This study was conducted to determine the abundance and symbiotic efficiency of native rhizobia nodulating common bean in Kisumu and Kakamega, Kenya. Soil sampling was carried out in three farms that had been used for growing common bean for at least two seasons and one fallow land with no known history of growing common bean or inoculation. Abundance of soil rhizobia and symbiotic efficiency (SE) were determined in a greenhouse experiment. Native rhizobia populations ranged from
Many countries in sub-Saharan Africa, including Kenya, are characterized by increased population growth and food insecurity, as well as increasing environmental and economic declines associated with conventional farming. These problems have necessitated the development and adoption of alternative food production practices. Sustainable soil fertility management is key to food production and environmental stability [
Integration of common bean into existing cropping systems has the potential to mitigate food insecurity in Kenya if challenges associated with its production are addressed. The national annual dry bean production is about 380,000 metric tons (
The study was carried out in Korando B, Kisumu district, Kenya (S 00° 05.167′, E 034° 41.613′), and Masinde Muliro University of Science and Technology (MMUST), Kakamega, Kenya (N 00° 17.104′, E 034° 45.874′). The sites were selected based on agroclimatic conditions and prevalence of common bean cultivation. MMUST is located at an altitude of 1585 metres above sea level within a high potential agroecological zone and has an annual rainfall of 1200–2100 mm while Korando B is located at an altitude of 1300 meters above sea level and has an annual rainfall of 1200–1300 mm. Soils in MMUST and Kisumu are generally classified as Nitisols and Arenosols, respectively [
Soil sampling was carried out in three farms with at least two seasons of growing common bean and one fallow farm with no history of common bean cultivation. The cropping systems in the farms consisted of bean, maize, maize-bean intercrop, napier, and fallow land. Using a 2.5 cm diameter soil probe, 20 soil cores were randomly collected from each farm to a depth of 15 cm and thoroughly mixed into a composite sample. Each sample was aseptically collected to avoid cross contamination between soils from different sampling points. The samples were then divided into two parts; one part was for the determination of rhizobia population in the soil while the other part was for the chemical and physical analyses. Soil samples were air-dried and passed through a 2 mm sieve for chemical analysis.
Soil pH was determined using a glass electrode pH meter in 1 : 2.5 soil : water suspension [
Abundance of rhizobia was determined using the most probable number (MPN) technique. Common bean (
After germination, two to three healthy seedlings were aseptically transplanted into each Leonard jar filled with sterilized vermiculite. After 7 days, seedlings were thinned to 1 plant per jar and then inoculated with the diluted soil samples [
Nodules were sampled during the late flowering and early pod setting stages. A total of 100 representative flowering bean plants were carefully uprooted from farms in Kisumu and MMUST after 7 weeks of emergence. After carefully washing the nodules, fresh and red nodules were carefully removed from the roots and wrapped in sterilized absorbent paper. The nodules were immersed in sterilized distilled water and let to imbibe water for one hour. Nodules were surface-sterilized in 1% NaOCl for 6 min, rinsed in several changes of sterile water, and then crushed with a flame-sterilized blunt-tipped pair of forceps. A loopful of the crushed nodule suspension was streaked across the surface of Petri dish containing yeast extract mannitol agar (YEMA) media containing Congo red and incubated in the dark at 26°C [
Each of the pure isolates was authenticated as root nodulating bacteria by reinoculating 1 mL of three-day-old pure YEM broth culture of the isolate on the host plant grown in a controlled environment in sterilized vermiculite in Leonard jar [
All data on root dry weight (RDW), SDW, tissue N concentration content per plant, and SE were subjected to analysis of variance (ANOVA) using General Linear Models Procedure of SAS software version 9.1. [
The soil chemical properties varied across farms at the two study sites (Table
Native rhizobia population and physicochemical characteristics of soils from the study farms in Kisumu and MMUST.
Soil properties | MMUST farms | KISUMU farms | ||||||
---|---|---|---|---|---|---|---|---|
Maize | Fallow | Bean | Napier | Bean | Maize | Fallow | Maize-bean | |
MPN (no. rhizobia in gm−1 of soil) | 7.8 × 101 | 1.98 × 102 | 4.102 × 103 | 3.9 × 101 | 1.25 × 104 | 3.5 × 104 | 3.2 × 101 | 1.25 × 104 |
pH (1 : 2.5 soil water ratio) | 5.12 | 5.01 | 4.98 | 5.4 | 6.1 | 6.06 | 5.98 | 5.24 |
EC (dS/m) | 0.3 | 0.7 | 0.7 | 0.4 | 0.2 | 0.2 | 0.4 | 0.4 |
Total |
0.35 | 0.18 | 0.24 | 0.31 | 0.11 | 0.13 | 0.15 | 0.2 |
Organic carbon (%) | 2.6 | 1.58 | 2.66 | 2.8 | 1.32 | 1.15 | 1.78 | 1.39 |
K (cmol/kg) | 1.75 | 0.75 | 0.93 | 1.2 | 1.39 | 1.42 | 1.39 | 1.42 |
Na (cmol/kg) | 0.6 | 0.5 | 0.7 | 0.8 | 0.6 | 0.7 | 0.6 | 0.8 |
Mg (cmol/kg) | 1.19 | 1.01 | 1.39 | 1.53 | 1.17 | 1.4 | 1.08 | 1.77 |
Ca (cmol/kg) | 2.74 | 2.84 | 3.08 | 3.7 | 3.02 | 2.66 | 2.56 | 5.31 |
Al (cmol/kg) | 2.8 | 1.9 | 2.6 | 2.5 | 0.6 | 0.7 | 0.6 | 0.9 |
Zn (ppm) | 7.2 | 4.5 | 7.6 | 20 | 9.8 | 6 | 6.2 | 1.7 |
Cu (ppm) | 5.6 | 5.4 | 5.1 | 5.6 | 1.6 | 1.8 | 1.4 | 1.6 |
Fe (ppm) | 16.1 | 13.3 | 10.1 | 24.6 | 22 | 12.5 | 20.4 | 43.2 |
Mn (ppm) | 72.4 | 43.3 | 75.2 | 91.1 | 38.3 | 48.6 | 32.3 | 94.1 |
P (ppm) | 24 | 6 | 28 | 35 | 35 | 62 | 11.8 | 12 |
Soil texture | Clay | Clay | Clay | Clay | SL | SL | SL | SL |
Note: SL: sandy loam.
Initial screening of the nodule bacteria on YEMA media with Congo red showed that the pure samples were Gram-negative with convex elevation. They failed to absorb Congo red or absorbed it lightly. On streaking on the YEMA-BTB media, the isolates acidified and turned the green media to moderately yellow and deep yellow color after 3 days of incubation in the dark. The colony diameter ranged between 1.0 mm and 5.7 mm. The colonies appeared either dull or shiny with entire margin and convex elevation on YMA plain media. The colony transparency was either opaque or translucent with firm, dry, or smooth viscous texture. The isolates were placed in 12 groups based on the differences on morphocultural characteristics (Table
Morphological and cultural characteristics of the rhizobia isolates from Kisumu and MMUST.
Characteristics | Isolates | |||||||||||
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Congo red absorption | ✓ | ✓ | ✓ | ✓ | ✓ | ✓ | ✓ | ✓ | ✓ | ✓ | ✓ | ✓ |
BTB |
✓ | ✓ | ✓ | ✓ | ✓ | ✓ | ✓ | ✓ | ✓ | ✓ | ✓ | ✓ |
Colony |
Cream yellow | Cream |
Cream white | Milky white | Milky white | Cream yellow | Cream white | Cream white | Milky white | Cream yellow | Milky white | Cream yellow |
Colony |
Opaque | Translucent | Opaque | Opaque | Translucent | Translucent | Opaque | Translucent | Opaque | Translucent | Opaque | Opaque |
Colony appearance | Shiny | Shiny | Shiny | Dull | Dull | Shiny | Shiny | Shiny | Shiny | Shiny | Dull | Dull |
EPS |
✓ | ✓ | ✓ | x | x | ✓ | ✓ | ✓ | x | ✓ | x | ✓ |
Colony |
Firm, |
Smooth, |
Smooth, viscous | Firm, dry | Firm, dry | Smooth, viscous | Smooth, viscous | Smooth, viscous | Smooth, viscous | Smooth, viscous | Firm, dry | Firm, dry |
Colony |
Circular | Oval | Oval | Circular | Circular | Circular | Oval | Oval | Circular | Circular | Circular | Circular |
Colony |
Convex | Convex | Convex | Convex | Convex | Convex | Convex | Convex | Convex | Convex | Convex | Convex |
Colony |
3.7 | 4.7 | 5.7 | 3.7 | 4.0 | 3.7 | 5.0 | 3.3 | 4.7 | 3.3 | 3.0 | 1.0 |
Gram stain | ✓ | ✓ | ✓ | ✓ | ✓ | ✓ | ✓ | ✓ | ✓ | ✓ | ✓ | ✓ |
Colony |
Entire | Entire | Entire | Entire | Entire | Entire | Entire | Entire | Entire | Entire | Entire | Entire |
BTB: bromothymol blue, EPS: exopolysaccharides, ✓: positive reaction, and x: negative.
Upon reinoculation of the host plant, all the isolates from Kisumu and four from MMUST initiated nodulation (Figure
Effect of isolate inoculation on root dry weight, shoot dry weight, N concentration, N content, and SE under controlled experiments in Korando B, Kenya.
Isolate | RDW |
SDW |
N concentration |
N content |
Symbiotic efficiency (%) |
---|---|---|---|---|---|
|
0.90ab | 1.59bc | 1.82ab | 2.89abc | 125.0abc |
|
0.94ab | 1.52c | 1.50abc | 2.31bc | 100.0bc |
|
0.96ab | 1.84bc | 2.06a | 3.94a | 170.0a |
|
0.82b | 1.64bc | 1.22c | 2.07bcd | 89.0bcd |
|
1.03ab | 2.01a | 1.45bc | 3.01ab | 130.0ab |
|
0.86ab | 1.65ab | 1.04cd | 1.72bcd | 74.0bcd |
|
0.86ab | 1.65bc | 1.31bc | 2.17bcd | 94.0bcd |
|
0.90ab | 1.88ab | 1.01cd | 1.89bcd | 81.0bcd |
Strain 446 | 1.07a | 1.69abc | 1.51abc | 2.56abc | 110.0abc |
CIAT 899 | 0.56c | 1.50c | 1.08c | 1.55cd | 67.0cd |
PCNTL | 1.07a | 1.67abc | 1.37bc | 2.32bc | 100.0bc |
NCTL | 1.00ab | 1.63bc | 0.46d | 0.75d | — |
LSD | 0.22 | 0.36 | 0.59 | 1.43 | 62.0 |
NCTL: negative control; PCNTL: positive control; LSD is the least significant difference of means; Strain 446 and CIAT 899 are reference commercial inoculants. Means within a column followed by the same letter(s) are not significantly different at
Effect of isolate inoculation on root dry weight, shoot dry weight, N concentration, N content, and SE under controlled experiments in MMUST, Kenya.
Isolate | RDW |
SDW |
N concentration |
N content |
Symbiotic efficiency (%) |
---|---|---|---|---|---|
|
0.89ab | 1.76a | 1.32b | 2.33b | 100.0b |
|
0.80b | 1.78a | 1.41b | 2.48b | 107.0b |
|
1.08a | 1.85a | 2.02a | 3.80a | 164.0a |
|
1.07a | 1.73a | 1.06b | 1.80bc | 78.0bc |
Strain 446 | 1.07a | 1.69a | 1.51ab | 2.56b | 110.0b |
CIAT 899 | 0.56c | 1.50a | 1.08b | 1.55bc | 67.0bc |
PCNTL | 1.07a | 1.68a | 1.37b | 2.32b | 100.0b |
NCTL | 1.00ab | 1.63a | 0.46c | 0.75c | — |
LSD (5%) | 0.24 | 0.39 | 0.56 | 1.13 | 49.0 |
NCTL: negative control; PCNTL: positive control; LSD is the least significant difference of means; Strain 446 and CIAT 899 are reference commercial inoculants; means within a column followed by the same letter(s) are not significantly different at
Correlation coefficients among shoot dry weight, nodule number, nodule dry weight, N concentration, N content, and SE in common bean.
Variables | Shoot dry weight | Nodule number | Nodule dry weight | Nitrogen concentration | Nitrogen content | SE |
---|---|---|---|---|---|---|
Root dry weight | 0.245* | 0.166 | −0.018 | 0.095 | 0.175 | 0.175 |
Shoot dry weight | 0.056 | 0.096 | 0.275* | 0.545** | 0.546** | |
Nodule number | 0.703** | −0.032 | −0.042 | −0.043 | ||
Nodule dry weight | −0.106 | −0.087 | −0.087 | |||
Nitrogen concentration | 0.948** | 0.948** | ||||
Nitrogen content | 1.000** |
Sample nodules formed by isolates from Kisumu (a) and MMUST (b).
The pH of soils did not vary so much across the study sites regardless of the land use system. Variation observed in rhizobia population in soils among the different farms could be due to the acidic pH. Lower pH increases the solubility of Al, Mn, and Fe in soil causing toxicity to plants in excess by slowing or stopping of root growth. Niste et al. [
Growth of the isolates on YEMA media confirmed morphological characteristics of
The development of legume inocula requires that the rhizobia must be highly effective in fixing nitrogen. At final harvest, inoculated beans had higher SDW compared to the control indicating that inoculation with native isolates improved the growth of plants and are therefore efficient in N fixation. These results are contrary to those described by Mungai and Karubiu [
This study has demonstrated the presence of native rhizobia that are potentially superior to the commercial inoculants and can be exploited to enhance bean inoculation programmes in the area. The identity of native rhizobia that improved the growth of common bean plants should be established and subjected to further greenhouse and field trials to ascertain their stability under different environmental conditions.
The authors declare that there is no conflict of interests regarding the publication of this paper.
This work was sponsored by SIDA and the Inter-University of Council for East Africa (IUCEA) through the Lake Victoria Research Initiative (VicRes). The authors are thankful to the Departments of Plant Sciences and Microbiology of Kenyatta University for the provision of greenhouse and laboratory space. Smallholder farmers along the Lake Victoria Basin, Kisumu, and Kakamega are further acknowledged for providing free access into their farms.