if there is any relationship between the values of each fitness component tested and the level of cyprodinil sensitivity of each isolate was for all, except the spore production in vivo, fitness components not significant (P > 0.05). This absence of significant correlation coefficient values suggests that the development of resistance to anilinopyrimidine fungicides did not affect the fitness of the resistant isolates. Competition of the resistant versus sensitive isolates was isolates-dependent, since in two of the isolate pairs the resistance frequency decreased significantly after five culture or disease cycles, while in the remaining two pairs resistance frequency increased significantly after five disease cycles or remained stable for one pair after five culture cycles on artificial nutrient media.     

Resistance to inhibitors of sterol biosynthesis in field isolates or laboratory strains of the eyespot pathogen Pseudocercosporella herpotrichoides

Strains of Pseudocercosporella herpotrichoides collected in France on winter wheat give either fast-growing mycelial colonies with regular margins or slow-growing mycelial colonies with irregular margins. Most of the fastgrowing isolates were sensitive to triadimenol (EC₅₀ below 2mg litre^?1), but some of them were resistant to this inhibitor of sterol C-14 demethylation. In contrast, all the slow-growing strains were highly resistant to triadimenol (EC₅₀ greater than 100 mg litre^?1). This resistance was also expressed in inhibition of germ-tube elongation. Positive cross-resistance was observed between most of the inhibitors of sterol C-14 demethylation, with the exception of some imidazole derivatives (clotrimazole, prochloraz). All the fast-growing strains were tolerant to fenpropimorph and fenpropidin whereas the slow-growing ones were susceptible; the reverse was true with piperalin and tridemorph. All the field isolates were inhibited to the same extent by the inhibitors of squalene-epoxidase, nafifine and terbinafine. Two types of mutant resistant to triadimenol have been induced under laboratory conditions from sensitive fast-growing strains. The most common mutants were resistant to all the inhibitors of sterol C–14 demethylation and also in some conditions to fenpropimorph, tridemorph and the inhibitors of squalene-epoxidase. The other mutants were characterised by a reduced spectrum of cross-resistance between triadimenol and the other inhibitors of sterol biosynthesis. The field isolates and laboratory mutants resistant to triadimenol and propiconazole were also resistant to each of the four enantiomers of these two fungicides.     

灰葡萄孢对氟啶胺的敏感性检测及敏感性降低菌株生物学性状研究

为评价灰霉病菌对氟啶胺的敏感性及抗药性风险,本试验于2020年-2021年在吉林、江西、湖北、山东、北京、湖南等地区的草莓、辣椒、四季豆、茄子和番茄上采集病叶、病茎、病花和病果,经单孢分离获得117个灰葡萄孢Botrytis cinerea菌株,采用菌丝生长速率法测定其对氟啶胺的敏感性。结果表明：有4株灰葡萄孢BJ14、BJ45、BJ46和BJ47对氟啶胺的敏感性显著降低,EC₅₀在0.113 7～0.394 6μg/mL,抗性倍数为4.7～16.3,MIC值>4μg/mL。其余113个菌株对氟啶胺的平均EC₅₀为0.025 1μg/mL。敏感性降低的4个菌株继代培养10代后,抗药性状稳定。交互抗性测定结果表明,对氟啶胺敏感性下降的菌株对腐霉利和咯菌腈2种杀菌剂表现为敏感,氟啶胺与腐霉利或咯菌腈没有交互抗性。生物学性状研究表明,4株敏感性下降菌株在PDA平板上的生长速率和在番茄果实上的致病力都显著低于敏感菌株,而菌丝生物量、产孢量和孢子萌发率与敏感菌株无显著差异。以上研究结果表明,田间已存在对氟啶胺敏感性降低的菌株,鉴于灰葡萄孢属于高风...     

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.     

Identification of W-type and R-type isolates of Pseudocercosporella herpotrichoides

W-type isolates of Pseudocercosporella herpotrichoides grown on a maize-based agar and exposed to near- ultra-violet radiation at c . 13°C produced a greenish black colour, whilst R-type isolates produced a pink or pale brown colour in the agar medium. More colonies from directly plated lesions or from spore suspensions could be recognized as P. herpotrichoides and could be more easily differentiated as W-type or R-type or as mixtures of both by colour production on maize agar (MA) than by colony morphology on potato dextrose agar (PDA), despite the presence of other fungi. Isolates with intermediate morphology on PDA were positively identified as W-type or R-type on MA; their pathogenicities to wheat and rye seedlings were usually similar to those of W-type or R-type isolates with typical colony morphology, confirming their identification on MA. Drops of mixed suspensions of W-type and R-type spores on PDA formed fast-growing colonies with smooth margins which sometimes had slow-growing sectors with feathery margins. Drops of the same mixtures on MA formed greenish black colonies which sometimes had pink or pale brown sectors. However, when these mixtures were spread onto MA, W-type and R-type colonies could easily be differentiated by colour.     

Differential binding of a N-phenylformamidoxime compound in cell-free extracts of benzimidazole-resistant and-sensitive isolates of Venturia nashicola,Botrytis cinerea and Gibberella fujikuroi

Venturia nashicola isolates with a high level of resistance to carbendazim showed either increased sensitivity or were resistant to theN-phenylformamidoxime compoundN-(3,5-dichloro-4-propynyloyphenyl)-N′-methoxyformamidine (DCPF). Isolates with an intermediate or low level of carbendazim resistance were resistant to DCPF. Increased sensitivity to DCPF was also associated with a high level of carbendazim resistance inBotrytis cinerea but not with a moderate resistance level. Increased sensitivity to DCPF was not observed in carbendazim resistant isolates ofGibberella fujikuroi.Binding of [¹⁴C]DCPF in cell-free mycelial extracts of the highly carbendazim-resistant and DCPF-sensitive isolate ofV. nashicola was higher than in those of DCPF-resistant isolates that were either highly-resistant, intermediately resistant, or weakly resistant to carbendazim or were sensitive to carbendazim. [¹⁴C]carbendazim binding in extracts of highly carbendazim-resistant isolates ofB. cinerea was lower than that in extracts of sensitive isolates, whereas [¹⁴C]DCFP binding was higher. A decreased [¹⁴C]carbendazim binding was also observed in extracts of carbendazim-resistant isolates ofG. fujikuroi, binding of [¹⁴C]DCPF, however, was similar in extracts of both carbendazim-resistant and sensitive isolates.     

8种杀菌剂对番茄灰霉病菌的毒力及田间番茄灰霉病菌对咯菌腈的敏感性

为了解决番茄灰霉病菌的抗性问题,筛选出理想的替代药剂,采用菌丝生长速率法测定了山东寿光和河北徐水两地番茄灰霉病菌对不同类型杀菌剂的敏感性。两地的番茄灰霉病菌对咯菌腈、氟啶胺均高度敏感,对其他不同作用方式的杀菌剂表现出不同的敏感性。用同样方法检测了不同地区的106株番茄灰霉菌对咯菌腈的敏感性,结果显示其EC50值分布在0.003 9～0.044 9μg/mL之间,平均EC50值为(0.014 2±0.008 1)μg/mL。咯菌腈可作为防治番茄灰霉病的候选药剂。     

A comparison of cultural, morphological and DNA markers for the classification of Pseudocercosporella herpotrichoides

Isolates of Pseudocercosporella herpotrichoides were classified by a variety of established techniques including colony morphology, colour, pathogenicity on seedlings and spore morphology. In all cases, certain isolates could not be clearly classified and correlation between the systems was incomplete. In particular, the typing system of Nirenberg in which two varieties of P. herpotrichoides (var. herpotrichoides and var. acuformis ) are recognized did not correlate with the widely used UK system based on colony morphology on PDA: fast, even (F E) and slow, feathery (S/F). Restriction fragment length polymorphisms in total DNA and rDNA indicated groupings related to morphology (and pathogenicity) with considerable polymorphisms in rDNA of F/E isolates. Generally, good correlation was found between RFLP markers and other tests.     

Dispersal of Pseudocercosporella herpotrichoides and Pyrenopeziza brassicae spores in splash droplets

Splash dispersal of conidia which differ in shape and size, those of Pseudocercosporella herpo ichoides (needle-shaped, 52×2 μm) and Pyrenopeziza hrassicae (cylindrical, 12×3 μm), was studisd by allowing 5 mm drops to fall 13 m on to suspensions (depth 0.5 mm, concentration 120, 000 spores/ml) of each. The resulting splash droplets were collected on photographic film. The pattern of spore dispersal with distance and the distribution of spore-carrying droplets and spores within droplet-size categories were similar for both fungi. Regressions of square root spore number) on droplet diameter gave lines with slopes of 0.0138 for P. herpotrichoides and 0.0167 for P. brassicae.     

Comparison of isoenzyme and DNA markers for differentiating W-, R- and C-pathotypes of Pseudocercosporella herpotrichoides

Biochemical differences between the wheat, rye and couch-grass pathotypes of the eyespot pathogen, Pseudocercosporella herpotrichoides, have been identified by comparing isoenzyme profiles and DNA markers from several isolates. The wheat (W) and rye (R) pathotypes were clearly differentiated by both techniques, with isoenzyme profiling also separating the couch-grass isolates from the W- and R-type isolates. The isoenzyme profiles separated the P. herpotrichoides pathotypes from the two related species, P. anguioides and P. aestiva. The isoenzyme profiles of P. anguioides were closer to those of the R-type isolates of P. herpotrichoides than the W-type isolates, whereas the banding pattern of P. aestiva was very different. The isoenzyme profiles of the two German isolates, originally obtained from Nirenberg's laboratory, P. herpotrichoides var. herpotrichoides and var. acuformis, were similar to those of the wheat and rye pathotypes, respectively, isolated from UK-infected material.