A Preliminary Study On The Function Of Symbiotic Bacteria In Melanotus Cribricollis Larvae

Insect microorganisms are diverse and complex,widely distributed in the host body.In the long-term process of coevolution,they are interdependent and mutualistic with host and play different roles in the host body.Therefore,the symbiotic bacteria have rich functional diversity.In addition to paying attention to symbiotic bacteria participating in the nutrient metabolism process,affecting growth,development,reproduction and behavior of the host,we pay more attention to the protective effect of symbiotic bacteria on host in the Integrated Pest Management.The larvae of Melanotus cribricollis are the major underground pest in bamboo forests in China.In recent years,this larvae have caused serious damage to more than 40bamboo species,including Phyllostachys praecox and P.edulis.M.cribricollis larvae harms the base of young bamboo shoots,and the wormholes are all over,which causes the bamboo shoots unable to grow,the quality decline,and affects the renewal of the bamboo forest.However,neither pesticide control nor biocontrol bacteria control has been able to effectively control the harm of M.cribricollis larva to bamboo forest.The symbiotic bacteria in the M.cribricollis larvae may play an irreplaceable role in resisting the adverse factors such as pathogenic microorganisms and pesticides.This study was based on the functional research of the symbiotic bacteria of M.cribricollis larvae.Firstly,the culturable symbiotic bacteria of M.cribricollis were isolated and purified.Next by co-culture with Metarhizium pingshaense,the symbiotic bacteria with antifungal activity were screened preliminarily,which was identified as Acinetobacter gyllenbergii by physiological and biochemical observation,morphological observation and gene sequence alignment.Then the effects of fermentation broth and volatiles of A.gyllenbergii,the dominant symbiotic bacteria,on conidial germination,mycelial growth and conidia production of M.pingshaense were measured.The SPEM-GC/MS method was used to analyze the active substances of volatiles to further verification of pure compounds with key effects.At the same time,the role of symbiotic bacteria in host resistance to pesticides was discussed through the degradation of common underground pest pesticides by A.gyllenbergii suspension.The main results are as follows:（1）In NA medium on cultured Melanotus cribricollis larvae symbiotic bacteria,43bacterial strains were isolated and purified,including 20 Acinetobacter.Four strains of Acinetobacter formed obvious inhibition zone when co-cultured with Metarhizium pingshaense,were identified as A.gyllenbergii by morphological observation,physiological and biochemical tests and gene sequence alignment.（2）Both A.gyllenbergii fermentation broth and volatiles showed strong inhibitory effect on conidial germination.After treatment with fermentation broth for 72 h,the conidial germination rate was only 29.86±4.953%,significantly lower than control group（100.0±0.000%）group（P<0.01,t=40.51）.After adsorbed volatiles for different time,the water agar medium was used to culture M.pingshaense for 72 h.The results showed that conidial germination of all treatment groups was significantly lower than that of control group(P<0.01,F_（3,16）=110.3).The inhibition of A.gyllenbergii fermentation broth on mycelial growth of M.pingshaense was enhanced with the increase of concentration.The mycelial growth was not affected by 10%of the fermentation broth,but was inhibited at 40%concentration.However,the inhibition effect lasted only 6 days;There was no significant difference in colony diameter of M.pingshaense under A.gyllenbergii volatiles treatment（P=0.125,t=1.71）;Both fermentation broth and volatiles had different effects on the conidia production of M.pingshaense.When M.pingshaense was treated with low concentration of fermentation broth（10%）,the difference of conidia production was not significant between treatment group and control group（P=0.712,t=0.38）.When the concentration was≥40%,the inhibition effect was significant;The conidia production of M.pingshaense treated with A.gyllenbergii volatiles was significantly lower than that of the control group（118.4±1.03）×10⁶/ml（P<0.01,t=12.15）.（3）The volatiles of A.gyllenbergii were further analyzed by GC-MS,and 10 kinds of volatiles were obtained,mainly including alkanes（70.29%）,aromatic compounds（12.24%）,olefins（10.84%）and aldehydes（5.90%）.Seven candidate compounds,includingα-pinene,aniline,nonanal,2,4-di-tert-butylphenol,limonene,1-dodecene and dimethyl trisulfide were screened according to the characteristics of volatiles.Among them,2,4-di-tert-butylphenol,dimethyl trisulfide and nonanal inhibited the growth of M.pingshaense.All the candidate volatiles had certain inhibitory effects on the mycelial growth of M.pingshaense,but the mycelial growth was not affected by the treatment of 1-dodecene at 10μL,100μL,150μL and limonene at 10μL.Except limonene,the other six candidate compounds had inhibitory effect on the conidia production of M.pingshaens.（4）The slow growth period of A.gyllenbergii was 0-10 h,the logarithmic growth period was 10-26 h,and then it entered the stable period after 26 h.among the screened four common pesticides for underground pests,A.gyllenbergii had a certain degradation ability to chlorpyrifos and fipronil,but had no degradation effect to clothianidin and phoxim.