Virulence Variation And Chemotaxis Of Soybean Cyst Nematode Heterodera Glycines

Soybean cyst nematode?SCN,Heterodera glycines Ichinohe?is a serious soybean?Glycine max L.?pathogen worldwide.Effective but highly toxic chemical pesticides can no longer meet the needs of sustainable development,and more effective and non-toxic methods are in demand.Host plant resistance is the most cost-effective and environmentally benign strategy against SCN.However,pathogenic races in filed change frequently and new races occur continuously.Therefore,developing resistance sources against multiple pathogenic races are very important.In addition,due to limited resistant varieties,further research about infection mechanism especially detecting chemical signals in the early stages of the host-parasite interactions is hot spot.Disrupting nematode-host recognition is the effective way to prevent nematode infection.Further,identifying signals that nematodes perceive is the premises.SCN Race 3?HG Type 0-?had been a predominant race in Heilongjiang province,the largest soybean producing region in China.Recently,increased virulence on resistant cultivars originally developed for resistance to SCN Race 3 was observed in fields.In order to identify new cultivars resistant to local SCN populations,four soil samples were collected from four counties?Harbin,Daqin,Anda and Wuchang?and single-cyst cultures?Anda and Wuchang?were maintained for more than five generations.Then 18 soybean genotypes were used to evaluate resistance response to the three identified pathogenic races.To further explore the signals attracting SCN to host roots,the important factors in the plant rhizosphere including acid,alkali,salts and amino acids secreted by plant and/or microbes were screened and dosage effect was considered.Nematodes were exposing to acetic acid,NaOH,salts and amino acids by using the 23%Pluronic F-127 gel system.And then the SCN transcriptome between the treatment and control was conducted through RNA-seq to identify the differentially expressed genes.The possible nematode genes involving in chemotaxis to acid,alkali and salts were identified.The results obtained from the study of virulence variation and chemotaxis of soybean cyst nematode are as follows:SCN races changed in Harbin,Daqing,Anda and Wuchang,indicating virulence variation.Soil samples from Harbin and Daqing were identified as SCN Race 6,Anda as Races 4 and Wuchang as Race 5.And two single-cyst cultures from the Anda sample were identified as Race 4?HG Type 1.2.3.5.6.7?and Race 14?HG Type 1.3?,and one single-cyst culture from Wuchang was identified as Race 5?HG Type 2.5.7?.Various levels of resistance or susceptibility to the three SCN populations were observed among18 genotypes.Both‘Kangxian12'and‘Kangxian13'showed resistance or moderate resistance to HG Type 2.5.7,HG Type 1.2.3.5.6.7 and HG Type 1.3.The breeding line‘09-138'was resistant to HG Type 1.3 and HG Type 1.2.3.5.6.7.Cultivars with‘Peking'-resistance were resistant or moderately resistant to HG Type 2.5.7 except for‘Kangxian8'.SCN showed attraction to HAc,NaOH,salts and amino acids with dosage effect.The optimal acidic pH range of SCN is 4.98⁵.46,while the alkaline pH ranges were8.40⁸.78 and 9.52⁹.99 for nematode attraction.The optimum concentrations of Na⁺and Cl⁺were 85.24²21.52 mM and 170.89²55.87 mM,respectively.Except 2.5 M KNO₃ and 100 mM histidine,all the tested inorganic salts and amino acids attracted SCN.The optimal concentration of aspartic and arginine were 20 mM and 100 mM in vitro,respectively.High concentrations of aspartic attracted nematodes to specific areas and killed the nematodes.A total of 37119 unigenes with annotation information were obtained by transcriptome analyses of nematode chemotaxis induced by acid,alkali and salt.The total 47?198 and 143 differentially expressed unigenes were found for nematode attraction to NaCl,HAc and NaOH,respectively.The Venn diagram shows that four differentially expressed genes shared in nematode chemotaxis to NaCl,HAc and NaOH.In addition,there were 7,10 and 48 differentially expressed genes shared between NaCl and HAc,NaCl and NaOH,HAc and NaOH,respectively.Finally based on the results of functional annotation,superoxide dismutase,cation transporting ATPase,annexin and phospholipid-transporting ATPase,ligand-gated ion channel and CBN-RSKN-2protein may be associated with nematode attraction.The reliability of transcriptome was verified by qRT-PCR.In summary,the findings suggest that the pathogenic races of soybean cyst nematode have been changed in the collection places of Heilongjiang province.Increased virulence on resistant cultivars was observed in fields and the resistance of most resistant varieties was reduced or lost,only‘Kangxian 12'and‘Kangxian 13'presented resistance/moderate resistance to all the three SCN populations.The studies would provide important theoretical foundation and applied value not only for SCN management but also for the SCN resistance breeding program against multiple SCN populations.It will provides valuable theoretical foundation for the study of nematode evolution.The studies of nematode chemotaxis determined nematode perceiving signals to locate the host,and the optimum dosage effect was detected for the first time.Through differential expression of RNA-seq,genes associated with nematode attraction to acid,alkali and salts were identified and further functional verification of these genes are ongoing.The understanding of nematode-host early interaction will lay a solid theoretical foundation for establishing new efficient,non-toxic and environmentally friendly control strategies.