Nitrogen-fixing azotobacters from mangrove habitat and their utility as marine biofertilizers

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Abstract

A dearth of information is available for nitrogen-fixing bacteria in coastal mangroves, and hence, the present study has been undertaken to analyse 44 root and associated soil samples, derived from a mangrove habitat of southeast coast of India. The root samples exhibit high counts of total heterotrophic bacteria and azotobacters along with high rates of nitrogen fixation, as compared to the rhizosphere soil samples. Among the plant species, Bruguiera cylindrica records high microbial counts and nitrogen fixation. From the samples analysed, three species of Azotobacter, viz., A. chroococcum, A. virelandii and A. beijerinckii were isolated, purified and identified. These species exhibit high growth, nitrogen fixation and in vitro production of phytohormone (Indole Acetic Acid, IAA) at NaCl salinity of 30 g l−1. The azotobacters, which were inoculated with Rhizophora seedlings, increased significantly the average root biomass up to by 98.2%, the root length by 48.45%, the leaf area by 277.86%, the shoot biomass by 29.49% as compared to controls and they also increased the levels of total chlorophylls and carotenoids up to by 151.0% and 158.73%, respectively. Thus, azotobacterisation is beneficial in raising vigorous seedlings of mangroves in coastal wetlands.

Introduction

Mangroves represent a unique and ecologically important coastal habitat in the tropical and sub-tropical belts (Chapman, 1984). A number of static and dynamic biological, physical and chemical factors are known to influence the development and stability of the mangrove community. These factors and their interactions play a significant role in the nutrient flows in the system and it is necessary to understand various processes interacting with them (Kathiresan and Bingham, 2001). Nitrogen is one of the most important limiting factors affecting the development of mangrove vegetation (Chandramohan, 1988). The nitrogen is present in low concentration in particulate matter, mainly in the mangrove plant detritus, which is exported from the ecosystem through tidal action. Despite the export of large quantities of the particulate matter, loss of nitrogen is relatively small (in the order of 3.7 g N−1 year−1) which is equivalent to 13% of average annual net primary production of mangrove forests Botto and Bunts, 1982, Botto and Robertson, 1990, Lakshmanaperumalsamy, 1987. In view of this, there is a need to determine the microorganisms involved with such nitrogen transformations and the rates at which these transformations occur in situ. In India, azotobacters have been isolated from the roots of several plants Sadasivam, 1963, Thomas, 1991, but only a few from saline soil Lakshmanaperumalsamy, 1987, Ravikumar et al., 2002. No such studies are available with salt tolerant azotobacters in mangroves. Generally, one would not expect high abundance of azotobacters in the mangrove sediment as the tannin-rich conditions in that sediment are unfavourable for the growth of bacteria (Ravikumar, 1995). Therefore, a thorough study is necessary on the occurrence, distribution and activity of halophilic azotobacters from the mangrove environment.

Strains of azotobacters have been reported to secrete growth promoting hormones such as auxins, gibberellins and cytokinin into their culture media Brown and Burlingham, 1968, Azeon and Barea, 1975. By virtue of this attribute, pre-treatment of seeds with a suspension of azotobacters has been shown to improve seed germination and plant growth Brown and Burlingham, 1968, Barea and Brown, 1974. These experiments have been done with crop plants, but not with mangrove seedlings which pose a serious problem of poor growth (Kathiresan and Veera Ravi, 1990) due to high salinity and anaerobic soil conditions (McKee, 1993). The present study has also made an attempt to improve the growth of mangroves by using azotobacters derived from mangrove habitat.

Section snippets

Isolation of bacteria and analysis of chemical constituents

Root and adjoining soil samples were collected from 16 plant species viz. Acanthus illicifolius L. (Acanthaceae), Aegiceras corniculatum L. Blanco (Myrsinaceae), Avicennia marina (Frosk.) Vierh. (Avicenniaceae), Avicennia officinalis L., Bruguiera cylindrica L. Bl. (Rhizophoraceae), Ceriops decandra (Griff.) Ding Hou (Rhizophoraceae), Excoecaria agallocha L. (Euphorbiaceae), Lumnitzera racemosa Willd, (Combretaceae), Rhizophora mucronata Poir. (Rhizophoraceae), Rhizophora apiculata Blume

Bacterial counts and chemical parameters

Total heterotrophic bacterial (THB) counts ranged from 2.24 to 68.65×106 g−1 in rhizosphere soil and they varied from 10.02 to 150.25×107 g−1 in the root samples. Azotobacter counts varied from 0.22 to 36.67×101 g−1 in rhizosphere soil samples and they ranged from 0.46 to 66.35×102 g−1 in root samples. In general, counts of THB and azotobacters were higher in root samples than those in their corresponding soil samples. Among the plant species, the bacterial counts were the highest in B.

Discussion

Generally, the root samples exhibit higher rates of nitrogen fixation than rhizosphere soil. This nitrogen fixation among plant species was positively correlated with bacterial counts. There were about 10-fold higher bacterial counts and nitrogen fixation of the root surface than those of rhizosphere soil Table 1, Table 4. Similar observations have been made in crop plants, attributing the reason to the growth promoting substances exuded from the roots, which in turn influence microbial counts

Conclusion

Among the species, A. chroococcum has been found to be the most efficient in fixing nitrogen and in producing phytohormone (IAA). The same species is very effective in enhancing growth characteristics of R. apiculata and R. mucronata. The azotobacterisation also enhances the levels of phytosynthetic pigments. Thus, azotobacteriazation is beneficial in raising vigorous seedlings of mangroves, which will be useful in mangrove restoration practices.

Acknowledgements

The authors are grateful to the authorities of Manonmaniam Sundaranar University, Tirunelveli and Annamalai University for providing necessary facilities and to University Grants Commission, New Delhi, India for providing financial assistance. [SS]

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