Characterization and genetic analysis of laboratory mutants of <Emphasis Type="Italic">Cochliobolus heterostrophus</Emphasis> resistant to dicarboximide and phenylpyrrole fungicides

 Laboratory mutants of Cochliobolus heterostrophus resistant to iprodione were obtained after chemical mutageneses. All the mutants were able to grow on the medium amended with iprodione 100 μg/ml. They showed positive cross-resistance to procymidone and fludioxonil and were sensitive to high osmolarity. Crosses between the mutant and a wild-type strain revealed that the fungicide resistance and osmotic sensitivity traits were inherited by their offspring in a 1 : 1 mutant/wild type ratio, indicating that the mutant phenotypes in these strains were due to alteration at a single gene locus. Results from allelism tests indicated that three genes (Dic1, Dic2, Dic3) conferred the mutant phenotypes. Among them, Dic1 mutant strains were classified into three types on the basis of their phenotypes. The first type was moderately resistant to the fungicides and less sensitive to osmotic stress than the other Dic1 mutant strains. The second type showed moderate fungicide resistance, but growth was inhibited under lower osmotic stress (50 mM KCl). The other Dic1 mutant strains grew well on medium containing iprodione and fludioxonil even at a concentration of 100 μg/ml and were highly sensitive to osmotic stress. The Dic2 and Dic3 mutant strains had moderate resistance to the fungicides with low-level osmotic sensitivity. The Dic1 gene was epistatic to Dic2 and Dic3 for fungicide resistance and hypostatic to them for osmotic sensitivity. These results suggest that the osmoregulatory system is involved in fungicide resistance in laboratory mutants of C. heterostrophus. Received: March 14, 2002 / Accepted: August 13, 2002     

Characterization of genetic and biochemical mechanisms of fludioxonil and pyrimethanil resistance in field isolates of Penicillium digitatum

Genetic and biochemical mechanisms of fludioxonil and pyrimethanil resistance in isolates of Penicillium digitatum were evaluated and compared to those characterized in other fungi. Resistant isolates were naturally occurring in packinghouses and were not associated with crop losses. For the phenylpyrrole fludioxonil, EC50 values were 0.02 to 0.04 microg/ml for sensitive, 0.08 to 0.65 microg/ml for moderately resistant (MR), and > 40 microg/ml for highly resistant (HR) isolates. Two fludioxonil-sensitive isolates evaluated were also significantly more sensitive to the unrelated dicarboximide fungicide iprodione, that also disrupts osmotic regulation, than the MR and HR isolates. There was no consistent relationship, however, between the HR and MR isolates and their sensitivity to iprodione or osmotic stress. Although, two nucleotide substitutions were found in a sequence analysis of the N-terminal amino acid repeat region of the os-1-related histidine kinase gene among isolates of P. digitatum, these were not correlated with fludioxonil resistance. In mycelia not exposed to fludioxonil, the amount of phosphorylated OS-2-related protein (PdOS-2) was higher in fludioxonil-sensitive isolates and lowest in the HR isolate. An increase in PdOS-2 was observed for sensitive and resistant isolates after exposure to fludioxonil. In addition, glycerol content in untreated mycelia of the fludioxonil-sensitive isolate was significantly higher than in resistant isolates. After exposure to fludioxonil, glycerol concentrations significantly increased in the sensitive and MR isolates, but not in the HR isolate. Thus, our studies indicate that the mode of action of fludioxonil in P. digitatum is probably the mitogen-activated protein kinase pathway that stimulates glycerol synthesis in sensitive and MR isolates. The general suppression of this pathway in resistant isolates was supported by the fact that growth and sporulation of MR and HR isolates were significantly reduced from that of sensitive isolates. In studies on the mode of action of anilinopyrimidines (AP), EC50 values for mycelial growth of P. digitatum and the previously characterized Botrytis cinerea were determined for cyprodinil and pyrimethanil using a defined culture medium without and with the addition of selected amino acids and homocysteine. The addition of amino acids resulted in a reduced toxicity of the two AP fungicides in both fungi, but the effect of each additive was significantly lower for P. digitatum than for B. cinerea. This suggests that methionine biosynthesis is not the primary target site of APs in P. digitatum.     

四川猕猴桃灰霉病菌对4种杀菌剂的抗药性检测

为明确猕猴桃灰霉病菌对常见杀菌剂的抗药性状况, 本试验于2016年-2018年调查统计了四川猕猴桃生产中防控灰霉病所用的药剂种类和施药频次, 并从四川8个猕猴桃主产区采集病叶?病花和病果, 经单孢分离获得122个灰葡萄孢 Botrytis cinerea 菌株, 采用最低抑制浓度法(MIC)测定其对4种化学药剂嘧霉胺?咯菌腈?腐霉利和异菌脲的抗药性?结果表明:用于四川猕猴桃灰霉病防控的药剂主要有8种, 其中嘧霉胺?异菌脲和腐霉利3种药剂施用频次最高; 猕猴桃灰霉病菌对嘧霉胺?咯菌腈?腐霉利和异菌脲的抗性频率分别达95.08%?86.07%?80.33%和5.74%, 且不同产区的灰霉病菌对药剂的抗药性频率不同; 所测菌株对4种杀菌剂的敏感性类型共有8种, 其中以Pyr RFlu RPcm RIpr S(对嘧霉胺?咯菌腈?腐霉利表现抗性, 对异菌脲表现敏感)类型为主, 该类型的菌株占65.57%, 对4种杀菌剂均表现抗性的菌株为7株?表明四川省猕猴桃灰霉病菌对嘧霉胺?咯菌腈?腐霉利和异菌脲已经产生了抗药性, 对异菌脲抗药性较低, 迫切需要筛选新的杀菌剂防治猕猴桃灰霉病?     

Impact of fludioxonil resistance on fitness and cross-resistance profiles of Altrenaria solani laboratory mutants

Sensitivity and inherent resistance risk of Alternaria solani to fludioxonil, cross-resistance profiles and the potential implications of resistance mutations on fitness parameters were investigated. Fludioxonil was highly effective against a wild type A. solani field strain both in vitro (EC₅₀?=?0.05 μg/mL) and in preventive applications on artificially inoculated tomato fruit. Mutants with low [Resistance factor (Rf): 15 based on EC₅₀], medium (Rf: 150–300) and high (Rf: > 1000) levels of phenylpyrrole resistance were isolated from the wild type strain at high frequencies following mutagenesis with UV irradiation and selection on fludioxonil containing medium. Resistant isolates retained their resistance levels even after 9 subcultures on fungicide-free growth medium while they could express their resistant phenotypes in planta. Investigation of cross-resistance relationships showed that fludioxonil resistance mutations also reduce the sensitivity of mutant strains to the aromatic hydrocarbon fungicide quintozene as well as the dicarboximides iprodione and vinclozolin. No cross-resistance was observed between fludioxonil and fungicides with different modes of action such as the sterol biosynthesis inhibitors (DMIs) imazalil and flusilazole and the carboxamide boscalid. All fludioxonil resistant isolates were more sensitive to the anilinopyrimidine pyrimethanil, while only two isolates were less sensitive to the QoI pyraclostrobin compared to the wild-type strain. Study of fitness determining parameters showed that resistance mutation(s) had no adverse effects on mycelial growth, conidial germination and sensitivity to osmotic stress while they had a pleiotropic effect on virulence and conidia production in resistant mutants. Results of the present study indicate that fludioxonil is a highly effective fungicide against A. solani, while the risk of resistance development to this fungicide is considered to be medium making fludioxonil an ideal alternative to high risk fungicides such as boscalid and pyraclostrobin whose performance against early blight has already been compromised by resistance development.

     

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.     

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.     

Induction of OS-2 phosphorylation in Neurospora crassa by treatment with phenylpyrrole fungicides and osmotic stress

Neurospora crassa is sensitive to phenylpyrrole fungicides and to high osmolarity. By contrast, the osmosensitive mutant os-2 is resistant to this class of fungicides. To understand the relationship between the phenylpyrroles fungitoxicity and the osmoregulatory pathway, we investigated the regulation of the mitogen-activated protein kinase (MAPK) OS-2 in Neurospora. The proteomic tools were used for the characterization of the OS-2 activation and for the measurement of the MAPK cascade induction in situ. Finally, the identification of the genes controlled by os-2 was attempted by comparing the proteomes of the wild type and the os-2 mutant strains. The results indicated that OS-2 is regulated by a dual phosphorylation on a Thr-X-Tyr motif. Furthermore, OS-2 undergoes a rapid phosphorylation after treatment with phenylpyrroles (fenpiclonil and fludioxonil) and under elevated salt concentrations in the surrounding medium. Finally, different enzymes involved in the metabolism of sugars and amino acids were identified as os-2-dependent.     

Characterization of fludioxonil-resistant and pyrimethanil-resistant phenotypes of Penicillium expansum from apple

Penicillium expansum is the primary cause of blue mold, a major postharvest disease of apple. Fludioxonil and pyrimethanil are two newly registered postharvest fungicides for pome fruit in the United States. To evaluate the potential risk of resistance development in P. expansum to the new postharvest fungicides, one isolate of each of thiabendazole-resistant (TBZ-R) and -sensitive (TBZ-S) P. expansum was exposed to UV radiation to generate fungicide-resistant mutants. Four fludioxonil highly-resistant mutants (EC(50) > 1,000 microg/ml) and four pyrimethanil-resistant mutants (EC(50) > 10 microg/ml) were tested for sensitivities to thiabendazole, fludioxonil, and pyrimethanil, and fitness parameters including mycelial growth, sporulation on potato dextrose agar (PDA), sensitivity to osmotic stress, and pathogenicity and sporulation on apple fruit. The stability of resistance of the mutants was tested on PDA and apple fruit. Efficacy of the three fungicides to control blue mold incited by the mutants was evaluated on apple fruit. Six fungicide-resistant phenotypes were identified among the parental wild-type isolates and their mutants based upon their resistance levels. All four fludioxonil highly-resistant mutants were sensitive to pyrimethanil and retained the same phenotypes of resistance to TBZ as the parental isolates. All four pyrimethanil-resistant mutants had a low level of resistance to fludioxonil with a resistance factor >15. The two pyrimethanil-resistant mutants derived from a TBZ-S isolate became resistant to TBZ at 5 microg/ml. After 20 successive generations on PDA and four generations on apple fruit, the mutants retained the same phenotypes as the original generations. All mutants were pathogenic on apple fruit at both 0 and 20 degrees C, but fludioxonil highly-resistant mutants were less virulent and produced fewer conidia on apple fruit than pyrimethanil-resistant mutants and their parental wild-type isolates. Compared with the parental isolates, all four fludioxonil highly-resistant mutants had an increased sensitivity to osmotic stress on PDA amended with NaCl, while the pyrimethanil-resistant mutants did not. Pyrimethanil was effective against blue mold caused by fludioxonil-resistant mutants at both 0 and 20 degrees C. Pyrimethanil and fludioxonil reduced blue mold incited by pyrimethanil-resistant mutants during 12-week storage at 0 degrees C but were not effective at 20 degrees C. TBZ was not effective against pyrimethanil-resistant mutants derived from TBZ-S wild-type isolates at room temperature but provided some control at 0 degrees C. The results indicate that: (i) a fitness cost was associated with fludioxonil highly resistant mutants of P. expansum in both saprophytic and pathogenic phases of the pathogen but not pyrimethanil-resistant mutants; (ii) pyrimethanil possessed a higher risk than fludioxonil in the development of resistance in P. expansum; and (iii) triple resistance to the three apple-postharvest fungicides could emerge and become a practical problem if resistance to pyrimethanil develops in P. expansum populations.     

不同抗性型灰葡萄孢菌Botrytis cinerea对不同作用机制杀菌剂的敏感性研究

不同类型杀菌剂对灰葡萄孢菌菌株的离体、活体毒力测定结果表明 ,苯并咪唑类的多菌灵、甲基硫菌灵之间 ,二甲酰亚胺类的腐霉利、异菌脲、菌核净之间表现交互抗性 ;苯并咪唑类和 N -苯氨基甲酸酯类乙霉威表现有负交互抗性趋势。离体测定中 ,对不同类型菌株以吡咯类杀菌剂咯菌腈 (EC50 200mg/ L) ,咯菌腈以及甾醇抑制剂戊唑醇、氟硅唑和丙环唑均表现出较高的活性。     

番茄叶霉病菌异菌脲抗药性突变体的诱导与生物学性状

测定了苯并咪唑类杀菌剂敏感-乙霉威抗性(BenS-DieR)、苯并咪唑类杀菌剂抗性-乙霉威敏感(BenR-DieS)和苯并咪唑类杀菌剂抗性-乙霉威抗性(BenR-DieR)3种类型的番茄叶霉病菌Cladosporium fulvum菌株对不同类型药剂的敏感性。结果表明,蕃茄叶霉病菌对供试药剂的敏感性与其对苯并咪唑类杀菌剂及乙霉威的敏感性无关。根据药剂对3类菌株EC50值的平均值, 16种杀菌剂抑制菌丝生长的活性依次为腐霉利＞乙烯菌核利＞异菌脲＞戊唑醇＞百菌清＞嘧霉胺＞醚菌酯＞代森锰锌＞8-羟基喹啉铜＞丙环唑＞苯醚甲环唑＞嘧菌酯＞灭锈胺＞烯酰吗啉＞烟酰胺＞三唑酮;抑制孢子萌发的活性依次为醚菌酯＞腐霉利＞百菌清＞乙烯菌核利＞灭锈胺＞8-羟基喹啉铜＞异菌脲＞代森锰锌＞嘧菌酯＞烟酰胺＞嘧霉胺＞戊唑醇＞丙环唑＞苯醚甲环唑＞三唑酮＞烯酰吗啉。通过紫外诱变共获得17株抗异菌脲突变体,突变频率为4.5×10-7。其中低抗、中抗和高抗菌株分别占 17.65%、70.59%和11.75%。这些突变体对腐霉利和乙烯菌核利表现交互抗性,对苯并咪唑类、脱甲基抑制剂(DMIs)、QoIs等药剂的敏感性与亲本菌株之间没有显著性差异,与亲本菌株在生长、产孢、致病能力等方面也无显著差异,但对渗透胁迫的敏感性要显著高于亲本。     

A Point Mutation in the Two-Component Histidine Kinase BcOS-1 Gene Confers Dicarboximide Resistance in Field Isolates of Botrytis cinerea

ABSTRACT Partial DNA fragments of Botrytis cinerea field isolates encoding the putative osmosensor histidine kinase gene (BcOS1) were cloned by polymerase chain reaction amplification and the predicted amino acid sequences were compared between dicarboximide-sensitive and resistant field isolates. The predicted BcOS1p is highly homologous to osmosensor histidine kinase OS1p from Neurospora crassa including the N-terminal six tandem repeats of approximately 90 amino acids. Four dicarboximide-resistant isolates of B. cinerea (Bc-19, Bc-45, Bc-682, and Bc-RKR) contained a single base pair mutation in their BcOS1 gene that resulted in an amino acid substitution in the predicted protein. In these resistant isolates, codon 86 of the second repeat, which encodes an isoleucine residue in sensitive strains, was converted to a codon for serine. The mutation of Botrytis field resistant isolates was located on the second unit of tandem amino acid repeats of BcOS1p, whereas the point mutations of the fifth repeat of OS1p confer resistance to both dicarboximides and phenylpyrroles and also osmotic sensitivity in Neurospora crassa. These results suggest that an amino acid substitution within the second repeat of BcOS1p is responsible for phenotypes of field resistant isolates (resistant to dicarboximides but sensitive to phenylpyrroles, and normal osmotic sensitivity) in B. cinerea.     

Effect of null mutations in the AbNIK1 gene on saprophytic and parasitic fitness of Alternaria brassicicola isolates highly resistant to dicarboximide fungicides

Alternaria brassicicola field isolates highly resistant to dicarboximide fungicides (DCF) and carrying non‐sense codons or micro‐deletions in the AbNIK1 gene encoding a group III histidine kinase were characterized at the protein level using specific antibodies. These isolates, as well as laboratory mutants expressing the same resistant phenotype, were shown to correspond to null mutants. The fitness of these isolates was compared to that of wild‐type isolates by measuring i) mycelial growth, ii) sporulation and iii) conidial germination under standard conditions. None of these factors were affected in the resistant isolates. Mycelial growth was also measured under stress conditions. Unlike suboptimal incubation temperatures or exposure to the superoxide ion generator menadione, osmotic stress generated by high sorbitol concentrations in the culture medium significantly affected the growth of AbNIK1‐null mutants compared to wild‐types. The effects of these mutations on parasitic fitness were estimated under both controlled and field conditions after inoculation of radish with individual isolates, to measure their aggressiveness, or with mixed conidial suspensions, to measure their competitive ability against wild‐type isolates. These tests on radish seedlings revealed that DCF‐resistant isolates were still aggressive. Despite this, the data obtained in the competition experiments showed that the loss of the ability to synthesize AbNIK1p in A. brassicicola DCF‐resistant mutants was linked to a severe decrease in their competitiveness.     

我国人参产区灰霉病菌抗药性研究初报

人参灰霉病给人参生产造成巨大损失。本研究选取人参生产上常用的4种杀菌剂,对东北人参产区分离的102株灰霉菌进行抗性检测。结果表明,灰霉菌对多菌灵carbendazim(C)、嘧霉胺pyrimethanil(P)、异菌脲iprodione(I)、咯菌腈fludioxonil(F)的抗性频率分别为100%、86.27%、73.53%和30.39%,辽宁和吉林分离菌株对嘧霉胺、异菌脲的抗性高于3省平均水平。分离菌株对4种杀菌剂共有7种抗性类型,即C_RP_RI_RF_S、C_RP_RI_RF_R、C_RP_RI_SF_S、C_RP_SI_SF_S、C_RP_SI_RF_S、C_RP_RI_SF_R和C_RP_SI_SF_R,其所占比例分别为39.22%、26.47%、14.71%、7.84%、5.88%、4.90%和0.98%,未发现对4种杀菌剂均敏感的灰霉菌菌株。研究结果对于指导人参产区防治灰霉病杀菌剂的合理选用,有效降低人参农药残留,延缓灰霉菌抗性产生具有重要意义。     

马铃薯早疫病菌对咯菌腈的敏感基线及其对不同药剂的交互抗性

采用菌丝生长速率法测定了河北省马铃薯主产区的122株早疫病菌Alternaria alternata对咯菌腈的敏感性,并测定了紫外诱导的15个抗咯菌腈突变菌株及其3个敏感亲本菌株对咯菌腈、异菌脲、腐霉利、苯醚甲环唑、吡唑醚菌酯的敏感性。田间马铃薯早疫病菌群体对咯菌腈保持高度敏感,122株早疫病菌对咯菌腈的EC₅₀值在0.0025~0.0857 μg/mL之间,平均值为0.0404±0.0171 μg/mL,相差34.87倍,呈近似正态分布,可作为早疫病菌对咯菌腈的敏感基线。此外,咯菌腈与吡唑醚菌酯和苯醚甲环唑之间无交互抗性关系,而与异菌脲和腐霉利之间存在正交互抗性关系。在田间药效试验中,50%咯菌腈WP施药量为105、115 g/hm²时,对马铃薯早疫病防效达80%以上,显著高于常规药剂80%代森锰锌WP 1 350 g/hm²和25%丙环唑EC 37.5 g/hm²的防效。表明咯菌腈可作为吡唑醚菌酯和苯醚甲环唑的替代药剂用于马铃薯早疫病的防治。     

Activity of fungicides and modulators of membrane drug transporters in field strains of Botrytis cinerea displaying multidrug resistance

In Botrytis cinerea, multidrug resistant (MDR) strains collected in French and German vineyards were tested in vitro, at the germ-tube elongation stage, towards a wide range of fungicides. Whatever the MDR phenotype, resistance was recorded to anilinopyrimidines, diethofencarb, iprodione, fludioxonil, tolnaftate and several respiratory inhibitors (e.g., penthiopyrad, pyraclostrobin). In MDR1 strains, overproducing the ABC transporter BcatrB, resistance extended to carbendazim and the uncouplers fluazinam and malonoben. In MDR2 strains, overproducing the MFS transporter BcmfsM2, resistance extended to cycloheximide, fenhexamid and sterol 14α-demethylation inhibitors (DMIs). MDR3 strains combined the overexpression of both transporters and exhibited the widest spectrum of cross resistance and the highest resistance levels. The four transport modulators, amitriptyline, chlorpromazine, diethylstilbestrol, and verapamil, known to affect some ABC transporters, were tested in B. cinerea. In our experimental conditions, the activity of several fungicides was only enhanced by verapamil. Interestingly, synergism was only recorded in MDR2 and/or MDR3 isolates treated with tolnaftate, fenhexamid, fludioxonil or pyrimethanil, suggesting that verapamil may inhibit the MFS transporter BcmfsM2. This is the first report indicating that a known modulator of ABC transporters could also block MFS transporters.     

禾谷镰刀菌对苯基吡咯类杀菌剂咯菌腈的抗性机制

由禾谷镰刀菌引起的小麦赤霉病是世界小麦生产上的重要真菌病害。为了进一步明确禾谷镰刀菌对苯基吡咯类杀菌剂咯菌腈产生抗性的机制,本文以前期室内通过药剂驯化方式得到的4株禾谷镰刀菌对咯菌腈的高水平抗性突变体(其抗性倍数在318.2～782.9之间)为主要研究材料,采用生物测定及分子生物学等方法开展了禾谷镰刀菌对咯菌腈的抗性机制研究。结果表明：供试禾谷镰刀菌抗咯菌腈突变体对小麦幼穗的致病力降低了约50%,部分菌株(2XZ-4R)甚至完全丧失了对小麦的致病能力;抗性突变体对渗透胁迫(0.5 mol/L NaCl,1.0 mol/L MgCl₂, 1.0 mol/L葡萄糖或1.0 mol/L甘露醇)高度敏感,且菌丝生长抑制率较敏感菌株降低约50%以上,表明其环境适合度显著下降。同时,抗性突变体中苯丙氨酸解氨酶(PAL)、过氧化物酶(POD)和多酚氧化酶(PPO)活性较敏感菌株均升高2倍以上。分子生物学分析发现,供试抗性突变体中候选靶标基因(FgOs1和FgOs5)的表达量显著下调(P<0.05),推测FgOs1和FgOs5可能参与了禾谷镰刀菌对咯菌腈抗性的形成过程。总之...     

Evolution of an Osmosensing Histidine Kinase in Field Strains of Botryotinia fuckeliana (Botrytis cinerea) in Response to Dicarboximide Fungicide Usage

ABSTRACT DNA sequence polymorphisms in the putative two-component histidine protein kinase encoded by the Daf1 gene have been identified within a sample of 5 sensitive and 27 dicarboximide-resistant field strains of Botryotinia fuckeliana (anamorph Botrytis cinerea). The gene of 3948 bp is predicted to encode a 1315-amino acid protein comprising an N-terminal region, an amino acid repeat region, which has been hypothesized to be the binding site for dicarboximide fungicide, and a C-terminal region encompassing kinase and response regulator domains. Two amino acid variants were distinguished among the sensitive strains characterized by alanine (group 1), or threonine (group 2), at position 1259 in the C-terminal region. All resistant strains could be classified into either group 1 or group 2 but, in addition, all showed changes in the second amino acid repeat region. On the basis of the differences in this repeat region, four classes of resistant strains were recognized; class 1 characterized by an isoleucine to serine mutation, class 2 by an isoleucine to asparagine mutation, class 3 by an isoleucine to arginine mutation (all at position 365), and class 4 by an isoleucine to serine mutation (position 365) as well as a glutamine to proline mutation (position 369). All classes showed similar low levels of resistance to iprodione and to vinclozolin, except for class 3 and class 4 strains, which show low resistance to iprodione but moderate (class 3) or high (class 4) resistance to vinclozolin. The classes as a group did not differ from sensitive strains in osmotic sensitivity measured as mycelial growth response, but some class 1 strains showed an abnormal morphology on osmotically amended medium. The evolution of the amino acid differences is discussed in relation to field observations. It is proposed that class 1 and class 2 strains arose by single mutations within the sensitive population, whereas classes 3 and 4 arose by single mutations within a resistant population.     

Survey of mutations of a histidine kinase gene BcOS1 in dicarboximide-resistant field isolates of Botrytis cinerea

Previously, we cloned a putative osmosensing histidine kinase gene (BcOS1) and revealed that a single amino acid substitution, isoleucine to serine at codon 365, conferred dicarboximide resistance in field isolates of Botrytis cinerea. This point mutation (type I) occurred within the restriction enzyme TaqI site of the wild-type BcOS1 gene. Thus, a procedure was developed for detecting the type I mutation of the BcOS1 gene using a polymerase chain reaction (PCR) in combination with restriction fragment-length polymorphism (RFLP). Diagnosis by PCR-RFLP was conducted on the 105 isolates isolated from 26 fields in Japan. All dicarboximide-sensitive isolates (49 isolates) had the wild-type BcOS1 gene, and the 43 isolates with the type I mutation were resistant to dicarboximides without exception. These data indicate that dicarboximide-resistant isolates with type I mutation are widespread throughout Japan. However, other types of dicarboximide resistance were detected among isolates from Osaka; among the 24 resistant isolates from Osaka, 12 had the BcOS1 gene without the type I mutation. BcOS1 gene sequencing of these resistant isolates classified them into two groups, type II and type III. The type II isolates have three amino acid substitutions within BcOS1p (³⁶⁸Val to Phe, ³⁶⁹Gln to His, and ⁴⁴⁷Thr to Ser). The type III isolates have two amino acid substitutions within BcOS1p (³⁶⁹Gln to Pro and ³⁷³Asn to Ser). These amino acid changes are located on the amino acid repeat domain in BcOS1p. The three types of resistant isolates were all moderately resistant to dicarboximides without significant osmotic sensitivity, and their pathogenicity on cucumber leaves was also very similar to that of the wild-type isolate.     

Sequence variation in the two-component histidine kinase gene of Botrytis cinerea associated with resistance to dicarboximide fungicides

The complete two-component histidine kinase gene (Bos1) was sequenced from eight dicarboximide-resistant (D^R) and six-sensitive (D^S) field isolates of Botrytis cinerea. Comparisons in the predicted amino acid sequences of Bos1 showed that each two D^R isolates had a single point mutation at amino acid position 365 from an isoleucine to serine (I365S) or to an asparagine (I365N). Three D^R isolates were characterized with a change from glutamine to proline at position 369 (Q369P) as well as an asparagine to serine at the position 373 (N373S). These mutations located within the 90-amino-acid repeats of Bos1 have been reported previously. One new mutation, however, was found in the D^R isolate 65-E8. In this isolate, a null mutation at the amino acid position 1040 in the Bos1 was detected. Inoculation tests showed that this isolate was nearly nonpathogenic to cucumber. After the Bos1 gene from the sensitive isolate 38B1 was transferred into the resistant isolate 65-E8, all transformants tested were sensitive to iprodione and capable of infecting cucumber. DNA fingerprint generated by micro-satellite primed-PCR showed that isolates were not clustered based on their sensitivity to iprodione. Results from this study indicate that mutations in the Bos1 gene associated with dicarboximide resistance are diverse in B. cinerea, and the Bos1 gene is associated with virulence of B. cinerea.