Isolation And Identification Of Nematicidal Constituents From Plants And Their Nematicidal Mechanism Against Bursaphelenchus Xylophilus

Pine wilt disease(PWD)as a highly destructive epidemic of pine trees can spread very quickly from the infected trees to normal ones.The disease has been reported to affect dozens of pine species in several countries including China,and become a severe worldwide threat to forest resources.Pine wood nematode(PWN),Bursaphelenchus xylophilus,is considered the causal pathogen of PWD,and controlling of PWN is crucial for PWD management.So far synthetic nematicides are still most effective reagents to prevent PWN,which causes more and more concerns about possible drug resistance,high toxicity and residues,environmental pollution and threats to non-target organisms.Therefore,natural ecofriendly nematicides are urgently needed.Phytonematicide related to PWN development has been a hot research topic in recent years since there are abundant plant resources in nature.Some plant materials with inexpensive and abundant resources or some by-products of the plant-related industries were chosen to be tested in the research,including fennel(Foeniculum vulgare Mill.)seeds,fennel roots,Ficus carica L.leaves,Ficus carica L.roots,Zanthoxylum bungeanum Maxim.,Chrysanthemum coronarium L.roots,the roots of Angelica pubescens Maxim.f.biserrata Shan et Yuan,Xanthium sibiricum Patrin ex Widder,Areca catechu L.,Carpesium abrotanoides L.seeds,the leaves of Artemisia argyi Levl.et Vant,Quisqualis indica L.seeds,pomegranate(Punica granatum L.)rind,Stemona japonica,and Tribulus terrester L.seeds.Nematicidal assays against PWN of the ethanol extracts of the above plant materials showed that the ethanol extracts of F.carica L.leaves,F.carica L.roots,the roots of A.pubescens Maxim.f.biserrata Shan et Yuan and pomegranate rind had stronger nematicidal activities.By bioassay-guided fractionation,nine nematicidal compounds were obtained with repeated column chromatographies,preparative high performance liquid chromatography and recrystallization from the nematicidal extracts,and identified as psoralen 1,bergapten 2,osthole 3,columbianadin 4,xanthotoxin 5,ellagic acid 6,punicalin 7,punicalagin 8,corilagin 9 by mass spectrum and nuclear magnetic resonance spectroscopy analysis.The nematicidal activities against PWN of compounds 2-9 were reported for the first time.Psoralen 1,bergapten 2,osthole 3,columbianadin 4,xanthotoxin 5 and punicalagin 8exhibited much stronger activities against PWN among the purified compounds with LC50 values in 72 h of 463.32 ?M,430.08 ?M,489.17 ?M,406.74 ?M,435.66 ?M and307.08?M,respectively.According to the chemical structures and nematicidal activities against PWN of psoralen1,bergapten 2,osthole 3,columbianadin 4,xanthotoxin 5 and punicalagin 8,2H-1-benzopyran-2-one,the core moiety of coumarins was considered to be the key structure contributing to nematicidal activity.In addition,gallagyl moiety and hexahydroxydiphenoyl moiety of punicalagin 8 were supposed to synergisticly contribute to nematicidal activity.?,?-unsaturated carbonyl,the mutual moiety of the above nematicidal compounds was speculated to be closely related to nematicidal activity.Nematicidal mechanisms against PWN of psoralen 1,bergapten 2,osthole 3,columbianadin 4,xanthotoxin 5 and punicalagin 8 were studied in the research.The effects of six nematicidal compounds on PWN morphologies and physiological characters were illustrated as follows:(1)According to the observations by stereo microscope,PWNs showed similar morphologies treated with psoralen 1,bergapten 2,osthole 3,columbianadin 4,xanthotoxin 5,respectively.PWNs moved much more slowly accompanied by severe curling and twisting at first,and then stretched gradually till rigidity.Abnormal bubbles or cavities were observed in most of dead PWN bodies.However,PWNs treated with punicalagin 8 twisted hard initially,then moved more and more slowly till rigidity.Serial blebs were observed in many dead PWN bodies.(2)Observation by scanning electron microscope indicated that the surfaces of the treated PWNs by the six nematicidal compounds significantly shriveled and they were more fragile than the control nematodes through fixation treatment.(3)The micrographs by transmission electron microscope on PWNs showed that there were abnormal cavums between the body wall and internal structure of the treated nematodes in contrast with the control ones,and cells collapsed at varying levels.The effects of six nematicidal compounds on amylase,cellulase,acetylcholinesterase(Ach E)and glutathione S-transferase(GST)from PWNs were summarized as follows:(1)In vitro,the six compounds could all inhibit amylase,cellulase and AchE,and punicalagin 8 showed strongest inhibitory effects on three enzymes with IC50 values of0.96 m M,1.24 m M and 0.60 m M,respectively.In addition,punicalagin 8 had greater inhibitory effect on GST than columbianadin 4 while other four nematicidal compoundshad no distinct effect.(2)In vivo,the six nematicidal compounds had more significant influences on Ach E and GST than that on amylase and cellulase.To learn more about the nematicidal mechanism of punicalagin 8,the most potential nematicial compound in the research,transcriptome analysis was carried out on punicalagin-treated PWNs and control ones by aid of Illumina sequencing technology.Results showed that 2575 differently expressed genes(DEGs)were found between the two libraries,among which 1428 DEGs were up-regulated and 1147 DEGs were down-regulated.Based on comprehensive analysis of Nr,GO,KOG,KEGG annotation,it was speculated as follows:(1)PWNs gave response to the stimulus from punicalagin 8 through endocytosis,phagosome,peroxisome and MAPK signaling pathways.(2)The genes encoding some proteins involved in respiratory electron-transport chain such as NADH dehydrogenase,cytochrome c oxidase,cytochrome b and electron transfer flavoprotein proteins,the enzymes related to energy metabolism such as ATP synthase and ?-glucosidase,heat shock proteins,twitchin and nematode cuticle collagen could be crucial regulation targets of punicalagin 8,which might also own to its nematicidal activity against PWN.The expression levels of some annotated DEGs from PWNs treated with punicalagin 8were validated by q RT-PCR,and the results were consistent with that of transcriptome analysis.The studies could provide important material and theoretical clues for the development of phytonematicides against PWN.