Sequence Of Spheroidin Genes Of Grasshopper Entomopoxvirus And The Effect Of OaEPV Mixed With Metarhizium Anisopliae Or Chemical Insecticides On Grasshoppers

In this thesis, The complete nucleotide sequences of spheroidin genes of Calliptamus italicus entomopoxvirus (CiEPV), Gomphocerus sibiricus entomopoxvirus (GsEPV) and Dociostarus kraussi EPV (DkEPV) were determined. The enhancement of metarhizium inffection in grasshoppers by Oedaleus asialicus entomopoxvirus (OaEPV) was tested. The effect of 7 chemical insecticides mixing with OaEPV on Oedaleiis infernalis was studied. These works would impact the CiEPV. GsEPV. DkEPV and OaEPV to be used as biological control factor.The CiEPV, GsEPV and DkEPV spheroidin genes respectively harbored ORFs of 2922 bps, 2967 bps and 2922 bps. that were capable of coding polypeptides of 109.2, 111.1 and 109.2 kDa. Computer analysis indicated that CiEPV, GsEPV and DkEPV spheroidins shared less than 20% aa identities with lepidopteran AmEPV and coleopteran AcEPV spheroidins, but more than 80% aa identities with orthopteran OaEPV, MsEPV and AaEPV spheroidins. The CiEPV, GsEPV and DkEPV spheroidins respectively contained 19, 21 and 19 cysteine residues that were particularly abundant at the C-termini, as is the case with those of the other orthopteran EPV spheroidins. The promoter regions of the there spheroidin genes were highly conserved (99%) among the orthopteran EPVs and also contained the typical very A+T rich and TAAATG signal mediating transcription of poxvirus late genes. We also sequenced an incomplete ORF downstream of the pheroidin gene of CiEPV, GsEPV and DkEPV. The ORF was in the opposite direction to the spheroidin gene and was homologous to MSV072 putative protein of MsEPV.The mortalities of 3-instar nymphs of Oedaleiis infemalis and Locusta migraloria manilensis inoculated with the mixture of OaEPV (1.67 × 105OBs per larva)and metarhizium(1.67× 105 spores per larva)were higher than of those inoculated the metarhizium (1.67 × 105 spores per larva) alone. The rates of enhancement were 46.7% . 32.5% in the 7th day. In addition, the enhancing activity was relation to the does of OaEPV. The enhancing activity increased as the does of OaEPV increased. Oedaleiis asialicus entomopoxvirus contained a factor, which enhanced the infecton of Metarhizium flaroviride in the grasshopper, Oedaleus infemalis. When the spheroids of OaEPV dissolved with 2× SDS spheroid alkaline solution and centrifuged, the enhancing activity of the supernatant was 2.5 times greater than the pellet. The enhancing activity of spheroid proteins depended on the alkali used to dissolve the spheroids. The greatest activity occurred with spheroid alkaline solution, followed with 0.02 mol/L NaOH, and none with 8 mol/L urea. Spheroid proteins were fractionated on a sephadex G-200 column. Two fractions were obtained and the second fraction had the enhancing activity. SDS-PAGE indicated the molecular weight of the active fraction was 40 kDa.The LC50 of 7 chemical insecticides mixed with OaEPV on Oedaleiis infemalis are lower than that of the insecticides alone. The ratios of decrease of LC50 of Flufenoxuron and Chlorfluazuron are 49.0% and 42.8%.