Research On Screening And Identification Of Biological Control Bacteria Against Aflatoxins And Mechanism Of Strain JPP1for Biocontrol

A variety of biological carcinogens exist in the environment，for exampleAspergillus flavus is widely present in the soil and infects the host under appropriateconditions to produce aflatoxins （AF）. AF can cause acute and chronic poisoning,but also has carcinogenic, teratogenic and mutagenic effects on humans and animals,even could affect ecological safety. Peanut is an important oil and economic crop inChina， but the aflatoxin content in peanut exceeds the standard seriously.Traditional control method was to use chemical pesticides, but a large number ofhighly toxic pesticides continuous use will result in agricultural non-point sourcepollution and harm to the soil, water and atmosphere. Biological control technologyis friendly to the environment and effective to control AF contamination in peanuts.Microbial pesticide is one of the important means of biological control.Using several kinds of media,236strains of endophytic bacteria weresuccessfully isolated from hulls of peanut plants growing in6provinces in China.By visual agar plate assay to screen,72strains of endophytic bacteria significantlydecreased AFs production. Using tip-culture method24strains were determined tohave decreased mycelia growth levels by>90%and54strains decreased AFsproduction levels by>90%.A total of72isolates of endophytic bacteria were sequenced for their16SrDNA，combined with morphological observation and some physiological andbiochemical characteristics to analyze their phylogenetic relations. The majority ofendophytic bacteria in Bacillaceae belonged to the genus Bacillus （48），which wasdominant and could be classified into seven species, B.amyloliquefaciens, B.subtilis, B. methylotrophicus, B. pumilus, B. tequilensis, B. fusiformis and B. cereus.The only one strain BPM12-1affiliated with the family Planococcaceae, andexhibited99%similarity with the strain of Lysinibacillus xylanilyticus. There were18strains affiliated with the family of Enterobacteriaceae with the two genera ofEnterobacter and Serratia. Among them,7strains belonging to the genus ofEnterobacter could be classified into three species, E. ludwigii, E. asburiae, and E.cloacae. The other11strains belonging to the genus of Serratia were related to thesame species Serratia marcescens. There were3strains which affiliated with thefamily of Xanthomonadaceae belonged to the same genus of Stenotrophomonas andshowed sequence similarity value of99%to Stenotrophomonas maltophilia. Thetwo strain affiliated with the family Pseudomonadaceae and exhibited99% similarity with the strain of Pseudomonas mediterranea.By transparent ring method22strains of identified endophytic bacteriadegraded chitin on the chitin medium plate. Using DNS method to determine theenzyme activity of represent bacteria, the strains which showed stronger chitinolyticactivity belonged to the genera Serratia and Stenotrophomonas. A strain with higherenzyme activity and exhibited remarkable biocontrol effect was selected. Combinedwith morphological, physiological, biochemical characteristics and phylogeneticrelations, the strain was identified as Serratia marcescens and designated S.marcescens JPP1. Polymerase chain reaction （PCR） amplification of the chitinasegene was performed and obtained a1,789bp nucleotide sequence, its ORF was1,500bp and encoded499amino acids. After similarity alignment and phylogeneticanalysis, the chitinase produced by strain JPP1was determined as ChiB. Themolecular weight and the isoelectric point of ChiBjp were55480.3Da and5.93,respectively. It was a fat-soluble protein and relatively stable and hydrophilic. Itspredicted secondary structure was31.66%alpha helix,20.64%extended strand and47.70%random coil. Its predicted tertrary structure exhibited similarity of98.6%with ChiB from S. marcescens wildtype in complex with catalytic intermediate.Using Plackett-Burman design and central composite design, the mediumformula for chitinase production of S. marcescens JPP1was optimized. The formulawas （g/l）: colloidal chitin12.70, glucose7.34, peptone5.00,（NH4）2SO₄1.32,K2HPO₄0.7, MgSO₄·7H2O0.5. By central composite design, the maximumresponse enzyme activity of established model was31.96U when the fermentationconditions of volume, rotary speed and inoculum size were separately23.2ml,116rpm and4.3%. The result of flask fermentation experiment was close to themaximum enzyme activity.Stability test of chitinase exhibited better thermal stability and the relativeenzyme activity was more than80%at pH5⁸. The metal ions of Zn²⁺, Cu²⁺andFe3+showed significant inhibitory effect on the chitinase activity, while Ca²⁺andMn²⁺had positive effect, Na+and K+had little effect on chitinase activity. Proteinprotectants of EDTA, mercaptoethanol and Tween80could protect the chitinaseactivity in certain degree, while denaturant of SDS significantly reduced thechitinase activity.SEM analysis showed major damage of the mycelia and the cell wall of A.parasiticus was partially degraded in the presence of chitinase produced by S.marcescens JPP1. Using tip-culture method the chitinase concentration withantifungal ratio and antiaflatoxigenic ratio exhibited satisfied linear relationship.TLC experiment showed that toxigenic A. parasiticus treated with chitinase had not visibly produced AFs. RT-PCR results showed that ChiBjp repressed thetranscription of aflR, aflC （pksL1） and aflO （dmtA） genes. Therefore, the chitinaseproduced by the strain played the biocontrol effect.The formulation of biological seed coating agent was determined, polyvinylalcohol and carboxymethyl cellulose sodium （4:1, v/v） were mixed to yield thecomplex filmogen. The glycerin （1%） acted as plastifier, then the whole bacteriummedium and the filmogen （1:4, v/v） were fully mixed. After coating, the peanutseeds were treated with CaCl2（2%） as crosslinker. The formulation did not inhibitthe growth of biocontrol bacterium. After coating treatment, the germination rate ofpeanut seeds was up to98%. The seed coating agent significantly promoted the seedgermination and in vitro the antiaflatoxigenic ratio was up to88.5%, its antagonisticeffect was better than the agricultural fungicide of carbendazim.