Characterization of moderate resistance to QoI fungicides in Pestalotiopsis longiseta and polymorphism in exon–intron structure of cytochrome b gene

Qo inhibitor (QoI) fungicides are used to control gray blight caused by Pestalotiopsis longiseta in Japanese tea cultivation. However, field isolates of P. longiseta highly resistant to QoI fungicides were found in 2008, resulting in failure of QoI fungicidal control. This resistance was attributed to a mutation in the cytochrome b gene (cytb) in which alanine was substituted for glycine at position 143 (G143A). In 2009–2010, we detected field isolates that had an intermediate reaction between sensitive and resistant isolates in a preliminary assay. These isolates showed intermediate sensitivity to azoxystrobin and kresoxim-methyl on PDA plates. The intermediate reaction to azoxystrobin was also confirmed on detached tea leaves. Consequently, they were considered moderately resistant to QoI fungicides. Nucleotide sequencing of cytb showed that moderate resistance correlated with a single point mutation; leucine was substituted for phenylalanine at amino acid position 129 (F129L). Sequence analysis also revealed two types of cytb, with or without an intron between codons 131 and 132, in P. longiseta. F129L and G143A mutations were detected in both types of cytb according to their QoI resistance. This result suggests that G143A and F129L mutations have each occurred at least twice in the P. longiseta population.     

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.     

Identification of the G143A mutation associated with QoI resistance in Cercospora beticola field isolates from Michigan,United States

BACKGROUND: Cercospora leaf spot (CLS), caused by the fungus Cercospora beticola, is the most serious foliar disease of sugar beet (Beta vulgaris L.) worldwide. Disease control is mainly achieved by timely fungicide applications. In 2011, CLS control failures were reported in spite of application of quinone outside inhibitor (QoI) fungicide in several counties in Michigan, United States. The purpose of this study was to confirm the resistant phenotype and identify the molecular basis for QoI resistance of Michigan C. beticola isolates. RESULTS: Isolates collected in Michigan in 1998 and 1999 that had no previous exposure to the QoI fungicides trifloxystrobin or pyraclostrobin exhibited QoI EC₅₀ values of ?0.006 µg mL^?1. In contrast, all isolates obtained in 2011 exhibited EC₅₀ values of > 0.92 µg mL^?1 to both fungicides and harbored a mutation in cytochrome b (cytb) that led to an amino acid exchange from glycine to alanine at position 143 (G143A) compared with baseline QoI‐sensitive isolates. Microsatellite analysis of the isolates suggested that QoI resistance emerged independently in multiple genotypic backgrounds at multiple locations. A real‐time PCR assay utilizing dual‐labeled fluorogenic probes was developed to detect and differentiate QoI‐resistant isolates harboring the G143A mutation from sensitive isolates. CONCLUSION: The G143A mutation in cytb is associated with QoI resistance in C. beticola. Accurate monitoring of this mutation will be essential for fungicide resistance management in this pathosystem. Copyright © 2012 Society of Chemical Industry     

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

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

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.     

Evaluation of the incidence of the G143A mutation and cytb intron presence in the cytochrome bc-1 gene conferring QoI resistance in Botrytis cinerea populations from several hosts

BACKGROUND: Previous studies have shown that resistance of Botrytis cinerea to QoI fungicides has been attributed to the G143A mutation in the cytochrome b (cytb) gene, while, in a part of the fungal population, an intron has been detected at codon 143 of the gene, preventing QoI resistance. During 2005–2009, 304 grey mould isolates were collected from strawberry, tomato, grape, kiwifruit, cucumber and apple in Greece and screened for resistance to pyraclostrobin and for the presence of the cytb intron, using a novel real‐time TaqMan PCR assay developed in the present study. RESULTS: QoI‐resistant phenotypes existed only within the population collected from strawberries. All resistant isolates possessed the G143A mutation. Differences were observed in the genotypic structure of cytb. Individuals possessing the intron were found at high incidence in apple fruit and greenhouse‐grown tomato and cucumber populations, whereas in the strawberry population the intron frequency was lower. Cultivation of QoI‐resistant and QoI‐sensitive isolates for ten culture cycles on artificial nutrient medium in the presence or absence of fungicide selection showed that QoI resistance was stable. CONCLUSIONS: The results of the study suggest that a high risk for selection of QoI‐resistant strains exists in crops heavily treated with QoIs, in spite of the widespread occurrence of the cytb intron in B. cinerea populations. The developed real‐time TaqMan PCR constitutes a powerful tool to streamline detection of the mutation by reducing pre‐ and post‐amplification manipulations, and can be used for rapid screening and quantification of QoI resistance. Copyright © 2011 Society of Chemical Industry     

Molecular and biochemical characterization of Colletotrichum gloeosporioides isolates resistant to azoxystrobin from grape in China

Anthracnose, caused by Colletotrichum gloeosporioides, is one of the most important diseases in grape-growing regions worldwide. In Jiangsu Province of China, quinone-outside inhibitor fungicides (QoIs) have been extensively sprayed as disease control for more than 10 years. A spore germination assay of 64 isolates obtained from 32 commercial vineyards was used to assess isolate sensitivity to azoxystrobin and 62 were found to be resistant to azoxystrobin. The biological fitness of QoI-resistant (QoI^R) isolates was significantly lower than the sensitive isolates (QoI^S) in terms of mycelial growth and conidiation. Nucleotide sequence alignment of CgCytb genes from the QoI^R and QoI^S isolates revealed that two point mutations (F129L and G143A) are involved in the QoI resistance. Isolates with the G143A mutation expressed high resistance to azoxystrobin, whereas isolates carrying the F129L mutation exhibited moderate resistance. Positive cross-resistance was observed between azoxystrobin and kersoxim-methyl, pyraclostrobin, or benzothiostrobin, but not with fluazinam. This study provides important information for management of QoI^R populations of C. gloeosporioides in the field.     

Detection and characterization of QoI resistance in Pyrenophora tritici-repentis populations causing tan spot of wheat in Argentina

Tan spot, caused by the fungus Pyrenophora tritici-repentis (Ptr), is a disease that has become more prevalent and intense in wheat crops in Argentina in recent years. Failure to control the disease with strobilurin fungicides, which were once effective, has been observed in different zones where wheat is grown. However, whether or not true resistance is present in the pathogen population in the region is not scientifically confirmed. This study evaluated the sensitivity of numerous Ptr isolates to representative QoI fungicides used in Argentina through in vitro and in planta assays, as well as through molecular analysis. Eighty-two monosporic isolates obtained in different locations in the north and south of Buenos Aires province in 2014, 2016, and 2018 were tested to determine sensitivity to selected QoI fungicides in conidial germination and mycelial inhibition assays, as well as in molecular analysis. Conidial germination was not inhibited at 1 µg/ml of azoxystrobin, trifloxystrobin, and pyraclostrobin. On the other hand, mycelial growth was inhibited by 59%, 56%, and 86% at 100 µg/ml of azoxystrobin, trifloxystrobin, and pyraclostrobin, respectively. The molecular analysis detected the G143A mutation in the cytb gene of all the 82 Ptr isolates, but the F129L and G137R substitutions were not present. This study documents the G143A mutation conferring QoI resistance in Ptr in South America. The findings of this study are key for future decisions regarding use of fungicide and rotation in the region.     

Analysis of Colletotrichum musae populations from Brazil reveals the presence of isolates with high competitive ability and reduced sensitivity to postharvest fungicides

In this study, the sensitivity of 218 isolates of Colletotrichum musae to imazalil and thiabendazole was evaluated, as well the fitness and competitive ability of less sensitive isolates. There was a positive correlation between the sensitivity to the two fungicides, but the isolates were more sensitive to imazalil. The estimated effective concentration of the fungicide able to inhibit mycelial growth by 50% (EC₅₀) was used to select four isolates with the lowest and the highest values for both fungicides, which were considered as sensitive (S) and less sensitive (LS), respectively. The level of sensitivity was maintained after 10 successive transfers on fungicide-free medium. Both fungicides were effective in controlling the disease caused by S isolates of C. musae in detached banana fruit when recommended doses were used. However, only imazalil was able to control the disease caused by LS isolates. For both fungicides, analysis of fitness-related variables (mycelial growth, sporulation, germination, and virulence) showed no difference between the groups of S and LS isolates, but a large variation was observed within the group. The LS isolates to thiabendazole that showed a mutation (F200Y) in the β-tubulin gene did not have fitness penalties. Our results allow a better understanding of the sensitivity and fitness of isolates of C. musae from Brazil, and demonstrate the importance of periodic monitoring to determine the frequency of LS isolates in populations, aiming at more effective management of anthracnose in banana orchards in Brazil.     

Genetic analysis and molecular characterisation of laboratory and field mutants of Botryotinia fuckeliana (Botrytis cinerea) resistant to QoI fungicides

BACKGROUND: QoI fungicides, inhibitors of mitochondrial respiration, are considered to be at high risk of resistance development. In several phytopathogenic fungi, resistance is caused by mutations (most frequently G143A) in the mitochondrial cytochrome b (cytb) gene. The genetic and molecular basis of QoI resistance were investigated in laboratory and field mutants of Botryotinia fuckeliana (de Bary) Whetz. exhibiting in vitro reduced sensitivity to trifloxystrobin. RESULTS: B. fuckeliana mutants highly resistant to trifloxystrobin were obtained in the laboratory by spontaneous mutations in wild‐type strains, or from naturally infected plants on a medium amended with 1–3 mg L^?1 trifloxystrobin and 2 mM salicylhydroxamic acid, an inhibitor of alternative oxidase. No point mutations were detected, either in the complete nucleotide sequences of the cytb gene or in those of the aox and Rieske protein genes of laboratory mutants, whereas all field mutants carried the G143A mutation in the mitochondrial cytb gene. QoI resistance was always maternally inherited in ascospore progeny of sexual crosses of field mutants with sensitive reference strains. CONCLUSIONS: The G143A mutation in cytb gene is confirmed to be responsible for field resistance to QoIs in B. fuckeliana. Maternal inheritance of resistance to QoIs in progeny of sexual crosses confirmed that it is caused by extranuclear genetic determinants. In laboratory mutants the heteroplasmic state of mutated mitochondria could likely hamper the G143A detection, otherwise other gene(s) underlying different mechanisms of resistance could be involved. Copyright © 2012 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.     

Occurrence and distribution of resistance to QoI fungicides in populations of Podosphaera fusca in south central Spain

Cucurbit powdery mildew caused by Podosphaera fusca limits crop production in Spain. Since its management is strongly dependent on chemicals, the rational design of control programmes requires a good understanding of the fungicide resistance phenomenon in field populations. Fifty single-spore isolates of P. fusca were tested for sensitivity to three quinone-outside inhibiting (QoI) fungicides: azoxystrobin, kresoxim-methyl and trifloxystrobin. Minimum inhibitory concentration (MIC) values for QoI-sensitive isolates were found to range from 0.25 to 10 μg ml⁻¹ for azoxystrobin to 5–25 μg ml⁻¹ for kresoxim-methyl, using a leaf disc-based bioassay. High levels of cross-resistance to QoI fungicides were found. Eleven isolates showed resistance to the three QoI fungicides tested with MIC and EC₅₀ values >500 μg ml⁻¹ resulting in RF values as high as >715 and >1000 for trifloxystrobin and azoxystrobin, respectively. A survey of P. fusca QoI resistance was carried out in different provinces located in the south central area of Spain during the cucurbit growing seasons in 2002, 2003 and 2004. Examination of a collection of 250 isolates for QoI resistance revealed that 32% were resistant to the three fungicides tested; the provinces of Ciudad Real, Córdoba and Murcia being the locations with the highest frequencies of resistance (44–74%). By contrast, no resistance was found in Badajoz, and relatively low frequencies were observed in Almería and Valencia (10–13%). Nearly 50% of resistant isolates were collected from melon plants. Based on these data, recommendations about the use of QoI fungicides for cucurbit powdery mildew management in the sampled areas are made.     

不同类型Monilinia fructicola菌株的温度适应性及其对苯并咪唑类杀菌剂和乙霉威的交互抗性

褐腐病菌Monilinia fructicola是引起多种果树褐腐病的重要病原菌,前期研究发现该病原菌对甲基硫菌灵的抗性与Tub2蛋白的多个氨基酸变异有关。为明确不同类型菌株的温度适应性及乙霉威是否对所有抗性类型菌株均具有抑菌活性,本研究测定了敏感型菌株S及3种抗性类型包括R_(E198A)、R_(E198Q)及R_(F200Y)型菌株的温度敏感性及其对甲基硫菌灵、多菌灵和乙霉威的敏感性差异,同时分析了不同突变类型菌株对苯并咪唑类药剂和乙霉威的交互抗性。结果表明,4种类型菌株的最适生长温度为21~22 ℃,其中R_(E198Q)型菌株的最适生长温度最低,且其在低温13 ℃和高温28 ℃条件下,生长速率显著低于其他3类菌株,而S、R_(E198A)及R_(F200Y)型菌株在所有温度条件下的生长速率均无显著差异。3种抗性类型菌株对甲基硫菌灵的敏感性有所差异,其中R_(E198Q)型表现较为敏感,R_(F200Y)型在低浓度下与R_(E198A)型无明显差异,但在高浓度下对甲基硫菌灵表现较为敏感;对多菌灵的敏感性差异与甲基硫菌灵相似;只是R_(E198Q) 和R_(F200Y)型菌株对多菌灵比甲基硫菌灵更为敏感。乙霉威的敏感性测定结果表明,R_(E198A)型菌株表现敏感,S类型表现为不敏感性,R_(E198Q)及 R_(F200Y)两种类型均表现为抗性,且抗性水平差别不大。线性相关分析结果表明,仅R_(E198A)型菌株和S型菌株对苯并咪唑类杀菌剂和乙霉威敏感性存在显著负相关性 (甲基硫菌灵和乙霉威:r = ?0.992,p = 0.000;多菌灵和乙霉威:r = ?0.982,p = 0.001)。综合分析结果表明,在3类抗性菌株中,仅R_(E198A)型菌株对苯并咪唑类杀菌剂和乙霉威存在负交互抗性。     

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.     

Genotyping of benzimidazole-resistant and dicarboximide-resistant mutations in Botrytis cinerea using real-time polymerase chain reaction assays

Botrytis cinerea, an economically important gray mold pathogen, frequently exhibits multiple fungicide resistance. A fluorescence resonance energy transfer-based real-time polymerase chain reaction assay has been developed to detect benzimidazole- and dicarboximide-resistant mutations. Three benzimidazole-resistant mutations-(198)Glu to Ala (E198A), F200Y, and E198K-in beta-tubulin BenA were detected using a single set of fluorescence-labeled sensor and anchor probes by melting curve analysis. Similarly, three dicarboximide-resistant mutations-I365S, V368F plus Q369H, and Q369P-in the histidine kinase BcOS1 were successfully distinguished. Unassigned melting profiles in BenA genotyping assay resulted in the identification of a new benzimidazole-resistant BenA E198V mutation. This mutation conferred resistance to carbendazim as do E198A and E198K mutations. The isolates with BenA E198V mutation showed a negative cross-resistance to diethofencarb, but to a lesser extent than the E198A mutants. A survey of 210 B. cinerea field isolates revealed that most of benzimidazole-resistant isolates possessed the E198V or E198A mutation in the BenA gene, and the I365S mutation in the BcOS1 gene was also frequently observed in Japanese isolates. However, benzimidazole-resistant isolates with BenA F200Y or E198K mutations, which confer the diethofencarb-insensitive phenotype, were rare. Our BenA and BcOS1 genotyping is a rapid and reliable method that is suitable for monitoring the fungicide-resistant field population.     

Field resistance to QoI fungicides in Podosphaera fusca is not supported by typical mutations in the mitochondrial cytochrome b gene

BACKGROUND: A single nucleotide polymorphism in the mitochondrial cytochrome b gene confers resistance to strobilurin (QoI) fungicides in phytopathogenic fungi. Recent studies have revealed worrying levels of resistance to strobilurins in Podosphaera fusca (Fr.) U Braun & N Shishkoff comb. nov. [ = Sphaerothecafusca (Fr.) S Blumer], the main causal agent of cucurbit powdery mildew in Spain. In the present study the underlying resistance mechanism to QoI fungicides in the Spanish populations of P. fusca was investigated. RESULTS: Analysis of the Q(o) domains of cytochrome b in a collection of isolates revealed that none of the typical mutations conferring resistance to QoI, including the G143A and F129L substitutions, was present in the QoI-resistant isolates. Moreover, although different amino acid polymorphisms were observed in the two regions spanning the Q(o) site, none of them consistently distinguished QoI-resistant from QoI-sensitive strains. Exposure to salicylhydroxamic acid (SHAM), a specific inhibitor of alternative oxidase, in the presence of trifloxystrobin did not have any effect on QoI resistance, ruling out alternative respiration as the mechanism of resistance. Sensitivity tests to a battery of respiration inhibitors revealed high levels of cross-resistance to all Qo-inhibitors tested but not to Qi-inhibitors, these features resembling those of a target-site-based resistance. CONCLUSIONS: The results indicate that the mechanism responsible for QoI resistance in P. fusca is not linked to typical mutations in cytochrome b gene and that the absence of the G143A substitution cannot be explained by an intron following codon 143. These are important observations, especially in relation to the possible molecular diagnosis of resistance.     

Genetic analysis and PCR-based identification of major <Emphasis Type="Italic">Fusarium</Emphasis> species causing head blight on wheat in Japan

Identifying the Fusarium species cause Fusarium head blight (FHB) and produces mycotoxins in wheat and other cereal is difficult and time consuming because of confusing phenotypic classification systems. In Japan, the F. graminearum complex, F. culmorum, F. avenaceum, and Microdochium nivale predominantly cause FHB. The internal transcribed spacer (ITS) and 5.8S of rDNA, a partial sequence of β-tubulin and mitochondrial cytochrome b (cytb) genes of the four species were PCR-amplified and analyzed. On the basis of the ITS, β-tubulin and cytb sequences, F. avenaceum and M. nivale are distinct from the F. graminearum complex and F. culmorum, whereas the F. graminearum complex is closely related to F. culmorum. Moreover, thiophanate–methyl-resistant isolates of the F. graminearum complex and F. culmorum did not have an amino acid substitution at amino acid codon 198 or 200 of β-tubulin. In contrast, very highly or highly thiophanate–methyl-resistant isolates of M. nivale had Glu (GAG) substituted with Ala (GCG) or Lys (AAG) at codon 198, respectively. The allele-specific PCR assay was used to identify the F. graminearum complex and F. culmorum, and these Fusarium species could be distinguished rapidly.     

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.

     

Identification of the mutation responsible for resistance to QoI fungicides and its detection in Ascochyta rabiei (teleomorph Didymella rabiei)

This study characterized a fragment of the cytochrome b gene from Ascochyta rabiei isolates collected in North Dakota, USA, that varied in sensitivity to quinone‐outside inhibitor (QoI) fungicides. The sequenced genomic DNA fragment contained a group I intron immediately after codon 131. The size of the cytochrome b gene was estimated to be over 4·6 kb. Multiple alignment analysis of cDNA and protein sequences revealed a mutation that changed the codon for amino acid 143 from GGT to GCT, introducing an amino acid substitution from glycine to alanine (G143A), which is frequently associated with QoI resistance. Based on this mutation, a diagnostic PCR assay was developed using an approach called mismatch amplification mutation assay. This method was successfully validated by testing a total of 70 A. rabiei isolates, of which 38 isolates were found to be QoI‐resistant. This fast and accurate PCR assay provides a very useful and simple screening method for QoI resistance in A. rabiei isolates.     

Characterization of the cytochrome b (cyt b) gene from Monilinia species causing brown rot of stone and pome fruit and its significance in the development of QoI resistance

BACKGROUND: Quinone outside inhibitor (QoI) resistance as a consequence of point mutations in the cytochrome b (cyt b) gene has been reported in numerous plant pathogenic fungi. To examine the potential for QoI resistance development in those Monilinia species causing brown rot of stone and pome fruits [Monilinia fructicola (G Winter) Honey, M. laxa (Aderhold & Ruhland) Honey and M. fructigena (Aderhold & Ruhland) Honey], an examination was made of the sequence and exon/intron structure of their cyt b genes for the presence of any point mutations and/or introns commonly associated with resistance to QoIs in fungal plant pathogens. RESULTS: None of the point mutations typically linked to QoI resistance was present in any of the Monilinia isolates examined. Furthermore, the cyt b genes from M. fructicola and M. laxa, but not M. fructigena, possessed a group‐I‐like intron directly after codon 143. Based on the results obtained, a simple PCR assay using a single primer pair was developed, allowing discrimination between the three Monilinia species without the need for culturing. CONCLUSIONS: Results suggest that resistance to QoI fungicides based on the G143A mutation is not likely to occur in M. fructicola or M. laxa. Conversely, M. fructigena may be at higher risk for developing QoI resistance owing to the absence of a G143‐associated intron. Copyright © 2010 Society of Chemical Industry