Studies On Identification And Pathogenicity Of Nematode Species Of Bursaphelenchus In Sichuan Province

Bursaphelenchus,especially Busaphelenchus xylophilius is an important kind of forestry pathogeny。The diseases have abroad host range,spread quickly and are difficult to protect,so we called it "cancers on pines".when protecting and quarantining, we must check it up quickly and exactly.But some other species of Bursaphelenchus are discovered from imported coniferous wrapper besides Bursaphelenchus xylophilus.meanwhile,that makes many problems to identification.This research takes Bursaphelenchus as materials which were atained from blasted pines in Sichuan province.The morphological,PCR-RFLP and ITS sequence identification of the nematode were conducted.The main results are as follows:(1) We separated and cultured Bursaphelenchus gained from blasted pines; measured and calculated the numerical value of V,A,b and so on;and described modality.The findings shows:we gained six kinds of Bursaphelenchus,they are Busaphelenchus xylophilius(Steiner & Buhrer,1934)Nickle,B.mucronatus Mamiya, B.rainulfi Braassch,B.aberrans,B.hofumanni,and B.thailandae..(2) A ploymerse chain rection-restriction tragment length polymorphism (PCR-RFLP)analysis was used to discriminated of isolation of Bursaphelenchus spp. ITS regions of Bursaphelenchus spp.were amplified using universal primers.We used the forward primer:P1:5'—CGTAACAAGGTAGCTGTAG—3'at ITS₁ region, the reverse primer P2:5'—TTTCACTCGC CGTTACTAAGG—3'at ITS₂ reglon. The amplification of isolation of Bursaphelenchus yielded one fragment of approximately 900bp.Four restriction edonucleases were used to digest the ITS-PCR products of Bursaphelenchus spp.The results showed that Aluâ… ,Hhaâ… ,Draâ… could distinguish Busaphelenchus xylophilius from B.mucronatus,and they could even distinguish it from other 4 groups of Bursaphelenchus.The products of PCR-amplification of rDNA's ITS₁,5.8S,ITS₂ of Busaphelenchus xylophilius and B.mucronatus were cloned and analyzed by sequence and homology analysis,the identification showed that YA,LS groups belonged to Busaphelenchus xylophilius and DZ,SN,LX were B.mucronatus which were accordant with the morphological identification.(3) The 3 or 4-year-old Pinusm assoniana were inoculated by Bursaphelenchus at the concentration of 3000-population per plant in the study of pathogenicity of Bursaphelenchus,and the observation samples were collected every months after the inoculability.The result showed that YA and LS(Barsaphelenchusx ylophilus) groups have pathogenicity,the symptoms could be described as follow:browning happened from inoculability point to blade tip of the needle,and the browning length was less than half of the blade length,and the disease grade was up to 2.The other 4 groups of Barsaphelenchusx ylophilus did not have any symptoms of pathogenicity.It indicated that the other groups of Pinusm assoniana didn't have pathogenicity or only had weak pathogenicity,and the results may be influenced by the wet climate and low quantity of solar radiation of Ya'an where the Bursaphelenchus were proved to be pathogenic.