Phenotypic and Molecular Characterization of Pectobacterium and Dickeya Species Associated With Blackleg And Soft Rot Of Potato in Kenya

Abstract

Blackleg and soft rot of potato, caused by Pectobacterium and Dickeya spp., are major production constraints in many potato-growing regions of the world. Despite advances in the understanding of the causative organisms, disease epidemiology and control, blackleg remains the principal cause of down- grading and rejection of potato seed in classification schemes across the world. Although symptom recognition is relatively straightforward and is applied universally in seed classification schemes, attributing disease to a specific organism is problematic and can only be achieved through the use of diagnostics. In Kenya Pectobacterium and Dickeya spp have not been characterized thus preventing early disease diagnosis and management. The purpose of this study was to isolate and characterize the pectolytic bacteria occurring in potato in Kenya. The methods included collection of symptomatic potato plants and tubers from four major potato production regions of Kenya which were: Nakuru, Nyandarua, Narok and Elgeiyo Marakwet. The samples were prepared and analysed using phenotypic methods which included plating on semi selective media(crystal violet polypectate(CVP) and nutrient agar with manganeseIIchlorite tetrahydrate(NGM), biochemical tests, pathogenicity tests and molecular based methods which included real time and conventional PCR as well as sequencing of 16S rDNA housekeeping gene using the sequencing primers. Sequence evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura-Nei model. Phylogeny was done using neighbor joining method with 1000 boostraps to determine the relationship of the species. Results of obtained from the sequences were used to compare the strains occurring in Kenya and those present in other countries. The results from phenotypic and molecular analysis identified 65 % isolates as Pectobacterium carotovorum subsp. Carotovorum, 19% as Pectobacterium wasabiae and 8% of the isolates as Pectobacterium atrosepticum. However, Dickeya spp. primer pair produced unexpected band size compared to the positive control, Dickeya dianthicola and therefore presented need for further investigation. The findings of this study confirmed presence of new pathogens; Pectobacterium atrosepticum and Pectobacterium wasabiae in potato fields in Nakuru and Elgeiyo Marakwet regions respectively for the first time in Kenya. There is therefore need for further studies to determine the origin and distribution of these two stains of Pectobacterium spp. in all potato growing regions of Kenya. There is also need for awareness creation to the potato farmers in order to understand economic importance of Pectobacterium and Dickeya spp. so as to initiate prevention and control measures.