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.     

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.     

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.     

Pyraclostrobin reduces germ tube growth of QoI‐resistant Mycosphaerella graminicola pycnidiospores and the severity of septoria tritici blotch on winter wheat

The effect of the quinone outside inhibitors (QoI) azoxystrobin and pyraclostrobin on yields of winter wheat where QoI resistant Mycosphaerella graminicola isolates were dominant was investigated in field trials in 2006 and 2007. Pyraclostrobin significantly increased yields by 1·57 t ha^?1 in 2006 and 0·89 t ha^?1 in 2007 when compared to the untreated controls, while azoxystrobin only provided a significant increase of 1·28 t ha^?1 in 2006. These yield increases were associated with reduction in septoria tritici blotch (STB) development as determined by weekly disease assessments over a 7 week interval. The effect of pyraclostrobin on STB was studied in controlled environment experiments using wheat seedlings inoculated with individual M. graminicola isolates. Pyraclostrobin significantly reduced STB symptoms by up to 62%, whether applied 48 h pre‐ or post‐ inoculation with resistant M. graminicola isolates containing the cytochrome b mutation G143A. Extremely limited disease (<1%) was observed on similarly treated seedlings inoculated with an intermediately resistant isolate containing the cytochrome b mutation F129L, while no disease was observed on seedlings inoculated with a wild‐type isolate. Germination studies of pycnidiospores of M. graminicola on water agar amended with azoxystrobin or pyraclostrobin showed that neither fungicide inhibited germination of spores of resistant isolates containing the mutation G143A. However, pyraclostrobin significantly reduced germ tube length by up to 46% when compared with the untreated controls. Although the QoIs can no longer be relied upon to provide effective M. graminicola control, this study provides an insight into why QoIs still provide limited STB disease control and yield increases even in situations of high QoI resistance.     

Sensitivity of mitochondrial respiration to different inhibitors in Venturia inaequalis

The sensitivity of Venturia inaequalis field isolates to inhibitors of the cytochrome bc1 complex at the Qo site (QoIs) was characterised at the molecular, biochemical and physiological level, and compared to other respiration inhibitors. Comparison of a sensitive and a QoI-resistant isolate revealed very high resistance factors both in mycelium growth and spore germination assays. Cross-resistance was observed among QoIs such as trifloxystrobin, azoxystrobin, famoxadone, strobilurin B and myxothiazol. In the mycelium growth assay, antimycin A, an inhibitor of the cytochrome bc1 complex at the Qi site, was less active against the QoI-resistant than against the sensitive isolate. The mixture of QoIs with salicylhydroxamic acid (SHAM), an inhibitor of the alternative oxidase, exerted synergistic effects in the spore germination but not in the mycelium growth assay. Thus, the cytochrome and the alternative respiration pathways are assumed to play different roles, depending on the developmental stage of the fungus. Induction of alternative oxidase (AOX) by trifloxystrobin was observed in mycelium cells at the molecular level for the sensitive but not the resistant isolate. Following QoI treatment, respiration parameters such as oxygen consumption, ATP level, membrane potential and succinate dehydrogenase activity were only slightly reduced in Qo-resistant mycelium cells, and remained at much higher levels than in sensitive cells. In contrast, no difference was observed between sensitive and resistant isolates when NADH consumption was measured. Comparison of the cytochrome b (cyt b) gene of the sensitive and resistant isolates did not reveal any point mutations as is known to occur in resistant isolates of other plant pathogens. It is assumed that QoI resistance in V inaequalis may be based on a compensation of the energy deficiency following QoI application upstream of the NADH dehydrogenase of the respiratory chain.     

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.     

Field efficacy of pyraclostrobin against populations of Pyrenophora teres containing the F129L mutation in the cytochrome b gene

Journal of Plant Diseases and Protection - Resistance of plant pathogenic fungi towards QoI fungicides is mainly caused by two mutations in the cytochrome b gene, the F129L and G143A. In...     

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.     

QoI resistance emerged independently at least 4 times in European populations of Mycosphaerella graminicola

BACKGROUND: QoI fungicides or quinone outside inhibitors (also called strobilurins) have been widely used to control agriculturally important fungal pathogens since their introduction in 1996. Strobilurins block the respiration pathway by inhibiting the cytochrome bc1 complex in mitochondria. Several plant pathogenic fungi have developed field resistance. The first QoI resistance in Mycosphaerella graminicola (Fuckel) Schroter was detected retrospectively in UK in 2001 at a low frequency in QoI-treated plots. During the following seasons, resistance reached high frequencies across northern Europe. The aim of this study was to identify the main evolutionary forces driving the rapid emergence and spread of QoI resistance in M. graminicola populations.RESULTS: The G143A mutation causing QoI resistance was first detected during 2002 in all tested populations and in eight distinct mtDNA sequence haplotypes. By 2004, 24 different mtDNA haplotypes contained the G143A mutation. Phylogenetic analysis showed that strobilurin resistance was acquired independently through at least four recurrent mutations at the same site of cytochrome b. Estimates of directional migration rates showed that the majority of gene flow in Europe had occurred in a west-to-east direction.CONCLUSION: This study demonstrated that recurring mutations independently introduced the QoI resistance allele into different genetic and geographic backgrounds. The resistant haplotypes then increased in frequency owing to the strong fungicide selection and spread eastward through wind dispersal of ascospores.     

Characterization of QoI resistance in Botrytis cinerea and identification of two types of mitochondrial cytochrome b gene

Botrytis cinerea field isolates collected in Japan were screened for resistance to Qo inhibitor fungicides (QoIs). Of the 198 isolates screened, six grew well on a medium containing azoxystrobin, a QoI, when salicylhydroxamic acid, an alternative oxidase inhibitor, was present. The resistance mutation in the cytochrome b gene ( cytb ) was characterized. All QoI-resistant isolates had the same mutation (GGT to G C T) in cytb that led to the substitution of glycine by alanine at position 143 of cytochrome b , which is known to confer QoI resistance in plant pathogens. To detect this mutation, a hybridization probe assay based on real-time PCR amplification and melting curve analysis was developed. Using DNA samples prepared from aubergines coinfected with QoI-resistant and QoI-sensitive B. cinerea isolates, two similar peak profiles with their corresponding melting temperatures were obtained. This result suggests that QoI-resistant and QoI-sensitive isolates may compete equally in terms of pathogenicity, and the assay may be used to assess the population ratio of mutant and wild-type isolates. However, the hybridization probe did not anneal to PCR products derived from the DNA samples of some QoI-sensitive isolates. Structural analysis of cytb revealed that B. cinerea field isolates could be classified into two groups: one with three introns and the other with an additional intron (Bcbi-143/144 intron) inserted between the 143rd and 144th codons. All 88 isolates possessing the Bcbi-143/144 intron were azoxystrobin-sensitive, suggesting that the QoI-resistant mutation at codon 143 in cytb prevents self-splicing of the Bcbi-143/144 intron, as proposed in some other plant pathogens.     

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     

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.     

Biological and molecular characterization of laboratory mutants of Cercospora beticola resistant to Qo inhibitors

The resistance to strobilurin-related fungicides and its molecular basis in laboratory mutant isolates of Cercospora beticola was investigated. After ultraviolet mutagenesis, mutants with high, moderate or low resistance levels to pyraclostrobin were isolated from a wild-type strain of C. beticola. Fungitoxicity tests on the response of resistant isolates on medium containing pyraclostrobin and salicylhydroxamate (SHAM), a specific inhibitor of cyanide-resistant (alternative) respiration, indicated that the biochemical mechanism of alternative oxidase was not responsible for the reduced sensitivity to pyraclostrobin for half of the mutants. Cross-resistance studies with other inhibitors of the cytochrome bc ₁ complex of the mitochondrial respiratory chain showed that the mutation(s) for resistance to pyraclostrobin also reduced the sensitivity of mutant strains to other Qo inhibitors such as azoxystrobin and fenamidone, but not to the Qi inhibitor cyazofamid. No effect of pyraclostrobin-resistant mutation(s) on fungitoxicity of the carboxamide boscalid, the triazoles epoxiconazole and flutriafol and to the benzimidazole benomyl, which affect other cellular pathways or other steps of the respiratory chain, was observed. Study of fitness parameters showed that most mutants had a significant reduction in sporulation and pathogenicity compared to the wild-type parental isolate. However, experiments on the stability of the resistant phenotype did not show a significant reduction of the resistance for half of the mutants when grown for at least four generations on pyraclostrobin-free medium. Molecular analysis of cytochrome b cDNA, isolated from the wild-type and the pyraclostrobin-resistant mutant isolates, revealed two novel amino acid replacements at positions involved in Qo resistance in other species. The glycine (GGT) to serine (AGT) replacement at position 143 (G143S) was found in the isolate with the highly resistant phenotype. The second amino acid change was the replacement of phenylalanine (TTC) by valine (GTC) at position 129 (F129V), which was found in a mutant strain with the moderately resistant phenotype. Four additional mutations located in conserved regions of the mitochondrial cytochrome b gene (I154L, N250D, E256G and V261D) were detected in some mutant isolates of C. beticola but their possible role in Qo-resistance needs further investigation. This is the first study reporting C. beticola strains resistant to Qo inhibitor fungicides due to the biochemical mechanism of target-site modification, resulting from amino acid changes in the mitochondrial cytochrome b␣gene.     

Site‐directed mutagenesis of the cytochrome b gene and development of diagnostic methods for identifying QoI resistance of rice blast fungus

BACKGROUND: It is possible that a single nucleotide polymorphism (SNP) (G143A mutation) in the cytochrome b gene could confer resistance to quinone outside inhibiting (QoI) fungicides (strobilurins) in rice blast fungus because this mutation caused a high level of resistance to fungicides such as azoxystrobin in Pyricularia grisea Sacc. and other fungal plant pathogens. The aim of this study was to survey Magnaporthe oryzae B Couch sp. nov. isolates in Japan for resistance to QoIs, and to try to develop molecular detection methods for QoI resistance. RESULTS: A survey on the QoI resistance among M. oryzae isolates from rice was conducted in Japan. A total of 813 single‐spore isolates of M. oryzae were tested for their sensitivity to azoxystrobin using a mycelial growth test on PDA. QoI fungicide resistance was not found among these isolates. The introduction of G143A mutation into a plasmid containing the cytochrome b gene sequence of rice blast fungus was achieved by site‐directed mutagenesis. Molecular diagnostic methods were developed for identifying QoI resistance in rice blast fungus using the plasmid construct. CONCLUSION: As the management of rice blast disease is often dependent on chemicals, the rational design of control programmes requires a proper understanding of the fungicide resistance phenomenon in field populations of the pathogen. Mutation of the cytochrome b gene of rice blast fungus would be specifically detected from diseased leaves and seeds using the molecular methods developed in this study. Copyright © 2009 Society of Chemical Industry     

Field Resistance to Strobilurin (Q(o)I) Fungicides in Pyricularia grisea Caused by Mutations in the Mitochondrial Cytochrome b Gene

ABSTRACT Gray leaf spot caused by Pyricularia grisea is a highly destructive disease of perennial ryegrass turf. Control of gray leaf spot is dependent on the use of preventative fungicide treatments. Strobilurin-based (Q(o)I) fungicides, which inhibit the cytochrome bc(1) respiratory complex, have proven to be very effective against gray leaf spot. However, in August 2000, disease was diagnosed in Q(o)I-treated perennial ryegrass turf on golf courses in Lexington, KY, Champaign, IL, and Bloomington, IL. To determine if resistance was due to a mutation in the fungicide target, the cytochrome b gene (CYTB) was amplified from baseline and resistant isolates. Nucleotide sequence analysis revealed an intronless coding region of 1,179 bp. Isolates that were resistant to Q(o)I fungicides possessed one of two different mutant alleles, each of which carried a single point mutation. The first mutant allele had a guanine-to-cytosine transition at nucleotide position +428, resulting in a replacement of glycine 143 by alanine (G143A). Mutant allele two exhibited a cytosine-to-adenine transversion at position +387, causing a phenylalanine-to-leucine change (F129L). Cleavable amplified polymorphic sequence analysis revealed that neither mutation was present in a collection of baseline isolates collected before Q(o)I fungicide use and indicated that suspected Q(o)I- resistant isolates found in 2001 in Indiana and Maryland possessed the F129L mutation. The Pyricularia grisea isolates possessing the G143A substitution were significantly more resistant to azoxystrobin and trifloxystrobin, in vitro, than those having F129L. DNA fingerprinting of resistant isolates revealed that the mutations occurred in just five genetic backgrounds, suggesting that field resistance to the Q(o)I fungicides in Pyricularia grisea is due to a small number of ancestral mutations.     

Molecular Characterization and Diagnosis of QoI Resistance in Cucumber and Eggplant Fungal Pathogens

ABSTRACT The molecular mechanism of QoI fungicide resistance was studied using isolates of cucumber Corynespora leaf spot fungus (Corynespora cassiicola) and the eggplant leaf mold (Mycovellosiella nattrassii). In both pathogens, a mutation at position 143 from glycine to alanine (G143A) was detected in the cytochrome b gene that encodes for the fungicide-targeted protein. Moreover, the nucleotide sequence at amino acid position 143 was converted from GGT or GGA in sensitive (wild-type) to GCT or GCA in resistant (mutant-type) isolates. The methods of polymerase chain reaction restriction fragment length polymorphism commonly used for QoI resistance monitoring were employed successfully, leading to the amplified gene fragment from resistant isolates being cut with the restriction enzyme ItaI. However, heteroplasmy (the coexistence of wild-type and mutated alleles) was found when the resistant isolates of C. cassiicola, M. nattrassii, and Colletotrichum gloeosporioides (strawberry anthracnose fungus) were subcultured in the presence or absence of QoI fungicides. QoI resistance of cucumber powdery and downy mildew isolates persisted for a few years following the removal of the selection pressure imposed by the fungicide under both laboratory and commercial greenhouse conditions. The proportion of mutated sequences in cytochrome b gene decreased over time in the pathogen population. The protective efficacy of the full dose of azoxystrobin decreased when the populations of powdery and downy mildews contained resistant isolates at 10%. Using FMBIO, a fluorescence bio-imaging analyzer, the mutant allele from the QoI-resistant isolates could be detected at the level of 1%, whereas the detection sensitivity of ethidium-bromide-stained gels was approximately 10 times lower.     

Assessment of QoI resistance in Plasmopara viticola oospores

QoI fungicides, inhibitors of mitochondrial respiration at the Qo site of cytochrome b in the mitochondrial bc(1) enzyme complex, are commonly applied in vineyards against Plasmopara viticola (Berk. & MA Curtis) Berl. & De Toni. Numerous treatments per year with QoI fungicides can lead to the selection of resistant strains in the pathogen population owing to the very specific and efficient mode of action. In order to evaluate the resistance risk and its development, two different methods, biological and molecular, were applied to measure the sensitivity of oospores differentiated in vineyards, both treated and untreated with azoxystrobin, from 2000 to 2004. Assays using oospores have the advantage of analysing the sensitivity of bulked samples randomly collected in vineyards, describing accurately the status of resistance at the end of the grapevine growing season. Both methods correlated well in describing the resistance situation in vineyards. QoI resistance was not observed in one vineyard never treated with QoI fungicides. In the vineyard where azoxystrobin had been used in mixture with folpet, the selection of QoI-resistant strains was lower, compared with using solely QoI. In vineyards where QoI treatments have been stopped, a decrease in resistance was generally observed.     

Metyltetraprole activity against plant pathogens with relatively rare cytochrome b haplotypes for azoxystrobin resistance

Journal of General Plant Pathology - Metyltetraprole is a novel quinone outside inhibitor (QoI) fungicide designed to avoid cross-resistance in cytochrome b G143A-harboring QoI-resistant...     

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.     

浙江省果蔬灰霉病菌对嘧菌酯的抗药性研究

采用菌丝生长速率法,连续监测了2010—2012年间浙江省果蔬灰霉病菌对QoI类杀菌剂嘧菌酯的敏感性变化。 结果表明:病菌群体中的低敏感性亚群体的比例明显上升,EC50值>5 mg/L 菌株的比例分别为12.5%、15.8%和28.3%;在菌丝生长阶段和孢子萌发阶段,旁路氧化在灰霉病菌对嘧菌酯敏感性中的平均相对贡献值(F)分别为2.91±0.89和5.72±2.82;嘧菌酯抗药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无显著差异。抗药性分子机制研究表明,灰霉病菌中存在2种类型的cyt b基因:Ⅰ型cyt b基因在第143位密码子后紧跟内含子;Ⅱ型cyt b基因在第143位密码子后没有紧跟内含子。大多数的灰霉病菌菌株属于Ⅱ型。Ⅰ型菌株均为嘧菌酯敏感菌株,Ⅱ型菌株为嘧菌酯敏感菌株或抗性菌株。抗性菌株的cyt b 基因的第143位密码子由甘氨酸(GGC)突变为了丙氨酸(GCC),抗药性机制为G143A。