Aromatic Hydrocarbon Fungicides Research Articles

The Group III Two-Component Histidine Kinase AlHK1 is Involved in Fungicides Resistance, Osmosensitivity, Spore Production and Impacts Negatively Pathogenicity in Alternaria longipes

Members of group III histidine kinases from different filamentous fungi were previously shown to mediate osmoregulation and resistance to dicarboximide, phenylpyrrole and, aromatic hydrocarbon fungicides. In this study, we report the disruption of the gene encoding group III histidine kinase, AlHK1, in the economically important plant pathogen Alternaria longipes. The AlHK1 gene disruption had pleiotropic effects on this fungus. Besides the expected osmosensitivity and fungicides resistance, AlHK1 participated in the spore production process. In addition, the ΔAlHK1 strains had stronger aggressive ability to infect their host plant than that of their parental strain, the wild-type strain C-00, suggested that AlHK1 was involved in the pathogenicity of A. longipes and performed in this function by a negative manner. This is the first report to our knowledge.

Field strains of Stemphylium vesicarium with a resistance to dicarboximide fungicides correlated with changes in a two-component histidine kinase

In Stemphylium vesicarium, four phenotypes were recognized according to their in vitro responses to dicarboximide fungicides: S (sensitive), S+ (low resistant to iprodione and procymidone but moderately resistant to vinclozolin), R1 (moderately resistant to iprodione and vinclozolin but highly resistant to procymidone), R2 (highly resistant to all dicarboximides). Cross-resistance was observed between dicarboximides and aromatic hydrocarbon fungicides in all cases while cross-resistance to phenylpyrroles was only detected in R2 phenotype. Moreover, no changes were noted in sensitivity to oxidative and osmotic stress inducers. An osmosensing histidine kinase gene, homologous to OS1 from Neurospora crassa, was sequenced from several field isolates of Stemphylium vesicarium. This gene is predicted to encode a 1,329 amino acid protein, comprising a conserved histidine-kinase domain in the C-terminal region and six tandem repeats of about 90 amino acids at the N-terminal end. In S+ and R1 phenotype isolates, a single amino acid substitution was observed in the first amino acid repeat; F267L and L290S respectively. For the R2 isolates, the exchanges T765R or Q777R were located within the histidine-kinase domain.

Impact of fungal drug transporters on fungicide sensitivity, multidrug resistance and virulence

Drug transporters are membrane proteins that provide protection for organisms against natural toxic products and fungicides. In plant pathogens, drug transporters function in baseline sensitivity to fungicides, multidrug resistance (MDR) and virulence on host plants. This paper describes drug transporters of the filamentous fungi Aspergillus nidulans (Eidam) Winter, Botrytis cinerea Pers and Mycosphaerella graminicola (Fückel) Schroter that function in fungicide sensitivity and resistance. The fungi possess ATP-binding cassette (ABC) drug transporters that mediate MDR to fungicides in laboratory mutants. Similar mutants are not pronounced in field resistance to most classes of fungicide but may play a role in resistance to azoles. MDR may also explain historical cases of resistance to aromatic hydrocarbon fungicides and dodine. In clinical situations, MDR development in Candida albicans (Robin) Berkhout mediated by ABC transporters in patients suffering from candidiasis is common after prolonged treatment with azoles. Factors that can explain this striking difference between agricultural and clinical situations are discussed. Attention is also paid to the risk of MDR development in plant pathogens in the future. Finally, the paper describes the impact of fungal drug transporters on drug discovery.

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.

The cAMP Signal Transduction Pathway Mediates Resistance to Dicarboximide and Aromatic Hydrocarbon Fungicides in Ustilago maydis

The cAMP signal transduction pathway mediates the switch between yeast-like and filamentous growth and influences both sexual development and pathogenicity in the smut fungus Ustilago maydis. Signaling via cAMP may also play a role in fungicide resistance in U. maydis. In particular, the adr1 gene, which encodes the catalytic subunit of the U. maydis cAMP-dependent protein kinase (PKA), is implicated in resistance to the dicarboximide and aromatic hydrocarbon fungicides. In this study, we examined the sensitivity of PKA to vinclozolin and could not demonstrate direct inhibition of protein kinase activity. However, we did find that mutants with disruptions in the ubc1 gene, which encodes the regulatory subunit of PKA, were resistant to both vinclozolin and chloroneb. We also found that these fungicides altered the morphology of both wild-type and ubc1 mutant cells. In addition, strains that are defective in ubc1 display osmotic sensitivity, a property often associated with vinclozolin and chloroneb resistance in other fungi.

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.

Significance of Insensitivity ofSclerotinia minorto Iprodione in Control of Sclerotinia Blight of Peanut.

Four hundred isolates of Sclerotinia minor were obtained from diseased peanut plants (Arachis hypogaea) in field plots untreated or treated three times with iprodione (1.12 kg/ha) in 1987. On glucose-yeast extract agar containing iprodione at 2 μg/ml, only 6% of the isolates grew. These isolates produced more mycelial growth than corresponding isolates not previously exposed to the fungicide in agar, indicating that insensitivity developed in vitro. In field microplots artificially infested with sclerotia of the insensitive isolates and planted to peanut for 7 yr, iprodione provided significant disease suppression even though insensitive isolates could still be recovered. In a separate 3-yr microplot study, the pathogenicity of an isolate (B-83-T2) of S. minor suspected of possessing field resistance to dicarboximide fungicides was compared to a sensitive isolate (S-2) after treatment of peanuts with dicarboximide fungicides (iprodione or vinclozolin), aromatic hydrocarbon fungicides (dicloran or pentachloronitrobenzene [PCNB]), and an experimental fungicide (fluazinam). Without fungicide treatment, disease incidence (stem lesions per microplot) at harvest averaged 19.9 in plots infested with isolate S-2 and 18.3 in plots infested with isolate B-83-T2 (no statistical difference), but yields were significantly lower (10%) in microplots infested with isolate S-2. When averaged across isolates, disease incidence was suppressed 96, 63, 42, 41, and 2O% by fluazinam, vinclozolin, iprodione, PCNB, and dicloran, respectively. Insensitivity in S. minor to the dicarboximide fungicides appears low but persistent and does not pose a threat to the continued use of dicarboximides or related fungicides in peanut production

Characterization and Genetic Analysis of Laboratory Mutants ofUstilago maydisResistant to Dicarboximide and Aromatic Hydrocarbon Fungicides

Mutants of Ustilago maydis resistant to dicarboximide (DCOF) and aromatic hydrocarbon fungicides (AHF) were isolated after chemical mutagenesis or by selection of spontaneous mutants on fungicide-amended agar medium. A wide range of mutants, with low to high levels of resistance, were isolated. The resistance factor compared to the wild type was greater than 150 for three mutants isolated on tolclophos-methyl and greater than 50 for three mutants isolated on vinclozolin. Growth rates for these six most tolerant isolates were similar to that of the wild type. The phenomenon of cross-resistance between these two groups was confirmed. None of the mutants were resistant to the ergosterol biosynthesis inhibitor propiconazole. Two mutants, VR43 and VR44, demonstrated the same osmotic sensitivity when grown in the presence of NaCl (1-8%) as the wild type under these conditions

Similarities in the antifungal activities of fenpiclonil, iprodione and tolclofos‐methyl against Botrytis cinerea and Fusarium nivale

AbstractFenpiclonil [4‐(2,3‐dichlorophenyl)pyrrole‐3‐carbonitrile], a new agricultural fungicide, is a structural analogue of the antibiotic pyrrolnitrin used as an antimycotic in animals. Its spectrum of antifungal activity is similar to that of iprodione and tolclofos‐methyl, which are representatives of dicarboximide and aromatic hydrocarbon fungicides. Laboratory mutants of Botrytis cinerea and Fusarium nivale were simultaneously resistant to fenpiclonil, iprodione and tolclofos‐methyl and they showed an increased susceptibility to high osmotic pressure (sodium chloride, glucose). Strains of B. cinerea, collected in Champagne vineyards, which were resistant to dicarboximides were not resistant to fenpiclonil and tolclofos‐methyl. The antioxidant α‐tocopheryl acetate, and the cytochrome P‐450 inhibitors piperonyl butoxide and tebuconazole antagonized the fungitoxicity of fenpiclonil and iprodione in a wild‐type strain of B. cinerea. The uncoupling activity of fenpiclonil, iprodione and tolclofos‐methyl was detected in mitochondria from both a wild‐type strain and a laboratory mutant of B. cinerea. The concentrations effective towards this process were greater than those inhibiting the mycelial growth of wild‐type strains. These results suggest that fenpiclonil, iprodione, tolclofos‐methyl and related fungicides have the same biochemical mode of action. However, a primary effect on respiration, previously considered for pyrrolnitrin, seems to be improbable.

Isolation, characterization and genetic analysis of mutants of Aspergillus nidulans resistant to the herbicide dichlobenil

Thirty one spontaneous mutants of Aspergillus nidulans were recovered from 5·2 × 10 6 conidia incubated on synthetic medium supplemented with 100–200 μg dichlobenil ml −1 . The mutants varied considerably in morphology, growth rate, conidiation, osmotic sensitivity and degree of resistance to the herbicide. All mutants were cross-resistant to the aromatic hydrocarbon fungicides quintozene and tolclofos methyl, and the dicarboximide fungicide vinclozolin. Both the herbicide and the fungicides enhanced growth and conidiation of some mutants. Genetic analysis of seven mutants with diverse phenotypes showed that they each carried a single gene for dichlobenil resistance that was tightly linked to methH on linkage group III. The mutant alleles were dominant to the wild-type gene.

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.