5.2.70
THE DEVELOPMENT OF A BIOLOGICAL CONTROL AGENT FOR BACTERIAL WILT RACE 3 IN KENYA, AND ITS APPLICATION WITHIN POTATO SYSTEMS

JJ SMITH1, LC OFFORD1, SA SIMONS2, G ODOUR2, GN KIBATA3, ZK MURIMI3, A TRIGALET4 and GS SADDLER1

1CABI Biosciences, Egham, UK; 2CABI International Africa Regional Centre, PO Box 633, Nairobi, Kenya; 3KARI NARL, PO Box 14733, Nairobi, Kenya; >4INRA-CNRS, BP 27, 31326 Castanet-Tolosan Cedex, France

Background and objectives
In Kenya, potato cultivation occurs in the highlands between 1200 and 2800 m and over 75000-100000 ha per year. Socio-economic pressures result in near-continuous potato cultivation. This practice is favourable to the build-up of diseases and pests, particularly bacterial wilt (Ralstonia solanacearum). Potato seed tubers harbouring latent infection of R. solanacearum are the main source of disease, and ensure carryover into the subsequent growing season and to new regions. Accordingly, the production of clean seed is central to effective control strategies; however, this has proved problematic within Kenyan agricultural systems, and control strategies need to embrace additional approaches that are appropriate to small-scale farmers. One such approach is biological control. In developing a control strategy, a knowledge of the indigenous pathogenic population is essential. Bacterial wilt of potato is known to be caused by a specific race of R. solanacearum, race 3 biovar 2a, which is adapted to cooler climates. This race is highly homogeneous and has a host range restricted to potato and, to a lesser extent, tomato. This research addresses the potential of biological control of bacterial wilt in Kenya and explores the potential of small on-farm seed production plots as a window for its application. The approach developed describes the use of pre-inoculation of potato with non-pathogenic mutants of the wild-type organism.

Results and conclusions
Indigenous populations of R. solanacearum affecting potato in Kenya were characterized by a multi-faceted approach assessing traits for pathogenicity and rhizosphere competence, pathogenicity per se and genomic fingerprinting by macro-restriction of genomic DNA resolved by pulsed field gel electrophoresis (MR-PFGE). From these studies, phenotypic differences were not apparent, however MR-PFGE identified isolate types with varying frequency [1]. This information provided a robust knowledge on the structure of R. solanacearum populations indigenous to Kenya at the regional level and allowed the rational selection of isolates for development as biocontrol agents.

Representatives of these isolate types were mutated to a non-pathogenic form by (i) insertion of a DNA omega element into the Hrp gene cluster [2], or (ii) deletion mutation through the insertion and eviction of the sacB gene of Bacillus subtilus [3]. Inoculation of potato plants with the non-pathogenic omega mutants under growth-room conditions has been shown to reduce disease in the order of 30%. Preliminary findings indicate that this protection may operate through an induced host-resistance mechanism, opening up the possibility of synergistic host/strain interactions. Non-pathogenic sacB mutants are yet to be evaluated, but could have a significant advantage as these possess no exogenous DNA; a facet that may make the end product more marketable.

The potential of non-pathogenic omega mutants of R. solanacearum as biocontrol agents has been established previously on tomato affected by race 1 [2]; however, transfer of the technology to potato represents a challenging problem due to the soilborne nature of the crop. Ongoing studies aim to assess whether this protection operates under field conditions, and to evaluate the potential of applying the biocontrol agent on intensively managed small on-farm seed production plots that meet farmers' seed potato requirements.

References
1. Smith JJ, Offord LC, Holderness M, Saddler GS, 1995. Applied and Environmental Microbiology 61, 4263-4268.
2. Frey P, Prior P, Marie C et al., 1994. Applied and Environmental Microbiology 60, 75-3181.
3. Hynes MF, Quandt J, O'Connell MP, Pühler A, 1989. Gene 78, 111-120.