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

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.

Key words:plant growth-promoting rhizobacteria;Cercidiphyllum japonicum;Burkholderia pyrrocinia;whole genome analysis;plant growth-promoting properties;heavy metal resistance