稻油轮作土壤多功能促生菌的鉴定及其对油菜生长和根际细菌群落的影响

doi:10.13560/j.cnki.biotech.bull.1985.2024-0823

摘要/Abstract

摘要：

目的筛选具备解磷及生长促进功能的菌种，提升土壤中有效磷的丰度，促进植物及其根系的健康生长与发育。方法从浙江省宁波市长期水稻-油菜轮作的土壤中筛选出一株能够在碱性环境下生长的解磷促生菌株，通过菌落形态、生理生化特征及16S rDNA序列进行鉴定，对其培养和解磷条件进行优化，然后通过盆栽试验验证其对油菜的促生效果，并采用高通量测序技术对油菜根际细菌群落结构进行分析。结果筛选出的菌株LM1-2解磷能力可达493.23 μg/mL，IAA的合成量为9.41 μg/mL，鉴定为中间克吕沃尔菌（Kluyvera intermedia）。摇瓶试验结果表明，菌株的最佳培养条件是初始pH为10、装液量为培养容器体积的30%、25℃培养7-9 d，最佳碳源、氮源、磷源分别为葡萄糖、胰蛋白胨、磷酸三钙，在最佳单因素优化条件下菌株的解磷能力最强可达587.03 μg/mL。油菜盆栽试验结果表明，与对照相比，接种菌株LM1-2的处理土壤速效磷含量增加了45.29%，油菜根系总长度、根直径、根分叉数和根交叉数分别显著增加了40.78%、18.97%、55.30%和87.82%，油菜植株的地上部鲜重和干重分别增重15.51%、26.82%。接种LM1-2改变了油菜根际细菌群落结构，显著提高了α多样性的Simpson指数，其中醋杆菌门（Acidobacteriota）是优势菌门，其相对丰度提高了23.66%。结论菌株LM1-2在解磷、油菜促生方面表现优良，在农业生产过程中有广阔的应用范围。

关键词: 油菜, 解磷菌, 中间克吕沃尔菌, 植物根际促生菌, 细菌群落结构

Abstract:

Objective This study aims to identify efficient phosphate-solubilizing growth-promoting strains to increase the available phosphorus content in soil and promote the growth and healthy development of plants and their root systems. Method A phosphate-solubilizing growth-promoting strain capable of thriving in an alkaline environment was isolated from a long-term rice-oilseed rape rotation in Ningbo, Zhejiang province, China. The strain was identified using a combination of the morphological, physiological and biochemical characteristics, along with 16S rDNA molecular analysis. The incubation and phosphate solubilizing conditions were optimized, and the growth promotion effects on rapes were verified through a pot experiment. Subsequently, high-throughput sequencing techniques were also employed to analyze the structure of the bacterial communities in the rhizosphere soil of oilseed rapes (Brassica napus). Result The isolated strain LM1-2 showed a strong capacity for phosphorus solubilization. The amounts of solubilized phosphorus and indole-3-acetic acid (IAA) synthesized were 493.23 and 9.41 μg/mL, respectively. Strain LM1-2 was identified as Kluyvera intermedia. The shake flask experiments demonstrated that the optimal conditions were an initial pH of 10, a liquid volume 30% of the container's volume, and a culture period of 7-9 d at 25℃, with glucose, tryptone, and tricalcium phosphate serving as carbon, nitrogen, and phosphorus sources, respectively. Strain LM1-2 had the strongest phosphorus-solubilizing ability, reaching 587.03 μg/mL. The pot experiment revealed that, compared to the control group, strain LM1-2 increased available phosphorus by 45.29%. The root length, root diameter, root forks and root crossings of oilseed rape treated with LM1-2 significantly increased by 40.78%, 18.97%, 55.30%, and 87.82%, respectively. Additionally, the fresh and dry weights of the oilseed rape increased by 15.51% and 26.82%, respectively. Inoculation with LM1-2 changed the structure of the rhizosphere bacterial community and significantly increased the Simpson index of alpha diversity, with Acidobacteriota being the dominant phylum and its relative abundance increased by 23.66%. Conclusion Strain LM1-2 demonstrates the excellent performance in phosphorus solubilization and growth promotion of oilseed rape, indicating its potential for diverse applications in agricultural production.

Key words: oilseed rape, phosphate-solubilizing bacteria, Kluyvera intermedia, plant growth promoting rhizobacteria, bacterial community structure

夏馨媛, 薛道晟, 李鑫静, 龙俊杰, 陆开形, 丁沃娜, 李梦莎. 稻油轮作土壤多功能促生菌的鉴定及其对油菜生长和根际细菌群落的影响[J]. 生物技术通报, 2025, 41(4): 289-301.

XIA Xin-yuan, XUE Dao-sheng, LI Xin-jing, LONG Jun-jie, LU Kai-xing, DING Wo-na, LI Meng-sha. Identification of Multi-function Growth-promoting Bacteria in Rice-oilseed Rape Rotation Soil and Its Effect on the Growth and Rhizosphere Bacterial Community of Oilseed Rape (Brassica napus)[J]. Biotechnology Bulletin, 2025, 41(4): 289-301.

图/表 12

图1 菌株LM1-2的菌落图（A）和革兰氏染色图（B）

Fig. 1 Colony diagram (A) and Gram staining diagram (B) of strain LM1-2

表1 LM1-2菌株的生理生化特性

Table 1 Physiological characteristics of strain LM1-2

项目 Item	结果 Result	项目 Item	结果 Result
革兰氏染色	-	V-P试验	-
好氧性试验	好氧	淀粉水解	-
接触酶试验	+	明胶液化	+
甲基红（M.R）试验	+	吲哚试验	+

图2 菌株LM1-2 16S rDNA基因序列的系统发育树

Fig. 2 Phylogenetic tree of 16S rDNA gene sequence of strain LM1-2

图3 不同培养条件对菌株LM1-2生长情况（OD600）的影响不同字母表示不同条件对LM1-2生长情况的影响（P<0.05）

Fig. 3 Effects of different culture conditions on the growth status (OD600) of strain LM1-2Different letters indicate the effects of different conditions on the growth status of LM1-2 (P<0.05)

图4 不同培养条件对菌株LM1-2解磷能力的影响不同字母表示不同条件对LM1-2解磷能力的影响（P<0.05）

Fig. 4 Effects of different culture conditions on the phosphorus solubilization ability of strain LM1-2Different letters indicate the effects of different conditions on the phosphate solubilization capacity of LM1-2 (P<0.05)

图5 LM1-2对油菜盆栽土壤速效磷（A）、植株（B）和根系（C）的影响*和**分别表示处理间在0.05和0.01水平差异显著

Fig. 5 Effects of LM1-2 on the soil available phosphorus (A), plants (B) and roots (C) of oilseed rapes in the pot experiment* and ** indicate significant differences between treatments at 0.05 and 0.01 levels, respectively

图6 LM1-2处理油菜盆栽幼苗各指标之间的主成分分析SAP：土壤速效磷；ABFW：地上部生物量鲜重；ABDW：地上部生物量干重；UBFW：地下部生物量鲜重；UBDW：地下部生物量干重；RL：根总长；RSA：根表面积；RD：根直径；RV：根体积；RT：根尖数；RF：根分叉数；RC：根交叉数；下同

Fig. 6 Principal component analysis of various indicators among potted oilseed rape seedlings under LM1-2 treatmentSAP: Soil available phosphorus; ABFW: above-ground biomass fresh weight; ABDW: above-ground biomass dry weight; UBFW: underground biomass fresh weight; UBDW: underground biomass dry weight; RL: total root length; RSA: root surface area; RD: root diameter; RV: root volume; RT: number of root tips; RF: number of root forks; RC: number of root crossings. The same below

图7 LM1-2处理油菜盆栽幼苗各指标之间的相关性分析* P<0.05，** P<0.01，*** P<0.001

Fig. 7 Correlation analysis among various indicators of potted oilseed rape seedlings under LM1-2 treatment

图8 LM1-2处理土壤细菌群落Veen图（A）和Simpson指数（B）*表示处理间在0.05水平差异显著

Fig. 8 Veen plot (A) and Simpson index (B) of soil bacterial community treated with LM1-2* indicates significant difference between treatments at 0.05 level

表2 不同处理根际土壤细菌群落α多样性指数

Table 2 Alpha diversity indexes of rhizosphere soil bacterial communities under different treatments

处理

Treatment

Ace指数

Ace index

Chao指数

Chao index

Shannon指数

Shannon index

Simpson指数

Simpson index

Coverage指数

Coverage index

PD whole tree指数

PD whole tree index

图9 油菜根际土壤细菌群落在门（A）、纲（B）、目（C）、科（D）和属（E）分类水平上的组成

Fig. 9 Composition of bacterial communities in the rhizosphere soil of oilseed rapes at the phylum (A), class (B), order (C), family (D) and genus (E) levels

图10 LEfSe分析差异物种图

Fig. 10 LEfSe analysis of differential species

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