江西农业大学学报2025，Vol.47Issue(4)：961-970,10.DOI:10.3724/aauj.2025082
岩生型硬叶兜兰内生固氮细菌的筛选及其生物学特性
Screening and biological characteristics of endophytic nitrogen-fixing bacteria of lithophytic Paphiopedilum micranthum
摘要
Abstract
[Objective]Endophytic nitrogen-fixing bacteria play a crucial role in enhancing nitrogen absorption and utilization in host plants,as well as in their adaptive evolution.This study aims to explore the functional diversity of endophytic nitrogen-fixing bacteria in lithophytic Paphiopedilum micranthum,understand their biological characteristics,and provide resources for the development of microbial agents.[Method]This study used the lithophytic P.micranthum as the experimental material,using tissue homogenization to isolate endophytic bacteria from leaves and roots.Bacterial identification was performed through morphological and 16S rRNA molecular techniques.Endophytic nitrogen-fixing bacteria were screened using nitrogen-free culture media.Five strains of nitrogen-fixing bacteria were selected based on their nitrogen fixing ability and growth amount,their ability to secrete IAA and produce siderophores was assessed using the Salkowski colorimetric method and plate assays,respectively.The culture conditions for five strains of nitrogen-fixing bacteria were optimized through single-factor experimental design,focusing on carbon sources,nitrogen sources,and inorganic salt.[Result]The results of isolation and identification showed that a total of 102 endophytic bacteria were isolated from the leaves and roots of P.micranthum,belonging to 15 genera across 4 families,with 32 species.The dominant genus was Bacillus,accounting for 66.67%of the total isolated strains.The preliminary screening results of nitrogen-fixing bacteria indicated that 90 strains belonging to 20 species had the ability of biological nitrogen fixation,accounting for 88.24%of the total isolated strains.Among them,the nitrogen-fixing ability and growth amount of NY-6(Fictibacillus enclensis),NY-21(Priestia megaterium),NY-47(Peribacillus frigoritolerans),NG-6(Priestia aryabhattai)and NG-44(Bacillus mycoides)are relatively high.The 5 species were preliminarily identified as endophytic nitrogen-fixing bacteria,and five representative strains among them were selected as test strains for subsequent experiments..The plant growth-promoting traits of the strains showed that all five nitrogen-fixing strains were capable of secreting IAA and producing siderophores.Among them,strains NY-6,NG-6,and NG-44 exhibited the high IAA production,reaching(6.07±0.01)µg/mL,(9.29±0.03)µg/mL,(10.76±0.04)µg/mL when 3 strain were cultured in media without tryptophan.IAA production reaching(19.08±0.01)µg/mL,(20.69±0.05)µg/mL,(20.83±0.04)µg/mL when 3 strain were cultured in media with tryptophan,which were 3.14,2.23 and 1.94 times higher than the strains without tryptophan,respectively.The strain NY-6 exhibited the strongest siderophore production,with a soluble index of 6.37±0.40,which was significantly higher than other nitrogen-fixing bacteria.Single-factor experiments revealed that the optimal inorganic salt for all 5 strains of nitrogen-fixing bacteria was CaCO3,and their preferred carbon and nitrogen sources varied.For strains NY-6 and NG-44,the optimal carbon and nitrogen sources were soluble starch and peptone,strains NY-21 and NG-6 preferred sucrose and beef extract,strain NY-47 preferred sucrose and peptone.[Conclusion]32 species belonging to 15 genera of endophytic bacteria were identified from 102 strains obtained from leaves and roots of P.micranthum.Among them,Calidifontibacillus erzurumensis,Heyndrickxia ginsengihumi,Niallia taxi,Neobacilluscucumis,and Metabacillus idriensis were isolated from orchid plantsfor the first time.A total of 20 species(90 strains)of endophytic bacteria were found topossess biological nitrogen-fixing capabilities.Among these,strains NY-6,NY-21,NY-47,NG-6,and NG-44 were preliminarily identified as nitrogen-fixing bacteria.All five strains of nitrogen-fixing bacteria have the ability to secrete IAA and siderophore production.Five strains exhibiting high utilization efficiency for beef extract and peptone.Strains NY-6,NG-6 and NG-44 exhibit notable plant growth-promoting abilities in vitro and could be used to investigate their promoting mechanisms in enhancing nitrogen absorption and assimilation in orchids.关键词
岩生型硬叶兜兰/内生固氮细菌/分泌IAA能力/产铁载体能力/培养条件优化Key words
lithophytic Paphiopedilum micranthum/endophytic nitrogen-fixing bacteria/IAA secretion ability/siderophore production ability/culture condition optimization分类
农业科技引用本文复制引用
阮月红,李宗艳,李银,胡家雪,伍倩,窦国蓉..岩生型硬叶兜兰内生固氮细菌的筛选及其生物学特性[J].江西农业大学学报,2025,47(4):961-970,10.基金项目
国家自然科学基金项目(32260414) Project supported by the National Natural Science Foundation of China(32260414) （32260414）
