致病疫霉拮抗菌株YR-7的分离鉴定及其活性物质

Isolation and identification of the strain YR-7 against Phytophthora infestans and study on its antibiotic substances

【目的】从黄河边的农田土壤中分离筛选拮抗致病疫霉的粘细菌,鉴定目标菌株,分析其发酵上清液的稳定性及对马铃薯晚疫病菌的抑制效果,为活性物质分离鉴定及抗马铃薯晚疫病菌生物农药的研发奠定基础。【方法】采用兔粪诱导法分离菌株,通过平板对峙法筛选对马铃薯晚疫病菌有拮抗作用的粘细菌,通过形态特征、生理生化特征以及16S r RNA基因序列分析对菌株进行鉴定。采用称重法测定菌株生长曲线,通过平皿法测定菌株不同生长时期发酵上清液对致病疫霉的菌丝生长抑制率和浓缩发酵上清液的稳定性。通过马铃薯离体叶片涂布浓缩发酵上清液和接种病原菌孢子悬浮液法,测定该菌株对马铃薯晚疫病的防病作用。【结果】从土壤样品中共分离获得7株粘细菌,其中4株拮抗致病疫霉,拮抗效果最强的为YR-7菌株,菌丝的生长抑制率为96%,该菌株被鉴定为Myxococcus xanthus。培养7 d后,菌株发酵上清液对致病疫霉的抑制活性趋于稳定。浓缩发酵上清液经30-50°C处理后,对致病疫霉菌丝的生长抑制率可达50.90%,高于50°C时抑菌活性逐渐下降,90°C处理后菌丝的生长抑制率仍可达25.45%。浓缩发酵上清液在p H 4.0-9.0条件下比较稳定,保持40.21%以上菌丝的生长抑制率,当p H<4.0或p H>9.0时,抗菌活性显著降低。活性物质不能被蛋白酶降解,其抗菌活性不受紫外线、自然光照射的影响。对马铃薯离体叶片的生防效果检测表明,YR-7的浓缩发酵上清液处理组叶片相对病斑面积仅为0.35%,对照组的相对病斑面积高达68.19%。【结论】粘细菌菌株YR-7可以产生抗马铃薯晚疫病菌的次生代谢产物,抗菌活性物质具有较好的稳定性,可以有效抑制致病疫霉侵染马铃薯叶片,具有开发成抗马铃薯晚疫病生物农药的潜在价值。

[Objective] The purpose of this study is to isolate and identify antagonistic myxobacteria against Phytophthora infestans from soil samples collected from a farmland near the Yellow River and analyze the stability of the fermentation supernatant of the antagonistic strain and the inhibition effect on the pathogen of potato late blight preliminarily. These findings may lay a foundation for the isolation and identification of the antibiotic substances and the development of new biological pesticides resistant to potato late blight. [Methods] The strain was isolated by the rabbit dung baiting method. The antagonistic strains were screened by plate confrontation assay and identified by its morphological, physiological, and biochemical characteristics, and its 16 S r RNA gene sequence. The growth curve of strain YR-7 was tested by a weighing method. Inhibitory rate of the fermented supernatant at different stages on the mycelial growth of P. infestans and stability of the fermented supernatant was tested by a plating method. Effect of the fermented supernatant of strain YR-7 on disease prevention for detached potato leaves was evaluated by spraying concentrated fermentation supernatant on the detached potato leaves. [Results] Seven strains of myxobacteria were isolated and four of them presented antagonistic activity against P. infestans. The strain YR-7 with the strongest antagonistic activity showed an inhibition rate of 96% to P. infestans and was identified as Myxococcus xanthus. After the strain YR-7 was cultured for 7 days, inhibitory effect of its fermented supernatant on the mycelial growth of P. infestans tended to be stable. Its activity could be maintained from 30 ℃ to 50 ℃（inhibition rate of 50.90%） and decreased when the temperature exceeded 50 ℃. Its activity still remained an inhibition rate of 25.45% after treated at 90 ℃ for 1 h. The concentrated fermentation supernatant was stable from p H 4.0 to 9.0 and its inhibition rate was more than 40.21%. Its activity significantly decreased when the p H value was less than 4.0 or greater than 9.0. The active substances could not be degraded by protease and their activity was not affected by UV and natural light exposure. The relative lesion area of potato leaves declined from 68.19% to 0.35% when the concentrated fermentation supernatant was sprayed on the leaves before the spore suspension of P. infestans was inoculated. [Conclusion] The strain YR-7 can produce secondary metabolites against the pathogen of potato late blight. The antibiotic substances have good stability and could effectively inhibit P. infestans from infecting potato leaves, which have the potential value for developing biological pesticides resistant to potato late blight.