Phenotypic And Genotypic Diversity Of Paenibacillus Mucilaginosus

Paenibacillus mucilaginosus, a potassium- and phosphate-dissolving and nitrogen-fixing bacterium in soil, has been widely used as biological fertilizer in China. To study the vital factor affecting the function of P. mucilaginosus, 27 P. mucilaginosus strains from different origins and their diversities in phenotype, genotype were investigated in this study.Firstly, the ERIC-PCR was carried and the glycosyltransferase group I gene(GTFs I), playing a vital role in polysaccharide biosynthesis, was cloned in genotypic diversity analysis. In the ERIC-PCR test, the P. mucilaginosus strains produced rich and stable bands, which was different from that of strain P. edaphicus VKPM B-7517. Each strain produced 7^-14 bands, of which 6 were shared by many P. mucilaginosus strains. The ERIC pattern was analyzed with the NTSYS-pc software package, and the dendrogram showed that the P. mucilaginosus strains were grouped into 4 clusters according to their origins. Strain YC12 was located in one group by itself. The RAPD-PCR and gyr B gene gave the similar results. However, the numbers of diversity bands present in the ERIC-PCR fingerprintings were greater than that of RAPD-PCR analysis. This result was coincident with great distinguish ability of ERIC-PCR, which can be used to identify intraspecific species.Four functional gene fragments were cloned from 11 special RAPD amplification bands, which included the gene of GTFs I (960 bp). GTFs I was positively amplified from 26 P. mucilaginosus strains but not from strains YC12 and P. edaphicus VKPM B-7517. The results of ERIC-PCR and GTFs I gene analysis indicated that strain YC12 was different from other P. mucilaginosus strains, which need further research.Secondly, the phenotypic characteristics of Paenibacillus strains, including the sizes of capsules and colonies, productions of polysaccharides and acid, P and K dissolution, and nitrogen fixation capability, were investigated. The Paenibacillus strains produced 2.04^-13.12 mg·ml^-1 polysaccharide production. Twelve of the strains had a polysaccharide production greater than 9 mg·ml^-1, however, strains CGMCC 1.2326, KNP413,CGMCC 1.3714 and VKPM B-7517 produced only 2.04-4.72 mg·ml^-1 polysaccharide. The sizes of the capsules and colonies cultured on the nitrogen free medium for 5 d were 200.07-857.23μm2 and 2.00-5.72 mm, respectively. The fermented liquid viscosity varied between 45.40-8.77 mPa.s. Therefore, the correlations between polysaccharide levels and colony and capsule sizes, and the fermented liquid viscosity were calculated, and the correlation coefficients were 0.824, 0.714, 0.923 respectively. The acid production ability was different noticeably between the strains, which could be demonstrated from the pH decrease of 1.21-2.62 in the zymotic fluid. Strains SCS3, SCS10, YC8, YC15, YC17, YC1, YC12, YC13, KNP414 and VKPM B-7519 appeared to produce more acid.The capacities of P, and K-dissolution and N-fixation were also various between the strains. the concentration of soluble P and K increased by 0.05^-102.72 mg·l^-1 and 0.02-27.92 mg·l^-1, respectively. The maximum increase of P was observed in strain YC17 (102.72 mg·l^-1), followed by strains YC15, SCS16, SCS17, the concentration of soluble P increased were 96.09 mg·l^-1,84.95 mg·l^-1,79.49 mg·l^-1 respectively. The maximum increase of soluble K was observed in strain SCS10(27.92 mg·l^-1), followed by strains KNP414, YC9, YC15. A positive correlation was obtained between acid production and solubilized P and K, with correlation coefficients of 0.777 and 0.778, respectively. The correlation coefficient between polysaccharide production and solubilized P and K were 0.462 and 0.526, respectively. Obviously, both acid and polysaccharide could be the biomarker of P and K dissolution. All the strains appeared to fix nitrogen, and the maximum N-fixing was observed in strain VKPM B-7519 (18.28 mg·l^-1), followed by strains NYY2, YC17, YC15, YC10 and SCS10 with nitrogen increment of 8.15 mg·l^-1, 8.04 mg·l^-1, 7.64 mg·l^-1, 7.52 mg·l^-1 and 7.08 mg·l^-1, respectively. The correlation coefficients between N and polysaccharides and acid production were 0.290 and 0.395, respectively.Interestingly correlation analysis revealed a clear relationship between polysaccharide diversity and molecular (gyrB gene and RAPD) diversity. In the case of polysaccharide diversity, each of the two groups in the phylogenetic trees based on ERIC and RAPD was divided into two branches. One branch contained the strains with higher polysaccharide production, and the other branch contained the strains with lower polysaccharide production. In the case of acid production, a similar relationship can be found. The relationship between molecular markers and phenotypic traits is accordingly apparent.In conclusion, there were large diversities observed in the phenotype and genetic traits of P. mucilaginosus strains. The diversity of genotypic showed good correspondence with that of acid and polysaccharide productions. Colony, capsule sizes, and the fermented liquid viscosity may be the biomarker of polysaccharide production and thus could be used conveniently during preparatory isolation of functional strains on a large scale.