Yong-Xiu Xing

Endophytic nitrogen-fixing Klebsiella variicola strain DX120E promotes sugarcane growth

Klebsiella variicola bacteria are found in association with plants. Little is known about their colonization patterns, roles, and mechanisms during association with the plant hosts. Here, we identified a nitrogen-fixing bacterium, DX120E, which was isolated from surface-sterilized roots of the ROC22 sugarcane cultivar, as K. variicola by phylogenetic analyses of its 16S rRNA gene, RNA polymerase β-subunit gene, and DNA gyrase subunit A gene sequences. gfp-tagged DX120E was found to colonize at the roots and aerial parts of micropropagated sugarcane plantlets by fluorescence microscopy and confocal microscopy. DX120E was able to survive in soils and colonize in root epidermal cells, intercellular spaces in root cortices, and leaf mesophyll and vascular tissues. DX120E preferentially colonized at root maturation and elongation zones and entered roots via cracks at the emergence site of lateral roots and at disrupted epidermis. DX120E may penetrate root epidermal cells with the aid of their cellulose-degrading enzymes. 15N isotope dilution assay demonstrated that DX120E was able to fix N2 in association with ROC22 sugarcane plants under gnotobiotic condition. DX120E was also able to promote GT21 cultivar growth and plant uptake of N, P, and K under greenhouse condition. Together, this study for the first time shows that a K. variicola strain is able to colonize in its sugarcane plant hosts, to fix N2 in association with plants, and to promote plant growth.

Identification of a new nitrogen fixing endo-bacterium strain isolated from sugarcane stalk

A bacterial strain B8S, with nitrogen fixation ability, was isolated from the stalk of sugarcane variety RB867515 introduced from Brazil. This strain was identified based on morphological, physiological and biochemical characteristics and 16S rRNA gene sequence analysis. The 16S rRNA gene sequence analysis showed that the fragment sequence length was 1403bp. When compared withAgrobacterium tumefaciens (AY51349, AB116668.1 and AJ1307119) in GenBank, the homology of the nucleotides was 100%. Certain physiological and biochemical characteristics of new bacterium strain B8S were almost the same as that ofAgrobacterium tumefaciens. Based on its nitrogen fixation ability, the strain B8S was named asAgrobacterium diazotrophicus.

Genetic Diversity of Nitrogen-Fixing and Plant Growth Promoting Pseudomonas Species Isolated from Sugarcane Rhizosphere

The study was designed to isolate and characterize Pseudomonas spp. from sugarcane rhizosphere, and to evaluate their plant- growth- promoting (PGP) traits and nitrogenase activity. A biological nitrogen-fixing microbe has great potential to replace chemical fertilizers and be used as a targeted biofertilizer in a plant. A total of 100 isolates from sugarcane rhizosphere, belonging to different species, were isolated; from these, 30 isolates were selected on the basis of preliminary screening, for in vitro antagonistic activities against sugarcane pathogens and for various PGP traits, as well as nitrogenase activity. The production of IAA varied from 312.07 to 13.12 μg mL−1 in tryptophan supplemented medium, with higher production in AN15 and lower in CN20 strain. The estimation of ACC deaminase activity, strains CY4 and BA2 produced maximum and minimum activity of 77.0 and 15.13 μmoL mg−1 h−1. For nitrogenase activity among the studied strains, CoA6 fixed higher and AY1 fixed lower in amounts (108.30 and 6.16 μmoL C2H2 h−1 mL−1). All the strains were identified on the basis of 16S rRNA gene sequencing, and the phylogenetic diversity of the strains was analyzed. The results identified all strains as being similar to Pseudomonas spp. Polymerase chain reaction (PCR) amplification of nifH and antibiotic genes was suggestive that the amplified strains had the capability to fix nitrogen and possessed biocontrol activities. Genotypic comparisons of the strains were determined by BOX, ERIC, and REP PCR profile analysis. Out of all the screened isolates, CY4 (Pseudomonas koreensis) and CN11 (Pseudomonas entomophila) showed the most prominent PGP traits, as well as nitrogenase activity. Therefore, only these two strains were selected for further studies; Biolog profiling; colonization through green fluorescent protein (GFP)-tagged bacteria; and nifH gene expression using quantitative real-time polymerase chain reaction (qRT-PCR) analysis. The Biolog phenotypic profiling, which comprised utilization of C and N sources, and tolerance to osmolytes and pH, revealed the metabolic versatility of the selected strains. The colonization ability of the selected strains was evaluated by genetically tagging them with a constitutively expressing GFP-pPROBE-pTetr-OT plasmid. qRT-PCR results showed that both strains had the ability to express the nifH gene at 90 and 120 days, as compared to a control, in both sugarcane varieties GT11 and GXB9. Therefore, our isolated strains, P. koreensis and P. entomophila may be used as inoculums or in biofertilizer production for enhancing growth and nutrients, as well as for improving nitrogen levels, in sugarcane and other crops. The present study, to the best of our knowledge, is the first report on the diversity of Pseudomonas spp. associated with sugarcane in Guangxi, China.

Colonization of Nitrogen Fixing Bacterial Strain Klebsiella sp. DX120E Labeled with Green Fluorescent Protein(GFP) Gene within Sugarcane Plants

To explore the invasion patterns of N2-fixing bacteria strain Klebsiella sp. DX120E and its colonization in sugarcane, and to determine the optimum concentrations of inoculum, we inoculated tissue culture seedlings of two sugarcane varieties(GT21 and B8) with different concentrations of Klebsiella sp. bacteria strain DX120E labeled by green fluorescent protein(GFP) gene, and with ddH2O as control. The results showed that the strain DX120E propagated in root, leaf sheath and leaf of sugarcane seedlings, and bacteria quantities in different parts were root leaf sheath leaf. There were no differences in the maximum propagation quantity under various inoculating concentrations, with the optimum concentration of 1×102 CFU mL–1. Fluorescence microscope observation indicated that the bacteria could invade root through the surface cracks, generating sites of main root and lateral root, and root fracture, and intensively propagate in intercellular spaces and cells of the root, also transfer to mesophyll cells and vascular bundle sheath cells in leaf.

Identification and Efficiency of a Nitrogen-fixing Endophytic Actinobacterial Strain from Sugarcane

Sugarcane production in China mostly uses a high amount of chemical nitrogen (N) as compared to the average use of other countries. A microbe-based alternative may help to manage this problem in ecofriendly manner. Therefore, the present study focused on the characterization of a sugarcane growth-promoting nitrogen-fixing endophytic actinobacteria. The actinobacterial strain WZS021 was identified on the basis of morphology, biology and sequencing of 16S rRNA, gyrB and acdS gene amplification. The nitrogenase activity was determined with acetylene reduction assay and effects of different nutritional conditions were evaluated, and 15N isotope dilution method was used to determine the N-fixing capability under sugarcane seedlings in the pot. The strain was identified as Streptomyces chartreusis, by three genes (16S rRNA, gyrB and acdS), but nifH gene showed similarity with Bacillus and these results showed nitrogen fixation ability and 1-aminocyclopropane-1-carboxylate deaminase enzyme production ability at the molecular level. Moreover, strain WZS021 showed maximum nitrogenase enzyme production under pH 8 and sustained at higher temperature. Different carbon sources have less influence on nitrogenase activity. However, small amounts of N source can stimulate the nitrogenase activity, but after certain concentration it starts to inhibit the enzyme activity. Next to this, in the 15N isotope pot assay strain WZS021 significantly enhanced the dry weight of shoot and root by 20 and 75%, and N content increased by 30 and 36%, respectively. The N-fixation percentage of strain WZS021 under sugarcane seedlings was 9.16%. Overall, N-fixing ability of Streptomyces chartreusis strain WZS021 was characterized, and sugarcane biomass enhancement and substantial %Ndfa indicated that the promising strain could be applied in sugarcane as a growth promoter.