Isolation and characterization of plant growth promoting endophytic bacteria and their effect on tomato (Lycopersicon esculentum) and chilli (Capsicum annuum) seedling growth

Abstract

The endophytic bacteria known to reside in plant tissues have often been shown to promote plant growth. The present study deals with the efficacy of eight isolates of putative endophytes from surface-sterilized seeds of the crop plants tomato and chilli. Two isolates were Gram negative and six were Gram positive. All isolates were able to grow in up to 6% NaCl, and isolate BETL13 grew in up to 12% NaCl. The isolates were identified using a microbial identification system (Biolog). Of the eight isolates, six belong to the genus Bacillus and two are Serratia. The partial 16S rRNA gene sequences of BECS7, BETS14 shared 99% and 98% similarity with Bacillus sp. BETS11 showed 97% similarity to Bacillus subtilis and BECS1 showed 98% similarity to Arthrobacter sp. Under in vitro conditions, all eight isolates showed antagonistic activity against Sclerotium rolfsii, Fusarium oxysporum, Colletotrichum capsici and Pythium sp. The endophytes were also found to produce indole acetic acid and siderophore and four isolates solubilized inorganic phosphate. The plant growth promotion properties were demonstrated through a chilli- and tomato-based bioassay under greenhouse conditions. Isolates BETL13, BETL9, BECS1, BECL8 and BECS7 showed plant growth promotion in terms of an increase in root and shoot length and the number of secondary roots with respect to their host as well as in cross inoculation.

Introduction

Bacteria of several taxonomic classes are found in the crop rhizosphere and soil, and many of them can increase plant growth and productivity. Attempts to introduce beneficial bacteria into the rhizosphere of agricultural crops have generally met with varying degrees of failure due to the difficulties of incorporating non-resident bacterial species into established and acclimated microbial communities. One strategy that may help to establish pre-selected beneficial bacteria in root zone soil is early establishment of selected communities of endophytic microorganisms within root systems. Native bacteria isolated from a specific plant rhizosphere may contain highly efficient genotypes to promote plant growth and may perform this function better than exotic strains.

Endophytes can be beneficial to their host by promoting plant growth and also acting as biocontrol agents (Mercado-Blanco and Bakker 2007; Ryan et al. 2008). Despite their different ecological niches, free-living rhizobacteria and endophytic bacteria use the same mechanisms to promote plant growth and control phytopathogens (Compant et al. 2005). For example, they can affect plant growth by producing auxins such as indole-3-acetic acid (IAA), or cytokinins; or by degrading the ethylene precursor ACC by ACC deaminase (Long et al. 2008; Ryan et al. 2008). Application of bacterial inoculants as biofertilizers has resulted in improved growth and increased yield of cereal crops (Arcand and Schneider 2006; Lucy et al. 2004). In the present study isolation of plant growth promoting (PGP) endophytic bacteria from tomato and chilli cultivated in the tropical region of the Andaman and Nicobar Islands, and their characterization on the basis of morphological and physiological studies using Biolog analysis is presented. The effects of these endophytic bacteria on inoculated tomato and chilli seedlings are also discussed.

Materials and methods

Isolation of endophytic bacteria

The plants used for bacterial isolation were collected from tropical regions of Andaman and Nicobar Islands, India. Processed samples were serially diluted, spread plated on nutrient agar and incubated at 28°C for 48 h. A total of 87 different colonies isolated on nutrient agar were purified with repeated culturing and maintained in 20% glycerol at −20°C. Potential isolates were selected on the basis of results of screening for antagonistic and PGP properties.

Antagonistic activities against plant pathogenic fungi

The antagonistic effects of endophytic bacteria were tested against four fungal plant pathogens viz., Sclerotium rolfsii, Pythium sp., Fusarium oxysporum and Colletotrichum capsici. For this the bacterial isolates were streaked at a distance of 3.5 cm from the rim of individual Petri dishes containing potato dextrose agar (PDA) medium. A 6-mm mycelial disc from a 7-day-old PDA culture of fungal pathogens was then placed on the other side of the Petri dish and the plates were incubated at 28°C for 4 days. The percent inhibition was calculated by using the formula: I = (C − T)/C × 100, where I is the percent inhibition of mycelial growth over the control, C is mycelial growth of fungal pathogen in the control plate and T is mycelial growth of fungus in endophytic bacteria inoculated plate. The experiment was carried out in three independent replicates.

Production of secondary metabolites

Indole 3-acetic acid production

The bacterial cultures were inoculated in nutrient broth with tryptophan (5 μg/ml), and incubated at 28 ± 2°C for 5 days. Cultures were centrifuged at 3,000 rpm for 30 min. Two ml of the supernatant was mixed with two drops of orthophosphoric acid and 4 ml Salkowski’s reagent (50 ml, 35% perchloric acid; 1 ml 0.5 FeCl₃). Development of red color indicates Indole 3-acetic acid (IAA) production; the optical density (OD) was read at 530 nm using a spectrophotometer. The level of IAA produced was estimated from a standard IAA graph and expressed as micrograms per milliliter (Patten and Glick 1996).

Siderophore production

Bacterial culture (2 days old) was streaked on King’s B medium amended with an indicator dye. The tertiary complex chromazurol-S (CAS)/Fe³⁺/hexadecyl trimethyl ammonium bromide served as an indicator. A change in the blue color of the medium surrounding the bacterial growth to fluorescent yellow indicated production of siderophore. The reaction of each bacterial strain was scored either positive or negative in the assay (Schwyn and Neilands 1987).

Phosphate solubilization

All bacterial isolates were screened for inorganic phosphate solubilization according to the method developed by Verma et al. (2001). A loopful of fresh bacterial culture was streaked onto Pikovaskaya’s medium amended with inorganic phosphate and plates were incubated at 28 ± 2°C for 4 days. A clear halo around the bacterial colony indicated solubilization of mineral phosphate.

Characterization, identification and sequence analysis

Preliminary biochemical characterization of endophytic bacteria was carried out using standard methodologies (Collins and Lyne 1980). The identity of the isolates was revealed on the basis of Biolog carbon source utilization, and isolates not showing similarity based on Biolog were validated by 16S rRNA gene sequencing. Bacterial suspensions were inoculated into Biolog GENIII Micro plates as described in the manufacturer’s instruction manual and incubated at 30°C for 24 h. The results were interpreted using Biolog Micro Log™, Release 4 software (Biolog, Hayward, CA). The universal primers pA and pH were used for the partial sequencing of the 16S rRNA gene, and the sequences obtained were submitted to GenBank (accession numbers GU569857, GU569858, GU569872 and GU569880).

Plant growth promotion ability of endophytic bacteria on tomato and chilli

Tomato and chilli seeds were sterilized with 70% ethanol for 2 min and in 2% sodium hypochlorite for 2 min, followed by washing ten times in sterile tap water. For this experiment, pure cultures were grown in nutrient broth at 28°C and diluted to a final concentration of 10⁸ colony-forming units (CFU) ml⁻¹ in sterile saline water (0.85%). The surface-sterilized seeds were immersed in appropriate PGP endophytic suspension for 1 h, air-dried, and sown immediately. The following treatments were investigated, with three replicates of two individual experiments: (9) control (without bacterial inoculation), (1) Bacillus sp. BETS11 (2) Bacillus cereus BETS14 (3) Bacillus sp. BETL9 (4) Bacillus pumilus BETL13 (5) Arthrobacter sp. BECS1 (6) Serratia marcescens BECL8 (7) Serratia marcescens BECS6 and (8) Bacillus megaterium BECS7. Pots were sterilized with 20% sodium hypochlorite solution and filled with sterile loam soil. The tomato and chilli seeds (50 seeds in each pot) were sown in plastic pots filled with 1 kg sterile field soil. On days 12 and 18 after sowing, the tomato and chilli seedlings were thinned to one plant per hole. The pots were arranged in a completely randomized factorial design. The seedlings were grown in a glasshouse at a temperature of 28–32°C and 85% relative humidity, on a day-night cycle of 13–14 h natural light. The pots were watered to 50% water-holding capacity and were maintained at this moisture content by watering to weight every day. For each species and treatment, the plants of three pots were harvested 3 weeks after the emergence of seedlings, washed and morphological characteristics viz., root length, shoot length, dry and wet weight of stem and root, and total number of secondary roots of each plant was recorded.

Statistical analysis

Data were analyzed statistically by analysis of variance using the general linear model software Agres and Agdata, and means were compared using the least significant difference (LSD) method; P ≤ 0.05 was considered significant.

Results

Isolation and characterization of bacterial isolates

A total of 87 endophytic bacteria were isolated from tomato and chilli plants. These isolates were evaluated for their antagonistic and PGP traits. Eight bacterial strains showing potential antagonistic and PGP activities were selected for characterization; two were Gram negative and six were Gram positive. All isolates could tolerate up to 6% of NaCl concentration (w/v), and isolate BETL13 tolerate up to 12% NaCl. The isolates were characterized phenotypically and biochemically using Biolog-based carbon source utilization, and variations in properties were observed. The partial 16S rRNA gene sequences of BECS7, BETS14 showed 99% and 98% similarity with Bacillus sp. BETS11 showed 97% similarity with B. subtilis and BECS1 showed 98% similarity with Arthrobacter sp. sequences available in public domain databases.

Anti-phytopathogenic activity and metabolite production

The isolates BETS14, BETL13, BECS6 and BECS7 exhibited the most obvious antagonistic activity in vitro and showed significant growth inhibition activity against S. rolfsii, F. oxysporum, C. capsici and Pythium sp., followed by BECS1, BECL8 and BETS11, which showed significant growth inhibition against three pathogens. All isolates were positive for siderophore production, with BETS14, BETL9 and BETL13 being potent siderophore producers. The isolates BETS11, BETL9, BECL8 and BECS6 showed a prominent zone of solubilization around the bacterial colony. The production of IAA by the different isolates ranged from 15.0 to 59.2 μg ml⁻¹ (Table 1).

Table 1 Antagonistic and plant growth promoting (PGP) activities of endophytic bacteria, and their Biolog identification. Values are mean of three replications

Plant yield parameters

The endophytic bacteria isolated from tomato and chilli were tested for their influence on growth parameters and showed considerable influence on both crops. Tomato and chilli plants inoculated with tomato isolates BETL13 and BETL9 both showed increased root and shoot length. Similarly, isolates from chilli viz., BECS1, BECS7 and BECL8 also showed significantly increased shoot and root length. A corresponding significant increase in the root and shoot biomass was also observed in endophyte applied plants. Seed bacterization resulted in greater enhancement of shoot growth, as compared with the root growth and dry biomass weight. The number of secondary roots in bacterized plants increased compared to controls (Tables 2, 3).

Table 2 Effect of inoculation of endophytic bacteria on growth parameters of chilli seedlings. Results obtained shown are the mean of triplicates. Data were analyzed using one-way analysis of variance and treatment means were compared (values with different lower case letters are significantly different; P ≤ 0.05%)

Table 3 Effect of inoculation of endophytic bacteria on the growth parameters of tomato seedlings. Results shown are the mean of triplicates. Data were analyzed using one-way analysis of variance and treatment means were compared (values with different lower case letters are significantly different; P ≤ 0.05%)

Discussion

The tropical regions of the Andaman and Nicobar Islands have a unique climate encompassing different degrees of rainfall and humidity, which favors a number of diseases in these islands. Such temporal and climatic selection processes are bound to aid the evolution of a variety of living organisms, including endophytic bacteria, that are highly adapted to changing extremities of weather, and the islands are known to be a rich source of microbial diversity (Greenland and Losleben 2001). Bacterial plant growth promotion is a well-established and complex phenomenon that is often accomplished via the activities of more than one PGP trait exhibited by the associated bacterium (Lifshitz et al. 1987). In this study, we found that endophytic bacterial isolates of tropical crops possessed multiple PGP traits such as P-solubilization, IAA production (15.0 to 59.2 μg ml⁻¹) and siderophore production. All the endophytic bacteria exhibited clear antifungal activity in vitro and showed significant growth inhibitory activity against the tested phyto-pathogenic fungi.

This study has demonstrated the plant growth promotion and biocontrol potential of strains BETS11, BETS14, BETL9, BETL13 and BECS7, i.e., members of the genus Bacillus spp. BECL8 and BECS6 members of the genus Serratia sp. and BECS1 member of the genus Arthrobacter sp. Serratia marcescens has been used as a plant growth promoter and to control plant diseases in blue berry (Xu et al. 2007), tea (Chakrabortya et al. 2010), banana (Ting et al. 2008), rice (Gyaneshwar et al. 2001) and maize (Koo and Cho 2009). The plant growth promoting phenomenon can be attributed to the ability of the isolate to produce IAA, as IAA positively influences root growth and development, thereby enhancing nutrient uptake (Khalid et al. 2004). It is a well-established fact that improved phosphorous nutrition influences overall plant growth and root development (Jones and Darrah 1994). In the present study, it was observed that all the isolates had the ability to produce siderophore. Siderophore production by the isolate assumes significance for iron nutrition of plants grown under iron-deficient conditions (Pieterse et al. 2001) as well inhibition of phytopathogens. Worldwide, there is a profound need to explore varied agro-ecological niches for the presence of native beneficial microorganisms. Many studies have been undertaken to understand the nature and properties of these unique microbes, which harbor potential PGP traits. With increasing awareness of the problems of chemical-fertilizer-based agricultural practices (Ahmad et al. 2008), it is important to search for region-specific microbial strains that can be used as a potential PGPS to achieve desired products. All eight isolates in this study stimulated the growth of chilli and tomato seedlings under pot culture conditions. The increased nutrient uptake parameters could be attributed to the enhancement of root growth and development. The differences in plant growth promotion among the isolates are attributed to their individual competencies. Future studies will be required to investigate the PGP nature of these isolates and to harness their potential as bio-inoculants in agriculture.