兰州大学机构库 >草地农业科技学院
梭梭和云杉根际溶磷细菌的特性研究及溶磷机制解析
Alternative TitleCharacteristics of Phosphate-Solubilizing Bacteria from the Rhizosphere of Haloxylon Ammodendron and Spruce and the Phosphate Solubilization Mechanism
李慧萍
Subtype博士
Thesis Advisor张金林 ; 赵祺
2023-05-31
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name农学博士
Degree Discipline草学
Keyword梭梭 Haloxylon ammodendron 云杉 Spruce 溶磷细菌 PSB 多相分类 Polyphasic taxonomy 菌株 AWH-NS6 Serratia sp. AWH-NS6 促生作用 Growth promotion 溶磷机制 Mechanism of mineral phosphate solubilization pqqE pqqE
Abstract

磷(P)是植物生长发育过程中不可缺少的大量营养元素之一。在天然土壤和农业土壤中,磷以无机和有机两种形式存在,且含量丰富。然而,植物对磷获取的有效性却非常低,唯一能为植物直接所用的只有正磷酸盐。因此,在农业生产中,磷养分的供需矛盾极为突出。作物的高产优质离不开磷肥的施用,但磷肥的过量施用导致磷矿石这类不可再生资源的浪费,也造成了一系列环境问题。溶磷细菌(Phosphate-Solubilizing Bacteria,PSB)作为一类具有溶解难溶性磷能力的细菌,可以提高土壤有效磷水平,进而促进植物生长。利用PSB帮助植物高效获取土壤中的磷已成为植物营养学研究的重点课题之一。不同宿主植物根际能招募并形成特色微生物。旱生植物梭梭是我国西北荒漠草原地区的优势建群种之一,拥有发达的根系,是极端荒漠环境中微生物的天然栖息地。定殖于梭梭根际的PSB菌株,在长期磷匮缺的荒漠环境中,高效转化利用难溶性磷的机制尚不明确。

本研究以磷匮缺荒漠生境中的优势植物(梭梭)和磷正常生境中的祁连山优势植物(云杉)根际细菌为研究对象,分析二者根际可培养细菌多样性,分离和筛选PSB野生菌种资源。在此基础上,分析比较不同PSB菌株的促生效果,获得了一株具有较强促生特性的高效溶磷菌株AWH-NS6;通过全基因组、转录组等组学分析,结合菌株溶磷关键基因突变体的构建,系统研究了菌株AWH-NS6的溶磷机制。取得如下主要研究结果:

两种优势植物根际土壤可培养细菌的多样性存在较大差异。祁连山地区的土壤肥力状况优于巴丹吉林沙漠,具有较高的全磷、速效磷、土壤含水量和土壤有机碳含量,土壤微生物多样性也较为丰富。从梭梭根际土壤中共分离到187株可培养细菌,门水平上主要分布于:厚壁菌门(Firmicutes)、γ-变形菌门(γ-Proteobacteria)、放线菌门(Actinobacteria)、α-变形菌门(α-Proteobacteria)和拟杆菌门(Bacteroidetes);属水平上主要分布于:芽孢杆菌属(Bacillus)、赖氨酸芽孢杆菌(Lysinibacillus)、链霉菌属(Streptomyces)、假单胞菌属(Pseudomonas)和副球菌属(Paracoccus)。从云杉根际土壤中共分离到203株可培养细菌,门水平上主要分布于:厚壁菌门、γ-变形菌门、放线菌门、α-变形菌门、拟杆菌门和β-变形菌门(β-Proteobacteria);属水平上主要分布于:假单胞菌属、黄杆菌属(Flavobacterium)、节杆菌属(Arthrobacter)、芽孢杆菌属和寡养单胞菌属 (Stenotrophomonas)等。

采用表型观察、生理生化测定、分子生物学鉴定、系统进化分析和基因组学分析等结合的多相分类方法,将一株分离自云杉根际(海拔3150米)的新种候选菌株LC-T2T进行了分类鉴定。根据综合分析结果,菌株LC-T2T属于类芽孢杆菌属(Paenibacillus),命名为Paenibacillus monticola LC-T2T。此外,菌株LC-T2T不仅是该属的新种代表菌株,还具有优良的促生性能,其挥发性有机化合物(Volatile organic compounds,VOCs)可促进模式植物拟南芥根系发育;接种菌株LC-T2T可显著促进豆科牧草白三叶(Trifolium repens L.)的生长。

从两地速效磷含量最高且可培养细菌较丰富的根际土壤样品中共分离到18株PSB菌株,分布于9个种。平板定性结果初步表明:菌株AWH-NS6、LT-4、LT-T17、LT-A20、F1-P7和LT-0具有较强的溶磷能力。其中,菌株AWH-NS6分离自梭梭根际,属于沙雷氏菌属(Serratia),其余菌株分离自云杉根际土壤,属于假单胞菌属。进一步采用液体定量法筛选得到5株菌株AWH-NS6、LT-4、LT-T17、LT-A20和F1-P7,均具有较强溶磷、分泌吲哚-3-乙酸(Indole-3-acetic acid,IAA)和固氮能力。植物接种实验结果表明,上述5株PSB菌株均不同程度促进了紫花苜蓿(Medicago sativa. L)的生长,以菌株AWH-NS6的效果最佳。

采用二代和三代测序技术相结合的方法,分析了菌株AWH-NS6的全基因组数据,初步挖掘得到了45个与促生相关和38个与非生物胁迫相关的功能基因。进一步通过系统进化和比较基因学分析,表明菌株AWH-NS6属于沙雷氏菌属的新种候选菌株,将其初步命名为Serratia sp. AWH-NS6。

外源可溶性磷(2 mM K2HPO4)能显著促进菌株AWH-NS6对Ca3(PO4)2的溶解(P < 0.05)。采用原核转录组学对不同处理组的菌株AWH-NS6进行分析,结果显示:添加2 mM K2HPO4时,在1和5 g·L-1 Ca3(PO4)2处理组中,菌株AWH-NS6分泌的有机酸所释放的H+有利于吸附态磷转化为有效磷,该过程受硫代谢的影响。在异化硫酸盐还原(Dissimilatory sulfate reduction,DSR)过程中,一些有机物质被氧化,相关基因上调表达,导致pH降低,从而影响H+的释放和磷酸盐的溶解。然而,菌株AWH-NS6在15 g·L-1 Ca3(PO4)2处理组中的磷酸盐溶解和硫代谢均受到抑制。进一步通过加权基因共表达网络分析(Weighted gene co-expression network analysis,WGCNA)发现,同化硫酸盐还原(Assimilatory sulfate reduction,ASR) 过程中的半胱氨酸代谢途径在yellow模块中显著富集,而半胱氨酸基序是有机酸分泌相关基因pqqE的重要组成部分。pqqE基因在5 g·L-1 Ca3(PO4)2处理组中均上调表达,表明其表达受中浓度Ca3(PO4)2的诱导。

采用分子克隆、同源重组和三亲本杂交实验,获得了菌株AWH-NS6有机酸相关基因pqqE的缺失突变体和回补菌株。pqqE基因的缺失与回补均减弱了菌株AWH-NS6对Ca3(PO4)2的溶解能力,也在一定程度上影响了菌株AWH-NS6原有的生理生化特性以及对资源的利用效率。植物接种实验表明,pqqE基因缺失突变体菌株对紫花苜蓿无明显促生作用,其促生效果显著低于野生型菌株AWH-NS6(P < 0.05);回补菌株恢复了部分溶解Ca3(PO4)2的能力,并且对紫花苜蓿有明显的促生效应。

以上结果表明,梭梭和云杉的根际微生物资源存在差异。梭梭根际蕴藏着优良的PSB菌株,云杉根际也蕴藏着优良的PGPR新种资源。对梭梭根际沙雷氏属PSB菌株AWH-NS6溶磷机制的系统解析可为极端环境中PSB菌株的溶磷机制研究提供理论依据,也可为磷高效微生物菌肥的开发提供优良的工程菌株。

Other Abstract

Phosphorus (P) is an essential macronutrient for plant growth, development and reproduction. In soils, P exists in various forms that can generally be categorized as insoluble organic phosphorus (Po) and insoluble inorganic phosphorus (Pi). However, the available P for plants is very low, and orthophosphates (H2PO4 or HPO42–) are the main forms of phosphorus that is taken up by plants. Therefore, the contradiction between supply and demand of P nutrients is turning increasingly prominent in agricultural production. Chemical phosphate fertilizer has traditionally been employed to obtain the high yield and quality of crops, but its application has been limited by the non-renewability of raw materials and the adverse influence on the ecological health of the environment. Phosphate solubilization is one of the important pathways of plant growth-promoting rhizobacteria (PGPR) to promote plant growth. How to fully and effectively use phosphate-solubilizing bacteria (PSB) to mobilize unavailable forms of soil P for plants has become a hot spot in the fields of plant nutrition and ecology. Different host plants can recruit and form characteristic microbes in the rhizosphere. Haloxylon ammodendron, a dominant species in China’s north-west desert, has superior drought resistance and barren tolerance. Its rhizosphere lacks soluble P for a long time, making it challenging to fulfil plant growth requirements. Moreover, the relevant mechanism of efficient conversion and utilization on phosphate dissolution by PSB in extreme habitats remains unclear.

In light of this, the aim of this study is to excavate and compare the culturable bacteria, mainly PSB resources, in the rhizosphere soil of Haloxylon ammodendron with P deficiency, collected at Badain Jaran Desert, Alxa region, Inner Mongolia; and the rhizosphere soil of spruce with P normal, the dominant tree species in typical forest ecosystem of Qilian Mountains. We obtained an excellent strain AWH-NS6 with the strong phosphate solubilization capability, belonging to the genus Serratia by growth promotion experiments. And the mechanism of P-solubilization of AWH-NS6 was systematically studied by genomics, transcriptomics and other omics analysis, combined with the construction of key gene mutants of strain AWH-NS6. The main findings are as follows:

There were great differences in culturable bacteria in rhizosphere soil of two dominant plants. The soil fertility of Qilian Mountains was much better than that of Alxa region, especially the total phosphorus (TP), available phosphorus (AP), soil water content (SWC) and soil organic carbon (SOC), which resulted in a greater abundance of soil microorganisms than that in Badain Jaran Desert. 187 strains of culturable bacteria were isolated from the rhizosphere soil of H. ammodendron, belonging to 5 phyla: Firmicutes、γ-Proteobacteria、Actinobacteria、α-Proteobacteria and Bacteroidetes, mainly distributed in Bacillus、Lysinibacillus、Streptomyces、Pseudomonas and Paracoccus etc. 203 strains of culturable bacteria were isolated from Qilian Mountain, belonging to 6 phyla: Firmicutes、γ-Proteobacteria、Actinobacteria、α-Proteobacteria、β-Proteobacteria and Bacteroidetes, mainly distributed in Pseudomonas, Flavobacterium, Arthrobacter, Bacillus, and Stenotrophomonas.

A novel PGPR species in the genus Paenibacillus was isolated from spruce forest at the height of 3,150 m in the Qilian Mountains based on phenotypic, phylogenetic, genotypic, chemotaxonomic data, and inoculation experiment, for which the name Paenibacillus monticola sp. nov. was proposed. The volatile organic compound (VOC) emitted from strain LC-T2T could significantly improve the growth of Arabidopsis. It is worth noting that strain LC-T2T could also significantly increase plant growth of legume crops like white clover.

9 species of 18 PSB strains were obtained from the rhizosphere soil with the highest content of AP and abundant culturable bacteria in the two regions. The plate qualitative analysis showed that strains AWH-NS6, LT-4, LT-T17, LT-A20, F1-P7 and LT-0 had strong phosphate-solubilizing ability. Among them, strain AWH-NS6 was isolated from the rhizosphere of H. ammodendron, belonging to the genus Serratia, while the other strains were isolated from spruce forests, belonging to the genus Pseudomonas. Inoculation of strain AWH-NS6, LT-4, LT-T17, LT-A20 and F1-P7 with strong growth promoting characteristics (phosphate solubilization, nitrogen fixation and secretion of IAA) can promote the growth of Medicago sativa in different degrees, especially strain AWH-NS6.

The whole genome data of strain AWH-NS6 were obtained and analyzed by the combination of second- and three-generation sequencing technology. 45 genes related to growth promotion and 38 functional genes related to abiotic stress were found in the genome. Strain AWH-NS6 was determined as Serratia sp. AWH-NS6 based on phylogenetic and comparative genomic analysis.

Adding 2 mM K2HPO4 can maximize the phosphate-solubilizing ability of strain AWH-NS6. Transcriptomic analysis of AWH-NS6 on Ca3(PO4)2 at different concentrations mediated by 2 mM K2HPO4 showed that: the liberation of H+ from weak acids secreted by strain AWH-NS6 can convert fixed P into accessible forms for plant in 1 and 5 g·L-1 Ca3(PO4)2 treatment groups, which affected by sulfur metabolism. Some organic substances were oxidized and related genes were upregulated, resulting in a decrease in pH, which affected the release of H+ and phosphate solubilization during dissimilatory sulfate reduction (DSR). However, the sulfur metabolism and phosphate solubilization were inhibited in the 15 g·L-1 Ca3(PO4)2 groups. Further, cysteine metabolism pathway was enriched in the yellow module based on weighted gene co-expression network analysis (WGCNA). And cysteine motif was a critical component of the organic acid-related gene pqqE. The expression of pqqE gene was up-regulated in the 5 g·L-1 Ca3(PO4)2 groups.

Knockout mutant and complementary strain of pqqE gene of strain AWH-NS6 were successfully constructed by molecular cloning, homologous recombination and tri-parental conjugation. The knock and complement of pqqE gene could affect the ability of strain AWH-NS6 to phosphate solubilization, the original physiological and biochemical characterizations, and the utilization efficiency of resources. Inoculation experiments demonstrated that the mutant strain had no obvious effect on the growth of Medicago sativa, and its growth promotion effect was significantly lower than that of wild type strain AWH-NS6 (P < 0.05). The complementary strain can partially restore the phosphate-solubilizing ability and can significantly increase plant growth of Medicago sativa (P < 0.05).

This study indicated that there are differences in the rhizosphere microbial resources of dominant plants between the two regions. There possess excellent PSB strains in the rhizosphere of H. ammodendron and outstanding PGPR resources in the rhizosphere of spruce. The molecular mechanism of phosphate solubilization of Serratia sp. AWH-NS6 is elucidated. The findings will provide the theoretical basis for the investigation of phosphate solubilization mechanism of PGPR in extreme environments, and explore a new pathway for efficient use of phosphorus in soil-plant system.

Subject Area植物与微生物互作
MOST Discipline Catalogue农学 - 草学
URL查看原文
Language中文
Other Code262010_120190900100
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/535664
Collection草地农业科技学院
Affiliation
兰州大学草地农业科技学院
Recommended Citation
GB/T 7714
李慧萍. 梭梭和云杉根际溶磷细菌的特性研究及溶磷机制解析[D]. 兰州. 兰州大学,2023.
Files in This Item:
There are no files associated with this item.
Related Services
Recommend this item
Bookmark
Usage statistics
Export to Endnote
Altmetrics Score
Google Scholar
Similar articles in Google Scholar
[李慧萍]'s Articles
Baidu academic
Similar articles in Baidu academic
[李慧萍]'s Articles
Bing Scholar
Similar articles in Bing Scholar
[李慧萍]'s Articles
Terms of Use
No data!
Social Bookmark/Share
No comment.
Items in the repository are protected by copyright, with all rights reserved, unless otherwise indicated.