Materials and Methods

5.2.1 Study site and parental selection

Parental selection was conducted in the greenhouse at Kabete Field Station. Kabete is located at coordinates 01^o14’59.7”S; 036^o44’28.8”E with an altitude of 1820 m above sea level. The area receives an average rainfall of 1046 mm annually, with a mean maximum temperature of 23^oC and mean minimum temperature of 12^oC. The soils are dark red or brown friable clay.

5.2.2 Parental selection

A total of 182 genotypes were screened for resistance to angular leaf spot. They included 159 Kenyan landraces sourced from the Kenyan Genebank, and 23 Rwandan landraces sourced from ECABREN.

The 182 genotypes were planted on 8^th November 2010 in five pots (size, 18x18x18 cm) each, two seeds per pot and replicated three times. The soil in the pots was collected from Kabete Field Station and mixed with chicken manure at a ratio of 3:2. The genotypes were inoculated with a mixture of the P. griseola races (where pathogen population is not defined) (isolated as indicated in Chapter 3, section 3.2.3 of this Thesis) at the V3 stage of development (where the first trifoliate leaf is open and the second trifoliate leaf appears). The first trifoliate leaf was inoculated on both sides of the leaf until runoff, using a hand sprayer. The plants were then covered for 4 days using clear polythene to increase the relative humidity and allow for the pathogen to infect the plants. On symptom appearance, data were collected on disease severity four times at 3 day intervals. The score on the last day was used in the analysis. Disease severity was based on scores of between 1 and 9, where 1 was resistant and 9 was susceptible.

The scores were further classified as follows: 1-3 was resistant, 4-6 intermediate resistant and 7-9 susceptible (Van Schoonhoven and Pastor-Corrales, 1987).

Thirty intermediate resistant genotypes (disease score 4-6) were selected and planted on 30^th March 2011 in five pots each, two seeds per pot and replicated three times. These were then screened again for ALS resistance using a mixture of P. griseola races. Four intermediate resistant genotypes, two from each common bean gene pools (Andean and Mesoamerican),

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were subsequently selected and used as parents to develop an inter-gene pool double cross segregating population.

5.2.3 Development of the inter-gene pool double cross population and advancement to F

3

The four selected parents were planted on 28^th June 2011 at the Kabete nethouse, in single row plots of 10 plants, spaced at 15 cm between plants and 50 cm between rows and replicated three times. The parents were crossed as follows: Parent A x Parent B and Parent C x Parent D to produce single crosses, F_{1 (AB)} and F_{1 (CD)}. The single crosses were planted on 11^th October 2011 at the Kabete nethouse in pots (18x18x18 cm). The F₁ single cross progeny were then crossed as F_{1 (AB)} X F_{1 (CD)} (Figure 5.1) to generate the double cross population, F_{1 (ABCD)} and subsequent generations were developed as shown in Table 5.1.

Figure 5.1: Successful cross F_1(AB) x F_1(CD)

Successful crosses in white tags

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Table 5.1: Methodology used to accumulate minor genes of resistance to angular leaf spot

Season Cross Cross Description

Season 1 AxB

F_1(AB)

CxD F_1(CD)

Four parents (with intermediate resistance to ALS and from different gene pools) were selected and single crosses produced (heterogametic parents) Season 2 F_1(AB) x F_1(CD)

F_1(ABCD)

Hybridization was done to produce a double cross population (assumed to have accumulated minor genes of resistance)

Season 3 F_1(ABCD) F₂

F_{1 (ABCD)} was planted and evaluated for resistance to angular leaf spot. The intermediate resistant plants were advanced to F₂.

Season 4 F2

F₃

The F2 seeds were planted in a plant to progeny row and evaluated for ALS resistance.

Intermediate resistant plants within each selected family were harvested, bulked and advanced to F₃ Season 5 F₃

F₄

Selected families were planted in rows and screened for resistance to ALS. Selection of resistant plants within and between families was carried out, and they were advanced to F₄

Season 6 Evaluation of F₄ The selected F₄ plants from the population, their parents (A, B, C, D), and selected market class varieties were evaluated for resistance, yield and other agronomic traits under three replications.

NB: Only single plant selection was done from F1 to F4

The double cross F1(ABCD) seed was planted on 21^st June 2012 in 3 m single row plots of 20 plants, spaced at 15 cm between plants and 50 cm between rows (Figure 5.2). They were inoculated with a mixture of P. griseola races (isolated as indicated in Chapter 3, section 3.2.3 of this Thesis) at the V3 stage of development. The intermediate resistant lines were selected and advanced to F₂. The F₂ seed was planted on 10^th October 2012 as plant to progeny rows in 3 m single row plots of 20 plants per row spaced at 15 cm between plants and 50 cm between rows. They were inoculated with a mixture of P. griseola races and intermediate resistant plants within each selected family were harvested, bulked and advanced to F_3. The selected F₃ families were planted on 16^th January 2013 in 3 m single row plots of 20 plants spaced at 15 cm between plants and 50 cm between rows. They were inoculated with a mixture of P. griseola isolates and ALS resistant plants selected and advanced to F_4.

127 Figure 5.2: F_1(ABCD) planted at Kabete nethouse

5.2.4 Experimental design and evaluation of F

4

lines

Eleven F₄ lines, the four parental genotypes used in the double cross and seven market class varieties were evaluated in the Kabete nethouse. The market class varieties included Super- rosecoco, GLP 24, GLP 2, GLP 585, New-rosecoco, GLP X92 and KAT 69. The experiment was set up in a randomized complete block design with three replications. The common beans were planted on 26^th April 2013 in five pots (18x18x18 cm in size) per genotype, two seeds per pot. The plants were inoculated with a mixture of P. griseola races, at the V3 stage of development, on both sides of the first trifoliate leaf until runoff using a hand sprayer. On symptom appearance, disease severity data was recorded four times at three day intervals. The score on the last day was used for the analysis. Severity scores were on a scale of 1-9, where 1-3 was resistant, 4-6 intermediate resistant and 7-9 susceptible (Van Schoonhoven and Pastor-Corrales, 1987). Data were recorded on: seed yield (g plant^-1), days to physiological maturity (days after planting where 50% of the plants have pods, and 50% of the pods have lost their pigmentation and begin to dry. At this stage the seeds begin to develop their typical varietal colour), 100-seed weight^-1, seed colour and growth habit.

5.2.5 Data analysis

Data were analysed using Genstat 12^th edition statistical package (Payne et al., 2009).

Separation of means was carried out using Tukey’s studentized range test.

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