Screening And Identifiacation Of Endophytic Bacteria Isolated From Peanut Seeds And Population Diversity Analysis

Peanut infection by aflatoxin becomes one of the most serious problems in theworld, which not only does harm to people’s health, but also leads to internationaltrade disputes. This study use Aspergillus parasiticus mutant NFRI-95as indicatedstrain, screening endophytic bacteria strains with antiaflatoxin activities isolatedfrom peanut seeds using fungal antagonism assay and tip culture assay. Theendophytic biocontrol bacteria isolated from peanut seeds will provide us plentifulof species and bioactive substance resources to control the aflatoxin contaminationin peanut effectively.The study obtained541endophytic bacteria strains isolated from different speciesof peanut seeds collected from different province. The results of fungal antagonismassay showed that65.6%stains which could inhibit the mycelial growth and36.1%stains which could inhibit the aflatoxin production of Aspergillus parasiticus havebetter effects on the PGY medium than on the GY medium. Then197strains withthe inhibitory rate of above50%were selected to test their fermentation supernatantinhibitory ability of mycelial growth and the accumulation of NA by tip culturemethod. It indicated that5endophytic bacteria strains could completely inhibit thegrowth of mycelial, and86endophytic bacteria strains could completely inhibit theaccumulation of NA. What’s more,68.53%of the fermentation solution had a highmycelia inhibition rate which was more than80%and80.20%of the fermentationsolution showed more than80%inhibition rate of NA acuumulation. Strains withhigh bioactivity were selected to detect the effects of pH and OD600on inhibitionrate of mycelial growth and NA accumulation. The results demonstrated that thefermentation solution with low pH (pH<7.0) had significant inhibition rate tomycelial growth and NA accumulation while OD600showed little impact on theinhibition rate and fluctuated between1.5and2.0.The classification of133strains with high antiaflatxin activities was achieved bymorphological identification and16S rDNA sequences analysis. The resultsdisplayed that66.92%of the strains were Gram-positive while32.08%wereGram-negative. According to the phylogenetic analysis,133strains were separatedinto12groups.47strains were identified as Bacillus amyloliquefaciens,36strainsas Serratia marcescens,13strains as Bacillus subtilis,6strains as Bacillus pumilus,10strains as Bacillus cereus,3strains as Alcaligenes faecalis,2strains asBurkholderia cepacia, and the other four groups only had one strain belonged tothem, and they were Paenibacillus polymyxa, Stenotrophomonas pavanii, Pseudomonas fluorescens and Achromobacter sp.66.7%of the endophytic bacteriastrains belonged to Bacillus which became dominant bacteria.This study took advantages of fungal antagonism assay and tip culture assay toscreen biocontrol endophytic bacteria strains isolated from peanut seeds, which notonly provides rich species resource for aflatoxin biocontrol bacteria, but alsoestablishes the theoretical foundation for peanut aflatoxin contamination in fields.