Inhibitory effect of isoprothiolane on metabolism of a phosphoramidate by isolates of Pyricularia oryzae cav. in relation to fungicide sensitivity

The inhibitory effect of isoprothiolane(diisopropyl 1,3-dithiolan-2-ylidenemalonate), a fungicide for rice blast control, on the metabolism of dibutyl N-methyl-N-phenylphosphoramidate (BPA) by 20 isolates of Pyricularia oryzae was examined in relation to sensitivity of the isolates to the reference fungicide IBP(S-benzyl diisopropylphosphorothiolate). The isolates were divided into five groups based on the modes of BPA metabolism and the inhibition of BPA metabolism by isoprothiolane. Every isolate in groups I and II, which was either a field isolate or a stock culture, decomposed BPA rapidly and produced both hydroxylated and N-demethylated BPA as metabolites. BPA decomposition by these isolates was strongly inhibited by isoprothiolane, resulting in the decreased production of both metabolites in group I and of the hydroxylated metabolite in group II. These isolates were almost equally sensitive to isoprothiolane. Isolates in groups III, IV, and V were all obtained from selection of the fungus mutants found growing on media containing isoprothiolane. Isolates in group III, derived by plating large numbers of conidia, did not decompose BPA to any extent. Mutants of groups IV and V were obtained from fast-growing sectors on agar containing isoprothiolane. Both these groups decomposed BPA, but isolates belonging to group IV produced copious amount of N-demethylated BPA whereas isolates in group V did not. BPA metabolism by these in vitro mutants in groups III, IV, and V was not inhibited by isoprothiolane. Thus, the inhibitory effect of isoprothiolane on BPA metabolism was correlated with sensitivity of an isolate to isoprothiolane. The inhibitory effect of IBP on BPA metabolism was not always correlated with the sensitivity of an isolate to IBP.     

Metabolism of a phosphoramidate by Pyricularia oryzae in relation to tolerance and synergism by a phosphorothiolate and isoprothiolane

Metabolism of dibutyl N-methyl-N-phenylphosphoramidate (BPA) by mycelial cells of Pyricularia oryzae was studied to elucidate the mechanism of synergism and negatively correlated cross-resistance in fungicidal action between phosphoramidates and phosphorothiolate derivatives. Rapid metabolism of BPA by a wild-type strain through hydroxylation and N-demethylation was observed. The metabolism was inhibited by diisopropyl S-benzyl phosphorothiolate (IBP; Kitazin P) and by isoprothiolane (diisopropyl 1,3-dithiolan-2-ylidenemalonate; Fuji-One). This inhibition of BPA metabolism is probably the mechanism of synergistic fungicidal action between the phosphoramidate and the thiol derivatives. The metabolism was, however, not inhibited by S-1358 (S-butyl S′-p-tert-butylbenzyl N-3-pyridyldithiocarbonimidate; Denmert) or triarimol [α-(2,4-dichlorophenyl)-α-phenyl-5-pyrimidinemethanol; EL-273], both of which are considered to be inhibitors of hydroxylation of a methyl radical in ergosterol biosynthesis. The metabolism of BPA by P. oryzae was much slower when mutants selected for IBP resistance and for isoprothiolane resistance were used. This phenomenon probably explains the differential sensitivity to phosphoramidate of wild-type strains and mutants resistant to the thiol derivatives.     

Failure to detect highly resistant strains in dicarboximide resistant field populations of Botrytis cinerea1

Field isolates of Botrytis cinerea with moderate levels of resistance to dicarboximide fungicides (ED50 1.0–4.9 μg ml^?1) and to dicloran were obtained from glasshouses where vinclozolin and iprodione failed to control grey mould. From sensitive and moderatcly-resistant cultures, laboratory isolates were selected on dicarboximide-amended medium, which were highly resistant to these fungicides (ED50 125->3000 μg ml^?1). Conidia of all the resistant isolates germinated well on media amended with 100 μg ml^?1 of the dicarboximides vinclozolin, iprodione, procymidone and myclozolin and with 5 μg ml^?1 of metomeclan. However, the spores of the moderately resistant isolates did not germinate on 100 μg ml^?1 metomeclan while the spores of the highly resistant isolates germinated well. Using media with 100 μg ml^?1 of metomeclan to distinguish between the two phenotypes, no highly resistant strain was detected among 312 resistant samples from five cucumber glasshouses with a high frequency of moderately resistant strains. From air-borne inoculum of five glasshouses with 100% resistant populations, 1604 colonies were recovered on vinclozolin-amended (100 μg ml^?1) medium and none on metomeclan-amended (100 μg ml^?1) medium. It is concluded that strains of B. cinerea highly resistant to dicarboximides are absent from field populations.     

稻瘟病菌对稻瘟灵抗性遗传研究

在离体条件下就稻瘟病菌对稻瘟灵抗性的诱导、抗性水平和遗传进行了研究。结果表明,供试３个小种（ＺＡ_４９、ＺＦ_１和ＺＤ_１）４个菌株分别经稻瘟灵５０μｇ／ｍｌ、１００μｇ／ｍｌ和稻瘟灵１００μｇ／ｍｌ＋亚硝基胍０.５μｇ／ｍｌ的３种处理诱变,均得到了抗稻瘟灵突变株,在含稻瘟灵的培养基中加入诱变剂亚硝基胍可显著提高稻瘟病菌对稻瘟灵抗性的突变率。上述３个不同处理获得的突变株的抗性水平相似,为野生型亲本的２.８～８.８倍。突变株对稻瘟灵的抗性在单分生孢子无性系后代可以稳定遗传。     

辽宁省稻瘟病菌对稻瘟灵的敏感性监测

 利用菌丝生长速率法,测定了自辽宁省主产稻区2009-2010年分离的87株稻瘟病菌对稻瘟灵的敏感性。结果表明,稻瘟灵的抑制中浓度EC₅₀值为0.35~7.01 μg/mL,敏感性差异达20倍;且2009年稻瘟病菌对稻瘟灵的敏感性高于2010年。根据野生敏感型病原群体对药剂敏感性呈正态分布的原理,辽宁省稻瘟病菌对稻瘟灵的敏感性基线定为(1.77±0.80)μg/mL,辽宁省主要稻区均有抗稻瘟灵菌株出现,除2010年在铁岭稻产区监测到1株中抗菌株外,大部分稻瘟病菌株都表现为低抗水平。稻瘟病菌不同生理小种群对稻瘟灵的敏感性无明显差别。     

水稻稻瘟病菌对烯肟菌胺的抗性风险评估及抗性机制初探

 采用菌丝生长速率法测定了100株采自我国主要水稻产区的水稻稻瘟病菌对烯肟菌胺的敏感性, 结果表明, 其EC₅₀分布于0.011 1~0.295 6 μg·mL^-1, 平均EC₅₀=(0.078 6±0.056 1) μg·mL^-1。供试菌株对烯肟菌胺的敏感性分布呈单侧峰曲线, 未出现抗药性亚群体, 可将该曲线作为稻病瘟菌对烯肟菌胺的敏感性基线。通过室内药剂驯化获得了7株抗药突变体, 突变频率为1.11×10^-4, 其中2株高抗突变体NJ0811-I和A10的抗性水平大于1 000倍, 抗药性性状能稳定遗传, 致病力显著弱于其亲本菌株;5株低抗突变体抗性水平在2.05～4.55倍之间, 抗药稳定性差, 适合度与亲本无显著性差异。交互抗药性结果表明, 烯肟菌胺与嘧菌酯存在正交互抗药性, 与田间防治稻瘟病常用药剂稻瘟灵、异稻瘟净无交互抗药性。综合分析表明, 稻瘟病菌对烯肟菌胺可能存在低到中等抗性风险。进一步克隆了抗药突变体及其亲本的cytb基因, CYTB氨基酸序列比对结果表明, 2株高抗突变体均在143位由甘氨酸突变为丝氨酸(G143S), 建立了高抗菌株的AS-PCR分子检测方法;而5株低抗突变体cytb基因未发生点突变, 推测可能存在其他的抗性分子机制。     

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.     

Resistance profiles of Botrytis cinerea populations to several fungicide classes on greenhouse tomato and strawberry in Lebanon

Tomato and strawberry are the most important protected crops in Lebanon and are seriously affected by grey mould disease, caused by Botrytis cinerea. In the present study, the fungicide sensitivity assays revealed medium to high frequencies of B. cinerea isolates resistant to benzimidazoles, dicarboximides, and anilinopyrimidines on tomato and strawberry. Fludioxonil- and boscalid-resistant mutants were uncommonly found at generally low frequency on both crops. Resistance to fenhexamid was detected in only one site on tomato but in most sites on strawberry with high frequencies, and the occurrence of resistance to QoI fungicides was ascertained on both crops. The majority of the tested isolates (>90%) exhibited multiple fungicide resistance, and isolates resistant to the seven antibotrydial fungicide classes were detected on strawberry in three locations. A high level of resistance was shown by B. cinerea mutants resistant to boscalid, fenhexamid, and QoI fungicides, while two levels of moderate and high resistance to anilinopyrimidines were identified. Genetic analysis revealed point mutations in the target genes commonly associated with resistance in B. cinerea isolates, with all mutants resistant to dicarboximides, fenhexamid, boscalid, and QoI fungicides carrying single-nucleotide polymorphims in BcOS1 (I365S/N, Q369P, and N373S), Erg27 (F412V/I), SdhB (H272R/Y), and cytb (G143A) genes, respectively. The general incorrect use of fungicides has caused the development and spread of fungicide resistance as a widespread phenomenon on protected tomato and strawberry in Lebanon. The implementation of appropriate antiresistance strategies is highly recommended.     

Recovery and characterization of asexual recombinants ofMagnaporthe grisea

Eleven nitrate non-utilizing (nit) mutants were recovered from six field isolates ofMagnaporthe grisea that had different degrees of sensitivity to the blasticide kitazin P (iprobenfos, IBP). Allnit mutants were resistant to chlorate and there were no significant differences in hyphal growth rate, conidial production, sensitivity to IBP, and pathogenicity betweennit mutants and their parent isolates.nit phenotypes and IBP-resistance were used as two independent genetic markers to study asexual recombination inM. grisea. Asexual recombinants were recovered from the heterokaryotic mycelium of three inter-strain pairings, namely, DY2-3(nitl-LR) + A7-3 (nitM-S); A7-3 (nitM-S) + F4-2 (nitl-MR); and F4-2 (nitl-MR) + DY2-4 (nitA-LR), at a frequency of 7.31%, 14.00% and 8.63%, respectively. The growth rate, conidial production and pathogenicity of asexual recombinants were similar to those of parental strains. We concluded that asexual recombination resulting from hyphal fusion might contribute to variability inM. grisea. http://www.phytoparasitica.org posting Nov. 14, 2005.     

Biological and molecular characterization of laboratory mutants of Cercospora beticola resistant to Qo inhibitors

The resistance to strobilurin-related fungicides and its molecular basis in laboratory mutant isolates of Cercospora beticola was investigated. After ultraviolet mutagenesis, mutants with high, moderate or low resistance levels to pyraclostrobin were isolated from a wild-type strain of C. beticola. Fungitoxicity tests on the response of resistant isolates on medium containing pyraclostrobin and salicylhydroxamate (SHAM), a specific inhibitor of cyanide-resistant (alternative) respiration, indicated that the biochemical mechanism of alternative oxidase was not responsible for the reduced sensitivity to pyraclostrobin for half of the mutants. Cross-resistance studies with other inhibitors of the cytochrome bc ₁ complex of the mitochondrial respiratory chain showed that the mutation(s) for resistance to pyraclostrobin also reduced the sensitivity of mutant strains to other Qo inhibitors such as azoxystrobin and fenamidone, but not to the Qi inhibitor cyazofamid. No effect of pyraclostrobin-resistant mutation(s) on fungitoxicity of the carboxamide boscalid, the triazoles epoxiconazole and flutriafol and to the benzimidazole benomyl, which affect other cellular pathways or other steps of the respiratory chain, was observed. Study of fitness parameters showed that most mutants had a significant reduction in sporulation and pathogenicity compared to the wild-type parental isolate. However, experiments on the stability of the resistant phenotype did not show a significant reduction of the resistance for half of the mutants when grown for at least four generations on pyraclostrobin-free medium. Molecular analysis of cytochrome b cDNA, isolated from the wild-type and the pyraclostrobin-resistant mutant isolates, revealed two novel amino acid replacements at positions involved in Qo resistance in other species. The glycine (GGT) to serine (AGT) replacement at position 143 (G143S) was found in the isolate with the highly resistant phenotype. The second amino acid change was the replacement of phenylalanine (TTC) by valine (GTC) at position 129 (F129V), which was found in a mutant strain with the moderately resistant phenotype. Four additional mutations located in conserved regions of the mitochondrial cytochrome b gene (I154L, N250D, E256G and V261D) were detected in some mutant isolates of C. beticola but their possible role in Qo-resistance needs further investigation. This is the first study reporting C. beticola strains resistant to Qo inhibitor fungicides due to the biochemical mechanism of target-site modification, resulting from amino acid changes in the mitochondrial cytochrome b␣gene.     

Identification of a novel point mutation in the <Emphasis Type="Italic">β</Emphasis>-tubulin gene of <Emphasis Type="Italic">Botrytis cinerea</Emphasis> and detection of benzimidazole resistance by a diagnostic PCR-RFLP assay

The molecular basis of resistance to benzimidazole fungicides with laboratory and field mutant isolates of Botrytis cinerea was investigated. After chemical mutagenesis with N-methyl-N-nitrosogouanidine (NMNG) two different benzimidazole-resistant phenotypes were isolated on media containing carbendazim or a mixture of carbendazim and diethofencarb. The mutant isolates from the fungicide-mixture-containing medium were moderately resistant to carbendazim with wild-type tolerance to diethofencarb while mutant isolates from carbendazim-containing medium were highly resistant to carbendazim but sensitive to diethofencarb. The studied field isolates were highly resistant to benzimidazoles and sensitive to diethofencarb. Study of fitness characteristics of benzimidazole highly-resistant isolates showed that the resistance mutation(s) had no apparent effect on fitness-determining parameters. Contrary to this, the moderately benzimidazole-resistant strains, with no increased diethofencarb sensitivity, had a significant reduction in certain ecological fitness-determining characteristics. Analysis of the sequence of the β-tubulin gene revealed two amino acid replacements in the highly benzimidazole-resistant mutants compared to that of the wild-type parent strain. One was the glutamic acid (GAG) to alanine (GCG) change at position 198 (E198A), identified in both laboratory and field highly benzimidazole-resistant isolates, a mutation previously implicated in benzimidazole resistance. The second was a novel benzimidazole resistance mutation of glutamic acid (GAG) to glycine (GGG) substitution at the same position 198 (E198G), identified in a highly benzimidazole-resistant laboratory mutant strain. Molecular analysis of the moderately benzimidazole-resistant strains revealed no mutations at the β-tubulin gene. A novel diagnostic PCR-RFLP assay utilising a BsaI restriction site present in the benzimidazole-sensitive (E198) but absent in both resistant genotypes (E198G and E198A) was developed for the detection of both amino acid replacements at the β-tubulin gene.     

Sensitivity of populations ofBotrytis cinerea to triazoles,benomyl and vinclozolin

Sensitivity of field isolates (121) ofBotrytis cinerea from France (1992), Germany (1979–1992), Israel (1990) and the Netherlands (1970–1989) to the triazoles tebuconazole and triadimenol, the benzimidazole benomyl and the dicarboximide vinclozolin were tested in radial growth experiments. Resistance to benomyl (in 21 to 100% of isolates tested) and vinclozolin (in 25 to 71% of isolates tested) was common in most countries. EC₅₀s (concentrations of fungicides inhibiting radial mycelial growth ofB. cinerea on B5-agar by 50%) for tebuconazole and triadimenol ranged between 0.01–1.64 and 0.4–32.6g ml^–1, respectively, and were log-normally distributed. The variation factor (ratio between EC₅₀s of the least and most sensitive isolate tested) amounts 164 and 82 for tebuconazole and triadimenol, respectively. These values are comparable to those for azole fungicides applied in control of other pathogens. Hence, variation in sensitivity to triazoles can probably not explain limited field performance of triazoles towardsB. cinerea. Isolates from south west Germany (1992) were significantly less sensitive to tebuconazole than isolates collected earlier in Germany, Israel and the Netherlands. Such less sensitive populations may contribute to the limited field performance of DMI fungicides towardsB. cinerea. The sensitivity of isolates from south west Germany to tebuconazole was similar to that of DMI-resistant mutants generated in the laboratory. These mutants displayed stable resistance with Q-values (ratio between EC₅₀ of resistant mutant and wild type isolate) between 5 and 20. Sensitivity of field isolates and laboratory mutants to tebuconazole and triadimenol was correlated.     

番茄灰霉病菌对咯菌腈的抗性诱变及抗药突变体的生物学特性

为评估番茄灰霉病菌Botrytis cinerea对咯菌腈的抗性风险,就室内经紫外照射获得抗药突变体的方法及抗性突变体的生物学性状进行了研究。结果表明:番茄灰霉病菌分生孢子的紫外照射亚致死时间为90~120 s;经亚致死时间紫外照射后,4个亲本菌株中有2个菌株共产生了6个抗咯菌腈的突变体,其EC50值是亲本菌株的310倍以上,抗性突变频率为3.13×10-7;经紫外照射诱变获得的所有抗性突变体在菌丝生长速率、产孢量、产菌核能力及其在番茄果实上的致病性方面均比其亲本菌株明显降低。相关分析显示,所得抗咯菌腈突变体对氟啶胺、啶菌唑、啶酰菌胺和嘧霉胺无交互抗性。表明番茄灰霉病菌对咯菌腈的抗药性风险较低。     

灰葡萄孢多药抗性菌株的筛选和鉴定

从浙江杭州市售草莓上分离得到83个灰葡萄孢菌株,测定了这些菌株对苯醚菌酯、多菌灵和异菌脲的敏感性,筛选出对这3种药剂同时产生了抗性的两个菌株HZ021和HZ054,其在马铃薯葡萄糖琼脂(PDA)平板上的菌丝生长和产孢量与敏感菌株相比无显著差异,在黄瓜叶片上均表现出很强的致病力。结果表明,HZ021和HZ 054有很高的适合度。通过对抗性菌株中细胞色素b (CYT b)、双组份组氨酸激酶(OS-1)和β-微管蛋白(TUB 2)基因序列进行分析发现,HZ021和HZ054对多种药剂的抗性是由于其药剂靶标基因上的点突变所致。     

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.     

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.     

Rational Management of Didymella rabiei in Chickpea by Integration of Genotype Resistance and Postinfection Application of Fungicides

ABSTRACT Various aspects of the integration of genotype resistance and chemical control of Ascochyta blight (caused by Didymella rabiei) in chickpea were examined in field experiments from 1993 to 1999 and in greenhouse experiments. Four commercially available chickpea cultivars representing a range of resistance to D. rabiei were used. The efficacy of chemical control in a highly susceptible cultivar was significantly (P < 0.01) related to the conduciveness of the environment to the pathogen. Adequate disease suppression (>80% control) was achieved when weather supported mild epidemics, but insufficient control (<20%) was achieved when weather supported severe epidemics. The contribution of genotype resistance to disease suppression in a moderately susceptible cultivar varied from <10% when weather supported severe epidemics to approximately 60% when weather supported mild epidemics. Spraying a moderately resistant cultivar resulted in 95% control when weather supported mild epidemics, but only 65% control was achieved when weather supported severe epidemics. The existing level of resistance in a moderately resistant cultivar resulted in 70% control when weather supported severe epidemics; fungicides improved control efficacy significantly to >95%. Under mild epidemics, moderate resistance alone provided >95% control. The level of genotype resistance available in a highly resistant cultivar was sufficient to suppress the disease under all weather conditions, even without application of fungicides. The possibility of relying on postinfection rather than prophylactic application of fungicides was tested in the greenhouse and in four field experiments. Activity of the systemic fungicide tebuconazole was detected when the fungicide was applied up to 3 days postinfection, and application of tebuconazole or difenoconazole in the field as a postinfection treatment (i.e., after rain or overhead irrigation) suppressed the disease as effectively as preventive applications and required fewer sprays. In two experiments, the interaction between genotype resistance and chemical control at various amounts of irrigation applied via overhead sprinklers (as a simulation of rain) was tested. The results show that both the level of genotype resistance and the quantity of water should be taken into account in deciding whether to apply a postinfection spray.     

Sensitivity profiles of Mycosphaerella graminicola and Phytophthora infestans populations to different classes of fungicides

Prior to the use of fungicides, the baseline sensitivity of individuals in a pathogen population may already differ by a factor of 10 to 100 between the least and the most sensitive isolates. In Mycosphaerella graminicola populations, this factor, measured in vitro, was 5 to 20 for both the strobilurin analogue azoxystrobin (baseline) and the triazole cyproconazole which has been in use for several years. In Phytophthora infestans populations, this factor, measured in a leaf disc assay, was about 100 for azoxystrobin (baseline), up to 1000 for the cyanoacetamide cymoxanil and >10000 for the phenylamide oxadixyl; both of the latter have been used for many years. In M. graminicola, cross-sensitivity was present between all azole fungicides for the majority of the isolates, whereas no correlation was found between triazoles and azoxystrobin. Despite the existence of cross-sensitivity between azoles, ‘box-and-whiskers’ plots revealed large variations in the sensitivity profiles of some triazoles; isolates resistant to triazoles have not been detected in M. graminicola populations. In P. infestans populations, the proportion of the phenylamide-resistant sub-population increased during the season more rapidly in treated than in untreated fields, but it was low at the beginning of the next season in all fields. During disease epidemics, the fitness of phenylamide-resistant P. infestans isolates, as characterised by lesion size, was higher than that of the sensitive isolates, but after the overwintering period, the recovery of resistant isolates was apparently lower. The presence of both A1 and A2 mating types of P. infestans in European populations, although at different frequencies, allows sexual recombination and increased genetic diversity, affecting sensitivity and fitness. Such mixed populations can still be adequately controlled by using sound anti-resistance strategies. ©1997 SCI     

Sensitivity ofFusarium graminearum to fungicide JS399-19:In vitro determination of baseline sensitivity and the risk of developing fungicide resistance

The baseline sensitivity ofFusarium graminearum Schwade [teleomorph =Gibberella zeae (Schweinitz) Petch] to the fungicide JS399-19 (development code no.) [2-cyano-3-amino-3-phenylacrylic acetate] and the assessment of risk to JS399-19 resistancein vitro are presented. The mean EC₅₀ values for JS399-19 inhibiting mycelial growth of three populations of wild-typeF. graminearum isolates were 0.102±0.048, 0.113±0.035 and 0.110±0.036 μg ml⁻¹, respectively. Through UV irradiation and selection for resistance to the fungicide, we obtained a total of 76 resistant mutants derived from five wild-type isolates ofF. graminearum with an average frequency of 1.71 × 10⁻⁷% and 3.5%, respectively. These mutants could be divided into three categories of resistant phenotypes with low (LR), moderate (MR) and high (HR) level of resistance, determined by the EC₅₀ values of 1.5–15.0 μg ml⁻¹, 15.1–75.0 μg ml⁻¹ and more than 75.0 μg ml⁻¹, respectively. There was no positive cross-resistance between JS399-19 and fungicides belonging to other chemical classes, such as benzimidazoles, ergosterol biosynthesis inhibitors and strobilurins, suggesting that JS399-19 presumably has a new biochemical mode of action. Although the resistant mutants appeared to have comparable pathogenicity to their wild-type parental isolates, they showed decreased mycelial growth on potato-sucrose-agar plates and decreased sporulation capacity in mung bean broth. Nevertheless, most of the resistant mutants possessed fitness levels comparable to their parents and had MR or HR levels of resistance. As these studies yielded a high frequency of laboratory resistance inF. graminearum, appropriate precautions against resistance development in natural populations should be taken into account. http://www.phytoparasitica.org posting August 7, 2008.     

Biological and genetic characterization of Phytophthora capsici mutants resistant to flumorph

Baseline sensitivity to flumorph, a carboxylic acid amide (CAA) fungicide used to control some oomycetes, was examined using 83 Phytophthora capsici isolates, resulting in a unimodal distribution of effective concentration for 50% inhibition of mycelial growth ranging from 0·716 to 1·363, with a mean of 1·033 ± 0·129 μg mL^?1. To assess the potential risk of developing flumorph resistance, 13 flumorph‐resistant mutants of P. capsici were obtained using ultraviolet irradiation. Most of these mutants and their progeny had high levels of fitness, including mycelial growth, sporulation and virulence. The resistance to flumorph changed slightly, either increasing or decreasing, after 10 transfers on agar media. Cross‐resistance was found between flumorph and other CAA fungicides (dimethomorph and iprovalicarb), but not between flumorph and non‐CAA fungicides (cymoxanil, metalaxyl, azoxystrobin and cyazofamid). To investigate the genetics of the flumorph resistance, 619 progeny were obtained by self‐crossing and sexual hybridization. Segregation of sensitivity to fungicide was measured as a ratio of sensitive (S) to resistant (R) isolates. Segregation of the progeny, from self‐crossed isolate PCAS1 (flumorph resistant), was 1:15 in the first generation; and 0:1 or 1:15 in the second generation. In sexual hybridization, segregation of progeny was 0:1 and 1:7 for R × R hybridization; and 1:3 for R × S hybridization. Therefore, the resistance of P. capsici against flumorph was controlled by two dominant genes.