| The citrus long-horned beetle,Anoplophora chinensis(Forster),is major trunk pest infected Casuarina coastal shelter forest in southeast coastal areas,and is also one of national dangerous forest pests,which has a wide range of hosts.Its larva feeds on the ground trunk of hosts and damages the transport of nutrient and water to the trees.That will weak the tree vigor,even make trees die.A.chinensis is widespread in the Casuarina coastal shelter forest in Fujian province.Average ratio of infested trees in Casuarina forest can reach 20~30%,the worst-hit parts even can achieve over 80%,which results in weak efficiency of shelter forest.The emergence of adult A.chinensis usually lasts a long time and the larva feeds on the tree trunk,therefore,chemical control is not a high effective or an environment friendly method to A.Chinensis.Metarhizium spp.is one of the most widely-used entomogenous fungi.It has good spreading effect,long time survival in the forest and no resistant reaction of host insect.Futhermore,it is quite easy to produce.Therefore,Metarhizium spp.play an important role in the pest control of agriculture and forestry.In this paper,artificial diets for rearing A.chinensis larva and relevant technologies were studied in the laboratory,which was beneficial to provide abundant standardized larvae for other researches.Then pathogenicity of different Metarhizium strains against A.chinensis larvae were tested,and the conditions for solid-state fermentation of Metarhizium were optimized.Furthermore,effects of different Metarhizium preparations for controlling A.chinensis were tested in the field.Meanwhile,the transcriptomes were compared among A.chinensis larvae infected by M.anisopliae strain MaZPTR-01 with different incubation time and control larvae by use of RNA-Seq technology.The immune response of A.chinensis larvae against infection by M.anisopliae was analysed.The main results are as follows:1.Effects of four kinds of artificial diets and different temperature regimes on the development of A.chinensis was compared through three successive generations(three years).The results showed that A.chinensis could complete life cycle on all tested diets,adults resulting from the artificial diets could mate and lay eggs as usuall,then the hatched larvae could also complete life cycle on artificial diets.But the pupation and emergence rhythm were different among the indicviduls from different diets.Diet A and Diet D based on sawdust of C.equisetifolia and wheat bran had better performance.Feeding with Diet A,pupation rates of three different generations were 100%,90% and 95%,respectively,and emergence rates were 100%,83% and 90%,respectively.As to Diet D,pupation rates were 87%,78% and 87% and emergence rates were 87%,67% and 80%,the pupation and emergence rates were significantly lower than that from Diet B and C.At room temperature,the pupation rate and the emergence rate were higher and more synchronized than those at constant temperature.When rearing at room temperature,the duration of larvae pupation and that of adult emergence were something like those in the field and almost all individuals completed development between April and May.These results showed that fluctuating temperature had important influences on the development of A.chinensis larvae.A larval chill period does increase the pupation rate and developmental synchronization.2.Colony growth and sporulation of different M.anisopliae strains and their pathogenicity to Anoplophora chinensis larvae were investigated in laboratory.Results showed that colony growth and sporulation of different strains were significantly different.Colony diameter of MaZPTR-01 was 5.9 cm and its spore yield reached 1.1×108 spores/cm2 15 days after inoculated on PPDA medium.Bioassay results showed that pathogenicity of different M.anisopliae strains were significantly different,the mortality rates of the larvae varied from 40% to 96.7%,20 days after inoculation with sporulation suspension.The mortality rate and muscardine cadaver rate of the larvae inoculated with MaZPTR-01 were 96.7% and 86.7%,respectively,which were higher than those inoculated with other strains.The median lethal time(LT50)of MaZPTR-01 treatment was 5.71 days,which was shorter than other strain treatments.Overall,MaZPTR-01 was an excellent strain with high pathogenicity against A.chinensis larvae.3.Pathogenicity of MaZPTR-01 against A.chinensis larvae was evaluated by using timedose-mortality(TDM)model.The results showed that after treatment with different concentrations spore suspensions,the mortality of A.chinensis larvae increased with time extending and the increasement of suspension concentration(spore content).The mortality of the larvae reached 100% on the 7th and the 9th days after inoculated with 1.0×109 and 1.0×108 spores/mL,respectively.The mortality was 93.3%,70.0% and 56.7%,respectively on the 15 days after inoculated with 1.0×107,1.0×106,1.0×105 spores/m L.These experimental results were analyzed by using the time-dose-mortality model.Afterthat,the lethal dose and lethal time of MaZPTR-01 against A.chinensis larvae were evaluated.as The LD50 and LD90 decreased with inoculation time extending,which implied the dose effect became stronger with time expand.On the other hand,the LT50 decreased with increasement of suspension concentrations,it meant the time effect became stronger.The pathogenicity of MaZPTR-01 varied among different-instar A.chinensis larvae.On the whole,the death rate,the LT50 and the LT90 of MaZPTR-01 against different-instar A.chinensis larvae were getting lower with the instar increasment.Values of the LT50 corresponding to the newly-hatched(1st instar larvae without feeding),1st,2nd,3rd and 4th instar larvae were 2.46 d,4.53 d,4.39 d,5.82 d and 8.92 d,respectively.It showed that MaZPTR-01 strain had high pathogenicity against the 1st,2nd and 3rd instar larva.5.The condition optimization for solid-state fermentation of MaZPTR-01 strain showed that the optimized medium was composed of corn flour(1 portion,volume ratio,the same below),wheat bran(3 portions),rice flour(1 portion),rice hull(3 portions).The conidial production in the treatment which the medium was sterilized after adding water was higher than the treatment which the solid-state medium was sterilized,then adding sterile water.The medium with 5cmthickness composed of 500 g solid medium and 400 g water had the highest sporulation.Field tests for controlling A.chinensis by using Ma ZPTR-01 showed that 13 days after adults were contacted with the spores on fungi strips,the mortality of A.chinensis adults reached 100%,the median lethal time(LT50)was 5.93 days,average survival time were 7.1 days which was significantly shorter than the control(26.1days).It demonstrated that it was very effective in controlling A.chinensis adults in the field by using fungi strips.The field tests on the larvae control showed that the lower of instar,the shorter of tunnel,then the better of the control effect.The short-cut logs with new-hatched A.chinensis larvae were sprayed with MaZPTR-01 spore suspension,the mortality of larvae exceeded 90%.Combining fungi strips with spraying spore suspension,only 5.3% trees damaged by A.chinensis in treating Casuarina coastal shelter forest.In the control forest 38.9% trees were infected,so the decresae rate reached 86.3%.This method possessed of popularization and application value.5.High-throughput sequencing was employed in the transcriptome analysis of A.chinensis larvae of control,infected 12 h(Ma12),infected 24 h(Ma24)and infected 36 h(Ma36)with MaZPTR-01.Differentially expressed genes(DEGs)and their functions,classifications and signaling pathways were analyzed using bioinformatic tools,then the responsive genes of the A.chinensis larvae against infection by M.anisopliae were screened.The results showed that compared with the de novo sequence of A.chinensis larvae,there were 3487(2284 up-regulated,1203 down-regulated),3476(2265 up-regulated,1211 down-regulated)and 4440(3067 upregulated,1373 down-regulated)DEGs corresponded to the infected 12 h(Ma12)sample,24 h(Ma24)and infected 36 h(Ma36)sample,respectively.Compared with background genome,GO annotations analysis on DEGs of the larvae with different infected time showed that 1756 DEGs in Ma12 sample enriched in 825 GO terms with 15 significantly enriched GO terms(P < 0.05),1746 DEGs in Ma24 sample enriched in 858 GO terms with 5 significantly enriched GO terms and 2584 DEGs in Ma36 sample enriched in 1076 GO terms with 22 significantly enriched GO terms.The DEGs were enriched in the terms related to insect immune were mostly up-regulated,while the DEGs enriched in the terms related to metabolism were mostly down-regulated.KEGG pathway analysis indicated that total 41 significantly enriched(Q-value≤0.05)pathways were identified from 3 different time samples(12h,24 h and 36h).Among them 8 pathways appeared in all 3 time points samples,which were lysosome,long-term depression,protein processing in endoplasmic reticulum,glutathione metabolism,other glycan degradation,pentose and glucuronate interconversions,phagosome,antigen processing and presentation.The 41 pathways were mainly related to immune,disease,metabolism,transcription and translation.Most of DEGs participating in the immune and disease pathways were up-regulated.Those DEGs participating in the metabolism pathways were downregulated.Based on GO and KEGG pathway analysis,genes related to insect immune reaction were screened out,including pattern recognition receptors such as peptidoglycan-recognition protein(PGRP),beta-1,3-glucan recognition protein(βGRP),C-type lectin precursor,TEP3,scavenger receptor protein、Gram-negative bacteria binding protein(GNBP),genes related to signal modulation such as serpin,serpin55,serpin peptidase inhibitor 3,serine protease subfamily D,signal conduction such as toll-like receptor 13,cactus isoform 2,dorsal interacting protein 3 which were key proteins in Toll pathway,and immune factors such as immune-related Hdd11、defensin 1 precursor 、 cecropin precursor,lysozyme-like protein 1 precursor,lysozyme,prophenoloxidase activating factor,coleoptericin 2.All these gene’s expression in A.chinensis larvae were up-regulated after being infected by M.anisopliae.Results showed that activation of prophenoloxidase cascade and antibacterial peptides expression regulated by Toll pathway played an important role in the immune reaction of A.chinensis larvae. |
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