| Lecanicillium lecanii is an important entomopathogenic fungus that is mainly used to control pests on greenhouse vegetables and flowers.Since the mid-1970s,biologists have been studying L.lecanii in depth.Up to now,not much research and development has been carried out in China on the preparation of mealybug formulations,and even less on the analysis of secondary metabolite activity and application compared to foreign countries.In this paper,the secondary metabolites were extracted and their effects on the enzymatic activities of peach aphid and mealybug moth were determined;then the differences in virulence of the three strains of L.lecanii were compared and analysed based on transcription-metabolomics to provide a reference basis for the production and application of L.lecanii.The main conclusions of the experiment are as follows.1.Effect of secondary metabolites of L.lecanii JMC-01 on the partial enzyme activities of the peach aphid and the small vegetable mothThe secondary metabolites of L.lecanii JMC-01 were extracted with ethyl acetate after 8 d of fermentation.The indoor toxicity of the secondary metabolites and the activity of detoxification enzymes(GST,Carboxylesterase(CarE)),Acetylcholinesterase(AchE)and Peroxidase(POD)in the test insects were measured using wingless peach aphid and third instar larvae.The results showed that JMC-01 secondary metabolites showed the highest virulence of 94.25%and 85.41%with LC50 of 37.62 mg·L-1 and 43.42 mg·L-1 against peach aphid and small vegetable moth,respectively;CarE activity reached 0.81 U·mgpror-1,AchE activity reached 0.1173 U·mgprot-1 and GST activity reached 22.52 U·mgprot-1 after treating aphids.GST activity reached 22.52 U·mgprot-1 and POD activity was 5.29 U·mgprot-1;after treatment with Chlorella,CarE,AchE,GST and POD activities reached 0.85 U·mgprot-1,0.1176 U·mgprot-1,27.5 U·mgprot-1 and 13.25 U·mgprot-1,respectively.2.Transcriptome-based analysis of differentially virulent strains of L.lecaniiIn this study,the transcriptome was de novo assembled by Trinity using the Illumina NovaSeq high-throughput sequencing platform,and a total of 88265 Unigene transcripts were obtained.225 differential genes were obtained between J-1_vs_J-2,of which 50 were up-regulated and 175 were down-regulated;2464 differential genes were obtained between J-1_vs_v-1,1253 up-regulated and 1211 down-regulated;3077differential genes between J-2_vs_v-1,1347 upregulated and 1730 down-regulated.GO annotation and KEGG enrichment analysis of differentially expressed genes showed that they were mainly associated with metabolic pathways,membranes and catalytic activity,all enriched with the most significant pathway being ABC transporter protein.3.Metabolome-based analysis of differentially virulent strains ofL.lecaniiIn this study,59 differential metabolites were identified in J-1_vs_J-2 and 75 differential metabolites were identified in J-1_vs_v-1 using broadly targeted metabolomics techniques.A search and annotation of the KEGG database and metabolic pathway enrichment revealed that Jl_vs_J-2 was enriched to a total of 43 metabolic pathways,of which eight more significant metabolic pathways were obtained at p<0.01,namely phenylalanine metabolism,tryptophan metabolism,unsaturated fatty acid biosynthesis,arachidonic acid metabolism,tyrosine metabolism,phenylalanine,tyrosine and tryptophan biosynthesis,and valine,leucine and isoleucine biosynthesis and ketone body synthesis;a total of 43 metabolic pathways were enriched in J-1_vs_v-1,of which 11 significantly different metabolic pathways were obtained at p<0.01,namely phenylalanine metabolism,tryptophan metabolism,arachidonic acid metabolism,tyrosine metabolism,phenylalanine,tyrosine and tryptophan biosynthesis,unsaturated fatty acid biosynthesis synthesis,aminoacyl-tRNA biosynthesis,nicotinic acid and nicotinamide metabolism,butyric acid metabolism,α-linoleic acid metabolism,and cyanamide metabolism.4.Combined transcription-metabolism-based analyses of virulence differences in L.lecaniiIn this study,three metabolic pathways were screened for significant enrichment.Thirteen differential metabolites were obtained in the phenylalanine metabolic pathway,of which nine were up-regulated and four were down-regulated,and six differential genes were obtained,of which two were up-regulated and four were down-regulated.And the genes with the largest differential ploidy with metabolites c75905.graph_c0 and salicylic acid were obtained;in the tyrosine metabolic pathway,differential genes and metabolites c70947.graph_c0 and 3Hydroxyphenylacetic acid were obtained;In the ABC transporter protein enrichment 64 differential genes were obtained,of which the ABCC1 subfamily The most enriched genes were the ABCC1 subfamily and the significantly different metabolite Methyl beta-D-galactoside.5.Study on the active components of the secondary metabolites of L.lecanii JMC-01In this study,the secondary metabolites of L.lecanii JMC-01 were isolated and purified by column chromatography and thin layer chromatography,and the activity was tested by microdrop method using peach aphid as the test insect.The two active substances were identified as dibutyl phthalate and oleic acid by 1H NMR,13C NMR and ESI-MS techniques and compared with literature.6.Formulation development and evaluation of dispersible oil dispersion of L.lecanii JMC-01 against aphidsThe formulation of two dispersible oil suspensions was optimized by screening suitable auxiliaries using the response surface method,and the quality standards were determined and the efficacy of the formulation against peach aphid was evaluated.The results showed that the two formulations with 5%(4.87×109 spores/g)mass fraction of spore powder,1#(mass fraction)of emulsifier(A:B=4:1)20%,calcium lignin sulfonate 1%,organic bentonite 3%,sodium fluorescein 1%,soybean oil supplemented to 100%,had the appearance of yellow-green oily liquid;2#of emulsifier(OP-10:Span 80=3:2)20%,Tween-80 1%,organic bentonite 3%,sodium fluorescein 1%,methyl oleate to 100%,its appearance is a light green liquid;1#and 2#OD of trash bacteria<5%and<3%,suspension rate of 93.3%and 93.6%,pH 6.74 and 6.85,after washing residue 0.62%and 0.23%,wet sieve test>98.9%and>99.12%and spore survival after six months storage:88.27%and 72.05%,both meeting national quality control standards.The indoor virulence of the two formulations against peach aphid increased with time,with mycelium-coated insects visible by the second day after treatment and mortality rates above 90%by the fifth day,with LC50 of 1.99×105 cfu/mL and 1.17×105 cfu/mL,respectively.Greenhouse efficacy increased significantly by the fifth day,reaching a maximum(96.92%)by the 14th day.The two oil dispersion formulations were of good quality,stable and had a good virulence effect on peach aphid. |
PDF Full Text Request