Phylogenetic diversity of phosphate solubilizing bacteria

Title: Phylogenetic diversity of phosphate solubilizing bacteria from P-rich soil in the Dianchi Lake drainage area and their tricalcium phosphate solubilizing abilities

Authors: Ma Li1 ♦, Yang Pei Xiang1◆，Su Hongyan2, Chen Ming Hui1, Xi Jia Qin3, He Feng1,

Duan Chang Qun1, Mo Ming He1

( 1:Laboratory for Conservation and Utilization of Bio-Resources & Key Laboratory for Microbial Resources of the Ministry of Education, Yunnan University, Kunming, 650091, P. R. China;

Department of Biology and Chemistry, Dali College Dali 671000, P. R. China;

Zhengzhou Tobacco Research Institute of CNTC, Zhengzhou，450001, P. R. China)

Abstract Isolation and characterization of PSB has been extensively studied in field soils and plant rhizosphereic soils more than a century ago. However, limited researches on the naturally colonized PSB in P-rich soils have been undertaken. This study is a first to analyze the phylogenetic diversity of a PSB community isolated from P-rich soils in the Dianchi Lake drainage area, southwestern of China and evaluate their TCP activity.

Firstly, 100 soil samples were collected and 1,328 bacteria strains were isolated, among these, 377 (28.39%) isolates showing different degrees of tricalcium phosphate (Ca3(PO4)2)-solubilizing activities isolated from NBRIP plates and bromophenol blue assay were screened as PSB strains. A total of 123 PSB isolates showing relative high TCP activity in quantitative assay according to their P content >54.00 mg/L, were selected for cloning of 16S rRNA gene sequences. These clones were further analyzed by restriction fragment length polymorphism (RFLP) and 32 unique RFLP patterns were obtained with three restriction enzymes. Of these, 62 representative clones possessing distinct RFLP patterns selected for subsequent 16S rRNA sequencing and phylogenetic analysis. The phylogenetic trees showed that all the sequences were clustered into 3 divisions of bacteria, namely Proterobacteria containing 10 genera in beta and gamma subdivision, Firmicutes containing 2 genera, Actinobacteria containing 1 genus. Among these, twenty-six RFLP patterns representing 107 clones were closely to the members of the phylum of Proterobacteria, which was the most abundant group. Our analysis showed that the diversity of PSB screened from P-rich soils of Zhujianzui phosphate mine was diverse not only the richness of the bacterial genus but as well in members of each bacterial species. This research will extend our understanding of PSB diversity in P-rich soils and expected to lead to the development of an environmental-friendly biophosphate fertilizer prepared by screened PSB strains as targets.

Key words Phylogenetic diversity, phosphate solubilizing bacteria, P-rich soil, phosphate solubilizing abilities Introduction

Dianchi Lake, the sixth largest freshwater lake in China, located in Kunming, Yunnan Province, southwest of China. Basin of Dianchi Lake is famous P-rich zone (PRZ) and also is one of the biggest bases of P mine in China (Luo et al 2004). The lake is reputed to be the Mother Lake of Kunming City for its vital functions to regulate climate, control flood, supply the agro-industrial water and partial drinking water. However, most bodies of the lake have lost their water function for years due to eutrophication. Currently, Dianchi Lake is under threat on an accelerated eutrophication as a consequence of urbanization and industrialization of this city (Wu

♦ These authors contributed equally to this work.