INTRODUCTION

The marine flora and fauna are considered as a special “bio-reservoir” of bioactive metabolites (Manilal et al., 2010a). Among them, marine bacterial vegetation constitutes an eminent resource of untapped novel metabolites (Selvin et al., 2009; Manilal et al., 2010b; Reddy et al., 2011). The biosynthesis of antimicrobial metabolites by marine bacteria isolated from different marine environment has been known for a long time (Burkholder et al., 1966). The majority of these bacterial strains were sourced from seawater and marine sediments. Bacteria associated with marine organisms have reported to display significant bioactivity including antifouling, antibacterial, cytotoxic activities, anticoagulant and cardioactive potentials (Burgess et al., 1999; Sponga et al., 1999; Bernan et al., 1997; Imamura et al., 1997; Jensen and Fenical, 1994; Burgess et al., 1991; Austin, 1989; Marderosian, 1969). Moreover, deep studies of marine natural products envisages that many bioactive compounds previously found in marine animals and plants possess notable similarities to metabolites produced from their associated microorganisms (Unson and Faulkner, 1993; Schupp et al., 1999; Davidson et al., 2001; Luesch et al., 2001; Proksch et al., 2002).

Seaweeds can host verdant varieties of heterotrophic bacteria and many of these bacteria play a cardinal role in maintaining the health of the host organism (Bolinches et al., 1988) by producing unique bioactive secondary metabolites. Hence, epibiotic bacteria become an incredible source of new natural products (Holmstrom and Kjelleberg, 1999). These microbial bioactives could be utilized as a source of lead compounds for the biomedical and biopharmaceutical industry (Donia and Humann, 2003; Anand et al., 2006). Isolation of these epibionts is relatively very easy as compared to other bacteria. Thus, seaweed-associated bacteria may be useful in the development and production of bioactive compounds and biocontrol agent.

Deployment of natural antibiotic and probiotic (biocontrol) offers a hopeful alternative to the synthetic antibiotics for fish and shrimp aquaculture system. The development of a suitable probiotics needs empirical and fundamental research, full scale trials, as well as the development of appropriate monitoring tools. The initial-step in the development of a probiotic involves isolation of appropriate bacteria from natural sources and optimization of its antagonistic activity. In this background, the present study is initiated to explore the antagonistic potential of marine culturable bacteria associated with seaweeds/seaweed habitat as a suitable source for the development of shrimp probiotics.

MATERIALS AND METHODS

Collection of seaweeds: Seaweeds were sourced from the Southwest coast of India (Kollam coast; 08°54’N and 76°38’E). Four species of seaweed specimens were handpicked during the lowest tide of the chart datum. Seaweed samples were identified by eminent algologist Dr. M.V.N. Panikkar, Director of Research, Department of Botany, Sree Narayana College, Kollam, Kerala, India.

Isolation of seaweed-associated microbes: The bacterial strains associated with seaweeds were obtained by using the following procedure. The seaweeds were rinsed repeatedly with sterile seawater in order to remove the freely attached bacteria. A small zone (1 cm²) of the cleaned thallus of each seaweed species were separately scrubbed with a sterile cotton swab and plated on the entire surface of 180 mm Petri dishes containing ZoBell Marine Agar (ZMA) or Nutrient Agar (NA) in duplicate until the colonies were observed. The media were supplemented with nystatin and cyclohexamide at 25 and 10 μg mL^-1, respectively to minimize contamination with fungi and 10 μg mL^-1 nalidixic acid was applied to minimize bacterial contaminant growth (Takizawa et al., 1993; Ravel et al., 1998). After 48 h at 28°C, different colonial morphologies of bacteria were chosen and purified by successive re-streaking. The pure bacterial cultures obtained were maintained on NA slants.

Screening of potential antagonistic bacteria against shrimp pathogens by cross streak method: The potential antagonistic bacteria were selected by screening the seaweed isolates against a battery of shrimp pathogens including six MTCC type culture and nine shrimp vibrio isolates (Table 1). The shrimp Vibrio isolates previously isolated from infected P. monodon collected from the shrimp ponds of Manroe Island, Kollam, South India were used for the inhibitory assay (Manilal et al., 2010a). All the test organisms were maintained on nutrient agar slants at 4°C before being used in the inhibitory assays. Determinations of antimicrobial activity of the seaweed associated bacteria are performed by cross-streak method (Egorov, 1965). Mueller-Hinton agar plates were prepared and inoculated with seaweed isolates by a single perpendicular streak of inoculum in the centre of the petridish and incubated at 27°C for 4 days. The plates were seeded with test organisms by a perpendicular streak at a 90° angle to the line of the seaweed isolates. Antagonism was observed based on the inhibitory interaction between the seaweed isolates and test organisms. The reference standard (Nalidixic acid) was used for the verification of strain sensitivity/resistance pattern. Inhibition activities and colonization effect were noticed at 24, 48, 72 and 96 h. All the tests were conducted in triplicates.

Screening of potential antagonistic bacteria against shrimp pathogens by agar well diffusion method: The bacterial strains which exhibiting the antagonistic activity in cross-streak method was further confirmed with the agar well-diffusion method (Manilal et al., 2010c). The seaweed associated bacterial strains were grown in a nutrient broth (Merck, Germany) incubated at 25°C for 18 h and adjusted to an approximate concentration of 10⁸ cfu mL^-1. The Mueller Hinton agar plates were prepared and uniformly spread with appropriate shrimp pathogens. Thereafter, wells with 5 mm of diameter were prepared using a sterile cork borer. The resultant wells were carefully filled with 120 μL of the appropriate microbial cell suspension. The well with broth used for culture was considered as negative control. The assay was performed in triplicates of individual Petri dishes. The presence of antagonistic metabolites/inhibitory compounds produced by the isolates suppressed the growth of the test pathogen producing a zone of inhibition around the well. The diameter of the inhibition halo after 24 h of incubation at 37°C was considered to be indicative of bioactivity. Zones of inhibition were scaled when bacterial growth was visible. The net halo diameter was calculated after subtracting the diameter of the well (5 mm) (Manilal et al., 2010b).

Molecular identification of active seaweed isolates (SWI-24): Molecular identification of the active seaweed isolate was based on partial 16S ribosomal RNA gene sequencing. The active bacterial strain was cultured in marine broth at 25°C and the total genomic DNA was extracted by CTAB/NaCl method. PCR amplification of bacterial 16S ribosomal RNA gene was performed using the universal eubacterial 16S rRNA gene primers. The amplified 16S ribosomal RNA gene product was cloned by TA cloning method using a TOPO TA cloning kit as per the manufactures instruction (Invitrogen) for sequencing. The resultant 16S ribosomal RNA gene sequence from the active isolate SWI-24 was compared with other bacterial sequences by using BLAST (Basic Local Alignment Search Tool) (Altschul et al., 1990) to analyse pair wise homology. The sequence used in the analysis was deposited in GenBank, EMBL in Europe and the DNA Data Bank of Japan with an accession No. EU432052.

Statistical analysis: One-way ANOVA using SPSS was used to analyze the difference between treatments and controls. Values of p<0.05 were considered significantly different.

RESULTS

Description of seaweed collection site-Kollam coast: Kollam coast is situated at the lower portion of Southwest coast of India. The Kollam coast harbours a great diversity of species including micro and macroalgae, fishes, clams, crustaceans, sponges, sea anemones, sea cucumbers, sea urchins, soft corals and other sessile invertebrates (Manilal et al., 2010c). Bioactivity of marine organisms collected from the Kollam coast has been extensively reported (Manilal et al., 2009, Manilal et al., 2010a, 2010b, 2011). Hitherto, the antagonistic properties of seaweed-associated microbes from the Kollam coast have not been explored. The seaweed specimens collected from the study area are appended in the Table 2.

Table 1:	Panel of shrimp pathogens used for antimicrobial assay
*Microbial type culture collection, **Isolated from moribund shrimp

Table 2:	Bacterial strains isolated from different species of seaweeds
SWI: Seaweed isolates, ZMA: ZoBell marine agar, NA- Nutrient agar

Epiphytic microorganisms isolated from different species of seaweeds: According to the colony morphology, totally 27 epibiotic bacteria from the four species of seaweed are isolated and sub-cultured to obtain pure cultures. The isolates sourced from seaweed were abbreviated as SWI (Table 2). The highest number of bacterial strains was isolated from A. orientalis (33.3%), followed by S. wightii (25.9%), P. tetrastromatica (22.2%) and S. marginatum (18.5%). Among the 27 seaweed isolates, 8 were obtained on Nutrient agar and the remaining 19 were isolated on ZoBell Marine Agar (ZMA).

Antagonistic bacteria isolated from different species of seaweeds: In seaweed isolates, of 27 bacteria isolated, only four bacterial strains (SWI-8, SWI-17, SWI-24 and SWI-26) showed inhibitory effects against at least one of the tested strains. The activity of these strains was further re-confirmed with agar well-diffusion method. The results of agar well diffusion method showed that SWI-24 and SWI-26 exhibited 100% inhibitory activity against all the tested shrimp pathogens. Spectrum of activity was narrow and weak for other two isolates. The seaweed isolate SWI-24 showed a strongest inhibition range of 7 to 21 mm against the MTCC shrimp pathogens whereas, the activity was in the range of 7 to 14 mm against the shrimp vibrio isolates (Fig. 1 and 2). The Culture Suspension (CS) of SWI-26 produced a mean zone inhibition ranged between 8 to 12 mm against the MTCC whereas, 6 to 12 mm against the shrimp vibrio isolates (Fig. 1 and 2). None of the zones of inhibition exceeded 14 mm, indicating that shrimp vibrio isolates are resistant or only slightly susceptible to the action of seaweed isolates. Therefore, the strain SWI-24 was considered as the most active isolate of this group and therefore, subjected to further molecular examination.

The taxonomic affiliation of the 16SrRNA sequences of the SWI-24 was retrieved from classifier programme of Ribosomal Database Project II version 9.0. The 16S rRNA sequence of the isolate was blasted using megablast tool of GenBank (http://www.ncbi.nlm.nih.gov/). This revealed that the isolate was a Pseudoalteromonas sp. (Fig. 3).

Fig. 1:	Zone of inhibition of seaweed isolates against shrimp vibrio isolates

Fig. 2:	Zone of inhibition of seaweed isolates against MTCC cultures

Fig. 3:	Neighbor joining phylogenetic tree of the seaweed isolate SWI24 and their closest NCBI (megaBLAST) relatives based on the 16S rRNA gene sequences (Accession No. EU432052)