Lack of cross-resistance to a novel succinate dehydrogenase inhibitor, fluopyram, in highly boscalid-resistant isolates of Corynespora cassiicola and Podosphaera xanthii

BACKGROUND: Recently in Japan, isolates resistant to boscalid, a succinate dehydrogenase inhibitor (SDHI), have been detected in Corynespora cassiicola (Burk. & Curt.) Wei and Podosphaera xanthii (Castaggne) Braun & Shishkoff, the pathogens causing Corynespora leaf spot and powdery mildew disease on cucumber, respectively. Resistant isolates of C. cassiicola are widely distributed and represent a serious problem in disease control at present. Novel SDHI fungicides, including fluopyram, are now under development. RESULTS: The growth of very highly boscalid‐resistant, highly resistant and sensitive isolates of C. cassiicola was strongly suppressed on fluopyram‐amended YBA agar medium. Although boscalid and another SDHI, penthiopyrad, hardly controlled Corynespora leaf spot and powdery mildew on cucumber plants when very highly or highly boscalid‐resistant isolates were employed for inoculation, fluopyram still exhibited excellent control efficacy against these resistant isolates as well as sensitive isolates of C. cassiicola and P. xanthii. CONCLUSION: Differential sensitivity to boscalid, penthiopyrad and fluopyram, clearly found in these two important pathogens of cucumber, may indicate involvement of a slightly distinct site of action for fluopyram from the two other SDHIs. This finding may lead to the discovery of unique SDHIs in the future. Copyright © 2011 Society of Chemical Industry     

Screening and characterization of resistance to succinate dehydrogenase inhibitors in Alternaria solani

Field isolates of Alternaria solani, which causes early blight of potato in Idaho, USA were evaluated in vitro for their sensitivity towards the succinate dehydrogenase inhibitor (SDHI) fungicides boscalid, fluopyram and penthiopyrad. A total of 20 isolates were collected from foliar‐infected tissue in 2009, 26 in 2010 and 49 in 2011. Fungicide sensitivity was tested using the spiral‐gradient end point dilution method. The frequency of boscalid‐resistant isolates (>50% relative growth when using a spiral dilution gradient starting at 507 mg L^?1) drastically increased over the duration of this study (15% in 2009, 62% in 2010 and 80% in 2011). Increasing resistance to fluopyram and penthiopyrad was observed. However, cross‐resistance was only observed between boscalid and penthiopyrad. The target site of this fungicide class is the succinate dehydrogenase (SDH) enzyme complex, which is vital for fungal respiration. Sequence analysis of the SDH complex revealed mutations in the subunits B and D that were correlated with the emergence of boscalid resistance in potato fields in Idaho. In particular, H277R and H133R were identified in SDH subunits B and D, respectively. The presence of restriction sites in the gene sequences allowed the development of a rapid PCR‐RFLP method to assess boscalid sensitivity in A. solani populations.     

Mutations in sdh genes in field isolates of Zymoseptoria tritici and impact on the sensitivity to various succinate dehydrogenase inhibitors

Zymoseptoria tritici is the causal agent of septoria tritici blotch (STB), a foliar wheat disease important worldwide. Succinate dehydrogenase inhibitors (SDHIs) have been used in cereals for effective control of STB for several years, but resistance towards SDHIs has been reported in several phytopathogenic fungi. Resistance mechanisms are target‐site mutations in the genes coding for subunits B, C and D of the succinate dehydrogenase (SDH) enzyme. Previous monitoring data in Europe indicated the presence of single isolates of Z. tritici with reduced SDHI sensitivity. These isolates carried mutations leading to amino acid exchanges: C‐T79N, C‐W80S in 2012; C‐N86S in 2013; B‐N225T and C‐T79N in 2014; and C‐V166M, B‐T268I, C‐N86S, C‐T79N and C‐H152R in 2015. The current study provides results from microtitre and greenhouse experiments to give an insight into the impact of different mutations in field isolates on various SDHIs. In microtitre tests, the highest EC₅₀ values for all tested SDHIs were obtained with mutants carrying C‐H152R. Curative greenhouse tests with various SDHIs confirmed the findings of microtitre tests that isolates with C‐H152R are, in general, controlled with lower efficacy than isolates carrying B‐T268I, C‐T79N and C‐N86S. SDHI‐resistant isolates of Z. tritici found in the field were shown to have cross‐resistance towards all SDHIs tested. So far, SDHI‐resistant isolates of Z. tritici have been found in low frequencies in Europe. Therefore, FRAC recommendations for resistance management in cereals, including a limited number of applications, alternation and combination with other MOAs, should be followed to prolong SDHI field efficacy.     

Non‐target site SDHI resistance is present as standing genetic variation in field populations of Zymoseptoria tritici

BACKGROUND

A new generation of more active succinate dehydrogenase (Sdh) inhibitors (SDHIs) is currently widely used to control Septoria leaf blotch in northwest Europe. Detailed studies were conducted on Zymoseptoria tritici field isolates with reduced sensitivity to fluopyram and isofetamid; SDHIs which have only just or not been introduced for cereal disease control, respectively.

RESULTS

Strong cross‐resistance between fluopyram and isofetamid, but not with other SDHIs, was confirmed through sensitivity tests using laboratory mutants and field isolates with and without Sdh mutations. The sensitivity profiles of most field isolates resistant to fluopyram and isofetamid were very similar to a lab mutant carrying SdhC‐A84V, but no alterations were found in SdhB, C and D. Inhibition of mitochondrial Sdh enzyme activity and control efficacy in planta for those isolates was severely impaired by fluopyram and isofetamid, but not by bixafen. Isolates with similar phenotypes were not only detected in northwest Europe but also in New Zealand before the widely use of SDHIs.

CONCLUSION

This is the first report of SDHI‐specific non‐target site resistance in Z. tritici. Monitoring studies show that this resistance mechanism is present and can be selected from standing genetic variation in field populations. © 2017 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.     

樱桃褐腐病菌对啶酰菌胺的敏感性及其对4种琥珀酸脱氢酶抑制剂的交互抗性

采用菌丝生长速率法测定了樱桃褐腐病菌Monilinia fructicola对啶酰菌胺的敏感性,同时研究了不同敏感性菌株的生物学性状,探究了琥珀酸脱氢酶B亚基的氨基酸突变与其对啶酰菌胺产生抗性的相关性,并分析了樱桃褐腐病菌对啶酰菌胺与其他3种琥珀酸脱氢酶抑制剂(SDHIs)氯苯醚酰胺、氟唑菌苯胺和氟吡菌酰胺之间的交互抗...     

Biological activity of the succinate dehydrogenase inhibitor fluopyram against Botrytis cinerea and fungal baseline sensitivity

BACKGROUND: Succinate dehydrogenase inhibitors (SDHIs) constitute a fungicide class with increasing relevance in crop protection. These fungicides could play a crucial role in successful management of grey mould disease. In the present study the effect of fluopyram, a novel SDHI fungicide, on several developmental stages of Botrytis cinerea was determined in vitro, and the protective and curative activity against the pathogen was determined on strawberry fruit. Furthermore, fungal baseline sensitivity was determined in a set of 192 pathogen isolates. RESULTS: Inhibition of germ tube elongation was found to be the most sensitive growth stage affected by fluopyram, while mycelial growth was found to be the least sensitive growth stage. Fluopyram provided excellent protective activity against B. cinerea when applied at 100 µg mL^?1 96, 48 or 24 h before the artificial inoculation of the strawberry fruit. Similarly, fluopyram showed a high curative activity when it was applied at 100 µg mL^?1 24 h post‐inoculation, but, when applications were conducted 48 or 96 h post‐inoculation, disease control efficacy was modest or low. The measurement of baseline sensitivity showed that it was unimodal in all the populations tested. The individual EC₅₀ values for fluopyram ranged from 0.03 to 0.29 µg mL^?1. In addition, no correlation was found between sensitivity to fluopyram and sensitivity to other fungicides, including cyprodinil, fenhexamid, fludioxonil, iprodione, boscalid and pyraclostrobin. CONCLUSIONS: The obtained biological activity, baseline sensitivity and cross‐resistance relationship data suggest that fluopyram could play a key role in grey mould management in the near future and encourage its introduction into spray programmes. Copyright © 2011 Society of Chemical Industry     

Biological and molecular characterization of field isolates of <Emphasis Type="Italic">Alternaria alternata</Emphasis> with single or double resistance to respiratory complex II and III inhibitors

Field isolates of Alternaria alternata collected from tomato processors were characterized for sensitivity to respiration inhibitors using in vitro mycelial growth assays. Pyraclostrobin (QoI), boscalid, fluopyram and isopyrazam (SDHIs) mean EC₅₀ values were 0.32, 1.43, 2.21, and 3.53 μg/ml respectively. Of the 42 isolates, 36 were sensitive to all respiration inhibiting fungicides tested whereas three isolates were less sensitive to boscalid, one to pyraclostrobin and two were simultaneously resistant to both inhibitors and isopyrazam. Correlation analysis between fungicide sensitivities revealed a positive cross-resistance between pyraclostrobin and tebuconazole, and between cyprodinil and mancozeb. There was no cross-resistance between QoIs, SHDIs or any other mode of action. Sequencing of the QoI and SDHI targets revealed the G143A cytochrome b resistance mutation in all pyraclostrobin-resistant isolates while analysis of the succinate dehydrogenase coding gene revealed point mutations in two of three of the gene subunits analyzed in boscalid-resistant isolates. Specifically, two isolates carried the H277Y and three the H133Q resistance mutations located in the sdhB and sdhD subunits of the respiration complex II, respectively. Isolates bearing the H277Y mutation also carried the G143A cytochrome b resistance mutation. Boscalid and pyraclostrobin-resistant isolates exhibited greater pathogenicity and sporulation compared to sensitive isolates, respectively. Isolates with cross-resistance exhibited greater pathogenicity and sporulation but slower mycelial growth compared to sensitive isolates. This is the first report of field isolates of A. alternata with single or double resistance to QoIs and SDHIs in Greece and should be considered in planning and implementing effective anti-resistance strategies.     

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     

五种琥珀酸脱氢酶抑制剂类杀菌剂与灰葡萄孢琥珀酸脱氢酶的结合模式及抗性机制分析

为探究琥珀酸脱氢酶抑制剂 (SDHI) 类杀菌剂与灰葡萄孢Botrytis cinerea琥珀酸脱氢酶 (SDH) (以下简称BcSDH) 的结合方式,阐明BcSDH对SDHI类杀菌剂产生抗性的结构生物学机制,通过同源建模构建了BcSDH的三维模型,通过分子对接预测了5种SDHI (异丙噻菌胺、氟吡菌酰胺、氟唑菌酰胺、吡噻菌胺和啶酰菌胺) 与野生型和突变型BcSDH的亲和力及结合模式之间的变化,分析其抗药性机制,对相关突变位点进行保守性预测,并分析突变类型。结果表明:5种SDHI与BcSDH 具有较强的亲和力,其中酸部分插入BcSDH活性腔底,胺部分在活性腔口,能够形成牢固的疏水作用、氢键、卤键、π-π堆积作用和π-阳离子等相互作用。B-P225F氨基酸残基突变 (以下简称突变) 会造成活性腔口变窄,使得SDHI酸部分不能进入活性腔;B-P225L突变会造成异丙噻菌胺、氟吡菌酰胺和吡噻菌胺与靶标蛋白的结合模式发生变化,亲和力降低;B-H272R突变后,活性腔底变窄,与SDHI的亲和力下降。另外,保守性分析结果表明,B-P225和B-H272均位于BcSDH的保守区域,B-P225F、B-H272R和B-H272L突变可能为随机突变。因此推测BcSDH的B-P225F和B-H272R突变可能是引起灰葡萄孢对5种杀菌剂产生抗性的主要原因,也可能是引起SDHI类杀菌剂之间交互抗性的主要原因之一;B-P225L突变可能降低灰葡萄孢对部分杀菌剂的敏感性,而不是引起BcSDH对SDHI类杀菌剂产生交互抗性的主要原因。在实际生产中,应采取合理有效的抗性监测治理策略来延缓灰葡萄孢对SDHI类杀菌剂抗性的产生,在SDHI分子设计时也应考虑该位点氨基酸残基突变,避免产生交互抗性。     

Distribution and molecular characterization of Corynespora cassiicola isolates resistant to boscalid

A total of 618 isolates of corynespora leaf spot fungus (Corynespora cassiicola) collected from 24 commercial cucumber greenhouses in 12 cities in Ibaraki Prefecture, Japan, were tested for their sensitivity to boscalid. Boscalid‐resistant isolates were detected in 17 out of 19 greenhouses with a history of use of this fungicide and detection frequencies of the resistant isolates exceeded 47% in nine greenhouses. Frequencies of very highly resistant (VHR) isolates with 50% effective concentration (EC₅₀) values of boscalid exceeding 30 μg mL^?1 were higher than those of moderately resistant (MR) isolates with EC₅₀ ranging from 2·0 to 5·9 μg mL^?1 in 11 greenhouses. Additionally, highly resistant (HR) isolates with EC₅₀ from 8·9 to 10·7 μg mL^?1 were first detected. Furthermore, molecular characterization of genes encoding succinate dehydrogenase (SDH) subunits (SdhA, SdhB, SdhC and SdhD) was carried out to elucidate the amino acid substitution responsible for the resistance to boscalid. All 23 VHR isolates had the same mutation from CAC to TAC in the SdhB gene leading to the substitution of histidine with tyrosine at amino acid position 278 (B‐H278Y). At the same position, the substitution to arginine conferred by a mutation to CGC (B‐H278R) was detected in all four HR isolates. Some MR isolates showed a substitution from serine to proline at position 73 in SdhC (C‐S73P), from serine to proline or from glycine to valine at position 89 (D‐S89P) and 109 (D‐G109V), respectively, in SdhD. There was no common mutation in SDH genes of all MR isolates.     

Amino acid substitutions responsible for different QoI and SDHI sensitivity patterns in Puccinia horiana,the causal agent of chrysanthemum white rust

Quinone outside inhibitors (QoIs) and succinate dehydrogenase inhibitors (SDHIs) are major groups of agricultural fungicides. However, resistance to some of these fungicides has been reported in a Japanese population of Puccinia horiana, the causal agent of chrysanthemum white rust disease. Because their mechanisms are not well understood, we investigated the existence of mutations in QoI and SDHI target protein-encoding genes. Eight out of nine isolates from cultivated chrysanthemum carried L275F and L299F amino acid substitutions in cytochrome b, the target protein of QoIs. These isolates showed 23- and 17-fold higher EC₅₀ values for the QoI fungicides azoxystrobin and kresoxim-methyl, respectively, in basidiospore germination inhibitory tests, while they were hypersensitive to another QoI, famoxadone. All nine isolates were resistant to SDHI oxycarboxin and carried the I88F substitution in SdhC. This substitution was orthologous to the SdhC-I86F substitution found in some Brazilian isolates of the soybean rust fungus, Phakopsora pachyrhizi, showing reduced sensitivity to some SDHIs. Although the rarity of wild-type sensitive isolates, the subsequent limited number of comparisons between wild types and mutants, and a difficulty in applying reverse genetic analysis to this obligate parasite, are obstacles in making definitive conclusions, L275F and L299F in cytochrome b and SdhC-I88F are suspected to be responsible for the different patterns of sensitivity to QoI and for oxycarboxin-resistance in P. horiana, respectively.     

Resistance to pyraclostrobin, boscalid and multiple resistance to Pristine® (pyraclostrobin + boscalid) fungicide in Alternaria alternata causing alternaria late blight of pistachios in California

Pristine® (pyraclostrobin + boscalid) is a fungicide registered for the control of alternaria late blight in pistachio. A total of 95 isolates of Alternaria alternata collected from orchards with and without a prior history of Pristine® sprays were tested for their sensitivity towards pyraclostrobin, boscalid and Pristine® in conidial germination assays. The EC₅₀ values for 35 isolates from orchards without Pristine® sprays ranged from 0·09 to 3·14 µg mL^?1 and < 0·01 to 2·04 µg mL^?1 for boscalid and Pristine®, respectively. For pyraclostrobin, 27 isolates had EC₅₀ < 0·01 µg mL^?1 and six had low resistance (mean EC₅₀ value = 4·71 µg mL^?1). Only one isolate was resistant to all three fungicides tested, with EC₅₀ > 100 µg mL^?1. Among 59 isolates from the orchard with a history of Pristine® sprays, 56 were resistant to pyraclostrobin; only two were sensitive (EC₅₀ < 0·01 µg mL^?1) and one was weakly resistant (EC₅₀ = 10 µg mL^?1). For the majority of these isolates EC₅₀ values ranged from 0·06 to 4·22 µg mL^?1 for boscalid and from 0·22 to 7·74 µg mL^?1 for Pristine®. However, seven isolates resistant to pyraclostrobin were also highly resistant to boscalid and Pristine® and remained pathogenic on pistachio treated with Pristine®. Whereas strobilurin resistance is a common occurrence in Alternaria of pistachio, this is the first report of resistance to boscalid in field isolates of phytopathogenic fungi. No cross resistance between pyraclostrobin and boscalid was detected, suggesting that Pristine® resistance appears as a case of multiple resistance.     

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.     

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

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

Fungicide resistance status and chemical control options for the brassica pathogen Pyrenopeziza brassicae

Pyrenopeziza brassicae causes leaf spot disease of Brassicaceae in Europe/Oceania (lineage 1) and North America (lineage 2). In Europe, fungicides currently used for disease management are sterol 14α-demethylase (CYP51) inhibitors (azoles), quinone outside inhibitors (QoIs), and succinate dehydrogenase inhibitors (SDHIs); methyl benzimidazole carbamates (MBCs) are no longer applied. In this study, in vitro screening revealed European populations (collected 2018–2020) had shifted towards decreased azole sensitivity, but the North American population (2014–2016) was highly sensitive. Genotyping revealed CYP51 substitutions G460S or S508T were prevalent in European populations, often with a CYP51 promoter insert. Compared to wildtype CYP51 isolates, those with G460S plus an insert (44/46/151/210/302 bp) were c.25–32-fold and c.50-fold less sensitive to tebuconazole and prochloraz, respectively; those with S508T plus an insert (44/46/151/233 bp) were c.9–15-fold and c.25–40-fold less sensitive to tebuconazole and prochloraz, respectively. Selection for G460S (quantified via pyrosequencing) under different fungicide regimes was investigated in UK field trials, but G460S levels were high (c.76%) before treatment, so further selection during the trials was unclear. Despite the high G460S frequency and low disease pressure, yield data indicated measurable benefit for both azole- and non-azole-based programmes. In vitro screening against the MBC carbendazim showed European populations were predominantly moderately resistant/resistant; the North American population was sensitive. European and North American populations were sensitive to QoI (pyraclostrobin) and SDHI (penthiopyrad) fungicides. Results support an azole plus QoI/SDHI mixing partner for robust disease control and decreased risk of resistance, with continued sensitivity monitoring to ensure optimal strategies are deployed.     

Molecular and biochemical characterization of boscalid resistance in laboratory mutants of Sclerotinia sclerotiorum

The plant‐pathogenic fungus Sclerotinia sclerotiorum has a broad host range and a worldwide distribution. Boscalid, an inhibitor of succinate dehydrogenase in the electron transport chain of fungi, is highly effective in controlling sclerotinia stem rot caused by S. sclerotiorum. The current study characterized the S. sclerotiorum boscalid‐resistant (BR) mutants obtained by fungicide induction. Among the bioactive fungicides against S. sclerotiorum, cross‐resistance was not detected between boscalid and dimethachlon, fluazinam or carbendazim; positive cross‐resistance was detected between boscalid and carboxin; and negative cross‐resistance was detected between boscalid and kresoxim‐methyl. Compared to their parental isolates, BR mutants had slower radial growth, no ability to produce sclerotia, lower virulence and oxalic acid content but higher mycelial respiration and succinate dehydrogenase (SDH) activity. Moreover, BR mutants had decreased sensitivity to salicylhydroxamic acid (SHAM) but not to oxidative stress. All the results indicated that the risk of resistance to boscalid in S. sclerotiorum is low to moderate. DNA sequence analysis showed that all of the BR mutants had the same point mutation A11V (GCA to GTA) in the iron sulphur protein subunit (SDHB). Interestingly, expression of the cytochrome b (cytb) gene was reduced to different degrees in the BR mutants, and this might be correlated with the negative cross‐resistance between boscalid and kresoxim‐methyl. Such information is vital in the design of resistance management strategies.     

Characterization of mutations in the membrane-anchored subunits AaSDHC and AaSDHD of succinate dehydrogenase from Alternaria alternata isolates conferring field resistance to the fungicide boscalid

The molecular basis of resistance to the fungicide boscalid in 25 Alternaria alternata field mutants exhibiting resistance to boscalid and previously tested negative for AaSDHB mutations conferring boscalid resistance was investigated by cloning and sequencing the A. alternata SDHC ( AaSDHC ) and SDHD ( AaSDHD ) genes from a boscalid-sensitive isolate. The SDHB and SDHC/SDHD genes encode the iron sulphur and two membrane-anchored subunits of succinate:ubiquinone oxidoreductase (SQR) that constitute the boscalid fungicide molecular targets. The deduced amino acid sequences exhibited low similarities with SDHC and SDHD peptides from other organisms, but residues essential to form the ubiquinone binding site or important in SQR assembly were particularly conserved. Sequence comparisons of the AaSDHC and AaSDHD genes between resistant mutants and wild-type isolates revealed that two highly conserved histidine residues implicated in the heme b ligation and located at codon 134 in AaSDHC (22 mutants) and codon 133 in AaSDHD (two mutants) were replaced by arginine residues (H134R and H133R). In another mutant, a substitution of an aspartate by a glutamic acid occurred at amino acid position 123 (D123E) in AaSDHD. Additional tests revealed that mycelial growth of boscalid-resistant isolates was reduced when isolates were subjected to oxidative stress. The identified mutations were confirmed using PCR-RFLP assays. This is apparently the first report of mutations located in the heme b ligands of the cytochrome II gene associated with carboxamide resistance.     

梨黑斑病菌抗药性检测及其对啶酰菌胺的敏感性基线

黑斑病是梨的主要病害之一,近年来不少地区反映多菌灵等传统常用杀菌剂对其防治效果已出现下降。作者从浙江、江苏和安徽3省分离了252株梨黑斑病菌Alternaria kikuchiana,采用菌丝生长速率法检测了其抗药性发生情况。结果发现:所检测的黑斑病菌群体(n=252)对苯并咪唑类杀菌剂多菌灵的抗性频率为57.1%,且全部为高水平抗性(HR);对二甲酰亚胺类杀菌剂异菌脲的抗性频率为46.8%,全部为低水平抗性(LR);对甾醇脱甲基抑制剂类杀菌剂苯醚甲环唑的抗性为低水平(LR)及中等水平(MR),抗性频率均为28.6%;表明梨黑斑病菌对常用杀菌剂已产生较为严重的抗性。供试252株梨黑斑病菌对琥珀酸脱氢酶抑制剂啶酰菌胺的EC50值分布在0.12~3.85μg/m L之间,平均EC50值为(1.21±0.12)μg/m L,且其分布呈近似正态的单峰曲线。研究表明,啶酰菌胺可作为潜在的梨黑斑病防治替代药剂,其平均EC50值(1.21±0.12)μg/m L可作为梨黑斑病菌对啶酰菌胺的敏感性基线。     

浙江省果蔬灰葡萄孢对啶酰菌胺的抗性

以2004—2006年从浙江、江苏等地采集的灰葡萄孢对啶酰菌胺的敏感性基线[EC₅₀ = (1.07 ± 0.11) mg/L]为依据,采用菌丝生长速率法连续监测了浙江省果蔬灰葡萄孢群体对啶酰菌胺的敏感性变化。结果表明:浙江省果蔬灰葡萄孢对啶酰菌胺的抗性发展迅速,2012—2013年和2017—2018年的平均EC₅₀值分别为 (5.23 ± 7.79) 和 (24.30 ± 49.33) mg/L。其中,2012—2013年的抗药性菌株频率为15.3%,且均为低水平抗性 (LR) 菌株;而2017—2018年的抗药性频率上升至53.2%,并出现了7.5%的中等水平抗性 (MR) 菌株和1.3%的高水平抗性 (HR) 菌株。啶酰菌胺抗性菌株的菌丝生长速率、产孢量、产菌核数和致病力与敏感菌株相比均无显著差异。抗药性分子机制研究表明:啶酰菌胺抗性菌株的琥珀酸脱氢酶B亚基 (SDH B) 均发生了点突变,共包括H272R、P225F和N230I 3种类型,其中H272R型突变占88.5%;其SDH A和SDH D均未发生点突变;而SDH C的突变 (G85A + I93V + M158V + V168I) 与对药剂敏感性之间无明显联系。