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Isolation and characterization of nitrogen-fixing bacteria nodulating phaseolus vulgaris in the lower rio grande valley of texas.

Article Excerpt
To establish a highly effective symbiosis between a legume plant and its bacterial symbiont it is essential to develop a successful program for the selection of superior strains to maximize production through [N.sub.2] fixation. Nitrogen-fixing bacteria from representative soil series of the Lower Rio Grande Valley region of southern Texas were examined for their symbiotic association with common 'Pinto' been (Phaseolus vulgaris L.). PCR-RFLP analyses of genes associated with nitrogen fixation indicated that the bacterial isolates belong to the recently described Rhizobium etli and none were identified as R. tropici. Examination of symbiotic responses of the rhizobial isolates under controlled, aseptic conditions, indicated that 20% produced effective nodula-tion, 35% partially effective, and the remaining 45% produced parastic or ineffective nodulation on the host legume. Strains UTPA-P 66, P82 and P80, each isolated from a different soil series, accounted for maximum plant response and nitrogen fixation activity. Although nodule morphology and size markedly varied among isolates, those with the highest tissue mass were associated with the highest production of school biomass. Electron micrographs of cross sections of 2-week old nodules indicated that effective bacterial cells were rod shaped, and more abundant than those enclosed in the peribacteroid membranes of ineffective nodules. To ensure adequate nodulation and [N.sub.2]-fixation, it is necessary to inoculate beans with locally isolated nitrogen-fixing bacteria, regardless of any previous history of bean cultivation.

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The inoculation of legume seeds with soil nitrogen-fixing bacteria (referred generically as 'rhizobia' or 'rhizobial cells') is a well-established technology of major economic and environmental importance. Natural populations of rhizobia can establish symbiotic associations with legumes. The success of the symbiosis, which is reflected by enhanced yield, depends largely on the performance of the rhizobial strains (Materon & Zibilske 2001). When needed, the symbiotic association could be optimized through seed inoculation (Materon & Ryan 1996). A well-nodulated common bean (Phaseolus vulgaris) crop can fix up to 100 kg of [N.sub.2] per haannually, which is equivalent to 208.3 kg of urea fertilizer (Somasegaran & Hoben 1994). This legume is cultivated in areas totaling 20,550 ha in the state of Texas (Smith & Anciso 2005). Most of this acreage is dedicated for commercial or processing purposes and approximately 60% of it is located in the Texas Winter Garden and in the High Plains region. The remaining production is obtained from small farms located in the southern part of the state (Smith & Anciso 2005).

Unfortunately, according to many researchers (Graham 1981; Graham et al. 2003), inoculation with introduced inoculatn rhizobial strains does not always lead to an abundant effective nodulation to secure high rates of nitrogen fixation and seed production (Graham 1981; Graham et al. 2002; Materon 1991; Materon & Zibilske 2001; Mrabet et al. 2005). This fact could be related to the promiscuity observed in P. vulgaris (cf. Michiels 1998) or to other limiting factors, like the high rate of nitrogen fertilizer used in intensive agriculture, which is particularly detrimental for this legume species (Materon & Ryan 1996).

In addition, this grain legume species also has a reputation for weakness in its ability to nodulate and efficiently fix nitrogen in symbiosis (Graham 1981; Graham & Halliday 1987; Graham et al. 2003). The diversity of rhizobial species that are able to nodulate P. vulgaris in relation to symbiotic effectiveness has been recently reported by various researchers (Amarger et al. 1997; Bal et al. 1982; Mostasso et al. 2002; Mrabet et al. 2005).

A clearer knowledge of indigenous nitrogen-fixing bacteria is thus essential for the understanding of the ecological consequences of native biodiversity for legume production (Tamimi 2002). Information is virtually lacking on the symbiotic properties of indigenous populations of rhizobia and their capacity to nodulate and fix nitrogen when in symbiosis with locally-adapted cultivars of Phaseolus and other legume species grown in southern Texas. Other important characteristics of selected elite strains include their ability to compete and survive in the inoculant carrier and in soil, to colonize the rhizosphere, and to migrate in soil (Materon 1991; Materon & Zibilske 2001; Tamini 2002). It is expected that further research on rhizobial ecology will illustrate the relationship between population size in the rhizosphere and nodulation. Local farmers should avoid the application of nitrogen fertilizers due to the inefficient nitrogen fixation capacity of the indigenous and introduced inoculant strains. In the present study, a wide variety of agricultural soils were sampled in southern Texas to identify and evaluate the presence of any of five rhizobial species known to nodulate P. vulgaris.

MATERIALS AND METHODS

Soil sampling procedures.--Soil samples were collected from 83 agricultural sites representing the different soil types of the region. The sites were selected using soil survey maps of four counties (Hidalgo, Cameron, Willacy and Starr) located in the lower Rio Grande Valley of Texas. The Hidalgo soil series is classified as fine-loamy, mixed, active, hyperthermic typic...

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