Journal of Applied Sciences and Environmental Management World Bank assisted National Agricultural Research Project (NARP) - University of Port Harcourt ISSN: 1119-8362 Vol. 11, Num. 4, 2007, pp. 63-66

Journal of Applied Sciences and Environmental Management, Vol. 11, No. 4, 2007, pp. 63-66

Antagonism of dominant bacteria in tea rhizosphere of Indian Himalayan regions

ANCHAL SOOD; SHIVESH SHARMA; VIVEK KUMAR*; RAM LAL THAKUR

Department of Microbiology, Division of Life Sciences, SBS (PG) Institute of Biomedical Sciences and Research, Balawala, Dehradun. 248161. Uttaranchal (India).

*Corresponding author: Department of Soil and Water, Public Authority of Agriculture Affairs & Fish Resources, PO Box 21422, Safat, 13075 Kuwait; email: vivekbps@yahoo.com

Code Number: ja07096

ABSTRACT: Some parts of the Indian Himalayan region are covered by established and abandoned tea bushes. Rhizospheric soils of these plants were studied for bacterial dominance and antagonism. Amongst bacteria, Bacillus (up to 45%) and Pseudomonas (up to 85%) were found to dominate the rhizosphere of established and abandoned tea bushes, respectively. Amongst the species, B. subtilis and B. mycoides appeared to be closely associated with roots of established tea bushes, and the rhizosphere of abandoned tea bushes was dominated by Pseudomonas putida. Four bacterial strains each of B. subtilis and P. putida were selected on the basis of maximum antibacterial activity. Incubation of indicator strains with different concentrations of bacteriocin exhibited bactericidal activity. Various species of Bacillus and Pseudomonas behaved antagonistically amongst themselves due to the production of bacteriocins under in vitro conditions.

The rhizosphere of established tea bushes have some specific characteristics, which are associated with long lived nature of tea plants viz. negative rhizospheric effect, lowering of soil pH, antagonistic activities among microbial communities and dominance of certain species (Lynch, 1987; Sood et al, 2007). The overall interactions amongst tea roots, microbes and environmental conditions prevailing in the tea rhizosphere seems to favor the growth of microbes, which are known to produce strong antibiotics with potential biocontrol agents. In rhizospheric niches, bacteria are constantly competing for nutrients and ecological space. As a consequence, bacteria have devised various offensive tools for intra and interspecies competition, such as antibiotic substances, bacteriolytic enzymes, and bacteriocins. In all likelihood, the bacteriocins constitute the most abundant and diverse class of antimicrobial agents (Parret et al, 2003). The purpose of this study was to establish whether or not tea rhizosphere Bacillus and Pseudomonas species produce bacteriocins like compounds and, if so, investigate their role in rhizosphere population dynamics of established and abandoned tea bushes.

MATERIALS AND METHODS

Isolation, enumeration and selection of dominant bacteria

A number of soil samples were taken at 0-15 cm depth from different tea gardens to isolate and enumeration of bacterial population, the counts were calculated by spread plate technique using Nutrient agar (HiMedia). These bacteria were identified up to genus level on the basis of their colony morphology and biochemical tests. Bacillus subtilis and P. putida strains due to their higher % age of occurrence have been studied for bacteriocin production. Four strains each of B. subtilis (HPAB7, HPAB12, HPAB13 and HPAB41) and P. putida (UAAP1, UAAP14, UAAP17 and UAAP122) were selected on the basis of maximum zone of antagonistic activities against indicator strains.

Detection of antimicrobial activity by the deferred agar spot test (DAS)

This method was carried out on tryptic soya agar or broth (TS) (pH 7.3 ± 0.2) (HiMedia) and Brain Heart Infusion Agar or broth (HiMedia) with 0.1 % glucose (BHIG) (pH 7.4 ± 0.2). Ten µl of culture of Bacillus and Pseudomonas test strains grown for 7 to 8 h in TS and BHIG broth were spot inoculated on the surface of TS and BHIG agar, respectively. After 18-24 h of incubation at 30°C, a soft overlay of media TS, BHIG, MRS or YDC (5 ml, 0.75 % agar), suitable for indicator strains, inoculated with 100 µl of indicator culture in the stationary phase (approximately 105 cells ml-1), were pored over the surface of spot inoculated TS or BHIG agar. Inhibition zones were observed after 24-48 h of incubation under appropriate conditions. Clear zones of inhibition with sharp edges around spots were considered as possible results.

Effect of bacteriocin on indicator strain

An exponentially growing culture of the indicator strain of B. mycoides and P. syringae (107 cells/ml) was suspended in 50 mM phosphate buffer (pH 6.0) and exposed for a maximum of 140 min to various concentrations of bacteriocin (0, 25, 40 and 75 µg/ml). At different times the survival count of bacteria (cfu/ml) was determined using the AWD plate count method.

RESULTS AND DISCUSSION:

Various species belonging to the genus Bacillus were observed and B. subtilis and B. mycoides dominated the rhizosphere in established tea bushes. Apart from these two species, a number of other species of Bacillus were also observed, B. polymyxa 15.1 % and B. cereus 14.8 % (Table 1).

Tea bushes exhibit several remarkable features, e.g. the negative rhizosphere effect, strong antagonistic activities amongst microbial communities in the rhizosphere (Pandey and Palni, 1996; Pandey et al, 1997). These factors collectively, may have helped in the development of a particular bacterial population which is well adapted in the tea rhizosphere. Apart from these dominant isolates a number of other unidentified isolates besides species of Azotobacter, Agrobacterium, Phosphate solublizers etc. were also isolated but were not used for further studies, as their occurrence percentage was very low. These observations are indicative of microbial diversity in tea rhizosphere.

To determine whether bacteriocin had a bactericidal or bacteriostatic effect, various concentrations of the bacteriocin (produced by selected dominant bacteria strains) was added to the indicator B. mycoides and P. syringae strains. The survival count of the bacteria was determined using the DAS method at different time intervals of bacteriocin addition. The incubation of B. mycoides cells with bacteriocin decreased the cfu count; therefore, it indicates the bactericidal activity of bacteriocin (Fig. 1 and 2). With the increase in bacteriocin concentration and incubation time the bactericidal effect increased. The proteinaceous nature of bacteriocin seems to be responsible for this activity, since there was loss of bacteriocin activity after treating with proteolytic enzymes (Ward and Somkuti, 1995).

Most strains of B. subtilis were antagonistic to B. mycoides and its growth was often inhibited. Bacillus subtilis, which was the most dominant bacteria of the tea rhizosphere exhibited best antagonistic activity against B. mycoides and inhibited its growth on dilution plates as well as after obtaining pure cultures. Two strains of B. subtilis also inhibited the B. polymyxa and B. cereus. Pseudomonas putida also behaved antagonistically towards each other. The species P. syringae was maximum inhibited by P. cepacia and P. fluorescens (Table 2). This antagonism by B. subtilis and P. putida was due to the production of bacteriocin (Riley and Gordon, 1999). In some other cases specific inhibitions have been observed due to various non specific compounds including hydrogen peroxide, lactic acid and ammonia (Vidaver, 1983). In this way, in established tea bushes, the best adapted species of Bacillus (due to spore formation) is also the most dominant species (due to antagonistic activities) in tea rhizosphere compared to the other Bacillus species. In abandoned tea bushes Pseudomonas putida due to its antagonistic behavior side lined other Pseudomonas species. It is interesting to note that dominant microbial species were those which are known to produce antimicrobial metabolites like bacteriocin. This may be a result of the constant interaction between the root exudates, soil microorganisms and triggered by different environmental signals.

REFERENCES

• Lynch, J M (1987). Biological control with microbial communities of the rhizosphere. In: Fletcher M and Gray TRG (eds) Ecology of microbial communities. Cambridge University Press: Cambridge; 55-82.
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• Ward D J and Somkuti G A (1995). Characterization of a bacteriocin produced by Stereptococcus thermophilus ST134. Appl. Microbiol. Biotechnol. 43: 330-335.

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