Classification and identification of yeast strain A822 and its inhibitory effect on Rhizopus stolonifer

Abstract

In recent years,with the increase of peach cultivation area and yield,the techniques for the peach postharvest disease control need to be improved.Since the 1980s,due to a variety of advantages,such as safety,effectiveness and without causing antibiotics resistance,biological control has become more and more important for fruits and vegetables postharvest preservation.Although many kinds of biological control agents have been developed,antagonism yeasts still have wide application prospects because of their convenient use. This study aimed at systematic classification and identification of the yeast strain A82-2 and studied the antagonism effect on peach soft rot diseases Rhizopus stolonifer. To study the inhibitory effect of strain A82-2 on R.stolonifer,four different experiments were designed,including the antifungal test on plates,the mixed strains culture experiments,the antibacterial test using fermentation broth and inhibitory studies on peach fruit in vivo.Regarding to classification and identification of strain A82-2,we used general classification methods such as observation of the yeasts morphology,and characterization of physiology and biochemistry,supplemented with molecular biology methods to analyze 26S rDNA sequence. First of all,using four different antifungal experiments step-by-step demonstrated the obvious inhibitory effect of yeast A82-2 on R.stolonifer.The inhibitory test of yeast A82-2 on plates showed the notably antagonistic action on R.stolonifer;the mixed strains culture experiments indicated the inhibitory effect of yeast A82-2 on R.stolonifer growth and there is obviously positive correlation with concentration of yeast A82-2;the suppressive test of yeast A82-2 fermentation broth demonstrated that there was no antifungal substance secreted outside yeast A82-2 cell,and further investigation results showed that the inhibitory action was achieved through nutritional competition and spatial competition between strain A82-2 and R.stolonifer,rather than yeast A82-2 producing any antifungal substances to inhibit the growth of R.stolonifer;In vivo studies showed that the 1×108 cfu/mL concentration of yeast A82-2 has a significant inhibitory effect on peach fruits which were inoculated with the 1×105 spores/mL concentration of R.stolonifer. Secondly,through general classification methods including observation of the yeasts morphology,and characterization of physiology and biochemistry,it was found that the characteristics of the strain A82-2 was closest to Pichia sp.Furthermore,molecular methods was used to analyze the 26S rDNA sequence of strain A82-2,and by sequence comparison in GenBank,the antagonistic yeast A82-2 was ultimately determined as Pichia anomala. As safe and effective antagonistic fungi,yeasts have been highly concerned,researchers have been discovered many yeast species with bio-control effect.However,at present there is no report that P.anomala had antagonistic effect on R.stolonifer.This study firstly identified that P.anomala A82-2 had an inhibitory effect on R.stolonifer,and its inhibitory mechanism was clarified.Based on the above results,we concluded that P.anomala A82-2 significantly inhibited R.stolonifer,therefore it has an important application potential in biological control and post harvest preservation of fruit and vegetable.