Cowpea (Vigna unguiculata (L.) Walp) is an important multipurpose legume crop grown in arid semiarid areas of sub-Saharan Africa. The crop associates with a wide diversity of high ecological value rhizobia bacteria, improving biological soil fertility and crop production. Here, we evaluated the symbiotic efficiency (SE) and genetic diversity of native rhizobia isolated from root nodules of cowpea genotypes cultivated in semiarid areas of Kenya.  Rhizobia trapping and SE experiments were done in the greenhouse while genetic diversity was evaluated based on 16S rRNA gene sequencing. Ninety four bacterial nodule isolates were purified from root nodules and placed into 20 groups based on morphological characteristics. SE of the native isolates varied (p<0.0001) significantly. Remarkably, two isolates, M2 and M3 recorded higher SE of 82.49 % and 72.76 % respectively compared to the commercial strain Bradyrhizobia sp. USDA 3456 (67.68 %). The isolates closely resembled bacteria belonging to the genus Rhizobium and non-rhizobial endophytes Enterobacter, Strenotrophomonas, Pseudomonas, and Paraburkholderia. For the first time, we report the presence of an efficient native nodule isolate Paraburkholderia phenoliruptrix BR3459a in Kenya. Our results form an important step towards the development of efficient microbial inocula which are pertinent for sustainable food production in African agriecosystems.

Ten farms were selected in the semi-arid areas of Embu (Mbeere South sub-county) latitude 1^o 10’S longitude 37^o 47’E and Kitui (Kitui West sub-county) latitude 0^o46’S longitude 37^o39’E counties in lower Eastern Kenya. Five farms were selected per county making a total of ten farms. Soil samples were collected from 20 points diagonally and across each farm keeping a radius of at least 6 m from each point. In the process, 20 kg of soil were obtained from each farm at a depth of 5-30 cm after clearing soil debris from the soil surface. The soils from each farm were then mixed thoroughly to obtain a homogenous soil sample, packed separately and transported to Kenyatta University for greenhouse experiments. Soil samples that were not used immediately were stored at 4^oC.

Greenhouse experiment

Objectives: (i) To trap and isolate bacterial isolates from root nodules of cowpea plants

                 (ii) To authenticate the bacterial isolates and determine their symbiotic efficiency

                 (iii) To determine the genetic diversity of rhizobial and non rhizobial endophytes isolated from cowpea root nodules

Procedure used for rhizobia trapping in the greenhouse

Pots accommodating three kilograms of soil were sterilized using 70% ethanol. Then the soils from each farm were potted separately in the sterilized pots. Cowpea varieties (K80, M66, KVU 27-1, and Kikamba, (which is a preferred cowpea landrace by farmers in the study area) were used in the trapping experiment. The genotypes are recommended by Kenya Agricultural and Livestock Research Organization (KALRO) for ASAL areas in Kenya.The cowpea seeds were sterilized in 3 % sodium hypochlorite for 5 minutes and rinsed in 6 changes of sterilized distilled water. Three seedlings were planted per pot and later thinned to two after 5 days of germination. The pots were arranged in a randomized complete block design. Watering was carried out at one day interval. Each treatment (farm soil) was replicated four times with two plants in each pot (4 cowpea varieties x 4 replicates) making a total of 160 pots representing the ten farms. Harvesting was done after 45 days. The roots and shoots were separated. The roots were carefully washed in a stream of running water after which the nodules were carefully detached. The detached root nodules for each treatment were separately wrapped in absorbent tissue paper and left to dry at room temperature [1].

Isolation and purification of nodule isolates

Nodules representing treatment were immersed in sterile distilled water and allowed to imbibe for two hours. They were then dipped in 70% ethanol (v/v) for 30 seconds to remove air bubbles from the tissues and reduce surface tension. The nodules were then dipped in 3% sodium hypochlorite (v/v) for 3 minutes for further sterilization and then rinsed in six changes of sterile distilled water. The nodules were later crushed in a drop of distilled water with a sterile glass rod. A loop full of the suspension was streaked onto Yeast Extract Mannitol Agar (YEMA) plates containing 0.025 mg/l of Congo red and incubated at 27^oC in the dark. Colonies emerged after three days and after five days well isolated colonies were streaked on YEMA with Congo Red. Morphological characteristics that include colour change, colony elevation, shape, colony size, exo-polysaccharide gum, transparency and mucosity were used for presumptive identification of the rhizobia. For biochemical identification, Bromothymol Blue Test (BTB) and Gram staining procedures were carried out. For BTB the isolates were tested for Acid/Alkali production by growing them on YEMA with BTB (0.025 mg/L) indicator at pH 6.8. The cultures were then incubated at 28 °C in a rotating orbital shaker for up to 5 days. The isolates were allowed to grow and then grouped as acid-producing, alkali producing or neutral, depending on colour changes observed in the media [1].

Authentication of bacterial nodule isolates

The rhizobia isolates were tested under bacteriologically controlled conditions to confirm their nodule forming ability. Leonard jar assemblies were prepared and they comprised of a modified plastic cup with a diameter of 8 cm (brim) and a bottom diameter of 4 cm. A rectangular hole 1.5 cm² was made at the bottom of the cup and fit with a 20 cm long wick. Prior to fitting the cups were swabbed with 70% ethanol while the wick was sterilized in 3% sodium hypochlorite. A larger plastic vessel was decontaminated using 70% ethanol and used to suspend the cup assembly. Sterile nitrogen–free plant nutrient medium was prepared [2]. Five stock solutions were later autoclaved at 121^oC for 15 min. The rooting medium used was sterilized vermiculite. The vermiculite was soaked in water overnight, and then thoroughly washed for two days. For the final rinse, distilled water was used. The vermiculite was then autoclaved and later packed into the cups of the Leornard jar assemblies which were later covered with sterilized aluminum foil to maintain the sterile conditions of the assembly. The jars were then put in khaki bags for insulation [3].

Cowpea seeds of uniform size and shape were selected and surface sterilized in 3% (v/v) sodium hypochlorite for 5 minutes then rinsed in six changes of sterile distilled water. The seeds were then pre-germinated on sterile moist vermiculite packed in Kilner jars for 3 days at 28^oC. Three seedlings were transplanted into sterilized Leonard jars using sterile forceps and later thinned to two. Eight days after transplanting, the seedlings were inoculated with (1ml) broth culture of each representative rhizobia isolates. The rhizobia isolates were cultured in Yeast Extract Mannitol Broth (YEMB) for three days (to exponential phase) before inoculation. Leonard jars inoculated with commercial Bradyrhizobium sp. strain USDA 3456 were used as positive controls. Jars of un-inoculated seedlings were used as negative controls. The experiment was laid out in a randomized block design and each treatment replicated four times. The nitrogen free media was replenished every week. After 45 days the plants were harvested. Vermiculite and liquid medium were emptied out of the cup and Leonard jars. Plant roots were washed under running tap water to rinse off vermiculite and the attached wick removed. The plants were scored for presence and absence of nodules.  The presence of a single nodule in a Leonard jar for any plant was viewed as evidence that the isolate was rhizobia [3].

Determination of symbiotic efficiency of representative rhizobia isolates

Sterilization, pre-germination, transplantation and thinning of cowpea seeds in Leonard jars was done as described in the authentication experiment above. After eight days, one milliliter (1ml) broth culture of each authenticated rhizobia isolate and a reference strain Bradyrhizobium sp. strain USDA 3456 were inoculated onto the seedlings. Plants inoculated with the reference strain (Bradyrhizobium sp. strain USDA 3456) and those in Leonard jars supplied with nitrogen (sterile 1.0M KNO₃ solution) were used as positive controls. Non-inoculated plants in Leonard jars supplied with nitrogen free media were used as negative controls. The experiment was arranged in a complete randomized block design and each treatment was replicated five times. Nutrient medium in the jars was replenished every week. Harvesting was done after 45 days. Shoots were separated from the roots after which the roots were carefully washed with tap water. All the nodules were detached and counted. Apart from nodule number (NN), nodule dry weight (NDW), shoot dry weight (SDW), and root dry weight (RDW) were recorded. Symbiotic efficiency (SE %) was calculated by (dividing the total dry weight of the inoculated plants with the total dry weight of non-inoculated control plants supplemented with nitrogen (1.0 M KNO₃) × 100 [2, 3].

Data analyses or processing

For cowpea genotypes, data on the number of nodules, nodule dry weight, shoot dry weight and root dry weight was analyzed using two-way analysis of variance (ANOVA). Means were separated by Tukey’s HSD test at 5 % probability level. All ANOVAs and post hoc tests were carried out using SAS software version 9.2 [4].  Wherever feasible, data was log (x+1) transformed to fulfill the assumptions of ANOVA

Genomic DNA extraction of bacterial isolates to determine genetic diversity of rhizobial and non rhizobial endophytes isolated from cowpea root nodules

Rhizobia cultures were re-suspended in eppendorf tubes containing 400 µl of normal saline to remove polymerase chain reaction (PCR) inhibitors like exopolysaccharides (EPS). The mixture was vortexed for 20 seconds then centrifuged at 13,000 rpm for 10 minutes. The supernatant was poured out leaving the cell pellets. These pellets were washed four times with normal saline, then harvested and re-suspended in 400 µl of genomic lysis buffer. This mixture was incubated in a water bath set at 65 °C for 30 minutes. This was followed by centrifugation at 13,000 rpm for 5 minutes and the supernatant transferred into another sterilized eppendorf tubes. Four hundred microliters of isopropanol was then added to the supernatant and samples incubated at -20°C. The samples were then centrifuged at 13,000 rpm for 3 minutes and the isopropanol discarded. Thereafter, 400 µl of DNA buffer (70% alcohol) was added to the pellet, centrifuged at 13,000 rpm for 1 minute and the liquid phase decanted out gently. The pellets were the air-dried followed by dissolving the DNA in 50 µl of   elution buffer (TE). The quality and quantity of DNA was determined by resolving SYBR green stained DNA in 0.8 % agarose gel and observing the DNA bands in a UV trans illuminator. Extracted DNA was stored at -20°C [5].

PCR amplification of 16S rRNA

PCR was carried out in a 25 µl reaction volume containing 9.0 µl sterile PCR water, 1.25 µl of 10 µM primer 1492 R and 1.25 μl of 10 μM primer 27 F, 12.5 µl One Taq 2X master mix with standard buffer (Biolabs) and 1.0 µl of DNA template. Sequences for the forward primer was (27F 5'-AGAGTTTGATCCTGGCTCAG-3') while the reverse primer was (1492R 5'-GGTTACCTTGTTACGACTT-3'). The PCR reaction was carried out in a Techgene thermocycler, FTGENE5D model (Techne UK). The PCR conditions for amplification were as follows:  An Initial DNA denaturation at 94 °C for 2 min then denaturation at 94 °C for 45 seconds, annealing at 62 °C for 45 seconds and extension at 72 °C for 2 min (35 cycles). Final extension was carried out at 72 °C for 5 minutes. Amplified DNA was held at 4⁰C.

Gel electrophoresis

The PCR products were separated by gel electrophoresis in 1.4% agarose in 0.5X TBE buffer at 80 V for 30 minutes and stained with SYBR-Green. A 1kb DNA ladder (Biolabs) was used to estimate the molecular sizes of the bands. Gel visualization was done using a UV trans-illuminator and photographed using a digital camera.

PCR product purification and 16S rRNA gene sequencing

The PCR products were sent to Inqaba Biotech in (Pretoria) South Africa for purification and Sanger sequencing using a ABI 3730 DNA sequencer (Applied Biosystems, USA).

Data analyses

Base calling for sequenced data was done using FinchTv software [6]. Consensus sequences were created using Genestudio software [6] after which the sequences obtained were compared with sequences in the National Centre for Biotechnological Information (NCBI) GenBank database using Basic Local Arrangement Search Tool (BLAST) program. The sequence identities that were closely related to the sequences of the study isolates were retrieved from the NCBI sequence data repository. The contig and corresponding retrieved sequences were then aligned using clustal W. The evolutionary history of the isolates was then presented in the form of a phylogenetic tree by neighbor joining method using Molecular Evolutionary Genetics Analysis (MEGA) 7 software [7].

References

1. Muthini M, Maingi JM, Muoma JO, Amoding A, Mukaminega D, Osoro N, Mgutu A, Ombori O. 2014 Morphological assessment and effectiveness of indigenous rhizobia isolates that nodulate P. vulgaris in water hyacinth compost testing field in Lake Victoria basin. BJAST. 4, 718-738. (doi:10.9734/BJAST/2014/5757)
2. Broughton, W.J. and Dilworth, M.J. (1971). Control of leghaemoglobin synthesis in snake beans. Biochem J. 125, 1075-1080. (doi: 10.1042/bj1251075)
3. Somasegaran P, Hoben HJ. 1994 Quantifying the Growth of Rhizobia. In: Handbook for Rhizobia. Springer, New York; 47–57.  (doi:10.1007/978-1-4613-8375-8_5)
4. Yuan Y. 2011 Multiple imputation using SAS software. J Stat Softw. 45, 1-25.
5. Koskey G, Mburu SW, Kimiti JM, Ombori O, Maingi JM, Njeru EM. 2018 Genetic Characterization and Diversity of Rhizobium Isolated From Root Nodules of Mid-Altitude Climbing Bean (Phaseolus vulgaris L.) Varieties. Front Microbiol. 9,968 (doi:10.3389/fmicb.2018.00968)
6. Treves DS. 2010 Review of Three DNA Analysis Applications for Use in the Microbiology or Genetics Classroom. JMBE. 11,186–7. (doi:10.1128/jmbe.v11i2.205)
7. Kumar S, Stecher G, Li M, Knyaz C, Tamura K. 2018 MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms. Battistuzzi FU, editor. Mol Biol Evol. 35, 1547–1549. (doi:10.1093/molbev/msy096)