By Jonah K. Ngeno

1. 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 • Dr. G.N Chemining’wa • Prof. S.I Shibairo PhD Research proposal presentation, Ngeno J.K., 2012

2. Introduction • Importance of cowpea • Food for humans (seed contains 23% protein and 57% carbohydrate; leaves contain 27 - 34% protein & vitamins, also a fodder crop • Crop rotation programs- leave N-fixed deposit of 60-70kg ha-1 after cropping season • Cover crop & used as green manure • Medicinal uses e.g. management of epilepsy, skin infections PhD Research proposal presentation, Ngeno J.K., 2012

3. 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

4. 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

5. 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 N-fixed. PhD Research proposal presentation, Ngeno J.K., 2012

6. Problem statement/justification- cont’ • Genetic diversity of rhizobia varies in different regions, depending on: • history of legume cultivation & soil chemical characteristics – like pH. • Some of the diverse strains of rhizobia have nodulation specificity towards certain legumes, or cultivars of the same species (Pule-Meulenberg et al., 2010). • 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. • Such knowledge would provide basis for selecting rhizobia stains for manufacturing commercial inoculants. PhD Research proposal presentation, Ngeno J.K., 2012

7. 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

8. Study objectives • To determine the abundance and nodulation efficiency of native cowpea rhizobia in South western Kenya soils •  To determine the amount of N-fixed by different cowpea genotypes using 15N natural abundance technique and N-difference method • To characterize cowpea nodulating rhizobia using PCR-RFLP and sequencing of 16S-23S rDNA IGS region •  To determine the effect of rhizobia inoculation and nitrogen fertilizer on biomass production, nodulation, and yield of four cowpea genotypes in S.W Kenya.  • 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

9. 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 N-Fixed • 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

10. Materials and methods • Objective 1:Abundance and efficiency of indigenous cowpea nodulatingrhizobia 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 6-1), 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

11. 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

12. 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

13. 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

14. 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

15. 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-Mulenberget 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

16. 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

17. 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

18. 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 nodulatingrhizobia 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

19. Work plan

20. Budget