Resistance to diethofencarb (NPC) in benomyl-resistant field isolates of Botrytis cinerea

Based on the 'negative cross-resistance' phenomenon between N -phenylcarbamates and benzimidazole fungicides, the fungicidal mixture of carbendazim + diethofencarb (MBC + NPC) was introduced, to control Botrytis cinerea phenotypes which are either sensitive to benzimidazoles and resistant to NPC (wild-type: Ben^S NPC^R). or resistant to benzimidazoles and sensitive to NPC(Ben^R NPC^S). At one out of four sites where the MBC + NPC mixture was used in commercial cucumber greenhouses, grey mould control failed and a new phenotype of B. cinerea was found. The new phenotype was resistant to benzimidazoles, as was 100% of the population screened in the four sites, but retained resistance to NPC. Accordingly, the new phenotype was designated Ben^R NPC^R. It was pathogenic on cucumber seedlings.     

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).     

Use of Trichoderma harzianum in combination or alternation with fungicides to control cucumber grey mould (Botrytis cinerea) under commercial greenhouse conditions

A preparation of Trichoderma harzianum was sprayed on cucumber plants in greenhouses in order to control fruit and stem grey mould. Up to 90% control was achieved by the biocontrol agent (0·5–1·0 g/l) which in most experiments under commercial conditions was as effective as the dicarboximide fungicides iprodione or vinclozolin (0·5 g/l each) alone or alternated with diethofencarb + carbendazim (0·25 g/l each). However, in one experiment disease incidence in Trichoderma -treated plots did not differ significantly from the control. A mixture of T. harzianum with a dicarboximide fungicide resulted in up to 96% control of grey mould. In this case control was always significant ( P =0·05) but improvement of control compared with each treatment alone was not significant ( P =0·05). The alternation of sprays with the biocontrol preparation and with a dicarboximide fungicide was tested in three out of the five experiments and was found to be effective, thus enabling a reduction in the use of chemical sprays. Populations of T. harzianum were on a level of 3 × 10⁵-8 × 10⁵ c.f.u. per leaf and ten times lower on one fruit. They remained high after the second and third sprays. Conditions favouring the ability of T. harzianum to control grey mould were temperatures above 20°C and relative humidity between 80 and 97%.     

Influence of fungicidal control of cucumber and tomato grey mould (Botrytis cinerea) on fruit yield

The fungicides tebuconazole, tebuconazole + dichlofluanid, fenethanil, diethofencarb + carbendazim, and vinclozolin combined with chlorothalonil were tested for their ability to control grey mould (Botrytis cinerea Pers. ex Fr.) of cucumber and tomato grey mould in greenhouses under commercial conditions. In winter 1987/88 the number of diseased female fruits of cucumber (Cucumis sativus L.) was reduced by diethofencarb + carbendazim (2·5 mg dm^?3 each) by 93% and by tebuconazole (2·5 mg dm^?3)(phytotoxic when alone) or tebuconazole (1 mg dm^?3) + dichlofluanid (4 mg dm^?3) by 54–57%. Vinclozolin (5 mg dm^?3) + chlorothalonil (25 mg dm^?3) significantly reduced disease incidence on fruits by 40%. Infection foci on cucumber stems were significantly decreased by vinclozolin + chlorothalonil. A more pronounced decrease was obtained with diethofencarb + carbendazim, tebuconazole, or tebuconazole + dichlofluanid. During the season of winter 1988/89, tebuconazole + dichlofluanid (1·5 + 6 and 3 + 12 mg dm^?3) and RH7592 (1 mg dm^?3) significantly reduced diseased fruits by 30–71%. Grey mould of tomato (Lycopersicon esculentum Mill.) leaves was reduced by more than 90% and on fruits by 78–87% when tebuconazole + dichlofluanid (1·5 + 6 and 3 + 12 mg dm^?3) or diethofencarb + carbendazim (2·5 mg dm^?3 each) were applied. Yields of cucumber fruits of the common parthenocarpic cv. Kasem 292 were weighed. There was no correlation between disease level and yield in any experiment, plot or date except for two measurements. Compesation in fruit production by the plant may be regarded as the reason for no positive yield response to efficient control. The possibility of reducing fungicide application is discussed. Control of grey mould on tomato resulted in yield increase.     

Cross-resistance relationships between benzimidazole fungicides and diethofencarb in Botrytis cinerea and their genetical basis in Ustilago maydis

After nitrosoguanidine- or UV-mutagenesis, three different benzimidazole-resistant phenotypes were isolated on media containing benomyl or a mixture of carbendazim and diethofencarb from wild-type strains of Botrytis cinerea Pers. ex Fr. and Ustilago maydis (D.C.) Corda. Mutants of B. cinerea with moderate (MBr) or low (LBr) resistance to benzimidazoles and high resistance to diethofencarb (Dr) were isolated from the fungicide-mixture-containing medium in low frequency (7–1 × 10^?8). Only benzimidazole-resistant strains highly sensitive to diethofencarb (HBrDs) were identified on benomyl-containing medium at a frequency of 6.6 × 10^?6. Fitness-determining characteristics such as sporulation, germination and germ-tube elongation, were found to be reduced significantly in the mutants of B. cinerea that were resistant to both benzimidazoles and diethofencarb. However, pathogenicity of a MBrDr mutant strain on cucumber seedlings was equal to that of the wild type and a carbendazim + diethofencarb mixture was found to control grey mould caused by the wild type, but was not effective when the plant cotyledons were infected by the mutant strain. Three benzimidazole-resistant phenotypes (HBrDs, HBrDr, MBrDr) were isolated easily in U. maydis from a benomyl-containing medium. In contrast with B. cinerea, only one-tenth of the benzimidazole-resistant strains were sensitive to diethofencarb. Genetic analysis of benzimidazole resistance in U. maydis showed that the three benzimidazole-resistant phenotypes were due to three allelic mutations in a single gene and one of them was responsible for the negative cross-resistance between benzimidazoles and diethofencarb.     

北京地区番茄灰霉病菌的多重抗药性检测

2009年12月-2010年5月,在北京12个郊区县采集番茄病标样150份,分离纯化得到109个灰葡萄孢（Botrytis cinerea）单孢菌株,用最低抑制浓度法（MIC）测定了其对苯并咪唑类（多菌灵）、二甲酰亚胺类（腐霉利）和氨基甲酸酯类（乙霉威）杀菌剂的抗药性。结果表明：番茄灰霉病菌对多菌灵、腐霉利和乙霉威产生抗性菌株的频率分别为96.3%、80.7% 和58.7%;所测菌株对3类杀菌剂的抗性类型有BenRDicSNPCS、BenSDicSNPCR、BenRDicRNPCS和BenRDicRNPCR 4种,所占比例分别是19.3%、3.7%、21.1%和56.0%,表明北京地区番茄灰霉病菌对苯并咪唑类、二甲酰亚胺类和氨基甲酸酯类三类杀菌剂的抗药性严重,在生产中需慎用,应选择一些替代的新型杀菌剂和生物农药。     

咯菌腈对草莓灰霉病Botrytis cinerea的毒力及防效研究初报

用生长速率法测定了咯菌腈、多菌灵、硫菌·霉威、苯醚甲环唑4种药剂对草莓灰霉病Botrytis cinerea的毒力。结果表明,4种药剂对草莓灰霉菌菌丝的生长都有不同程度的抑制作用,随药剂浓度增加抑制作用增强,其EC50值分别为0.0472、95.7193、0.4854、1.3611 mg/L,其中咯菌腈的毒力最强。田间防治试验结果表明:2.5%咯菌腈FC每667 m2施药量120 mL时防效为83.7%,80 mL时防效为67.7%;65%硫菌·霉威WP每667m2施药量100 g时防效为65.3%。     

Negative cross resistance between benzimidazole and N-phenylcarbamate fungicides and control of Botrytis cinerea on grapes

Isolates of Botrytis cinerea resistant to benzimidazoles (Ben^R), dicarboximides (Dic^R), or both (Ben^R Dic^R) were found on table grapes in vineyards in Israel. In vineyards treated for one or two seasons with benomyl and dicarboximides, 41% of the isolates were benomyl-resistant, 18% were dicarboximide-resistant and 21 % were resistant to both fungicides. The frequency of resistant strains was very low in non-treated vineyards. Negatively correlated cross resistance (NCCR) was manifested by 46 Ben^R isolates which were sensitive to isopropyl N -(3,4-diethoxyphenyl) carbamate (NPC) and 73 benomyl-sensitive (Ben^s) isolates which were insensitive to NPC; NCCR was not influenced by sensitivity to dicarboximides. A mixture of Ben^s Dic^R plus Ben^R Dic^s isolates, inoculated on rose petals, was inhibited by mixtures of benzimidazoles plus NPC but not by benomyl, NPC or iprodione alone. Grey mould, incited on maturing grape berries by a vineyard population composed of Ben^s and Ben^R strains, was not controlled by benomyl, while the mixture of NPC plus carbendazim was effective.     

In vitro and In vivo Activity of Cyprodinil and Pyrimethanil on Botrytis cinerea Isolates Resistant to other Botryticides and Selection for Resistance to Pyrimethanil in a Greenhouse Population in Greece

No cross-resistance was observed between pyrimethanil or cyprodinil and the fungicides benomyl, iprodione or carbendazim + diethofencarb. In vitro, both anilinopyrimidine fungicides were effective against strains of Botrytis cinerea resistant to benzimidazoles and/or dicarboximides and against a wild type strain insensitive to diethofencarb (EC₅₀ values ranged between 0.03–0.19 and 0.006–0.054gml^–1 for pyrimethanil and cyprodinil, respectively). Preventive applications of anilinopyrimidines completely protected young cucumber plants and fruits that were inoculated with all strains of B. cinerea. The effectiveness of pyrimethanil against grey mould was studied in greenhouse grown tomatoes in relation to (a) the type of infection and the progress of the disease on different plant parts and (b) the response of the naturally occurring B. cinerea population to the selection pressure caused by eight successive applications of this fungicide. Pyrimethanil effectively controlled grey mould on leaves, fruits and stems but did not significantly reduce the number of dead plants and fruits with 'ghost spot' symptoms. The selection pressure caused by the consecutive applications of pyrimethanil resulted in reduction of its effectiveness on leaves that became apparent after the sixth application. This was correlated with a shift of the B. cinerea population (not previously exposed to anilinopyrimidines) towards reduced sensitivity, probably due to the development of a low level of resistance (R _L = 7.7). Pyrimethanil delayed the onset of the disease but it did not reduce the infection rate.     

多菌灵等4种杀菌剂对烟草灰霉病菌的室内生物活性

采用菌丝生长速率法和孢子萌发法,分别测定了烟草灰霉病菌对多菌灵、嘧霉胺、异菌脲和丙环唑的敏感性,同时通过离体叶片法评估了这4种杀菌剂对烟草灰霉病的保护和治疗作用。结果表明:4种杀菌剂对烟草灰霉病菌的菌丝生长和孢子萌发均表现出了不同程度的抑制活性,并对灰霉病同时具有保护和治疗作用。其中多菌灵对菌丝生长的抑制活性最强,EC₅₀平均值为0.06 mg/L,其次为丙环唑、嘧霉胺和异菌脲,EC₅₀平均值分别为0.36、0.53和0.60 mg/L;异菌脲和丙环唑对烟草灰霉病菌孢子萌发的抑制活性较强,EC₅₀平均值分别为2.05和2.21 mg/L,其次为嘧霉胺和多菌灵,EC₅₀平均值分别为10.56和131.23 mg/L。异菌脲和多菌灵对灰霉病的保护作用和治疗作用均最强,当药剂质量浓度为200 mg/L时,其对离体叶片的保护和治疗作用防效分别为100%、100%和98.3%、91.8%。研究结果可为烟草灰霉病的科学防治提供依据。     

Control of grey mould (Botrytis cinerea) with film-forming polymers

Several film-forming polymers reduced the amount of grey mould on various crops in a dew chamber and in a plastic house under natural conditions. The polymers Wilt Pruf, Biofilm and Colfix reduced germination of conidia and germ tube length of Botrytis cinerea . The most effective inhibition of linear growth of the pathogen on potato dextrose agar was obtained by Safe Pack and Biofilm. All polymers significantly reduced grey mould on detached leaves of Capsicum, Phaseolus , tomato, cucumber, rose and pelargonium. Grey mould on rose flowers was not controlled, apparently due to latent infection. The substances Biofilm and Vapor Gard were applied either alone or with chlorothalonil fungicide on cucumber plants in a commercial greenhouse. The polymers had no harmful effect to the host. Disease on senescing female fruits of cucumber was reduced by 46–67% with no additive effect to the mixture with fungicides. Stem infection also was reduced.     

Fungicide resistance inBotrytis cinerea isolates from strawberry crops in Huelva (southwestern Spain)

The severity of disease caused byBotrytis cinerea in strawberries is very high and chemical control is common practice; low residue levels of chemical products are required. Thus, it is important to be aware of the development of fungicide resistance in order to choose the best strategies of chemical control. In the present study we evaluated the response of 36B. cinerea isolates against eight different fungicides. The isolates were sampled twice, at the beginning and the end of the season, in 11 commercial strawberry fields located in the area of Huelva (Spain). In addition, two reference isolates, SAS56 and SAS405, were evaluated. The proportion of isolates resistant to benomyl was very high (86%). Resistance to dicarboximides was detected in 44% of the isolates and resistance to pyrimethanil in 25% of the isolates. Different degrees of sensitivity to captan and dichlofluanid were recorded. No resistance was found to diethofencarb plus carbendazim. http://www.phytoparasitica.org posting Sept. 18, 2002.     

Influence of spray schedules on fungicide resistant populations of Botrytis cinerea Pers. on grapevine

Treatments with dicarboximide fungicides provided only 20 to 40% control of grey mould on grape (cv. Moscato d'Asti) in vineyards where benzimidazole and dicarboximide resistant strains ofBotrytis cinerea Pers. constituted about 50% of the fungal population and a high disease pressure existed. The percentage of dicarboximide resistant strains increased after treatment. Fungicide combinations of a benzimidazole with diethofencarb, sprayed twice per season or once alternated with a dicarboximide, provided satisfactory control of grey mould and decreased the percetage of benzimidazole resistant strains. A combination of thiram and procymidone controlled grey mould, but increased dicarboximide resistance in the population.     

Detection and characterization of fungicide resistant phenotypes of Botrytis cinerea in lettuce crops in Greece

The development of resistance to chemical control agents needs continuous monitoring in Botrytis cinerea. 790 isolates from lettuce and other vegetable crops were collected from six widely separated sites in Greece and tested for their sensitivity to 11 fungicides from nine unrelated chemical groups. 44 of the isolates exhibited multiple resistance to fenhexamid (hydroxyanilides), azoxystrobin and pyraclostrobin (QoI’s), boscalid (SDHI’s), cyprodinil and pyrimethanil (anilinopyrimidines), fludioxonil (phenylpyrroles), carbendazim (benzimidazoles) and iprodione (dicarboximides). Thirty per cent of such phenotypes were detected in an experimental glasshouse with lettuce crops, the third year after commencing fungicide applications. The average resistance factor (R_f) for mycelial growth to fenhexamid, pyraclostrobin, boscalid, cyprodinil and fludioxonil, was over 40, 1,000, 100, 700 and 50, respectively. Some strains with high resistance to anilinopyrimidines (14 %) or moderate to fludioxonil (7 %) were detected even in isolates collected from vegetable crops prior to commercial use of these fungicides in Greece. Isolates with fludioxonil moderate resistance and fenhexamid high resistance, were detected for the first time in Greece. The results suggested the high risk in chemical control of grey mould due to development of resistance to most fungicides with site-specific modes of action. Isolates with resistance to fluazinam (phenylpyridinamines) and to chlorothalonil (phthalonitriles) were not found. The inclusion of appropriate multi-site inhibitors like chlorothalonil in fungicide anti-resistance strategies was indispensable.     

灰霉菌的抗药性与适合度测定

室内监测了北京、沈阳、保定等地区番茄灰霉菌 (Botrytis cinerea)对不同杀菌剂的抗药性 ,发现抗多菌灵菌株占监测总数的 74 .6 7% ,抗乙霉威菌株占 2 5.82 % ,还监测到 3株抗多菌灵、乙霉威菌株——即双抗菌株 ;利用菌落直径法和番茄叶片法测定了 3种抗药类型的番茄灰霉菌在离体和活体条件下的适合度 ,结果表明 ,不同抗药类型菌株在生长速率、致病力、繁殖力等方面差异不明显。     

French vineyards provide information that opens ways for effective resistance management of Botrytis cinerea (grey mould)

Resistance to fungicides is an evolutionary process resulting from the selection of advantageous genotypes in naturally diverse populations. Seven fungicide modes of action are authorised to control grey mould caused by Botrytis cinerea on grapevine in France, and five of them have encountered specific resistance, with variable frequencies in populations and possible consequences for field fungicide efficacy. Moreover, multidrug resistance is caused by fungicide efflux and allows a weak resistance towards six unrelated modes of action. Here, a review is given of the fungicide resistance status of B. cinerea in France, particularly in the vineyards of Champagne, which are the most affected. Recently developed resistance and recent findings concerning the associated resistance mechanisms are focused upon in particular. Finally, antiresistance strategies are presented, and examples of managed resistance are discussed in a more general manner with the aim of extending this knowledge to other crops and countries undergoing similar resistance problems. © 2013 Society of Chemical Industry     

Reduced sensitivity of Botrytis cinerea to two sterol biosynthesis-inhibiting fungicides: fenetrazole and fenethanil

Isolates of Botrytis cinerea having reduced sensitivity to the sterol biosynthesis-inhibiting (SBI) fungicides fenetrazole and fenethanil were obtained from one out of four sites from which isolates were tested. Reduced sensitivity was associated with poor disease control by fenetrazole, which had been applied with dichlofluanid. Conidial germination and hyphal growth of B. cinerea from the four sites were tested in vitro on media amended with the fungicides. Following fenetrazole or fenethanil treatment, at the site where control had failed, populations of B. cinerea were detected with higher EC₅₀ and EC₉₀ values than at the three other sites. Germination of conidia of B. cinerea was markedly inhibited by 1.0 μg/ml fenetrazole and 0.5–1.0 μg/ml fenethanil. The frequency of isolates insensitive to 1.0 μg/ml fenetrazole or to 0.5 μg/ml fenethanil was 3.4 and 1.8 times higher, respectively, at the site where control had failed, compared with another site where SBI fungicides had never been applied to control grey mould. Grey mould caused by selected isolates of B. cinerea which expressed the phenotype of low sensitivity to SBI fungicides in leaves of tomato, pepper and Senecio cineraria was not controlled by either fenetrazole or fenethanil (1 .5–3.0 μg/ml). However, up to 100% disease reduction was obtained when leaves infected by sensitive isolates were treated with the fungicides.     

设施蔬菜灰霉病菌对不同类型杀菌剂的抗性检测

为了明确设施蔬菜灰霉病菌Botryotinia fuckeliana的抗药性现状,采用菌丝生长速率法检测了20052006年采自浙江、江苏、山东和辽宁4省的144个菌株对6种常用防治药剂的敏感性。结果表明,灰霉病菌对百菌清已经产生了低水平的抗性,频率为5.56%,其对多菌灵的抗性非常严重,总的抗性频率为43.05%,高抗(HR)频率为27.08%;乙霉威的EC50值在0.137728.9μg/mL之间,平均为40.06μg/mL。其中多菌灵-乙霉威双抗频率为36.11%,且首次检测到了两种新的双抗类型。二甲酰亚胺类杀菌剂在生产上已经应用近20年,但灰霉病菌对异菌脲和腐霉利只有频率为20%左右的低水平抗性,没有检测到高抗菌株;苯胺嘧啶类杀菌剂嘧霉胺虽然只应用几年时间,但已经产生了抗性,其抗性菌株频率为4.16%。研究表明,设施蔬菜灰霉病菌对常用的6种防治药剂均产生了不同程度的抗性。     

Pervasive fungicide resistance in Botrytis from strawberry in Norway: Identification of the grey mould pathogen and mutations

Control of grey mould, caused by Botrytis spp., is a major challenge in open field strawberry production. Botrytis was isolated from plant parts collected from 19 perennial strawberry fields with suspected fungicide resistance in the Agder region of Norway in 2016. Resistance to boscalid, pyraclostrobin and fenhexamid was high and found in 89.1%, 86.0% and 65.4% of conidia samples, respectively. Multiple fungicide resistance was common; 69.6% of conidia samples exhibited resistance to three or more fungicides. Botrytis group S and B. cinerea sensu stricto isolates were obtained from 19 and 16 fields, respectively. The sdhB, cytb, erg27 and mrr1 genes of a selection of isolates were examined for the presence of mutations known to confer fungicide resistance to boscalid, pyraclostrobin, fenhexamid and pyrimethanil plus fludioxonil, respectively. Allele-specific PCR assays were developed for efficient detection of resistance-conferring mutations in cytb. Among B. cinerea isolates, 84.7%, 86.3% and 61.3% had resistance-conferring mutations in sdhB, cytb and erg27, respectively. A triplet deletion in mrr1, resulting in ΔL497, commonly associated with the multidrug resistance phenotype MDR1h, was detected in 29.2% of Botrytis group S isolates. High frequencies of resistance to several fungicides were also detected in Botrytis from both imported and domestically produced strawberry transplants. Fungicide resistance frequencies were not different among fields grouped by level of grey mould problem assessed by growers, indicating factors other than fungicide resistance contributed to control failure, a fact that has important implications for future management of grey mould.