Isolation And Identification Of Fungal Leaf Spot Disease From Carya Cathayensis Sarg. And Establishment Of LAMP Detection Method

Chinese hickory(Carya cathayensis Sarg.)is an important economic forest plant in the genus Carya of the Juglandaceae family.The planting of Chinese hickory has brought huge profits to farmers,and the leaves of Chinese hickory have high medicinal research value.In recent years,disease spots have appeared on the leaves of Chinese hickory,resulting in weakened tree vigor and reduced yield and quality of Chinese hickory.However,there are few detailed reports on the leaf spot disease of Chinese hickory.In the study,the diseased tissue was isolated from the diseased spots of hickory leaves collected from Lin’an and Anji,Zhejiang,and the purified strains were inoculated on the leaves of detached leaves and live hickory seedlings,respectively.Detached leaf inoculation and live seedling inoculation were both carried out to confirm the pathogenicity of the strains.The diseased tissue at the inoculation site was re-isolated,which verified the Koch postulates.Two strains with strong pathogenicity were finally isolated from the diseased leaves of Chinese hickory in Lin’an and Anji,Zhejiang.Through the morphological observation and multi-gene phylogenetic identification of the two pathogenic strains,the isolated pathogenic fungi on the leaves of Chinese hickory were identified as Colletotrichum fructicola and Alternaria alternata,and the proportions of the two pathogens isolated from the diseased leaves were 45% and 20%,respectively.This is the first report that C.fructicola and A.alternata can infect Chinese hickory.The TUB2 gene of C.fructicola was determined to be the detection target by comparing the genome.A set of LAMP primers were designed and screened,including two pairs of amplification primers(outer primers F3 and B3,inner primers FIP and BIP)and a pair of loop primers(Loop F and Loop B)and a LAMP system for detection of C.fructicola infecting Chinese hickory was initially established.The detection result can be judged by the color change after adding SYBR Green I dye at the end of reaction.When amplification was positive,that is,C.fructicola had been detected,the color of the reaction mixture changed to green,whereas the original orange color indicated nothing was amplified.The LAMP assay result can be confirmed by 2% agarose gel electrophoresis,and the typical ladder-like band pattern appeared in the positive LAMP product.The optimized reaction conditions showed that the optimum reaction temperature of the LAMP system was 64℃,and the optimum reaction time was 40 min.In the sensitivity test,the detection limit of the LAMP assay was10 pg/μL DNA template.In the specificity test,the LAMP method could specifically detect C.fructicola,not only had no specific amplification to other common pathogenic fungi,but also had no specific amplification to C.acutatum.In the test of the detection of artificially inoculated leaf spot,the LAMP assay could successfully detect C.fructicola on the hickory leaf spot 72 h after inoculation.It showed that the LAMP assay in this study is a simple,efficient,high-sensitive,specific and visualized rapid detection method,which is suitable for the detection of C.fructicola infecting Chinese hickory.This is also the first report of applying LAMP to detect Chinese hickory leaf diseases,which provides reliable technical support for early warning of Chinese hickory diseases.