一株拮抗病原真菌的固氮菌Paenibacillus spGD812

 【目的】筛选具有拮抗病原真菌功能的固氮菌，研究菌株的固氮酶活性、nifH、生理生化特征、菌株抗逆性与接种效果等，并对该菌株进行鉴定，为微生物肥料生产筛选菌种资源。【方法】采用无氮培养基富集、筛选固氮菌，用对峙法筛选拮抗菌；乙炔还原法测定固氮酶活性，PCR扩增16S rDNA和nifH；通过形态、生理生化特征和16S rDNA序列分析鉴定菌种；采用温室盆栽小白菜试验接种效果。【结果】筛选到1株固氮菌GD812，该菌株固氮酶活性达到30.661 nmol C2H4/h?mg蛋白，同时具有拮抗麦类赤霉病菌（Gibberella zeae）和棉花黄萎病菌大丽轮枝菌（Verticillium dahliae）功能，抑菌率分别达到59.5%和49.3%；根据形态、生理生化特征和16S rDNA序列分析结果，GD812被鉴定为类芽孢杆菌Paenibacillus sp.；该菌株的nifH基因长度300 bp，与Paenibacillus sp. Bs57 nifH基因序列相似性98%；GD812可以利用35种供试碳源中的20种，耐酸碱pH4—11，在4—50℃均可生长，盆栽试验接种比对照小白菜鲜重增加52%。【结论】固氮菌GD812被鉴定为类芽孢杆菌，该菌株利用碳源广泛，抗逆性强，具有较高的固氮酶活性和拮抗病原真菌能力，盆栽试验显示较好的接种效果，可望进一步研发成为优良的固氮微生物肥料生产菌种。

A Nitrogen-Fixing Bacterium Paenibacillus sp.GD812 Antagonistic Against Plant Pathogenic Fungi

【Objective】 The objective of this study is to screen nitrogen-fixing bacteria antagonistic against plant pathogenic fungi and research nitrogenase activity, nifH, physiological and biochemical characteristics, identification, stress resistance of the functional strains and inoculation effect on plant and to prepare strains for microbial fertilizer production. 【Method】 Nitrogen-free medium was used to isolate nitrogen-fixers and confrontation method was used to screen antagonistic bacteria against plant pathogenic fungus. Nitrogenase activity was determined with acetylene reduction assay. 16S rDNA and nifH were amplified with PCR. Strain identification was carried out based on the morphology, physiology, and biochemical test and 16S rDNA sequence analysis. Microbial inoculation effect on plant was tested with a pot culture in green house. 【Result】 One strain designated as GD812 was selected, which showed nitrogenase activity 30.661 nmol C2H4/h?mg protein, and strongly antagonistic against plant pathogenic fungi Gibberella zeae and Verticillium dahliae with the inhibition rate of 59.5% and 49.3%, respectively. nifH of GD812 is 300 bp sharing 98% sequence identity with that of Paenibacillus sp. Bs57. GD812 was identified as Paenibacillus sp. based on the results of morphology, physiology and biochemical test and 16S rDNA sequence analysis. Further investigations showed that GD812 could use 20 of the 35 tested carbon sources, could grow under temperature ranging from 4 to 50℃, and could grow in range of pH4 to11. Pot culture result showed that fresh weight of Chinese cabbage inoculated with GD812 increased by 52% compared with the control. 【Conclusion】 The newly selected Paenibacillus sp. GD812 showed high nitrogenase activity, strong antagonism against plant pathogenic fungi, extensive carbon source utilization, stress resistance and good inoculation effect, which is a candidate to be further developed for microbial fertilizer production.