DOI: 10.5061/dryad.c866t1g4x
title: Symbiotic efficiency and genetic characterization of bacteria associated with cowpea genotypes grown in semiarid tropics of Kenya

description: 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&lt;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.
description: Ten farms were selected in the semi-arid areas of Embu (Mbeere
 South sub-county) latitude 1<sup>o </sup>10’S longitude
 37<sup>o </sup>47’E and Kitui (Kitui West sub-county) latitude
 0<sup>o</sup>46’S longitude 37<sup>o</sup>39’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<sup>o</sup>C. 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]<b>.</b> 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<sup>o</sup>C 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 cm<sup>2</sup> 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<sup>o</sup>C 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<sup>o</sup>C. 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 <i>Bradyrhizobium</i>
 sp.<i> </i>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 <i>Bradyrhizobium</i> sp. strain USDA 3456
 were inoculated onto the seedlings. Plants inoculated with the reference
 strain (<i>Bradyrhizobium</i> sp. strain USDA 3456) and those
 in Leonard jars supplied with nitrogen (sterile 1.0M
 KNO<sub>3</sub> 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 KNO<sub>3</sub>) ×
 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
 (<i>x</i>+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 4<sup>0</sup>C. 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].
description: References 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. <i>BJAST</i>. 4, 718-738.
 (doi:10.9734/BJAST/2014/5757) Broughton, W.J. and
 Dilworth, M.J. (1971). Control of leghaemoglobin synthesis in snake beans.
 <i>Biochem J</i>. 125, 1075-1080. (doi:
 10.1042/bj1251075) Somasegaran P, Hoben HJ. 1994
 Quantifying the Growth of Rhizobia. In: Handbook for Rhizobia.
 <i>Springer,</i> New York; 47–57.  (doi:10.1007/978-1-4613-8375-8_5) Yuan Y. 2011 Multiple imputation using SAS software. <i>J Stat Softw</i>. 45, 1-25. 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 (<i>Phaseolus vulgaris</i> L.) Varieties. <i>Front Microbiol.</i> 9,968 (doi:10.3389/fmicb.2018.00968) Treves DS. 2010 Review of Three DNA Analysis Applications for Use in the Microbiology or Genetics Classroom. <i>JMBE</i>. 11,186–7. (doi:10.1128/jmbe.v11i2.205) Kumar S, Stecher G, Li M, Knyaz C, Tamura K. 2018 MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms. Battistuzzi FU, editor. <i>Mol Biol Evol</i>. 35, 1547–1549. (doi:10.1093/molbev/msy096)