尾矿先锋植物根际溶磷菌的分离鉴定与其促生研究

doi:10.16258/j.cnki.1674-5906.2023.01.018

生态环境学报 ›› 2023, Vol. 32 ›› Issue (1): 166-174.DOI: 10.16258/j.cnki.1674-5906.2023.01.018

尾矿先锋植物根际溶磷菌的分离鉴定与其促生研究

杨瑞¹^,²(), 孙蔚旻²^,^*(), 李永斌², 郭丽芳², 焦念元¹^,^*()

1.河南科技大学农学院/河南省旱地农业工程技术研究中心，河南洛阳 471023
2.广东省科学院生态环境与土壤研究所/华南土壤污染控制与修复国家地方联合工程研究中心/广东省农业环境综合治理重点实验室，广东广州 510650

收稿日期:2022-10-24 出版日期:2023-01-18 发布日期:2023-04-06
通讯作者: 孙蔚旻（1979年生），男，博士，研究员，研究方向为类金属砷/锑与MTBE等的微生物转化机制研究。E-mail: wmsun@soil.gd.cn
*焦念元（1974年生），男，研究方向为间套作资源高效利用及生理生态机理研究。E-mail: jiaony1@163.com；
作者简介:杨瑞（1996年生），男，研究方向为生态修复研究。E-mail: 764005863@qq.com
基金资助:
国家自然科学基金项目(U21A2035);广东省科学院打造综合产业技术创新中心行动资金项目(2022GDASZH-2022010106);广东省科技计划项目(2020B1212060048);广东省高层次人才项目(2017GC010570)

Isolation, Identification and Plant Growth Promotion of Rhizosphere Phosphorus-dissolving Bacteria from Tailings Pioneer Plants

YANG Rui¹^,²(), SUN Weimin²^,^*(), LI Yongbin², GUO Lifang², JIAO Nianyuan¹^,^*()

1. College of Agriculture, Henan University of Science and Technology/Henan Dryland Agriculture Engineering Technology Research Center, Luoyang 471023, P. R. China
2. Institute of Eco-environmental and Soil Sciences, Guangdong Academy of Sciences/Institute of Eco-environmental and Soil Sciences, Guangdong Academy of Sciences/National-Regional Joint Engineering Research Center for Soil Pollution Control and Remediation in South China/Guangdong Key Laboratory of Integrated Agro-environmental Pollution Control and Management, Guangzhou 510650, P. R. China

Received:2022-10-24 Online:2023-01-18 Published:2023-04-06

摘要/Abstract

摘要：

尾矿重金属环境污染严重，由于有毒金属的浸出严重污染了附近的栖息地，矿山尾矿引起了越来越多的关注。然而，养分限制，特别是磷（P）缺乏，会阻碍植物生长，破坏尾矿的植物修复。溶磷菌能通过分泌质子、有机酸等方式把土壤中难溶性的无机磷活化，提高土壤有效磷含量，促进植物对磷的吸收利用。从湖南锡矿山和广东凡口两个尾矿的先锋植物根际土中采用解磷固体培养基筛选溶磷菌，分离得到5株具有溶磷特性的溶磷菌，根据16S rRNA基因序列分析，对分离出的溶磷菌分别命名为Gordonia sp. G2、Intrasporangium sp. G16、Pseudomonas sp. Y1、Pseudomonas sp. Y22和Bacillus sp. Y38。分离筛选出的5株溶磷菌PSI范围为3.14—5.01之间，其中G2溶磷能力最强，Y38相比其他菌株溶磷能力最弱。共有3个菌株G16、G2和Y22具备固氮酶活性，其中G16的固氮酶活性最高（6808.34 μmol·h^-1·mL^-1）。同时对菌株的最适pH研究表明，大部分菌株可以在中性偏酸性环境下生长良好。为进一步验证尾矿溶磷菌的植物促生潜力，进行盆栽试验，结果表明，与对照组相比，接种G2、G16、Y38显著促进了植物干质量和鲜质量、株高的增加，分别提高了196.7%、96.7%、80.0%，而接种Y1、Y38显著增加了植株磷含量，相比对照组显著提高了74.12%和30.25%。研究结果表明，尾矿溶磷菌具有较大的植物促生潜力，其中G2和Y1菌株在尾矿生态修复中发挥重要作用，有助于推动新型生物肥料的研究，为尾矿绿色生态修复提供新的途径。

关键词: 磷, 溶磷菌, 尾矿, 植物促生, 根际土, 金属抗性

Abstract:

Heavy metal pollution from mine tailings is a serious environmental problem. The leaching of toxic metals has severely contaminated nearby habitats, and mine tailings remediations have received increased attention. However, nutrient limitations, particularly phosphorus (P) deficiency, can impede plant growth and disrupt the plant restoration of tailings. Phosphate-solubilizing bacteria can activate insoluble inorganic phosphorus in soil by secreting protons and organic acids, which enhances phosphorus availability to plants during phytoremediation. In this study, phosphate-solubilizing bacteria were isolated from the rhizosphere soil of pioneer plants in tailings from the Xikuangshan and Fankou mines in Hunan and Guangdong, respectively. Five strains of phosphate-solubilizing bacteria were isolated by using a phosphorus- solubilizing solid medium. The five strains were named Gordonia sp. G2, Intrasporangium sp. G16, Pseudomonas sp. Y1, Pseudomonas sp. Y22, and Bacillus sp. Y38, respectively, based on their 16S rRNA sequences. The phosphorus solubilizing index (PSI) range of the five isolates was 3.14-5.01, with G2 exhibiting the strongest phosphate-solubilizing ability, and Y38 having the weakest ability compared to the other strains. Three strains (G16, G2 and Y22) exhibited nitrogenase activity, with G16 having the highest nitrogenase activity (6808.34 μmol·h^-1·mL^-1). The optimal pH range for most strains was found to be in the slightly acidic to neutral range. The pot experiments were conducted to further verify the plant-promoting potential of these phosphate-solubilizing bacteria. Compared to the control group, inoculation with G2, G16, and Y38 significantly increased plant dry weight and fresh weight, as well as plant height, with increases of 196.7%, 96.7%, and 80.0%, respectively. Inoculation with Y1 and Y38 significantly increased plant phosphorus content, with increases of 74.12% and 30.25%, respectively. The current study indicates that tailings phosphate-solubilizing bacteria have significant plant-promoting potentials, especially G2 and Y1 strains, which have beneficial contributions to plant growth under mine tailing conditions, and are potential bio-fertilizers for phytoremediation practices.

Key words: phosphorus, phosphorus solubilizing bacteria, tailings, plant promotion, rhizosphere soil, metal resistance

中图分类号:

X172

杨瑞, 孙蔚旻, 李永斌, 郭丽芳, 焦念元. 尾矿先锋植物根际溶磷菌的分离鉴定与其促生研究[J]. 生态环境学报, 2023, 32(1): 166-174.

YANG Rui, SUN Weimin, LI Yongbin, GUO Lifang, JIAO Nianyuan. Isolation, Identification and Plant Growth Promotion of Rhizosphere Phosphorus-dissolving Bacteria from Tailings Pioneer Plants[J]. Ecology and Environment, 2023, 32(1): 166-174.

图/表 11

表1 溶磷菌在LB固体培养基中的菌落特征

Table 1 Colony characteristics of phosphate solubilizing bacteria in LB solid medium

菌名	形态	颜色	表面形态
G2	圆形	淡黄色	湿润
G16	圆形	淡黄色	干燥
Y1	圆形	白色	湿润
Y22	圆形	淡黄色	湿润
Y38	圆形	白色	湿润

表2 所选溶磷菌生理和生物化学特性

Table 2 Physiological and biochemical characteristics of the selected phospholysis bacteria

菌名	固氮酶活性/ (μmol·h^-1·mL^-1)	产吲哚乙酸能力	产铁载体	溶磷圈/ cm
G2	584.81	+	+	5.01
G16	6808.34	+	-	4.03
Y1		+	-	4.57
Y22	4600.68	+	+	4.89
Y38		+	+	3.14

图1 溶磷菌水解圈

Figure 1 Phosphorus solubilizing bacteria hydrolysis circle

图2 不同pH对溶磷菌生长变化（OD600）的影响

Figure 2 Effect of pH on the growth of phosphorus solubilizing bacteria

表3 不同浓度As3+对菌株的生长影响

Table 3 Effect of heavy metal As3+ on the growth of strain

菌名	As³⁺的浓度/( mmol·L^-1)
菌名	0 (CK)	20	30	40	50	60
G2	++	++	++	++	+	-
G16	++	++	++	+	-	-
Y1	++	++	++	++	+	-
Y22	++	++	++	++	+	-
Y38	++	++	++	+	-	-

表4 基于16S rRNA基因序列的溶磷菌的鉴定

Table 4 Identification of phospholysis bacteria based on 16S rRNA gene sequences

菌名	类型	特性/%	登录号
G2	Gordonia amicalis (T3)	99.86	EU427321.1
G16	Intrasporangium calvum (DSM 43043)	98.73	NR_042185.1
Y1	Pseudomonas putida (F1-1-2)	100.00	KX349990.1
Y22	Pseudomonas monteilii (B45)	99.65	KT380512.1
Y38	Bacillus huizhouensis (JZY2-12)	99.79	MT071364.1

图3 基于16S rRNA序列的系统发育进化树

Figure 3 Phylogenetic evolutionary tree based on 16S rRNA sequences

图4 解磷菌对鬼针草促生效果

Figure 4 Effect of phosphorus solubilizing bacteria on growth of pilosa

图5 解磷菌对鬼针草生物量的影响数值代表重复的平均值（n=3）±标准偏差。“*”表示处理组与对照组统计学上的显著差异（P<0.05）。下同

Figure 5 Effect of phosphorus-disintegrating bacteria on biomass of pilosa

图6 解磷菌对鬼针草生物量的影响

Figure 6 Effect of phosphorus-disintegrating bacteria on biomass of pilosa

图7 解磷菌对鬼针草磷含量的影响

Figure 7 Effect of phosphorus-disintegrating bacteria on phosphorus content in pilosa

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尾矿先锋植物根际溶磷菌的分离鉴定与其促生研究

Isolation, Identification and Plant Growth Promotion of Rhizosphere Phosphorus-dissolving Bacteria from Tailings Pioneer Plants

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