•Fitness of B. cinerea strains resistant to SDHIs estimated using sdhB recombinant mutants.•Different sdhB mutations lead to variable resistance costs, affecting distinct fitness ...components.•Inhibition of SDH activity not always consistent with fitness defect and mutant frequency in the field.•Compensatory system probably operating in natura or in genetic background of field strains.
Botrytis cinerea is a pathogenic ascomycete fungus that causes gray mold on many crops. Chemical control remains the principal method for curbing this disease. However, fungicide efficacy may be compromised by the selection of resistant strains. Assessments of the fitness of resistant strains is important, to evaluate the risk of their establishment in populations. Strains resistant to boscalid, the first succinate dehydrogenase inhibitor (SDHI) registered for the treatment of gray mold on grapevine in France, have recently been detected in the field. Most of these strains harbor mutations of the sdhB gene, encoding subunit B of SDH. In this study, we used sdhB recombinant mutants to investigate the impact of mutations conferring SDHI resistance on the fitness of B. cinerea. We have shown that sdhB mutations (except for the sdhBH272Y mutation) affect SDH activity and respiration rate. Our results suggest that different sdhB mutations have different effects on fitness. In particular, mutants displaying an inhibition of SDH activity do not suffer the same effects on fitness. We discuss the results in the context of mutant frequencies in field populations and the possible occurrence of compensatory mechanisms that modulate fitness losses.
In French and German vineyards, Botrytis cinerea isolates with multiple fungicide resistance phenotypes have been observed with increasing frequencies. Multidrug resistance (MDR) results from ...mutations that lead to constitutive overexpression of genes encoding drug efflux transporters. In MDR2 and MDR3 strains, overexpression of the major facilitator superfamiliy transporter gene mfsM2 has been found to result from a rearrangement in the mfsM2 promoter (type A), caused by insertion of a retroelement (RE)-derived sequence. Here, we report the discovery of another, similar RE-induced rearrangement of the mfsM2 promoter (type B) in a subpopulation of French MDR2 isolates. MDR2 isolates harboring either type A or type B mutations in mfsM2 show the same resistance phenotypes and similar levels of mfsM2 overexpression. RE sequences similar to those in mfsM2 were found in low copy numbers in other but not all B. cinerea strains analyzed, including non-MDR2 strains. Population genetic analyses support the hypothesis that the two rearrangement mutations have only occurred once, and are responsible for the appearance and subsequent spread of all known MDR2 and MDR3 strains in French and German wine-growing regions.
The grey mould fungus Botrytis cinerea causes losses of commercially important fruits, vegetables and ornamentals worldwide. Fungicide treatments are effective for disease control, but bear the risk ...of resistance development. The major resistance mechanism in fungi is target protein modification resulting in reduced drug binding. Multiple drug resistance (MDR) caused by increased efflux activity is common in human pathogenic microbes, but rarely described for plant pathogens. Annual monitoring for fungicide resistance in field isolates from fungicide-treated vineyards in France and Germany revealed a rapidly increasing appearance of B. cinerea field populations with three distinct MDR phenotypes. All MDR strains showed increased fungicide efflux activity and overexpression of efflux transporter genes. Similar to clinical MDR isolates of Candida yeasts that are due to transcription factor mutations, all MDR1 strains were shown to harbor activating mutations in a transcription factor (Mrr1) that controls the gene encoding ABC transporter AtrB. MDR2 strains had undergone a unique rearrangement in the promoter region of the major facilitator superfamily transporter gene mfsM2, induced by insertion of a retrotransposon-derived sequence. MDR2 strains carrying the same rearranged mfsM2 allele have probably migrated from French to German wine-growing regions. The roles of atrB, mrr1 and mfsM2 were proven by the phenotypes of knock-out and overexpression mutants. As confirmed by sexual crosses, combinations of mrr1 and mfsM2 mutations lead to MDR3 strains with higher broad-spectrum resistance. An MDR3 strain was shown in field experiments to be selected against sensitive strains by fungicide treatments. Our data document for the first time the rising prevalence, spread and molecular basis of MDR populations in a major plant pathogen in agricultural environments. These populations will increase the risk of grey mould rot and hamper the effectiveness of current strategies for fungicide resistance management.
Trehalose is a non-reducing disaccharide found at high concentrations in Aspergillus nidulans conidia and rapidly degraded upon induction of conidial germination. Furthermore, trehalose is ...accumulated in response to a heat shock or to an oxidative shock. The authors have characterized the A. nidulans tpsA gene encoding trehalose-6-phosphate synthase, which catalyses the first step in trehalose biosynthesis. Expression of tpsA in a Saccharomyces cerevisiae tps1 mutant revealed that the tpsA gene product is a functional equivalent of the yeast Tps1 trehalose-6-phosphate synthase. The A. nidulans tpsA-null mutant does not produce trehalose during conidiation or in response to various stress conditions. While germlings of the tpsA mutant show an increased sensitivity to moderate stress conditions (growth at 45 degrees C or in the presence of 2 mM H(2)O(2)), they display a response to severe stress (60 min at 50 degrees C or in the presence of 100 mM H(2)O(2)) similar to that of wild-type germlings. Furthermore, conidia of the tpsA mutant show a rapid loss of viability upon storage. These results are consistent with a role of trehalose in the acquisition of stress tolerance. Inactivation of the tpsA gene also results in increased steady-state levels of sugar phosphates but does not prevent growth on rapidly metabolizable carbon sources (glucose, fructose) as seen in Saccharomyces cerevisiae. This suggests that trehalose 6-phosphate is a physiological inhibitor of hexokinase but that this control is not essential for proper glycolytic flux in A. nidulans. Interestingly, tpsA transcription is not induced in response to heat shock or during conidiation, indicating that trehalose accumulation is probably due to a post-translational activation process of the trehalose 6-phosphate synthase.
In filamentous ascomycetes, HOG-like signal transduction cascades are involved in the resistance to hyper-osmotic conditions and to dicarboximides and phenylpyrroles. The histidine kinase (HK) Bos1 ...and the mitogen-activated protein kinase (MAPK) Sak1 are important for the adaptation to hyper-osmotic and oxidative stress, development, and pathogenicity in the phytopathogenic fungus
Botrytis cinerea. However,
bos1Δ and
sak1Δ mutants created previously, also presented different phenotypes, especially the
sak1Δ mutants were not resistant to high fungicide concentrations. Since both single mutants were constructed in different parental strains, phenotypic variations due to the genetic background might be suspected. In order to establish the relationship between both protein kinases, we analyzed Sak1 phosphorylation under the control of the Bos1 HK and we realized epistasis analysis between
bos1Δ and
sak1Δ mutations through the construction of isogenic single and double mutants. Our results show that Bos1 negatively regulates Sak1 phosphorylation and that Bos1 regulates certain phenotypes independently of Sak1. They include fungicide susceptibility, adaptation and conidiation on high neutral osmolarity.
Filamentous ascomycetes contain large numbers of histidine kinases (HK) that belong to eleven classes. Members of class III from different species were previously shown to be involved in ...osmoregulation and resistance to dicarboximide and phenylpyrrole fungicides. We have inactivated the gene encoding the single group III HK, BOS1, in the economically important plant pathogen Botrytis cinerea. BOS1 inactivation had pleiotropic effects on the fungus. Besides the expected osmosensitivity and resistance to fungicides, null mutants presented additional characteristics indicating that BOS1 is necessary for normal macroconidiation and full virulence. On standard culture media, null mutants very rarely formed conidiophores and those few conidiophores failed to produce conidia. This defect could be partially restored with 1 M sorbitol, suggesting that another BOS1-independent signal cascade may be involved in macroconidiation. The mutants were not found to be hypersensitive to various oxidative stresses but were more resistant to menadione. Finally, pathogenicity tests showed that bos1-null mutants were significantly reduced in the ability to infect host plants. Appressorium morphogenesis was not altered; however, in planta growth was severely reduced. To our knowledge, this is the first class III HK characterized as a pathogenicity factor in a plant-pathogenic ascomycete.
Forty three Chilean Botrytis cinerea isolates of different fenhexamid sensitivities, obtained from table grapes, were phenotypically analyzed and sequenced for the erg27 gene that encodes the ...3-ketoreductase enzyme. Fifteen isolates were highly resistant to fenhexamid (HydR3⁺) with conidial germination EC₅₀ values >5 μg·mL⁻¹ and colony growth EC50 values >2 μg·mL⁻¹. Five isolates had slight to moderate resistance levels (HydR3⁻) with conidial germination EC50 values between 0.7 and 2.6 μg·mL⁻¹ and colony growth EC50 values between 0.4 and 3 μg·mL⁻¹. Twenty-three isolates were fenhexamid sensitive (HydS) (conidial germination and colony growth EC50 values <0.1 μg·mL⁻¹). Resistance to anilinopyrimidine (phenotype AniR1), benzimidazole (phenotype BenR1) and dicarboximide fungicides (phenotype ImiR1) was common among isolates tested. When HydR3⁻ and HydR3⁺ sequences were compared with fenhexamid-resistant French isolates, it was verified that all the HydR3⁺ had a modification in the C-terminal at position 412 of the protein, close to the putative transmembrane domain responsible for fenhexamid resistance. The HydR3⁻ isolates showed six specific amino acid changes in the sequenced region of the erg27 gene, between positions 199 and 408 of the protein, with three of these described for the first time.
Summary
The role of cAMP signalling during germination of asexual spores (conidia) of the filamentous fungus Aspergillus nidulans was investigated. A. nidulans
strains defective for adenylate cyclase ...(CyaA) or for the functionally overlapping
cAMP‐dependent protein kinase (PkaA) and newly characterized SchA protein kinase,
homologous to Saccharomyces cerevisiae Sch9, show altered trehalose mobilization
and kinetics of germ tube outgrowth, in addition to other defects in colony formation.
cAMP‐dependent trehalose breakdown is triggered by the addition of a carbon source
independently of further catabolism, suggesting that cAMP signalling controls early
events of conidial germination in response to carbon source sensing. Additional results
suggest that cAMP has targets other than PkaA and SchA and that PkaA retains activity
in the absence of cAMP. Conversely, PkaA regulates cAMP levels in A. nidulans
because these are elevated by ≈ 250‐fold in a strain that lacks PkaA. Furthermore,
analysis of mutant strains impaired in both adenylate cyclase and RasA GTPase previously
implicated in the control of A. nidulans spore germination suggested that
RasA and cAMP signalling proceed independently during germination in A. nidulans.
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