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.     

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

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

四川猕猴桃灰霉病菌对4种杀菌剂的抗药性检测

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

福建省番茄灰霉病菌对氟啶胺敏感基线建立及与不同杀菌剂的交互抗性

为明确中国福建省番茄灰霉病菌对氟啶胺的敏感性及其与不同杀菌剂的交互抗性,采用菌丝生长速率法测定了106株采自福建省主要番茄产区的番茄灰霉病菌对氟啶胺的敏感性。结果表明,氟啶胺对福建省番茄灰霉病菌菌丝生长的EC50值在0.0037～0.0452 μg/mL之间,平均值为（0.0221 ±0.0098）μg/mL,其敏感性频率分布呈连续单峰曲线,符合正态分布,因此可将该EC50平均值（0.0221±0.0098）μg/mL作为福建省番茄灰霉病菌对氟啶胺的敏感基线,用于其田间抗药性监测。从106株菌株中选择15株对氟啶胺敏感性不同的菌株,测定了其对嘧霉胺、异菌脲、腐霉利和啶氧菌酯的敏感性。结果表明,供试5种杀菌剂对15株番茄灰霉病菌菌丝生长的平均抑制活性依次为氟啶胺 >异菌脲 >腐霉利 >啶氧菌酯 >嘧霉胺,氟啶胺与异菌脲、腐霉利、啶氧菌酯和嘧霉胺之间均不存在交互抗性。     

Studies on the Inherent Resistance Risk to Fenhexamid in Botrytis cinerea

After chemical mutagenesis with N-methyl-N-nitrosoguanidine (MNNG) two phenotypes that were highly or moderately resistant to fenhexamid, were isolated from a wild-type strain of Botrytis cinerea, at a mutation frequency of 0.9 × 10^–5. Resistance factors, based on EC⁵⁰ values, were 460–570 and 10–15, respectively. The mutation(s) for resistance to fenhexamid did not affect the sensitivity of mutant strains to the benzimidazole benomyl, the phenylpyridinamine fluazinam, the anilinopyrimidine cyprodinil, the guanidine iminoctadine or to the sterol-biosynthesis-inhibiting fungicides fenarimol, fenpropimorph and tridemorph. On the contrary, an increased sensitivity (EC⁵⁰ ratios of 0.2–0.6) of fenhexamid-resistant strains to the phenylpyrrole fludioxonil and the dicarboximide iprodione was observed. Study of fitness parameters of fenhexamid-resistant isolates of both phenotypic classes showed that these mutation(s) had no effect on mycelial growth and sensitivity to high osmolarity, but they did affect one or more of some other characteristics, such as sporulation, conidial germination and sclerotia production. In tests on cucumber seedlings under greenhouse conditions, all highly fenhexamid-resistant isolates tested presented decreased infection ability compared with the wild-type. Preventive applications of a commercial formulation of fenhexamid, Teldor 50 WP, were effective against lesion development on cotyledons by the wild-type, but ineffective, even in high concentrations, against disease caused by the fenhexamid-resistant isolates. The risk of resistance problems arising during commercial use of fenhexamid is discussed.     

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

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

Resistance to polyoxin AL and other fungicides in Botrytis cinerea collected from sweet basil crops in Israel

A total of 568 B. cinerea isolates were collected from diseased sweet basil plants and the air in 10 sweet basil greenhouses. Mycelial growth tests were used to evaluate the sensitivity of these isolates to benomyl, fenhexamid, iprodione, polyoxin AL and pyrimethanil. EC₅₀ values for polyoxin, the main botryticide on sweet basil in Israel, ranged from 0.4 to 6.5 μg ml^?1 and had a bimodal distribution; the EC₅₀ values for sensitive isolates ranged from 0.4 to 1.5 μg ml^?1 and the EC₅₀ values for low-level resistant isolates ranged from 4 to 6.5 μg ml^?1. Among populations that had not been exposed to polyoxin treatments, 20 to 35 % of the collected isolates were low-level resistant for polyoxin. Polyoxin treatments in an experimental greenhouse shifted the equilibrium in favour of low-level resistant isolates, and the change occurred rapidly: from a frequency of 20 % low-level resistant isolates in the population that had never been treated with polyoxin to a frequency of 72 % after a few treatments over two seasons. Prolonged use of polyoxin in Israeli basil crops (in some sites for more than 10 years) does not appear to have led to the development of high-level resistance, but low-level resistant isolates were found in commercial greenhouses with the frequency of up to 73 %. High-level resistance to benzimidazoles was common (60 to 80 % of isolates) in greenhouses with a history of benzimidazole treatments; whereas 15–25 % of the isolates from greenhouses in which fungicides were not used were resistant. Low-level resistance to dicarboximides was fairly widespread (frequency of 30 to 80 % depending on the greenhouse) and a few cases of moderate resistance to dicarboximides were also noted (frequency of 0 to 9 %). Neither high- nor low-level resistance to anilinopyrimidines was common in sweet basil commercial greenhouses (0 to 7 %). However, 34 % of the isolates were strongly resistant in the experimental greenhouse, following a few treatments with anilinopyrimidine fungicides during the previous season. Before those treatments, the proportion of anilinopyrimidines resistant isolates had been 1 %. About 3 % of the isolates exhibited low-level resistance to fenhexamid and no isolates were found to be strongly resistant to fenhexamid. Low-level resistance to one fungicide was often associated with low-level resistances to other fungicides. Thirty-two phenotypes exhibiting resistance to one or more of the tested fungicides were noted among B. cinerea isolates. Resistant isolates showed similar or reduced fitness parameters in comparison to wild-type isolates.     

Multiple resistance of Botrytis cinerea from kiwifruit to SDHIs,QoIs and fungicides of other chemical groups

BACKGROUND: Botrytis cinerea Pers.: Fr. is a high‐risk pathogen for fungicide resistance development that has caused resistance problems on many crops throughout the world. This study investigated the fungicide sensitivity profile of isolates from kiwifruits originating from three Greek locations with different fungicide use histories. Sensitivity was measured by in vitro fungitoxicity tests on artificial nutrient media. RESULTS: Seventy‐six single‐spore isolates were tested for sensitivity to the SDHI fungicide boscalid, the QoI pyraclostrobin, the anilinopyrimidine cyprodinil, the hydroxyanilide fenhexamid, the phenylpyrrole fludioxonil, the dicarboxamide iprodione and the benzimidazole carbendazim. All isolates from Thessaloniki showed resistance to both boscalid and pyraclostrobin, while in the other two locations the fungal population was sensitive to these two fungicides. Sensitive isolates showed EC₅₀ values to boscalid and pyraclostrobin ranging from 0.9 to 5.2 and from 0.04 to 0.14 mg L^?1 respectively, while the resistant isolates showed EC₅₀ values higher than 50 mg L^?1 for boscalid and from 16 to > 50 mg L^?1 for pyraclostrobin. All QoI‐resistant isolates carried the G143A mutation in cytb. Sensitivity determinations to the remaining fungicides revealed in total eight resistance phenotypes. No isolates were resistant to the fungicides fenhexamid and fludioxonil. CONCLUSION: This is the first report of B. cinerea field isolates with resistance to both boscalid and pyraclostrobin, and it strongly suggests that there may be a major problem in controlling this important pathogen on kiwifruit. Copyright © 2010 Society of Chemical Industry     

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

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

     

A rapid and simple method for determining fungicide resistance in Botrytis

A simple test based on the germination of conidia of Botrytis on agar media augmented with various fungicides has been developed. Average concentrations causing a 50% reduction of germ-tube growth (EC₅₀) of highly sensitive isolates were determined on 1% malt extract agar (thiophanate-methyl 0.090 ppm; iprodione 0.566 ppm; fludioxonil 0.026 ppm; fenhexamid 0.144 ppm), 1% malt extract agar with 100 ppm salicyl hydroxamic acid (QoI fungicides, viz. trifloxystrobin 0.009 ppm; pyraclostrobin 0.013 ppm; azoxystrobin 0.087 ppm), 0.5% yeast extract agar (boscalid 0.069 ppm) and 0.5% sucrose agar (cyprodinil 0.053 ppm). In order to detect different levels of resistance against these various fungicides, two discriminatory concentrations were identified for each compound. A routine assay method was developed in which drops of a conidial suspension harvested directly from diseased plant material or sporulating cultures were incubated on each of 20 different agar media. Because of a very short time-span of 24–48 h between sample collection and evaluation of results, field-specific information on the occurrence, frequency and types of resistance of Botrytis against common botryticides in soft-fruit production may be generated prior to the main fungicide spray season at blossom time.

     

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

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

我国人参产区灰霉病菌抗药性研究初报

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

Recovery of fungicide-resistant <Emphasis Type="Italic">Botrytis</Emphasis> isolates from strawberry nursery plants

In a four-year survey of strawberry nursery plants, 340 Botrytis isolates were collected and examined for fungicide resistance. High percentages of isolates with resistance to members of all fungicide classes registered on strawberries in Germany were found, i.e. trifloxystrobin (90.3%), boscalid (53.8%), cyprodinil (41.5%), fludioxonil (28.2%) and fenhexamid (15.6%). Of these isolates, 10.3% possessed multiple resistance to all compounds. At 5.0%, resistance to the recently registered fungicide fluopyram was low. Dutch nursery material harboured significantly higher proportions of isolates with resistance to any or all fungicides than plants produced in Germany. These data point to nursery plants as a possible route of introducing fungicide-resistant Botrytis strains into commercial strawberry fields.     

湖南省草莓灰霉病菌对4种杀菌剂的抗药性检测

为明确湖南省草莓灰霉病菌菌株的抗药性状况,2013-2015年从湖南省不同地区草莓灰霉病病果或病叶上经单孢分离获得草莓灰霉病菌454株,采用最小抑制浓度法(MIC)检测不同地区草莓灰霉病菌株对多菌灵、腐霉利、异菌脲、嘧霉胺的抗药性。结果表明:草莓灰霉病菌对多菌灵、腐霉利、异菌脲、嘧霉胺的抗性频率分别为55.73%,77.31%、3.52%和77.31%;所测菌株对4种杀菌剂的敏感性类型有Cbz~RPcm~RIpd~RPmt~R、CbzSPcm~RIpd~RPmt~R、Cbz~RPcm~RIpdSPmt~R、Cbz~SPcm~RIpdSPmt~S、Cbz~RPcm~SIpd~SPmt~R、Cbz~SPcm~RIpd~SPmt ~R等6种,其所占比例分别为33.26%、5.07%、41.41%、4.63%、3.96%、11.67%,未发现对4种杀菌剂均敏感的类型,表明湖南省草莓灰霉病菌已对多菌灵、腐霉利和嘧霉胺产生抗药性,对异菌脲的抗药性较低。     

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.     

马铃薯早疫病菌对咯菌腈的敏感基线及其对不同药剂的交互抗性

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

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.     

五种杀菌剂对烟草立枯病菌的生物活性

由立枯病丝核菌引起的烟草立枯病是我国烟草苗床上危害最严重的病害之一。本研究评价了5种杀菌剂 (嘧菌酯、啶酰菌胺、氟啶胺、丙环唑和嘧霉胺) 对立枯丝核菌菌丝生长、菌核形成和萌发的影响,以及其对烟草立枯病的防治效果。结果表明:立枯丝核菌菌丝对氟啶胺和嘧菌酯的敏感性高于丙环唑和啶酰菌胺,而对嘧霉胺的敏感性较低;嘧菌酯对菌核形成的抑制作用强于丙环唑、氟啶胺、啶酰菌胺和嘧霉胺;5种杀菌剂对立枯丝核菌菌核萌发均无抑制作用。在离体烟叶的保护活性方面,12.5和50 mg/L的嘧菌酯和啶酰菌胺对立枯病的保护作用优于氟啶胺、丙环唑和嘧霉胺;在治疗活性方面,50和200 mg/L的嘧菌酯的治疗作用优于其他4种杀菌剂。因此,供试的5种杀菌剂中嘧菌酯最适合用于烟草立枯病的防治。     

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.     

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。