Population Dynamics and Diversity of Endophytic Bacteria Associated with Soybean ( Glycine max (L) Merril)

NK the analyses of the study. JD carried out the designed research work managed the literature searches and performed the statistical analysis. the the ABSTRACT Aim: Endophytic bacterial population and their diversity in soybean were investigated. Study Design: Endophytic population was assessed during different growth stages of soybean (CV JS 335) viz., vegetative and reproductive stages. Place and Duration of Study: Microbiology Research Laboratory, Department of Microbiology, R. A. Mahavidyalaya, Washim (MS), India, during the cultivation period of June-December 2010. Methodology: Healthy plants of soybean were screened from the different locations of Washim district (M. S., India). Samples represent each growth stage viz., vegetative (V1-V5) and reproductive (R1-R8) were collected. Population densities were expressed as log 10 colony forming units (CFU) g -1 fresh weight. The isolates were identified to genus level according to Bergey’s Manual of Determinative Bacteriology on the basis morphological, cultural and biochemical characteristics. Results: The maximum endophytic population was recorded for vegetative stage at V5 and V4 (5.74 and 5.01 log 10 CFU g -1 fresh weight) and for reproductive stage at R2, R1 and R3 (5.84, 5.80 and 5.74 log 10 CFU g -1 fresh weight). A total of 572 (35.50 %) from vegetative growth stages and 1039 (64.50 %) from reproductive growth stages bacterial isolates were obtained. The endophytic isolates were identified as members belonging to the genera Pseudomonas, Bacillus, Enterobacter, Klebsiella, Acetobacter Burkholderia, Rhizobium and Xanthomonas . Conclusion: As soybean development progresses endophytic population increased. At maturity, the high population density was observed and thereafter the population declined.


INTRODUCTION
Plant-microbial interactions have been a premier area of research interest; as plants present an excellent ecosystem for microorganisms where different niches are exploited by a wide variety of bacteria [1]. Microbial endophytes are typically defined as plant associated microbes that colonize living internal tissues of plants without causing any visible symptoms or immediate over-negative effects and can be isolated from surface disinfected plant tissue [2,3,4]. The occurrence of endophyte is ubiquitous and usually may include bacteria, fungi and actinomycetes [5]. These microorganisms include both commensal species and mutualistic symbionts [6].
Endophyte-plant associations have been found to improve plant health and may help host plant to rescue from various biotic and abiotic stresses [7,8]. They may also provide fitness benefits to host plants such as tolerance to herbivory, heat, salt, disease, and drought and increased below and aboveground biomass etc. [9,10]. Thus, endophytic colonization improves the ecological adaptability of the host. Hence endophytes may be regarded as a true companion of host.
The unique ecological niche has made endophytic bacteria as attractive and potentially promising tool for agricultural applications especially, for those bacteria having commercial features such as plant growth promotion and activation of plant defense mechanisms [11]. Several bacterial endophytes have been reported as potential biocontrol agents that may improve and promote plant health [12]. Various reports indicate that these bacteria exist in a variety of tissue types within numerous plant species, suggesting a ubiquitous existence in most plants [13]. The bacterial endophytic population may originate from the usual soil bacterial community and a great diversity has been reported in diverse plant community [14].
Soybean (Glycine max (L.) Merril) is an Asiatic leguminous plant, occupying large acreages of land worldwide for its oil and protein [15]. In recent years, soybean has assumed important position in India. It has well adapted to black soils of central and peninsular India. Major soybean producing states in India including, Madhya Pradesh, Maharashtra and Rajasthan, contribute about 97% to total area and 96% production of soybean in the country [16]. Maharashtra is the second largest soybean producing state in India. It accounts for 34% of the India's bean production. Soybean is gaining popularity on account of its unique characteristics and adaptability to vary agro-climatic conditions [17]. Washim is an important soybean producing area of this region, occupying 2095 ha of area with production of 2987 tones during 2010-2011 [18].
The study of the structure of endophytic microbial populations, their distribution, interaction and functions within their host is important for understanding their ecological role [19]. Analysis of the structure of microbial populations has practical importance; the results can be used to assess the fate of released strains and their impact on resident microbial communities [6]. Hence, the present investigation was carried out to study variation in distribution and diversity of endophytic bacterial population colonizing soybean.

Study Area
Washim (Latitude: 20º 05'58.90" N, Longitude: 77º 08'11.82" E, Altitude: 600M MSL) is situated in central Vidarbha region of Maharashtra state of India. The climate of this region is hot moist semi arid and the soil type is deep clayey to shallow black soil (Fig. 1).

Sample Collection
Healthy plants of soybean were screened from six different locations of Washim district during the cultivation period of June-December 2010. Within each location, randomly 3 different sub-locations were selected for sample collection. From each sub-location 3-5 healthy plants were selected. The growth stages of soybean were identified as specified by McWilliams et al. [20]. Sample represent of each growth stage viz., vegetative (V1-V5) and reproductive (R1-R8) were collected. The plants were uprooted, sealed into plastic bags and labeled. All samples were processed immediately after collection.

Isolation and Identification Endophytic Bacteria
The collected plants were washed under tap water to remove soil and separated into root, stem and leaf. All root, stem and leaf samples were washed twice in distilled water then surface sterilized as described by Hung and Annapurna [3] and Pimentel et al. [5]. Surface sterilization was carried out by immersion for 1 minute in 70% (v/v) ethanol, in 0.1% HgCl 2 upto 3 minutes out for roots and nodules, whereas, upto 5 minutes for leaves and stems respectively. The tissue was then washed ten times using sterile distilled water. Sterility checks were carried out by monitoring the rinse wash water for the presence or absence of microbial growth on nutrient media for 6 days at 30ºC. The absence of growth was taken into consideration as positive test for surface sterilization.
One gram of surface disinfected samples was macerated [3]. The slurry was filtered through sterile cotton cloth. The filtrate was serially diluted and spreaded onto nutrient agar supplemented with nistatin (200g/mL). For any slow growing bacteria the filtrate was enriched in nutrient broth for 24 hrs. at 30ºC and further processed for isolation. Plates were dried in a laminar flow cabinet for 15 min before incubation at 28-30ºC for 2-3 days. Bacterial population densities were expressed as log 10 colony forming units (CFU) g -1 fresh weight [21]. From culturable population a total of 1611 distinct bacterial isolates were isolated and identified upto genus level on the basis of morphological, cultural and biochemical characterization according to Bergey's Manual of Determinative Bacteriology [22].

Statistical Analysis
The results were analyzed by ANOVA using data analysis in MS-Excel.

Variation of Endophytic Bacterial Population
Endophytic bacterial population was found to increase with plant development (Table 1). However, at onset of reproductive stages viz., R1-R8 the endophytic population started to decrease and attained a constant value at R8 stage. Thus, it was observed that endophytic population declined, as plant Senescenced. The maximum endophytic population was recorded for vegetative stage at V5 (5.74 log 10 CFU g -1 fresh weight) followed by V4 (5.01 log 10 CFU g -1 fresh weight) and for reproductive stages at R2 (5.84 log 10 CFU g -1 fresh weight) followed by R1 (5.80 log 10 CFU g -1 fresh weight) and R3 (5.74 log 10 CFU g -1 fresh weight).

Isolation of Endophytic Bacteria
A total of 1611 endophytic bacterial isolates were isolated; 572 (35.50 %) from vegetative growth stages and 1039 (64.50 %) from reproductive growth stages (Table 2). Based upon the plant parts, 709 (44.00 %) were isolated from roots, 431 (26.75 %) from leaves and 471 (29.23 %) from stems. It was found that the number of isolates obtained from vegetative stages is larger than that of reproductive stages. As a primary site of entry, the roots showed maximum population, followed by leaves and stems respectively.
The endophytic isolates were found to be member of the genera Pseudomonas, Bacillus, Enterobacter, Klebsiella, Acetobacter, Burkholderia, Rhizobium, Xanthomonas and some unidentified genera (Table 3).

Morphological
Gram Reaction

Pseudomonas Xanthomonas Enterobacter Rhizobium Klebsiella Acetobacter Burkholderia Burkholderia
Our findings are in congruence with Zinniel et al. [5], who reported that the population size of endophytic bacteria in agronomic plants including soybean varied between 0-6.0 log 10 CFU/sample tissue. Endophytic bacterial population is affected by the growth stages of plant. At reproductive maturity (R1-R3) is population maximum as compared to other growth stages. Kuklinsky-Sobral et al. [1] also observed significant differences in bacterial population densities and found to be influenced by soybean growth phase and type of tissue sampled.
A decline in endophytic population was observed as plant aged. The decrease in endophytic population from vegetative to reproductive stages could have been due to unavailability of essential nutrients during maturation and senescence of plants [6,23]. As plants mature all the nutritional requirements for bacteria are optimum and a stable endophytic population is obtained. Thus, there appear to be coincidence of plant maturity and endophytic population.
It thus seems that endophytic population is influenced by several factors that include the developmental stages environmental conditions and geographical location of the experimental site [1]. Moreover, genotype or cultivar of plant also affects significantly the endophytic population. Conn and Franco [24] reported that soil type to a large extent, determines the endophytic population.

CONCLUSIONS
It appears from the present study that as soybean development progresses; endophytic population increased. However, at maturity, the high population density was observed and thereafter the population declined. Thus, endophytic population is influenced by developmental stages of soybean plant. Moreover, research on endophytic bacteria offers an innovative and enlivening opportunity for the discovery of novel strains with biotechnological utility.