红树林植物内生菌分离鉴定及其抑菌活性研究

Isolation,Identification and Antibacterial Activity of Endophytic Bacteria from Mangrove Plants

[目的]从红树林植物组织中分离出对动物病原菌具有广谱抑菌活性的植物内生菌。[方法]以铜绿假单胞菌、多杀性巴氏杆菌、产气肠杆菌、粪肠球菌4种常见的致病菌作为指示菌,采用M10和P3培养基从红树林植物根、茎、叶组织中分离内生菌,以固体琼脂打孔法筛选具有抑菌活性的菌株,提取菌株的基因组DNA,PCR扩增16S rDNA基因并测序,通过邻接法构建系统发育树。[结果]从红树林植物组织中共分离857株可培养的内生菌,有145株菌有抑菌活性,占分离菌株的16.6%。其中通过M10培养基从红树林植物叶组织分离的菌株32-5对铜绿假单胞菌、产气肠杆菌都具有最强抑菌活性,其抑菌圈直径分别为38和42 mm;通过P3培养基从红树林植物茎组织分离的菌株12-4对粪肠球菌具有最强抑菌活性,其抑菌圈直径为56 mm;通过M10培养基从红树林植物茎组织分离的菌株13-2对多杀性巴氏杆菌具有最强抑菌活性,其抑菌圈直径为64 mm。有抑菌活性菌株分为10个属,其中芽孢杆菌属是优势菌属。[结论]该试验初步揭示红树植物根、茎、叶组织中植物内生菌的抑菌活性和多样性,为开发新型抗菌药物及应用提供理论依据。

[Objective]To isolate plant endophytes with broad-spectrum antibacterial activity from mangrove plant tissues.[Method]Pseudomonas aeruginosa,Pasteurella multocida,Enterobacter aerogenes and Enterococcus faecalis were selected as indicator bacteria.M10 and P3 solid medias and agar-well diffusion were used to isolate and screen endophytes with antibacterial activity from root,stem and leaf tissues of mangrove plants.Extracted DNA were used as template for PCR amplification of the 16S rDNA gene and sequenced to construct a phylogenetic tree by neighbor-joining method.[Result]A total of 857 culturable bacteria were isolated from mangrove plant tissues and 145 strains had inhibitory activity,accounting for 16.6%of the isolates.The strain 32-5,isolated from mangrove plant leaf tissue using M10 solid medium,had the strongest inhibitory activity against Pseudomonas aeruginosa and Enterobacter aerogenes,with an inhibition circle diameter of 38 mm and 42 mm,respectively.Strain 12-4 isolated from mangrove plant stem tissue using P3 solid medium showed the strongest inhibitory activity against Enterococcus faecalis with an inhibition circle diameter of 56 mm.Strains 13-2 isolated from mangrove plant stem tissues using M10 medium showed the strongest inhibitory activity against Pasteurella multocida with an inhibition circle diameter of 64 mm.Bacteria with antibacterial activity were classified into 10 genus,of which Bacillus was the dominant genus.[Conclusion]This experiment initially revealed the antibacterial activity and diversity of endophytic bacteria in mangrove plant roots,stems and leaf tissues,providing a theoretical basis for the development and application of new antibacterial drugs.