Study On Early Diagnosis And Rapid Detection Of Bursaphelenchus Xylophilus And Differential Proteome Analysis Of B. Xylophilus And B. Mucronatus

Pine wilt disease is one of the most important pine tree diseases,and its causal agent is pinewood nematode(Bursaphelenchus xylophilus,PWN) which is a great threat to the pine forests of many countries and has caused great economic loss every year as well as imponderable ecological damage.Thus,B.xylophilus is a quarantine organism at the top of the list of the pathogenic species in many countries,and also listed on A2 in China.However,it is difficult to differentiate B.xylophilus from the closely-related species B.mucronatus,which is not pathogenic to pine trees under microscope for their morphological similarity,which makes the quarantine and prevention very difficult to perform.In this study,we focused on the improvement of the rapid detection and early diagonosis techniques of B.xylophilus,and developed several application systems and methods for port quarantine and pine forest management.We also carried on differential proteome analysis of B.xylophilus and B.mucronatus.Using the Rapid Isolating Machine and molecular detection methods,an application system for rapid detection of pinewood nematodes was developed,which took only 90 min to go through the whole procedure from nematode sample collecting to the final identification result.This specific and sensitive system would greatly save quarantine detection time and save the clearance delay for cargoes.Combining early diagnosis tube and equal-length double-stranded fluorescence probe real-time PCR assay,a system for early diagnosis of pine wilt disease was developed,which can effectively detect PWN-infested pinetrees in the first stage,and help to wipe out the infectants and prevent the further spread of PWN.Another application was developed for investigation of the Monochamus alternatus's nematodes carrying situation,which could help to solve the problem that the rotten nematodes collected from trapped Monochamus alternatus were extremely difficult to identify.In this study,we first applied two-dimensional electrophoresis and MALDI-TOF-MS/ MS to study the differential proteome of B.xylophilus and B. mucronatus.We found that their differences included metabolism pathway,signal transduction and cytoplasm transport mechanism.We supported the theory that B. xylophilus is the sole pathogen to pine wilt disease and discussed its pathological mechanism.Our study will help to develop an immunological detection technique for PWN and further uncover the virulence mechanism of pinewood nematodes.