Screening Of Antagonistic Bacteria YS-1of Postharvest Diseases Of Citrus Fruits And Study On Antimicrobial Mechanism And Properties Of The Antibacterial Crude Extract Of The Strain YS-1

Citrus spp., the largest fruit crops worldwide, are cultivated in140countries and regions, mainly in China, Brazil, USA and Mediterranean countries. China occupies a prominent position in citrus industry due to the largest area and yield. We keep pace with the world in the aspect of biotechnology, part of the research in the leading level. But we are behind the USA, Brazil, Spain and other countries in the biocontrol of postharvest diseases of citrus mainly because of late starting and no enough attention. Fortunately, we are strengthening the power equipment and research efforts about the aspect along with the public’s growing concern for food safety and environmental protection. We expect that the research will be a breakthrough in the next few years.A strain of bacteria (named YS-1) that has high biocontrol efficacy on postharvest diseases of blue mold and green mold on citrus fruits was isolated and identified to be Paenibacillus brasilensis. The efficacy of YS-1for the controlling of postharvest diseases of citrus in vitro and in vivo, the optimization of the culture conditions of YS-1, the conditions selection of YS-1fermentation broth pretreatment were studied. The solubility and stability of antimicrobial substance produced from YS-1were also studied. The results are as followed:1. A total of191isolates from the soil, the surface of fruits and leaves, the wound of fruits were screened for antagonism against Penicillium italicum and Penicillum digitatum. Bacterial YS-1having the highest biocontrol efficacy was isolated from the surface of kumquat fruit by intial selection and the farther selection. It was identified as Paenibacillus brasilensis by the Biolog microbial identification system combined with16S rDNA sequence analysis, biochemical and physiologic characteristics and rpoB sequence analysis. The Gen Bank is JQ677085.2. There was inhibition of fermentation supernatant and cell suspension of strain YS-1on the pathogens of blue mold and green mold. The inhibition of the cell suspension on Penicillum digitatum showed significant advantages compared with the supernatant of strain YS-1, nevertheless the efficacy of the supernatant for the controlling of postharvest Penicillium italicum was better than that of the cell suspension. There was significant difference between the two treatments in vivo test, and the fruit disease incidence and lesion diameter were33.74%and4.71mm, respectively.3. The antibacterial mechanism of strain YS-1were investigated, the results showed that it was concerned with not only the secretion of antibiotics but also the competition of nutrition and space. In addition, the strain YS-1also had a direct inhibitory effect. The antagonism spectrum of YS-1showed that the strain YS-1inhibited not only fungal diseases such as blue mold, green mold and anthracnose but also bacterial diseases such as citrus canker.4. Biological potency of active substances produced by strain YS-1was measured and calculated from the inhibition diameter. And the formula is: Y=l0(x+I0.695)/8.904. Optimization of culture medium and fermentation conditions was studied through the combination of one-factor-at-a-time test and orthogonal design test. The optimal medium was consisted of soluble starch7%, yeast extract2.5%, NaCl0.6%, MgSO40.2%, K2HPO40.2%; the optimal fermentation conditions was4%inoculum, fermentation temperature30℃, initial pH of fermentation6.0, culture time was72h; the best carbon source and nitrogen source was soluble starch and yeast extract, respectively.5. The methods of fermentation broth pretreatment were discussed by heating and adjustment acidity. The results showed that the suitable pretreatment methods were adjustment the pH of the fermentation broth to3.0and heat treatment for15min at80℃.6. The solubility of the antagonistic substances of the strain YS-1in different solvents were studied. The result showed that the material is a kind of water-soluble substances being of high polarity.7. The stability of YS-1antagonistic substances was studied. The results showed that there was resistance to high temperature, acid and ultraviolet radiation. The antagonistic substances showed better antagonistic activity at121℃for20min, pH low to3.0, Ultraviolet irradiation for24h and low temperature storage for a month. But it was sensitive to chloroform.