水稻内生菌Acinetobacter soli JKDY-3分离鉴定及其促生作用

Isolation and identification of an endophytic Acinetobacter soli JKDY-3 from rice and its plant growth-promoting effects

  • 摘要: 【目的】鉴定水稻内生菌株并分析其对水稻的促生和定殖能力,为丰富水稻内生菌资源库、发掘优质内生菌资源及水稻促生菌的应用提供参考依据。【方法】采用划线法从水稻根、茎、叶组织中分离纯化内生菌。用功能培养基检测内生菌的促生功能,包括解钾、溶磷、产铁载体、固氮和分泌吲哚乙酸(IAA)能力。对筛选获得的菌株通过形态特征、生理生化特性及分子生物学方法(16S rRNA序列)进行菌种鉴定;通过测定菌株的生长曲线;筛选不同碳、氮源培养基优化菌株培养条件;通过测定菌株生物膜、果胶酶的形成能力并进行定殖试验,研究菌株的定殖能力;通过种子萌发水稻幼苗促生试验评估菌株的促生效果。【结果】从水稻叶片中分离得到菌株JKDY-3,该菌株解钾黄色晕圈直径为26.0 mm、溶磷圈直径为13.7 mm、产铁载晕圈直径为17.7 mm,IAA分泌量为58.44 mg/L,兼具固氮能力。系统发育树分析结果显示,菌株JKDY-3为Acinetobacter soli。JKDY-3菌株快速生长对数期在2~14 h,培养基的最佳碳、氮源分别为麦芽糖和酵母浸粉。菌株JKDY-3有较强形成生物膜的能力,溶解生物膜后,空白对照组OD570 nm为0.065,菌株JKDY-3处理组OD570 nm为0.196;且该菌能产生果胶酶。接种7 d后,菌株JKDY-3在水稻根、茎、叶中均可有效定殖,定殖量分别为3.75×10~3、7.26×10~3和6.76×10~3 CFU/g。种子萌发试验结果显示,菌株JKDY-3菌悬液浸种可极显著提高种子的发芽势、活力指数和萌芽后的芽长(P<0.01),并显著提高种子的发芽指数和萌芽后的根长(P<0.05,下同),5个指标分别增加12.29%、30.05%、28.22%、16.42%、26.80%。在难溶钾环境中,菌株JKDY-3处理水稻幼苗可显著增加单株地下部分干重,达36.96%,说明菌株JKDY-3能显著促进水稻幼苗根系生长。【结论】从水稻叶片中分离得到1株水稻内生菌株JKDY-3,鉴定为Acinetobacter soli,该菌株具有较好促生功能,能有效定殖于水稻植株内,可用于微生物肥料开发。

     

    Abstract: Objective To identify the rice endophytic strain and analyze its plant-promoting and colonization abilities,which could enrich the rice endophytic bacteria resource library,explore high-quality endophytic bacteria resources,and provide reference for the application of rice plant-promoting bacteria.Method The streak plate method was used to isolate and purify endophytic bacteria from rice root,stem and leaf tissues.Functional media were used to determine the plant-promoting functions of endophytic bacteria,including the ability to solubilize potassium and phosphorus,produce siderophores,fix nitrogen,and secrete indoleacetic acid(IAA).The screened strains were identified by morphological characteristics,physiological and biochemical properties and 16S rRNA sequence molecular biology method.The growth curve of the strain was determined,and different carbon and nitrogen source media were screened to optimize the culture conditions.The colonization ability of the strain was studied by determining the capacity to form biofilm and pectinase,and by conducting colonization tests.The plant-promoting effect of the strain was evaluated through seed germination and rice seedling promotion experiments.ResultThe strain JKDY-3 was isolated from rice leaves.The diameter of the yellow halo formed by potassium solubilization of the strain was 26.0 mm,the diameter of the phosphorus solubilization halo was 13.7 mm,the diameter of the siderophore-producing halo was 17.7 mm,the IAA secretion amount was 58.44 mg/L,and it had nitrogen fixation ability.Phylogenetic tree showed that strain JKDY-3 was Acinetobacter soli.The rapid growth logarithmic phase of the strain JKDY-3 occurred from 2 to 14 h,and the optimal carbon and nitrogen sources of the medium were maltose and yeast extract respectively.Strain JKDY-3 had a strong biofilm formation ability.After dissolving the biofilm,the OD570nm value of the blank control group was 0.065,while that of the strain JKDY-3 treatment group was0.196.The strain was able to produce pectinase.Strain JKDY-3 could effectively colonize in rice roots,stems,and leaves at 7 d after inoculation,with colonization amounts of 3.75×103,7.26×103,and 6.76×103 CFU/g respectively.The results of the seed germination test showed that soaking seeds with strain JKDY-3 bacterial suspension could extremely significantly improve the germination potential,vitality index and bud length after germination(P<0.01),and significantly improve the germination index and root length after germination(P<0.05),the 5 in deyes increasing by 12.29%,30.05%,28.22%,16.42% and 26.80% respectively.In the insoluble potassium environment,treating rice seedlings with strain JKDY-3 could significantly increase the dry weight of the underground per plant by 36.96%,the 5 indexes indicating that strain JKDY-3 could significantly promote the root growth of rice seedlings.Conclusion The rice endophytic strain JKDY-3 is isolated from rice leaves and identified as Acinetobacter soli.The strain has good plant-promoting functions,can effectively colonize in rice plants,and can be used for the development of microbial fertilizers.

     

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