MANGROVES ISOLATION OF CYANOBACTERIA. SCREENING FOR PLANT GROWTH PROMOTING ABILITIES Nitrogen fixation Phosphate Solubilization IAA production

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1 Mangrove, a tropical or subtropical community of highly adapted trees and shrub species which are capable of growing in intertidal estuarine and secluded marine areas. Mangroves act as physical barrier to mitigate the effects of coastal disasters like tsunami, hurricanes, and waves. Mangroves create unique niche that hosts rich agglomeration of species diversity. Even though mangrove ecosystem is one of the valuable ecosystems, this is the most threatened one at present (Ellison and Farnsworth, 1997; Kathiresan, 2000; Adeel and Pomeroy, 2002). Mangroves have immense ecological value and play important role in protecting and stabilizing coastlines (Lewis, 1990; Field, 1999; Rajiv, 1999). They improve the quality of coastal waters by exporting large amounts of carbon and nitrogen, yielding commercial forest products, and support coastal fisheries (Kannan, 1990; Vidy, 2000; Alongi et al., 2005; Romigh et al., 2006). Mangrove ecosystem serves the mankind in many ways and it can be classified into three types viz., economical, social, and ecological (Sundararaman et al., 2007). The economical and social uses exclusively cater to human population directly. However, the ecological uses tend to maintain the balance in the ecosystem. In a way, most of the benefits derived from mangrove forest contribute to the degradation too. Food and Agriculture Organization (FAO) denoted the reduction of about 5 million hectares in global area of mangrove forest. According to the survey, most comprehensive data on state of the world s mangrove forests revealed that the mangrove area worldwide had fallen below 15 million hectares at the end of 2000 down from an estimated 19.8 million hectares in 1980 (FAO, 2003). The deforestation is because of many reasons especially anthropogenic effects rather than natural calamities (Kairo et al., 2001). This human infringement of habitat destruction includes exploitation of land for urbanization, agriculture, aquaculture and mining, and clearing forest for fire wood and timber (Ellison and Farnsworth, 1997; Kairo et al., 2001; Adeel and Pomeroy, 2002). All such uses can be made in to sustainable outcome from 1

2 mangrove forest, if the management considers the balance between the exploitation and conservation of the system. For example, leaving the juvenile plants to grow and/or planting new seedlings periodically can conserve mangrove area though the plants are cut off for the timber production. The submerged part of mangrove roots, trunks, and branches serve as islands of habitat that may attract rich epifloral and faunal communities including bacteria, fungi, macroalgae, and invertebrates. Despite low nutrient levels, mangroves grow efficiently in this environment (Sengupta and Chaudhuri, 1991; Alongi et al., 1993; Vazquez et al., 2000; Bashan and Holguin, 2002) through efficient recycling of available nutrients by the activity of microorganisms (Alongi et al., 1993; Kathiresan, 2000; Holguin et al., 2001; Bashan and Holguin, 2002). Bacteria have significant role in the recycling of nitrogen in mangrove environments. Cyanobacteria, a group of photosynthetic prokaryotes, are vital component of the microbiota ranging from unicellular colonial to filamentous contribute a source of nitrogen in every mangrove ecosystems (Kathiresan and Bingham, 2001). This is one of the ignored groups where only a very few studies have been conducted. The studies on cyanobacteria associated with mangroves are very important not only because of their abundance, but also of their high capability for nitrogen fixation, which are natural candidates for reforestation and rehabilitation of destroyed mangroves (Bashan et al., 1998). The study of cyanobacterial biodiversity is paradoxical because these bacteria differ in cytological characters even though they are having uniform metabolism. The cyanobacterial diversity has been studied in various natural environments, in recent years mostly by means of molecular techniques. The cyanobacterial taxonomy is mainly following two different approaches; the traditional botanical approach and the considerably more recent bacteriological approach. However, methods based on nucleic acids and proteins are now well-established and widely used. The 16S rrna gene has proven to be a useful marker for investigating phylogenetic relationships and is the most commonly used marker for differentiating identities between prokaryotic organisms at the genus level. However, this is also paradoxical as the structural genes of rrna are too conservative for a large phylum. For filamentous cyanobacteria the more variable hetr gene were shown to distinguish organisms even at the strain level. 2

3 The internal transcribed spacer (ITS) between the 16S and 23S rrna genes has also been used as a complement to 16S rrna analysis for diversity studies in environmental samples (Taton et al., 2003). The ITS is a variable sequence and has been successfully used to distinguish between cultured strains (Scheldeman et al., 1999; Iteman et al., 2000; Boyer et al., 2002; Iteman et al., 2002). Cyanobacteria have been explored for their ability to fix nitrogen, exclusively. Even though nitrogen-fixing activity of cyanobacteria on mangrove is well studied, the nature of relationship between them is still to be explored. By seeing ostensibly, it is clear that the interaction is non-pathogenic but there is no explicit studies taken to reveal the relationship between cyanobacteria and mangroves. There is paucity in revealing the role of cyanobacteria other than nitrogen fixation like plant growth promoting factors with respect to mangrove community. Cyanobacteria are also known to take up and assimilate organic compounds (Prasanna et al., 2004) and produce plant growth promoting substances (Sergeeva et al., 2002). Phytohormones play a key role in regulation of growth and development in plants. Auxins, among them in particular indole-3-acetic acid (IAA), are the most studied plant growth regulators. Though the production of phytohormones is considered as a feature of the plant kingdom, the existence of phytohormone production is evidenced in soil and plant-associated prokaryotes (Costacurta and Vanderleyden, 1995). Phytohormones produced by plant-associated bacteria are concerned as key determinants in stimulation of plant growth, in plant pathogenesis and in associative or plant microbe symbiotic interactions (Christiansen-Weniger, 1998). The association between plant and cyanobacteria in the aspects of plant growth promotion is least studied. Exploring the plant growth promoting abilities of cyanobacteria will add more value for the exploitation towards mangrove conservation and restoration. After the threat elicited by tsunami in Southeast Asia, the global awareness of the importance of mangrove forests has spread rapidly (Kathiresan and Rajendran, 2005). Hence, the worldwide scientific and social endeavours have been promoted on the afforestation. The establishment of nursery of coastal plants especially well-adopted mangroves will help in such process of resilience to mitigate the effect of coastal disasters. Nursery approach is one of the best traditional methods of conserving 3

4 mangroves and prerequisite for mangrove afforestation. Experiments and field trials were performed on this approach and the feasibility of this approach was already recognized (Field, 1999; Rajiv, 1999; Kairo et al., 2001; Toledo et al., 2001; Clarke and Johns, 2002). There were many considerations to succeed in this approach with proper nursery technique (Toledo et al., 2001; Clarke and Johns, 2002). Modern biotechnological approach is considered as a necessity for development of nursery. The establishment of nursery is essential in reforestation programs, provides good quality of seedlings at the right quantity in time (Melana et al., 2000; Kairo et al., 2001). Since, the availability of seedlings is limited to seasons in many mangrove species, it is important to maintain a nursery to make the seedlings available throughout the year (Rajiv, 1999). Already cyanobacteria were exploited in agriculture as biofertilizer, especially on rice fields. However, biofertilizers are more advantageous over chemical fertilizers as they are cost effective, eco friendly and not harmful to plants. Moreover, mangrove plants are always associated with cyanobacteria of diverse species. Although the cyanobacteria are considered as a major component of mangrove ecosystem, there are only very few attempts made to use cyanobacteria in mangrove plant growth (Bashan et al., 1998; Selvakumar and Sundararaman, 2001; Toledo et al., 2001). Application of plants with plant-growth-promoting bacteria (PGPB) is a successful method to increase crop yield. However, several studies reported that plant-growth promotion by beneficial bacteria have the advantage of using mixed cultures of microorganisms over pure cultures (Bashan and Holguin, 1997a; Bashan and Holguin, 1997b). The consortium of N 2 -fixing bacterium Phyllobacterium sp. and phosphate-solubilizing bacterium Bacillus licheniformis which are isolated from the mangrove rhizosphere showed 300% increase in N 2 fixation rate by Phyllobacterium sp. when compared to N 2 fixation by pure culture (Holguin et al., 2001). The ex situ conservation of mangroves by rearing nursery is a new concept for future afforestation programs. Thus, the present works is aimed at exploring plant growth potentials of mangrove associated cyanobacteria and its application in the development of mangrove nursery. The diversity of cyanobacteria associated with mangrove roots and pneumatophores are planned to study in detail. Further, the research was designed to 4

5 screen the cyanobacteria for plant growth promoting potential in the aspects of Phosphate solubilization, Nitrogen fixation and IAA production. The potent cyanobacteria selected from preliminary screening were used as a biofertilizer and tested on the growth of selected mangrove plant including endangered species. The overall view of the work is depicted in the following flowchart (Fig. 1). Objectives: The objectives of the present work is as follows, To enumerate the aquatic epiphytic Cyanobacterial diversity of mangrove plants. To isolate and identify the Cyanobacteria from mangroves. To screen isolated mangrove associated cyanobacteria for plant growth promoting potentials such as Nitrogen fixation, phosphate solubilization and IAA production. To formulate a consortium of plant growth promoting cyanobacteria. To test the plant growth promoting ability of consortium on mangrove plants in green house. To test the effect of consortium on nursery reared endangered mangrove species in the field. MANGROVES ANALYSIS OF CYANOBACTERIAL DIVERSITY OF ROOT ASSOCIATE ISOLATION OF CYANOBACTERIA SCREENING FOR PLANT GROWTH PROMOTING ABILITIES Nitrogen fixation Phosphate Solubilization IAA production PHYLOGENETIC ANALYSIS USING 16SrRNA & RFLP PREPARATION OF CYANOBACTERIAL CONSORTIUM Studies on antagonistic activity Testing saline tolerance APPLICATION OF CYANOBACTERIAL CONSORTIUM ON MANGROVE PLANT At Laboratory and Greenhouse Avicennia marina At Nursery Level Bruguiera gymnorrhiza At field level- Bruguiera gymnorrhiza Figure 1. Outline of the research activity 5