Entomopathogenic Nematodes Resources Of Gansu Province And Its Adaptation To Abiotic Stress

Based on the analysis of entomopathogenic nematodes circumscription in Gansu province, thelocal EPN s infectivity against grubs（Holotrichia oblita Fald and Melolntha hippocastanica Menetries）were conducted by bioassays and field experiments in Linxia natural pasture.Under the background ofabiotic stress conditions, the paramenters of biological and ecological characteristics were measured,combined with measurement of contents of energy material such as protein, lipid and sugar, andadaptation mechanism of entomopathogenic nematodes to abiotic stress were discussed. The mainresearch results and conclusions were as follows:1. Soil samples were collected from different areas in Gansu Province and the entomopathogenicnematodes (EPN) were extracted with Galleria mellonella trapping method in order to clare the speciesand distribution in this areas.Combined with the vegetation and soil type relationship between EPN sdistribution, the relationship between nematode distribution and soil,habitat were analyzed, the results asfollow:EPN resources were relatively rich for the arid climate in Gansu Province,51populationsbelong to8species were isolated from soil samples,among which5species are Steinernema, while3species are Heterorhabditis. The result showed that3species of Steinernema (S. affine、S. krussei、S.karil) and1species of Heterorhabditis (H. marelatu) are new species in China.The dominant species isSteinernema feltiae,and distribution of Steinernema is relatively widely than Heterorhabditis in Gansuprovince.It was determined that occurrence of EPN was closely related to vegetation and soil types. Theprevalence of EPN in silty-loam and loam were higher than that in clay.2. Using indigenous isolates of EPN from Gansu,the bioassays and field experiments were conductedin laboratory and meadows against grubs（Holotrichia oblita Fald and Melolntha hippocastanicaMenetries）,the results showed all isolates showed infectivity to H.diomphalia,especially Steinernemafeltiae0619HT and Heterorhabditis megidis0627M. In pot experiments, S.feltiae0619HT killed thehost within a short time while H.megidis0627M caused greater mortality as time goes on,and the rightdoes of EPN in assay about it against H.oblita is3200IJs/H.Similar results were observed in fieldexperiments2weeks after nematode application,the revision insects reduced rate of Melolnthahippocastanica in which used S. feltiae0619HT and H. megidis0627M field were83.8%and79.5%respectively. These experiments overall suggest S. feltiae0619HT and H. megidis0627M to be apromising biocontrol agent against grubs.3. In order to eliminate influence of abiotic stress on entomopathogenic nematodes biological andbehavioristics,UV-B radiation,thermobiosis and anhydrobiosis simulation experiments were conducted.Life span,pathogenicity and migration ability were analyzed in different simulation abiotic stressexperiments, the results showed that:3.1Entomopathogenic nematodes isolated from Gansu province has a certain tolerance to UV-Bradiation.UV-B radiation had a little effects on its survival under20W intensity for30min,but as theradiation time increases, the EPN s survival rate will decline,and significantly decreased after120minradiation. When the time reaches6hours, EPN s mortality rate reached100%.3.2UV-B radiation has influence on pathogenicity of entomopathogenic nematodes, and there havedifference among different species and different strains of the same species. After UV-B radiation,thepathogenicity of H.brevicaudis0641TY,H.megidis0627M,S.krussei0663HT, S.krussei0657L,S. felitiae0661YM,S. felitiae0619HT is reduced,while that of S. felitiae0622L has no significant difference,andwhat was shocking those of Steinernema felitiae0639C and0663TD were increased.And what s more,the offspring of S.felitiae0663TD after UV-B radiation treatment,whether ultraviolet radiation again,itspathogenicity is higher than either parent.Although there were no significant difference, in supplementedUV-B treatment, Steinernema felitiae0663TD s reproduction had a lower trend than control, indicated that UV-B radiation had no effect on reproduction of entomopathogenic nematodes.UV B radiation hason influence on offspring s reproduction,while with the radiation time increases, their reproductionreduced.3.3UV-B radiation and sunlight had no effect on EPN s tropism for host insects, but had effects on itsmigration ability.H.megidis0627M and S.krussei0657L s movement reduced after UV-B radiation,whileS.felitiae0619HT s has little change,and its migration ability is stronger than others.If there has the hostinsect,their movement would been stimulated,during the test time, UV-B radiation time had no effect ontheir migration ability.With UV-B radiation effects on pathogenicity and the ability to migrate,EPN’smigration ability changes was the main factor of UV-B radiation influence on its pathogenicitychanges.3.4Thermobiosis influenced entomopathogenic nematodes infection,and various biological processes ofEPN are similarly influenced by temperature.EPN lost its pathogenicity to Galleriam ellonella L whentemperature at35℃.The thermal niche breadths of H. megidis0627M is narrower, so high temperatureand low temperature influenced its pathogenicity,while S. feltiae0619HT has broad temperature range,so its adaptability to high and low temperature is higher.whereas S. kraussei0657L s temperaturerange tend to high temperature,its adaptability to high temperature is relatively higher than that to lowtemperature.3.5Differences in the desiccation tolerance among different species and strains of entomopathogenicnematodes have been demonstrate,and infective juveniles of Steinernema.spp were more tolerant ofdesiccation than those of Heterorhabditis.spp, S.feltiae were more tolerant to rapid desiccation thanother species tested.Drought-stress have influence on survival and penetration of entomopathogenicnematodes,but have little influence on pathogenicity.The survival rate of S.feltiae0619HT,S. bicornutum0631PG, S.affine0619M,S.kraussei0657L,S.karii0663YC were higher than that of H.megidis0627M,H.brevicaudis0641TY and H.marelatu0639G s after desiccation.Although the penetration rate of5Steinernema species to Galleriam ellonella L have varying degrees of decline, only S. affine0619M sfatality rate get down, the rest have little change.4. The colorimetric analysis method were used to study the acclimation and response of EPN to UV-Bradiation, drought-stress, adaptation and dissipation characteristics of energy reserves on the UV-Bradiation, drought-stress were discussed. The results showed that:4.1All kinds of material content from large to small in turn is lipid,protein and sugar,lipid is majorenergy reserves of entomopathogenic nematodes.Neutral fat are minority of the total lipids, and thesoluble sugar content> trehalose content> glycogen content.The amount varies with nematodespecies,about the tested strains of nematodes, Heterorhabditis megidis0627M has total protein contentand soluble sugar,while Steinernema feltiae0663TD s neutral lipids content and total lipids content arehighest.4.2The EPN’s resistance to UV-B radiation is depending on its total lipid content and ability ofprotein accumulation under UV-B stress. UV-B radiation caused the energy reserve changes, anddegree varies with nematode species or strain. UV-B radiation influence the protein and lipids content ofSteinernema feltiae0663TD strongly, that mean the protein content increased, and neutral lipids contentincreased with the increase of UV B radiation time, and higher than other strains,while total lipidscontent reduced but has been higher than the other strains.The soluble sugar content of Heterorhabditismegidis0627M,Steinernema kraussei0622L,Steinernema feltiae0663TD can been increased by UV-Bradiation,and gaining different addition under different radiation length. All of those showed there have the correlation between the nematodes resistance to UV-B radiation and its lipids content; proteins mightprovide extra muscle for locomotion and infection for as long as possible prior to be used as an energysource,so under the UV-B stress protein accumulation ability determines IJs ability of migration, thusaffecting the the pathogenicity of EPN under UV-B radiation.4.3UV-B radiation not only have influence on parental s substance content, but also has effect on F1andF2generation s. After radiation, the protein, neutral lipids, polarity, glycogen and soluble sugar contentof F1generation have little change, and F2were elevated and has nothing to do with radiation again; UV-B radiation length has impact to the nematodes offspring s trehalose content, the trehalose of F1and F2IJs(except for30min)gradually accumulated as the radiation time increases.4.4Nematode’s resistance to anhydrobiosisi is depending on the accumulation of lipid and solublesugar under stress,the stronger ability of accumulating,the stronger resistance toanhydrobiosisi.The results also showed that low humidity stress can cause Heterorhabditis megidis0627M,Steinernema kraussei0622L and Steinernema feltiae0663TD s protein, polarity, neutral lipids,soluble sugar content increased.4.5The trehalose accumulation under abiotic stresses research showed that UV-B radiation causednematode trehalose accumulation, while low humidity stress caused trehalose content reduce.