Isolation, Screening And The Inhibition Effects Of Antagonists In The Biocontrol Of Fruit Postharvest Diseases

Blue mold and black spot rot are important postharvest diseases of citrus and pear fruits. Pathogens attacks are main causes of postharvest losses. Generally, synthetic fungicide was considered to be an effective means for controlling postharvest diseases. However, its disadvantages such as chemical residues in foods, environmental pollution, and the occurrence of fungicide-tolerant strains of pathogens, have accelerated the development of alternative approaches. Among these, biological control using harmless antagonistic microorganisms was proved to be a promising strategy for managing fruit diseases.The aim of this research is to isolate and screen effective antagonists against blue mold and pear black spot rot, and identify them. The inhibition effects and their population dynamics in wounds were evaluated after the antagonists were screened. Main conclusions were drawn as follows.1. There were 102 strains isolated from the wounds and the surface of fruits, leaves, and rhizosphere soil using the method of serial dilution. After primary and further screening in vitro, 10 strains exhibited inhibitory activity to Penicillium italicum and 9 strains exhibited inhibitory activity to Alternaria kikuchiana. Among screened strains, it was found that the isolates YF1, YF4 and BF2 were effective antagonists against P. italicum and A. kikuchiana on fruits through Fruits-inoculation test. The inhibition rate of isolates YF1, YF4 and BF2 got to 79.12%, 62.5% and 66.67% at 25℃.2. Identification of the antagonists: According to the morphological, nutritional and physiological character, we found YF1 belonged to Candida sp., and YF4 belonged to Kloeckera apiculata. In order to determine the taxonomic placement, the identification of BF2 was performed with traditional bacterial systemic identification method. And then the molecular identification of BF2 was performed. Through the comparative 16S rDNA sequences analysis and phylogenetic analysis, we found BF2 belonged to Bacillus cereus.3. The effects of antagonists increased with the increase of antagonists cell population when the antagonists at the concentrations of 106-108 CFU/mL. Fruits were treated with antagonists suspension at 1×108 CFU/mL at various time intervals (48 h, 24 h, 2 h, 0 h) before and after challenge-inoculated with pathogenic spore suspension at 104 spores/mL. The efficacy of antagonists against A. kikuchiana and P. italicum was maintained when it was applied simultaneously with or before the pathogens. However, the efficacy decreased when applied after the spores. The result of inoculation-time test showed that the efficacy of biocontrol increased significantly with the time antagonists applied before pathogen inoculated. In mixed-antagonists inoculation test, the blue mold infection rate was reduced to 12.50% when the two yeasts, YF1 and YF4, suspensions were applied simultaneously. This meant that the efficacy of the mixture excelled they applied individually and indicated significant discrepancy between them. The infection rates of black spot rot were 17.5%, 10.00% and 15.83% when isolates YF1, YF4 and BF2 were mixed with each other. It suggested that the efficacy of the two mixtures was better than that of YF1, YF4 and BF2. In the antangonists-enhancement test, the result showed that CaCl2 and low synthetic fungicide elevated biocontrol efficacy of antagonists. But when antagonists combined with SA, there is no different from antagonist used alone.4. Rapid colonization of isolates YF1 and YF4 in wounds was observed whether pathogenic spores inoculated. The population of YF1 increased 123.26 folds after 48 h. When P. italicum spores inoculated simultaneously, the population increased 144.17 folds after 96 h. The population of YF4 increased almost 200 folds after 72 h. While it was 344.54 folds compared with the start point at 72 h when P. italicum spores co-inoculated, and it was 294.12 folds compared with the start point at 72 h when A. kikuchiana spores co-inoculated.5. When the citrus fruits were soaked in the two yeast cell suspensions of 1×108 CFU/mL, the activities of phenlalanine ammonialyase (PAL) and peroxidase (POD) in fruits of citrus fruits raised remarkably, while polyphenol oxidase (PPO) reduced prominently and activities ofβ-1,3-glucanase and exochitiase almost did not change. But when P. italicum and the two yeasts added into the wounds of the fruit simultaneously, the activities ofβ-1, 3-glucanase and exochitiase remarkably raised by being induced. These indicate that both of the yeasts can induce diseases resistance in citrus.