Three new lytic bacteriophages were found to effectively control the pathogen Ralstonia solanacearum, a quarantine bacterium in many countries, and causative agent of bacterial wilt, one of the most important vascular plant diseases. Bacterial wilt management has been carried out with fluctuating effects, suggesting the need to find alternative treatments. In this work, three lytic phages were isolated from environmental water from geographically distant regions in Spain. They proved to specifically infect a collection of R. solanacearum strains, and some of the closely related pathogenic species Ralstonia pseudosolanacearum, without affecting non-target environmental bacteria, and were able to lyze the pathogen populations within a wide range of conditions comprising environmental values of water temperatures, pH, salinity, and lack of aeration found in storage tanks. The three bacteriophages displayed high efficiency in controlling R. solanacearum, with reductions of the bacterial populations of several orders of magnitude in just a few hours, and proved to be able to survive in freshwater for months at environmental temperatures keeping activity on R. solanacearum, pointing out their suitability for field application through irrigation. Concerning their biocontrol potential, they were effective in reducing high populations of the pathogen in environmental water, and bacterial wilt incidence in planta by watering with either one phage or their combinations in assays with more than 300 plants. This is the first report on effective R. solanacearum biocontrol by applying single or combined bacteriophages through irrigation water in conditions mimicking those of the natural settings. The three phages belong to the Podoviridae family and are members of the T7likevirus genus. They are the first isolated phages from river water with activity against R. solanacearum, showing the longest persistence in natural water reported until now for phages with biocontrol potential, and consistently being able to control the disease in the host plant under environmental conditions. Consequently, the use of these bacteriophages for the prevention and/or biocontrol of the bacterial wilt disease caused by R. solanacearum has been patented. Evidence provided reveals the suitability of these waterborne phages to be effectively considered as a valuable strategy within the frame of sustainable integrated management programs.

Survival strategies exhibited over 4 years by Ralstonia solanacearum phylotype (ph) II biovar (bv) 2 in environmental water microcosms were examined. The bacterium is a devastating phytopathogen whose ph II bv 2 causes bacterial wilt in solanaceous crops and ornamental plants. Outbreaks of the disease may originate from dissemination of the pathogen in watercourses, where it has to cope with prolonged nutrient limitation. To ascertain the effect of long-term starvation on survival and pathogenicity of R. solanacearum in natural water microcosms, survival experiments were conducted. Microcosms were prepared from different sterile river water samples, inoculated separately with two European strains of ph II at 10⁶ c.f.u. ml⁻¹ and maintained at 24 °C for 4 years. In all assayed waters, starved R. solanacearum remained in a non-growing but culturable state during the first year, maintaining approximately the initial numbers. Thereafter, part of the population of R. solanacearum progressively lost the ability to form colonies, and non-culturable but metabolically active cells appeared. During the whole period, the bacterium remained pathogenic on host plants and underwent a transition from typical bacilli to small cocci which tended to aggregate. Some starved R. solanacearum cells filamented and formed buds. Starvation response, viable but non-culturable state, morphological changes and aggregation have not previously been reported for this pathogen as survival mechanisms induced in oligotrophic conditions. The potential existence of long-starved pathogenic cells in environmental waters may raise new concerns about the epidemiology of bacterial wilt disease.

Bacterial wilt diseases caused by Ralstonia solanacearum, R. pseudosolanacearum and R. syzygii subsp. indonesiensis (former R. solanacearum species complex) are among the most important plant diseases worldwide, severely affecting a high number of crops and ornamentals. Difficulties of bacterial wilt control by non-biological methods are related to effectiveness, bacterial resistance and environmental impact. Alternatively, a great many biocontrol strategies have been carried out, with the advantage of being environmentally friendly. Advances in bacterial wilt biocontrol include an increasing interest in bacteriophage-based treatments as a promising re-emerging strategy. Bacteriophages against the bacterial wilt pathogens have been described with either lytic or lysogenic effect but, they were proved to be active against strains belonging to R. pseudosolanacearum and/or R. syzygii subsp. indonesiensis, not to the present R. solanacearum species, and only two of them demonstrated successful biocontrol potential in planta. Despite the publication of three patents on the topic, until now no bacteriophage-based product is commercially available. Therefore, there is still much to be done to incorporate valid bacteriophages in an integrated management programme to effectively fight bacterial wilt in the field.

The presence of Ralstonia solanacearum biovar 2 in the watercourses of European countries is increasing, but little is known about its ecology in aquatic habitats. The detection of this pathogen in 2000 in one Spanish river led us to study its population density at different locations on the river over a period of 3 years. During 2000 and 2001, the pathogen was recovered at low densities (10 to 80 CFU/ml) by direct plating on modified SMSA agar from water samples at 14°C or higher, but its isolation was usually unsuccessful at temperatures below 9°C. To monitor the pathogen's abundance in winter, we used two liquid selective media for enrichment (at 29 and 35°C) and compared them by using spiked river water samples: modified Wilbrink broth (MWB) was more efficient than modified SMSA broth for double-antibody-sandwich indirect enzyme-linked immunosorbent assay (DASI-ELISA) detection of R. solanacearum . Enrichment in MWB at both temperatures allowed us to recover R. solanacearum cells that were nonculturable on solid media up to 25 days after their entry into the viable but nonculturable state. When we applied this technique to water samples during the cold months of 2001 and 2002, we obtained the best detection results by the most-probable-number method after enrichment at 35°C with MWB. The enrichment protocol was combined with DASI-ELISA and validated by Co-PCR to detect both naturally and artificially starved and cold-stressed cells in water, which were still infective. Overall, the data from this study demonstrate the effects of temperature variation on the population and culturability of R. solanacearum cells on solid media and their survival at low temperatures.

Ralstonia solanacearum phylotype II biovar 2 causes bacterial wilt in solanaceous hosts, producing severe economic losses worldwide. Waterways can be major dissemination routes of this pathogen, which is able to survive for long periods in sterilized water. However, little is known about its survival in natural water when other microorganisms, such as bacteriophages, other bacteria, and protozoa, are present. This study looks into the fate of a Spanish strain of R. solanacearum inoculated in water microcosms from a Spanish river, containing different microbiota fractions, at 24°C and 14°C, for a month. At both temperatures, R. solanacearum densities remained constant at the initial levels in control microcosms of sterile river water while, by contrast, declines in the populations of the introduced strain were observed in the nonsterile microcosms. These decreases were less marked at 14°C. Lytic bacteriophages present in this river water were involved in the declines of the pathogen populations, but indigenous protozoa and bacteria also contributed to the reduced persistence in water. R. solanacearum variants displaying resistance to phage infection were observed, but only in microcosms without protozoa and native bacteria. In water microcosms, the temperature of 14°C was more favorable for the survival of this pathogen than 24°C, since biotic interactions were slower at the lower temperature. Similar trends were observed in microcosms inoculated with a Dutch strain. This is the first study demonstrating the influence of different fractions of water microorganisms on the survival of R. solanacearum phylotype II released into river water microcosms.

Ralstonia solanacearum causes bacterial wilt in numerous plant species worldwide. Although biovar 2 mostly affects solanaceous crops, identification of new hosts remains a matter of concern since there is still no clear-cut distinction between host and nonhost plants. In this work we provide data based on histological studies on the status of 20 plant species, most of them of potential interest in crop rotation. Plants were watered with a β-glucuronidase-expressing derivative of R. solanacearum biovar 2, and after a month of incubation, sections of roots and stems were analyzed to localize the pathogen on surface, in cortex and/or xylem. Depending on whether the xylem was colonized or not, plants were classified as hosts or nonhosts, respectively. Hosts generally affected in a few xylem vessels or occasionally in all xylem bundles were classified as tolerant. These included some cabbage, kidney bean, and rutabaga cultivars, and the weed bittersweet nightshade (Solanum dulcamara). Nonhosts were the cultivars tested of alfalfa, barley, black radish, carrot, celery, colocynth, fennel, fiber flax, field bean, field pea, horseradish, maize, and zucchini. However, barley and maize, though nonhosts, may act as reservoirs for the pathogen. The present work constitutes a basis for further studies on cropping systems in fields where R. solanacearum has been detected.