African Journal of Microbiology Research Vol. 5(30), pp. 5343-5353, 16 December, 2011 Available online at http://www.academicjournals.org/ajmr ISSN 1996-0808 ©2011 Academic Journals DOI: 10.5897/AJMR11.826

Full Length Research Paper

Multilocus sequence analysis of the from five woody legumes in southern China

Junkun Lu1, Lihua Kang1, Xinhua He2 and Daping Xu1*

1Research Institute of Tropical Forestry, Chinese Academy of Forestry, Guangzhou, Guangdong 510520, China.. 2State Centre of Excellence for Ecohydrology and School of Plant Biology, University of Western Australia, Crawley, WA 6009, Australia.

Accepted 9 November, 2011

A total of 11 rhizobial strains from five woody legumes (Acacia confusa, Dalbergia odorifera, Dalbergia fusca, Erythrophleum fordii and Pterocarpus macarocarpus) in Southern China were identified, using 16S rRNA gene sequence analysis and multilocus sequence analysis (MLSA) of three housekeeping genes (recA, glnII and dnaK). Among the 11 isolated rhizobia, seven were classified as Agrobacterium tumefaciens, Bradyrhizobium elkanii, Ensifer adhaerens and multihospitium and four were within the genus Rhizobium that might be novel species when considering their clustering independently in topology tree and relatively low recA gene sequence similarities (<94%) to the recognized species. In each gene phylogeny, the 16S rRNA gene was found to be incongruent with each house-keeping gene, indicated that this gene should not be used as a single marker in rhizobial . In addition, more robust topology trees were produced through the analysis of a concatenation of three housekeeping genes than through each housekeeping gene and 16S rRNA gene trees, with high bootstrap support (≥55%). Overall, our results support that the MLSA has higher discrimination potential in rhizobial taxonomy than the 16S rRNA gene and is suitable to estimate phylogenetic relationships among rhizobia species.

Key words: Multilocus sequence analysis, 16S rRNA, rhizobia and woody legume.

INTRODUCTION

Southern China is one of the most important result, investment has triggered large scale woody hardwood-timber produce regions in China. The legume plantations in southern China. biodiversity of precious woody legumes is exceptional in Legumes are able to establish mutualistic symbiosis this region and many of them, such as Dalbergia species with rhizobia to fix atmospheric dinitrogen into ammonia, a (e.g. Dalbergia balansae, Dalbergia fusca, Dalbergia primary source of nitrogen for plant growth, which reduces mimosoides, Dalbergia obtusifolia, Dalbergia odorifera the need for chemical N fertilizer in agriculture and etc), are precious because of the quality and beauty of the agro-forestry (Roux et al., 2009; Peoples et al., 1995; heartwood (Gao et al., 1994; Lin et al., 2009). However, Zahran, 1999). Studies of tropical or subtropical woody such precious heartwoods are seriously over-exploited legume species have revealed an unexpected diversity of due to the high demand by the furniture and construction symbionts as various new species of rhizobia were industries with the recent economic boom in China. As a observed in southern China (Chen et al., 1997; Gao et al., 1994; Lin et al., 2009). However, detailed information regarding taxonomic features of native rhizobia in southern China is not available. *Corresponding author. E-mail: [email protected]. Tel: +8620 The 16S rRNA gene has been widely applied to 87031626. Fax: +86 020 87031622. rhizobial taxonomy as a molecular marker (Kwon et al., 5344 Afr. J. Microbiol. Res.

Table 1. List of primers, and sizes of PCR products.

Gene Primer Sequence(5’-3’) Size (bp) Reference 1492R AGA GTT TGA TCA TGG CTC AG 16S rRNA 1450 DeLong (1992) 27F TAG GGT TAC CTT GTT ACG ACT T

recA-41F TTC GGC AAG GGM TCG RTSA TG recA 508 Islam et al. (2008) recA-640R ACA TSA CRC