| Fungal pathogens have been thought as the main factors leading to crop loss. Chemical pesticides, which have been wildly used, cause great damage to the environment and human health. Furthermore, there are other defects such as the high cost of developing new chemical pesticides and the rapid emergence of drug-resistant microbes. We have drawn our attention to biological pesticides. Above all, antifungal protein provides important information for the development of new antifungal substances, because antifungal protein is widely-distributed and has unique mechanism of action. Besides, the rate of discovering new antimicrobial substances from land-base is on the decline, so it is imperative for us to explore new resources. The sea provides a special ecological environment, which may led to the development of new metabolic pathways of marine microorganisms. Then, marine microorganisms are more likely to produce new antimicrobial substances. These antimicrobial substances will play a huge role in the control of plant pathogens and the development of bioactive substances. So, exploring marine microorganisms to find antifungal protein has important academic significance.In order to find new and efficient natural products with antagonistic activity against plant pathogens from the oceans, this study used flat plate dilution method and diffusion method to screen bioactive strains. By using a variety of culture medium with different formulation, a huge number of aboriginal microorganisms were isolated from marine samples which were collected from different marine environment such as the Indian Ocean, the South Atlantic Ocean, the Arctic and Antarctic ocean. About300fungal strains and a large number of marine bacteria were isolated, including many bioactive strains with antagonistic activity. Then, seven plant pathogenic fungi and seven pathogenic bacteria were used as indicator microorganisms for rescreening. Seven plant pathogenic fungi include Paecilomyces variotii, Colletotrichum gloeosporioides, Fusarium oxysporum, Trichoderma viride, Rhizoctonia solani Kuhn, Alternaria longipes and Sclerotinia sclerrotiomm. Seven pathogenic bacteria include Staphylococcus aureus, Bacillus subtilis, Micrococcus lysodeikticus, Vibrio parahaemolyticus, Vibrio alginolyticus, Aeromonas nigdrophila and Xanthomonas Campestris. In this study,60bioactive strains with strong antagonistic activity were isolated and identified, including53bioactive fungi species and seven bacteria species. The statistics about the antagonistic activity and sample source of the bioactive strains may contribute to the study of marine microbial resources and the development of new antimicrobial substances.Moreover, Penicillium citrinum W1was purified from the sediment samples from the Southwest Indian Ocean. The supernatant of the fungal strain W1cultured in modified YTM liquid medium showed remarkable inhibitory activity against various pathogenic fungi. An antifungal protein, named PcPAF, had been isolated from marine Penicillium citrinum W1fermentation supernatant by purification process mainly including ammonium sulfate precipitation, dialysis, lyophilization, anion exchange chromatography, cation exchange chromatography, ultrafiltration concentration. The active component showed only a single band on a SDS-PAGE gel with an estimated molecular weight of10kDa. MALDI-TOF-TOF mass spectrometry and N-terminal amino acid sequence analysis were used to identify PcPAF. Both analysis results showed that PcPAF might be an unknown antifungal protein. The purified PcPAF protein was tested against different pathogenic fungi with the paper disc dilution method. PcPAF showed strong antifungal activity against Trichoderma viride, Fusarium oxysporum, Paecilomyces variotii and Alternaria longipes with MIC of1.52,6.08,3.04and6.08μg/disc respectively. The physicochemical properties of PcPAF had been detected. The activity of the purified PcPAF did not decrease at all while incubated at the temperature of80℃for20min. The inhibition activity of PcPAF remained above75%of control’s activity when it was treated with20mM Mn2+. In the presence of acidic environment, the PcPAF activity retained well. The highest antifungal activity of PcPAF was found to be at pH5. Furthermore, PcPAF activity retained well when it was treated with ultraviolet radiation, organic solvents, surfactants and proteases. PcPAF can maintain good inhibition activity in general conditions. There are certain differences between PcPAF and reported antifungal proteins in partial sequences and characters, so PcPAF might be a novel antifungal protein of fungal origin. The fermentation optimization of marine Penicillium citrinum W1was studied. The antifungal activity of the fermentation supernatant can be increased by about62%after adding glycine into the fermentation medium. In addition, PcPAF had been applied for crops diseases control and shown good antidisease effect, in which the control efficient of wheat scab reached52%. In conclusion, an antifungal protein, named PcPAF had been found in this study. PcPAF has good antifungal activity, broad antimicrobial spectrum and stability. The antifungal activity of fermentation product had been improved by nutrition supplement. Moreover, PcPAF has certain effect to protect crops against fungal disease in practical applications. The results suggested that PcPAF may represent a novel antifungal protein with potential application in controlling plant pathogenic fungal infection. |
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