CHARACTERIZATION OF HALO BLIGHT RACES ON DRY BEANS IN SOUTH AFRICA

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Isolation and identification of isolates

Diseased plant material was collected from major bean production areas in South Africa and Malawi, Lesotho and Zimbabwe. Leaves were rinsed under running tap water for 10 min, surface-disinfested for 3 min in 3.5% sodium hypochlorite and then rinsed twice in sterile water for 1 min each. Leaf material was macerated in a droplet of sterile water and streaked onto yeast-extract-dextrose-calcium-carbonate (YDC) agar (Schaad & Stall 1988). Plates were incubated at 25°C. Following 72 hr incubation, yellow-pigmented colonies typical of Xanthomonas spp. were purified on YDC agar by a series of single colony transfers. Production of brown diffusible pigment on YDC differentiated Xapf from Xap isolates (8asu & Wallen 1967). Agglutination of antiserum specific to Xap and Xapf, obtained from Adgen Agrifood Diagnostics, Auchincruive, Scotland, was used to identify isolates. Pathogenicity tests on susceptible cultivar Teebus were done to confirm identity of isolates.

Pathogenicity tests

Seed from eight tepary lines previously reported to differentiate between Xap and Xapf races (Table 1) (Opio et a/., 1996), were obtained from Dr. DP Coyne, University of Nebraska, Lincoln, USA and multiplied from a single seed in a greenhouse to ensure genetically uniform material. The tepary differential set was expanded to include resistant genotypes, XAN 159, GN #1 Nebr. sel 27, Wilk 2, Wilk 6, Vax 4, Vax 5 and Vax 6. Resistance in these lines are all tepary derived. Cultivar Teebus was included as susceptible check. Five seeds of each genotype were planted in 15-cm-diameter plastic pots in sterile soil and maintained in a greenhouse at 18°C night/28°C day. Seedlings were thinned to four plants per pot after emergence. One pot per differential was used per isolate, each plant representing a replicate. Pots were randomised prior to inoculation. Experiments were repeated to confirm reactions of isolates.
One hundred and forty three isolates from 44 localities in four countries of southern Africa were selected for the study (Table 2). Four isolates received from the International Centre for Agriculture in the Tropics (CIAT) were included as reference cultures. Isolates used for each experiment were regenerated from storage at -72°C, because loss of pathogenicity was encountered by sub-culturing. Inoculum was prepared by suspending 48- to 72-h-old cultures in sterile distilled water and adjusting it turbidimetrically to contain approximately 108 CFU/ml. Fourteen to 20-day-old plants with fully expanded first trifoliate leaves were used for inoculation. Plants were inoculated using the multiple-needle inoculation method (Andrus 1948). Control plants were inoculated with sterile distilled water. Inoculated plants were kept in a greenhouse at 18°C nightl28°C day. Plants were rated for infection 14 days after inoculation on a 1 to 9 scale (Aggour et a/. 1989). Plants, rated 1 to 3, were classified as resistant (incompatible) and ratings of 4 to 9 considered susceptible (compatible).

Isolation of bacterial DNA

Eight isolates (two Xap and six Xapf) from southern Africa, one Xapf isolate from CIAT and a non-pathogenic Xanthomonas isolate (Table 3) were used in genetic studies. These isolates were selected based on their geographic origin. Isolates were cultured in 50 ml nutrient broth for 24-48 hr at 25°C prior to DNA isolation. Bacterial cells were collected by centrifugation at 5 000 rpm for 10 min. Cells were washed three times by resuspending in 5 ml 1 M NaCI and centrifugation at 5 000 rpm for 10 min, followed by two wash steps in 5 ml sterile distilled water. Washed cells were resuspended in 10 ml warm (55°C) extraction buffer containing 0.2 M Tris.HCI (tris (hydroxymethyl) aminomethane), pH 8.0; 10 mM EDTA (ethylenediaminetetraacetate), pH 8.0; 0.5 M NaCI; 1 % (w/v) SDS (sodiumdodecylsulfate) and 10 fl9.ml -1 Proteinase K. Resuspended cells were incubated in a water bath at 55°C for one hr and half a volume 7.5 M ammonium acetate was added. The suspension was mixed by gentle inversion and incubated at room temperature for 10 min. Phase separation was enhanced by adding 100 fll TE buffer (10 mM Tris.HCI, pH 8.0; 1 mM EDTA, pH 8.0). Phases were separated by centrifugation at 14 000 rpm for 15 min. The upper aqueous layer was transferred to a fresh tube containing an equal volume of isopropanol, mixed by gentle inversion and incubated at room temperature for a minimum of 2 hr to overnight. DNA was collected by centrifugation at 14 000 rpm for 15 min . The precipitated DNA was washed twice in 1 ml ice-cold 70% (v/v) ethanol, the pellet air-dried at room temperature, and resuspended in 10 fll TE buffer. The DNA was treated with RNase for two hours at 37°C and concentration and purity estimated by measuring absorbances at A260 and A28o . DNA samples were diluted to a working solution of 200

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CHAPTER 1. GENERAL INTRODUCTION
CHAPTER 2. DISTRIBUTION AND SEVERITY OF BACTERIAL DISEASES ON DRY BEANS (PHASEOLUS VULGARIS L.) IN SOUTH AFRiCA
Abstract
Introduction
Material and Methods
Results
Discussion
References
CHAPTER 3. CHARACTERIZATION OF HALO BLIGHT RACES ON DRY BEANS IN SOUTH AFRICA
Abstract
Introduction
Material and Methods
Results
Discussion
References
CHAPTER 4. PATHOGENIC AND GENETIC VARIATION IN XANTHOMONAS AXONOPODIS PV. PHASEOLI AND X. AXONOPODIS PV. PHASEOLI VAR. FUSCANS IN SOUTHERN AFRICA 
Abstract
Introduction
Material and Methods
Results
Discussion
References
CHAPTER 5. SUSCEPTIBILITY OF SOUTH AFRICAN DRY BEAN CUL TIVARS TO BACTERIAL DiSEASES  
Abstract
Introduction
Material and Methods
Results
Discussion
References
CHAPTER 6. COMMON BACTERIAL BLIGHT: A DEVASTATING DISEASE OF DRY BEANS IN AFRICA
Introduction
Symptomology
Distribution and Economic Importance
The Pathogen
Disease Development
Epidemiology
Disease Management
Conclusion
References
CHAPTER 7. IMPROVEMENT OF COMMON BACTERIAL BLIGHT RESISTANCE IN THE SOUTH AFRICAN DRY BEAN CULTIVAR
TEEBUS
Abstract
Introduction
Material and Methods
Results
Discussion
References
CHAPTER 8. APPLICATION OF MOLECULAR MARKERS IN BREEDING FOR BEAN COMMON BLIGHT RESISTANCE IN SOUTH AFRiCA 
Abstract
Introduction
Material and Methods
Results
Discussion
References
CHAPTER 9.YIELD LOSS ASSESSMENT IN SOUTH AFRICAN DRY BEAN GENOTYPES CAUSED BY COMMON BACTERIAL BLIGHT 
Abstract
Introduction
Material and Methods
Results
Discussion
References.
CHAPTER 10. GENERAL DiSCUSSiON
SUMMARY.

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