Identification Of Root-knot Nematodes Parasitizing Vegetables And Fruits In Southern China And Phylogenetic Analysis Based On Their Mitochondrial Genomes

The root-knot nematode(Meloidogyne spp.)is a kind of sedentary endoparasitic nematode with various species,wide host range,great pathogenicity and wide distribution.Investigation and identification of root-knot nematodes is the basis of prevention and control.Therefore,this study identified root-knot nematodes parasitizing fruit and vegetable crops in seven provinces in the south of China.Meantime,mitochondrial genomes from Meloidogyne spp.identified were sequenced.Based on the mitochondrial genomes,phylogenetic trees were constructed,which provided a basis for the classification and evolution research of root-knot nematodes.In addition,according to the mitochondrial genomes and other sequences,the duplex-PCR and qPCR systems were explored to identify M.aberrans and M.enterolobii.The main results are as follows.1.According to morphology,combining with isozymes and molecular analysis,four species of root-knot nematodes were identified from 89 Meloidogyne populations.Of these,62 populations are M.incognita,12 pupolations are M.enterolobii,11 populations are M.javanica,and 4 populations were identified as a new species named M.aberrans n.sp.The proportion of M.incognita,M.enterolobii,M.javanica and M.aberrans in the total samples is 70.8%,13.5%,11.2% and 4.5% respectively.The results showed that M.incognita is the dominant species and carambola and grape are new hosts of M.enterolobii.2.One new root-knot nematode species named Meloidogyne aberrans n.sp.was described.Meloidogyne aberrans sp.n.can be differed from all other Meloidogyne spp.by morphology,isozymes and molecular characters.It is characterized by prominent posterior protuberance,round and faint perineal pattern and a medium-length stylet(13.6-15.5?m)characterized females.Males with stylet 18.2-19.6 ?m long,spicules 22.7-36.8?m long and11-15 lateral lines.J2 s were characterized by a smooth lip region with distinct protruded medial lips and a depression in outline at the oral perture,a relatively long stylet(15.9-16.8?m),four incisures in the lateral field and a very short,even not clearly defined,hyaline tail terminus(2.2-5.5 ?m).The new is close to M.ichinohei,but can be differed from M.ichinohei by the longer female,male and J2 stylet,the larger male length,the lower DGO of male,the shorter male tail,more lateral lines in males and fewer incisures in the J2 lateral field.The isozyme electrophoretic analysis of M.aberrans n.sp.showed a rare EST phenotype,S2,two Est bands at Rm = 40.5% and 44.5%.The band of MDH peheotype of M.aberrans n.sp.was N1 phenotype.In this study,the sequences of SSU,LSU D2D3,ITS,and cox2-16 S rRNAs of this new species were also obtained to construct the phylogenetic tree that displayed a closed relationship to M.ichinohei on the SSU and LSU D2D3,to M.megadora on the ITS,and to M.camaelliae on the cox2-16 S rRNAs.Histopathological observations on M.aberrans n.sp.indicated that it can induce the formation of 4-6 large multinucleate feeding cells known as giant cells.3.The complete mitochondrial genomes of M.aberrans,M.enterolobii,M.incognita and M.javanica were sequenced by long PCR amplifications.The full length of mitochondrial genomes were 17,004 bp,17,494 bp,17,543 bp and 18,392 bp for M.aberrans,M.enterolobii,M.incognita and M.javanica,respectively.22 tRNA genes of these four Meloidogyne spp.all encode the typical cloverleaf tRNA secondary structure and are all single copy.The arrangement of twelve protein-coding genes are the same,but the arrangement of tRNA is not always same.All genes in mitochondrial genomes have the same transcription direction.In addition,the number and length of non-coding regions vary slightly,for example,there are three non-coding regions in M.enterolobii,but two in other three Meloidogyne species.Based on twelve protein-coding genes,phylogenetic relationships between nematodes were inferred by Bayesian inference(BI)and Maximum like-hood(ML)methods respectively.The phylogenetic trees showed that all Meloidogyne are positioned in the same clade,in which M.incognita,M.javanica,M.arenaria and M.enterolobii cluster together and M.graminicola and M.chitwoodi are in another clade,and M.aberrans is positioned in the basal clade that sister to other Meloidogyne.The clade comprising Meloidogyne is the closet to the root-lesion nematode,Pratylenchus vulnus,butfar from the cyst nematode.4.Based on the mitochondrial genomes,the conserved primers Mt-RKN-F/Mt-RKN-R for detecting root-knot nematodes,the specific primers Mab-Mt-F/Mab-Mt-R for detecting M.aberrans and the specifc primers Me-Mt-F/Me-Mt-R for the detection of M.enterolobii were developed to construct the duplex-PCR systems,which can detect single nematode.Meanwhile,based on the rDNA-ITS region and SCAR(Sequence characterized amplified region)fragment,the specific primers Mab-qF/Mab-qR and the probe Mab-Probe for detecting M.aberrans and the specific primers Me-qF/Me-qR and the probe Me-Probe for detecting M.enterolobii were designed respectively.Based on these primers and probes,the qPCR systems were developed.The qPCR assays could detect the DNA template 1000-fold diluted of a single nematode.In order to evaluate the application of the duplex and real-time PCR systems as diagnostic tool for the detections of M.aberrans and M.enterolobii,rhizosphere soil samples comprising root-knot nematodes and other nematodes were used.The results displayed that the qPCR system can successfully detect the target nematodes,M.aberrans and M.enterolobii,in all samples,but the duplex PCR can not detect the target nematodes in those samples including the low number of target nematodes.