Effects of Klebsiella michiganensis LDS17 on Codonopsis pilosula growth, rhizosphere soil enzyme activities, and microflora, and genome-wide analysis of plant growth-promoting genes

Abstract

Codonopsis pilosula is a perennial herbaceous liana with medicinal value. It is critical to promote Codonopsis pilosula growth through effective and sustainable methods, and the use of plant growth-promoting bacteria (PGPB) is a promising candidate. In this study, we isolated a PGPB, Klebsiella michiganensis LDS17, that produced a highly active 1-aminocyclopropane-1-carboxylate deaminase from the Codonopsis pilosula rhizosphere. The strain exhibited multiple plant growth-promoting properties. The antagonistic activity of strain LDS17 against eight phytopathogenic fungi was investigated, and the results showed that strain LDS17 had obvious antagonistic effects on Rhizoctonia solani, Colletotrichum camelliae, Cytospora chrysosperma, and Phomopsis macrospore with growth inhibition rates of 54.22%, 49.41%, 48.89%, and 41.11%, respectively. Inoculation of strain LDS17 not only significantly increased the growth of Codonopsis pilosula seedlings but also increased the invertase and urease activities, the number of culturable bacteria, actinomycetes, and fungi, as well as the functional diversity of microbial communities in the rhizosphere soil of the seedlings. Heavy metal (HM) resistance tests showed that LDS17 is resistant to copper, zinc, and nickel. Whole-genome analysis of strain LDS17 revealed the genes involved in IAA production, siderophore synthesis, nitrogen fixation, P solubilization, and HM resistance. We further identified a gene (koyR) encoding a plant-responsive LuxR solo in the LDS17 genome. Klebsiella michiganensis LDS17 may therefore be useful in microbial fertilizers for Codonopsis pilosula. The identification of genes related to plant growth and HM resistance provides an important foundation for future analyses of the molecular mechanisms underlying the plant growth promotion and HM resistance of LDS17.

Importance: We comprehensively evaluated the plant growth-promoting characteristics and heavy metal (HM) resistance ability of the LDS17 strain, as well as the effects of strain LDS17 inoculation on the Codonopsis pilosula seedling growth and the soil qualities in the Codonopsis pilosula rhizosphere. We conducted whole-genome analysis and identified lots of genes and gene clusters contributing to plant-beneficial functions and HM resistance, which is critical for further elucidating the plant growth-promoting mechanism of strain LDS17 and expanding its application in the development of plant growth-promoting agents used in the environment under HM stress.

Keywords: ACC deaminase; Klebsiella michiganensis; heavy metal resistance; microbial functional diversity; plant growth-promoting bacteria; plant growth-promoting property; soil enzyme activity; whole-genome analysis.

Fig 1

Scanning electron microscope photo of Klebsiella michiganensis LDS17 isolated from Codonopsis pilosula rhizosphere.

Fig 2

The abilities of Klebsiella michiganensis LDS17 to dissolve insoluble P by determining the soluble P concentration in the culture supernatant of LDS17 (A), produce siderophore on the CAS agar plate (B), and inhibit plant pathogenic fungi (C and D). (A) Different letters in the column indicate significant differences (P < 0.05). (D) Values are means ± SD, different letters indicate significant differences (P < 0.05).

Fig 3

Inoculation effects of Klebsiella michiganensis LDS17 on Codonopsis pilosula seedlings. (A) Growth of Codonopsis pilosula seedlings after Klebsiella michiganensis LDS17 inoculation. (B–E) Seedling height, ground diameter, biomass, and chlorophyll content of Codonopsis pilosula seedlings after Klebsiella michiganensis LDS17 inoculation. Different letters in the column indicate significant differences (P < 0.05).

Fig 4

Soil enzyme activities in Codonopsis pilosula rhizosphere soil after Klebsiella michiganensis LDS17 inoculation. (A) Invertase activity. (B) Urease activity. (C) Catalase activity. Different letters in the column indicate significant differences (P < 0.05).

Fig 5

Culturable microorganism numbers in Codonopsis pilosula rhizosphere soil after Klebsiella michiganensis LDS17 inoculation. (A) Culturable bacteria. (B) Culturable actinomycetes. (C) Culturable fungi. Different letters in the column indicate significant differences (P < 0.05).

Fig 6

Klebsiella michiganensis LDS17 genome circle map of Klebsiella michiganensis LDS17. From outside to inside, the circle is represented as follows: circle 1: genome size; circles 2 and 3: gene distribution in positive and negative chains; circle 4: ncRNA (black), tRNA (blue), and rRNA (red); circle 5: GC ratio; and circle 6: GC skew.

Fig 7

Phylogenetic tree based on housekeeping genes recA, gyrB, rpoA, rpoB, rpoC, and rpoD indicates the phylogenetic position of the strain LDS17 and its relationship with related species. The number on each node represents the bootstrap value (1,000 replications). The GenBank accession numbers of the housekeeping genes are shown in Table S3.

Fig 8

Gene clusters associated with plant growth promotion and HM resistance.

Fig 9

Speculative pattern diagram of plant growth promotion and HM resistance mechanisms in Klebsiella michiganensis LDS17.

Fig 10

The growth curves of Klebsiella michiganensis LDS17 in LB media containing various concentrations of HM salts. (A) CuSO₄; (B) ZnSO₄; and (C) NiCl₂.

Fig 11

Amino acid sequence alignment of LuxR solo family proteins. The source of each LuxR and its corresponding gene accession number are as follows. XccR: Xanthomonas campestris pv. campestris 8004, XC_1295; XagR: Xanthomonas axonopodis pv. glycines 12-2, A9D66_16010; OryR: Xanthomonas oryzae pv. oryzae PXO86, AZ54_18140; XocR: Xanthomonas oryzae pv. oryzicola RS105, ACU12_06325; PsoR: Pseudomonas fluorescens Pf5, PFL_5298; NesR: Sinorhizobium meliloti 1021, SMc04032; PipR: Pseudomonas sp. GM79, PMI36_04623; PDO_04282: Rhizobium sp. PDO1-076, PDO_04282; PMI03_04239: Rhizobium sp. AP16, PMI03_04239; PMI11_06415: Rhizobium sp. CF142, PMI11_06415; PMI09_05533: Rhizobium sp. CF122, PMI09_05533. All of the above belong to LuxR solos in PAB. LuxR: Aliivibrio fischeri ES114, VF_A0925; TraR: Agrobacterium tumefaciens C58, Atu6134. Both LuxR and TraR are typical LuxR in bacterial QS systems.

Fig 12

Gene arrangement on both sides of koyR in Klebsiella michiganensis LDS17.