Coring method of sampling potato tubers to detect ralstonia solanacearum.

Bacterial wilt caused by Ralstonia solanacearum is considered among the most damaging diseases of potato in Sub-Saharan Africa. In Kenya, majority of farmers visually select and save seed from harvested potato tubers and reuse the same tubers for several seasons. Latently infected seed tubers which cannot be identified by visual inspection during certification further compounds the situation compelling the need for laboratory testing. The study evaluated the effectiveness of coring tuber samples to improve sampling efficiency for onward laboratory diagnosis. In this study, the coring method of sampling potato tubers for detection R. solanacearum was evaluated. Coring involves taking multiple tuber samples direct from the stolon attachment site into a collection tube containing extraction buffer that provides the extract for further diagnostic tests. Coring was assessed using field samples from different potato growing regions of Kenya including, Koibatek, Molo, Uasin Gishu, Bungoma and Kisii and tested using Nitrocellulose Membrane (NCM) ELISA. These results were compared to PCR, qPCR and LAMP. Coring method was statistically reliable (p>0.05) when compared to the standard sampling method used in Kenya to detect R. solanacearum. The coring of potato tubers is a reliable and quicker method of sampling that reduces the turnaround time of testing hence improving efficiency

Phylogenetic Distribution of Ralstonia solanacearum Species Complex Populations in Potato in Kenya

Ralstonia solanacearum is a pathogen causing bacterial wilt disease of potato, resulting in 70% potato production losses in Kenya. A study was conducted to determine the diversity of R. solanacearum species complex strains within the main potato-growing regions of Kenya. Potato tubers were collected in different potato-growing regions of Kenya from visibly wilted potato plants as well as samples of tomato, irrigation water, and cultures for pathogen isolation. Genomic DNA was isolated from 135 purified cultures of RSSC isolates and PCR-amplified using multiplex and sequevar primers targeting the endoglucanase (egl) partial gene sequences. Pathogenicity tests using R. solanacearum strain (phylotype II sequevar I) were done on the cultivars Kenya Karibu, Shangi, Chulu, Wanjiku, and MoneyMaker. Phylogenetic analysis of the partial egl gene identified two genospecies, R. pseudosolanacearum sp. nov. (1.5%) and R. solanacearum (98.5%). All R. solanacearum strains clustered in sequevar I and were distributed in all the potato-growing regions surveyed. The cultivars were grown in a greenhouse for two cycles in a randomized complete block design and inoculated with R. solanacearum strain. The severity scores were assessed and the area under the disease progress curve (AUDPC) was determined. All the cultivars tested for pathogenicity exhibited wilting symptoms at varying intervals after infection, with none showing complete resistance to R. solanacearum. Cultivar Shangi exhibited minimum disease severity and progression of 41.14% and AUDPC of 1041.7, respectively, while 'Kenya Karibu' was the most susceptible with a high progression rate of 68.24% and AUDPC of 1897.5, respectively. 'MoneyMaker', 'Chulu', and 'Wanjiku' showed no significant difference in disease severity, depicting a simultaneous rate of infection among them. These findings provide valuable information to better understand the pathogen genetic diversity in Kenya and how it spreads

Comparative evaluation of LAMP, qPCR, conventional PCR, and ELISA to detect ralstonia solanacearum in Kenyan potato fields

Bacterial wilt caused by Ralstonia solanacearum is considered among the most damaging diseases of potato in Sub-Saharan Africa and the most significant biotic constraint of potato production alongside late blight. Unlike late blight, which can be managed by chemical means, R. solanacearum can only be managed through cultural methods and clean seed. Laboratory testing to certify seed before planting is required to confirm the absence of the pathogen in Kenya. A loop-mediated isothermal amplification (LAMP) assay was developed using the UDP-(3-O-acyl)-N-acetylglucosamine deacetylase gene (IpxC) to screen seed potato for R. solanacearum strains. The assay was assessed using DNA extracted from R. solanacearum and other soil and potato pathogens to demonstrate specificity and sensitivity. The LAMP assay was validated using field samples from different potato growing regions of Kenya collected over two growing seasons and compared with established nucleic acid and protein-based assays. The IpxC LAMP assay was found to be specific and sensitive to R. solanacearum, detecting as low as 2.5 pg/µl of R. solanacearum DNA. Of the 47 potentially infected field samples collected, both IpxC LAMP and quantitative polymerase chain reaction (PCR) detected R. solanacearum DNA in 90% of the samples, followed by conventional PCR (86%) and ELISA (75%). This IpxC LAMP assay is a promising diagnostic tool to rapidly screen for R. solanacearum in seed potato with high sensitivity in Kenya