Introduction

Soil-dwelling bacteria are important components of soil health status as they play crucial roles in recycling and transforming nutrients so that they are available to plants [15]. A diverse group of soil bacteria known as plant growth promoting rhizobacteria (PGPR) isable to inhabit the area close to plant roots and provide beneficial effects on the growth of the plant [16]. Plant growth promotion by PGPR involves direct mechanisms which improve nutrient uptake and regulation of phytohormones [6, 10]. PGPR facilitate nitrogen, phosphorus and iron uptake via N2-fixation, inorganic phosphate solubilization and production of iron chelators known as siderophores, respectively [30]. PGPR are able to modulate phytohormones by synthesizing the auxin, indole-3-acetic acid (IAA) and producing 1-aminocyclopropane-1-carboxylate (ACC) deaminase which decreases ethylene levels [1]. Indirect mechanisms of plant growth promotion mainly involve biocontrol and reducing inhibitory effects on plant growth [10]. Biocontrol by PGPR is achieved by competition for nutrients and sites, reduction of virulence factors, production of antifungal compounds and induced systemic resistance [18]. The beneficial features that bacteria exert on plants, while the two are in association can be harnessed by isolating the relevant bacteria and re/introducing them into crop agricultural environments at favourable conditions, i.e., at increased population numbers or in a conducive substrate.

The majority of Namibians based in the North and North Eastern regions depend on subsistence agriculture for their livelihood [36]. Land degradation and environmental threats that are linked to the long-term impacts of climate change can lead to disastrous situations [38]. Therefore, it is important to identify adapted solutions in order to realise sustainable land management practices for the benefit of using natural biological resources for crop farming [38]. The sustainable agriculture system which comes to mind is one in which, instead of synthetic inputs, natural resource management is used to maximise crop production [34]. Agricultural soils used by small scale subsistence farmers lack sufficient amounts of nutrients to support ideal crop growth [10]. By incorporating native plant-associated bacteria into agricultural farming systems, it is possible to improve sustainability and productivity of crop farming in Namibia. This study was done to isolate rhizospheric bacteria from grasses along the Kavango River and screen the isolates for the plant growth promoting characteristics.

Materials and Methods

Sampling

The samples were taken from a total of 8 sites located in the Kavango River in the East Kavango region of Namibia. Half of the sites were situated in pristine lands (17°53′38.49″S; 20°09′08.97″E, 17°52′40.49″S; 20°10′31.03″E, 17°53′33.62″S; 20°14′56.13″E and 17°52′30.82″S; 20°15′21.88″E) and the other half were located in small subsistence farmers’ crop fields (17°55′00.13″S; 20°06′16.14″ E, 17°53′49.75″S; 20°09′07.07″E, 17°53′43.80″S; 20°14′05.26″E and 17°54′04.40″S 20°14′14.34″E). Convenience sampling coupled with a plant health scale that was based on the physical appearance of the plants was used in subsistence farming fields. Plants were ranked on three levels: 0 = wilted/pale yellow leaves, 1 = wilted/green leaves and 2 = not wilted/green leaves. Only those with a healthy appearance level 2 were selected. A systematic sampling method, where samples were selected along a 30–50 m transect line at 10-m intervals, was used to select plants from pristine areas. Samples were collected by digging up a whole plant, removing the surrounding soil followed by aseptically detaching the root system from the plant and then stored at 4 °C.

Isolation of Bacteria

For each sample, one gram of root material was rinsed with 2 ml sterile distilled water and transferred into 9 ml phosphate buffer saline (Oxoid Ltd.) solution, followed by vortexing. Thereafter, 1 ml of the mixture was transferred into 9 ml of enrichment media i.e., combined carbon medium [3] and synthetic malate (SM) [25], and incubated at 30 °C for 14 days. Serial dilutions up to dilution factor of 10−6 were prepared from the incubated tubes followed by spread plating on VM-ethanol agar [26]. The inoculated plates were incubated at 30 °C for 5–7 days. After incubation, single colonies were subcultured onto fresh VM-ethanol agar plates and incubated at 30 °C for 5–7 days.

Inorganic Phosphate Solubilization

The ability to solubilize inorganic phosphate was tested by growing the bacterial isolates on Pikovskaya agar (Oxoid Ltd) dyed with bromophenol blue (Sigma-Aldrich Co.) [14] for 10 days at 30 °C. The formation of more transparent zones around the bacterial colonies was indicative of inorganic phosphate solubilization on Pikovskaya agar. The diameter of the zones and the diameter of the colonies were measured and recorded. This test was done in triplicate. The solubilization index was calculated using the published formula [7].

Siderophore Production

Chromeazurol-S (Chromeazurol S; Sigma-Aldrich Co.) [33] agar was used to determine if the bacteria were able to produce iron chelating siderophore complexes. After growing the bacteria at 30 °C for 5 days on CAS agar [33], plates were observed for the formation of orange halos around the bacterial colonies indicating siderophore activity. This test was done in triplicates.

Quantitative Valuation of Indole-3 Acetic Acid Production

The quantitative valuation of indole-3 acetic acid (IAA) was done according to [14], with slight modifications. Bacterial isolates were grown in VM-ethanol broth and adjusted to a concentration of OD600 = 1. The broth cultures were used to prepare 10 ml suspensions (10 % v/v) in VM-ethanol broth supplemented with 5 mM l-tryptophan (Sigma-Aldrich Co.) and placed in a shaking incubator under dark conditions at 30 °C for 2 days. Cultures were centrifuged at 4,400 rpm for 15 min followed by removing and mixing the supernatant with Salkowski’s reagent in the ratio of 1:2. The mixture was incubated at room temperature for 30 min. Absorbance values of the supernatant and Salkowski reagent (Sigma-Aldrich Co) mixtures were measured at 530 nm. Non-inoculated broth was kept as a control. This test was done in triplicates. The quantity of IAA produced by the bacterial isolates was determined by comparing absorbance values with those from a standard curve. Pure IAA (Sigma-Aldrich Co.) was used to prepare standard concentrations of 0, 5, 10, 15, 20, 25, 30, 35, 40 and 45 mg l−1 [39].

Amplification of Nitrogenase nifH Gene Amplicons

The gene that codes for the Fe protein polypeptide of the nitrogenase enzyme is the nifH gene. Thus, the detection of the nifH gene in the genomic DNA sample of a bacterial isolate would label that particular isolate as a nitrogen fixer. In order to amplify the nifH gene, a nested PCR using outer primers FGPH19 (5′-TACGGCAARGGTGGNATHG-3′), PolR (5′-ATSGCCATCATYNTCRCCGGA-3′) and inner primers PolF (5′TGC GAYCCSAARGCBGACTC-3′) and AQER (5′-GACGATGTAGATYTCCTG-3′) [31] was performed in a Esco Swift™ MaxPro Thermal Cycler. In the first step, a 25 µl PCR mixture consisted of 12.5 µl DreamTaq Green PCR Master Mix 2× (ThermoScientific), 2 µl Genomic DNA prepared with Zymo Research Fungal/Bacterial DNA MiniPrep™, 0.5 µM of each primer and 10 µl nuclease-free water (ThermoScientific). The reaction was carried out as follows: initial denaturation at 94 °C for 4 min, 30 cycles at 94 °C for 1 min, 55 °C for 1 min and 72 °C for 2 min, and final extension at 72 °C for 5 min. In the second step, a 25 µl PCR mixture consisted of 12.5 µl DreamTaq Green PCR Master Mix 2 × (ThermoScientific), 2 µl template from the first step, 0.5 µM of each primer and 10 µl Nuclease-free water. The reaction was carried out as in the first step except the annealing temperature was set to 56 °C instead of 55 °C. A strain of Enterobacter sp. MOP 1-1 [4] was used as a positive control. The PCR products were separated by electrophoresis in 1.2 % agarose gels stained with ethidium bromide (Sigma-Aldrich) and visualized under UV light.

PCR Amplification of 16S rRNA and Sequence Analysis

Genomic DNA from each isolate was used as a template for amplifying the 16S RNA gene fragment by PCR using forward primer 27F (5′-GAGTTTGATCMTGGCTCAG-3′) and reverse primer 1492r (5′-GGTTACCTTGTTACGACTT-3′). The 50 µl PCR mixture consisted of 25 µl DreamTaq Green PCR Master Mix 2 × (ThermoScientific), 2 µl Genomic DNA, 1 µM of each primer and 21 µl nuclease-free water. The reaction was carried out according to Grönemeyer et al. [13] in a Esco Swift™ MaxPro Thermal Cycler. More specifically, PCR cycle steps were as follows: initial denaturation at 95 °C for 4 min, 35 cycles at 95 °C for 1 min, 50 °C for 30 s and 72 °C for 1 min, and final extension at 72 °C for 10 min and holding at 4 °C. The products were separated by electrophoresis in 1 % agarose gels stained with ethidium bromide and visualized under UV light. Purification and sequencing of PCR fragments were carried out at Inqaba Biotec (Pretoria, South Africa). The Sequences were analysed using the Basic Local Alignment Search Tool (BLAST) to determine which organisms the sequences are most similar to. The BLAST programme is available in the National Center for Biotechnology Information website (NCBI-http://www.ncbi.nlm.nih.gov/gene bank).

Results

Bacteria Isolated from Grasses

A total of 21 bacterial isolates were isolated from the rhizospheres of Pennisetum glaucum (12) and Sorghum bicolor (5) of subsistence farmers; and from rhizospheres of Sporobolus sp. (1) and Phragmites australis (1). One of the bacteria was isolated from both Vetiveria nigritana and Sporobolus sp. and the last was a common isolate from P. glaucum and Sorghum bicolor plants, see Table 1. The majority of the isolates were isolated from the fields of subsistence farmers.

Table 1 Molecular identification of bacterial isolates based on 16S rDNA sequences
Full size table

Plant Growth Promoting Characteristics

Bacterial isolates LCM1-14 (Pseudomonas veronii), ACM2-32 (Pseudomonas stutzeri) and WSS2-47 (Bacillus megaterium) were the only phosphate solubilizing isolates as tested via Pikovskaya agar plate assay. The solubilisation index was used to assess the relative phosphate solubilisation of the bacteria, see Table 2. Isolate ACM2-32 (index = 3.5) had a desirable index value of greater than 3, while WSS2-47 and LCM1-14 had indexes of 1.4 and 2.8, respectively.

Table 2 Relative quantification of inorganic phosphate solubilized zone
Full size table

The qualitative measure of siderophore production depends on an iron mobilization reaction which causes a change in pH thus bringing about a colour change in the medium. Four of the isolates, LCS2-11, LCM1-14, WSS2-47 and LSM1-65, were able to produce iron siderophore complexes in CAS agar. As with phosphate solubilization, only bacteria isolated from P. glaucum and S. bicolor crops had the ability to chelate iron.

The bars in Fig. 1 show the amount of IAA produced by isolates with the capability to produce indole in broth supplemented with l-tryptophan. Isolate FCM2-50 (E. cloacae) produced the highest value of 8.98 ± 0.46 mg IAA L−1, whilst the isolate LCS2-11 (S. maltophilia) produced the lowest value of 3.63 ± 0.54 mg IAA L−1 amongst the selected isolates. The other isolates that produced IAA were LCM1-14 (P. veronii), LSS1-21 (P. validus), ASM1-59 (B. subtilis), FCM1-66 (B. licheniformis), LSE-68 (Bacillus sp.) and LSM1-61 (B. amyloliquefaciens).

Fig. 1
figure 1

Mean average indole-3-acetic acid (IAA) produced (milligrams per litre) by eight selected bacterial isolates. Error bars denote one standard error around the mean

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Detection of nifH Gene from Genomic DNA

The nifH gene fragment was amplified from the genomic DNA of 5 isolates (Fig. 2). The amplified fragments were as expected ~320 bp long. The nifH was detected in DNA from isolates LCS2-11(S. maltophilia), WCM1-41 (B. cereus), WSS2-49 (B. cereus), FCM2-50 (E. cloacae) and LSE-68 (Bacillus sp.). The presence of the nifH gene is an indication of N2-fixation capability by the isolates.

Fig. 2
figure 2

Agarose gel of nifH amplicons from the DNA of five isolates and Enterobacter sp. MOP 1-1, which was used as a positive control

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Discussion

The resilient spore producing Bacilli and the versatile Pseudomonas spp., is predominant genera in the rhizosphere [23, 24]. The ubiquitously occurring Stenotrophomonas spp. are mainly found in soils and are known to be in association with plants. Beneficial relationships between Stenotrophomonas spp. and plants have been reported by Ryan et al. [27]. The actinomycete isolate Kocuria sp. LSM1-65 had demonstrated siderophore producing capability. Kocuria turfanensis strain 2M4 isolated from the rhizospheric soil was found to be an IAA producer, siderophore producer and phosphate solubilizer [11]. Enterobacter bacteria are prevalent in the rhizosphere as well as the endorhizosphere; they have been isolated from the rhizosphere of various plants [17, 21, 32]. Enterobacter spp. belong to a group (Enterobacteriaceae) which have rhizosphere colonization advantage [2]. Most of the isolates considered as plant growth promoting bacteria are known human pathogens. Reassuringly, we are aware that the pathogenic strains come into being via induced stress factors and selective pressures [5, 35]. Avoiding the risk to human health requires the use of a multidisciplinary approach to better understand interactions that take place between the microbes plant associates, the host plant, and the environment to accomplish proper screening so that the clinical strains are distinguished from the agricultural ones [8].

Phosphate solubilizing bacteria can play an essential role in maintaining soil phosphorus balance. Pseudomonas spp. are amongst the most effective phosphate solubilizing bacteria [17, 19, 37]. Siderophore activity by isolate P. veronii LCM1-14 may be attributed to pyoverdine, pyochelins and/or quinolobactin [20, 29], whilst S. maltophilia LCS2-11 is believed to secrete siderophores of the catechol-type [9]. Bacillus spp. are known to produce hydroxamate-type siderophores [22]. Tryptophan introduced as an external source initiates IAA production by the bacteria. Bacteria that reside in the rhizosphere produce plant hormones that can positively influence plant growth [12], particularly the root system development [28]. Fixing of atmospheric nitrogen is a valuable feature of diazotrophic PGPR. These bacteria should be regarded as vital components in inoculation formulations for the reason that other than chemical fertilizers, nitrogen-fixing bacteria offer the only practical alternative to solving the problem of nitrogen deficiency in agricultural soils.

Thirteen bacterial isolates with various plant growth promoting characteristics were isolated from the rhizospheres of grasses along the Kavango River. Efforts aimed at discovering native microorganisms that can improve crop growth are encouraged. A diverse collection of plant-beneficial bacteria is accommodated in the rhizospheres of a wide variety of plants. Native PGPR bacteria inoculants have the potential to alleviate challenges of local chemical fertilizer production and low cereal grain production. Using bacteria as a bio-fertilizer for sustainable soil health improvement and increased crop production can be achieved in a moderately cheap and ecologically safe manner.