Identification of Ralstonia Solanacearum in Kyrgyzstan's Potato Fields and the Possibility of Using Biocontrol Agents Against this Pathogen

Ralstonia solanacearum is a soil-borne pathogen that naturally infects roots. It exhibits a strong and tissue-specific tropism within the host, specifically invading and highly multiplying in the xylem vessels (Smith, 1896, Yabuuchi et al., 1995). It causes a wilt disease in more than 450 plant species of 54 botanical families across the globe (Allenet al., 2005, Agrios, 1997, Stevenson, 2001). Ralstonia solanacearum has been studied intensively both biochemically and genetically, and has long been recognized as a model system for the analysis of pathogenicity (Staskawicz et al., 2001). It is well adapted to life in soil in the absence of host plants (Granada and Sequeira, 1983), thereby providing a good system to investigate functions governing adaptation to such an ecological niche. Considering the genetic diversity among the strains responsible for wilting disease in different plants, the pathogen is now termed as Ralstonia solanacearum species complex (Genin and Denny, 2012). In a traditional way, this pathogen has been classified into five races with respect to their host specificity and six biovars according to their biochemical properties (French et al., 1995). 

The first signs of the disease are shown in the beginning of the flowering and tuber formation. Plants suddenly wilt; the leaves turn yellow, shrivel, and droop. The lower basal part of the stem softens and rots. A typical feature of brown rot is the splitting of the stems; the crosscut of them follows a drop of bacterial exudates. Subsequently, the bacteria penetrate into the stolon, then into young tubers, causing browning of the vascular ring. From sections of the affected vessels and tubers follows brown mucus (Daughtrey, 2003). Bacterial wilt occurs mainly in tropic, sub-tropic and warm temperature zones (Jaunet and Wang, 1999). However, this disease has spread to more temperate areas (Kim et al., 2003). 

Ralstonia solanacearum is a b-Proteobacterium, whose complete genome sequence was presented by analysis of strain GMI1000. The 5.8-megabase (Mb) genome is organized into two replicons: a 3.7-Mb chromosome and a 2.1-Mb megaplasmid. The genome encodes many proteins potentially associated with a role in pathogenicity (Salanoubat et al., 2002).

 As the disease is widely distributed, it has a wide host range and is mainly soil-borne; it is difficult to control with chemicals and cultural practices (Grimaultet al., 1993). Biological control strategies may either help development of alternative management measures or be integrated with other practices for effective disease management at the field level. Several microorganisms have been tried out with variable success for biological control of bacterial wilt (Shekhawat et al., 1993). Effective microorganisms (EM) are a mixture of beneficial microorganisms, which can increase the crop yield and also protect against plant pathogens (Higa, 1999, Lwin and Ranamukhaarachch, 2006). It is a mixed culture of photosynthetic bacteria, Azotobacter, Streptomyces, and Lactobacillus spp., which improve crop yield by increasing photosynthesis, nitrogen fixation, controlling soil diseases, and accelerating decomposition of lignin material in the soil (Hussain et al., 1993). Page | 146 International Journal of Environmental & Agriculture Research (IJOEAR) ISSN:[2454-1850] [Vol-2, Issue-5, May- 2016] Biocontrol of bacterial wilt by plant growth-promoting rhizobacteria provided disease control and increased yields in greenhouses (Guoet al.,2004, Lemessa and Zeller, 2007). Research on microbial antagonists, such as Candida ethanolica has shown promise for bacterial wilt control (Lwin and Ranamukhaarachch, 2006). Toyota and Kimura (1996) reported the suppressive effect of some antagonistic bacteria on R. solanacearum. Three antagonists isolated from soil (Bacillus megaterium, Enterobacter cloacae, Pichia guillermondii) and Candida ethanolica showed high potential for disease suppression and also increased fruit weight, biomass, and plant height (Nguyen and Ranamukhaarachchi,2010). 

In Kyrgyzstan, the potato (Solanum tuberosum) is a staple product for the population. Recently, farms in different regions have started to grow the varieties such as Picasso, Sante, and Nevskiy, which were imported from Russia and other countries of the world, besides local potato varieties. Approximately 32% of potato yields are lost every year due to viral, bacterial, fungal, and pest attacks to potato tubers and potato plants [An overview of the emergence and spread of major pests and diseases, 2011). Brown, slimy bacterial bacterios of potatoes (bacterial wilt, or wilt) caused by Ralstonia solanacearum potatoes (RS) is a relatively a new disease in the fields of Kyrgyzstan. There are still no data and records of the scientists and experts on the biology and distribution of this disease in the potato crop regions of Kyrgyzstan. There are suggestions that this bacterial disease was brought with imported planting material to Kyrgyzstan from neighboring countries. So, the disease has been found in Russia in 1999 by a quarantine inspection only in an area of 0.06 hectares, planted with the imported variety Sante, then the infestation of potato was found in many regions of Russia: in the Urals, Far East, and Western and Eastern Siberia (http://www.kartofel.org/bolezn/bacteria/bacwilt.htm). There is a particular threat to potato production (especially seed production) because of asymptomatic cases of these bacterial diseases, as apparently healthy tubers have a margin hidden (latent) infection and pose a threat to crops the following year. This makes it important to be able to identify the disease in the contaminated material. Still, the prevalence and host range of races and biovars of Ralstonia solanacearum is unknown in the potato-cultivated regions of Kyrgyzstan, but it is becoming increasingly clear that this species causes disease in the vegetation period and in storage after harvesting. Biological control of Ralstonia solanacearum is still in its research phase in Kyrgyzstan. The objective of this study was to distinguish the biovars of Ralstonia solanacearum by using biochemical and ELISA tests, PCR analysis, to determine the prevalence of pathogen races in commercial potato fields of Kyrgyzstan and to develop the biocontrol agents to reduce the harmfulness of latent infection of seed tubers.