一株连香树根际促生细菌LWK2的分离鉴定及其全基因组序列分析

Isolation, identification, and whole-genome sequence analysis of a plant growth-promoting bacterium LWK2 from Cercidiphyllum japonicum rhizosphere

摘要:

【背景】植物根际促生细菌是一类位于植物根际并能对植物生长产生促进作用的有益菌，在微生物肥料领域具有重要的应用价值。【目的】对濒危植物连香树根际的植物根际促生细菌进行分离筛选和连香树接种效应评价，挑选对连香树生长促进作用最为显著的菌种进行促生特性分析、菌种鉴定及全基因组序列测定与促生相关基因分析。【方法】利用相应筛选培养基对连香树根际土壤中解有机磷、溶无机磷和解钾细菌进行分离筛选，通过根际接种验证各菌株对连香树实生苗的促生能力。从中选取促生作用最为显著的细菌，进行解钾能力、产吲哚乙酸(indole-3-acetic acid, IAA)和1-氨基环丙烷-1-羧酸(1-aminocyclopropane-1-carboxylate, ACC)脱氨酶能力测定。利用菌体形态观察、16S rRNA基因序列分析及全基因组序列的平均核苷酸一致性比对进行菌种鉴定。最后利用基因组功能注释和比较基因组学分析对该菌株中的植物促生及重金属抗性相关基因进行解析。【结果】从连香树根际土壤中共筛选得到3株解有机磷细菌、2株溶无机磷细菌和2株解钾细菌，其中解钾细菌LWK2对连香树实生苗的生长促进作用最为显著。该菌株能够产IAA和ACC脱氨酶。经鉴定菌株LWK2为吡咯伯克霍尔德氏菌(Burkholderia pyrrocinia)。LWK2全基因组包括2条染色体和1个质粒，大小分别为3 713 209、3 026 422和880 277 bp，GC含量分别为66.50%、66.37%和65.69%。其基因组包括IAA、铁载体、硝吡咯菌素合成，以及ACC脱氨酶和溶磷相关基因。上述植物促生相关基因在另外13株植物促生性伯克霍尔德氏菌中普遍存在，但每株菌所含有的IAA合成途径种类及催化各反应相关酶的种类却存在显著的多样性。此外，LWK2基因组还含有大量铜、钴-锌-镉和砷等重金属抗性基因，重金属抗性实验表明该菌株对CuSO₄、ZnSO₄、CdCl₂和CoCl₂均具有抗性，对这4种重金属盐的最高耐受浓度分别为4、10、3和1 mmol/L。【结论】分离自连香树根际的吡咯伯克霍尔德氏菌LWK2具有多种植物促生特性，能显著促进连香树实生苗的生长，对于濒危植物连香树微生物肥料的开发具有重要应用价值。LWK2全基因组序列的测定，丰富了目前为数不多的植物促生性吡咯伯克霍尔德氏菌基因组数据库，其基因组序列中植物促生及重金属抗性相关基因的分析，对进一步深入揭示该菌株的植物促生机制，扩展其在重金属胁迫环境下植物促生菌剂中的应用具有重要意义。

Abstract:

[Background] Plant growth-promoting rhizobacteria are beneficial bacteria inhabiting the rhizosphere of plants, with a promising prospect of application in the development of microbial fertilizers. [Objective] We isolated the plant growth-promoting rhizobacteria from the rhizosphere of the endangered plant Cercidiphyllum japonicum and evaluated their effects on C. japonicum seedlings. We then chose the strain with the greatest growth-promoting effect on C. japonicum seedlings, characterized its plant growth-promoting properties, and carried out strain identification, whole genome sequencing and plant growth-promoting related gene analysis. [Methods] The plant growth-promoting rhizobacteria capable of solubilizing organophosphate, inorganic phosphorus, and potassium were isolated with the corresponding selective media. The inoculation assay was employed and the strain with the greatest growth-promoting effect on C. japonicum seedlings was selected to verify the abilities for potassium solubilization, IAA and ACC deaminase production. The strain was identified based on cell morphology observation, 16S rRNA gene sequence analysis and the average nucleotide identity of the whole genome sequence. Furthermore, the genes conferring plant growth promotion and heavy metal resistance in the genome of this strain were identified by genome functional annotation and comparative genomics analysis. [Results] Three, two, and two strains capable of solubilizing organophosphate, inorganic phosphate, and potassium, respectively, were isolated from the rhizosphere soil of C. japonicum. Among them, the potassium-solubilizing bacterial strain LWK2 had the best performance of promoting the growth of C. japonicum seedlings. Strain LWK2 can produce IAA and ACC deaminase and was identified as a strain of Burkholderia pyrrocinia. The whole genome of LWK2 consisted of two chromosomes and a plasmid, with the lengths of 3 713 209, 3 026 422, and 880 277 bp and the GC proportions of 66.50%, 66.37%, and 65.69%, respectively. The genes associated with IAA, siderophore, pyrrolnitrin biosynthesis, ACC deaminase production, and phosphorus solubilization were identified in the LWK2 genome. These genes are ubiquitous in the other 13 strains of Burkholderia spp. with plant growth-promoting activities, while the IAA synthesis pathways and the enzymes catalyzing the reactions vary between different strains. In addition, the LWK2 genome carried the genes involved in the resistance to heavy metals, including copper, cobalt-zinc-cadmium, and arsenic. Heavy metal resistance experiments showed that LWK2 was resistant to CuSO₄, ZnSO₄, CdCl₂, and CoCl₂, with the maximum tolerance concentrations of 4, 10, 3, and 1 mmol/L for these four heavy metal salts, respectively.[Conclusion] B. pyrrocinia LWK2 isolated from C. japonicum rhizosphere has plant growth-promoting properties. It can significantly promote the growth of C. japonicum seedlings, demonstrating the potential of application in the development of microbial fertilizers for the endangered plant C. japonicum. The whole genome sequencing of LWK2 enriches the genome data of plant growth-promoting B. pyrrocinia. The identification of the genes associated with plant growth promotion and heavy metal resistance in the LWK2 genome is critical for further elucidating the plant growth-promoting mechanism of LWK2 and expanding its application in the development of plant growth-promoting agents used in the environment with heavy metal stress.