| Soybean anthracnose caused by Colleloilrichum truncatunm is an important disease,impacting on the growth of soybean and causing a serious reduction.Verticilium albo-atrum and V.dahliae can infect alfalfa,cotton and other crops.What’s more,they are important plant quarantine fungus in China.So,it is urgent for us to improve the early detection technology of C truncatum、V.albo-atrum and V.dahliae to reduce the economic loss.Basiing on loop-mediated isothermal amplification(LAMP),we established three specific systems for the detection of the tliree pathogens respectively.Using Rpb1(the large subunit of RNA polymerase Ⅱ)as the newly target gene,we developed a LAMP technology system for rapid detection of C.truncatum,allowing amplification at 62℃ over 60 min.After addition of SYBR Green Ⅰ to LAMP reaction products,a yellow-green color(detectable by the unaided eye)developed only in the presence of C.truncatum.The detection limit of the LAMP assay was 100 pg·μL-1.Using this LAMP assay,we have rapidly and successfully detected C.truncatum in field samples collected from Jiangsu,Anhui and Hubei provinces of China,and in soybean seeds from farmers’ markets.Selecting Tub(β-tublin,beta tubulin gene)as the target gene,we established a LAMP assay to rapidly detect V.albo-atrum.This assay efficiently amplified the target gene at 62℃ over 70 min and the result can be directly determined using naked eye by adding HNB(Hydroxynaphthol blue)before the amplificatioin.A positive color(sky blue)was only observed in the presence of V.albo-atrum,whereas all other isolates showed purple color.The detection limit of the LAMP assay for V.albo-atrum was 1 pg·μL-1 and its sensitivity in soil and seeds were respectly 10 conidiospores in 0.25 g soil and 50 conidiospores in 10 g seeds.We have successfully detected V albo-atrum in 3 diseased alfalfa of 7 suspect diseased samples collected from Xinjiang.This method provides a new technology for the diagnosis and quarantine of V.albo-atrum.By comparing V.dahliae with approximate species among different target sequences,we selected Gpd(glyceraldehyde-3-phosphate dehydrogenase)as the target gene,developed a simple,rapid and sensitive method for the detection of-V dahliae.The LAMP assay efficiently amplified the target gene in 70 min at 62℃.The result cain be directly determined using naked eye by adding HNB(Hydroxynaphthol blue)before the amplification.The positive reaction was sky blue whereas the negative reaction was purple color.The detection limit of the LAMP assay for V.dahliae was 100 pg·μL-1 and its sensitivity for detecting the pathogen in soil was 10 conidiospores in 0.25 g soil.V dahliae was detected irn 11 diseased cotton from 24 suspect diseased samples collected from Jiangsu and Shandong provinces.Tlhis method provides a new technology for the detection of V dahliae.Soybean root rot caused by a dozen of species of pathogens is a worldwide disease that causes serious damage to soybean production in Clina.Often these diseases have similar symptoms,not easy to distinguish,and difficult to accurately diagnose.In order to clarify the pathogen species and dominant species of soybean root rot in Huaing Huai area,we detected Fusairium oxysporum、F.equiseti、F.proliferatum、F.culmorum、F.graminearum、F.solani、Calonectria ilicicola、Phytophthora sojae、Rhizoctonia solani and Macrophomina phaseolinafrom 165 soybean root rot samples collected from Shandong、Jiangsu and Anhui provinces using the ten LAMP systems developed in our laboratory.The results of LAMP detection showed that the positive ratios were respectively 26%of Fusarium oxysporum、28%of F.equiseti、12%of F.proliferatum、18%of F.culmorum、13%of F.graminearum、16%of F.solani、17%of Rhizoctonia solani、8%of Macrophomina phaseolina、38%of Calonectria ilicicola and 44%of Phytophthora sojae.Complex infection was a conmmon phenomenon in Huang Huai area,and its ratio was 75%.The complex infection were mostly caused by two or three pathogens.And Calonectria ilicicola and Phytophthora sojae were the dominant pathogens in Huang Huai area. |
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