一株旱稻联合固氮菌株的分离鉴定及促生作用  

Isolation,identification and growth-promoting characteristics of endophytic nitrogen-fixing bacteria in upland rice roots

作　　者：胡应平[1] 林冬梅[1,2] 胡弘正 吴颖 林佳丽 林占熺[1,2] 刘斌[1,2] HU Ying-ping;LIN Dong-mei;HU Hong-zheng;WU Ying;LIN Jia-li;LIN Zhan-xi;LIU Bin(Fujian Agriculture and Forestry University,Fuzhou 350002,China;National Engineering Research Center of JUNCAO Technology of Fujian Agriculture and Forestry University,Fuzhou 350002,China)

机构地区：[1]福建农林大学,福州350002 [2]福建农林大学国家菌草工程技术研究中心,福州350002

出　　处：《西南农业学报》2024年第4期780-788,共9页Southwest China Journal of Agricultural Sciences

基　　金：中国援巴新菌草、旱稻技术项目(271/KH200039A)。

摘　　要：[目的]旱稻是一种重要的种质资源,对成熟期旱稻根系内生固氮菌进行筛选与鉴定,以获得对旱稻有较好促生作用的菌株。[方法]采用联合固氮固体培养基培养方法,从成熟期旱稻根系中分离出内生固氮菌,通过扩增固氮酶nifH基因和16S rRNA基因进行分子鉴定和系统进化分析生化指标测定和形态学鉴定;采用透明圈法进行溶磷、产铁载体能力验证;以褐球固氮菌为阳性对照,利用ELISA测定试剂盒测定菌株分泌固氮酶活性、1-氨基环丙烷-1-羧酸(ACC)脱氨酶,采用Salkowski比色法测定生长素(IAA)含量,并研究其生长特征。将分离的固氮菌株以1%浓度(约8×10~7CFU/mL)浸泡旱稻谷种进行催芽,研究其促生效果。[结果]分离的菌株URR45为固氮菌,属于嗜温型微生物,最适生长温度为32~50℃,最适pH为6.0~6.5,不耐盐。其分泌的固氮酶含量为220.15 U/L,ACC含量为898.69 ng/mL,生长素含量为14.91 mg/L,固氮酶含量和ACC含量显著高于对照组褐球固氮菌。该菌株能够利用葡萄糖、麦芽糖等碳源,属于发酵型细菌,具有水解明胶、溶磷和产铁载体特性。该菌株经16S rRNA基因测序后通过NCBI BLAST比对,鉴定为Herbaspirillum seropedicae,隶属于Pseudomonadota门、Betaproteobacteria纲、Burkholderiale目、Oxalobacteraceae科、Herbaspirillum属,革兰氏染色呈阴性。促生试验结果表明,在主根长度、侧根长度以及测根数方面均显著高于对照组。[结论]该菌株对旱稻幼苗具有一定的促生作用,在农业上产上可作为功能性内生固氮菌开发利用。[Objective]Upland rice was an important germplasm resource.The present study aimed to obtain strains with good growth-promo-ting effects on upland rice through isolation,screening and identification of endophytic nitrogen-fixing bacteria in the roots of mature upland rice.[Method]The combined nitrogen fixation solid culture medium was used for cultivation.Isolation of endophytic nitrogen-fixing bacteria from the roots of mature upland rice,molecular identification and phylogenetic analysis were conducted by amplifying the nitrogenase nifH gene and 16S rRNA gene,measuring biochemical indicators,and identifying their morphology.The capacities of phosphorus dissolution andi ron carriers production were verified by using the transparent circle method.Using nitrogen fixing bacteria Azotobacter chroococcum as a control(CK),the activities of nitrogenase and 1-aminocyclopropane-l-carboxylic acid(ACC)deaminase secreted by the strains were measured u-sing an ELISA assay kit.Determination of auxin(IAA)content using Salkowski colorimetric method and its growth characteristics were stud-ied.The upland rice seeds were soaked in separated nitrogen-fixing strains at a concentration of 1%(about 8 x107CFU/mL)to promote ger-mination and study their growth-promoting effects.[Result]The isolated strain URR45 was a nitrogen-fixing bacterium,belonging to a ther-mophilic type of microorganism.The optimal growth temperature was 32-50 C,the optimal pH was 6.0-6.5,and it was not salt tolerant.It secreted a nitrogenase content of 220.15 U/L,an ACC content of 898.69 ng/mL,and an auxin content of 14.91 mg/L.The nitrogenase and ACC content were significantly higher than those of the control.The strain could utilize carbon sources such as glucose and maltose and belonged to the fermentation-type bacteria.It had the characteristics of hydrolyzing gelatin,dissolving phosphorus,and producing iron carri-ers.After 16S rRNA gene sequencing and NCBI BLAST comparison,the strain was identified as Herbaspirillum seropedicae belonging to the Pseudomonadot

关 键 词：旱稻 联合固氮菌 菌株鉴定 固氮能力 促生作用 

分 类 号：S182[农业科学—农业基础科学]