Characterization of Xanthomonas Campestris Pv.Vesicatoria In African Nightshades (Solanum Scabrum Mill.) and its Control Using Extracts of Bidens Pilosa L. and Euphorbia Hirta L.

Abstract

African nightshades (Solanum scabrum Mill.) are an important vegetable in terms of food security, medicinal values and income for most communities in Kenya. Its productivity is majorly affected by bacterial leaf spot disease caused by Xanthomonas campestris pv.vesicatoria which reduces yield by 40-70%. The disease is controlled by application of synthetic pesticides which have adverse effect on the environment. Botanicals including Bidens pilosa and Euphorbia hirta have been used to control Phytophthora infectans and Xanthomonas citri plant pathogens hence the reason why they were selected for this study. There is no information available on the isolation and characterization of Xanthomonas campestris pv.vesicatoria and use of B. pilosa and E. hirta to control this disease. The main objective of this study was to characterize Xc. pv.vesicatoria in Solanum scabrum and to evaluate its control using B. pilosa and E. hirta leaf and root extracts. Six isolates (WJOUST 1, WJOUST 2, WJOUST 3, WJOUST 4, WJOUST 5 and WJOUST 6) from diseased S. scabrum leaves were characterized using colony morphology and simple staining. Catalase test, oxidase test, KOH solubility test, Gram staining and hydrogen sulphide gas production test were carried out. Molecular characterization was done using 16S rRNA. Phylogenetic relationship between Xc pv.vesicatoria and other Xanthomonas species was determined. Bidens pilosa and Euphorbia hirta plant materials were collected within and around Jaramogi Oginga Odinga University of Science and Technology and extracted with ethanol and water to yield the crude extract. Antibacterial activity of extracts by agar disc diffusion method was carried out.The six isolates were subjected to B. pilosa and E. hirta leaf and root water extracts (5%, 10% 15%, 25%, 50% and 100%) and ethanol extracts (12.5mg/ml, 25mg/ml, 50mg/ml, 100mg/ml, 150mg/ml, and 200mg/ml) and inhibition zones measured. In the greenhouse plants were inoculated with the pathogen and treated with the various extracts. A synthetic chemical Ridomil® was used as standard and sterile water as control.Stem diameter, plant height, leaf weight and disease severity index was determined. The experiments were laid out in completely randomized design. Data was subjected to Analysis of variance and treatment means were separated and compared by Fisher’s Least Significant Differences (LSD p≤0.05). The isolates were rod shaped and showed yellow and mucoid colonies on modified Tween B Media and Yeast Dextrose Calcium carbonate media. The six isolates were found to be Gram negative, catalase positive, oxidase negative and produced hydrogen sulphide gas. 16S RNA of the isolates had 1400 bp and was identified as Xanthomonas campestris. Phylogenetic tree indicated that the isolates had 99% maximum identity with Xanthomonas vesicatoria obtained from NCBI sequence database. The B. pilosa and E. hirta water extract had no significant effect (p˃0.05) on zone of inhibition while the inhibition zones of ethanol extracts were significantly (p≤0.05) different. Water and ethanol root extracts of B. pilosa had significantly larger zones of inhibition than leaf extracts. Bidens pilosa and E. hirta water extract significantly reduced disease severity. Generaly water and ethanol extracts of B. pilosa and E. hirta increased stem diameter, plant height and leaf weight. This was probably due to active compounds that inhibited the development of the disease or improved the growth of the plant.The morphological, biochemical and molecular characterization identified the six isolates as Xanthomonas campestris, and they were found susceptible to B. pilosa and E. hirta leaf and root extracts.The similarity of the isolates suggests endemism of this pathogen. The B. pilosa and E. hirta 100% water extract and 200mg/ml ethanol extract effectively reduced disease severity. Therefore, both B. pilosa and E. hirta extracts can be used as an alternative to synthetic chemicals to control bacterial leaf spot disease.