Survey of benomyl resistance in Fusavium species on winter wheat in England and Wales in 1986

In 1986, samples from 109 winter wheat crops in England and Wales were examined for benomyl resistance in Fusarium species. Of the 704 isolates obtained, 581 were identified as Fusarium nivale, of which 92.1 % were resistant to benomyl. Of the remaining isolates, 82 were identified as F. avenaceum, 40 as F. culmorum and one as F. poae; none of these isolates were resistant to benomyl. The vast majority of isolates of Fusarium spp. were obtained from superficial nodal or internodal lesions on the stems.     

Survey of benomyl resistance in Pseudocercosporella herpotrichoides on winter wheat and barley in England and Wales in 1983

Surveys of 528 randomly selected commercial fields in England and Wales in 1983 showed that isolates of Pseudocercosporella herpotrichoides resistant to benomyl were common and widespread on winter wheat and winter barley. During June and July resistant strains were recovered from 16 to 66% of fields and the proportion of resistant isolates was 37–52%. The frequency of occurrence of resistant isolates was positively correlated with the number of applications of carbendazimgenerating (MBC) fungicides during the previous 8 years. Benomyl resistance was found in 21–48% of fields where it was claimed that MBC had never been used, and 7–28% of isolates from those fields, were resistant. Resistance was more frequent in R-type than in W-type isolates of the pathogen. The proportion of R-type isolates decreased as the number of previous winter wheat crops rose and increased with the number of winter barley crops grown and with the number of MBC sprays applied to crops in previous years. The implications of these findings for the control of eyespot in England and Wales are discussed.     

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.     

Phenotypic and genotypic diversity in Phytophthora infestans on potato in Great Britain, 1995–98

A total of 2691 single-lesion isolates of Phytophthora infestans was established from samples of late-blight disease from 354 commercial and garden/allotment sites in Scotland, England and Wales over four growing seasons, 1995–98. The A2 mating type was rare (3·0% of isolates) and was detected at only 34 sites. In vitro tests of sensitivity to the phenylamide fungicide metalaxyl showed that 316 sites yielded isolates with some insensitivity (resistant and/or intermediate); these were more often commercial sites than garden/allotment sites. Over the four seasons, the frequency of isolates with intermediate fungicide sensitivity increased, while the frequency of resistant phenotypes decreased. Resistant isolates were always of A1 mating type. A subset of 1459 isolates from 326 sites was analysed for molecular diversity. Mitochondrial DNA (mtDNA) haplotype Ia predominated (91·0% of isolates); haplotype IIa was present at 54 sites and both haplotypes at 33 sites. The multilocus RFLP probe RG57 detected 30 fingerprints. Four fingerprints were particularly common (RF002, RF006, RF039 and RF040) and 10 were unique to a single site in a single year. The three commonest fingerprints (RF039 > RF002 > RF006) were of A1 mating type and the fourth (RF040) was A2. RF002 isolates were resistant to the phenylamide metalaxyl and were more common in Scotland than in England and Wales. Small sample sizes limited the usefulness of estimates of diversity. Although approximately half of all sites appeared to be colonized by RF039 genotypes, some sites (both commercial and garden/allotment) showed a higher diversity, having both common and unique genotypes. The genotypic diversity within isolates collected from commercial sites and those from garden/allotment sites were similar. The contributions of sexual reproduction and alternatives to sex to the generation of variation are discussed.     

Carbendazim resistance in the eyespot pathogen Pseudocercosporella herpotrichoides

Mycelial isolates (115) of Pseudocercosporella herpotrichoides were obtained from five field sites in England. Carbendazim-resistant isolates were detected by their mycelial growth on agar containing 1 μg/ml carbendazim. Resistant isolates were found at two of the five sites examined and one of these had never been treated with benzimidazole fungicides. Amongst the carbendazim- resistant isolates there was a predominance of isolates with pale mycelium, an irregular colony margin and a relatively slow growth rate; however, this association was not absolute. Large differences in the effects of carbendazim on mycelial growth of sensitive and resistant isolates were demonstrated; growth of sensitive isolates was completely inhibited at 0.5 μg /ml carbendazim whilst five of the six resistant isolates examined grew on agar containing 1000 μg/ml fungicide. The carbendazim-resistant isolates were cross-resistant to benomyl, thiophanate-methyl and to a Icsser degree thiabendazole, but not to prochloraz. Conidia of carbendazim-resistant isolates were as resistant. Carbendazim-resistant isolates were just as pathogenic to wheat as sensitive isolates. The implications of these results and other reports of benzimidazole resistance in P. herpotrichoides are discussed in relation to disease control.     

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.     

Phenotypes of Phytophthora infestans collected in England and Wales from 1985 to 1988: mating type, response to metalaxyl and isoenzyme analysis

In a survey of 1864 isolates derived from samples of Phytophthora infestans obtained from potato and tomato collected in England and Wales between 1985 and 1988, the A2 mating type was identified in approximately 10% of samples. There was no evidence for an overall increase in the frequency of the A2 mating type over this period. In agar culture, A2 isolates exhibited a distinct lumpy-colony morphology. Lumps were made up of intensely dichotomously branched hyphae. The incidence of resistance to metalaxyl among samples increased from 41 to 60% between 1986 and 1988. Some of the A2 isolates were resistant to metalaxyl and like other A2 isolates gave rise to progeny from in vitro matings with Al isolates. Except in one case, Al and A2 isolates were uniformly homozygous at an isoenzyme locus for glucosephosphate isomerase but isolates exhibited some variation at a peptidase locus. The source of the A2 isolates could not be established from these data.     

Occurrence and distribution of Raspberry bushy dwarf virus in commercial Rubus plantations in England and Wales

Serological surveys for Raspberry bushy dwarf virus (RBDV) made between 1995 and 1997 and covering ≈ 10% of the commercial farms growing Rubus (red raspberry and hybrid berries) in England and Wales showed that this virus was present on approximately one-quarter of all farms and in approximately one-sixth of all plots tested. RBDV was found in all of the four main raspberry cultivars being grown at that time (Autumn Bliss, Glen Moy, Glen Prosen and Leo), in Loganberry and in Tayberry. Fifteen RBDV genotypes (including two that appeared to be mixed) were identified using RT-PCR/RFLPs, but the majority of genotypes were found only rarely. Of the RBDV isolates tested, two genotypes each comprised 12·5% and another 46·4%. None of the three most common genotypes was associated solely with single Rubus cultivars and vice versa . It is suggested that two separate outbreaks of RBDV are occurring in England and Wales. One outbreak comprises the most frequent genotype combined with one of the moderately frequent genotypes; this outbreak is largely confined to the main growing areas and is either spreading between farms or coming from multiple local sources. Circumstantial evidence suggests that these isolates (and hence this first outbreak) are of the RB pathotype. The second outbreak consists of the other moderately frequent genotype and those genotypes which are less common. These genotypes appear to be more scattered across England and Wales and seem more likely to be coming from local sources and not to be spreading naturally between commercial farms.     

Distribution and fitness of isolates of Botrytis cinerea with multiple fungicide resistance in Spanish greenhouses

Forty-nine greenhouses of vegetable crops were surveyed in southeast Spain at the beginning of the disease season in December 1992 to estimate frequencies of resistance to benzimidazoles, dicarboximides and N -phenylcarbamates (NPC) in B. cinerea . Out of 261 isolates collected, 28% were sensitive to both benzimidazoles and dicarboximides, 15% were benzimidazole-resistant and dicarboximide-sensitive, 8% were benzimidazole-sensitive and dicarboximide-resistant and 46% were benzimidazole- and dicarboximide-resistant. Resistance to benzimidazole, dicarboximide and N -phenylcarbamate was determined by measuring the ability of each isolate to grow in the presence of carbendazim, procymidone and diethofencarb fungicides respectively. Carbendazim- or procymidone- resistant isolates were found in all surveyed greenhouses. Three isolates were found with resistance to carbendazim, procymidone and diethofencarb collected in two adjacent greenhouses that were sprayed with the carbendazim and diethofencarb mixture. All other isolates were sensitive to the mixture because they were either sensitive to carbendazim and resistant to diethofencarb or vice versa. Fitness of 31 isolates of B. cinerea was determined in vivo by measuring their sporulation and lesion growth rate on leaf disks. No fitness costs were associated with resistance to iprodione (dicarboximide) and benomyl (benzimidazole). Isolates with EC₅₀ values higher than 101 mg/L for benomyl and 1.6 mg/L for iprodione were considered to be field resistant (they caused visible lesions on cucumber leaf disks treated with each fungicide).     

Occurrence of Corynespora cassiicola isolates resistant to boscalid on cucumber in Ibaraki Prefecture, Japan

A total of 651 isolates of cucumber corynespora leaf spot fungus ( Corynespora cassiicola ) collected from cucumber in Japan, either with (438 isolates) or without (213 isolates) a prior history of boscalid use, were tested for their sensitivity to boscalid by using a mycelial growth inhibition method on YBA agar medium. Additionally, seven isolates of C. cassiicola obtained from tomato, soybean, eggplant (aubergine) and cowpea in different locations in Japan were tested before boscalid registration. Minimum inhibitory concentration (MIC) and 50% effective concentration (EC₅₀) values for 220 isolates from crops without a prior history of boscalid use ranged from 0·5 to 7·5 μg mL⁻¹ and from 0·04 to 0·59 μg mL⁻¹, respectively. Two hundred and fourteen out of 438 isolates collected from ten cucumber greenhouses in Ibaraki Prefecture, Japan, which received boscalid spray applications showed boscalid resistance, with MIC values higher than 30 μg mL⁻¹. Moreover, resistant isolates were divided into two groups: a moderately resistant (MR) group consisting of 189 isolates with EC₅₀ values ranging from 1·1 to 6·3 μg mL⁻¹, and a very highly resistant (VHR) group consisting of 25 isolates with EC₅₀ values higher than 24·8 μg mL⁻¹. MR isolates were detected from all ten greenhouses, but VHR isolates were detected from only three. As a result of fungus inoculation tests which used potted cucumber plants, control failures of boscalid were observed against resistant isolates. Efficacy of boscalid was remarkably low against VHR isolates in particular. This is the first known report on boscalid resistance in Japan.     

拟轮枝镰孢对多菌灵的敏感性及抗性菌株生物学性状和交互抗性

为评价玉米穗腐病主要致病菌拟轮枝镰孢Fusarium verticillioides对多菌灵的敏感性,采用菌丝生长速率法测定了采自山东、河北、河南、四川、甘肃、辽宁及宁夏7省(自治区)的168株玉米穗腐病拟轮枝镰孢对多菌灵的敏感性,并对经药剂驯化获得的拟轮枝镰孢抗多菌灵菌株主要生物学性状和交互抗性进行了研究。结果表明:多菌灵抑制拟轮枝镰孢菌丝生长的EC₅₀值在0.013 2~0.774 0 mg/L之间,平均EC₅₀值为(0.220 8 ± 0.143 7) mg/L。敏感性频率分布显示,供试病原菌群体中已出现对多菌灵敏感性下降的亚群体,但其中仍有35.71%的菌株对多菌灵的敏感性频率呈正态分布,因此可将此部分菌株的平均EC₅₀值 (0.081 4 ± 0.028 9) mg/L作为拟轮枝镰孢对多菌灵的相对敏感基线。药剂驯化共获得6株抗性菌株,抗性倍数在5.05~12.22之间。抗性菌株的菌丝生长速率及菌丝干重均低于亲本菌株,表明其抗性菌株的生物适合度有所降低,同时发现其抗药性均不能稳定遗传。室内交互抗性测定结果表明,拟轮枝镰孢对多菌灵的抗性与咪鲜胺、戊唑醇、三唑酮及吡唑醚菌酯之间均不存在交互抗性关系。     

番茄叶霉病菌对多菌灵、乙霉威及代森锰锌抗性检测

报道了番茄叶霉病菌Fulvia fulva对多菌灵、乙霉威及代森锰锌的敏感性基线,以及抗性频率和抗性水平。离体条件下,多菌灵、乙霉威及代森锰锌对番茄叶霉病菌敏感菌株的平均EC50值分别为0.101、2.475、9.067 μg/mL;最低抑制浓度(MIC)值分别为0.5、5、50 μg/mL 。山西晋南地区番茄叶霉病菌对3种杀菌剂的抗性频率最高,分别达到97.4%、70.5%、98.7%;山西吕梁地区与太原地区相对较低,但该病菌对3种杀菌剂的抗性频率也都超过了30%。辽宁沈阳、山东寿光、河北保定番茄叶霉病菌对多菌灵的抗性频率均为100%;对乙霉威的抗性频率前两地为100%,保定为10%;对代森锰锌的抗性频率都超过90%。所有抗性菌株对多菌灵均属于高抗类型,抗性指数超过5000,测不出MIC值;对乙霉威有50%的高抗菌株,抗性指数在100以上;对代森锰锌各地均未发现高抗菌株,低抗和中抗菌株所占比例较大,其抗性指数集中在50左右。对多菌灵与乙霉威具有双重抗性的菌株占测试菌株总数的49.9%,并且首次在田间发现了对3种杀菌剂都具有抗性的番茄叶霉病菌多抗菌株。     

Effects of fungicides on in vitro spore germination and mycelial growth of the phytopathogens Leptosphaeria maculans and L. biglobosa (phoma stem canker of oilseed rape)

BACKGROUND: Phoma stem canker, caused by the coexisting related fungal pathogens Leptosphaeria maculans (Des.) Ces. & de Not and L. biglobosa Shoemaker & H Brun, is a major disease of winter oilseed rape in the UK. Annually, over 90% of UK crops receive at least one foliar application of fungicide, but little is known about the sensitivity of the more damaging L. maculans and the less damaging L. biglobosa to these fungicides. The effects of flusilazole, tebuconazole and Methyl Benzimidazole Carbamate (MBC) fungicides (benomyl and carbendazim) on the germination of ascospores, conidia and germ tube growth of both species were examined. Isolates collected from different oilseed rape crops in England and Wales were assessed for their mycelial growth on fungicide‐amended medium, and ED₅₀ values were calculated. RESULTS: Leptosphaeria maculans and L. biglobosa differed in their sensitivity to fungicides. Conidial germination of L. maculans was more sensitive to these fungicides than that of L. biglobosa. Isolates of L. maculans had smaller ED₅₀ values for mycelial growth for all fungicides tested than isolates of L. biglobosa. CONCLUSION: These results suggest that fungicide applications might affect the structure of L. maculans/L. biglobosa populations in UK winter oilseed rape crops. Copyright © 2009 Society of Chemical Industry     

Characterization of Fusarium spp. responsible for causing dry rot of potato in Great Britain

A 3-year survey was undertaken to establish the relative frequency of different Fusarium spp. present as inoculum on potato tubers collected from four regions of Great Britain. A total of 219 samples (comprising 10 950 tubers) were collected from the 2000, 2001 and 2002 crops and processed to recover dry rot-producing isolates. In total, 228 isolates of Fusarium spp. were recovered. Most (94·7%) of these isolates were attributed to one of four Fusarium species: F. coeruleum, F. avenaceum, F. culmorum and F. sambucinum (formerly F. sulphureum) . The incidence of the combined Fusarium spp. increased the further south the crops had been grown. Fusarium coeruleum was the most commonly isolated species in each survey year, comprising 37 to 52% of the total Fusarium species. Selected isolates of each species were evaluated for their ability to produce rots in potato tubers. Fusarium sambucinum was a more aggressive pathogen than the other Fusarium species in eight out of 10 cultivars. Fusarium avenaceum and F. culmorum were relatively weaker pathogens. However, these species were aggressive on some cultivars, notably Hermes. The selected isolates were also assessed for their sensitivity to the fungicides thiabendazole and imazalil. Using in vitro tests, 65% of F. sambucinum isolates were resistant to thiabendazole and 7% of F. avenaceum isolates were resistant to imazalil. Tubers treated with imazalil yielded a higher proportion of isolates of F. avenaceum than those that were untreated. Similarly, a higher proportion of F. sambucinum isolates were recovered from tubers treated with thiabendazole than from those that were not treated.     

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.     

中国华北地区瓜类尖孢镰刀菌对咪鲜胺的敏感性及抗药突变株生物学性状研究

为明确中国华北地区瓜类尖孢镰刀菌Fusarium oxysporum Schl.对咪鲜胺的抗药性现状及抗药突变株的生物学性状,采用菌丝生长速率法,分别测定了采自北京、山东、河北等地未使用过咪鲜胺的112株瓜类尖孢镰刀菌对咪鲜胺的敏感性,并通过药剂驯化的方法获得尖孢镰刀菌抗咪鲜胺突变株。结果表明:咪鲜胺对112株瓜类尖孢镰刀菌的平均EC50值为（0.030 1±0.030 4）μg/mL,菌株频率呈连续单峰曲线分布,未发现敏感性明显下降的亚群体,因此,可将该EC50值作为瓜类尖孢镰刀菌对咪鲜胺的敏感基线。药剂驯化共获得7株抗咪鲜胺突变株,其抗性倍数介于6.2~26.8之间;突变株在菌丝生长速率、菌丝干重和致病力等方面均明显低于亲本菌株,差异显著;仅突变株HG13052701-R1的抗药性可以稳定遗传,其他6株抗咪鲜胺突变株的抗药性均不能稳定遗传。室内交互抗性测定结果表明,咪鲜胺仅与戊唑醇之间有交互抗性,与多菌灵和齅霉灵之间均无交互抗性关系。研究表明,瓜类尖孢镰刀菌在药剂选择压下可以形成抗咪鲜胺群体,具有低等抗药性风险。     

Phylogenetic relationships and pathogenicity of Colletotrichum acutatum isolates from grape in subtropical Australia

The identity of Colletotrichum acutatum as the causal pathogen of grape ripe-rot, which causes yield loss and a bitter taint that lowers wine quality in Australian subtropical wine-grape regions, was confirmed using species-specific primers. Cultural, morphological and molecular methods (RAPD-PCR and sequencing of parts of the 5·8S-ITS regions and the β-tubulin-2 gene) were used to determine the phylogenetic relationships of Australian C. acutatum isolates from wine grapes and other horticultural crops. A combination of RAPD-PCR and β-tubulin-2 gene data showed that all wine-grape ripe-rot isolates from northern regions of New South Wales (NSW) and Queensland belong to a proposed new C. acutatum group (A9), together with isolates from Australian strawberry, mango, blueberry and olive. The 5·8S-ITS sequences for these grape pathogens were identical to published sequences for an isolate from Cyclamen (the Netherlands) and differed by 1 bp from isolates from Capsicum (Taiwan) and orange (Costa Rica). The grape ripe-rot isolates from the Shoalhaven Valley (southern NSW) were clustered within two other C. acutatum groups: A2 and A5. In vitro infection studies showed that Australian C. acutatum isolates from almond, blueberry, chilli, grape, mango, olive, strawberry and tomato were able to infect grape and could also infect blueberry and strawberry, indicating a lack of host specificity. This lack of host specificity, the genetic similarity with non-grape isolates, and the fact that many of the non-grape hosts were isolated from wine-growing regions, suggest the potential for cross-infection between grape and other horticultural crops.     

Biological control of fusarium crown and root rot of tomato with antagonistic bacteria and integrated control when combined with the fungicide carbendazim

Two bacterial isolates, Bacillus megaterium (c96) and Burkholderia cepacia (c91), demonstrated to be antagonistic against Fusarium oxysporum f.sp. radicis-lycopersici , the causal organism of fusarium crown and root rot of tomato, were evaluated as biocontrol agents alone and when integrated with the fungicide carbendazim. In an initial screening, these isolates reduced disease incidence by 75 and 88%, respectively. In vitro , both biocontrol agents were highly tolerant to the fungicide carbendazim, commonly used to control fusarium diseases. Carbendazim reduced disease symptoms by over 50% when used at > 50  µ g mL⁻¹, but had little effect at lower concentrations. Combination of the bacterial isolates and carbendazim gave significant ( P  ≤ 0·05) control of the disease when plants were artificially inoculated with the pathogen. Application of carbendazim at a low concentration (1  µ g mL⁻¹) in combination with B. cepacia c91 reduced disease symptoms by 46%, compared with a reduction of 20% obtained with the bacterium alone and no control with the chemical treatment alone. A combination of B. megaterium c96 with an increased application rate of 10  µ g mL⁻¹ carbendazim significantly reduced disease symptoms by 84% compared with inoculated controls and by 77% compared with carbendazim treatment alone. In this experiment, the integrated treatment also slightly outperformed application of 100  µ g mL⁻¹ carbendazim, and bacteria applied without fungicide also provided good disease control.     

Metalaxyl sensitivity of isolates of Phytophthora erythroseptica in Prince Edward Island

Isolates of Phytophthora erythroseptica , causal agent of pink rot of potato, were obtained from diseased tubers collected in Prince Edward Island (PEI), Canada. In an in vitro assay of field and single-zoospore isolates, all isolates of the pathogen from PEI were sensitive to metalaxyl (EC₅₀ < 0·5  µ g mL⁻¹), unlike moderately or highly resistant reference isolates obtained from Maine, USA. Allozyme-banding patterns at the glucose-6-phosphate isomerase ( Gpi ) locus were identical (91/91) for all isolates of P. erythroseptica examined from PEI and Maine, but could be used to distinguish isolates of P. erythroseptica from local isolates of Phytophthora infestans . Inoculation of potato tubers from plants treated with foliar applications of metalaxyl in the field indicated that compounds inhibitory to metalaxyl-sensitive isolates of P. erythroseptica were present in the periderm, even after 4 months' storage. By contrast, moderately or highly resistant isolates of the pathogen caused significantly ( P  ≤ 0·05) more disease than sensitive isolates in tubers from plants treated with metalaxyl. The effectiveness of metalaxyl in controlling pink rot in a particular region will depend on resistance levels of local populations of P. erythroseptica . Preventing the development and spread of resistant strains of P. erythroseptica will be critical in maintaining metalaxyl as an effective chemical for control of this pathogen.