Research & Reviews: Journal of Microbiology and Biotechnology
MenuE- ISSN: 2320 - 3528
P- ISSN: 2347 - 2286
E- ISSN: 2320 - 3528
P- ISSN: 2347 - 2286
1Jomo Kenyatta University of Agriculture and Technology, School of Biological Sciences, P.O. Box 62000-00200 Nairobi, Kenya
2Pure and Applied Sciences, Technical University of Mombasa, Kenya
3Embu University College, Department of Biological Sciences, P.O. Box 6-60100 Embu, Kenya
Received date: 06/05/2015 Accepted date: 02/16/2016 Published date: 02/26/2016
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Banana being the world’s third most important starchy food and widely grown in developing countries requires large amounts of nitrogen fertilizers for commercial cultivation which are costly and hazardous to the environment when used in excess. Plant growth promoting rhizobacteria are bacteria found in the rhizosphere in association with roots and can enhance the growth of the plant. We isolated and identified the rhizobacteria associated with bananas (Musa spp.) from rhizosphere soil samples collected from seven banana farms in Juja, Kenya and evaluated their potential as biofertilizers. We obtained 20 bacterial isolates and 19 had the potential to fix nitrogen as observed from acetylene reduction assay while 18 had phosphate solubilisation activities. Phylogenetic analysis clustered the isolates into three phyla represented by six different genera: Pseudomonas, Bacillus, Staphylobacterium, Chryseobacterium, Streptomyces and Paenibacillus spp. This work has shown that banana rhizospheric bacterial isolates harbor plant growth promoting traits that can be utilized as biofertilizers in agriculture to promote the growth as well as production of bananas.
Rhizobacteria; Rhizosphere; Biofertilizers; Musa spp.
Rhizosphere is the thin layer of soil that immediately surrounds the plant roots [1] and has abundant nutritious elements because of the continuous rot activities of the plants. This zone is mixed with solid particles and an active community of free living beneficial bacteria [2], usually known as plant-growth-promoting rhizobacteria (PGPR).
The banana plant (Musa spp.) has adventitious and horizontal roots proliferating the topsoil and cannot get water and nutrients from the deeper soil profile unlike other fruit crops. The undeveloped root system inhibits the utilization of essential mineral nutrients thus limiting the large scale production of bananas under adverse tropical soil conditions. In the recent past there has been an increasing interest in soil microorganisms due to their importance in maintaining soil fertility since it has been shown that plant growth may be stimulated by vitamin-related and pathogen suppressing phytohormones produced by rhizospheric bacteria [3]. The mutual association where the bacteria obtain the nutrients released from the roots of the plants for growth as they in turn secrete metabolites to the rhizosphere [4], is perceived to augment plant growth by the rhizobacteria directly acting as biofertilizers to provide nutrients to the host plant or indirectly by their positive influence on the growth and morphology of roots [2].
Previously, Ngamau et al. and Reena et al. have demonstrated diverse banana endophytic bacteria with potential in utilization of the limiting nitrogen and phosphorous nutrients in banana plant growth [5,6]. We postulate that the tropical banana rhizosphere harbor’s a wide diversity of PGPR that may not only aid in beneficial symbiotic relationships but may stimulate the plant growth by suppressing pathogenic organisms. Since biofertilizers are widely accepted as a source of fertilizers with significant increase in crop yields [7], we set herein to characterize the PGPR associated with banana roots and determine their potential use as a biofertilizer in banana growth.
Sample collection, isolation and profiling
Soil samples from the rhizosphere region were collected during the month of June 2012 from the cooking and ripening varieties of Musa spp. from seven farms in Juja, Kenya. They were collected by digging at 20-40 cm surrounding the banana plant roots and scrubbing the soil attached to the roots. The rhizosphere soil was scrubbed off the roots, serial diluted fivefold and inoculated on nitrogen-free media, yeast extract mannitol agar and nutrient agar (HIMEDIA) for 24 hours at 30ºC. Individual colonies were aseptically picked and streaked on sterile fresh media to purify them and generate pure cultures for morphological, biochemical and molecular characterization. The pure cultures were Gram classified and tested for: hydrogen sulphide production, catalase utilization, urease production, nitrate reduction, IMViC tests, starch, casein and gelatin hydrolysis using standard methods as described by Cappuccino and Sherman (2002) [8].
Characterization of rhizobacteria for plant growth promoting traits
The isolates were assessed for nitrogenase activity based on their ability to reduce acetylene (C2H2) to ethylene (C2H4) by acetylene reduction assay by growing an isolate for 72 hours at 30ºC in 5 ml nitrogen free semisolid agar in 10 ml vials. Acetylene was added at 12% v/v concentration and the ethylene produced was determined on a Shimadzu Gas Chromatograph (GC-9A, Japan) after 12 hours [9]. Using a syringe, 1 ml of ethylene produced was injected into the GC machine and the retention time for each isolate measured.
The isolates were assessed for nitrogenase activity based on their ability to reduce acetylene (C2H2) to ethylene (C2H4) by acetylene reduction assay by growing an isolate for 72 hours at 30ºC in 5 ml nitrogen free semisolid agar in 10 ml vials. Acetylene was added at 12% v/v concentration and the ethylene produced was determined on a Shimadzu Gas Chromatograph (GC-9A, Japan) after 12 hours [9]. Using a syringe, 1 ml of ethylene produced was injected into the GC machine and the retention time for each isolate measured.
Genomic DNA extraction and amplification of the 16S rRNAs
The 16S rRNA gene of the bacterial isolates was amplified by polymerase chain reaction using universal primers 27F 5’-GAGTTTGMTCCTGGCTCA-3’ and 1492R, 5’-TACGGYTACCTTACGACT-3’ (Bioneer, USA) as described by Embley and Stackebrandt (1994) [12] using an Eppendorf AG, model 22331 thermal cycler (Hamburg, Germany).
The PCR mixture comprised of 0.2 Units of Taq polymerase, 20 pmol of 27 F forward primer, 20 pmol of 1492R reverse primer, 1.25 mM dNTPs mix (QIAGEN), 10x PCR buffer (QIAGEN), 1 ml of template DNA and 29.8 ml of PCR water. The thermal program was initial denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, primer annealing at 43°C for 2 min, chain extension at 72°C for 1.5 min for 35 cycles, followed by final extension at 72°C for 5 min. The amplicons were purified with the Gene JET Purification kit (Thermo Fisher Scientific Inc.) and subjected to DNA sequencing according to the manufacture’s protocol (Macrogen, South Korea) using 27 F (5’-GAGTTTGMTCCTGGCTCA-3’) and 1492R (5’-TACGGYTACCTTACGACT-3’) primers.
The sequences were processed using the Chromas Pro software (www.technelysium.com.au) and aligned using CLUSTAL Omega program (http://www.clustal.orgused). They were used in a BLAST search against sequences hosted at the National Center for biotechnology Information (NCBI) database to determine their closest neighbours using (BLASTn algorithm, http://www.ncbi.nih. gov as described by Altschul et al. [13]. A phylogenetic tree was constructed using neighbor-joining method [14] by using MEGA V5.10 package (www.megasoftware.net) while evolutionary distances were computated using the Maximum Composite Likelihood method [15]. To obtain statistical support values for the branches, bootstrapping was conducted with 1000 replicates and all sites, including gaps in the sequence alignment, were excluded pairwise in the phylogenetic analysis [16]. The partial sequences for the 16S rRNA genes were deposited in the GenBank sequence database and assigned accession numbers KP797914 to KP797931. The taxonomic assignment was confirmed using the naïve Bayesian rRNA classifier on the RDP website at a 95% confidence level [17].
Assessment of growth enhancement effect on tissue culture banana plantlets
Based on plant growth promoting traits screened previously, five strains namely Chryseobacterium spp., Bacillus megaterium, Bacillus flexus, Pseudomonas monteilii and Streptomyces spp. were assessed for plant growth enhancement under controlled greenhouse conditions. Bacterial cultures prepared as previously described by Mia et al. were infected on tissue culture banana plantlets by dipping the 3 month old plantlets into the specific bacterial isolate treatment for 48 hours at room temperature while distilled water was used for the control experiment. The plantlets were grown in a sterile nutrient inert Cocopeat media (Amiran, Kenya) supplemented with nitrogen, phosphorus and potassium (NPK) slow release fertilizer at 5 levels (0 gms, 2.5 gms, 5 gms, 7.5 gms and 10 gms) in a random block design in three replicates [18].
Agronomical features of the plantlets: height, diameter and number of green and dry leaves were assessed at 14 days intervals for 3 months [19] after which the plants were destructively sampled by gently uprooting, washing off the media under running tap water and measuring the fresh weight. Dry weight was taken after oven drying the plantlets at 80ºC for 14 days then essential nutrient levels of nitrogen, phosphorus, potassium, calcium, iron and magnesium was assessed according to Plank [20].
Statistical analyses
The agronomical data, mean ± SD from three experimental replicates were collected for analysis. Differences in agronomical parameters among the bacterial treatments were tested by analysis of variance procedure (ANOVA) using SPSS software. The student t-test was used to assess the variation between each of the treatments taken against the control experiment (Walpole 1986). A probability of 5% was considered significant (P ≤ 0.05).
Isolation and profiling of banana rhizosphere isolates
After isolation and culturing, 20 pure isolates were obtained from 67 soil samples based on colony morphological and biochemical characterization revealing relative bacterial diversity. Nitrogenase activity was seen in 19 isolates, 18 (90%) solubilized phosphates while none of the bacterial strains produced the auxin indole acetic acid hormone. Catalase activity was observed in all the isolates (Table 1).
Table 1. Morphology, physiological and biochemical characterization of rhizobacteria isolated from banana farms in Juja, Kenya.
The Bacillus spp. and Pseudomonas spp. isolates showed strong phosphorus solubilization activity than the rest of the isolates. Pseudomonas spp. had phosphate solubilization activity of 1.6 being the highest, followed by Bacillus spp. having 1.5 and 1.45 on the phosphate solubilization index on agar assay (Table 2). Phosphate solubilization ability of the plant growth promoting rhizobacteria is considered to be one of the most important traits associated with plant phosphorous nutrition. Soil or seed inoculations with phosphate solubilizing microbes (PSM) have been used for the improvement of crop growth and production by the solubilization of fixed and applied phosphates [21].
All Bacillus isolates were found to have nitrogenase activity and one of the Bacillus spp. showing the highest concentration of 0.7547 μl/ml followed by another with 0.1515 μl/ml indicating their potential in fixing nitrogen (Table 2). From table 2, Chryseobacterium spp. showed a nitrogenase activity of 0.069 μl/ml and 0.0145 μl/ml which could be a potential nitrogen fixer since members of these genus exhibit plant-growth promoting activities as they are universal symbionts of some higher plants [22,23].
Table 2. The extent of phosphate solubilisation on agar and concentration of ethylene produced by the bacterial isolates.
Two Staphylococcus spp. with nitrogenase and phosphate solubilisation abilities were identified which to our knowledge has not been isolated from banana rhizosphere. Cases of this genus promoting plant growth have rarely been documented. Nitrogen is a crucial element for bananas as it may be a limiting factor in the production and growth of bananas. Ahemad and Khan demonstrated that nitrogen is among the nutrients commonly required by the banana plant for their optimal growth and productivity [1,24].
The spore forming PaeniBacillus xylanilyticus identified showed promising plant growth promoting nitrogenase and phosphate solubilisation activities. Some species of PaeniBacillus have consistently shown ability to fix atmospheric nitrogen in vitro [25].
Though reported to be one of the dominant species in banana rhizosphere, the present study isolated only one Pseudomonas monteilii with nitrogenase and phosphate solubilisation ability. This bacterium has been suggested to belong to the sub group of P. putida which are ecologically important in colonizing the banana plant roots rhizosphere [26]. Only two Streptomyces spp. were identified despite being shown as important microorganisms present in the banana root rhizosphere that have antagonism to phytopathogenic fungi [27]. Streptomyces strains promote banana plantlets’ growth by supplying iron to them.
Bacterial identification using the amplification of the 16S rRNA
Phylogenetic relationship of the bacterial isolates sequenced was inferred using the Neighbour Joining method. Eighteen (18) sequences of the 20 isolates in this study were included while two (2) were excluded in subsequent analysis since they had chimeras. The bacterial isolates clustered with sequences from different genera: three isolates were categorized as Staphylococcus spp., two (10%) as Streptomyces spp., two (10%) as Chryseobacterium jejuense strain NW50, while 11 (55%) were categorized as belonging to the genus Bacillus. With 100% sequence similarity, one isolate was characterized as PaeniBacillus spp., and also Pseudomonas monteilii strain IHB B 2329 (Figure 1).
Figure 1. Neighbour joining phylog enetic tree showing the position of the banana rhizospheric isolates from Juja, Kenya. Eighteen strains from seven different farms in Juja that were isolated in this study are indicated in bold font. The accession numbers of the sequences are indicated in parantheses. The bootstrap values are the numbers seen at the nodes based on 1000 replications. The 16S rRNA gene sequence of Methanoculleus thermophiles was an outgroup used.
Assessment of growth enhancement effect on tissue culture banana plantlets
Inoculation of the plants with five selected isolates had a positive growth influence on tissue cultured banana plantlets treated with 5 gms, 7.5 gms and 10 gms levels of NPK fertilizer levels in height, diameter, number of green leaves and mass in dry weight compared with their respective controls. The direct effect of the inoculants on the plantlets could not be established because of the confounding fertilizer level effect. The mean plant diameter (cm) and number of green leaves in isolates plantlets treated with Pseudomonas monteilii and Streptomyces spp. were higher than their controls, as similarly observed in the mean diameter for plantlets treated with Chryseobacterium spp., Bacillus megaterium and Bacillus flexus strains at 10 gms fertilizer level. Plantlets treated with Streptomyces spp. isolate registered a significantly higher growth in all the parameters (p<0.05). Height and dry weight were significantly higher in all the five strains tested (p=0.000) except Pseudomonas monteilii in height (p=0.007). Diameter was significantly higher on plants treated with Streptomyces spp. (p=0.000) at 10 gms fertilizer level (Figure 2). The total accumulation of nitrogen, phosphorus, magnesium and calcium was significantly higher (p=0.00000684, 0.000163, 4.46E-09 and 3.77E-05 respectively) in all the treatments following inoculation with the isolates. This could be attributed to the effect of the inoculation on the uptake of these nutrients. However, the intake of potassium, zinc and iron was not influenced by inoculants since there were no differences observed between the treatments and the controls. Plant growth promoting rhizobacteria inoculation on bananas substantially increases the development and growth of tissue-cultured banana plantlets [19].
Figure 2. Effect of selected bacteria isolates from Juja on banana plant growth (A: Mean diameter, B: Mean height, C: Mean number of green leaves, D: Mean number of dead leaves, E: Mean mass in dry weight). An asterisk (*) means significantly different to the control (p<0.05).
The present study shows that banana rhizospheric bacterial isolates from Pseudomonas, Bacillus, Staphylobacterium, Chryseobacterium, Streptomyces and PaeniBacillus genera in Juja, Kenya harbor plant growth promoting traits and therefore these isolates can be used as biofertilizers in agriculture to promote the growth as well as production of bananas.
This study was supported by a research grant from the Research, Production and Extension Division (RPE) of Jomo Kenyatta University of Agriculture and Technology-Kenya. We wish to acknowledge the departments of Horticulture and Botany (JKUAT) and their technical staff for technical support and availing their laboratory facilities for this study.
Special appreciation goes to Mr. Zachary Ndung’u (JKUAT) for his support during sample collection and green house experiments.