水稻内生固氮菌分离鉴定、生物特性及其对稻苗镉吸收的影响

doi:10.3864/j.issn.0578-1752.2016.19.008

摘要/Abstract

摘要： 【目的】分离鉴定湖南水稻内生固氮菌，研究水稻内生固氮菌的系统发育，分析测定分离菌株的生物学特性，探讨接种水稻内生固氮菌对稻苗镉（Cd）吸收的影响。【方法】表面灭菌水稻植株样品后采用低氮培养法分离水稻内生细菌，采用PCR扩增、测序检测菌株nifH基因确认分离物是固氮菌，通过16S rRNA基因序列测定、比对初步鉴定菌株，分析菌株系统发育，通过温室盆栽试验探讨接种水稻内生固氮菌对稻苗Cd吸收的影响。【结果】从8个湖南水稻植株样品中分离到19株内生固氮菌，这些菌株在系统发育地位上属于Bacillus aryabhattai、B. cereus、B. idriensis、B. indicus、B. licheniformis、B. megaterium、B. methylotrophicus、B. subtilis、B. tequilensis、Brevibacterium halotolerans、Fictibacillus phosphorivorans、Paenibacillus barcinonensis、P. lautus 4属13种。分离到的19株内生固氮菌中有大约1/3的菌株产生蛋白酶和纤维素酶的能力较强，在48℃生长良好，在产孢液体培养基上生长良好(OD＞1.0)，在固体产孢培养基上产孢率高(60%—90%)，产碱能力也相对较强(pH 8.5—9.0)。有1/6的内生固氮菌（3个菌株）分别对立枯丝核菌、禾谷镰孢、拟枝孢镰孢具有拮抗性，抑菌率为42%—55%。有大约2/3的菌株对抗生素相对比较敏感，对杀菌剂耐性强。测定的4个代表菌株对检测过的78种碳源中的7种利用较好，它们是乳酸钠、蔗糖、葡萄糖、甘油、苹果酸、丙氨酸、葡萄糖醛酰胺。试验的19株内生固氮菌中有6个菌株促进水稻苗期Cd吸收，与对照相比植株Cd含量增加6.41%—38.45%；其他13个菌株抑制水稻苗期Cd吸收，与对照相比植株Cd含量减少2.06%—34.46%。【结论】从湖南水稻分离到19株内生固氮菌，系统发育地位属于 Bacillus、Brevibacterium、Fictibacillus、Paenibacillus 4属13种。部分菌株产碱能力强，产孢率高，可在48℃高温下生长，产蛋白酶、纤维素酶，拮抗立枯丝核菌、禾谷镰孢、拟枝孢镰孢，具有良好应用前景。接种水稻内生固氮菌可以显著影响水稻苗期Cd吸收，提示采用微生物方法阻控稻田Cd污染是一个非常值得研究、探讨的途径。

关键词: 水稻, 内生固氮菌, 生物特性, Cd

Abstract: 【Objective】The objective of this study is to isolate, identify and analyze phylogenetics of endophytic diazotrophs from rice planted in Hunan province, test the biological characteristics of the isolates, and to explore the influences of diazotroph inoculation on rice seedlings Cd accumulation. 【Method】Surface sterilization and low nitrogen medium were used to isolate endophytic diazotrophs. nifH detection was conducted based on PCR amplification to confirm the isolates as nitrogen-fixing bacteria. 16S rRNA was amplified with PCR, blasted in EzTaxon after sequencing, and analyzed with Clustalx-MEGA to make phylogenetic tree. Greenhouse trials were conducted to investigate the influence of diazotroph inoculation on rice seedling Cd accumulation. 【Result】Nineteen endophytic diazotrophs were isolated from root, stem and leaf of 8 rice samples. These 19 strains phylogenetically belong to 4 genus 13 species of Bacillus aryabhattai, B. cereus, B. idriensis, B. sindicus, B. licheniformis, B. megaterium, B. methylotrophicus, B. subtilis, B. tequilensis, Brevibacterium. halotolerans, Fictibacillus phosphorivorans, Paenibacillus barcinonensis, P. lautus. Biological tests showed that about 1/3 of the 19 strains produce protease and cellulose, grow well at 48℃, form spores well with percentage 60%-90%, produce alkali with final pH 8.5-9.0. About 1/6 of the 19 strains are antagonistic against plant pathogenic Rhizoctonia solani ACCC36246, Fusarium graminearum ACCC36249 and Fusarium sporotrichioides ACCC37402 with rate of 42%-55%. About 2/3 of the 19 strains showed sensitive to antibiotics and resistant to fungicide. Four representative strains of the 19 could utilize 7 of the 78 carbon sources, sodium lactate, sucrose, dextrose, glycerol, malic acid, alanine and glucuronic acid amide. Greenhouse trials showed that 6 of the 19 strains promoted rice seedling Cd absorption with increase of 6.41%-38.45%, and other 13 strains decreased rice seedling Cd absorption with 2.06%-34.46% compared with control.【Conclusion】Nineteen endophytic diazotrophs were isolated from rice planted in Hunan. These 19 strains phylogenetically belong to 4 genus 13 species of Bacillus, Brevibacterium, Fictibacillus and Paenibacillus. Partial strains produce protease and cellulose, grow well at 48℃, form spores well, antagonistic against plant pathogenic Rhizoctonia solani, Fusarium graminearum and Fusarium sporotrichioides, have good prospects of application. Inoculation of diazotroph can significantly affect rice seedling Cd absorption. The results suggest that application of microbial method to control paddy Cd is a very worthwhile pathway.

Key words: rice, endophytic diazotrophs, biological property, Cd

袁梅，谭适娟，孙建光. 水稻内生固氮菌分离鉴定、生物特性及其对稻苗镉吸收的影响[J]. 中国农业科学, 2016, 49(19): 3754-3768.

YUAN Mei, TAN Shi-juan, SUN Jian-guang. Isolation and Biological Properties of Endophytic Diazotrophs from Rice and Their Influences on Rice Seedling Cd Accumulation[J]. Scientia Agricultura Sinica, 2016, 49(19): 3754-3768.

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