| Rice bakanae disease is a major disease in the rice producing areas,and widely distributes in our country,often bringing serious losses.Fusarium root rot of soybean is caused by a variety of Fusarium spp.,inducing roots and stem base rot as the main symptoms,seriously restricts the quality and yield of soybean,which has become an important disease of soybean producing areas in Northeast and Huanghuaihai region in our country.Phytophthora lateralis and P.cambivora are entry plant quarantine pests of China,which usually cause destructive diseases to the host plants.Seed-borne pathogen is one of the important primary source of infection of many crop diseases,and is a significant risk of crop seed storage diseases,field seedling and growth period diseases.The detection of seed-borne pathogens on seeds is the guarantee of seed quality,an important means to prevent seed-borne diseases.In order to improve the rapid detection level of F.verticillioides causing rice bakanae disease,and F.culmorum causing Fusarium root rot of soybean,and to diagnose the two diseases in the early infection,to reduce the economic losses,we established two specific LAMP systems for the detection of F.verticillioides and F.culmorum respectively using loop-mediated isothermal amplification.We used Pgk(the gene encoding 3-phosphoglycerate kinase)gene as the target gene,and a LAMP assay to rapidly detect F.verticillioides was developed,allowing amplification at 62? over 70 min.The result could be inspected directly using naked eyes by adding HNB(Hydroxynaphthol blue)before the amplification.The color of the reaction mixture changed from purple to sky blue for positive amplification,whereas the color retained purple when nothing was amplified.The detection limit of the LAMP assay is 100 pg·?L-1.The LAMP assay could be used to detect the pathogen in rice tissues directly.And we have rapidly and successfully detected F.verticillioides in field samples of rice bakanae disease which were collected from Nanjing and Zhenjiang of Jiangsu.We selected Cyp51c(the third subunit of the gene encoding cytochrome P450 lanosterol C-14a-demethylase)as the target gene,and a LAMP assay for the rapid detection of F.culmorum was developed.The amplification reaction was conducted at 63 ? over 60 min and the result could be determined using naked eyes directly by adding HNB before the amplification.The color of the reaction mixture changed from purple to sky blue for positive amplification,whereas the color retained purple when nothing was amplified.The detection limit of the LAMP assay is 100 pg·?L-1 and 10 conidiospores in 0.25 g soil.The LAMP assay could be used to detect the pathogen in soybean tissues directly,and we have detected F.culmorum in suspected diseased soybean samples from various regions in China successfully.In order to prevent the spread of P.lateralis and P.cambivora,rapid and accurate detection methods are needed.We choosed Yptl(the gene encoding GTP binding protein)as the target gene,and two LAMP assays were developed to rapidly detect of P.lateralis and P.cambivora,Ypt1-Pl-LAMP and Yptl-Pc-LAMP.The amplification reaction was conducted at 62? over 80 min and the result can be determined using naked eyes directly by adding HNB before the amplification.The color of the reaction mixture changed from purple to sky blue for positive amplification,whereas the color retained purple when nothing was amplified.The detection limit of the two LAMP assays are 100 pg ·?L-1,The assays reported here provide new methods for the rapid detection of P.lateralis and P.cambivora.In order to know the situation of soybean seed-borne pothogens in Huang-huai area,13 species of seed-borne pothogens on soybean seed samples of 29 cultivars in the area were detected using the 13 LAMP systems developed by our team for the detection of F.oxys porum,F.equi seti,F.proliferatum,F.culmorum,F.graminearum,F.solani,Phomapsis longicolla,Colletotrichum truncaltum,C.gloeosporioide,Rhizoctonia solani,Macrophomina phaseolina,Calonectria ilicicola and Phytophthora sojae.The results of LAMP detection showed that there are 20 soybean cultivars carring 7 of these seed-borne pothogens,and the dominant population is C.truncatum,followed by P.longicolla,F.equiseti,F.culmorum,F.oxysporum,C.gloeosporioide,and F.proliferatum.The pathogens carried with seeds of different cultivars are various,the number of pathogens detected changes from 1 to 6.A simple,rapid and sensitive method for detecting soybean seed-borne pothogens was developed in this study,and the situation of soybean seed-borne pothogens in Huang-huai area was revealed with the method. |
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