| Fungi are an important class of entomopathogenic microorganisms,up to more than 400entomopathogenic fungi have been reported so far,and more than 200 pathogenic fungi that parasitize insects.Entomopathogenic fungi invade into the host body mainly via the insect body wall and can sometimes also be infected via the respiratory and digestive tracts.In the invasion of host pests by entomopathogenic fungi,hosts also mount stress responses through auto humoral immunity and cellular immunity,which provide defense against fungal invasion.Epidemics caused by fungi in nature can naturally constrain the population size of insects,so the use of entomopathogenic fungi for biological control has been favored and valued.Simplicillium species belong to the family Cordycipitaceae of the order Hypocreales and is a class of endoparasitic fungi that play a very important role in biological control and application of biologically active compounds.Simplicillium species are widely distributed in natural ecosystems,they can usually parasitize plant tissues,soil,nematodes and mushrooms.Currently there are more than ten species in Simplicillium,among which S.lanosoniveum is considered as plant symbiotic fungi and insect pathogenic fungi,especially as insect pathogenic fungi used in biological control,which has high economic and ecological value.In this study,firstly,S.lanosoniveum was isolated and identified,and the dominant virulence strains were selected.Then explore its pathogenicity to insects and biological characteristics.Finally,measure the changes of protective enzyme activity of 3rd instar of Bombyx mori which infected with highly pathogenic strains.The main tests and results are as follows:1.Isolation and identification of S.lanosoniveum and screening the dominant virulent strainSix fungal strains were isolated and purified from the soil by selective culture method and were morphologically preliminarily identified as Simplicillium genus.The ribosomal ITS sequences of six fungal strains were amplified,cloned and sequenced by PCR,and the sequence length was 616 bp.Sequences from the NCBI database were searched for comparison and phylogenetic trees were constructed,and the results showed that the six obtained fungi were located in the same branch as the registered S.lanosoniveum(Gene Bank NO.KT878334 and MT001191),with 99.9%～100%similarity.Then the dominant virulent strain was selected by spore impregnation method by infesting the 3rd instar of B.mori with a concentration of 1×10~8spores/m L(Beauveria bassiana and Metarhizium anisopliae positive control groups).The results showed that the highest cumulative corrected mortality rate was92.30%and the lowest 30.78%for each treatment.Among them,the cumulative corrected mortality of S.lanosoniveum-Sl05 and Sl06 strains after 7 days of inoculation were 84.62%and 89.29%respectively,and the median mortality was 3.96 days and 3.73 days respectively,with the strongest pathogenicity.Therefore,the two dominant virulence strains were selected for follow-up study.2.The study of the pathogenicity of S.lanosoniveum-Sl05 and Sl06 against insectsThe 3rd instar of B.mori was infected with S.lanosoniveum-Sl05 and Sl06 strains with different concentration gradients.The pathogenicity of the strains to B.mori was analyzed by time-dose-mortality model(TDM model).The results showed that the cumulative mortality of3rd instar of B.mori increased with the increase of spore concentration.The lethal concentration and lethal time of S.lanosoniveum-Sl06 were smaller than S.lanosoniveum-Sl05 under the same spore concentration treatment,indicating that S.lanosoniveum-Sl06 had higher pathogenicity to B.mori.Strains S.lanosoniveum-Sl05 and S.lanosoniveum-Sl06 were applied to the 5th instar of B.mori and observe the process of holometamorphosis.The results showed that with the increase of spore concentration,the mortality of B.mori increased and the pupation rate decreased.Further observation showed that the B.mori that had completed cocooning could not complete the development from pupae to moths under the influence of S.lanosoniveum as well as the B.bassiana treatment group,indicating that the S.lanosoniveum affected the process of holometamorphosis of B.mori.At the same time,S.lanosoniveum-Sl05 and S.lanosoniveum-Sl06 were applied to Menopon gallinae for in vitro parasiticide test,and it was found that with the spore concentration increasing,the survival time of M.gallinae in vitro was shorter.These results suggested that the S.lanosoniveum isolates show potential as biological control agents for insects.3.The study of the biological properties of S.lanosoniveum-Sl06Five commonly used fungal media were selected for screening the optimum medium for S.lanosoniveum-Sl06,and the results showed that the strain grew best on PDA medium,with colony diameter up to 65.52 mm,average growth rate was 4.49 mm/d and spore production reach 2.92×10~8spores/cm~2.The strain grew best at 27℃,with colony diameter up to 66.87mm,average growth rate was 4.35 mm/d and the spore production reach2.83×10~8spores/cm~2.The strain showed the highest growth data at p H 6.0,with colony diameter up to 68.40 mm,average growth rate reach 4.47 mm/d and the spore production was 3.12×10~8spores/cm~2.Carbon source screening revealed that the strain had good utilization of glucose and lactose,while the most suitable nitrogen source was ammonium sulfate.The strain grew up to 64.43mm in diameter at 15 d in plate culture,and growth rate reach 4.10 mm/d and sporulation rate was 3.62×10~8spores/cm~2.4.Effects of S.lanosoniveum-Sl06 infection on the activity of protective enzymes in the3rd instar of B.moriTo investigate the pathogenic effect of S.lanosoniveum-Sl06 on the 3rd instar of B.mori,the 3rd instar of B.mori was treated with a suspension at a concentration of 1×10~8spores/m L using the maceration method,and measure the activity changes of superoxide dismutase(SOD),catalase(CAT)and peroxidase(POD)of 3rd instar of B.mori every 10 h using the absorbance method of the kit.The results showed that the activities of the three protective enzymes all showed rising at first and then falling with time passing.SOD plays the first role in the fungal infection process,and POD mainly plays a role in the late stage of infection,and the decreasing trend of CAT activity in the late stage of infection is more obvious,while the decreasing trend of SDO and POD activity is relatively gentle.These results illustrate that the protective enzymes that play a major role during the infection of B.mori by S.lanosoniveum-Sl06 are mainly SOD and POD,while CAT plays a secondary role. |
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