Species Distribution Of Root-knot Nematodes In Northeast China And Analysis Of Chloride Channel Genes In Meloidogyne Incognita

Root-knot nematode(RKN)is a kind of plant parasitic nematodes distributed all over the world,which seriously threatens agricultural production.There are many species of RKNs in China,most of which are distributed in the southern region of China.There is no comprehensive investigation of RKNs in the northeast of China.Therefore,this study systematically investigated species and distribution of RKNs in the three provinces of the northeast China.The results were as follows:In 2018 and 2019,174 samples were collected in Liaoning,Jilin and Heilongjiang provinces,and RKNs were found in 56 samples.Three specieses of RKNs were identified by morphological observation,sequence analysis of r DNA-ITS and r DNA-SSU,and sequence characterized amplification region markers,namely Meloidogyne incognita,M.hapla and M.arenaria.Among of them,M.incognita was found in greenhouse vegetables in the three provinces,which was the dominant species in greenhouses.It was distributed in all protected vegetable bases of Liaoning province,but only distributed in greenhouse vegetable base of Changchun city and Songyuan city of Jilin province,and Daqing city of Heilongjiang province.The main hosts of M.incognita were tomato,eggplant and cucumber.Here,M.incognita had been found new distribution in greenhouses of Fushun city,Liaoyang city,Dandong city and Panjin city of Liaoning province,as well as in Songyuan city of Jilin province.The results indicated that M.incognita had spread to all protected vegetable bases of Liaoning province,then extended to Jilin province.Meanwhile,M.hapla was found in greenhouses in Dandong city and Jinzhou city of Liaoning province,as well as in field vegetables and plants in Fushun city,Huludao city and Shenyang city.And M.hapla was the dominant species in the fields.That was first report that M.hapla could infect strawbarry in greenhouse in Dandong city,as well as infect eggplant in the field in Fushun city.We also first discovered that M.arenaria infected open field peanuts in Liaozhong county,Shenyang city,and this indicated that M.arenaria had also spread to the northeast of China and survived through the frigid winter.ITS and SSU sequences of M.incognita were used to construct phylogenetic tree analysis.The results showed that the nematodes of M.incognita in Liaoning province were generally divided into two branches: the nematode populations of M.incognita in Shenyang,Tieling,Fushun,Benxi,Anshan,Yingkou,Dandong and Dalian cities were classified as one branch,and another in Jinzhou,Fuxin,Panjin,Chaoyang and Huludao cities was classified as additional branch.M.incognita populations in Changchun and Songyuan of Jilin province and Daqing in Heilongjiang province were closely related to that of Tieling and Shenyang in Liaoning province.It was speculated that the M.incognita in Jilin province and Heilongjiang province maight have spread from Liaoning province.Resistant populations of M.incognita to Avermectin was collected from the greenhouse vegetable in Dalian city,Liaoning province.The resistance of resistant populations was 5.13 times higher than that of the sensitive populations.Glu Cl(glutamate-gated chloride channel)is the target of avermectin acting on nematodes.The primers were designed and cloned and sequenced to obtain the 1259 bp Glu Cl gene in M.incognita.The two populations of Glu Cl genes were obtained by cloning and sequencing.Nucleic acid and amino acid alignment of the Glu Cl genes of the two populations showed that 18 synonymous mutations and 2 nonsynonymous mutations occurred in the base of the resistant populations.Amino acid alignment showed that aspartic acid was deleted at position 22 and lysine was replaced by glutamine at position 110(Q110K)in the resistant populations.Through the prediction of secondary structure of Glu Cl protein,it was found obviously difference that the number of ? helix in secondary structure in the resistant populations was more than that of sensitive populations,and the number of extension,also the random coil was relatively less.The tertiary structure of Glu Cl protein was predicted that the mutation point of the resistant populations was located at Q110 K site outside the Glu Cl subunit pentamer,which may be related to the resistance to Avermectin.In this study,3 species of RKNs in Northeast China were identified,mainly distributed in all protected vegetable bases of Liaoning province,while a few also distributed in greenhouses of Jilin province and Heilongjiang province.We also found M.arenaria of peanuts in Liaoning province.The resistant populations of M.incognita to Avermectin was found,and the gene sequence,amino acid and protein structure changes of Glu Cl were analyzed.The research results provide important data for guiding the prevention and control of RKNs in northeast China,especially for the detection of drug-resistant nematode populations.