一株Fusarium oxysporum f. sp. niveum拮抗菌的筛选、鉴定及其抑菌特性

Screening, identification, and characteristics of an antagonistic strain against Fusarium oxysporum f.sp. niveum

为筛选西瓜枯萎病拮抗菌株并探究其抑菌特性,以齐齐哈尔市青昕蔬菜基地根际土为材料,采用平板对峙法筛选到1株能高效抑制西瓜专化型尖孢镰刀菌( Fusarium oxysporum f.sp.niveum,Fon)的拮抗菌株,通过形态特征、16S rDNA、 gyrA 和 gyr B序列分析鉴定其菌种;通过盆栽试验验证拮抗菌株对西瓜枯萎病的抑制作用;采用生长速率法研究拮抗菌株不同生长时期的无菌发酵滤液及其不同贮存条件下的抑菌能力;利用扫描电子显微镜(SEM)和共聚焦激光扫描显微镜(CLSM)观察并分析无菌发酵滤液对Fon孢子形态和膜完整性的影响。结果表明,拮抗菌株WD为 Bacillus amyloliquefaciens,能有效抑制西瓜枯萎病的发生,抑制率达到57.57%,其无菌发酵滤液在衰亡期有较高的抑菌能力,对Fon菌丝生长抑制率为54.32%;无菌发酵滤液对温度、pH具有较高的耐受性,30 ℃、pH 7时抑菌活性最强,抑菌率可达58.36%;在4 ℃贮存至少45 d,且紫外光照120 min内对其抑菌活性没有影响;Fon孢子暴露在WD无菌发酵滤液下,孢子表面皱缩凹陷,膜完整性被破坏。综上所述,Bacillus amyloliquefaciens WD是1株高效的拮抗菌株,为西瓜枯萎病生防菌剂的研发与利用奠定了基础。

In order to screen an antagonistic strain against Fusarium oxysporum f.sp. niveum(Fon), the rhizosphere soil from vegetable crops on Qingxin farm in Qiqihar City was collected and used in this study. The antagonistic strain was screened out using plate confrontation method, and identified by morphological, 16 S rDNA, gyrA and gyrB sequence analysis. Pot experiment was conducted to verify the control effect of antagonistic strains on Fusarium wilt of watermelon. Growth rate method was used to study antifungal activities of sterile fermentation filtrate from different growth stages of antagonistic strain and the stability of sterile fermentation filtrate. Fon conidia morphology and cell membrane integrity were analyzed by scanning electron microscopy(SEM) and confocal laser scanning microscopy(CLSM), respectively. The results showed that the antagonistic strain WD was Bacillus amyloliquefaciens, which could effectively inhibit the occurrence of watermelon wilt, and the inhibition rate reached 57.57%. The sterile fermentation filtrate had higher antifungal ability in the decline period, and the growth inhibition rate of Fon mycelium was 54.32%. Sterile fermentation filtrate from B. amyloliquefaciens WD had high tolerance to temperature and pH. The antifungal activity was the strongest at 30 ℃ and pH 7, the inhibition rate reached 58.36%;The sterile fermentation filtrate could store at 4 ℃ for at least 45 days, and UV light for 120 min had no effect on its antifungal activity. The Fon conidia were exposed to the sterile fermentation filtrate from B. amyloliquefaciens WD, the surface of the conidia collapsed and the membrane integrity was destroyed. In conclusion, B. amyloliquefaciens WD had high-efficiency antifungal activity on Fon, the results laid a foundation for development and utilization of biocontrol agent to Fusarium wilt of watermelon.