| Two pathogens causing anthracnose on Cymbidium goeringii in Hunan were isolated and identified, called CCH and CCB. Their virulence was tested, and the impact of nutritional and non-nutritional factors on its growth was analyzed. On these basis, three strains antifungal microorganism were isolated and identified, and the active ingredients from ZY721 strain fermentation broth were characterized. The details as follows:1. Isolation and identification of the pathogens and analysis of their biological characteristics.CCH colonies was round, gray, and aerial hyphae was rich, loose flocculent; There are separated mycelium, conidia colorless, unicellular, smooth surface, long cylindrical, rounded at both ends. One end rounded, slightly truncated at one end of the base, was umbilical. Size range 13.2-20.1 ?m×4.0-6.1 ?m, L/W=3.2. The CCH was identified as Colletotrichum gloeosporioides (Penz.) Penz. & Sacc based on its morphological characteristics and phylogenetic analysis of ITS and 28S rDNA.CCB colonies was round, white, velvet-like tile; There are separated mycelium, conidia colorless, unicellular, smooth surface, long cylindrical, one end rounded, slightly truncated at one end of the base, umbilical. Size range 9.1-18.2 ?m×4.6-6.5 ?m, L/W=2.5. The CCB was identified as Colletotrichum boninense Moriwaki, Sato & Tsukiboshi. based on its morphological characteristics and phylogenetic analysis of ITS and 28S rDNA.Virulence test results showed:In the case of stabbing, CCH and CCB more likely to infect Cymbidium goeringii and Cymbidium longibracteatum, can infect Cymbidium ensifolium, Cymbidium hookerianum, Cymbidium floribundum, but less likely. The pathogens can infect a variety of Cymbidium (Orchidaceae) plants by its wound.By analyzing the impact of nutritional factors and non-nutritive factor on the growth of pathogens CCH and CCB, The results showed that the strains CCH and CCB grew well on media of PDA, PSA and CSA. For carbon sources, CCH and CCB can effectively utilize a variety of carbon sources, and CCH preferentially use glucose, while CCB grew better in glucose than in sucrose, maltose or fructose. Yeast extract, tryptone and KNO3 were their better organic and inorganic nitrogen sources, respectively. The optimal acidity of culture medium was pH 5.0-8.0, light or darkness. Its hyphae could extend at 15-35?, and the optimum temperature of CCH and CCB was 28? and 25? respectively. Their lethal temperatures of hyphae and spores were 48? and 50? in 20 min, respectively.2. Isolation and identification of biocontrol strainsTo combat the pathogens CCH and CCB, three antifungal microbial were isolated from two different habitats, they belong to three major groups of microorganisms, ie fungi, actinomycetes and bacteria, and they were temporarily named as ZQ967, ZL966 and ZY721 respectively.ZQ967 had obviously inhibitory effect on pathogens CCH and CCB, it was identified as Penicillium oxalicum based on its morphological characteristics and its phylogenetic analysis of ITS sequence. ZL966 had significant inhibitory effect on pathogens CCH and CCB, according to the culture characteristics on Gause's medium and its typical characteristics in scanning electron microscope, strain ZL966 was identified as Streptomyces purpurascens combined with its phylogenetic analysis of 16s rDNA sequence. ZY721 had very significant inhibitory effect on pathogens CCH and CCB, ZY721 colonies on NA medium was white, large, umbrella, surface wrinkled, central projection; cells were rod-shaped in microscope, size ranges (0.7-0.8) ?m × (2.0-3.0) ?m. According to the culture characteristics on NA medium, source of this strain, and its physiological and biochemical characteristics, combined with its phylogenetic analysis of 16s rDNA sequence, strain ZY721 was identified as Bacillus subtilis (Ehrenberg) Cohn.3. Antifungal effect of strain ZY721 fermentation brothAntifungal effect of strain ZY721 fermentation broth on pathogens CCH and CCB were measured in PDA medium with different proportions of fermentation broth. The antifungal ratio was 100% on PDA medium with 1/2 (v/v) ZY721 fermentation broth; and its inhibition rate decreased with reducing the concentration of ZY721 fermentation broth in PDA medium. Cultured for 3 days, their inhibition rates to CCH and CCB on PDA medium with 1/3,1/5, 1/9,1/17 (v/v) ZY721 fermentation broth were 54.27,48.37,35.98,27.64% and 52.31,37.95, 19.23,14.36% respectively. The inhibitory effect of ZY721 on CCH was superior to on CCB.4. Identification of active ingredients in ZY721 fermentation brothThe active ingredients in ZY721 fermentation broth was extracted with yield of 1% (m/v) by acidified method. The antifungal effect of extracts on pathogen CCH and CCB was similar to its fermentation broth, its minimum inhibitory concentration (MIC) was 0.15625 mg/mL.The maximum absorption wavelength of methanol extract is 203 run; Infrared spectroscopy showed that the main groups are proteins and lipids; The result of UPLC-ESI-MS showed that the main ion peaks (m/z) were clustered in two regions:that were 1000-1100 and 1450-1550, and the former was absolute predominance. Five peaks in 1000-1100 (m/z) region were homologues of fatty acid chains differed by one methylene (-CH2), they were homologues of lipopeptide Iturin C, namely C13-IturinC?C14-IturinC? C15-IturinC?C16-IturinC?C17-IturinC; And the main peaks in 1450-1550 (m/z) region were three homologs of Fengycin A and seven homologs of Fengycin B. |
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