枸骨益生菌的筛选及其抑菌作用

Screening probiotic endophytic bacteria from medicinal plant Ilex cornuta and the phytopath-ogen-inhibiting effect

采用涂布平板法分离药用植物枸骨可培养内生细菌,采用对峙法和发酵滤液培养法筛选对植物病原菌具高抑制作用的菌株,通过镜检和显微摄影观察处理组菌丝的变化,对16S r DNA PCR产物进行测序和同源性分析菌株的系统发育关系,通过形态、生理生化特征和16S r DNA序列比对确定菌株的分类地位.从枸骨健康植株的根、茎、叶及果实中分离得到85株内生细菌,筛选出对烟草赤星病菌、稻瘟病菌、棉花枯萎病菌等有较强抑菌作用的10株菌,初步鉴定为4属7种.其中,抑菌效果最强的GG78(60.3%)、GG31(48.1%)、GG3(61.0%)分别属于阴沟肠杆菌、路德维希肠杆菌和蜡样芽孢杆菌.受抑制病原菌菌丝均发生畸形、扭曲,局部膨大形成原生质浓缩球、菌丝基部出现毛发状细分支等现象,可能与内生菌分泌到胞外具有一定抑菌或杀菌作用的代谢物质如抗生素、水解酶类、生物碱等有关.

Culturable endophytic bacteria were isolated from medicinal plant Ilex cornuta by platespreading method,strains with strong inhibitory effect on phytopathogen were screened by confrontation culture and fermentation filtrate culture methods,and the morphological changes of phytopathogen hyphae treated with endophytic bacteria were examined by microscopy and micrograph. Their phylogenetic relationships were determined by homology analysis of the 16 S r DNA sequences of PCR products and the taxonomic status of the selected strains was determined based on their morphology,physiology,biochemical test results and 16 S r DNA sequence analysis. A total of 85 endophytic bacteria were isolated from the healthy roots,stems,leaves and fruits of I. cornuta,and 10 strains of them showed strong inhibitory effect on Alternaria alternata,Magnaporthe grisea,Fusarium oxysporum,and were preliminarily identified belonging to four genera and seven species. Three strains with the strongest inhibitory effect,GG78（ 60. 3%）,GG31（ 48. 1%） and GG13（ 61. 0%） belonged to Enterobacter cloacae,Enterobacter ludwigii and Bacillus cereus,respectively. Microscopic analyses showed that the inhibited phytopathogen hyphae became deformed,distorted,and partially expanded forming plasma concentration and hair-like branch on the hyphae base. These morphological changes could be caused by the extracellular metabolic substances secreted by the endophytic bacteria,such as antibiotics,hydrolytic enzymes,alkaloids and so on.