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1.
Flávia E. de Mello Valéria S. Lopes-Caitar Sheila A. Xavier-Valencio Helen P. da Silva Sören Franzenburg Andreas Mehl Joseph-Alexander Verreet Maria I. Balbi-Peña Francismar C. Marcelino-Guimaraes Cláudia V. Godoy 《Plant pathology》2022,71(2):373-385
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. 相似文献
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BACKGROUND: Carbendazim (MBC) has failed to control wheat scab, caused by Fusarium graminearum Schwabe, on the eastern coast of China in recent years after about 30 years of application. RESULTS: MBC resistance was found to be common in pathogen populations on the eastern coast and along areas of the Yangtze River. EC(50) and minimum inhibitive concentration (MIC) values of MBC inhibiting mycelium growth of wild-type isolates were less than 0.9 and 1.4 microg mL(-1) respectively, while EC(50) values of resistant collections averaged 7.02 +/- 11.86 microg mL(-1). The slope of the MBC dosage-response curve (DRC) for resistant isolates of F. graminearum was flat: 1 < b < 2.8 for resistant isolates and 3.5 < b < 11 for sensitive isolates). Both field resistant and sensitive MBC strains shared similar temperature sensitivity, fitness and virulence on ears. Field resistant strains and UV-induced mutants showed positive cross-resistance to other benzimidazole derivatives and were mainly at intermediate MBC resistance level. Highly resistant field MBC strains rarely appeared, but only some of the highly resistant MBC UV mutants were insensitive to N-phenylaminecarbamates. No mutation in beta-tubulin was found in F. graminearum, in contrast to mutation in this tubulin which has led to MBC resistance in other plant pathogens. CONCLUSION: MBC(R) isolates have high fitness and competition in field, conferred by a novel molecular mechanism. 相似文献
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香蕉条斑病毒LAMP快速检测方法的建立 总被引：1，自引：0，他引：1
环介导等温扩增（loop-mediated isothermal amplification,LAMP）是一种特异、灵敏、快速的新型基因检测技术。本研究以香蕉条斑病毒（Banana streak virus,BSV）ORF3保守区域为基础针对6个特定区域设计并筛选了4条LAMP扩增引物,通过对LAMP反应中MgSO4、dNTPs、Betaine等主要试剂浓度进行优化,建立了香蕉BSV的LAMP检测方法,63℃反应90 min后通过在反应产物中添加SYBR Green Ⅰ染料后颜色的变化,肉眼即可判断检测结果。LAMP具有极高的检测特异性和灵敏性,其检测下限约为3.2 ng·μL-1,是PCR检测灵敏度的25倍,能快速、准确地对疑似样品进行检测,本研究对华南地区部分疑似样品的检测结果显示LAMP阳性检出率比PCR检出率高。本文建立的BSV LAMP检测方法是对BSV检测方法的拓展和延伸,为香蕉病毒的快速检测提供技术保障。 相似文献
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油菜菌核病菌对多菌灵和乙霉威的抗药性机理 总被引：1，自引：0，他引：1
生物测定结果表明,油菜菌核病菌田间菌株对多菌灵(MBC)敏感性表型呈多样性,即存在MBCS、MBCLRLR、MBCHRHR和MBCVHR表型.而对乙霉威(DIE)则只检测到DIES和DIEHR表型.MBCS、MBCLR和MBCHR菌株中除JD2-3菌株为DIES外,其余菌株均为DIEHR,MBCVHR菌株对DIE表现为DIES.MBC和DIE之间存在典型的负相关交互抗性.序列分析结果表明,表型为MBCVHRDIES菌株的β-微管蛋白基因,第198位氨基酸由Glu(GAG)突变为Ala(GCG);表型为MBCHRDIEHR菌株的β-微管蛋白基因的突变位点在第200位,由Phe(TTC)突变为Tyr(TAC);而表型为MBCSDIES和MBCLRDIEHR的菌株在所扩增的β-微管蛋白基因片段中未发生突变.初步表明,β-微管蛋白基因198和200位氨基酸的突变是引起油菜菌核病菌对多菌灵抗药性呈多样性的分子机理. 相似文献
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陕西省小麦赤霉病菌对多菌灵敏感性研究 总被引：2，自引：0，他引：2
The mycelium growth rate method was used to test the sensitivity to carbendazim (MBC) at distinctive concentrations in 136 isolates of Fusarium graminearum from 19 counties of 6 districts in Shaanxi Province in 2008. The distinctive MBC concentration was 4 mg / L for testing of resistance and sensitivity. The results showed that average 50% effective concentration (EC50 ) of 136 tested sensitive isolates were (0. 908 6 ± 0. 062 3) mg / L. All the isolates were sensitive to MBC. The fungicide of MBC could be continually applied wheat production in Shaanxi. 相似文献
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PIRA-PCR ( p rimer- i ntroduced r estriction a nalysis PCR) was developed to detect isolates of Fusarium graminearum with moderate resistance to carbendazim, a methyl benzimidazole carbamate (MBC)-group fungicide. Two primer pairs were designed and synthesized according to the nucleotide sequence of the β 2 -tubulin gene from F. graminearum. Fragments of 164 bp were amplified by nested PCR from isolates differing in carbendazim sensitivity. A Hin dIII restriction enzyme recognition site was introduced artificially by inner primers to detect a mutation at codon 167, and Taa I ( Tsp 4CI) restriction enzyme was used to detect a mutation at codon 200. The sensitivity of isolates to carbendazim was determined by analyzing electrophoresis patterns of the resulting PCR products after simultaneous digestion with both Hin dIII and Taa I. Results from PIRA-PCR and a conventional method (mycelial growth on agar) were identical but PIRA-PCR required only 7–8 h while the conventional method required 5–7 days. This study demonstrates that PIRA-PCR not only monitors the appearance of moderately resistant isolates, but can be useful for detecting genotypes of F. graminearum with moderate resistance to carbendazim. 相似文献
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ABSTRACT Colonization of watermelon root and stem tissues by Fusarium oxysporum f. sp. niveum race 1 and its relationship to the apparent resistance to Fusarium wilt was investigated. In each of 2 years, 17 differentially susceptible watermelon cultivars and one accession were tested in the greenhouse, and 7 cultivars also were tested in the field. Colonization by a chlorate-resistant marked isolate of the fungus was assayed by plating homogenized tissue samples on a selective medium. Six days after inoculation, seedlings of highly resistant, moderately resistant, and susceptible cultivars had F. oxysporum f. sp. niveum race 1 CFU counts in the lower stems of 10(2), 10(3), and 10(4) CFU/g of fresh tissue, respectively. Percent wilt (Y) of the seedlings was positively correlated with colonization (X) by F. oxysporum f. sp. niveum race 1 in roots (Y = 21.2 ln [X + 1] - 140.7, R(2) = 0.85) or lower stems (Y = 17.3 ln [X + 1] - 78.6, R(2) = 0.86). Percent wilt (Y) also was correlated with the ratio (X(r), 0 to 1 values) of lower stem to root colonization (Y = 34 ln X(r) + 112, R(2) = 0.36). Field evaluations confirmed these relationships, and a link between cultivar resistance and a reduced rate of spread of the fungus in primary stems during a season was observed. Fruit yield decreased with increased tissue colonization at linear rates of 9.9 to 12.7 t/ha per ln (CFU/g + 1) (R(2) >/= 0.58). The greenhouse seedling stem colonization assay described may be utilized as a collaborative method to quantify Fusarium wilt resistance in watermelon. 相似文献
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This study reports the development of a loop-mediated isothermal amplification procedure (LAMP) for polymerase chain reaction (PCR)-based detection of 'Candidatus Liberibacter solanacearum', the bacterial causal agent of potato zebra chip (ZC) disease. The 16S rDNA gene of 'Ca. Liberibacter solanacearum' was used to design a set of six primers for LAMP PCR detection of the bacterial pathogen in potato plants and the psyllid vector. The advantage of the LAMP method is that it does not require a thermocycler for amplification or agarose gel electrophoresis for resolution. Positive LAMP results can be visualized directly as a precipitate. The LAMP strategy reported here reliably detected 'Ca. Liberibacter solanacearum' and the closely related species 'Ca. Liberibacter asiaticus', the causative agent of huanglongbing disease of citrus, in plant DNA extracts. Although not as sensitive as quantitative real-time PCR, LAMP detection was equivalent to conventional PCR in tests of ZC-infected potato plants from the field. Thus, the LAMP method shows strong promise as a reliable, rapid, and cost-effective method of detecting 'Ca. Liberibacter' pathogens in psyllids and field-grown potato plants and tubers. 相似文献
9.
水稻潜根线虫(Hirschmanniella oryzae)在水稻产区普遍存在,是水稻(Oryza spp.)的主要植物寄生线虫,在世界范围内造成产量损失。本研究通过比较GenBank数据库中潜根属线虫相关序列,以水稻潜根线虫ITS-rDNA序列为靶标,设计了水稻潜根线虫LAMP的一组特异性引物,包括一对外引物(F3:5′-ATCTTGTCCTTTGGCACG-3′,B3:5′-CGGTTGAACAAACAACGT-3′)和一对内引物 (FIP: 5′-CAGCATAGCAACAGAATGAATTCACGGTCGTAAACCTAATACGCG-3′,BIP:5′-TTGTACTACAATGGATTGTTTTCGCCTGATCCATCCACCCATG-3′);用琼脂凝胶电泳法和SYBR Green I染色法筛选合适的引物和扩增条件,建立了优化的水稻潜根线虫LAMP检测条件:扩增温度为57℃,反应时间为45 min,反应体系中dNTPs 和 MgSO4的浓度分别为1.4 mmol·L-1和7 mmol·L-1。本研究建立的检测方法可以检测鉴定水稻潜根线虫不同发育时期虫态(雌虫、雄虫、幼虫)个体,还可以从近缘种和其他植物寄生线虫混合的样品及水稻根组织样品中直接检测出水稻潜根线虫,且检测灵敏度达到1/1 000条成虫DNA。 相似文献
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Botrytis cinerea isolates obtained from apple orchards were screened for resistance to boscalid. Boscalid-resistant (BosR) isolates were classified into four phenotypes based on the levels of the concentration that inhibited fungal growth by 50% relative to control. Of the 220 isolates tested, 42 were resistant to boscalid, with resistant phenotypes ranging from low to very high resistance. There was cross resistance between boscalid and carboxin. Analysis of partial sequences of the iron-sulfur subunit of succinate dehydrogenase gene in B. cinerea (BcSdhB) from 13 BosR and 9 boscalid-sensitive (BosS) isolates showed that point mutations in BcSdhB leading to amino acid substitutions at the codon position 272 from histidine to either tyrosine (H272Y) or arginine (H272R) were correlated with boscalid resistance. Allele-specific polymerase chain reaction (PCR) analysis of 66 BosR isolates (including 24 additional isolates obtained from decayed apple fruit) showed that 19 carried the point mutation H272Y and 46 had the point mutation H272R, but 1 BosR isolate gave no amplification product. Analysis of the BcSdhB sequence of this isolate revealed a different point mutation at codon 225, resulting in a substitution of proline (P) by phenylalanine (F) (P225F). The results indicated that H272R/Y in BcSdhB were the dominant genotypes of mutants in field BosR isolates from apple. A multiplex allele-specific PCR assay was developed to detect point mutations H272R/Y in a single PCR amplification. Levels of boscalid resistance ranged from low to very high within isolates carrying either the H272R or H272Y mutation, indicating that, among BosR isolates, different BosR phenotypes (levels of resistance) were not associated with particular types of point mutations (H272R versus H272Y) in BcSdhB. Analysis of genetic relationships between 39 BosR and 56 BosS isolates based on three microsatellite markers showed that 39 BosR isolates and 30 BosS isolates were clustered into two groups, and the third group consisted of only BosS isolates, suggesting that the development of resistance to boscalid in B. cinerea likely is not totally random, and resistant populations may come from specific genetic groups. 相似文献
12.
环介导等温扩增技术快速检测麦根腐平脐蠕孢 总被引：1，自引：0，他引：1
为建立麦根腐平脐蠕孢简单快速的分子检测方法,基于环介导等温扩增(Loop-mediated isothermal amplification, LAMP)技术,以核糖体DNA的ITS为靶标序列,设计和筛选出一组麦根腐平脐蠕孢的特异性引物,成功开发可快速准确检测麦根腐平脐蠕孢的LAMP方法。甜菜碱作用测试显示LAMP体系中是否添加甜菜碱对扩增结果无明显影响;特异性测试显示该LAMP方法能够从14种植物病原菌中特异地检测出麦根腐平脐蠕孢;灵敏度测试显示该LAMP方法的检测极限为10-3 ng·μL-1,是常规PCR检测方法灵敏度的100倍;通用性测试显示该LAMP方法能够准确检测洛阳、安阳、开封和邯郸等不同地理来源的麦根腐平脐蠕孢菌株;发病组织检测显示该LAMP方法能够准确地从人工接种的小麦发病组织中检测出麦根腐平脐蠕孢,检出时间为侵染12 h及以上。这些研究结果表明所建立的麦根腐平脐蠕孢LAMP检测方法特异性好、灵敏度高、适用性强,可用于小麦根腐病的早期快速诊断。 相似文献
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本研究选取番茄溃疡病菌(Clavibacter michiganensis subsp.michiganensis,Cmm)致病岛上的chpC基因的部分序列,作为环介导等温扩增(loop-mediated isothermal amplification,LAMP)靶标片段进行LAMP引物设计。对反应体系优化后进行特异性测定,结果表明供试的89株番茄溃疡病菌中86株检测结果为阳性,3株为阴性,供试的14株非番茄溃疡病菌(其他重要植物病原细菌)均为阴性。检测番茄溃疡病菌菌悬液样品的阈值为4.8×10~5 CFU·mL~(-1),对DNA样品的检测阈值为1.8×10~(-2) ng·μL~(-1),并据此建立了番茄溃疡病菌的LAMP检测方法。将该方法应用于番茄种子携带Cmm的检测,通过提取种子浸提液样品的总DNA,实现了对番茄种子携带Cmm的直接检测。与普通PCR相比,该方法更加快捷简便,不依赖PCR仪等昂贵的仪器设备,可以丰富现有的番茄溃疡病菌分子检测体系,为口岸等检疫部门提供简单易行的检测初筛手段。 相似文献
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BACKGROUND: Bemisia tabaci, the sweetpotato whitefly, is a globally invasive pest that causes serious agricultural damage by transmitting plant viruses. This pest forms a cryptic species complex that displays morphologically indistinguishable biotypes. Among them, the B and Q biotypes are the most important pests worldwide. Because they have different levels of insecticide resistance, these biotypes must be identified in order to achieve proper pest control. Therefore, a convenient, rapid and specific detection method for identifying the two biotypes is necessary. RESULTS: Loop‐mediated isothermal amplification (LAMP) was employed for rapid identification of B. tabaci B and Q biotypes. By combining a quick DNA extraction method, identification of the two biotypes was achieved within 1 h of detection time. The LAMP assay was applied to study the dynamics of B. tabaci biotypes both in the field and in greenhouses. It was found that, while temperature may be important for population dynamics of the whitefly in the field, population dynamics in greenhouse conditions may be influenced by the types of insecticide. CONCLUSION: The newly designed LAMP assay is a simple, rapid and accurate method for identifying the B and Q biotypes. It can be conducted by non‐specialists and can contribute to pest management. Copyright © 2012 Society of Chemical Industry 相似文献
16.
Fusarium head blight, one of the most damaging plant diseases in Jiangsu province of China, is a leading cause of economic loss and toxin accumulation in the crop, including nivalenol, deoxynivalenol and its acetylated derivatives. Disease control by carbendazim (MBC) has been applicated for many years, and the resistance frequency increased steadily. Furthermore, resistance may trigger toxin growth. Here, a total of 7261 isolates were collected throughout Jiangsu province from 2010 to 2012 to determine their sensitivity to MBC and trichothecene chemotypes. We studied the relevance between trichothecene chemotype and MBC-sensitivity, and found that the MBC-sensitive isolates occupied more NIV chemotype proportion up to date; 15-AcDON chemotype only existed in MBC-sensitive isolates; and most MBC-resistant isolates secreted 3-AcDON in chemotype. Besides, trichothecene production analyses indicated that MBC resistance increased 3-AcDON yield and percentage, especially site-directed mutants at codon 167 in the β2-tubulin gene. 相似文献
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Cytochrome b from yeast (Saccharomyces cerevisiae Meyer ex Hansen) provides a convenient model system for the study of Qo-site inhibitor (QoI) resistance mutations from a variety of organisms. QoI resistance mutations from fungal plant pathogens (G143A and F129L), malaria agent Plasmodium sp (Y279C/S), and Pneumocystis carinii (L275F), an opportunistic pathogenic fungus of man, were introduced into yeast cytochrome b and their effect on the binding of a variety of natural (myxothiazol and stigmatellin) and synthetic (atovaquone, azoxystrobin and pyraclostrobin) inhibitors to the bc1 complex monitored. L275S (from a myxothiazol-resistant yeast) was also re-examined. Stigmatellin binding was relatively unaffected by the introduction of these mutations. Significant increases in resistance were observed for the strobilurin-class inhibitors myxothiazol, azoxystrobin and pyraclostrobin, with the largest increase in resistance conferred by G143A. In contrast, atovaquone binding was most effected by Y279C/S and L275S. Notably, F129L, G143A and L275S had a minor effect on bc1 activity, and so are unlikely to confer significant fitness penalties in vivo. These data are discussed in the light of the atomic structures for myxothiazol- and azoxystrobin-inhibited bovine bc1 which have recently become available. We propose that QoI resistance due to G143A arises from steric hindrance between the inhibitor and cytochrome b, whereas the mechanism of resistance for the other mutations is due to an increase in binding energy between the protein and inhibitor molecule. Site-directed mutagenesis was also used to model selected regions of the mammalian Qo site in yeast cytochrome b in order to further understand the differential efficacy of these QoI in the mammalian and pathogen bc1 complexes. 相似文献
19.
Banno S Fukumori F Ichiishi A Okada K Uekusa H Kimura M Fujimura M 《Phytopathology》2008,98(4):397-404
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. 相似文献
20.
Chengzhong Lan Hongcheng Ruan Xiujuan Yang Jinai Yao Junxi Jiang 《Phytoparasitica》2018,46(3):283-293
Fusarium wilt, caused by Fusarium oxysporum f. sp. cucumerinum Owen (FOC), is a destructive disease affecting cucumber production worldwide. Developing an accurate and reliable method for detection of FOC is important for disease prediction and control. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed and validated for specific and sensitive detection of FOC. Four LAMP primers were designed based on the sequence of the FOC-specific random amplified polymorphic DNA (RAPD) marker OPZ-12865. LAMP reactions were performed at different temperatures and for different durations, and the optimal temperature and duration were 63 °C for 60 min, respectively. Hence, a LAMP assay for detection of FOC was established. The specificity of the LAMP method was evaluated against 119 isolates of FOC and other pathogens, and only FOC isolates yielded positive results. In sensitivity tests, the lowest concentration of genomic DNA required for the LAMP assay was 10 fg in a 25 μL reaction. The LAMP assay was successfully applied to detect FOC in cucumber tissues and soil from infested fields, and the positive ratios of LAMP, PCR, and traditional tissue isolation for detecting FOC from diseased cucumber root samples were100%, 86.6 and 83.3%, respectively. Therefore, the LAMP assay developed herein should serve as a simple, cost-effective, rapid, highly specific, and sensitive tool for the visual detection of FOC and contribute to improved disease management. 相似文献