新疆优良耐盐促生菌的筛选及其耐盐促生机制的研究    

 王琦琦    

 20192306010    

 公开    

 chi    

 082803    

 工学 - 农业工程 - 农业生物环境与能源工程    

 博士    

 工学博士    

 学术学位    

 2024    

 石河子大学    

 生命科学学院    

 农业工程    

 祝建波    

 祝建波    

 石河子大学    

 林忠平    

 2024-03-01    

 2024-05-05    

 Screening of Excellent Salt-tolerant and Plant Growth-Promoting Rhizobacteria in Xinjiang and Study of The Salt-tolerant and Plant Growth-Promoting Mechanism    

 根际耐盐促生菌（Salt-tolerant and plant growth-promoting rhizobacteria ; ST-PGPR）能通过自身活动促进植物对养分的吸收 ; 增强作物抗胁迫能力 ; 促进植物生长 ; 筛选能够适应新疆当地生态环境和作物类型 ; 具有较好的耐盐促生效果的ST-PGPR ; 为开发在新疆适用广泛的微生物菌肥具有重要意义 ; 前期从新疆盐生植物根际筛选获得的耐盐菌株Bacillus pumilus BG-5 ; Glutamicibacter mishrai 1-3-3 ; Staphylococcus succinus 1X1-3以及Enterobacter ludwigii MH-F具有一定的促生效果 ; 但在盐胁迫下促生菌株能产生哪些耐盐促生活性物质 ; 菌株对植物代谢是如何调控的 ; 菌株是否能协助植物塑造有益的根际微生物群落 ; 以及菌株提高植物耐盐性 ; 促进植物生长的作用机制尚不明确 ; 因此 ; 本研究在前期工作的基础上 ; 对其复配组合开展了以下研究工作并得出主要结果如下： （1）通过盆栽试验 ; 探究4株耐盐促生菌及复配组合F ; G ; H对新疆重要的经济作物玉米 ; 油葵和棉花生长及耐盐性的影响以及对不同土质类型的适应性 ; 结果显示：在盐胁迫下 ; 菌株接种能显著增加玉米 ; 油葵和棉花的地上和地下生物量 ; 促进脯氨酸的积累 ; 提高抗氧化酶活性 ; 但不同菌株对不同作物及土壤类型适应性不同 ; 菌株1-3-3在不同作物间的耐盐促生效果产生了物种差异 ; 对油葵的生长作用更好 ; BG-5促生效果受土壤类型影响较大 ; 更适用于中度盐碱沙壤土 ; 复合接种比单一接种耐盐促生效果更显著 ; 其中1X1-3以及复合处理F ; G的耐盐促生效果最显著且适应性最强 ; （2）通过3年棉花田间试验 ; 探究菌株及其复配组合与不同施用量（0% ; 60% ; 80%和100%）的化肥联合施用 ; 对棉花整个生长发育期农艺性状及产量的影响 ; 结果显示：菌株接种处理显著增加棉花苗期株高 ; 叶面积 ; 蕾期的蕾数 ; 花铃期的地上鲜重和铃数 ; 增加收获期产量 ; 菌与化肥的联合施用在田间种植的棉花中表现出极显著的生长优势 ; 其中1X1-3和复配组合F增产效果最好 ; 联合60%的化肥时产量就达到了只施用（100%）化肥的效果 ; 三年平均产量为6835.33和6299.95 kg/hm2 ; 分别增加了26.08%和16.77% ; 通过盆栽试验及棉花田间试验 ; 筛选出一株适应性强 ; 作用范围广 ; 且实际应用效果稳定的优良ST-PGPR菌株1X1-3 ; 进行耐盐促生机制的探究 ; （3）通过非靶向代谢组学 ; 探究盐胁迫下菌株1X1-3对棉花根系分泌物的响应 ; 进而挖掘潜在的耐盐促生活性物质 ; 分析其可能的直接耐盐促生作用 ; 结果显示：盐胁迫下棉花根系分泌物显著诱导了菌株1X1-3产生5-羟基阿魏酸酯 ; 邻苯二甲酸盐 ; 顺式-（高）2-乌头酸酯 ; 它们分别被注释到苯丙烷生物合成 ; 甲烷代谢 ; ABC转运蛋白等代谢途径中 ; 我们推断 ; 菌株1X1-3感应到了盐胁迫下棉花根系分泌物组分的变化 ; 通过5-羟基阿魏酸酯的富集调控了苯丙烷这类抗氧化物质的生物合成 ; 减少了外界活性氧对自身的损伤 ; 增强了对盐胁迫的适应性 ; 通过顺式-（高）2-乌头酸酯的显著富集调控了甲烷代谢 ; 促进了棉花根际养分循环 ; 有利于棉花在盐胁迫下生物量的积累 ; 通过邻苯二甲酸盐的积累调控了ABC转运蛋白活性 ; 促进了菌株1X1-3与棉花之间的物质交流及信号感知 ; 增强了植物与菌株1X1-3之间的协同作用 ; （4）通过广泛靶向代谢组学 ; 探究菌株1X1-3对棉花根系分泌物的影响 ; 分析该菌株对棉花耐盐促生的直接作用 ; 结果表明：盐胁迫下 ; 菌株1X1-3接种使棉花根系分泌物中黄酮类化合物 ; 精氨酸和脯氨酸生物合成代谢通路显著增强 ; 我们推断 ; 盐胁迫下菌株1X1-3通过增加棉花根系分泌物中3-异丙基-6-亚甲基-1-环己烯等抗菌物质抑制了病原菌的生长 ; 提高了有益微生物的竞争优势 ; 通过高车前素和木犀草素等黄酮类物质积累 ; 激活了棉花体内抗氧化酶活性 ; 减少盐胁迫造成的氧化损伤 ; 通过无水肌酸的积累 ; 调控了棉花精氨酸和脯氨酸代谢 ; 有利于维持了棉花细胞渗透平衡 ; 也为根际微生物提供了营养物质 ; 改善了棉花根际微生态环境 ; 有利于PGPR菌群的塑造 ; （5）通过宏基因组学方法 ; 探究菌株1X1-3对棉花根际微生物群落结构及功能的影响 ; 分析其耐盐促生的间接作用 ; 结果表明：菌株1X1-3接种显著增加了盐胁迫下棉花根际Deltaproteobacteria_bacterium ; Hyphomicrobiales_bacterium和Myxococcales_bacterium等有益微生物菌群的显著富集 ; 促进了棉花生物量的积累 ; 提高了棉花抗氧化酶活性 ; 菌株1X1-3接种也增加了棉花根际微生物相关网络的复杂性 ; 促进了微生物之间的紧密交流 ; 有利于有益微生物菌群的塑造 ; （6）通过多组学联合分析 ; 拟构建菌株1X1-3-棉花-根际微生物之间的相互作用模型 ; 分析菌株1X1-3可能的耐盐促生作用机制 ; 结果表明：盐胁迫下 ; 菌株1X1-3特异性产生5-羟基阿魏酸酯 ; 邻苯二甲酸盐等差异代谢物与棉花根系分泌物中&gamma ; -氨基丁酸 ; 氯化胆碱等呈极显著的正相关关系 ; 棉花根系分泌物中这些差异代谢物促进Bradyrhizobium ; Cystobacter ; Myxococcus等有益微生物以及参与双组分系统 ; 群体感应系统相关通路的K00990 ; K02035基因显著富集 ; 增加了植物根际微生物群落之间的相互交流及生态位的竞争 ; 通过与ABC转运蛋白的K02015相关的基因的显著富集 ; 促进了根际微生物之间的物质信息交流与能量交换 ; 有利于形成更稳定的微生物群落结构 ; 通过增加参与抗生素的生物合成相关通路的K01714基因的富集 ; 抑制了病原微生物的生长 ; 增加了有益微生物的竞争优势 ; 因此 ; 我们提出了菌株1X1-3耐盐促生作用模型：盐胁迫下 ; 菌株1X1-3接种 ; 产生的活性代谢物引发了棉花根系分泌物的代谢重编程 ; 微生物感知了这些棉花根系分泌物中这些特性代谢物后 ; 通过群体感应或双组分系统相关的基因选择性的表达 ; 促进了微生物间的信号交流 ; 招募了有益微生物菌群 ; 诱导了盐胁迫下微生物组的选择性“组装” ; 形成彼此有利的生态位及竞争优势 ; 最终塑造了稳定的微生物组 ; 结论：本研究通过探究4株ST-PGPR菌株对不同作物及不同类型土壤的耐盐促生效果的适应性及稳定性 ; 筛选出了一株作用范围广 ; 适应性强 ; 且促生效果稳定的菌株S. succinus 1X1-3 ; 为适应新疆本土的微生物菌剂的开发提供了物质基础 ; 通过多组学联合分析揭示了菌株1X1-3对棉花的耐盐促生的生理生态机制 ; 为深入了解盐胁迫下菌株1X1-3与棉花的耐盐促生机制提供了新见解 ; 为深入挖掘及开发菌株1X1-3潜在的功能提供了理论依据 ;     

 Salt-tolerant and plant growth-promoting rhizobacteria (ST-PGPR) can promote the absorption of nutrients by plants ; enhance the ability of crops to resist stress ; and promote plant growth through its own activities. It is of great significance to screen ST-PGPR that can adapt to the local ecological environment and crop types in Xinjiang and have good salt tolerance and growth promotion effect ; so as to develop microbial fertilizers that are widely applicable in Xinjiang. The ST-PGPR strains of Bacillus pumilus BG-5 ; Glutamicibacter mishrai 1-3-3 ; Staphylococcus succinus 1X1-3 ; and Enterobacter ludwigii MH-F were obtained through preliminary screening from halophytes rhizosphere in Xinjiang ; and have good salt tolerant and plant growth promoting effects. What salt tolerant and growth promoting substances can PGPR strain produce under salt stress ; how PGPR strains regulated plant metabolism ; whether PGPR strain can assist plants in shaping beneficial rhizosphere microbial-communities ; and the mechanisms by PGPR strain improve plant salt tolerance and promote growth are still unclear. Therefore ; based on the previous work ; this study conducted the following research on its compound combinations ; and the main results are as follows: (1) Through pot experiments ; The effects of four salt-tolerant growth-promoting bacteria and their compound combinations F ; G and H on the growth and salt tolerance of maize ; oil sunflower and cotton in Xinjiang ; as well as their adaptability to different soil types were investigated. The results showed that under salt stress ; strain inoculation significantly increased the aboveground and underground biomass ; promoted the accumulation of proline ; improved antioxidant enzyme activity ; and enhanced salt tolerance and promoted growth of corn ; oil sunflower ; and cotton. But different strains have different adaptability to different crops and soil types. Strain 1-3-3 showed differences in salt tolerance and growth promotion among different crops species ; resulting in better growth-promoting effects on sunflowers. The growth-promoting effect of BG-5 was greatly affected by soil type ; and it was more suitable for moderate saline-alkali soil. Co-inoculation has a more significant effect on promoting salt tolerance and growth than single inoculation. 1X1-3 and the co-inoculation F and G had the most significant salt tolerance and adaptability. (2) Through a 3-year cotton field experiment ; we combined the salt-tolerant strains with different application amounts of chemical fertilizers (0% ; 60% ; 80% ; and 100%) to investigate the effects of 4 salt-tolerant bacteria and their combination on the agronomic traits and yield throughout cotton growth and development period. The results showed: the bacteria strain inoculation treatment significantly increased the plant height ; leaf area during the seedling stage ; number of buds during the budding stage ; aboveground fresh weight and number of bolls during the flowering and bolling stage ; and increased the yield during the bolls opening period. The combined application of bacteria and chem-fertilizers has shown significant growth advantages in cotton grown in the field. Among them ; 1X1-3 and co-inoculation F have the best yield increasing effect ; and the yield of only (100%) chemical fertilizer was achieved when 60% chemical fertilizer was combined ; and the three-year average yield was 6835.33 and 6299.95 kg/hm2 ; an increase of 26.08% and 16.77% ; respectively. Therefore ; we selected strain 1X1-3 as an excellent ST-PGPR for subsequent mechanisms research on salt tolerant and plant growth promoting. Through pot experiments and cotton field experiments ; an excellent ST-PGPR strain 1X1-3 with strong adaptability ; wide range of action and stable practical application effect was screened out to explore the mechanism of salt tolerance and growth promotion. (3) Through non-targeted metabolomics ; we investigate the response of 1X1-3 to cotton root exudates under salt stress ; and then explore the potential functional metabolites for salt tolerant and plant growth-promoting. The results showed: under salt stress ; cotton root exudates significantly induce strain 1X1-3 to produce 5-hydroxyferulic acid esters ; phthalates ; and cis-(Homo)-2-aconitate ; which are annotated into metabolic pathways such as linoleic acid metabolism ; ABC transporters ; methane metabolism ; and phenylpropanoid biosynthesis. We infer that strain 1X1-3 sensed changes in the composition of cotton root exudates under salt stress and regulated phenylpropanoid biosynthesis through the enrichment of 5-hydroxyferulic acid esters ; clears the reactive oxygen species in the cotton rhizosphere ; and also reduces the damage of external reactive oxygen species to itself ; The significant enrichment of cis-(Homo)-2-aconitate regulated methane metabolism and improved the absorption of nutrients by cotton under salt stress ; The accumulation of phthalates regulates the activity of ABC transporters ; promoted material exchange and signal perception between strain 1X1-3 and cotton ; and enhanced the synergistic effect between cotton and strain 1X1-3. (4) Through wide target metabolomics ; the effect of strain 1X1-3 on cotton root exudates was explored ; and the direct effect of this strain on salt tolerance and growth promotion of cotton was analyzed. The results showed: inoculation with strain 1X1-3 significantly promoted the accumulation of flavonoids in cotton root exudates and enhanced the biosynthetic metabolic pathways of arginine and proline metabolism. We speculated that strain 1X1-3 inhibits the growth of pathogens by increasing the secretion of 3-isopropyl-6-methyl-1-cyclohexene in cotton roots under salt stress ; reducing the niche competitiveness of harmful microorganisms with PGPR ; enhancing the competitive advantage of beneficial microbe ; Strain 1X1-3 activated the antioxidant enzyme activity in cotton ; reduces oxidative damage caused by salt stress through accumulating flavonoids such as resveratrol and luteolin ; Through the accumulation of creatine ; the metabolism of arginine and proline in cotton is regulated ; which is conducive to maintaining the osmotic balance of cotton cells ; providing nutrients for rhizosphere microorganisms ; improving the microecological environment of cotton rhizosphere ; shaping the PGPR microbial community. (5) Through metagenomics ; we investigated the effect of 1X1-3 inoculation on the microbial community structure and function of cotton rhizosphere under salt stress. The results showed: inoculation of strain 1X1-3 significantly increased the enrichment of beneficial microbial communities in the cotton rhizosphere under salt stress ; such as Deltaproteobacteria_bacterium ; Hypomicrobiales_bacterium and Myxococces_bacterium ; it promoted the accumulation of cotton biomass and increased the activity of antioxidant enzymes in cotton. At the same time ; it also increased the complexity of the common linear network of cotton rhizosphere microorganisms ; promoted close communication between microorganisms ; and facilitated the shaping of beneficial microbial communities. (6) Through multi omics joint analysis ; we construct an interaction model between strain 1X1-3-cotton-rhizosphere microorganisms to analyze the possible mechanism of salt tolerance and growth promotion of strain 1X1-3. The results showed: the specific metabolites produced by strain 1X1-3 (5-hydroxyferulicr ; phthalate ; cis - (Homo) 2-aconitic acid ester) were significant positive correlated with root exudates of cotton under salt stress ; such as &gamma ; -aminobutyric acid ; Choline (Bitartrate) ; Choline (Chloride) and 1-oleoyl-SN-glycerol-3-phosphatecholine. The changed of metabolites of cotton root exudates significantly increased the enrichment of beneficial microbial communities such as Bradyrhizobium ; Cytoactor ; Myxococcus ; Ramlibactor ; and also led to the significant enrichment of K00990 and K02035 genes involved in the two-component system and quorum sensing system related pathways ; that increased mutual communication and competition for ecological niches between microbial communities in cotton rhizosphere ; The significant enrichment of K02015 genes related to ABC transporter promotes material information communication and energy exchange among rhizosphere microorganisms ; which is conducive to the formation of a more stable and beneficial community structure of PGPR ; The increasing of the enrichment of K01714 gene involved in the biosynthesis pathway of antibiotics ; that inhibited the growth of pathogenic microorganisms ; and increased the competitive advantage of beneficial microorganisms. Therefore ; we propose an interaction model for strain 1X1-3 to enhance plant salt tolerance and promote growth: the inoculation of strain 1X1-3 produces active metabolites that trigger metabolic reprogramming of cotton root exudates. Microorganisms perceive these characteristic metabolites in cotton root exudates and promote signal exchange between microorganisms through gene selectivity expression related to quorum sensing or two-component systems ; recruiting beneficial microbial communities and inducing selective " ; assembly" ; of microbial communities under salt stress ; forming mutually advantageous ecological niches and competitive advantages ; shapes a stable microbial community ultimately. Conclusion: our study explored the adaptability and stability of 4 ST-PGPR strains to salt tolerance and plant growth-promotion effects on different crops and different types of soils through potting experiments ; and combine with practical application in cotton fields ; S. succinus 1X1-3 we screened out because of it&rsquo ; s wide range of effect ; strong adaptability ; and stable growth promotion effects. We provided a material basis for the development of microbial agents adapted to the local conditions of Xinjiang. The physiological and ecological mechanisms of strain 1X1-3 in promoting salt tolerance and growth of cotton were revealed through multi omics joint analysis ; providing new insights into the salt tolerance and growth promoting mechanisms of strain 1X1-3 in cotton under salt stress ; and providing theoretical basis for further exploring and developing the potential functions of strain 1X1-3.     

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 2024-07-01