| Plant parasitic nematodes(PPN)are recognized as economically important threat to worlds'agriculture.Nematodes alone or either combination with other soil microorganisms had been found to attack almost every part of the plant including roots,stems,leaves,fruits and seeds.Longidorid and Trichodorid nematodes are polyphagous root ectoparasitic group that represents the only families in Class Enoplea that proven to have a plant parasitic ability.Feeding by Longidorid and trichodorid nematodes causes substantial damage to a wide range of crop species.As importantly,some of the species in nematode genera Longidorus,Paralongidorus,Xiphinema,Trichodorus and Paratrichodorus are vectors ofimportant plant virusesIdentification of new or potentially harmful species of nematodes in this genus is important to the success of agriculture and aids in the development and evaluation of quarantine or regulatory procedures to minimize their spread.The main objective of this study is to provide an accurate identification of Longidorid and Trichodorid nematodes detected in China through integrative morphological and molecular characterization and present the phylogenetic status of species occurring in China within the taxa of Longidorids and Trichodorids reported worldwide.A total of 528 soil samples collected from Zhejiang,Hubei,Shandong,Anhui,Hainan,Shanxi,Yunnan,Guangdong and Beijingcovering various biotopes such as natural parks,forest and orchards including samples from quarantine port were investigated.The nematodes were extracted using modified Cobb's decanting and sieving technique.For morphological studies,fresh specimens were use for mophometric evaluation and molecular analysis while remaining nematodes were fixed in 4%formalin and processed through Seinhorst(1959)method for permanent slide.Morphological characterization was based on the important diagnostic characters derived from polytomous keys and with comparisons of-mophology to the original description of known species while all the species were molecularly characterized by 18S,D2-D3 region of 28S,ITS1 region of rDNA and COI gene of mtDNA.Furthermore,phylogenetic reconstructions were established to confirm the validity of species.Recent study revealed 13.3%of Longidorid and Trichodoridnematodes recovered from the soil samples.A total of sixty eight inter and intra species of family Longidoridae and Trichodoridae were recorded.About 46 populations(67.6%)of Longidorid nematodes and 22 populations(32.4%)of Trichodorid were recovered.Seven known(L.jonesi,L.pius,L.ferrisi,L.hangzhouensis,L.camelliae,L.diadectuurus,L.litchii and 1 new species(Longidorus n.sp)of Longidorus while 8 known(X.brevicolle,X.insigne,X.hunaniense,X.krugi,X.oxycaudatum,X.diffusum,X.americanum sensu lato,X.elongatum)species of Xiphinemawere recorded.From family Trichodoridae only Trichodorusand Paratrichodorus were found where 4 species(T.cedarus,T.nanjingensis,T.pakistanensis,T.yokooi)belongs to Trichodorus and 2 species(P.porosus and P.renifer)from Paratrichodorus.During present study genus Xiphinema found to be the most prevalent genus as compared to others.Out of 38 different populations of Xiphinema,19 were the intraspecies of Xbrevicolle,9ofX.hunaniense,5 ofX insigne,these 3 species are assumed to be distributed widely in China.For Longidorus no additional populations were found to study intraspecies variation.Among 22 recorded populations of Trichodoridae,7 were the intraspecies of T.cedarus,5 of T.nanjingensis and 7 of P.porosus.These three species mostly recovered from different localities of Zhejiang Province.Among all the known species of family Longidoridae and Trichodoridae,T.yokooi,L.pius,L.ferrisi and L.diadecturus are the newly recorded species from China.One new species of Longidorus isolated from the rhizosphere of Pittosporum tobira collected from Beijing is also described here.The new species can be characterized by relatively long body(L=4.1-6.6mm),wider lip 16-23?m wide,very posterior guide ring(70-93?m)long odontostyle(142-168?m),male spicule 100-123?m long with 10-13 ventromedian supplements.The identification code for new Longidorus species based on the polytomous key of Chen et al.,(1997)is A67,B34,C5,D12,E2,F23,G1,H1,and 12.The new species also molecularly characterized on the basis of 18S and D2-D3 28S rDNA sequences,typologically this new species is most similar to L.litchii and L.laicis,both originated from Asia.Molecular characterizations of 68 Longidorids and Trichodorids populations yielded 57 sequences of D2D3 expansion segment of 28S rDNA,17 sequences of 18S and 29 sequences of ITS1 region.Amplified DNA fragments of Longidorus spp.approximately ranges from 774-848bp in 28S,918-1183 bp in ITS 1 and 1722-1758 bp in 18S region.Sequence length of Xiphinema spp.obtained approximately 826-852 bp in 28S,1271-1362 bp in ITS1 and 1761-1768bp in 18S region.While the amplified DNA of Trichodorus and Paratrichodorus has sequence lengths of 698-765 bp in 28S,1149-1154 bp in ITS1 and 1425-1793 bp in 18S region.The near full length 18S and partial 28S D2/D3 and ITS sequences were compared with the related species available in GenBank using BLAST homology search program.The sequences were analysed and aligned using the program Clustal W and phylogenetic trees were generated with the Neighbour-joining(NJ)method.The Phylogenetic relationships of Longidoridae and Trichodoridae inter and intraspecies isolates in China showed a well supported clade(98-100%)with the known species or related species by forming a distinct molecular clade which confirms and support the morphological identity of the species under investigation.The findings of this study,provides foundation of the occurrence,distribution and host association of Longidorids and Trichodorids in China.The research further provides the morphological and molecular characterization of all the species which will be useful in awareness of the presence of harmful nematodes(virus vector)that may a pose threat to important crops in the future. |
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