Introduction

Phyllosphere or plant leaf surface is a habitat for many microorganisms ₁ (Raimen, 1961). The leaf surface microbes are important in several ways. Some of them are known to fix atmospheric nitrogen ₂,₃ (Murty, 1983; Favilli and Messini, 1990), produce plant growth regulators ₄ (Buckley and Pugh, 1971) and can control plant parasites either by stimulating plants to synthesize phytoalexins ₅ (Last and Warren, 1972) or by producing antibacterial (Mc Cormack et al., 1994) ₆ and antifungal compounds (Starmer et al., 1987). ₇ The epiphytic microflora are also known to produce sugars, aminoacids, peptides, enzymes, vitamins, organic acids and nucleotides which exert influence on the plant growth (Chandramohan and Mahadevan, 1968 ). ₈ The microbial communities on leaves vary drastically from one leaf to another and undergo constant change in size and composition (Hirane and Upper, 2000). ₉ The microflora are challenged with the tharsh condition of the leaf environment (Beattie and Lindow, 1995) including highly fluctuating water availability, exposure to UV radiation from sunlight and limited access to nutrients. ₁₀ The present study was undertaken to evaluate the bacterial including actinomycetes diversity of groundnut phyllosphere of different ages, seasons and kind of levees adopting culture dependent method.

Materials & Methods

Sample collection

Microbiological analysis

Statistical analysis

Results

Enumeration of total heterotrophic bacterial and actinomycetes population

The total heterotrophic bacterial (THBP) and actinomycetes population varied according to the season and the kind of leaves investigated. A strong positive correlation (r = 1.0) was observed between the population and the season, as well as kind of leaves. During summer season, bacterial population was found to be maximum in all kind of leaves, except diseased leaves. In the case of healthy leaves the bacterial population increased from 20 days old plant (17.6 x 10 ⁵ CFU/g) to 80 days old plant (13.2 x 10 ⁷ CFU/g) (r = 0.9812). But during khariff season, the bacterial population recorded at the age of 20, 40, 60 and 80 days were 9.3 x 10 ³ , 17.7 x 10 ³ , 23.1 x 10 ³ and 27.6 x 10 ³ CFU/g respectively (r = -0.5134) (Table 1). As observed for healthy leaves THBP was same or more or less similar at all ages as well as both seasons in lepidopteron-infested leaves. Irrespective of season, the aphids infested leaves recorded highest bacterial population and this was statistically significant (P < 0.05). In aphid-infested leaves, the bacterial population increased from 23.7 x 10 ⁷ CFU/g to 71.7 x 10 ⁸ CFU/g as the plant advanced from 20 to 80 days of age respectively in summer season. Similar trend was also observed during khariff season. But the diseased leaves consist of least bacterial population in both seasons as well as in all ages than the other kind of leaves recorded (Table 1).

Actinomycetes population fluctuated according to the season. The actinomycetes population at the age of 20, 40, 60 and 80 days recorded in healthy leaves during summer season were 6.4 x 10 ² , 27.4 x 10 ³ , 16.4 x 10 ³ and 29.0 x 10 ² CFU/g respectively but during khariff season actinomycetes population was not recorded at 20 and 40 DASE. Aphids infested leaves did not showed any effect on actinomycetes population as in bacteria. The diseased leaves of 40 DASE consist of 17.5 x 10 ¹ CFU/g in summer and the actinomycetes count was absent during khariff.

Table 1. shows bacterial and actinomycetes population from groundnut phyllosphere

Figure 1

Table 1: Enumeration of total heterotrophic bacterial, actinomycetes, fungal and yeast population (CFU/g) in phyllosphere of groundnut with different kinds and season

Bacterial composition

367 bacterial isolates belonging to different species were isolated from the phyllosphere of groundnut with different kinds of leaves. The bacterial composition has showed variation with respect to age, kind of leaves and season. Pseudomonas fluorescens, Chromobacterium violaceum and Bacillus cereus were reported at 20 days age of healthy leaves and B. polymyxa reported with the above three bacterial species at 40 and 60 days age. But P. fluorescens and B. cereus persisted on leaves of 80 days age. The same bacterial species with small differences occur in lepidopteron-infested leaves as in healthy leaves. Among the various bacterial species, P. fluorescens was found to be dominant at all ages followed by Chromobacterium violaceum at 20 days, B. cereus at 40, 60 and 80 days age respectively. But specific differences in colorization and persistence pattern could be observed in khariff season. Enterobacter sp. and Alcaligenes sp. were reported in all kind of leaves except diseased leaves. P. fluorescens, Enterobacter sp. and Alcaligenes sp. were reported in all ages in healthy and lepidopteron infested leaves. During this season, Enterobacter sp. was found to be dominant in all ages except 80 days followed by P. fluorescens.

The aphids infested leaves had shown more bacterial species than the other leaves during both seasons. During summer, P. fluorescens, Chromobacterium violaceum, Enterobacter sp., Alcaligenes sp., P. stutzeri, Klebsiella sp. and Erwinia herbicola were isolated from aphids infested leaves of 20 DASE. There was no marked difference in bacterial composition (P > 0.05), except the presence of B. polymyxa at 40 DASE and the absence of Chromobacterium violaceum at 60 and 80 DASE and Pseudomonas stutzeri at 80 DASE. As observed in healthy and lepidopteron infested leaves, Pseudomonas fluorescens was dominant in all the ages of groundnut followed by Klebsiella sp. at 20 DASE, B. polymyxa at 40 DASE and B. cereus at 80 DASE. As recorded in summer, P. fluorescens was found to be dominant in all ages of groundnut during khariff followed by P. stutzeri, B. polymyxa, Alcaligenes sp. and B. cereus at 20, 40, 60 and 80 DASE respectively. Irrespective of the season and ages, B. cereus was found to be dominant followed by B. polymyxa in the diseased leaves. The remaining bacterial species was not observed in the diseased leaves.(Table 2)

Table 2 shows generic composition of bacterial species iso.lated from groundnut phyllosphere

Figure 2

Table 2: Bacterial composition (%) of groundnut phyllosphere of different kinds, ages and season

Actinomycetes composition

127 actinomycetes were isolated which belonging to three genera such as Micromonospora, Streptomyces and Actinomyces also showed variations with respect to age, season and kind of leaves. Micromonospora reported in all kind of leaves during 20, 40 and 60 DASE except in diseased leaves. Except in diseased leaves, Actinomyces and Streptomyces recorded on 40 and 60 DASE in all kind of leaves. But during 80 DASE, Streptomyces sp. was only observed in healthy, aphids infested and lepidopteron infested leaves. The diseased leaves greatly influenced the actinomycetes composition. For instance, actinomycetes belonging to Micromonospora spp reported diseased leaves of 40 DASE. Other genera were not recorded at 60 and 80 DASE of both the season. Among the actinomycetes, Micromonospors spp. was found to be dominant in all ages except 80 days.(Table 3)

Table 3 shows generic composition of actinomycetes isolated from groundnut phyllosphere

Figure 3

Table 3: Actinomycetes composition (%) of groundnut phyllosphere of different kinds, ages and season

Discussion

The microbial communities of leaves are diverse and include many different genera of bacteria, actinomycetes, filamentous fungi, yeast and less frequently protozoa and nematodes (Brighna et al, 1997). ₁₅ The total heterotrophic microbial population varied according to the age of the crop, season and kind of leaves. Andrews et al. (1981), ₁₆ Blakeman and Parbery (1977) ₁₇, Kinkel (1997) ₁₈ exclusively studied the factors influencing the microbial communities in leaves. They found that host plants, leaf age, leaf position, physical environmental condition and availability of immigrant inoculum were involved in determining the microbes in the phyllosphere. Distinct difference could not be observed in bacterial and actinomycetes population and composition according to the age. But seasonal impact in all ages could be observed. Summer season recorded maximum bacterial and actinomycetes population than khariff season. Irrespective of season and age's aphids infested leaves supported maximum growth of bacteria. Stadler and Muller (1996) ₁₉ reported the effect of aphids infested leaves on the phyllosphere microflora of Picea abies (Linn.). They observed the presence of honeydew significantly increased the growth of bacteria, yeast and filamentous fungi. They further reported that honeydew have acted as a potential source of energy which might have promoted the growth of the microorganisms. Aphids present in Spraces, Beetch and Oak produced honeydew which was consumed by microorganism. As a result, bacteria, yeast and filamentous fungal population were increased three folds. Moreover leaf-feeding moth caterpillars also positively affected the growth of the microorganisms on leaves of Beetch and Oak (Stadler and Muller, 2000). ₂₀ But in the present study, the lepidopteron infested leaves didn't show the distinct bacterial population population as observed by Stadler and Muller, 2000.) Similarly the diseased leaves recorded very least bacterial and actinomycetes population. The pathogens obviously change the metabolism of the host plant following the infection of the pathogen. The obvious change is a rise in the host respiratory rate which is especially due to the increase in the pentose phosphate pathway. This produces 4 and 5 carbon sugars. In addition to these sugars, pathogens were also responsible for the synthesis of phenols, flavonoids, isoperenoids and teopenoids which have antifungal and antibacterial activities. ₂₁ These compounds might prevent the colonization of microflora..

In the present study, 367 bacterial isolates belonging to 9 bacterial species were isolated from different kinds of groundnut leaves during both summer and khariff. P. fluorescens was reported in all the ages of groundnut during both the season and in all kind of leaves except diseased leaves. Presence of Pseudomonas sp. in phylloplane of trees was reported by Jenny et al. (1989). ₂₂ Nitrogen fixing Bacillus polymyxa and B. macerans in cotton phyllosphere was reported by Oliveira et al. (1993). ²³ The production of indole-3 acetic acid by B. polymyxa was reported by Holl et al. (1988). ²⁴ The diseased leaves during both the seasons at all ages consist of only B. cereus and B. polymyxa. Remaining bacterial species were such as in health, Aphids and Lepidopteron pests infected leaves were not detected. This might be due to the antimicrobial compounds produced by the host plant after the entry of pathogens and may prevent the colonization of microflora. Barelle et al. (1992) ²⁵ reported that B. cereus is antagonist of leaf spot of peanut which can be used as a biological control agent with chitin as a amendment. Even though B. cereus was reported in diseased leaves, significant reduction of infection could not be observed.

Raujimakers et al (1995) ²⁶ reported that effective colonization and high population size of the introduced bacterial biological control agents on plant surfaces have been considered to be important factor in the successful control of plant diseases. Ji and Wilson (2003) ²⁷ also studied the enhancement of population size of a biological control agent and their bio-efficacy in the control of bacterial speck of tomato through salicylate and ammonium surface amendments. In future, trials will be carried out to control leaf spot diseases using B. cereus and B. polymyxa with suitable amendments. Aphids infested leaves consisted of more bacterial strains. For instance Pseudomonas stutzeri and Erwinia herbicola were recorded only in this kind of leaves in both the season and in all ages except P. stutzeri at the age of 80 DASE. As mentioned earlier by Stadler and Muller (1996), ₁₉ the aphids honeydew a carbon rich waste product might have promoted the growth of microorganisms. Bacterial composition also showed variation with respect to the season. Occurrence of Alcaligenes sp. and Enterobacter sp. were only reported in all kind of leaves except the diseased leaves. This may be due to the humid environment that favoured the colonization of these bacteria. Ercolani (1991) ²⁸ found a distinct pattern of microbial colonization at different times of the year. The actinomycetes compositions were found to be greatly changed according to the season as well as age of the groundnut. Actinomycetes were not observed in most of the ages and their presence on leaves of certain ages may be recorded occasionally in the physllosphere.

The present study simply explained the bacterial and actinomycetes composition of the phyllosphere of groundnut by culture dependent methods. In contrast, Ching Hung Yang et al. (2001) ²⁹ demonstrated that culture-independent methods revealed higher community complexity in the phyllosphere than conventional culture based methods. Their study revealed that majority of the 16s rRNA sequences recovered from the leaf washings of various bacterial species were not previously described as being found in the phyllosphere. Hence, for the better understanding of the groundnut phyllosphere both culture independent as well as dependent methods are essential in future.