DOI: 10.5061/dryad.1vhhmgqv3
title: Characterization and biodiversity of native Azotobacter in semi-arid agroecosystems of Eastern Kenya

description: Declining food production in the African agroecosystems is attributable to
 changes in weather patterns, soil infertility, and limited farming inputs.
 The exploitation of plant growth-promoting soil microbes could remedy
 these problems. Azotobacter, a free-living, nitrogen-fixing bacterium,
 confers stress tolerance, avails phytohormones, and aids in soil
 bioremediation. The study aimed to isolate, characterize and determine the
 biodiversity of native Azotobacter isolates from soils in semi-arid
 Eastern Kenya. The isolation was conducted on nitrogen-free Ashby’s agar
 and the morphological, biochemical and molecular attributes were
 evaluated. The isolates were sequenced using DNA amplicons of 27F and
 1492R primers of the 16SrRNA gene loci. The Basic Local alignment search
 Tool (BLASTn) analysis of their sequences, revealed the presence of three
 main Azotobacter species viz., Azotobacter vinelandii, Azotobacter
 salinestris, and Azotobacter tropicalis. Azotobacter vinelandii was the
 most dominant species. Kitui County had the highest number of recovered
 Azotobacter isolates (45.4%) with the lowest diversity index (0.8761).
 Tharaka Nithi County showed the lowest occurrence (26.36%) with a
 diversity index of (1.057). The diversity was influenced by the soil pH,
 texture, and total organic content. This study revealed the presence of
 native strains of Azotobacter species in Kenyan soils with the potential
 for utilization as a bioinoculant.
description: <b><i>Soil
 sampling and physical chemical analysis</i></b>
 Soil samples were collected
 from smallholder farms in Tharaka Nithi, Embu and Kitui counties of
 Eastern Kenya. The selected study areas received minimal rainfall and are
 categorized as semi arid. The farms in Tharaka Nithi County were situated
 in the Tunyai region (0°10’33” S, 37°50’12). In Embu County, soil was
 collected from farms in Karurumo area (0°29’12”, 37°41’50” E) where
 agriculture is practiced by 80% of the population. The area is
 predominantly hot and dry. The sampling site in Kitui County was located
 in Matinyani area (1°18’30.6” S37°59’30.2” E. These areas are in the lower
 midland agroecological region experiencing moisture stress with less than
 400mm rainfall per annum coupled with high temperatures that result in low
 crop production(1) Soil sampling was
 conducted during the dry, post harvest season in August of 2019. The soil
 samples were obtained from 30 farms within each of the three regions in
 the Counties; with 20 sampling points in each of the farms. Each sample
 was thoroughly mixed to create a composite soil sample then collected into
 sterile khaki bags and labeled. They were then air dried and sieved using
 2mm aperture sieve and stored in two portions, at room temperature for
 soil physicochemical analysis and another at 4°C for <i>Azotobacter
 </i>isolation. A
 physicochemical analysis of all the soil samples was conducted by
 determining the soil pH and soil texture using a glass electrode pH meter
 and the hydrometer method respectively. Subsequently, the soil samples
 were grouped as either sandy loam, loamy sandy or sandy clay loam. The
 total organic matter, total soil nitrogen and the available phosphorus
 content was determined by the Walkley and Black oxidation method, the
 macro-Kjeldahl method and Olsen extraction method
 respectively(2) <b><i>Isolation, biochemical and
 morphological analysis of isolates</i></b> Isolation of
 <i>Azotobacter</i> from the soil samples was conducted using
 Ashby’s nitrogen free selective media. One gram of soil sample was placed
 in a sterile test tube and suspended in 9ml of distilled sterile water
 then thoroughly agitated. Serial dilutions were prepared up to
 10<sup>-3 </sup>and aliquot of 10µl of each dilution spread on
 a plate containing Ashby’s Nitrogen-free selective medium (20 g mannitol,
 0.2 g NaCl, 0.2 g
 K<sub>2</sub>HPO<sub>4</sub>,0.100 g
 MgSO<sub>4</sub>, 0.010 g
 K<sub>2</sub>SO<sub>4</sub>, 5.000 g
 CaCO<sub>3</sub>,15g agar )(3).It was then incubated at 28 °C
 for 5 days. The
 <i>Azotobacter</i> isolates were characterized in accordance
 with Bergeys manual of determinative bacteriology and their culture
 morphology noted as follows; colony color, texture appearance, colony
 size, shape and margins, elevation on the agar and colony
 form(4). The production of
 pigment was evaluated by sub culturing the isolates on modified
 Ashby’s–benzoate(0.5% w/v) medium and the subsequent pigment production
 was recorded after incubation at 28 °C for 5 days according to
 Banerjee<i> et al</i>.(5) procedures. The production of
 acid in the media was evaluated by growing the isolates in Ashby’s media
 containing bromothymol blue as an indicator. <b><i>Molecular
 characterization</i></b> Two isolates per
 morphological group were selected randomly for DNA extraction; they were
 named P1 to P25.Bacterial cells isolated from the pure five day old plate
 culture were added to sterile normal saline and vortexed. They were then
 centrifuged to produce pellet whose DNA was extracted by use of the Zymo
 Quick -gDNA<sup>TM</sup>Mini Prep DNA extraction kit using the
 manufacturer protocol. Gel electrophoresis was conducted using 1.0%
 agarose gel and visualized on UV trans-illuminator and then stored for use
 at -20°C . PCR analysis was
 conducted by preparing a mixture with 1µl of isolated DNA from each of the
 25 purified cultures, 1µl dNTPs, 0.5µl of both forward and reverse
 primers, 0.125 µl of taq polymerase, 2.5µl of dream taq reaction buffer
 and 19.375 µl PCR water to top up to a total of 25µl. The universal
 primers used 27F (5’AGAGTTTGATCCTGGCTCAG 3’) and 1492R
 (5’TACGGCTACCTTGTTACGACTT 3’) are complimentary to the conserved regions
 of the bacterial 16SrRNA gene. The PCR reaction was carried out in a
 Techgene thermocycler (FTGENE5D model) having thermal cycling conditions
 set as follows; the initial denaturation step was at 95 °C for 3 minutes,
 35 cycles were ran (with the denaturation at 94°C for 45 seconds,
 annealing at 51.8 °C for 45 seconds, elongation at 72 °C for 2 minutes)
 and final extension step at 72°C for 5 minutes. The samples were finally
 stored at 4 °C. Gel
 electrophoresis of 3µl of the PCR product mixed with 2µl of loading dye
 containing SYBR (Thermo Fisher) green stain was loaded on 1.4% (w/v)
 agarose gel and electrophoresis done at 80v for 30 minutes. Subsequent
 visualization was done on a UV trans-illuminator; a 1 Kb DNA ladder was
 used as reference to estimate the bands molecular size. The PCR products
 were sent to Macrogen-Netherlands for the purification and Sanger
 sequencing. The sequencing primers used were 27F (5’AGAGTTTGATCCTGGCTCAG
 3’) and 1492R (5’TACGGCTACCTTGTTACGACTT 3’). <i>DATA ANALYSIS</i>
 After
 base-calling and creation of a consensus sequence the DNA sequences, were
 compared to available sequences in the Genbank using Basic Local Alignment
 Test (BLAST) software on the NCBI database. This was to identify the
 various strains isolated by comparing with the bacterial lineage standard
 sequences. The sequences with the highest hits on the 16SrRNA gene were
 extracted and aligned on the Clustal X program (6). Further, evolutionary
 analyses were conducted in MEGA X software; the Phylogenetic tree’s
 evolutionary distance depiction was computed using Jukes – Cantor method
 and to show the number of base substitutions per site. The analysis
 involved 33 nucleotide sequences. Codon positions included were
 1st+2nd+3rd+Noncoding. All ambiguous positions were removed for each
 sequence pair. There were a total of 1635 positions in the final
 dataset.(7). The influence of soil quality on
 the occurrence and biodiversity of recovered
 <i>Azotobacter</i> isolates was determined by examining the
 correlation through a redundancy analysis (RDA).This was achieved by
 comparing the recovered sequences against the soil physical chemical
 properties.
description: <strong><em>Note: Isolate
 sequence P4 ( Azotobacter salinestris), from morphogroup IsD,did not
 generate an accession number because the sequence was too short. Therefore
 its sequence and phylogenetic match has been included as a file
 (sequence_P4_(A. salinestris)_phylogenetic_match) in this
 data.</em></strong> <strong><em>References</em></strong> 1. Nyaga JW, Njeru EM. Potential of Native Rhizobia toimprove Cowpea Growth and Production in Semiarid Regions of Kenya. Front Agron. 2020; 2. Okalebo, R.J., Gathua, K.W. and Woomer PL. Laboratory methods of soil and plant analysis: A working manual. Second Edition. TSBF-CIAT and Sacred Africa, Nairobi, Kenya. 2002; 3. Stella M, Suhaimi M, Matthews S, Masduki S. Selection of suitable growth medium for free-living diazotrophs isolated from compost (Pemilihan medium pertumbuhan yang sesuai untuk bakteria pengikat nitrogen hidup bebas yang dipencil daripada kompos). J Trop Agric Fd Sc. 2010;38(2):211–9. 4. Beking J. The family Azotobacteraceae. Prokaryotes. In: Bergeys manual of systemic bacteriology. 2006. p. 759–83. 5. Banerjee A, Supakar S, Banerjee R. Melanin from the Nitrogen-Fixing Bacterium Azotobacter chroococcum: A Spectroscopic Characterization. PLoS One [Internet]. 2014 Jan 9;9(1):e84574. Available from: https://doi.org/10.1371/journal.pone.0084574 6. Thomson J, Higgins D. The clustal_x windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic acids res. 1997;48–82. 7. Tamura K, Dudley J, Mei N. MEGA4, a molecular evolutionary genetic analysis MEGA software version 4.0. Mol bio evol. 2007;1596–9.