Dynamics,variety Resistance And Invasion Mechanisms Of Rice Root-knot Nematode In Hainan

Rice root-knot nematodes are widely distributed in South Asia,Southeast Asia and North America.Meloidogyne graminicola are one of the most representative species and are considered to be the most important plant parasitic nematodes affecting rice yield and quality.In the past decade,the occurrence area of root-knot nematodes in main rice planting areas in China has been expanding year by year,which is probably being neglected due to its specific infecting and damaging way.Besides,lacking of control measures in production is also one of the reasons,with the result that serious loss frequently happens in some areas.Hence,rice root-knot nematode in Hainan was systematically studied and the main results are presented as follows:1.Occurrence dynamics and species identification of rice root-knot nematode in HainanThrough investigation in 16 cities and counties,rice root-knot nematodes in southern cities are more serious than those in northern cities generally,and the accumulation of root-knot nematodes is more there.Old disease centers such as Dingan,Qiongzhong and Sanya still have higher accumulation of root-knot nematodes amounts.The occurrence of seedling stage and tillering stage was slight in most of the investigated fields.Whereas the incidence and root knot index in the stubble stage increased hugely.The reason is that the accumulation of root-knot nematodes amounts in previous crops,and also the moisture and temperature of the soil and the number of new roots produced at the rice stubble stage are related.The most critical rice stump stage,taken as the accumulation and infection cycle of root-knot nematodes in the whole growing season,determines the amount of eggs in the soil.The rice-rice-vegetable planting pattern not only provides good host conditions for rice root-knot nematodes,but also controls the population size and achieves a certain ecological balance.Based on morphological characteristics,perineal pattern and molecular biological identification,the dominant pathogenic population of rice root-knot nematode in Hainan is identified as M.graminicola.2.Evaluation of rice varieties resistance to M.graminicolaThe comprehensive resistance of 21 conventional rice varieties was evaluated in the way of combining single index with comprehensive index.Three varieties(Huaxiang,Lujinzhan and Xiufengzhan5)with higher comprehensive resistance and three varieties(Boyou 225,Haixiuzhan 9 and L671)with high susceptibility to M.graminicola were screened out.Haifengnuo 1 was defined to be highly susceptible with root knot index,while the relative height of plant growth and aboveground fresh weight did not change much,it was finally defined to be a medium-level susceptible variety according to the comprehensive evaluation results of membership function.The rice varieties Haifengnuo 1,Hainonghong 1,L684,Xiufeng 5,L639 and L675 are quite different in the root knot coefficient and root knot index,namely,the root knot index of Hainonghong 1,L639 and L675 is higher,and the root knot coefficients of Haifengnuo 1,L684 and Xiufeng 5 are larger,indicating that disease resistance of rice varieties cannot be determined by single index.3.Cloning and function analysis of flp gene of M.graminicolaIn this research,two fragments of Mg-flp-1(Gen Bank accession No.MK886770)and Mg-flp-13(Gen Bank accession No.MK886771)of nematode amide polypeptide gene family were cloned,493 bp and 269 bp respectively.After ORF finder analysis,the cloned Mg-flp-1 contained a 315 bp ORF open reading frame,encoding 105 amino acids.Hydrophobic analysis of the amino acid sequence of the ORF open reading frame of Mg-flp-1 showed that the protein encoded by the ORF might be hydrophilic.The results of transmembrane domain analysis showed that the protein had no transmembrane domain and might be an extramembrane protein.Signal IP4.1 was used to predict signal peptide of the amino acid sequence of flp gene.It was found that the amino acid sequence encoded by Mg-flp-1 gene had no signal peptide.Situ hybridization technique was employed to locate the Mg-flp-1 and Mg-flp-13 genes of nematode.It was found that Mg-flp-1 and Mg-flp-13 genes were expressed in nematode ring and pharyngeal nerve ring of nematode.It was speculated that the gene might be related to the feeding behavior and swallowing activity of nematode.Mg-flp-1 gene is expressed in the head of nematode,speculating that this gene may be expressed in part of neurons in the head of nematode,thus controlling the infection of nematode to host plants and the secretion and feeding behavior of oral needle after infection.Mg-flp-13 gene was expressed near the basal bulb of nematode's mouth needle,which indicated that the gene was related to nematode's mouth needle activity or feeding.Meanwhile,the Mg-flp-1 gene was silenced by immersing with ds RNA synthesized in vitro.Semi-quantitative results showed that there was no silencing effect,which might be related to the inadequate accuracy of semi-quantitative detection.4.Proteomics analysis of rice root induced by M.graminicolaIn this research,i TRAQ technique was used to identify and analyze the root proteome of susceptible rice variety Boyou 225 root inoculated with M.graminicola after 3,7,15and 30 days respectively,and the difference was compared with that of rice root without inoculated at the same time.The results of proteomics analysis showed that the relative molecular weights of the identified proteins were different,most of which were concentrated between 20 and 70 k Da.Pathway analysis showed that 815 proteins were annotated on 114 pathways,including phenylpropanoid biosynthesis,plant-pathogen interaction and plant hormone signal transduction related to plant disease resistance.Statistical analysis of differentially expressed proteins showed that a certain number of differentially expressed proteins were obtained at each stage.The maximum number of differentially expressed proteins was 412 at 7 days after inoculation,of which 314 were up-regulated and 98 were down-regulated.5.Effectiveness evaluation of nematicide against rice root-knot nematodeIn order to screen out effective nematicides for M.graminicola,five nematicides were selected to test the virulence in laboratory and field.The results of toxicity test revealed that both 41.7%fluopyram SC and 1.8%abamectin EC showed high toxicity to M.graminicola.The LC₅₀ of 41.7%fluopyram SC to M.graminicola was 0.06106 mg/L and 0.00815 mg/L within 24 h and 48 h respectively;The LC₅₀of 1.8%abamectin EC was0.05078 mg/L and 0.01025 mg/L respectively to M.graminicola.The results of field experiment revealed that 41.7%fluopyram SC,20%fosthiazate EW,35%metham-sodium AS and 98%dazomet MG had good control effect.They could control the number of J2 in soil and effectively inhibit the formation of root knot;35%metham-sodium AS and 98%dazomet MG are fumigant and thus are suitable to be used in high temperature environment in Hainan;1.8%abamectin EC showed high toxicity in the laboratory,yet the control effect in the field was ordinary.