The Study Of Pathogens Identification And Rapid Detection Method By LAMP For Blueberry Stem Blight

The stem blight and twig dieback of blueberries is one of the most severe fungal diseases in blueberry production,which leads to the death of branches and whole plants,yield losses and quality decline of blueberry fruits.The stem blight and twig dieback of blueberry mainly caused by the infection of botryosphaeriaceous fungi,which leads to the highest death rate and severe economic losses.In China,the blueberry was first introduced and cultivated in China in 1981.The blueberry cultivation has developed rapidly after that,and the damage of blueberry stem blight have become more and more severer.The aims of this study are:to survey the botryosphaeriacous fungi causing diseases of blueberry the pathogens of blueberry blight in China,and to develop rapid detection and identification methods for them.From February to November in 2018,the pathogens of blueberry stem blight in Shandong,Fujian and Yunnan provinces were collected and isolated,and identified based on morphological characterization and phylogenetic analyses.Nines species of botryosphaeriacous fungi,viz.Botryosphaeria fujianensis and B.longispermatia,Lasiodiplodia,viz.Lasiodiplodia clavispora,L.fujianensis,L.nanpingensis and L.paraphysoides,Macrophomina viz.Macrophomina vaccini,Neofusicoccum,viz.Neofusicoccum lincangense,N.longiiconidium were recognized.In order to quarantine earlier and faster in the process of production,a visual quantitative detection method Loop-mediated isothermal amplification（LAMP）was developed to detect the pathogens of blueberry blight.In this study,LAMP primers were designed by the target gene tef-1α’s comparison among related species.Based on the visualization of SYBR GreenⅠ,the specificity and sensitivity of each set of primers were evaluated by experiments,only one set of LAMP primers were selected for the detection of nine target pathogens.The sensitivity of each set of primers was up to“ag”.In order to make LAMP efficient,the products of LAMP were monitored in by Real-time fluorescence quantitative PCR instrument.By analyzing the data,temperature,primers ratio,the concentration of betaine and Mg²⁺were optimized.Nine detection systems have successfully detected the field blueberry plants inoculated with pathogens for 5 days.It can be judge by observing the color change of the LAMP systems at a constant temperature of 63℃,50～70 min,so as to determine whether the blueberry stems contain target pathogens.It provides an important theoretical basis and technical support for the rapid identification and early quarantine of the pathogens of blueberry stem blight,which is of great significance for the timely and effective control of the disease.