Abstract
Angular leaf spot incidence and severity have increased in areas where beans are cultivated in Kenya and other Eastern Africa countries. Monitoring pathogenic diversity is critical for breeding programs aiming at genetic resistance. However, a comprehensive survey of bean diseases and race diversity of angular leaf spot in all major bean growing regions has not been conducted. The objective of this study was to conduct a survey of prevalent diseases and identify races of P. griseola in Kenya. Five economically important diseases causing major losses mainly (angular leaf spot, anthracnose, common bacterial blight, root rots and bean common mosaic virus) were sampled in all the agro-ecological zones. A collection of 62 samples of angular leaf spot, 55 of anthracnose, 9 of root rots, 121 of common bacterial blight and 32 of bean common mosaic virus were obtained. The survey covered 12 agro-ecological zones (AEZs) that included Upper Highland (UH2), Lower Highland (LH1, LH2, LH3 and LH4), Upper Midland (UM1, UM2, UM3, UM4 and UM5) and Lower Midland (LM1 and LM2). The pathogenic variability of 57 isolates of the P. griseola, collected from thirty-five districts in the bean growing regions of Kenya, was studied using the current 12 angular leaf spot differentials (Don Timoteo, Bolon Bayo, Montcalm, G05686, Amendoin, G11796, BAT 332, PAN 72, Cornell 49-242, MEX 54, Flor de Mayo and G02858). The first trifoliate leaf was inoculated with a 2 × 104 conidia ml-1. Plants were maintained at 22–28 °C and ≥ 95% relative humidity for 48 hours. Symptoms were evaluated 12, 17 and 21 days after inoculation. Twenty-three races of P. griseola were identified, 12 of which were represented by only one isolate. Only 11 races were found in two or more districts. Race 63-63 was the most virulent and caused leaf spots on all 12 common-bean differential genotypes, whereas race 63-55 was the most frequent (10 of 57 isolates), being widely distributed among the regions studied. Races 63-55, 63-63, 63-54 and 63-35 were found to be the most dominant races in areas studied. Two new races, 31-31 and 63-31 were reported for the first time in Kenya. The virulence phenotype indicated that 45 isolates studied were of the Mesoamerican group, with only 12 isolates of Andean group, suggesting coevolution of the pathogen with P. vulgaris in this host-pathogen interaction. The information generated on race diversity and distribution of P. griseola pathogen will facilitate targeted development of common bean varieties with resistance to these races and for specific regions.
Key words: Pseudocercospora griseola, Phaseolus vulgaris, differential cultivars, races, pathogenic variability.
1.0 Introduction
Angular leaf spot, incited by the imperfect fungus Pseudocercospora griseola (Sacc.) Crous and Braun, is the most wide spread disease of beans in Africa, particularly in tropical countries such as Kenya, Uganda, Tanzania and the Great lakes region where beans are produced (Stenglein et al., 2003; Namayanja et al., 2006; Mahuku et al., 2010). Angular leaf spot is rated as the second most significant among numerous biotic and abiotic constraints that afflict the bean crop in Africa (Aggarwal et al., 2004). In Kenya, angular leaf spot has been reported to cause significant losses of bean (Wagara et al., 2004) resulting in yield losses of up to 80% under favourable environmental conditions (Mahuku et al., 2010; Singh and Schwartz, 2010; Wahome et al., 2011). The disease affects foliage, petioles and pods throughout the growing season and is particularly destructive in areas where warm, moist conditions are accompanied by abundant inocula (Mahuku et al., 2010). Lesions on leaves start as small, brown or grey spots that become angular and necrotic, being confined by leaf veins while pod symptoms consist of circular to elliptical reddish-brown lesions.
In the recent past, angular leaf spot incidence and severity have increased in many areas where beans are cultivated (Stenglein et al., 2003), particularly in Kenya and other eastern Africa countries (Mwang‘ombe et al., 2007). Angular leaf spot is a significant constraint to bean production with annual losses estimated at 281,300 tonnes in eastern Africa (Wortmann et al., 1998). The disease also causes severe and premature defoliation resulting in shrivelled pods, shrunken seeds and reduced seed and pod quality (Stenglein et al., 2003). The disease is controlled mainly through the use of resistant varieties derived from monogenic and polygenic genes acting together or separately (Stenglein et al., 2003). Monogenic resistance is typically race-specific and controlled by single genes whereas polygenic resistance is race-non-specific and controlled by many genes (Stenglein et al., 2003). Most of the qualitative genes such as Phg-2, Phg-3, Phg-5 and Phg-6 are dominant or partially dominant and have a great phenotypic effect but might be overcome by virulent genes present in specific races of the pathogen (Caixeta et al., 2005). The angular leaf spot pathogen has been reported to have a high degree of pathogenic and genetic variability (Nietsche et al., 2001; Sartorato, 2002; Wagara et al., 2004; Mahuku et al., 2010). The race composition in any given area is continuously changing, with the emergence and introduction of new races, and needs to be constantly monitored. These races frequently vary in time and space; thus, a bean variety that is resistant in one year or location may be susceptible in another (Aggarwal et al., 2004). Pathogenic variation of P. griseola is expressed in different geographical and agro-ecological zones and on host genotypes. Hence identification of the P. griseola races present in an area and the understanding of their geographical distribution are critical in screening for resistance and deployment of resistant genes. Pathogenicity is differentiated in the symptom variation expressed in test plants as a result of pathogen variability. Race-typing the P. griseola population from Kenya will provide information on the amount of pathogenic variation that is maintained in this pathogen and the geographical distribution of the different pathotypes. Severity and incidence of bean diseases is dynamic and changes with localities and environmental conditions especially those associated with climate changes and variability. However, a comprehensive survey of bean diseases in major bean growing regions has not been conducted. The objective of this study was to conduct a survey of prevalent diseases and identify races of P. griseola in bean growing regions of Kenya.
2.0 Materials and methods
2.1 Collection of diseased material
A survey was conducted in all major common bean growing regions of Kenya. This survey covered the prevalence of angular leaf spot, anthracnose, common bacterial blight, Pythium root rot and bean common mosaic disease. Two surveys were carried out in December 2010 and June 2011 in 35 bean growing districts of Kenya during the short and long rains season, respectively. Samples were collected from naturally infected common bean cultivars and landraces from the major bean producing agro-ecological regions of Kenya. The agro-ecological zones (AEZs) covered were Upper Highland (UH2), Lower Highland (LH1, LH2, LH3 and LH4), Upper Midland (UM1, UM2, UM3, UM4 and UM5) and Lower Midland (LM1 and LM2). The five economic important diseases causing major losses mainly (angular leaf spot, anthracnose, common bacterial blight, root rots and bean common mosaic virus) were sampled in all the agro-ecological zones. Diseased leaf samples were preserved neatly between sheets of old newspapers and pressed with a wire mesh to dry and for transportation from the field to the laboratory for analysis. Leaves symptomatic to bean common mosaic virus were placed in ziplock polythene bags and preserved in ice flakes in cool boxes. Roots sampled with root rot symptoms were carried in khaki size-3 paper bags while soil was carried in 5 kg size nylon sacs. Seeds obtained from the survey with common bacterial blight symptoms were carried in khaki size-3 bags. The bean gene pools (Andean or Mesoamerican), plant growth habit (climber or bush) and variety identities from which the isolates were obtained were recorded whenever it was possible. The geographic coordinates were recorded for all the points where diseases were sampled using an eTrex® (Garmin™, Taiwan) global-positioning-system (GPS) recorder.
2.2 Pathogen isolation and preservation
Isolation and culturing was done aseptically in the Pathology Laboratory at University of Nairobi. Angular leaf spot pathogen was isolated by picking conidia from well-developed and sporulating lesions using a tiny piece of agar placed on the tip of a sterilized dissecting needle, and streaked onto tap water agar (TWA – 15g Agar powder and 1000ml double distilled water) and then incubated in darkness for 24h at 21 °C. The TWA plates streaked with conidia were then observed under a dissecting microscope to identify the germinated conidia. The individual germinated conidia were then transplanted onto V8 medium (200ml V8 juice, 3g CaCO3, 18g Bacto agar and 800ml ddH2O) to obtain monosporic cultures for each P. griseola isolate. Isolates were maintained on V8 juice agar and kept in a dark incubator at 21 °C for up to 21 days to promote sporulation (Fig. 3.2). V8 juice is a trademark name for commercial beverage products made from either eight vegetables or a mixture of vegetables and fruits and whose brand is owned by the Campbell Soup Company-USA. Anthracnose was isolated using small pieces of symptomatic tissue that were surface sterilized by immersing them in 0.5% NaOCl for 3 minutes. After which they were rinsed in sterile distilled water, blotted dry on sterile filter paper. Inorder to suppress bacterial growth, the antibiotic streptomycin sulphate (40ppm) was added onto the medium before plating.
The surface sterilized tissues were then plated in to potato dextrose agar (PDA), and were incubated for 14 days in darkness at 20-21 °C. Under sterile conditions the cultures were monitored and transferred into new potato dextrose agar plates to increase inocula. Monosporic isolates were characterized using a set of 12 host differential genotypes (Musyimi, unpubl. data) and were maintained on potato dextrose agar medium at 4 °C for short term storage, or maintained on fungus-colonized filter papers at -20 °C for long term storage. The inoculum of each bean common mosaic disease collected from farmers‘fields was prepared by making sap extracts from severely infected young leaves. This was done by grinding infected leaves with a mortar and pestle in hydrogen phosphate buffer containing 0.1% Tween 20. The supernatant was then sieved to eliminate plant debris. Healthy seedlings raised under greenhouse were inoculated at primary leaf stage by leaf rub method using carborundum (Kieselguhr) powder as an abrasive. Proper care was taken to avoid the contamination of isolates. Individual isolate was maintained on healthy seedlings of the susceptible variety G10909 and infected leaves collected from the artificially inoculated plants and stored in the refrigerator at -20 °C. Root rot isolations were accomplished by first washing soil from infected root samples in a jet-stream of tap water, rinsing twice in sterile distilled water, blotting dry on sterile paper towel, and placing infected root pieces cut from expanding lesions on the prepared selective medium (CMA) using flamed forceps. Petri plates with plant samples were observed after incubation for 24-48 h at room temperature (20-25 °C). The pathogen mycelia developing from the plant tissues were confirmed and transferred onto potato dextrose agar (PDA) slants.
2.3 Inoculation of test plants
Numerous methodologies were used to determine P. griseola physiological specialization in the past (Alvarez-Ayala and Schwartz, 1979; Buruchara, 1983; Correa-Victoria, 1987). In an effort to standardize the methodology for P. griseola pathotype identification, a set of 12 common bean differential cultivars was established (CIAT, 1995). Test plants from these cultivars were used for characterizing the virulence diversity of P. griseola in this study (Table 3.1). The differential genotypes consist of six Mesoamerican and six Andean bean lines. Seeds of the angular leaf spot differentials were obtained from CIAT, Kawanda in Uganda and Dr. Merion Liebenberg of Grain Crops Institute, Potchefstroom, South Africa. Some seeds were also obtained from Dr. Isabel Wagara of Egerton University, Kenya.
Kabete Field Station of University of Nairobi. There were five replicates of one pot each (a total of fifteen plants) for each genotype. Inoculum of each isolate was increased by culturing the pathogen onto several plates of V8 juice agar medium. Conidia were harvested from 12 day old cultures, suspended in sterile distilled water from petri plates by gently scraping the surface of sporulating colonies with a soft brush. The conidia suspension was filtered through a double layer of cheese cloth to remove mycelia mass. The number of spores was determined using a haemocytometer and concentration adjusted to 2 × 104 conidia ml-1. The aqueous conidial suspension was misted with a hand sprayer until run off onto both abaxial and adaxial surfaces of bean differential plants at first trifoliate leaf (14-day old seedlings) to induce disease. Inoculated plants were covered with polythene sheets and maintained in the humid chamber for four days after inoculation under high relative humidity (RH ≥ 95%). Plants were then uncovered and disease reactions evaluated for angular leaf spot symptoms (Fig. 3.3).
2.4 Disease evaluation and data analysis
Inoculation of test plants, disease evaluation and data collection was conducted between May 2011 and July 2012. Plants were maintained in greenhouse for 12 days and evaluations for symptoms performed 12, 17 and 21 days after inoculation, using 1 to 9 (Schoonhoven and Pastor-Corrales, 1987) standard scale where 1 = no symptoms; 2 = lesions on up to 3% of leaf area; 3 = lesions on up to 5% of leaf area, with no sporulation of the pathogen; 4 = lesions and sporulation on up to 10% of leaf area; 5 = lesions and sporulations with 2–3 mm in diameter on 11–15% of leaf area; 6 = lesions and sporulations >3 mm in diameter on 16–20% of leaf area; 7 = lesions and sporulations >3 mm in diameter on 21–25% of leaf area; 8 = lesions and sporulations >3 mm in diameter on 26–30% of leaf area; and 9 = lesions, frequently associated with early loss of leaves and plant death, on 90% of leaf area. Plants with scores ≤ 3 were considered resistant, 4 to 6, intermediate and 7 to 9 as susceptible. Reaction type categories were determined according to the averages of these symptom scores attributed for each plant-pathogen combination.
2.5 Angular leaf spot race identification
A scale of binary values proposed by Pastor-Corrales and Jara (1995) was used. The race designation was obtained by adding the binary value for each differential line that presented a compatible reaction. To determine races of isolates, two numbers separated by a dash were used, for example, race 63-31 for isolate Pg01BN from Bungoma. The first number, 63, was obtained by adding the binary values of the susceptible Andean differential cultivars, each of which was given a (+) sign denoting compatibility; 1 + 2 + 4 + 8 + 16 + 32 = 63. The second number, 31, was obtained by adding the binary values of the susceptible Middle American cultivars: assigned (+) sign; 1 + 2 + 4 + 8 + 16 = 31.
3.0 Results
3.1 Prevalence of major common bean diseases in Kenya
The disease prevalence was high for angular leaf spot, anthracnose, common bacterial blight and bean common mosaic disease, but very low prevalences were recorded for Pythium root rot (Table 3.1). Angular leaf spot was highly prevalent in the coastal, central and rift valley regions with a prevalence rate of >96%. The disease was recorded in all fields visited in the coastal region. Angular leaf spot was found to occur from the low altitudes (<1500 m asl) to the high altitudes (>2000 m asl) across all farms visited, and was observed from 1151 to 2371 m asl. Angular leaf spot was particularly more prevalent in the mid altitude (1500-2000 m asl) compared to low and high altitudes (Table 3.3). Anthracnose prevalence was high in central (95.6%), rift valley (72.4%) and coastal (75%) regions with lowest occurrences being reported in eastern region (16.7%). Unlike angular leaf spot, anthracnose was highly prevalent (96.4%) in the high altitude and low prevalence in low altitudes (58.1%). Common bacterial blight was also highly prevalent in the areas surveyed with a total of 121 samples being collected (Table 3.2). The disease was widespread across all the regions and occurred in all farms visited in the rift valley, nyanza and coastal regions. The lowest prevalence of common bacterial blight was observed in the central region (71.5%), and within the high altitude areas (74.5%) compared to the low altitude zones (100%). Bean common mosaic virus had the highest prevalence in the nyanza region (76.5%) and within the low altitudes (77.4%). The viral disease was however low in the rift valley (62.1%) and the mid altitudes (60.9%) (Table 3.1). Pythium root rot was the least prevalent disease with only western region recording a prevalence of 12.5% with negligible occurrence in all other regions and across altitude range. There Pythium root rot was only concentrated in a few hot spots in Kakamega and Busia. The other occurrences of root rot were mostly caused by other pathogenic agents and not the targeted Pythium root rot.
Table 3.1: Prevalence of major common bean disease in various regions and agro-ecological zones of Kenya.
Prevalence (%)
Region ALS ANT CBB BCMV RR
Central 97.9 95.6 71.5 63.6 9.1
Coastal 100 75 100 75 0
Eastern 75 16.7 83.3 75 0
Nyanza 64.7 52.9 100 76.5 0
Rift Valley 96.2 72.4 100 62.1 3.4
Western 68.8 56.3 93.8 75 12.5
Altitude
Low altitude 93.7 58.1 100 77.4 0.1
Mid altitude 97.1 66.7 98.1 60.9 0.1
High altitude 78.9 96.4 74.5 72.7 0
ALS=angular leaf spot, ANT=anthracnose, BCMV=bean common mosaic virus, CBB=common bacterial blight, RR=Pythium root rot
3.2 Isolation and preservation of P. griseola
Fifty-seven monosporic cultures were successfully isolated (Table 3.3). All the 57 isolated samples were maintained onto V8 medium and preserved at -20 ºC in Pathology Laboratory at University of Nairobi.
3.3 Races of angular leaf spot in Kenya
Fifty-seven isolates obtained from samples collected in bean growing regions of Kenya were classified based on their virulence reactions into 23 different physiological races of Pseudocercospora griseola (Table 3.7). Isolates exhibited a different virulence pattern when inoculated on the 12 bean differential genotypes through expression of symptomatic variation giving different disease severity scores as shown in Table 3.6. Races 63-55, 63-63, 63-54 and 63-35 were the most prevalent pathotypes observed among 57 isolates studied and were reported in 10, 7, 6 and 4 different locations respectively. Two new races, 31-31 and 63-31 were reported for the first time in Kenya. The new races included two isolates race-typed as race 31-31 originated from Kibirigwi (UM2) and Turbo (UM3), and two isolates characterised as race 63-31and obtained from Othaya (LH3) and Kakamega (UM1). The lower highland (LH) and upper midland (UM) recorded the highest number of the most virulent races observed.
Table 3.2: Angular leaf spot race identification based on the reaction of 12 differential cultivars inoculated with 57 isolates of Pseudocercospora griseola.
Differential cultivar1
Andean Mesoamerican
A B C D E F G H I J K L
Isolate Origin 12 2 4 8 16 32 1 2 4 8 16 32 Races
Pg01NY Nyeri +3 + + + + + + + + -4 + + 63-55
Pg02NY Nyeri + + + + + – + + + – – – 31-7
Pg02BR Bureti – + + – – – – – – – – – 6-00
Pg01WN Wundanyi + + + + + + + + + + + + 63-63
Pg02KK Kakamega – + + + + – – – + – – – 30-4
Pg01KG Kibirigwi + + + + + – + + + + + – 31-31
Pg01NK Nakuru + + + + + – – – – – – – 31-0
Pg01TY Othaya + + + + + + + + + + + – 63-31
Pg02KR Kericho – + + + + + + – + – – + 62-37
Pg01NJ Njoro + + + + + + + + + – + + 63-55
Pg01GC Gucha + + + + + + + + + – – – 63-7
Pg02SY Siaya – + + + – – – – – – – – 14-0
Pg02NH Nyahururu + + + + + + + + + – + + 63-55
Pg02BN Bungoma – + – + + – – – – – – – 26-0
Pg01TH Thika + + + + + + + + + + + + 63-63
Pg01MC Machakos + + + + + + + + – – – + 63-35
Pg01KS Kisumu + + + + – + – + – – – – 46-2
Pg02MG Mugirango + + + + + + + + + + + + 63-63
Pg02MR Meru Central + + + + + + – + + – + + 63-54
Pg02NK Nakuru + + + + + + + + + – + + 63-55
Pg04WN Wundanyi – + + + + – – + – – + – 30-18
Pg01KK Kakamega + + + + + + + + + – – + 63-39
Pg01MN Murang’a + + + + + + + + + – + + 63-55
Pg01KL Kitale + + + + + + + + – – – + 63-35
Pg01SY Siaya – + + + – + – + – – – – 46-2
Pg01NH Nyahururu + + + + + + + + + – + – 63-23
Pg01NV Naivasha + + + + + + + + + – + + 63-55
Pg06BR Bureti + + + + + + + + + – + + 63-55
Pg01LD Eldoret – + + + + + + + + – – – 62-7
Pg02GC Gucha + + + + + + – + + – + + 63-54
Pg02MS Meru – + + + – + – + – – – – 46-2
Pg04GC Gucha + + + + + + + + + + + + 63-63
Pg04BR Bureti + + + + + + + + + + + + 63-63
Pg01BM Bomet + + + – – + – – – – – – 39-0
Pg02TR Turbo + + + + + + + + + – + + 63-55
Differential cultivar1
Andean Mesoamerican
A B C D E F G H I J K L
Isolate Origin 12 2 4 8 16 32 1 2 4 8 16 32 Races
Pg01MS Meru + + + + + + – + + – + + 63-54
Pg01ST Sotik – + + + + + + – + – – + 62-37
Pg01TR Turbo + + + + + – + + + + + – 31-31
Pg01MG Mugirango + + + + + + + + + – + – 63-23
Pg01BN Bungoma – + + + + – – + – – + – 30-18
Pg03KK Kakamega + + + + + + + + + + + – 63-31
Pg02NV Naivasha – + + + + + + – + – – + 62-37
Pg01KM Kiambu + + + + + + + + + – + + 63-55
Pg02KY Kerugoya + + + + + + + + + – + – 63-23
Pg03GC Gucha + + + + + + + + + – + + 63-55
Pg02MC Machakos – + – + + + – + – – + – 58-18
Pg05GC Gucha + + + + + + + + – – – + 63-35
Pg01KR Kericho + + + + + – + + + – – + 31-39
Pg01KN Kangema + + + + + + – + + – + + 63-54
Pg03WN Wundanyi + + + + + + + + + – – + 63-39
Pg02MB Embu + + + + + + + + – – – + 63-35
Pg01BR Bureti + + + + + + + + + + – + 63-47
Pg01TG Tigoni + + + + + + – + + – + + 63-54
Pg02TG Tigoni + + + + + + – + + – + + 63-54
Pg01KB Kabete + + + + + + + + + + + + 63-63
Pg02KB Kabete + + + + + + + + + + + + 63-63
Pg03KB Kabete + + + + + + + + + – – + 63-39
1 Andean differential genotypes: A=Don Timoteo; B=G 11796; C=Bolon Bayo; D=Montcalm;
E=Amendoin; F=G5686. Mesoamerican differential genotypes: G=PAN 72; H=G2858;
I=Flor de Mayo; J=MEX 54; K=BAT 332; L=Cornell 49-242.
2 Binary values used to classify Pseudocercospora griseola races. 3 Compatible reaction (+)
4 Incompatible reaction (-)
Table 3.8: Distribution of angular leaf spot races based on regions, altitude range and agro-ecological zones of Kenya.
Region Races 1AEZ Races Altitude Races
Central 63-63 LH1 63-54 Low altitude 63-63
63-55 62-37 63-55
63-54 31-7 63-54
63-39 31-39 63-39
63-31 LH2 63-63 63-35
63-23 62-37 63-23
31-7 39-0 46-2
31-31 LH3 63-63 31-31
Coastal 63-63 63-55 30-18
63-39 63-39 14-0
30-18 63-31 Medium altitude 63-7
Eastern 63-54 62-7 63-63
63-35 LM1 46-2 63-55
58-18 14-0 63-54
46-2 LM2 46-2 63-47
Nyanza 63-7 30-18 63-39
63-63 UH2 63-55 63-35
63-55 63-23 63-31
63-54 UM1 63-7 63-23
63-35 63-63 62-37
63-23 63-55 58-18
46-2 63-54 39-0
46-2 63-47 31-7
14-0 63-39 31-39
Rift Valley 63-63 63-31 31-0
63-55 63-23 30-4
63-47 46-2 26-0
63-35 30-4 6-0
62-7 26-0 High altitude 63-55
62-37 UM2 63-55 63-54
39-0 63-35 63-23
31-39 31-31 62-7
31-31 30-18 31-31
31-0 UM3 63-63
6-0 63-55
Western 63-39 63-39
Seven isolates collected in six different locations were classified as race 63-63 (Table 3.7). Race 63-63 was the only race virulent to all the 12 differential cultivars. All the isolates studied in this work presented a pathogenic reaction of compatibility with either/both Andean and Mesoamerican cultivars. The two main pathogen groups; Andean and Mesoamerican, were also identified from this study. Out of the 57 isolates characterised, 12 races were classified as from the Andean pathogenic group while 45 races were from the Mesoamerican pathogenic group. Mesoamerican cultivars MEX 54, Cornell 49-242 and BAT 332, were the most resistant cultivars tested indicating their importance in bean breeding aimed at developing new common bean cultivars resistant to angular leaf spot disease. Different isolates collected at the same location showed differences in their patterns of virulence. For example, isolates Pg01GC, Pg02GC, Pg03GC, Pg04GC and Pg05GC (Table 3.5, 3.7), collected from Gucha, race-typed into races 63-7, 63-54, 63-55, 63-63 and 63-35 (Table 3.7), and caused compatible reactions on nine, 10, 11, 12 and nine differential genotypes, respectively.
Similar results were observed with isolates collected in other locations (Table 3.7). We detected five isolates that attacked only Andean beans (e.g., Pg01BM, Pg02SY and Pg02BN), although some isolates (e.g., Pg01SY, Pg02KK and Pg04WN) attacked some Middle American beans but were often more virulent on Andean cultivars and thus classified as Andean pathotypes (Table 3.7). Analysis of virulence phenotype of 57 isolates on a set of 12 differential cultivars using a hierachical cluster analysis method and tree option of Genstat program, generated a dendrogram, which divided all the study samples into two clusters: Mesoamerican and Andean groups (Fig. 3.6). The Mesoamerican cluster was composed of 45 isolates (78.9%), while the Andean cluster consisted of 12 isolates (21.1 %) (Fig. 3.6). Ten angular leaf spot isolates from Mesoamerican group were observed to share a high pathogenic similarity (Fig. 3.6). Rift valley region had the highest number of different races with 11 pathotypes being realized (Table 3.8). There were only 3 different races from the coastal region. Rift Valley region was also one of the two regions, the other being central region where a new race (31-31) was identified (Table 3.7). The most virulent pathotype (63-63) of angular leaf spot was reported in Central, Nyanza, Rift Valley and Coastal regions, but was absent in Eastern and Western regions. Mid altitude range had the most diverse races reaching 18 different pathotypes compared to 10 and 5 different races from low and mid altitudes respectively. The new race was however reported in the low and mid altitudes where pathogenic diversity was minimal (Table 3.7, 3.8). Eleven races were recorded in the upper midland 1 zone (UM1), with 5 and 4 races being identified in the UM3 and UM2 zones. The highly pathogenic race 63-63 was found in all agro-ecological zones surveyed except zones LM1 and UM2. Race 31-31, which is being reported in Kenya for the first time, was obtained from zones UM2 and UM3 (Table 3.7, 3.8).
4.0 Discussion
The major diseases of common bean surveyed in bean growing regions of Kenya were observed to be highly prevalent in the regions surveyed. This indicates the necessity of developing multiple disease resistance genotypes to counter the severe losses experienced when they infest susceptible cultivars. Angular leaf spot was observed to occur in all the regions of Kenya where common bean is cultivated. It was highly prevalent in the coastal, central and
rift valley regions with a prevalence rate of greater than 96%. The results confirm earlier findings by Mwangombe et al. (2007) who reported a prevalence rate of 89%, and observed the occurrence of angular leaf spot in all regions surveyed. Angular leaf spot was also recorded across all altitude ranges from 1151 to 2371 m asl. The prevalence among the altitude ranges
differed with high prevalence being recorded in mid altitude (1500-2000 m asl), with lower prevalences in low and high altitude ranges. This is attributable to the warm and moist conditions within the mid altitude and inhibition of growth and development of the pathogen in the low and high altitudes due to high temperatures and low temperatures respectively. Anthracnose was also highly prevalent across regions but was more prevalent
in the high altitudes where cool and humid conditions prevail. Common bacterial blight generally occurred in all regions and altitudes with high prevalence rates, but was dominant in rift valley, nyanza regions and in the low altitudes. There were very low occurrences of Pythium root rot in all areas surveyed except a few hot spots in western region. This could partly be attributed to the nature of the numerous causal agents that incite root rots, and the complexity of distinguishing them symptomatically. Pseudocercospora griseola isolates were examined to evaluate physiological diversity in an attempt to understand the distribution and pathogenic virulence structure of P. griseola present in Kenya. The P. griseola isolates characterized showed variation in virulence towards differential cultivars
demonstrating a high pathogenic variability of the pathogen in Kenya. There were 23 races identified from the 57 isolates obtained from major bean growing regions of Kenya. This occurrence of large pathogenic variation in P. griseola populations supports earlier findings by Mahuku et al. (2002a), Stenglein et al. (2003), Wagara et al. (2004) and Silva et al. (2008). The broad variation occurred whether isolates were collected from different geographical areas between and within districts or from a given location. Similar observations have been made with the Kenyan isolates by Wagara et al. (2004). Pathotypes of angular leaf spot observed adhered to the two Pseudocercospora griseola pathotype groups. Twelve isolates were classified as from the Andean pathotype group while 45 isolates were from the Mesoamerican pathotype group. There were different patterns of pathogenic virulence for isolates obtained within close proximity. In the rift valley region 11 different pathotpes were identified each with a divergent pathogenic virulence. This was largely because the range of pathogenic variation among P. griseola isolates found at a given location may be a function largely of the variability found among common bean genotypes grown in that location or genetically diverse pathotypes. These results confirmed the existence of pathogenic diversity within the population of angular leaf spot pathogen in Kenya. Races 63-55, 63-63, 63-54 and 63-35 were found to be the most prevalent pathotypes in areas studied. These races were observed to possess high pathogenic virulence to the differential cultivars, which may explain their ability to infect and spread widely within common bean growing areas. The identification of these races, particularly race 63-63 which breaks all resistance genes present in all differential cultivars, is of great importance to a breeding programme aiming at the development of angular leaf spot resistant cultivars. Two isolates were identified as the new race 31-31, which had not bee identified in Kenya before. New races have been identified from regions they were absent in the past. This necessitates the regular monitoring and race identification of angular leaf spot from time to time and the development of broad and durable resistance against the Pseudocercospora griseola. The results show that both Andean and Mesoamerican bean differential genotypes evaluated in this study are genetically highly variable in response to different races of P. griseola. Mesoamerican cultivars MEX 54, Cornell 49-242 and BAT 332, recorded high levels of resistance to majority of isolates characterised indicating their importance in bean breeding programmes aimed at developing new common bean varieties resistant to angular leaf spot disease. These results also show the need to improve local susceptible commercial varieties against the major common bean diseases and other constraints. Highly pathogenic isolates in both common bean gene pools (Andean and Mesoamerican) were observed. The existence of isolates from Mesoamerican and Andean origins has also been demonstrated in Brazil (Nietsche et al., 2001; Sartorato, 2002, 2004; Silva et al., 2008). Based on data from the pathogenicity tests, it was observed that all the P. griseola isolated in Kenya were pathogenic on common beans. These data confirmed also that the angular leaf spot symptoms previously observed on the sampled materials were due to P. griseola agent. Moreover, there was an important variability of the bean differential variety reaction following inoculation with the P. griseola isolates. In fact, for a given P. griseola race, it was noticed that there were differences in symptom expression and the disease severity level recorded on different bean varieties. There was evidence in variation of symptomatology in any given reaction between isolates and differential cultivars. The findings of this study further showed the distribution of different races among agro-ecological zones implying that targeted-resistant varieties can be developed for specific bean growing regions of Kenya. For instance, bean breeders in Kenya can now develop a resistant variety to race 63-63 that was found in all agro-ecological zones surveyed except zones LM1 and UM2. In conclusion, the identification of P. griseola races in Kenya will enhance control of angular leaf spot disease by using genetic resistance to improve commercial and popular varieties and monitor emerging pathotypes in different regions of Kenya.
Acknowledgement
Funding for this project was from Kirkhoust trust while facilities and support was provided by the University of Nairobi Bean Program
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