Isolation and identification of bacteria from root nodules of philippine legumes using 16S rRNA gene sequencing

Issue Date

3-2017

Abstract

A total of 39 different species of leguminous and nodule-forming plants were collected from different places in the Philippines. From this total, 364 bacterial colonies were isolated using rhizobium medium amended with Congo red and Kabicidin™. Rhizobial colonies were selected, purified and partially sequenced (620-710 base pairs, bp) using universal primers (8F and 1510R) for the 16S rRNA gene. Basic local alignment search tool (BLAST) similarities revealed that majority (92%) of the bacterial isolates and the six probable novel species were identified as members of the order Rhizobiales of the class Alphaproteobacteria and few as members of the classes Betaproteobacteria and Gammaproteobacteria. Majority (95%) of the leguminous plants were associated with the bacterial genera that are considered as nodulating and nitrogen-fixing or 'true' rhizobia. About 85% of the plants were associated with Rhizobium, Bradyrhizobium (62%), Ensifer (18%), Mesorhizobium (10%), and Burkholderia (3%). On the other hand, few (3-13%) leguminous plants were found to have minor associations with free-living nitrogen fixers such as Pleomorphomonas, Crabtreella, Herbaspirillum and Labrys. These free-living nitrogen fixers had been reported in previous studies as plant root endophytes incapable of inducing nodulation (Baldani et al. 1986; Elbeltagy et al. 2001; Valverde et al. 2003; Xie and Yokota 2005a, 2005b, 2006; Chou et al. 2007; Madhaiyan et al. 2013). Among the collected leguminous plants, root nodules of L. leucocephala and D. scorpiurus were found to host several genera of rhizobia. Rhizobium, Bradyrhizobium, Mesorhizobium and Ensifer were isolated from their root nodules obtained from different regions in the Philippines. Leguminous plants, like S. sesban and different species of Mimosa, hosted the species of Rhizobium. The almost complete sequences (∼1370 bp) of the 16S rRNA genes of the six probable novel isolates revealed 89.5-98.8% sequence similarities with known type species of rhizobia and phylogenetically within the order Rhizobiales, more specifically as members of the heterogenous family Rhizobiaceae. Strains designated as P5b, P-Ab, 56b, M30a, ELS-4, M9cR1, and T25a were isolated from the root nodules of Pterocarpus indicus Willd., Desmodium triflorum (L.) DC, Desmodium scorpiurus (Sw.) Desv., Aeschynomene indica L., Desmodium stryracifolium Merr., Cajanus cajan (L.) Millsp., and Vigna radiata (L.) R. Wilczeck., respectively. These nodule-forming plants were collected from different places in the Philippines. The distinct phenotypic features in terms of their utilization of lipids, amino acids and carbohydrates compared with the closely related Rhizobium phaseoli NBRC 14785T and Rhizobium leguminosarum IAM 12609T suggest that they are probable novel species of Rhizobium. The 99.9% 16S rRNA gene sequence similarity and the exact similarities in the phenotypic characteristics of strains M9cR1 and T25a suggest that they belong to a single probable novel strain of Rhizobium.

Source or Periodical Title

Philippine Agricultural Scientist

ISSN

0031-7454

Volume

100

Issue

1

Page

103-117

Document Type

Article

Physical Description

illustrations, tables

Language

English

Subject

16S rRNA, Leguminous plants, Novel species, Rhizobia, Root nodules

Digital Copy

yes

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