Study On The Movement Behavior Of Bursaphelenchus Xylophilus And The Acetylcholine As Neurotransmitters

Pine wood nematode disease is the most devastating disease of pine trees. Its pathogen Bursaphelenchus xylophilus is a kind of quarantinable pest. This disease distributes in Japan,Korea,America,Canada,Mexico and Portugal,and so on.In China it is first discoved in Nanjing province,and then spreaded to other provinces.B. xylophilus is a kind of migratory nematode.The movement of B. xylophilus which is controlled by nervous system related to the parasitism and pathogenicity of the nematodes. Based on the study of the movement of B. xylophilus and the acetylcholine as neurotransmitters,we have carried out lots of experiments.Firstly,we identified and kept four strains, BXRC1、NXY61、NXY67and BXZS from Pinus thunbergii collected in Shandong,Weihai, P. massoniana collected in Zhejiang, Ningbo, P. massoniana collected in Zhejiang Hangzhou and P. tabulaeformis collected in Shan’xi Zhashui. The morphological index of BXRC1、NXY61and NXY67was measured. Then we cultivated the three strains on PDA-botrytis to study their reproductive capacity, and using the branches of host trees such as P. thunbergii to study the migration abilities. Experimental results proved that the reproductive capacity is NXY61> NXY67> BXRC1. BXRC1has the highest transfer ability in the branches of P. thunbergii, P. densiflora and P. armand.In the branches of P. tabulaeformis NXY67has the highestt transfer ability. And in the branches of P. bungeana and Cedrus deodara all the three strains have low transfer ability.Secondly,based on the neural biology characteristics using acetylcholine as neurotransmitters,we studied the effects on the movement of B. xylophilus from anticholinergic and anticholinesterase drug. We used atropine sulfate which is a kind of anticholinergic drug to handle the nematodes. We find the abnormal motion of the larvae and adults showed the lower activities such as the smaller swing frequency of the head and body and the abnormal twists such as heliciform crispation and rinkly crispation.At the same time,the normal nematodes moved as S form. Neostigmine metilsulfate that is a kind of anticholinesterase drug was also used to handle the nematodes to observe their movements. The change of the swing frequency of the head and body was not obvious.But we can see a bigger swing range and faster movement,and so on. So the nematodes had a bigger energy consumption which accorded with the effect of anticholinesterase drug on nervous systm.Finally,we cloned the cholinesterase gene ace-1of strain BXRC1.Analysis showes that the gene contains a whole ORF of1869bp which encodes622amimo acid.And the molecular weight and isoelectric point of the predicted potein are71.231kDa and5.56. To compare the gene sequence with that of other B. xylophilus and other species,we find it has a99.89%similarity to the ace-1gene(Accession Number:GU166345) which is reported by Jae S K.And the predicted potein has99.84%similarity.The gene sequence also has an identity from57.06%to99.89%with the gene sequences from other species such as Dictyocaulus viviparous and Caenorthadits elegans. The phylogenetic tree built by neighbor-joining of MEGA4.1proves BXRC1is B. xylophilus.The acetylcholinesterase from BXRC1, Ditylenchus destructor, Meloidogyne incognita and M. javanica are on the same branch. BXRC1-ACE-1has the identity of71.86%、67.83%and67.22%to that of the species。...