Screening,Isolation And Identification Of Bacterial Strains Anti-Fungus

Fungi often have some direct or indirect contacts with human.Some of them are harmful to humans althougth most of them are beneficial to people.Some fungi can cause food mildew,or cause human disease,etc.At present there are mainly two ways to control the harm of fungi to human beings:one is the use of sorbitol,sodium acetate,fumaric acid and other chemicals inhibiting fungi;the second is using physical inhibiting fungi.But both have some disadvantages,such as pollution,at the same time may remain harmful substances on human,etc.So it is important to look for biological methods to inhibit fungi.This study,several infected fruits and vegetables were selected as experimental material,Finaly,three bacterial strains were isolated from peach and nectarine which could inhibit Aspergillus Niger by dilution plate method,E.coli and Bacillus Subtilis were controlled lines.We numbered the three bacterial strains 001,003,004 respectively,and 001 came from the pear,001 and 003 came from the nectarine.16SrRNA sequence analysis and blast on NCBI for each stain have been done.The results show that the sequence of 16SrRNA of 001 strain revealed 99%sequence similarity to that of Pantoea vagans,so we proposed that 001 stain belonged to Pantoea.The sequence of 16SrRNA of 003 strain revealed 99%sequence similarity to that of Pseufomonas aeruginosa,so we proposed that 003 stain belonged to Pseufomonas.The sequence of 16SrRNA of 004 strain revealed 99%sequence similarity to that of Enterobacter sp.,so we proposed that 004 stain belonged to Enterobacter.To determining species relationship of the three strains,the morphological and physiological and biochemical properties of them are identified respectively.The results were as follows:001 stain is Gram-negative,0.5-1.0 um long and 1-3 um in diameter.And most of strains produce yellow pigment.They are opaque,and are reaction positive to oxidase,urease and catalase,V-P,Nitrate,reaction positive and are reaction negative to indole Methyl red,Arginine double hydrolysis,Gelatin liquefaction and Hydrogen sulfide.It can use citrate as as a carbon source and can use glucose as fermentation material.Combining the 16SrRNA sequence analysis with the bacteria morphology,physiological and biochemical tests,we concluded that 001 strain belonged to Pantoea ananas.003 stain is Gram-negative,1.5-5.0 um long and 0.5-1 um in diameter.They are opaque and viscous,and are reaction positive to oxidase,catalase,nitrate and gelatin liquefaction,and are reaction negative to methyl red,V-P,indole,arginine,hydrogen sulfide negative.It can use citrate as as a carbon source.Combining the 16SrRNA sequence analysis with morphological,physiological and biochemical tests,we concluded that 003 strain belonged to the Pseufomonas.004 stain is Gram-negative,1.2-3.0 um long and 0.6-1.0 um in diameter.They are opaque and are reaction positive to oxidase,catalase,Urease,V-P,nitrate and gelatin liquefaction,and are reaction negative to methyl red,arginine and hydrogen sulfide.It could not use citrate as a carbon source.Combining the 16SrRNA sequence analysis with morphological,physiological and biochemical tests,we concluded that 004 strain belonged to the Enterobacter.We have identified antifungal active substances of the above three strains.We have used methanol and acetone to extract the secrect substance of the three strains,and found that 001 strain had no antifungal activity.So we infered that antifungal active substances may not be fat peptides.At the same time,we haveprecipitated the crude protein of 001,003 strains by using ammonium sulfate,and found the precipitation had antifungal activity which could inhibit the growth of aspergillus niger,so we proposed the antifungal active substances could be protein.