ABUNDANCE, GENETIC DIVERSITY AND NODULATION
EFFICIENCY OF COWPEA (Vigna unguiculata L.) RHIZOBIA
IN SOUTH WESTERN KENYA SOILS
By Jonah K. Ngeno
Research proposal submitted in partial fulfillment of the requirements for
the award of the degree of Doctor of Philosophy in Agronomy
Department of Plant Science and Crop Protection, Faculty of Agriculture,
College of Agriculture and Veterinary Sciences, University of Nairobi
Supervisors
1) Dr. G.N Chemining’wa
2) Prof. S.I Shibairo
PhD Research proposal presentation, Ngeno J.K., 2012
Introduction
o Importance of cowpea
o Food for humans (seed contains 23% protein and
57% carbohydrate; leaves contain 27 - 34%
protein & vitamins, also a fodder crop
o Crop rotation programs- leave N-fixed deposit of
60-70kg ha-1 after cropping season
o Cover crop & used as green manure
o Medicinal uses e.g. management of epilepsy, skin
infections
PhD Research proposal presentation, Ngeno J.K., 2012
Introduction – cont’
• Where grown
– Eastern province (85% of total production), Coast,
Central & Western - 15% of remainder
• Cowpea has wide ecological adaptation
– Altitude up to 2500M ASL; r.fall (450mm-1500mm)
; temp (25oC - 35oC), soil pH 5.5-7.5.
• Crop can grow well in S. Western Kenya (Kisii,
Bomet, Kipkelion, Bureti & Kericho),
especially in the short rains
PhD Research proposal presentation, Ngeno J.K 2012
Problem statement/justification
 N is a limiting element in Kenyan soils; can be
corrected by inorganic N fertilizer- but
expensive for small holder farmers
 Organic manures can be used, but not available in
adequate Qties; may be low in total N (Palm et al.,
2000)
 Biological N2 fixation by rhizobia in legume host
crop – best option for correcting N deficiency; it’s
cost effective (Ksh. 670/ha of cowpea crop; N
fertilizer costs Ksh. 3600/ha of cowpea crop- Brink
et al., 2006)
PhD Research proposal presentation, Ngeno J.K., 2012
Problem statement/justification- cont’
 Cowpea is inoculated by commercial rhizobia, if:
• Population (abundance) of native rhizobia in soils is
low, or inefficient in N2 fixation.
• Abundance & nodulation efficiency of native cowpea
rhizobia has not been established in the study area; this
study will fill this knowledge gap
• Nodulation efficiency of native cowpea rhizobia will
further be compared with commercial strains and N
fertilizer in field experiments & by quantification of Nfixed.
PhD Research proposal presentation, Ngeno J.K., 2012
Problem statement/justification- cont’
o Genetic diversity of rhizobia varies in different
regions, depending on:
o history of legume cultivation & soil chemical
characteristics – like pH.
o Some of the diverse strains of rhizobia have nodulation
specificity towards certain legumes, or cultivars of the
same species (Pule-Meulenberg et al., 2010).
o This study seeks to determine the genetic diversity of
cowpea nodulating rhizobia in S.W Kenya soils, and to
identify the strains that efficiently nodulate cowpea
genotypes.
o Such knowledge would provide basis for selecting rhizobia
stains for manufacturing commercial inoculants.
PhD Research proposal presentation, Ngeno J.K., 2012
Problem statement/justification- cont’
• Phosphorous (P) enhances nodulation in
legumes, But is deficient in Kenyan soils
• Soil acidity also a challenge to cowpea
production in the study area; enhances solubility
of Al3+, Mn2+ &Fe3+ which causes:
Toxicity in plants; P fixation & deficiency of Ca, Mo, N, mg
and K.
Restriction of growth &survival of rhizobia
• Liming and application of P is therefore expected
to:
– Enhance solubility of P & other nutrient elements,
hence enhanced symbiotic efficiency of native
rhizobia, biomass production & yield of cowpea
PhD Research proposal presentation, Ngeno J.K., 2012
Study objectives
o To determine the abundance and nodulation efficiency of native
cowpea rhizobia in South western Kenya soils
o To determine the amount of N-fixed by different cowpea genotypes
using 15N natural abundance technique and N-difference method
o To characterize cowpea nodulating rhizobia using PCR-RFLP and
sequencing of 16S-23S rDNA IGS region
o To determine the effect of rhizobia inoculation and nitrogen fertilizer
on biomass production, nodulation, and yield of four cowpea
genotypes in S.W Kenya.
o To determine the effect of P fertilizer application and liming on
nodulation, biomass production and yield of three cowpea genotypes
in S.W Kenya.
PhD Research proposal presentation, Ngeno J.K., 2012
Hypotheses
 Abundance and nodulation efficiency of native cowpea rhizobia is expected to
vary in S.W Kenya, depending on the site, history of cowpea cultivation and
soil physiochemical characteristics.
 Genotype and site differences would be expected in terms of quantities of NFixed
 There is wide genetic diversity of cowpea nodulating rhizobia in S.W Kenya
soils, and hence rhizobial strains x cowpea genotype’s symbiotic specificity are
expected.
 Rhizobia inoculation and N fertilizer are expected to have significant effects on
biomass production, nodulation and yield of cowpea, which could be genotype
or site dependent.
 P fertilizer application and liming are expected to enhance available P in soil,
nodulation, biomass production and yield of cowpea genotypes. Liming is also
expected to decrease the solubility of Al3+ ions, and enhance the population of
native cowpea rhizobia.
PhD Research proposal presentation, Ngeno J.K., 2012
Materials and methods
• Objective 1:Abundance and efficiency of indigenous
cowpea nodulating rhizobia in S.W Kenya soils
– Experimental site – greenhouse experiment-UON Kabete
Campus’ field station
– Soil samples to be collected from five farmer field from 5
geographic regions in S.W Kenya. From each farmer’s field,
soils will be collected from areas with and without history
of legume cultivation. 50 soil samples in total. 20g of each
sample to be diluted in 80ml sterile water, to make 10-1 soil
inocula, then used to make dilution series from 2-1 to 6-1.
– Treatments: Seven dilution levels (from 10-1 then 2-1 to 61), a positive control- with commercial rhizobia and a
negative control- with water.
• 1 ml of each diluent will be used to inoculate seedlings grown in
the Leonard jars under N free solution. Cowpea variety M66 will
be used to trap rhizobia. Sterilized sand will be propagation media
PhD Research proposal presentation, Ngeno J.K., 2012
Objective 1 – cont’
• Experimental design: Plants receiving each
treatment in Leonard jars will be laid down in
RCBD, treatments replicated thrice.
• Data collection:
– 46 days after treatment application, nodules counts
will be taken. Each nodule count will constitute a +ve
score, MPN technique will used to determine
population of rhizobia as desribed by Somasegaran
and Hoben, 1994.
– Nodule numbers and biomass, root & shoot dry
matter will also be taken. this will determine
nodulation efficiency of native cowpea rhizobia.
PhD Research proposal presentation, Ngeno J.K., 2012
Effects of Rhizobia inoculation and N fertilizer on nodulation, growth
and yield of cowpea &quantification of N-fixed- Objective ii & iv
• Experimental sites – two farmer fields in Kericho &
Bomet districts, for 2 seasons.
• Treatments– Four cowpea genotypes (M66, K80, KVU 27-1 & Ng’ombe)
x Rhizobia inoculation, Okg N ha-1, 20kg N ha-1, 40kg N ha-1
- Maize will be grown on adjacent plots- used as reference
plant for N-fixed determination.
• Experimental design- RCBD in a 4x4 factorial
arrangement.
• Data to be collected: Nodule numbers, LAI, Root &
shoot biomass, Tissue N, yield and yield components,
soil available N - after the experiment
PhD Research proposal presentation, Ngeno J.K., 2012
Quantification of N fixed
15N
natural abundance technique: shoot dry matter from field experiment will be
milled to fine powder
• Isotopic composition of 15N (∂15N ‰) of each sample will be determined,
then used to calculate Ndfa (nitrogen derived from fixation), which will be
used to calculate N-fixed. Procedures described by Pule-Mulenberg et al.,
2010 will be used, i.e:
– ∂15N ‰= [N15/N14 sample]- [N15/N14 standard]/ [N15/N14 standard]× 1000
– Sum of ∂15N for all the plant organs (∂ 15Nleg)is determined.
– %Ndfa = [(∂ 15Nref - ∂ 15Nleg) / (∂ 15Nref - B value)]*100
– N- fixed = [% Ndfa/100]* legume shoot N.
– The samples will be run on a stable light isotope mass spectrometer, joined to
an elemental analyser against an internal reference plant material to give
N15/N14
sample.
– In N difference method, Ndfa[kg/ ha ] = N fix - N contr; where N fix is total N in
legume, - N contr is total N in non fixing ref. plant
PhD Research proposal presentation, Ngeno J.K., 2012
Genetic diversity of cowpea rhizobia in S.W
Kenya
• Soil samples to be collected from 8 sites in each of
three Geographic regions of S.W Kenya. In each site,
soils will be collected from two areas, with and
without history of legume cultivation.
• Samples to be put in pots in g.hse, then cowpea
genotypes (K80, M66, KVU 27-1 & Ng’ombe) to be
grown in 3 replicates- to trap native rhizobia
• Nodule harvested after 46 days
• DNA of rhizobia to be extracted from nodules
following procedures described by Krasova–Wade
and Neyra (2007).
PhD Research proposal presentation, Ngeno J.K., 2012
Genetic diversity cont’
• Molecular characterization- to be done by PCR-RFLP of 16S-23S
rDNA spacer region, using two primers. Two restriction enzymes
will be used. Procedures of Pule-Mulenberg et al., 2010 to be
followed.
– Amplified DNA products to be separated by horizontal gel electrophoresis.
– DNA fragments to be analysed after migration in 3% agarose gel;
electrophoregrams with similar migratory products to be grouped
together into dirrerent IGS groups.
– Samples from each IGS group to be selected for sequencing of 16S-23S
rDNA IGS gene- using 3 primers and antiprimer.
• Gene Bank database to be scanned for related sequences
• closely related sequences to be included in Phylogenetic analysis
using CLUSTAL X software
• Phylogenetic trees to be constructed by neighbor joining method;
graphic representation of trees to be done using NJPLOT software .
PhD Research proposal presentation, Ngeno J.K., 2012
Effects of P fertilizer & liming on
nodulation, biomass & yield of cowpea
- Exp. Site – 2 farmer field in Kericho and Bomet
- Treatments: three cowpea genotypes (M66, KVU
27-1 & Ng’ombe); P fertilizer (0 kg P/ha, 25 kg
P/ha and 50 kg P/ha and liming (0 t/ha, 4 t/ha)
- Exp. Design – RCBD in a 2 x 3 x 3 factorial
- Data to be collected: nodule numbers, root&
shoot biomass, tissue N and P, yield & yield
components, available P, P-sorption xtics, soil pH
and exchangeable alumiminium.
PhD Research proposal presentation, Ngeno J.K., 2012
Data analysis
• Data from field & g.house experiments to be
subjected to Analysis of variance using
Genstat statistical program at 5% level of
significance.
• Treatment means to be compared using
Fisher’s LSD method
• Regression analyses will be used to compare
relationships Between parameters.
PhD Research proposal presentation, Ngeno J.K., 2012
Expected outputs
• Information on population size of native cowpea rhizobia
in the study area, which will justify the need for
commercial rhizobia inoculation
• Information on the genetic diversity of cowpea nodulating
rhizobia in S.W Kenya, and a guide to inoculant selection
• Identification of cowpea genotypes with the highest yield
and symbiotic efficiency with rhizobia, which will be
recommended to farmers
• Determination of P rate which gives high cowpea yield
• Information on response of cowpea genotypes to liming in
acid soils
• Papers for disseminating research findings through
workshops and journals
• Research thesis
PhD Research proposal presentation, Ngeno J.K., 2012
Work plan
Quarter 1
YEAR 1
Development of research proposal
Seminar presentation
Final proposal development
X
X
Quarter 2
Quarter 3
X
X
Pot experiment (determination of population of native rhizobia)
and data collection (Objective i)
X
Site identification, soil sampling & Setting up field experimentsseason I (Objective iv & v )
X
X
Data collection from field experiment- season I
X
X
Field experiments (Season II) and determination of N fixed
(objective ii)
YEAR 2
Data collection from field experiment- season II & Determination
of N-fixed
Pot experiment (Objective iii)
Quarter 4
X
X
X
X
Molecular characterisation of cowpea rhizobia (Objective iii)
X
YEAR 3
Molecular characterisation of cowpea rhizobia & data collection
Data analysis and thesis writing
Thesis submission for examination
Final thesis submission
Preparation of papers and submission for publication, and
graduation.
X
X
X
X
X
X
X
Budget
Item
Field experiment
Land preparation and
field layout
Lime
TSP
CAN
Soil sampling
Soil analysis
Crop management and
data collection
Plant N analysis
Plant P analysis
Analysis of N-Fixed
Pot experiment (abundance and
efficiency of native rhizobia)
Leonard jars
Nutrient free solution
Soil sampling
Data collection
Soil analysis
Molecular characterisation of
cowpea rhizobia (in a
laboratory with all the
necessary equipment)
DNA extraction
Electrophoresis
PCR
Taq polymerase
dnTPS
Restriction enzymes
Transport and accommodation
Total
10% contingencies
Grand Total
Unit cost (US Dollars)
Total cost (US Dollars)
375 (1site)
25
54
50
70
20 x 135 samples
625
7 x 204 samples
7 x 204
10 x 144 samples
750
50
108
100
210
2700
1250
1430
1430
1440
4 x 450
60
235
190
20 x 50
1800
60
235
190
1000
2 x 300 samples
1 x300
7 x 300
5 x 300
1 x 300
1 x 300
600
300
2100
1500
300
300
2500
2500
18,923
1892.30
20,815.30