防风内生生防细菌FFR3的筛选、鉴定及其抑菌物质特性研究

Screening, Identification of Endophytic Bacterium FFR3 from Saposhnikovia divaricate (Trucz.) Schischk. and Study on Characteristics of Its Antibacterial Substances

摘要: 植物内生细菌在植物的促生、生物防治、抗逆性以及生物医药、环境保护等方面具有重要的作用。本文从中草药防风(Saposhnikovia divaricata (Trucz.) Schischk.)根系中筛选出1株对多种病原细菌和病原真菌具有广谱抗性的内生细菌FFR3，并通过形态学和16SrRNA分子生物学鉴定，在此基础上研究了FFR3对病原真菌的形态影响和抑菌物质的特性。结果表明，FFR3为类芽孢杆菌(Paenibacillus spp.)，对梨火疫病菌、稻黄单胞菌水稻致病变种、苏云金芽孢杆菌、金黄色葡萄球菌、丁香假单胞杆菌桑致病变种等多种病原细菌以及核盘菌、禾谷丝核菌、串珠镰胞菌、辣椒疫霉、灰霉菌等多种病原真菌具有明显抑制作用。FFR3产生的胞外物质破坏病原真菌细胞壁结构，使菌丝脱水、皱缩、断裂，环状或U型打环，菌丝变短变细，达到抑制病原真菌的效果。硫酸铵沉淀、乙醇醇沉、不同有机溶剂分级萃取FFR3胞外抑菌物质，以金黄色葡萄球菌和丁香假单胞杆菌桑致病变种为指示菌，初步判定抑菌物质为蛋白质类(或肽类)物质、生物碱或者黄酮类物质。硫酸铵沉淀的蛋白质类(或肽类)物质在低于80℃时保持良好的抑菌活性；在蛋白酶K和胰蛋白酶处理后，抑菌活性明显下降；在pH 6~8即中性范围内对指示菌的抑制效果最好。类芽孢杆菌是一类重要的植物病害生物防治微生物，在环境污染治理、食品、医药等领域均有广阔的应用前景。本研究为植物内生菌及类芽孢杆菌的进一步开发应用提供一定的基础。

Abstract: Plant endophytic bacteria played important roles in plant growth, biological control, stress resistance, biomedicine and environmental protection. In this paper, an endophytic bacterium with broad spectrum resistance to many pathogenic bacteria and fungi, named FFR3, was isolated from the root of Saposhnikovia divaricata (Trucz.) Schischk. Based on the morphology and 16SrRNA molecular biology, strain FFR3 was identified. The morphological effects of strain FFR3 on pathogenic fungi and the characteristics of antibacterial substances were also studied. Results showed that strain FFR3 was identified as Paenibacillus spp. which had obvious antibacterial activities to a variety of pathogenic bacteria and pathogenic fungi, such as Erwinia amylovory, Xanthomonas oryzae pv. Oryzae, Bacillus thuringiensis, Staphylococcus aureus, Pseudomonas syringae pv. Mori and Sclerotinia sclerotiorum, Rhizoctonia zeae, Fusarium moniliforme, Phytophthora capsici, Botrytis cinerea. The extracellular substances, produced by FFR3, destroyed the cell wall structure of pathogenic fungi, caused the mycelium to dehydrate, shrink, break, ring or u-shaped ring, and shortened and thinned the mycelium, so as to inhibit the growth of pathogenic fungi. Using Staphylococcus aureus and Pseudomonas syringae pv. Mori as indicator bacteria, the antibacterial substances, extracted by ammonium sulfate, ethanol and different organic solvents, were preliminarily determined as proteins (or peptides), alkaloids or flavonoids. The proteins (or peptides), precipitated by ammonium sulfate, showed excellent heat stability, which could remain good antibacterial activities below 80˚C. After treatment with protease K and trypsin, the antibacterial activities decreased significantly. The antagonist could exert its excellent activities in the pH range from 6.0 to 8.0. Paenibacillus spp. was an important biological control microorganism of plant diseases, which had broad application prospects in the fields of environmental pollution control, food, medicine and so on. This study provided a basis for the further development and application of endophytic bacteria and Paenibacillus spp.