First New Host Report Of Ditylenchus Destructor In China And Establishment Of Rapid Detection Of Ditylenchus Destructor By LAMP

Ditylenchus?Filipjev,1936?is one of the important plant pathogens,and it is also hard to distinguish different species using traditional morphological method in this genus.D.destructor can cause serious harm to a variety of crops,and has been listed as quarantine nematodes by many countries and regions.In this study,a new host population of D.destructor was identified with morphological and molecular methods.In addition,a rapid detection of D.destructor by Loop-mediated Isothermal Amplification?LAMP?based on 28S rRNA sequences were also carried out.The main results are shown as follows.1.The D.destructor was first reported in carrot in China.The D.destructor extracted from carrot callus was first described with detailed morphological;L=896.3-1122.3?m;a=30.9-41.5?m;b=7.1-10.4?m;c=12.1-16?m;c`=3.4-4.8?m;v=79.3-82.3?m;Stylet=9.7-11?m,which overlapped with the reported values of this population.The ITS and 28S D2-D3 expansion region were amplified by molecular biology methods,and a 916 bp and779 bp sequences were obtained,respectively,which should 99%identity to the reported ITS sequences?EF208210?and 28S D2-D3 expansion region sequences?EU400636?of D.destructor on sweet potato in China.2.The loop-mediated isothermal amplification?LAMP?specific primers were designed based on the 28S D2-D3 expansion region sequences of D.destructor based on the new reported population and other cultured populations,and there primers were DdBIP,DdFIP,DdF3,DdB3 and DdLF.Optimization of the reaction conditions of LAMP was carried out,and the optimum conditions were to add 6.0 mmol·L^-1 Mg²⁺,1.0 mmol·L^-1dNTPs,0.2 U·?L^-1 Bst 2.0 DNA polymerase,and run at 65?and for 50 minutes.The results of LAMP reaction were detected by gel electrophoresis and SYBR Green I dye visualization respectively.3.The specificity,sensitivity and stability of the established LAMP detection of D.destructor were studied by gel electrophoresis and SYBR Green I dye visualization.The results showed that LAMP could detect different stage individuals?female,male,juvenile and single egg?and populations from different hosts and geographical origins of D.destructor.This technology also could directly detect D.destructor from samples mixed with many other nematode species,plant tissue samples and soil samples,the sensitivity of LAMP assay was evaluated to 1/1000 of single nematode DNA.This study provides a basis for monitoring and prevention of carrot nematode diseases in China,and provides a new and effective method for the rapid detection of D.destructor in quarantine inspections at port and dispatch and in field surveys and monitoring.All in all,it is of great significance to prevent the spread of D.destructor from causing more disasters.