Elsevier

Applied Soil Ecology

Volume 39, Issue 3, July 2008, Pages 311-320
Applied Soil Ecology

Evaluation of genetic diversity and plant growth promoting activities of nitrogen-fixing bacilli isolated from rice fields in South Brazil

https://doi.org/10.1016/j.apsoil.2008.01.006Get rights and content

Abstract

Several strains of bacilli, mainly species of the genera Bacillus and Paenibacillus, displaying important plant growth promoting (PGP) characteristics were isolated from seven distinct rice production zones of the Rio Grande do Sul State, Brazil. Of 296 isolates, 155 were from rhizospheric soil and 141 from bulk soil. In order to evaluate the diversity among the isolates of each bacterial population the Shannon index was used on a 70% similarity basis. Diversity indices varying from 2.27 to 5.51 were obtained. Using principal coordinate analysis (PCA) to correlate bacterial diversity with soil parameters, it was found that soil pH was the characteristic most closely related to bacilli diversity. The bacilli isolated were also analyzed for some PGP activities. Of those 296 isolates, 94 and 148 produced between 0.1 and 30 μg of indole-3-acetic acid (IAA) ml−1 in vitro after 72 and 144 h of incubation, respectively. Twenty-two isolates were able to solubilize phosphate and 32 isolates produced siderophores. Paenibacillus and Bacillus genera were the most prominent groups in the rhizosphere and soil populations analyzed. Paenibacillus borealis was the most frequent species in both locations. The isolate SVPR30, identified by 16S rRNA gene sequence analysis as a strain of Bacillus sp., was chosen for in vivo greenhouse experiments and proved to be very efficient in promoting a significant increase in the roots and shoot parts of rice plants. The identification and isolation of PGP bacilli from temperate and subtropical soils, which combine the ability to fix nitrogen with the production of substances capable to promote the plant growth, could significantly increase productivity of grain crops in Brazil.

Introduction

Previously confined to inoculation of legume seeds with Rhizobium, the management of the rhizosphere bacterial population has nowadays advanced toward the concept of plant growth promoting rhizobacteria (PGPR) (Lalande et al., 1989). These bacteria stimulate host plant growth, and have been the object of studies due to the effects produced in commercially important crops. The exact mechanism by which PGPR stimulate plant growth is not clearly established, although several hypotheses such as production of plant growth substances, suppression of deleterious organisms, and promotion of the availability and uptake of mineral nutrients are usually believed to be involved (Lalande et al., 1989). Albeit variations in the plant response to PGPR in laboratory and field assays are evident, the full potential of rhizobacteria and other microorganisms to promote plant growth should be more extensively investigated.

The widely studied Bacillus genus represents one of the most diverse genera in the Bacilli group (Garbeva et al., 2003). Numerous Bacillus and Paenibacillus strains express plant growth promoting (PGP) activities and a number of these strains have already been commercially developed as biological fungicides, insecticides, and nematicides or generic plant growth promoters. The use of these strains in agriculture has recently been reviewed (Gardener, 2004).

Rice is the most important staple crop in the developing world, and nitrogen is the most important input required for rice production. In order to make rice cultivation sustainable and less dependent on fertilizer nitrogen, it is extremely important to know how to use PGPRs that can biologically fix nitrogen and produce substances (for example, indole-3-acetic acid and siderophores) that can contribute to rice growth improvement (Verma et al., 2001).

The diversity of bacilli species in Southern Brazilian soils remains largely unknown, especially of those strains that, besides having several PGP properties, can also fix nitrogen. The objectives of this study were to (i) isolate the predominant nitrogen-fixing bacilli species from different rice crops, (ii) estimate their diversity, and (iii) evaluate their PGP activities in order to use them further as inoculant strains.

Section snippets

Sampling and sample preparation

Samples of rhizosphere and bulk soil were collected in seven distinct currently rice production zones of the Rio Grande do Sul State, Brazil: Guaíba [Gu (30°06′50′′S, 51°19′30′′W)], São Lourenço do Sul [SLS (31°21′55′′S, 51°58′42′′W)], Santa Vitória do Palmar [SVP (33°31′08′′S, 53°22′04′′W)], Hulha Negra [HN (31°24′14′′S, 53°52′08′′W)], Santana do Livramento [Li (30°53′27′′S, 55°31′58′′W)], Uruguaiana [U (29°45′18′′S, 57°05′16′′W)] and Osório [O (29°53′13′′S, 50°16′12′′W)]. Three c. 1 ha fields

Isolation and diversity of PGP bacilli

A total of 296 bacilli were selectively isolated based on their resistance to high temperature, growth on nitrogen-free medium under anaerobic conditions, and the presence of the nifH gene. Of those 296 isolates, 155 were from rhizospheric soil and 141 from bulk soil, representing, respectively, 52.4% and 47.6% of the bacilli isolated.

Strains of nitrogen-fixing bacilli belonging to different species were discriminated by the RFLP-PCR of the nifH gene approach using the two selected restriction

Isolation and diversity of PGP bacilli

RFLP-PCR of the nifH gene showed that certain genotypes were typical of the rhizosphere or of the bulk soil. Similar results were obtained by Bardgett et al. (1999), which suggested that plant species could affect the soil microbial community more than the physical or chemical soil properties. Elo et al. (2000) also indicated that the bacteria associated with the roots were selected by the plant from the bacterial pool in the soil. On the other hand, Gelsomino et al. (1999) resorted to PCR-DGGE

Conclusion

Although it is well known that many species of Bacillus and Paenibacillus can contribute to plant growth and health in many ways, there are only a few studies concerning the Gram-positive spore forming bacteria. In this work, several bacilli strains displaying important PGP characteristics were isolated, and one (SVPR30) proved to be very efficient in promoting the growth of rice plants. This strain could be useful in the formulation of new inoculants, improving the cropping systems into which

Acknowledgments

The authors are grateful to Dr. Fernanda Bered for its statistical support. This work was financed by a grant and fellowships from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq/Brazil).

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