Characterization and diversity of rhizobia nodulating selected tree legumes in Ghana

Symbiosis (2016) 69:89–99 DOI 10.1007/s13199-016-0383-1 Characterization and diversity of rhizobia nodulating selected tree legumes in Ghana Emmanuel Yaw Boakye 1 & Innocent Yao Dotse Lawson 1 & Seth Kofi Akyea Danso 1 & Samuel Kwame offei 1 Received: 22 April 2015 / Accepted: 5 February 2016 / Published online: 18 February 2016 # The Author(s) 2016. This article is published with open access at Springerlink.com Abstract The study was conducted to assess the characteristics and diversity of the rhizobia that nodulate some prominent tree legumes in three soils of Ghana. Five introduced and/or indigenous tree legumes were initially assessed for nodulation in three Ghanaian soils. After 12 weeks of growth in nursery pots the 200 rhizobial strains isolated from their nodules were characterized culturally, metabolically and phenotypically. Sixty of these isolates were selected randomly and their genotypic characteristics determined using PCR-RFLP of 16S rRNA and intergenic spacer (ITS) genes. Each tree legume was nodulated by isolates classified as fast or very fastgrowers or by isolates classified as slow- or very slowgrowers with 54 % of all the 200 isolates belonging to fastor very fast-growers. Morphologically, eighty five percent of the colonies formed on yeast extract mannitol agar were wet and gummy while 70 % were acid tolerant, i.e. they were able to grow at a pH of 3.5. Combined restriction of the 16S rRNA genes of the 60 rhizobial isolates with five restriction enzymes clearly distinguished seven different clusters at 80 % similarity level. The majority of A. lebbeck isolates were distinct from those of the Acacias and L. leucocephala. The M. thonningii isolates were related to L. leucocephala isolates. Simple PCR of the ITS DNA provided several distinct band sizes indicating great variation among the isolates and restriction of the ITS with three different enzymes did not yield many further differences. Molecular techniques revealed a great diversity among the rhizobia that nodulate tree legumes in the tropics * Emmanuel Yaw Boakye 1 College of Agriculture and Consumer Sciences, Department of Soil Science, University of Ghana Legon, P. O. Box 245, Legon, Ghana and this may explain why many introduced and/or indigenous trees are able to form nodules with indigenous rhizobia in this region. Keywords Characterization . Diversity . Rhizobium . Tree legumes 1 Introduction Rhizobia are usually defined as nitrogen-fixing soil bacteria capable of inducing the formation of root or stem nodules on leguminous plants in which atmospheric nitrogen is reduced to ammonia for the benefit of the plant. Although the majority of legumes form symbiosis with members of genera that belong to the class Alphaproteobacteria (Allorhizobium, Azorhizobium, Blastobacter, Bradyrhizobium, Devosia, Ensifer, Mesorhizobium, Methylobacterium, Rhizobium and Sinorhizobium), some legumes, such as those in the large genus Mimosa, are nodulated predominately by members of the class Betaproteobacteria in the genera Burkholderia and Cupriavidus (Gyaneshwar et al. 2011). However, a recent report by Bontemps et al. (2015) indicated that the endemic Mexican mimosas unlike their Brazillian counterparts were nodulated predominantly by Alphaproteobacteria from the genera Rhizobium and Ensifer. In the last few years, many studies investigating rhizobia isolated from tree legumes in Kenya and Sudan have revealed considerable phenotypic and genotypic diversity among strains, and several distinct groups have been identified and novel species described (Zhang et al. 1991; Odee et al. 1997, 2002; Nick et al. 1999; McInroy et al. 1999). These studies concluded that there is a large heterogeneity among the strains (Crow et al. 1981; De Lajudie et al. 1994; Dupuy et al. 1994; Jarvis 1983; Moreira et al. 1998; Zhang et al. 1991). This 90 indicates that trees can form nodules and fix nitrogen with several different groups of rhizobia (Crow et al. 1981; Jarvis 1983; Lindstrom et al. 1983; Padmonabhan et al. 1990). Of late, the assessment of diversity within rhizobial natural populations in various regions of the world has received increasing attention (Amann et al. 1995; Batzli et al. 1992; Brunel et al. 1996; Cartwright et al. 1995). Many attempts have been made to determine the actual composition and characteristics of indigenous strains isolated from different cultivated legumes (Chen et al. 1991; Crow et al. 1981) and also from less explored plants (Felsenstein 1985). The development of molecular genetic methods and the availability of sensitive and accurate PCR- based fingerprinting methods (Galtier et al. 1996; Gibson 1980; Gurtler 1993) has enabled the differentiation of closely related bacterial strains and the detection of high rhizobial diversity (Gurtler and Stanisich 1996; Jarvis 1983; Jarvis et al. 1982). In Ghana, with the exception of studies on cowpea rhizobia (Fening 1999) there is little knowledge of the diversity of rhizobia that nodulate other legumes especially tree legumes. The aim of the present study was to assess the phenotypic and genotypic characteristics of the rhizobia including their diversity that nodulate some indigenous and introduced tree legumes in Ghana. 2 Materials and methods 2.1 Soil and site characteristics The three soil types used for the studies were taken from the Accra plains (05o 39.6271 N, 0011.6191` W). The soils belong to the Toje, Hatso and Alajo series (Local Names) (Brammer 1967). The Toje series is classified as Rhodic lixiso, and the Hatso and Alajo series classified as Haplic lixisol and Calcic vertisol, respectively, according to FAO (2006). The three soils are widely cultivated by resource-poor farmers in the area and occur on the same soil catena with Toje series being at the top, Hatso and Alajo series being at the middle and the bottom slope, respectively. E.Y. Boakye et al. Ca, Mg, K and Na. Soil texture was determined based on particle size analysis using the modified Bouyoucos hydrometer method as describe by Day (1965). 2.3 Initial nodulation studies Seeds of one indigenous (Milletia thonningii) and four introduced (Albizia lebbeck, Acacia auriculiformis, Acacia mangium and Leucaena leucocephala) tree legumes were initially assessed for their nodulation potential in three different soil types by growing them in pots filled with two kilograms of each soil type for 12 weeks. Representative nodules were collected for rhizobia isolation. 2.4 Rhizobia enumeration in the three soils The population of indigenous rhizobia in the three different soils capable of nodulating the selected tree legumes (Albizia lebbeck, Acacia auriculiformis, Acacia mangium, Leucaena leucocephala and Milletia thonningii) were enumerated based on the most probable number (MPN) method (Vincent 1970) using plastic growth pouches (Weaver and Frederick 1972). Clean seeds of the tree legumes were surface sterilized in 70 % alcohol for 3 min and rinsed thoroughly in several chan (...truncated)