Evaluation of plant growth promoting and colonization ability of endophytic diazotrophs from deep water rice

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Abstract

A study of the diversity of endophytic bacteria present in seeds of a deepwater rice variety revealed the presence of seven types of BOX-PCR fingerprints. In order to evaluate the plant growth promoting potential the presence of nitrogenase, indole acetic acid production and mineral phosphate solubilization were estimated in the representative BOX-PCR types. The seven representatives of BOX-PCR types produced indole acetic acid, reduced acetylene and showed specific immunological cross-reaction with anti-dinitrogenase reductase antibody. Only four types showed mineral phosphate solubilizing ability. Comparison of cellulase and pectinase activities showed differences among different BOX-PCR types. PCR fingerprinting data showed that one strain isolated from the surface sterilized seeds as well as the aerial parts of the seedlings of rice variety showed low cellulase and pectinase but relatively high ARA. On the basis of 16S rDNA nucleotide sequence and BIOLOG system of bacterial identification, this strain was identified as Pantoea agglomerans. For studying the endophytic colonization this strain was genetically tagged with the reporter gene, gusA. Histochemical analysis of the seedling grown in hydroponics showed that the tagged strain colonized the root surface, root hairs, root cap, points of lateral root emergence, root cortex and the stelar region. Treatment of the roots with 2,4-D produced short thickened lateral roots which showed better colonization by P. agglomerans.

Introduction

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 the rice cultivation sustainable and less dependent on fertilizer nitrogen there is a need to use diazotrophic bacteria that can make biologically fixed nitrogen available for the growth of rice plants (Ladha et al., 1997). Although free living diazotrophic bacteria form natural associations with the roots and submerged portions of rice plants, their contribution of fixed nitrogen to rice plants is very low in comparison to that provided by rhizobia to the legumes (de Bruijn et al., 1995, Ladha and Reddy, 2000). The endophytic diazotrophs, however, occupy microniches in the plant tissues where they fix nitrogen without competition with other microorganisms and without the soil constraints present in the rhizosphere.

Some Brazilian sugarcane varieties, which are grown without supplementation of high concentrations of nitrogen fertilizers have been shown to derive a substantial percentage of their nitrogen requirements from N2 fixation (Boddey et al., 1995). The predominant endophytic nitrogen fixers in sugarcane belong to Acetobacter diazotrophicus and Herbaspirillum seropedicae (Cavalcante and Doebereiner, 1988, Baldani et al., 1986, James and Olivares, 1998) which are able to penetrate deeply inside the plant and spread systemically to reach aerial tissues. These endophytes have been investigated intensively for their potential in contributing biologically fixed nitrogen to their host plants.

The search for natural association and endophytic interaction of diazotrophs with rice is considered very promising especially in primitive rice varieties not bred to efficiently respond to N fertilizer (Barraquio et al., 1997, Stoltzfus et al., 1997). These rice varieties are considered excellent hosts to identify superior endophytes that may potentially impact agricultural rice production. Wild rice varieties, which are cultivated in wetlands, have been shown to harbor diazotrophic endophytic bacteria more frequently (Barraquio et al., 1997). The search for rice genotypes showing high biological nitrogen fixation, characterization of their endophytic nitrogen fixing bacteria and colonization of rice with rhizobia has attracted a great deal of attention in recent years. Presently, no information is available on the types of endophytic diazotrophs and their ability to colonize deep-water rice.

Deep-water rice is a valuable crop for the small, subsistence farmer, as it requires only small cash input. It is the staple food for over 100 million rural people, living in deep water rice areas, who are among the poorest in Asia (Catling, 1992). It is virtually the only crop that can be grown during the wet season in the deeply flooded areas in Asia and West Africa. Poor understanding of deep-water rice and its agro system had a negative effect on agricultural planning, which resulted in underestimating its importance as a staple food crop. The irreversible degradation of watersheds and the predicted rise in the world's sea level may lead to more extensive flooding and thus greater scope for deep water rice in future.

Out of nearly 9 million hectares of deep-water rice, which yields approximately 2 tons/hectare, 60% occurs in the Indian continent. Deep water rice is grown in areas usually flooding deeper than 50 cm for 1 month or longer during the growing season (Catling, 1992). The plant length usually reaches more than 140 cm. The floating rice usually start at 1 m water depth and survive chiefly by their extraordinary elongation ability, i.e. up to 5–8 cm per day for 7–10 days. The study of deep-water rice, especially the floating rice, represents the most extreme example of flood adaptability. The ratooning ability of deep-water rice represents a potential cropping pattern for deeply flooded areas.

The total nitrogen requirement for a good deep-water rice crop was estimated at 147 kg N/hectare (Rother et al., 1988). Although the aquatic ecosystems are able to meet part of their nitrogen demand, the rest needs to be supplemented. Poor farmers depending on the cultivation of deep-water rice cannot afford chemical fertilizers. The supplemented chemical fertilizer also has adverse effects, such as nitrate contamination of underground water and growth of non-target aquatic weeds. The possibility of meeting nitrogen requirements through biologically fixed nitrogen provides an ecofriendly and sustainable alternative. In view of this, diversity of endophytic diazotrophs was investigated in a traditional deepwater rice variety that grows up to a height of 20 feet in a lake not amended with N fertilizers. The aim of the study was to identify plant growth promoting endophytic bacteria, which can systemically spread, stably persist and fix nitrogen inside the rice tissues.

Section snippets

Rice genotype

Jaisurya or Desaria variety of rice is a floating type of rice, which represents one of the most extreme examples of flood adaptability. It shows a remarkable elongation ability which is needed in areas flooding regularly to depths of 3.0–3.5 m (Catling, 1992). It has been grown for the last 40 years in a large lake named Suraha Tal located in the Ballia district of Uttar Pradesh, India. The lake is not amended with chemical fertilizers, except with the surface run off from the adjacent

Diazotrophic bacteria in the seeds of deep water rice

Rice grown under water logged conditions has been shown to fix significant levels of nitrogen (Boddey et al., 1995, Wu et al., 1995). It has also been observed that cultivated wetland and wild rice genotypes harbor diazotrophic putative endophytic bacterial populations (Barraquio et al., 1997). In view of this we selected a deepwater traditional rice variety to study the biodiversity of culturable endophytic diazotrophs. A preliminary acetylene reduction assay was performed with the surface

Acknowledgements

This work was part of a frontier project on ‘Assessing opportunities of nitrogen fixation in rice: development of novel endophytic and symbiotic associations’ which was supported by Deutsche Gesellschaft fuer Technische Zusammenarbeit (GTZ). We thank S. Nordlund, K. Wilson, B. Reinhold-Hurek, A. Kumar and B. Dhar for their valuable cooperation and support. Technical assistance from Santosh Kumar Maurya is appreciated.

References (57)

  • W.L. Barraquio et al.

    Isolation of endophytic diazotrophic bacteria from wetland rice

    Plant Soil

    (1997)
  • F. Barras et al.

    Extracellular enzymes and pathogenesis of sot-rot Erwinia

    Ann. Rev. Phytopathol.

    (1994)
  • M.A. Bekri et al.

    Azospirillum irakense produces a novel type of pectate lyase

    J. Bacteriol.

    (1999)
  • R.M. Boddey et al.

    Biological nitrogen fixation associated with sugarcane and rice: contributions and prospects for improvement

    Plant Soil

    (1995)
  • V.A. Cavalcante et al.

    A new acid tolerant nitrogen fixing bacterium associated with sugercane

    Plant Soil

    (1988)
  • D. Catling

    Rice in Deepwater

    (1992)
  • F.-P. Chalifour et al.

    Indirect evidence for cellulase production by Rhizobium in pea root nodules during bacteroid differentiation: cytochemical aspects of cellulose breakdown in rhizobial droplets

    Can. J. Microbiol.

    (1989)
  • M. Chippaux

    Genetics of cellulases in Erwinia chrysanthemi

  • L.H. Clark et al.

    Observation on the root anatomy of rice (Oryza sativa L.)

    Am. J. Bot.

    (1981)
  • A. Costacurta et al.

    Synthesis of phytohormones by plant associated bacteria

    Crit. Rev. Microbiol.

    (1995)
  • F.J. de Bruijn et al.

    Potentials and pitfalls of trying to extend symbiotic interactions of nitrogen fixing organism to presently non-nodulated plants, such as rice

    Plant Soil

    (1995)
  • A. Fahraeus

    The infection of clover root hairs by nodule bacteria studied by simple glass slide technique

    J. Gen. Microbiol.

    (1957)
  • F. Gavini et al.

    Transfer of Enterobacter agglomerans (Beijerinck, 1888) Ewing and Fife 1972 to Pantoea gen. Nov. as Pantoea agglomerans comb. Nov. and description of Pantoea dispersa sp. Nov

    Intl. J. Syst. Bacteriol.

    (1989)
  • A.H. Goldstein

    Bacterial solubilization of mineral phosphates: historical perspective and future prospects

    Am. J. Alter. Agric.

    (1986)
  • A.H. Goldstein et al.

    Separating phosphate from ores via bioprocessing

    Bio/Technology

    (1993)
  • M. Goto

    Fundamental of Bacterial Plant Pathology

    (1992)
  • K. Haathela et al.

    Root associated N2 fixing (acetylene reduction) by Enterobacteriaceae and Azospirillum strains in cold-climate spodsols

    Appl. Environ. Microbiol.

    (1981)
  • J. Hallmann et al.

    Bacterial endophytes in agricultural crops

    Can. J. Microbiol.

    (1997)
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