| The entomopathogenic fungi Metarhizium and Beauveria have been developedinto different forms of fungal insecticides, and widely used in the biocontrol of insectpests, Currently, There are several fungal insecticides products have been developedto control locust, but the control effects were still unsatisfactory. Therefore, selectionof entogmopathogenic fungi with high virulence has been considered to be the basisof biological control of locusts. In this thesis, firstly, different genotypes of fungalstrains was classified through DGGE method. Secondly, for accurate identification ofthe strains, EF sequence-based PCR amplification and sequencing were conducted,and phylogenetic tree was constructed for each of the strains. Thirdly, virulence ofstrains was determined and selected through two bioassay methods of dipping andfeeding. Finally, biological characteristics of the selected highly pathogenic strainswere measured for good production traits. The main results were as follows:1. The molecular identification of strains.(1) Prelimary classification of the strains using DGGE method. the results ofDGGE profiles show that the tested27strains of Beauveria bassiana could beclassified to6genotypes, including A genotype: Bb854, B genotype: Bb1028, Bb6,Bb860, Bb125, C genotype: Bb292, Bb654, Bb1179, Bb2270, Bb321, Bb322, Bb458,Bb859, Bb1372, Bb1843, Bb2153, Bb2157, Bb2248, Bb2250, Bb2251, D genotype:Bb1519, Bb2253, Bb1906, Bb1975, E genotype: Bb1062, Bb2268, F genotype:Bb324.(2) Molecular identification of the fungal strains.6Beauveria spp. strains wereselected from the6genotypes, with one strain from every genotype. Together with theother4strains, totally10fungal strains were identified by molecular methods. DNAwas extracted respectively and PCR amplification was conducted using the primerpair of983F and2218R. The Amplified DNA was sequenced and blasted in GenBank,and homology among strains was analyzed. The other six genotypes are different.However, the six strains are all similar to the type strain of Beauveria bassiana intheir EF sequence from99%to100%. Therefore, the tested27strains could beidentified as Beauveria bassiana. Likewise, the other4strains of Vl325, Ic449, Bbr91and Ma63were identified as Lecanicillium psalliotae, Isaria cateniannulata,Beauveria brongniartii, and Metarhizium acridum. According to the results ofclassification and the spore production capacity of the fungal strains, the following strains were choosed for further bioassay experiments: Bb6, Bb860, Bb292, Bb654,Bb1179, Bb2270, Bb1519, Bb1906, Bb2253, Bbr91, Ma63.2. Locust breeding and Bioassay experiments.(1) Locust breeding. Locust eggs were hatched for6～12d, at30℃,50～75%RH, and12L:12D. The average hatching rate was65.0%, ranging from59.0to71.2%.The nymph of locust were raised at25～30℃,30～50%RH, and lighting12～16h.Average molting time was7~8d.27~36days were needed for the hatched nymphgrowing to third-instar for bioassay.(2) Bioassay experiments. The pathogenicity of eleven strains against the3thinstarnymphs of Locusta migratoria manilensis was bioassayed in laboratory byinsect-soaking method. Four Beauveria bassiana and one M. anisopliae strains withhigh mortality rate were chosen at the preliminary bioassay experiement. Thevirulences of the five strains against locust were in the order of Bb860＞Ma63＞Bb1906＞Bb2270＞Bb1519, with the corrected mortality of100%,93.3%,90%,86.7%,83.3%, and LT50of3.04d,4.17d,4.37d,4.50d,5.76d. Furtherly, fivestrains were choosed for bioassay with feeding method and the strains of Ma63,Bb860, Bb2270, Bb1906exibited highest virulence, with the LT50of4.81d,4.37d,5.00d and5.94d. When the strains were bioassayed by insect-soaking method, thevirulences of the five strains against locust were in the order of Bb860＜Ma63＜Bb1906＜Bb2270＜Bb1519, with the LC50of2.274×105conidia·ml-1,5.904×105conidia·ml-1,7.332×105conidia·ml-1,7.378×105conidia·ml-1,8.954×105conidia·ml-1.In the experiment of different temperature, the corrected mortality of Bb860, Bb1906,Bb2270, Ma63, Bb1519were75.9%,72.4%,69%,69%,55.2%, and LT50of6.77d,7.32d,7.45d,7.75d,9.44d at20℃. The corrected mortality of Bb860, Ma63,Bb1906, Bb2270, Bb1519were100%,100%,89.7%,89.7%,82.8%, with the LT50of3.13d,4.10d,4.53d,4.39d,5.87d at25℃。3. Biological characteristics of the selected strainsThe biological characteristics of the four strains of Ma63, Bb860, Bb1906,Bb2270were studied, including the colony shape, the shape of conidia, thesporogenous structure, the spore production quantity, the germination rate of sporesand the colony grow speed. the results showed that the colony shape of Bb860,Bb1906and Bb2270was loose, villiform, and conidia were single spore, nearlyspherical,1.5～2.8×1.5～2.4μm. The colonies of Bb860and Bb1906were white,and pale yellow at the reverse side. And the colonies of Bb2270were pale yellow, yellowish-brown at the reverse side. The strain Ma63was compact, villiform,single-spore, conidia3.8～5.3×2.4～3.3μm, the color of colony was yellow green,and at the reverse side. Spores production abilities of the strains were that Bb860＞Bb2270＞Ma63＞Bb1906, with2.8×107,2.3×107,2.0×107,1.3×107conidia/cm2,respectively. The germination rate were98.0%,96.7%,93.7%,96.0%respectively,and the daily growth rate on SDAY medium at25℃were18.36,14.94,14.66,14.89mm/d, respectively. |
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