防风内生细菌的分离鉴定及其促生特性分析

doi:10.13560/j.cnki.biotech.bull.1985.2025-0070

摘要/Abstract

摘要：

目的从健康防风根部中分离、筛选出具有促生特性的菌株，探究其对防风植株的促生效果，挖掘内生细菌开发利用的潜力。方法采用稀释涂布法从健康防风根部中分离内生细菌，并通过选择培养基筛选具有固氮、溶磷、产IAA和产铁载体的促生细菌；通过形态学、生理生化鉴定和16S rRNA基因测序对促生菌株进行分类归属；基于抗生素标记法和盆栽试验探究内生细菌在土壤中的定殖能力及其对防风植株生长的影响。结果在防风根部中共分离出202株内生细菌，26株具有固氮能力，21株具有溶磷能力，24株具有产IAA能力，27株具有产铁载体能力。其中2株内生细菌具有3种及3种以上不同的促生功能，分别为MX-56和MX-31，鉴定为密歇根克雷伯氏菌（Klebsiella michiganensis），韩国假单胞菌（Pseudomonas koreensis）。两株抗利福平标记菌株可稳定定殖于防风根围土壤中，且在盆栽试验中，与CK相比，MX-56处理下防风的株高、根长、根粗、地上鲜重、根鲜重和根干重增长最为明显，分别增加13.47%、43.04%、42.75%、31.43%、63.21%和77.12%（P<0.05），与CK相比，菌株MX-31和MX-56处理下防风植株中两种色原酮总含量分别提高了33.72%和30.23%。结论从健康防风根中分离筛选到2株具有较好促生效果的韩国假单胞菌MX-31和密歇根克雷伯氏菌MX-56，可以显著提高防风植株的生物量和药效成分含量。

关键词: 防风, 内生细菌, 密歇根克雷伯氏菌, 韩国假单胞菌, 促生作用

Abstract:

Objective The growth-promoting strains were isolated and screened from the healthy roots of Saposhnikovia divaricata, and their growth-promoting effects on S. divaricata were investigated. The potential of endophytic bacteria for development and utilization was mined. Method Endophytic bacteria were isolated from healthy S. divaricata roots by dilution coating method and growth-promoting bacteria with nitrogen fixation, phosphorus solubilization, IAA production and siderophore production were screened by selective medium. The growth-promoting strains were classified by morphological, physiological and biochemical identification and 16S rRNA analysis. The colonization ability of endophytic bacteria in soil and its effect on the growth of S. divaricata were investigated based on antibiotic labeling method and pot experiment. Result A total of 202 endophytic bacteria were isolated from the roots of S. divaricata, 26 strains had the ability of nitrogen fixation, 21 strains had the ability to dissolve phosphorus, 24 strains had the ability to produce IAA, and 27 strains had the ability to produce siderophores. Two strains of endophytic bacteria MX-56 and MX-31 had three or more different growth-promoting functions, were identified as Klebsiella michiganensis and Pseudomonas koreensis. Two rifampicin-resistant strains stably colonized in the rhizosphere soil of S. divaricata. In the pot experiment, compared with CK, MX-56 had the most significant effect on plant height, root length, root diameter, aboveground fresh weight, root fresh weight and root dry weight of S. divaricata, increased 13.47%, 43.04%, 42.75%, 31.43%, 63.21% and 77.12% (P<0.05). Compared to CK, the total contents of two chromones in S. divaricata plants treated with strain MX-31 and MX-56 respectively increased by 33.72% and 30.23%. Conclusion In this study, two strains of P. koreaensis MX-31 and K. michiganensis MX-56 with good growth-promoting effect are isolated and screened from healthy S. divaricata, it may significantly increase the biomass and the content of effective components of S. divaricata.

Key words: Saposhnikovia divaricata, endophytic bacteria, Klebsiella michiganensis, Pseudomonas koreensis, growth promotion

孙孟雪, 张艺潆, 徐鹏, 孙卓, 王云贺, 韩忠明. 防风内生细菌的分离鉴定及其促生特性分析[J]. 生物技术通报, 2025, 41(7): 299-311.

SUN Meng-xue, ZHANG Yi-ying, Xu Peng, SUN Zhuo, WANG Yun-he, HAN Zhong-ming. Isolation and Identification of Endophytic Bacteria from Saposhnikovia divaricata and Analysis of Its Growth-promoting Characteristics[J]. Biotechnology Bulletin, 2025, 41(7): 299-311.

图/表 17

图1 防风内生细菌固氮效果检测

Fig. 1 Detection of nitrogen-fixing effect of endophytic bacteria in S. divaricata

图2 防风固氮菌固氮酶活性图中不同字母代表显著性差异（P<0.01），下同

Fig. 2 Nitrogenase activity of nitrogen-fixing bacteria in S. divaricataDifferent letters in the figure indicate significant differences (P<0.01), the same below

图3 防风内生细菌溶解无机磷效果检测

Fig. 3 Effect of endophytic bacteria in S. divaricata on inorganic phosphorus-dissolving

图4 防风内生细菌溶解无机磷能力测定

Fig. 4 Determination of endophytic bacteria on inorganic phosphorus-dissolving ability in S. divaricata

图5 防风内生细菌溶解有机磷效果检测

Fig. 5 Effect of endophytic bacteria on organophosphorus-dissolving in S. divaricata

图6 防风内生细菌溶解有机磷能力测定

Fig. 6 Determination of endophytic bacteria on organophosphorus-dissolving ability in S. divaricata

图7 菌株产IAA定性染色效果

Fig. 7 Qualitative staining effect of IAA-producing strain

图8 防风内生菌产IAA能力测定

Fig. 8 Determination of endophytic strain on IAA-producing capacity in S. divaricata

图9 防风内生细菌产铁载体效果

Fig. 9 Effect of endophytic bacteria on siderophore-producing of S. divaricata

图10 防风内生细菌产铁载体能力测定

Fig. 10 Determination of endophytic bacteria on siderophore-producing ability in S. divaricata

图11 内生细菌菌株形态与革兰氏染色观察结果A：MX-56在NA培养基上的菌落形态；B：MX-56革兰氏染色显微观察图片；C：MX-31在NA培养基上的菌落形态；D：MX-31革兰氏染色显微观察图片。显微观察图片为徕卡DM 2500 100X镜拍摄后LAS X软件放大35%，未改变其分辨率

Fig. 11 Morphological and Gram staining observations of endophytic bacterial strainsA: Colony morphology of MX-56 in NA medium. B: Microscopic observation of MX-56 Gram staining. C: Colony morphology of MX-31 in in NA medium. D: Microscopic observation of MX-31 Gram staining; The microscopic image was taken with a Leica DM 2500 100X lens and magnified 35% by LAS X software without changing the resolution

图12 基于16S rRNA基因序列构建的菌株MX-31和MX-56系统发育树

Fig. 12 Phylogenetic tree of strain MX-31 and MX-56 based on 16S rRNA gene sequences

图13 防风生长形态图

Fig. 13 Growth morphology of S. divaricata

表1 内生细菌的生理生化鉴定结果

Table 1 Results of physiological and biochemical identification of endophytic bacteria

Item	MX-56	MX-31
木糖	+	+
葡萄糖分解	+	-
L-阿拉伯糖	+	+
D-甘露醇	-	+
甲基红试验	-	-
V-P测试	+	-
OF试验	氧化型	氧化型
硫化氢产生	-	-
明胶水解	-	-
淀粉水解	-	-
氧化酶	-	-
硝酸盐还原	+	-

表2 标记菌株在土壤中的定殖数量

Table 2 Amount of colonizing bacteria of marked strains in soil

标记菌株 Strain	定殖数量 Colonization quantity （1×10⁴ CFU/g）
标记菌株 Strain	0 d	4 d	7 d	10 d	15 d	20 d	30 d
MX-31	1 000	343.33±14.53b	172.67±4.63c	20±0.58e	513.33±12.02a	118.33±4.41d	45±2.89e
MX-56	1 000	245±2.89b	20.33±0.33e	510±11.55a	224.3±4.48c	108.6±1.45d	26.7±0.57e

表3 不同菌处理对防风形态指标的影响

Table 3 Effect of different bacterial treatments on the morphological indexes of S. divaricata

菌株

Strain

株高

Plant height （cm）

根长

Root length （cm）

根粗

Root diameter （mm）

地上鲜重

Shoot fresh weight （g）

根鲜重

Root fresh weight （g）

根干重

Root dry weight （g）

表4 不同菌处理对防风升麻素苷和5-O-甲基维斯阿米醇苷含量的影响

Table 4 Effects of different treatments on the contents of prim-O-glucosylcimifugin and 5-O-methylvisaminide

处理

Treatments

升麻素苷含量

Content of prim-O-glucosylcimifugin （%）

5-O-甲基维斯阿米醇苷含量

Content of 5-O-methylvisaminide （%）

药典规定总含量

Total content specified in pharmacopoeia （%）

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