Sensitivity of South African Ramulispora herpotrichoides isolates to carbendazim and ergosterol biosynthesis inhibitors

In a survey conducted during 1991–1992, single-spored isolates of the eyespot fungus from the Swartland area were characterized and tested for sensitivity to carbendazim and ergosterol inhibiting fungicides. The 100 isolates tested were all fast growing, even marginate, and designated as Ramulispora herpotrichoides . Fungal growth was completely inhibit on PDA amended with carbendazim (1 μg/ml), indicating that the local population of the fungus is still at baseline sensitivity to benzimidazoles. The mean concentration of prochloraz calculated to inhibited growth by 50% (IC₅₀ value) was 0.043 ± 0.029 μg/ml, which is comparable with the baseline sensitivity reported for European isolates. Of the 36 representative isolates screened against 2 μg/ml triadimenol, 44% were sensitive, while 36% were resistant. The triadimenol-resistant isolates were sensitive to propiconazole and flusilazole. However, four of the triadimenol-resistant isolates were also resistant to tebuconazole. These results indicate that South African isolates of R. herpotrichoides are sensitive towards carbendazim, prochloraz, propiconazole and flusilazole. They were found to differ, however, in sensitivity towards triadimenol and tebuconazole, where some isolates had an IC₅₀ value greater than 2 μg/ml.     

Dicarboximide resistance in Botrytis cinerea in protected lettuce

In surveys of protected lettuce crops in the Humberside area of the UK during 1984-5, 47.3% of Botrytis cinerea isolates were resistant to dicarboximides and 25% to benomyl. Dicarboximide-resistant isolates obtained direct from the field had a moderate degree of resistance with ED₅₀ values for mycelial growth on agar of 1.0-2.4 μg/ml vinclozolin for all except two of 30 isolates.
Isolates which were resistant to vinclozolin were also resistant to iprodione, myclozolin and procymidone. Thirty dicarboximide-resistant isolates obtained direct from the field were capable of infecting cucumber cotyledons sprayed with field-strength Ronilan (250 μg/ml vinclozolin) whereas sensitive strains were not. Strains showing different degrees of resistance to dicarboximides developed after incubation of sensitive and resistant strains on agar supplemented with vinclozolin. Some of these strains had ED₅₀ values for mycelial growth on agar of more than 100 μg/ml vinclozolin. No highly resistant isolates were obtained direct from the field. Resistance to dicarboximides in field isolates was stable.     

Resistance to benzimidazole fungicides in the gummy stem blight pathogen Didymelta bryoniae on cucurbits

Benzimidazoles were used successfully against gummy stem blight of cucurbits for only one growing season (1980). It was soon noticed in experiments in vivo that they were not effective against some isolates of the pathogen. In a survey during the 1981 crop season, 76 out of 102 isolates collected from 20 plastic greenhouses all over the main cucurbit-growing areas of Crete were resistant to benomyl. The average benomyl concentrations that reduced mycelial growth rates of seven resistant and seven sensitive isolates by 50% were 716 ± 49 and 1.2 ± 0.34μg/ml respectively. The average growth rate of resistant isolates was lower than that of sensitive isolates but there were no differences in spore production and spore germination. The pathogenicity of resistant isolates on young cucumber plants was lower than that of sensitive isolates. The application of benomyl, carbendazim, cypendazole and thiophanate methyl to young cucumber plants at the rate of 300μg a.i./ml 1 day before inoculation with 500 000 spores/ml of the resistant isolates did not affect the infection rate of the disease after 4 days. Sensitive isolates caused no onslight symptoms on similarly treated plants.     

Studies on resistance to dichlofluanid and other fungicides in Botrytis cinerea

Amongst 489 mass isolates of Botrytis cinerea obtained from protected crops in Humberside during 1989, 67.3% were resistant to benomyl and 45.6% to vinclozolin. Two isolates resistant to dichlofluanid were obtained with EC₅₀ values for mycelial growth on agar of 21.45 and 19.78 μg dichlofluanid/ml, respectively, compared with values of 1.55-2.56 μg/ml for sensitive isolates. EC₅₀ values for germ tube emergence and length were much lower than for mycelial growth. In population studies carried out on tomatoes in a polythene tunnel, resistance to dichlofluanid and to vinclozolin declined over a 12-week period in the absence of fungicides. Field isolates of B. cinerea resistant to dichlofluanid were also resistant to vinclozolin. but strains resistant to dichlofluanid and sensitive to vinclozolin were obtained during population studies. Examination of a number of features of dichlofluanid-resistant strains revealed a degree of lack of fitness as compared with sensitive strains. There is no indication that resistance to dichlofluanid poses a great threat to the control of grey mould in Humberside at present.     

应用电导仪测定番茄灰霉病菌对多菌灵抗药性的初步研究

1.0 mg/L和5.0 mg/L多菌灵盐酸盐溶液处理灰霉病菌多菌灵敏感菌株和抗性菌丝体,用电导仪测定溶液电导率的动态变化,结果表明:在敏感菌株存在时,多菌灵盐酸盐溶液2 h后的电导率显著降低,而抗性菌株存在的溶液中电导率则未见下降。     

Sensitivity of Pseudoeevcosporella herpotriehoides to the fungicide prochloraz

In a survey carried out in 1984, isolates of the eyespot fungus, Pseudocercosporella herpotrichoides , from cereals in the UK, Denmark and West Germany were tested in vitro for sensitivity to the fungicide prochloraz. Of 194 isolates tested, 191 showed no growth on agar amended with prochloraz at 2 μg/ml or more. The mean concentration of prochloraz calculated to inhibit colony growth by 50% (IG50) was 0 44+0−010 μg/ml. The three isolates that grew at 2 μg/ml displayed weak colony development and were clearly disrupted by the presence of the fungicide. R-type isolates appeared more sensitive to prochloraz than W-type isolates. Comparisons with similar data for isolates obtained from other years revealed no consistent change with time in sensitivity to prochloraz.     

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.     

Fungicide resistance in Cercospora species causing cercospora leaf blight and purple seed stain of soybean in Argentina

Cercospora species cause cercospora leaf blight (CLB) and purple seed stain (PSS) on soybean. Because there are few resistant soybean varieties available, CLB/PSS management relies heavily upon fungicide applications. Sensitivity of 62 Argentinian Cercospora isolates to demethylation inhibitor (DMI), methyl benzimidazole carbamate (MBC), quinone outside inhibitor (QoI), succinate dehydrogenase inhibitor (SDHI) fungicides, and mancozeb was determined in this study. All isolates were sensitive to difenoconazole, epoxiconazole, prothioconazole, tebuconazole, and cyproconazole (EC₅₀ values ranged from 0.006 to 2.4 µg/ml). In contrast, 51% of the tested isolates were sensitive (EC₅₀ values ranged from 0.003 to 0.2 µg/ml), and 49% were highly resistant (EC₅₀ > 100 µg/ml) to carbendazim. Interestingly, all isolates were completely resistant to azoxystrobin, trifloxystrobin, and pyraclostrobin, and insensitive to boscalid, fluxapyroxad, and pydiflumetofen (EC₅₀ > 100 µg/ml). The G143A mutation was detected in 82% (53) of the QoI-resistant isolates and the E198A mutation in 97% (31) of the carbendazim-resistant isolates. No apparent resistance mutations were detected in the succinate dehydrogenase genes (subunits sdhB, sdhC, and sdhD). Mancozeb completely inhibited mycelial growth of the isolates evaluated at a concentration of 100 µg/ml. All Argentinian Cercospora isolates were sensitive to the DMI fungicides tested, but we report for the first time resistance to QoI and MBC fungicides. Mechanism(s) other than fungicide target-site modification may be responsible for resistance of Cercospora to QoI and MBC fungicides. Moreover, based on our results and on the recent introduction of SDHI fungicides on soybean in Argentina, Cercospora species causing CLB/PSS are insensitive (naturally resistant) to SDHI fungicides. Insensitivity must be confirmed under field conditions.     

Influence of spray programmes on development of fungicide resistance in the eyespot pathogen of wheat, Pseudocercospovella herpotrichoides

A site with no known history of cereal growing or carbendazim fungicide use was inoculated in 1983 with an equal mixture of spores of R-type and W-type isolates of Pseudocercosporella herpotrichoides. all sensitive to carbendazim. Following three consecutive years of carbendazim sprays, more than 90% of isolates recovered were carbendazim-resistant. A majority of R-type isolates was recovered after 3 years and resistance was found to be more frequent in R-type than in W-type isolates. A mixture of carbendazim with prochloraz initially delayed the development of carbendazim resistance, but after 3 years the incidence of resistance was similar to that where carbendazim alone was applied. The best control of eyespot was given by the mixture and by a split application of prochloraz in the first year, by the mixture in the second year, and by the split prochloraz application in the final year. A greater proportion of R-type isolates was recovered from prochloraz-treated plots than from unsprayed plots; the proportion of these that were resistant to carbendazim was lower than that from carbendazim treatments. Throughout this study isolates resistant to prochloraz were not detected.     

禾谷镰孢菌对多菌灵的敏感性基线及抗药性菌株生物学性质研究

 引起小麦赤霉病的禾谷镰孢菌(Fusarium graminearum)在华东地区对多菌灵已出现了高水平抗药性。本文报道了F.graminearum对多菌灵的敏感性基线及其抗药性菌株生物学特性。离体条件下多菌灵对100个野生敏感型菌株的平均EC₅₀和MIC值分别为0.5748±0.0133 μg/ml和 < 1.4μg/ml。而对50个抗药性菌株的平均EC₅₀和MIC值则分别为9.2375μg/ml和 > 100 μg/ml。在麦穗上多菌灵对50个敏感菌株和抗药性菌株防效的平均EC₅₀分别为282.6 μg/ml和 > 2 000μg/ml。从田间获得的抗多菌灵菌株对苯菌灵、噻菌灵、甲基托布津表现交互抗药性,但不同于室内突变菌株,对乙霉威不表现负交互抗药性。抗药性菌株的无性和有性繁殖后代以及在无药培养基上菌丝体转代培养后,抗药水平保持不变。抗药性菌株的菌丝生长速率、产孢能力及致病力等与敏感菌株相比没有差异。     

Further studies of the inheritance of benomyl resistance in Venturia pirina isolated from pear orchards in Israel

Fourteen single-spore cultures of benomyl-resistant Venturia pirina were isolated from pear scab lesions at four sites in Israel. According to the ability of the isolates to germinate and grow at varying benomyl concentrations, four levels of resistance were determined in vitro : three isolates with low resistance (LR) grew at 0.5 but not at 5 μg/ml benomyl: five moderately resistant (MR) isolates grew at 5 but not at 50 μg/ml benomyl: five highly resistant (HR) isolates grew at 50 μg/ml but their hyphae were curled: and one isolate with very high resistance (VHR) grew unaffected at 50 μg/ml benomyl. The difference between the HR and the VHR phenotypes was clearly shown on medium amended with N -(3.5-dichlorophenyl) carbamate (MDPC): only the VHR isolate showed negative cross-resistance to 1 μ g/ml MDPC. whereas HR isolates grew unaffected. Crosses between resistant isolates and sensitive wild types, as well as between different resistant isolates, showed that the various levels of resistance are conferred by four allelic mutations that constitute a polymorphic series at a single locus.     

Persistence and fitness of carbendazim- and dicarboximide-resistant isolates of Monilinia fructicola (Wint.) Honey in flowers, shoots and fruit of stone fruit

Flowers on detached peach shoots and ripe fruit were inoculated under controlled conditions in order to estimate the competitive ability of Monilinia fructicola isolates sensitive and resistant to carbendazim and dicarboximide fungicides. Isolates varied considerably, but there was no consistent relationship between carbendazim resistance and competitive ability; there was, however, evidence of reduced competitive ability in the single dicarboximide-resistant and the dual dicarboximide/high carbendazim-resistant isolate examined. The ability to produce conidia on twig cankers inoculated in late spring 1989 was retained under field conditions by all sensitive and resistant isolates for at least 1 year. Dicarboximide-resistant isolates produced fewer conidia than the carbendazim-resistant and sensitive isolates on cankers. The production of conidia on mummified fruit inoculated in February 1990 decreased in the field during the winter and the following spring, but some conidia were produced by all isolates. Measures of pathogenicity, virulence and fitness for all isolates were similar to the original values after survival for 1 year. The evidence presented in this paper supports field observations that carbendazim-resistant isolates are likely to persist permanently in the M. fructicola population, whereas dicarboximide-resistant isolates are more likely to decline unless fungicide selection pressure is maintained.     

胶孢炭疽菌及两种疫霉菌对苯酰菌胺的敏感性与β-微管蛋白氨基酸突变的相关性分析

选择对多菌灵、乙霉威和苯酰菌胺具有不同敏感性的胶孢炭疽菌 Colletotrichum gloeosporioides、辣椒疫霉菌 Phytophthora capsici 及恶疫霉菌 P.cactorum,采用菌丝生长速率抑制法及氨基酸序列比对法分析了其 β-微管蛋白氨基酸突变与敏感性的关系。结果表明,胶孢炭疽菌对苯酰菌胺、多菌灵和乙霉威的敏感性与 β-微管蛋白198位或200位氨基酸突变有关:对多菌灵敏感、对苯酰菌胺和乙霉威不敏感的胶孢炭疽菌 β-微管蛋白氨基酸198位为谷氨酸(E),200位为苯丙氨酸(F);对多菌灵已产生抗性而对苯酰菌胺和乙霉威不敏感的菌株,其 β-微管蛋白氨基酸200位由苯丙氨酸(F)突变为了酪氨酸(Y);对多菌灵高抗、对苯酰菌胺和乙霉威敏感的菌株其 β-微管蛋白氨基酸198位由谷氨酸(E)突变为了丙氨酸(A)。辣椒疫霉菌和恶疫霉菌对苯酰菌胺敏感,对多菌灵和乙霉威均不敏感。检测疫霉菌菌株 β-微管蛋白未发现氨基酸突变,但发现其 β-微管蛋白氨基酸在196~200位与胶孢炭疽菌差异较大,这可能是导致苯酰菌胺仅对疫霉菌有抑制效果的原因。     

Laboratory studies on benzimidazole resistance in Septoria tritici1

A technique is described for testing isolates of Septoria tritici from winter wheat for resistance to benzimidazole fungicides. Secondary spores from 23 isolates were tested on Czapek Dox V-8 agar amended with benomyl at 1–10 μg ml. Twenty-one isolates were recovered from eight crops in England in 1984 and two (PBI isolates) were obtained in 1973. Thirteen isolates, including both PBI isolates, were sensitive to benomyl at 1 μg ml^?1 and nine were resistant at 10 μg ml^?1. The remaining isolate had a low proportion (1:3.7 x 10⁴) of resistant spores. The minimum inhibitory concentration for sensitive isolates was 0.2–0.4 μg ml ¹ benomyl and for resistant isolates was more than 1000 μg ml ¹. Benomyl-resistant isolates were cross-resistant to carbendazim, thiabendazole and thiophanate-methyl, but not to 12 other fungicides with different modes of action. The implications of these findings are discussed.     

PIRA-PCR for detection of Fusarium graminearum genotypes with moderate resistance to carbendazim

PIRA-PCR ( p rimer- i ntroduced r estriction a nalysis PCR) was developed to detect isolates of Fusarium graminearum with moderate resistance to carbendazim, a methyl benzimidazole carbamate (MBC)-group fungicide. Two primer pairs were designed and synthesized according to the nucleotide sequence of the β ₂-tubulin gene from F. graminearum. Fragments of 164 bp were amplified by nested PCR from isolates differing in carbendazim sensitivity. A Hin dIII restriction enzyme recognition site was introduced artificially by inner primers to detect a mutation at codon 167, and Taa I ( Tsp 4CI) restriction enzyme was used to detect a mutation at codon 200. The sensitivity of isolates to carbendazim was determined by analyzing electrophoresis patterns of the resulting PCR products after simultaneous digestion with both Hin dIII and Taa I. Results from PIRA-PCR and a conventional method (mycelial growth on agar) were identical but PIRA-PCR required only 7–8 h while the conventional method required 5–7 days. This study demonstrates that PIRA-PCR not only monitors the appearance of moderately resistant isolates, but can be useful for detecting genotypes of F. graminearum with moderate resistance to carbendazim.     

Strains of Botrytis cinerea resistant to dicarboximide and benzimidazole fungicides in New Zealand vineyards

In a survey of New Zealand vineyards at harvest 1985, isolates of Botrytis cinerea resistant to benzimidazole and to dicarboximide fungicides were common. The mean frequency of resistance in the major vine-growing districts ranged from 8 to 41% for benzimidazoles, and from 51 to 59% for dicarboximides. All benzimidazole-resistant isolates showed high levels of resistance (EC50 greater than 100 mg/l carbendazim based on radial growth response), and all dicarboximide-resistant isolates showed low levels of resistance. Two subgroups of dicarboximide-resistant isolates were recognized, distinguished in the first instance by their osmotic response. Low-level resistant isolates, which formed a dense margin on osmotically amended medium, exhibited an EC50 for mycelial growth on iprodione of c. 3-2 mg/l; ultra-low-level resistant isolates, which formed a fibrillose margin on osmotically amended medium identical to that of sensitive isolates, exhibited an EC50 of c. 1-3 mg/1. In agar culture, radial growth rate, and conidial and sclerotial production of both subgroups were similar to those of sensitive isolates. Virulence (lesion size) and conidial production on grape berries were highest in sensitive isolates, intermediate in ultra-low-level dicarboximide-resistant isolates, and lowest in low-level dicarboximide-resistant isolates. Evidence is presented indicating that ultra-low-level dicarboximide-resistant strains have progressively replaced low-level dicarboximide-resistant strains in the vineyard population. The presence of dicarboximide-resistant strains was linked with a partial loss of fungicide efficacy.     

Incidence of benomyl and iprodione resistance in isolates of Botrytis cinerea in tomato crops in England and Wales in 1986

In 1986, mass-mycelial isolates of Botrytis cinerea from 67 tomato crops in England and Wales were examined for benomyl and iprodione resistance. Of the 706 isolates obtained, 62.7% were resistant to benomyl at 2μg/ml and 43.2% were resistant to iprodione at 2 μg/ml. Iprodione resistance persisted in the absence of a dicarboximide spray programme. The incidence of benomyl resistance has not decreased since the last survey in 1984 in spite of a considerable reduction in the use of benzimidazole fungicides. There were no clear indications that the use of dichlofluanid influenced the incidence of benomyl or iprodione resistance. Disease control was poorer in crops with a higher incidence of iprodione resistance.     

Characterisation of benzimidazole resistance of Cercospora beticola in Serbia using PCR-based detection of resistance-associated mutations of the β-tubulin gene

A survey to detect and characterise benzimidazole resistance within populations of Cercospora beticola in Serbia was performed. From 52 field isolates collected from sugar beet and beet root, only eight were found to be benzimidazole-sensitive based on the inhibition of mycelial growth by discriminatory concentrations of carbendazim and thiophanate-methyl. Sensitivity tests revealed the presence of three resistant phenotypes among the tested isolates: high-resistance (HR), low-resistance (LR) and moderate-resistance (MR). The benzimidazole resistant isolates were characterised based on the DNA sequence of the β-tubulin gene and temperature sensitivity. The HR isolates showed no temperature sensitivity regardless of carbendazim concentration, whereas the LR and MR isolates were sensitive at lower temperatures. Analysis of the β-tubulin gene sequence revealed two amino acid replacements in the benzimidazole-resistant isolates of C. beticola. One was a glutamic acid to alanine change at position 198 (codon GAG to GCG) that was identified in HR isolates; this mutation has previously been reported to be associated with the development of benzimidazole resistance in C. beticola. The second replacement was a novel point mutation of phenylalanine (TTC) to tyrosine (TAC) at position 167, identified in low and moderate benzimidazole-resistant isolates, sharing a single LR/MR β-tubulin genotype. A diagnostic PCR-RFLP assay utilising a BsaI restriction site present in the benzimidazole sensitive and LR/MR genotypes but absent in the HR genotype was developed for the routine detection of high resistance. A mutation-specific PCR assay was developed for the diagnosis of LR/MR genotype based on a mutation from T to A at codon 167, which is unique to this genotype.     

Effect of epoxiconazole on rice blast and rice grain yield in China

Sensitivity to epoxiconazole of 90 single-spore isolates of Magnaporthe oryzae was determined. The EC₅₀ values for epoxiconazole in inhibiting mycelial growth of the 90 M. oryzae isolates were 0.11–0.86 μg/ml, with an average EC₅₀ value of 0.260?±?0.082 μg/ml. There was no correlation between sensitivity to epoxiconazole and sensitivity to carbendazim or iprobenfos. In protective and curative tests, epoxiconazole applied at 200 μg/ml provided over 70 % control efficacy, while tricyclazole exhibited better protective than curative activity. The results of four field trials performed in 2010 and 2011 at two sites showed that epoxiconazole at 112.5 g a.i/ha provided over 75 % control efficacy, which was similar to tricyclazole with 300 g a.i./ha and better than carbendazim with 562.5 g a.i./ha.     

Effets de divers herbicides sur la croissance mycélienne de souches de Botrytis cinerea pers et de Penicillium expansum Link, sensibles ou résistantes à certains fongicides

Effects of various herbicides on mycelial growth of strains of Botrytis cinerea. Pers. and Pénicillium expansum Link, sensitive or resistant to certain fungicides Of seventy herbicides tested, thirty-eight were slightly toxic to B. cinerea (CI50, concentration giving 50% inhibition of the speed of mycelial growth, exceeding or equal to 100g of herbicide/ml of nutrient solution) and, of those, ten had no effect even at 1000 g/ml. The most active products (CI50 less than 10 g/ml) were Chlorthiamid, dichlobénil, nitrofen, propyzamide and phenolic derivatives (DNOC, ioxynil, PCP). In most cases P. expansum was less sensitive than B. cinerea except to endothal, propachlor, prynachlor and certain substituted ureas. Strains resistant to the benzimidazole fungicides (carbendazim, thiabendazole etc.) show increased sensitivity to certain carbamade herbicides (barban, chlorbufam etc.), this indicates the existence of a negative cross resistance between these groups of antimitotic pesticides. Strains resistant to the cyclic-imide fungicides (iprodionc, procymidone, vinclozolin) and to various aromatic compounds (biphenyl. chloroneb, dicloran etc.) may also be resistant to bipyridilium, dinitroanilinc and diphenylethcr herbicides, to chlorthamid. dichlobénil and oxadiazon. This cross resistance between pesticides with different biochemical modes of action has yet to be explained.