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.     

Resistance of Pseudoperonospora cubensis to flumorph on cucumber in plastic houses

Plastic-house experiments were conducted over a 2-year period (2004–05) to estimate the effects of successive applications of flumorph or a mixture of flumorph with mancozeb to cucumber plants on selection for flumorph resistance in the downy mildew oomycete, Pseudoperonospora cubensis . Application of flumorph alone favoured the selection of resistant isolates of Ps. cubensis . Resistant populations were detected at a frequency of 2·5% after six successive applications of flumorph alone in a plastic house in 2004. Resistant isolates were also detected (4·8%) after eight successive applications of the mixture of flumorph and mancozeb in 2004, although the mixture gave significantly better disease control than flumorph alone and produced a slight delay in the development of resistance. In a second cucumber crop in the same plastic houses in 2004, the frequency of resistant isolates increased to 100% after three successive applications of flumorph or four of flumorph + mancozeb. Under laboratory conditions, most flumorph-resistant isolates showed high levels of resistance and their levels of pathogenicity and sporulation were as high as that of wild-type isolates. Flumorph showed cross-resistance with dimethomorph and iprovalicarb, but not with azoxystrobin, cyazofamid, cymoxanil or metalaxyl. These studies suggest a high risk for the occurrence of resistance to flumorph in Ps. cubensis in cucumber crops under plastic-house conditions.     

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     

Pathogenic and genetic variation among isolates of Corynespora cassiicola in Japan

In order to develop a method for discrimination of Corynespora cassiicola isolates pathogenic to sweet pepper among Japanese isolates, this study analysed pathogenic variations of 64 Japanese isolates of C. cassiicola on perilla, cucumber, tomato, aubergine and sweet pepper, and their multigene phylogeny. Japanese isolates were divided into seven pathogenicity groups (PG1–PG7). The virulence of isolates in PG1–PG5 was restricted to perilla, cucumber, tomato, aubergine and sweet pepper, respectively. Isolates in PG6 were virulent to sweet pepper, tomato and aubergine. Isolates in PG7 were avirulent to all tested plants. Multigene phylogenetic analysis of the isolates based on β‐tubulin, translation elongation factor 1‐α, calmodulin and actin genes showed three divergent clusters, MP‐A, MP‐B and MP‐C. These clusters included all isolates in PG1, PG2, PG8 and PG9 (MP‐A), PG3 and PG5 (MP‐B) and PG4 and PG6 (MP‐C). Isolates in PG7 were distributed amongst all clusters. Furthermore, random amplified polymorphic DNA (RAPD) analysis using universal primers, Q17 (5′‐GAAGCCCTTG‐3′) and Q13 (5′‐GGAGTGGACA‐3′), facilitated discrimination of isolates virulent on sweet pepper amongst isolates in MP‐B and MP‐C, respectively. Together, a combination of the multigene analysis and the RAPD technique allowed the discrimination of the isolates virulent to sweet pepper.     

Resistance of Corynespora cassiicola from soybean to QoI and MBC fungicides in Brazil

Fungicide sprays on soybean in Brazil have contributed to the selection of less sensitive isolates of Corynespora cassiicola. We collected 59 isolates of C. cassiicola from three Brazilian states and two isolates from Paraguay. We investigated their EC₅₀ to quinone outside inhibitors (QoI) and methyl benzimidazole carbamate (MBC), any cross-resistance to compounds within QoI and MBC groups, and characterized the polymorphisms in their cytb and β-tubulin genes. Local associations of polymorphisms identified in each gene were statistically correlated with assays results. In total, 79% and 74% of the isolates were classified as resistant to QoI and MBC fungicides, respectively. There was positive cross-resistance to active ingredients within QoI and MBC groups. For QoI, all isolates presented heteroplasmy in G143A of cytb gene; the mutations F129L and G137R were not found. For MBC, 63% of isolates possessed E198A and 21% possessed F200Y mutations, associated with reduced control by MBC fungicides. Heteroplasmy was identified in two and one isolates from Brazil with E198A and F200Y mutations, respectively. The resistance factor for isolates with E198A (10.9) was statistically similar to the isolate with F200Y (8.8) mutation. Genic association analysis of the in vitro assays using discriminatory doses proved them to be accurate. Reduced sensitivity of C. cassiicola to QoI and MBC was also identified in isolates from Paraguay and resistance to QoI and MBC was widely present in C. cassiicola isolates from the main soybean-producing states in Brazil. Thus, integrated management measures should be adopted to manage soybean target spot in these countries.     

两株橡胶多主棒孢霉对12种杀菌剂原药的敏感性

采用菌落生长速率法测定了两株橡胶多主棒孢霉［Corynespora cassiicola (Berk &; Curt.) Wei］对12种杀菌剂原药的敏感性。结果表明：两个菌株对杀菌剂的敏感性存在一定的差异;供试药剂中多菌灵、甲基硫菌灵、咪鲜胺和抑霉唑的EC50均小于5 μg/mL,抑菌活性高;在加入SHAM后嘧菌酯的抑菌活性也很高,与SHAM具有协同增效作用;其他供试杀菌剂均有较好的抑菌作用,依次为戊唑醇、腈菌唑、异菌脲、溴菌腈、代森锰锌、百菌清和三唑酮。     

Inheritance of resistance to angular leaf spot (Pseudomonas syringae pv. lachrymans) in cucumber and identification of molecular markers linked to resistance

Angular leaf spot is a common disease of cucumber ( Cucumis sativus ) caused by Pseudomonas syringae pv. lachrymans . Genetics of resistance to this disease was investigated using two sets of parameters: (i) disease severity, i.e. the number and size of necrotic and chlorotic lesions on the infected leaves, and (ii) presence or absence of a chlorotic halo around the necrotic spots on the infected leaves. Disease severity appears to be controlled by multiple genes and the heritability of the resistance was estimated to be 53%. The presence or absence of the chlorotic halo was determined to be governed by a single gene, with the presence of the halo (the susceptible phenotype) being a dominant character. A RAPD marker linked to the gene conferring the chlorotic halo was identified. Genetic distance between this marker, OP-AO07, a polymorphic 420 bp amplicon in the DNA of the susceptible plants, and the locus encoding the chlorotic halo was estimated to be 13 cM.     

Phylogenetic network analysis of South and North American Corynespora cassiicola isolates from tomato,cucumber, and novel hosts

European Journal of Plant Pathology - Corynespora cassiicola isolates display morphological, pathogenic, and ecological diversity, inducing target spot-like diseases in more than 500 hosts...     

巴西橡胶树品系对棒孢霉落叶病的抗病性鉴定

采用巴西橡胶树棒孢霉落叶病病原菌的分生孢子接种和其毒素接种分别测定了国内天然橡胶主产区9个主栽品系的抗病性,结果表明过滤灭菌毒素划伤接种橡胶离体小叶片可以产生与分生孢子接种相似的症状,而不划伤接种则不产生病害症状;121℃、20 min湿热灭菌毒素接种无论划伤与否均不产生病害症状;分生孢子和病原菌毒素两种接种方法所测橡胶品系的抗病性之间有很好的一致性,说明毒素接种可以用来代替病原菌接种测定橡胶品系的抗病性。抗病性测定结果显示,供试9个巴西橡胶树品系中,PB86、热研88-13、南华1、PR107、热研7-33-97对病原菌表现出高抗,而RRIM600、南强1-97、热研44-9则表现高感,GT1则表现中感。     

Sensitivity of Botrytis cinerea from vegetable greenhouses to boscalid

Between 2004 and 2006, 228 isolates of Botrytis cinerea from two regions in China were characterized for baseline sensitivity to boscalid, a new active ingredient that interferes with succinate ubiquinone reductase in the electron transport chain. The isolates showed similar sensitivity in different years and regions. Baseline sensitivities were distributed as unimodal curves with mean EC₅₀ values of 1·07 (± 0·11) and 0·42 (± 0·05) mg L⁻¹ for inhibition of mycelial growth and conidial germination, respectively. Laboratory studies were conducted to evaluate the risk of development of resistance to boscalid. Boscalid-resistant mutants were obtained by UV-treatment at lower frequencies and with smaller resistance factors than pyrimethanil-resistant mutants. All boscalid-resistant mutants were also significantly more sensitive to Qo inhibitors than their wild-type parents and showed reduced sporulation in vitro and pathogenicity on aubergine leaves. The results suggested that the risk of resistance developing for boscalid was lower than for pyrimethanil. However, as B. cinerea is a high-risk pathogen, appropriate precautions against resistance development should be taken. Synergism between the activity of boscalid and that of kresoxim-methyl was observed.     

Morphological and pathogenic characterization of Corynespora cassiicola isolates reveals specific genotypic interactions in soybean

Soybean target spot, caused by the fungus Corynespora cassiicola, is an important disease in northwestern Argentina (NWA). A cultural, morphological, and virulence characterization of 24 C. cassiicola isolates from different geographic localities in NWA was performed. The isolates were classified into five different cultural phenotypes, one of which has not been previously described. The ribosomal DNA internal transcribed spacer region sequence from five representative isolates (587 bp) exhibited 100% identity with C. cassiicola. A high variability of conidial morphology was observed among isolates, but no correlation with cultural phenotype was observed. When the soybean cultivar A 8000 RG was challenged with the 24 isolates, different degrees of virulence were observed, ranging from highly virulent to nonvirulent. No association of virulence with cultural or morphological characters was observed, but a relationship with geographical origin was demonstrated. Histopathological studies were performed on a nonvirulent and a highly virulent isolate. Microscopic observations of infected tissues of the former showed low mycelium development restricted to the upper epidermis, a thickening of the cuticle and primary wall of subepidermal cells, and accumulation of callose in phloematic vessels. In tissue infected with the latter, there was abundant mycelium development accompanied by cellular disorganization in mesophyll cells. Pathological challenges of isolates on nine different cultivars indicated that the degree of virulence of isolates depends on the plant genotype, demonstrating that the C. cassiicola–soybean interaction is highly specific. Understanding the genetic basis of this interaction will provide new information for better disease management and breeding strategies.     

Co‐infection with Cucumber vein yellowing virus and Cucurbit yellow stunting disorder virus leading to synergism in cucumber

Symptom expression and levels of the ipomovirus Cucumber vein yellowing virus (CVYV) and the crinivirus Cucurbit yellow stunting disorder virus (CYSDV) were compared in greenhouse cucumbers in single and mixed infections. Results were contrasted with those obtained for plants infected with the potyvirus Zucchini yellow mosaic virus (ZYMV) in single and in mixed infections with either CVYV or CYSDV. Cucumber showed leaf symptoms of each co‐infecting virus, except for the combination of CYSDV with ZYMV, where the typical CYSDV‐like symptoms of interveinal leaf yellowing were inconspicuous or absent. The progression of CVYV as quantified by real‐time RT‐PCR was similar in plants with single infections and in mixed infection with CYSDV between 15 and 60 days post‐inoculation (dpi). However, CYSDV was detected at significantly enhanced levels in plants when co‐infected with CVYV but not when co‐infected with ZYMV. In the latter case, ZYMV levels were reduced when compared with single infections. During mixed infections of ZYMV and CVYV, the titre levels of the ipomovirus were significantly lower when compared with single infections. Cucumber had reduced plant height, internode length, dry weight and fruit yield, positively correlated with the titre levels of CVYV and not of CYSDV during mixed infections. It is concluded that co‐infections with CVYV enhance the titre of CYSDV, which could have epidemiological significance.     

Occurrence of azoxystrobin‐resistant isolates in Passalora fulva,the pathogen of tomato leaf mould disease

Since 2007, serious damage to tomato from leaf mould caused by Passalora fulva has frequently been observed in commercial greenhouses in Gifu Prefecture, Japan. One of the factors relating to this damage was suspected to be a decrease in azoxystrobin sensitivity of the pathogen. Biological and molecular studies were conducted to characterize fungicide resistance. In in vitro sensitivity tests using mycelial homogenate placed on fungicide‐amended medium, the minimum inhibitory concentrations (MIC) of azoxystrobin for mycelial growth of the isolates divided into two ranges, 0.031–0.5 mg L^?1 and 8–32 mg L^?1. Isolates with MICs within the two ranges were considered as sensitive and resistant, respectively, to azoxystrobin because, in in vivo tests, the percentage protection conferred by this fungicide (100 mg a.i. L^?1) against these isolates was 89.7–100% and 4.5–31.1%, respectively. Resistant isolates had a replacement of phenylalanine with leucine at codon 129 (F129L) in cytochrome b. Forty‐five percent of the 271 isolates collected from 63 tomato greenhouses from 2007 to 2008 were resistant to azoxystrobin. In many greenhouses where the isolation frequency of resistant isolates was 80% or more, azoxystrobin had been used twice per crop for approximately 6 years. In 2012, 27% of the 405 isolates collected were resistant to azoxystrobin, and there was a marked difference in the frequency of occurrence of resistant isolates in the field populations between the three locations sampled. The occurrence of azoxystrobin‐resistant P. fulva isolates (F129L mutants) inflicted considerable damage on greenhouse tomatoes.     

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.     

Effect of microclimate and nutrients on development of cucumber gray mold (Botrytis cinerea)

Infection of young parthenocarpic cucumber fruits byBotrytis cinerea begins in the petals. Removing petals or washing nutrients from the flower significantly reduced infection. Germination of conidia occurred at relative humidity (r.h.) above 92%, but when water deposition on artificial surfaces was prevented, germination did not occur even at 98% r.h. Germination of conidia on petals is promoted by deposition of an aqueous film not visible on the petal surface by the bare eye (but demonstrable by CoCl₂). Provided there is a film of water on the surface of the host, germination and the infection process occur at a wide range of temperatures up to 25 °C. Pre-exposure of cucumber plants at temperatures as high as 30 °C or as low as 8 °C, prior to their infection and incubation under conditions conducive to gray mold, resulted in greater severity of the disease on young fruits or leaves as compared with plants previously incubated at 10-25 °C. The relevance of these results to cultural control of gray mold is discussed.     

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.     

Monitoring of Brazilian wheat blast field populations reveals resistance to QoI,DMI, and SDHI fungicides

Wheat blast is one of the most important and devastating fungal diseases of wheat in South America, South-east Asia, and now in southern Africa. The disease can reduce grain yield by up to 70% and is best controlled using integrated disease management strategies. The difficulty in disease management is compounded by the lack of durable host resistance and the ineffectiveness of fungicide sprays. New succinate dehydrogenase inhibitor (SDHI) fungicides were recently introduced for the management of wheat diseases. Brazilian field populations of the wheat blast pathogen Pyricularia oryzae Triticum lineage (PoTl) sampled from different geographical regions in 2012 and 2018 were shown to be resistant to both QoI (strobilurin) and DMI (azole) fungicides. The main objective of the current study was to determine the SDHI baseline sensitivity in these populations. Moderate levels of SDHI resistance were detected in five out of the six field populations sampled in 2012 and in most of the strains isolated in 2018. No association was found between target site mutations in the sdhB, sdhC, and sdhD genes and the levels of SDHI resistance, indicating that a pre-existing resistance mechanism not associated with target site mutations is probably present in Brazilian wheat blast populations.     

Involvement of two different types of Verticillium dahliae in lettuce wilt in Ibaraki Prefecture, Japan

Although wilt diseases of various plants by Verticillium dahliae occur worldwide, Verticillium wilt of lettuce is relatively new and rare. In 2009, this disease appeared suddenly in commercial fields in Ibaraki Prefecture, Japan. To investigate the cause of the disease, we characterized each lettuce isolate for pathogenicity, mating types, and genetic characteristics and found two types of V. dahliae. A population that included at least two types of V. dahliae was the putative infection source of the disease. Effective management systems against V. dahliae isolates of different races and having different host ranges are necessary.