Adaptability Of Meloidogyne Hapla To Temperature And Inorganic Compounds

The northern root-knot nematode,Meloidogyne hapla,is one of the most important nematode pathogens occurring in cold regions,that causes severe economic losses.In this paper,we worked on the adaptability of M.hapla to temperature,inorganic compounds and pH.Moreover,the physiological and metabolic responses of M.hapla to temperature stress and inorganic compounds stress were studied.These provides a basal gist for ecological control of M.hapla.The results are as follows:1.Determination for the adaptability of M.hapla to temperature:The M.hapla J2 exposed to various temperature to test survival.Results showed that the 50%survival temperature(S50)of low temperature was-2.22?,and S50 of high temperature was 40.28?;Meanwhile,the lethal low temperature of egg-mass was-18?,and hatching rate of egg-mass was 13.00%at 41?;Moreover,cryoprotective dehydration,cold shock and cold acclimation had significant effects on freezing survival of M.hapla J2.This indicated that the cold tolerance of M.hapla was moderate,but heat tolerance was weak.2.Determination for the adaptability of M.hapla to inorganic compounds and pH:The effects of 8 inorganic compounds and different pH on M.hapla were studied.Results showed that M.hapla were sensitive to NH4HCO3,FeCl3·6H2O,CuCl2·2H2O and CuSO4·5H2O with LC50 of 5.96 mM?0.77 mM?0.16 mM and 0.16 mM,respectively.And the suitable pH range for survival of M.hapla J2 was 4-12 which indicated that the adaptability of M.hapla to pH was strong.And the sensitive compounds inhibited the hatching of egg-mass for M.hapla.3.Successfully obtained the full-length transcripts for M.hapla under temperature and development stage treatments:The full-length transcript of 70730 were obtain in M.hapla by Iso-seq technology,which of 61600 were annotated.The prediction for alternative splicing were 1243,SSR were 26906,complete CDS were 38787,and IncRNA were 3320 based on the final transcript.4.Determination for the suitable reference genes of M.hapla:Eleven candidate reference genes of M.hapla were selected to evaluate gene expression stability under different conditions using ACq,geNorm,Normfinder,BestKeeperd and RefFinder,then validated by Mh-Hsp90.Results showed that Mdh and RpS6 could be used as suitable reference genes for M.hapla,and Mdh plus RpS6 were better.5.Determination for the physiological and biochemical characteristics of M.hapla responded to the temperature stress:Detection for the content of hydrolyzed amino acids,combined with analysis for the activity and gene expression of key enzymes on proline synthesis pathway in M.hapla J2.Found that the enzyme activity of P5CS increased first and then decreased with the extension of 4? stress time,as well as the elevation for proline content and expressions of MhP5CS and MhProC,indicated that the accumulation of proline mainly depended on the synthesis pathway of P5CS and ProC for temperature stress.Detection for the related protein content and gene expression of calcium,PI3K-AKT and MAPK signaling pathway.Found that the ascent for CaM content,expression of MhCaM and MhCa2+ATPase under low temperature stress,indicated that the Ca2+were regulated by CaM and Ca2+ATPase to transduce stress signals in M.hapla J2.Moreover,the rise for 14-3-3 protein content and the expressions of Mhl4-3-3,MhAKT,MhHsp90(except-12? treatment)and MhHsp70(except-12? treatment),indicated that 14-3-3 protein and AKT in PI3K-AKT signaling pathway directly inhibited pro-apoptotic protein(BAD)and FOXO.Meantime,the AKT and Hsp70 inhibited JNK(c-Jun N-terminal kinase)in the MAPK signaling pathway,and then further blocked the apoptotic pathway.Results showed that MAPK-AKT signaling pathway inhibited both BAD and FOXO to maintain normal cell survival.6.Determination for the physiological and biochemical characteristics of M.hapla responded to the inorganic compound stress:Detection for the content of hydrolyzed amino acids,combined with analysis for the activity and gene expression of key enzymes on proline synthesis pathway in M.hapla J2.Found that the increase for OAT enzyme activity and expression of MhOAT indicated that proline accumulation depended on OAT synthesis pathway treated by FeCl3·6H2O and CuSO4·5H2O.However,the reduction for proline,but the increase for PDH enzyme activity and expression of MhPDH indicated that the proline degraded by treated with NH4HCO3Detection for the related protein content and gene expression of calcium,PI3K-AKT and MAPK signaling pathway.Found that the descent for CaM content,expression of MhCaM and MhCa2+ATPase that revealed the Ca2+did not depend on the regulation of CaM and Ca2+ATPase in M.hapla J2.Moreover,the ascent for 14-3-3 protein content and expressions of Mh14-3-3,MhAKT and MhHsp90 in PI3K-AKT signaling pathway,as well as expressions of MhAKT and MhHsp70 in MAPK signaling pathway,which showed that these two pathways cooperatively inhibited BAD and FOXO to maintain survival for cells under NH4HCO3 treatment.However,the BAD were inhibited by 14-3-3 proteins under CuSO4·5H2O and FeCl3·6H2O treatmentsResults show that the north root-knot nematodes had a certain cold tolerance,but weak heat tolerance,and were sensitive to NH4HCO3,FeCl3·6H2O,CuCl2·2H2O and CuSO4·5H2O.Moreover,the proline synthesis pathways,calcium and MAPK-AKT signaling pathways were activated to respond to temperature and inorganic compound stresses for maintaining the normal level of physiological and biochemical levels in M.hapla.