METHOD FOR THE PREVENTION AND/OR THE BIOLOGICAL CONTROL OF BACTERIAL WILT CAUSED BY RALSTONIA SOLANACEARUM, VIA THE USE OF BACTERIOPHAGES SUITABLE FOR THIS PURPOSE AND COMPOSITIONS THEREOF

Abstract

A method is for prevention and/or biological control of wilt caused by Ralstonia solanacearum, by use of suitable bacteriophages. In addition a method uses the structural characterisation, genome sequence and activity of three specific lytic bacteriophages of R. solanacearum. Podovirus presents an elevated stability between 4 C. and 30 C. in an aqueous medium in the absence of a host. As a result of the high level of stability, lytic activity, elevated specificity towards R. solanacearum and the absence of activity against the microbiota associated with the plants to be protected, bacteriophages are used for the biological control of R. solanacearum in river courses and irrigation water, as well as in a method for preventing and/or controlling the wilt produced by the bacteria, in which at least one of the bacteriophages, or combinations thereof, are delivered to the plants and/or the soil in the irrigation water.

Claims

1. A bacteriophage capable of lysing cells of Ralstonia solanacearum selected from the group consisting of a) vRsoP-WF2 (DSM 32039), vRsoP-WM2 (DSM 32040), vRsoP-WR2 (DSM 32041), or b) a podovirus whose genome has the sequence of SEQ ID NO: 1 (corresponding to vRsoP-WF2), SEQ ID NO: 2 (corresponding to vRsoP-WM2) or SEQ ID NO: 3 (corresponding to vRsoP-WR2).

2. (canceled)

3. A composition comprising at least one of the bacteriophages of claim 1, or combinations thereof.

4. The composition of claim 3 comprising one of the following combinations of bacteriophages: a) vRsoP-WF2 and vRsoP-WM2; b) vRsoP-WF2 and vRsoP-WR2; c) vRsoP-WM2 and vRsoP-WR2; d) vRsoP-WF2, vRsoP-WM2 and vRsoP-WR2.

5. Composition according to claim 4, wherein each of the bacteriophages of the combination is present in the same concentration.

6. (canceled)

7. Composition according to claim 3, wherein the total concentration of bacteriophages capable of lysing R. solanacearum cells varies between 10.sup.2 and 10.sup.9 plaque forming units per millilitre (PFU/ml).

8. (canceled)

9. Composition according to claim 3, comprising an agronomically acceptable carrier and/or excipient.

10. Composition according to claim 3, further comprising a chemical agent for the control of R. solanacearum or a biocontrol agent of R. solanacearum different from: a) vRsoP-WF2 (DSM 32039), vRsoP-WM2 (DSM 32040), vRsoP-WR2 (DSM 32041), or b) a podovirus whose genome has the sequence of SEQ ID NO: 1 (corresponding to vRsoP-WF2), SEQ ID NO: 2 (corresponding to vRsoP-WM2) or SEQ ID NO: 3 (corresponding to vRsoP-WR2).

11. Composition according to claim 3, further comprising a biological control agent of R. solanacearum that is a lytic or lysogenic bacteriophage with activity against said bacteria.

12. Use of a method of using the bacteriophage of claim 1 or a composition comprising the bacteriophage, the method comprising controlling R. solanacearum in natural watercourses, channelled water streams, natural reservoirs of water, artificial water reservoirs, irrigation water and irrigation water reservoirs, which will be used to irrigate crops.

13. The method according to claim 12, wherein the bacteriophage of claim or the composition is added to a natural reservoir of water or to an artificial water reservoir, and wherein the water in the reservoir is maintained at a temperature between 4 C. and 30 C., or the average water temperature in the reservoir is between 4 C. and 24 C., both included.

14. (canceled)

15. (canceled)

16. The method according to claim 12, wherein the water pH is in the range of 6.5 to 9.0, both included.

17. Use of a method of using the bacteriophage of claim 1 or a composition for the control of comprising the bacteriophage, the method comprising controlling R. solanacearum in soil, by adding one or more bacteriophages or the composition to said soil via irrigation water with which the soil is irrigated, which is previously treated with one or more aforementioned bacteriophages or with the aforementioned composition.

18. A method for preventing or treating wilt caused by R. solanacearum in a plant, comprising the steps of: a) adding a composition of claim 3 to the water to be used to irrigate the plant; b) watering the plant with said water.

19. Method according to claim 18, wherein the water pH is in the range of 6.5 to 9.0, inclusive.

20. Method according to claim 18, wherein the water to which the composition has been added previously to irrigation, is maintained at a temperature between 4 C. and 30 C. or an average temperature of between 4 C. and 24 C., both included.

21. (canceled)

22. (canceled)

23. (canceled)

24. (canceled)

25. Method according to claim 18, wherein the irrigation system is a system of partial or total flooding, drip irrigation, subsurface irrigation via perforated pipes, by exudation via porous pipes, or spray irrigation.

26. Method according to claim 25, wherein irrigation is produced by partial flooding.

27. Method according to claim 18, wherein the plant is growing in a field, in a nursery, in a greenhouse or in hydroponics.

28. Method according to claim 18, wherein the plant is a species belonging to the Solanaceae family and susceptible to and/or tolerant of R. solanacearum or any other species susceptible to and/or tolerant of R. solanacearum.

29. Method according to claim 28, wherein the plant is selected from the group consisting of potatoes (Solanum tuberosum), tomatoes (Solanurn lycopersicum), sweet peppers (Capsicum annuum), aubergine (Solanum melongena).

30. Method according to claim 18, further comprising a prior step to the application of said method in which the irrigation water is subjected to one or more other strategies selected from the group consisting of: chemical, physical, and/or biological control for a same plant pathogen or others.

31. Method according to claim 18, comprising a step in which copper compounds, antibiotics and/or soil fumigants are applied to the soil where the plant is growing.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0065] FIG. 1 shows photographs of the culture medium dishes from the lytic activity tests on the bacteriophages isolated from river water against Ralstonia solanacearum. Darker areas correspond to areas of lysis and/or isolated plaques, which are bacteriophage breeding areas in the bacterial lawn, which allow the lysis of the bacteria to be observed in the culture medium, the massive growth of the aforementioned bacterium being seen in whitish and opaque areas.

[0066] FIG. 2 shows a photograph of a culture medium dish with bacterial lawn from strain IVIA 1602.1 of R. solanacearum on which lysis tests were performed. In each quadrant, the bacteriophage contained in the suspension added to the bacterial lawn is indicated; the location of the control quadrant without bacteriophages (upper left quadrant, marked with the name of the bacterial strain) is also indicated.

[0067] FIG. 3 shows a photograph of the bacteriophages of the present invention obtained by transmission electron microscopy after negative staining. It is observed that they present a non-enveloped, polygonal head (40 to 60 nm in diameter depending on the bacteriophage) and a short tail.

[0068] FIG. 4 is a photograph obtained after subjecting to electrophoresis the samples in which digestion of the DNA of bacteriophages vRsoP-WF2, vRsoP-WM2 or vRsoP-WR2 (as indicated at the top of the photograph) had been carried out with various restriction enzymes indicated above each column. The columns at the extreme right and left ends correspond to the pattern of molecular weights (M): phage DNA digested with HindIII.

[0069] FIG. 5 shows the area in which the sequence corresponding to the vRsoP-WM2 bacteriophage presents an insertion of 468 nucleotides to the sequences corresponding to the bacteriophages vRsoP-WF2 and vRsoP-WR2, as well as areas close to this sequence. The presence of a hyphen in a sequence indicates a position where a nucleotide is absent in the aforementioned sequence with respect to one of or both of the other sequences, such absence allowing continued alignment in the same area. In the lower line below the corresponding sequence lines, the presence of an asterisk indicates coincidence between the nucleotides situated at that position in the three sequences.

[0070] FIG. 6 shows the genomic organization of bacteriophages vRsoP-WF2, vRsoP-WM2, and vRsoP-WR2 as compared to bacteriophage T7. In various shades of grey or with weaves of parallel lines in different directions, the location of functional open reading frames (ORFs) that have been identified using the BLAST tool is indicated, as specified in the legends in the lower part of the figure. It is noted that the three bacteriophages possess a genomic organization and expression of the ORFs similar to bacteriophage T7 in part of the genome.

[0071] FIG. 7 shows the survival curves of bacteriophages vRsoP-WF2, vRsoP-WM2 and vRsoP-WR2, at incubated at 14 C. in the absence of host cells in water from the Tormes river (panel A, top) and the Turia River (panel B, bottom). Survival is expressed as the base 10 logarithm of the plaque forming units detected per millilitre (PFU/ml) in samples taken at the times indicated on the x/y graph.

[0072] FIG. 8 shows a graph of the lytic activity of the bacteriophage vRsoP-WF2 added to an initial concentration of 10.sup.3 plaque forming units per millilitre (PFU/ml) in sterile river water, to which 10.sup.6 colony forming units per millilitre (CFU/ml) of Ralstonia solanacearum were added. A decrease within time of the CFU/ml corresponding to the bacteria (expressed in the form of the base 10 logarithm, points indicated with a filled circle) and an increase in PFU/ml corresponding to the bacteriophage (also expressed as the base 10 logarithm, points indicated with a filled square) were observed.

[0073] FIG. 9 shows an illustrating scheme of the experimental procedure of the use of the bacteriophages of the invention on irrigation water developed by the present inventors for the ability to control bacterial wilt. At the bottom, photographs of the condition of the plants at the beginning of the test (time zero, top row of photographs) and after 1 month (1 month, bottom row of photographs) are shown for each of the combinations of R. solanacearum and the bacteriophage vRsoP-WF2 indicated.

[0074] FIG. 10 shows a bar graph in which reduction of bacterial wilt, expressed as a percentage, in two different trials in tomato plants are shown. Experiment (Exp.) 1, bacteriophage concentration: 10.sup.9 plaque forming units per millilitre (PFU/ml) and Exp. 2 bacteriophage concentration: 10.sup.6 PFU/ml. In both experiments, the concentration of bacteria was 10.sup.5 colony forming units per millilitre (CFU/ml). Vertical frame bars correspond to those plants treated only with bacteria, without bacteriophages; bars without weaving correspond to plants treated with bacteria and bacteriophage at the indicated concentrations; bars with horizontal weaving correspond to plants treated with bacteria and 1/10 dilutions of the aforementioned concentrations of bacteriophages.

[0075] FIG. 11 shows a bar graph that represents reduction of bacterial wilt caused by Ralstonia solanacearum, expressed as a percentage, in trials where tomato plants were watered with water containing the combinations of bacteria (RsoI) and bacteriophage indicated under the bars. The four cases located further to the right correspond to irrigation water with binary combinations (from left to right, vRsoP-WF2 with vRsoP-WM2, vRsoP-WF2 with vRsoP-WR2, or vRsoP-WM2 with vRsoP-WR2) or tertiary combinations (vRsoP-WF2, vRsoP-WM2 and vRsoP-WR2) of bacteriophages with the bacteria.

DETAILED DESCRIPTION OF THE INVENTION

[0076] As mentioned hereinbefore, the invention relates to novel specific bacteriophages of Ralstonia solanacearum (bacteriophages of the invention), the use of at least one of the bacteriophages of the invention, or combinations, for R. solanacearum control in natural watercourses, natural water reservoirs, irrigation water and irrigation water reservoirs, by adding one or more of these bacteriophages to the aforementioned water or reservoirs; also it refers to the use of these bacteriophages to control R. solanacearum in soil, by adding one or more of these bacteriophages to the aforementioned soil via treated irrigation water; and a method for preventing or controlling bacterial wilt caused by Ralstonia solanacearum in plants, a composition is added to water to be used for watering plants, the aforementioned composition comprising at least one of the bacteriophages known as vRsoP-WF2 (DSM 32039), vRsoP-WM2 (DSM 32040) or vRsoP-WR2 (DSM 32041), or combinations, and the aforementioned plants are watered with the aforementioned treated water.

[0077] In the present application, the word phage is used as an abbreviation for the word bacteriophage with the same meaning. Therefore, hereinafter the two terms will be used interchangeably. Bacteriophage refers to a virus capable of infecting bacteria, either by producing lysis (lytic cycle) or by inserting itself into the genome and replicating itself therewith without causing lysis (lysogenic cycle).

[0078] The bacteriophages of the invention have been isolated from river water from various regions of Spain, specifically Badajoz, Salamanca and the Alpujarras (Granada).

[0079] Morphological characterization by electron microscopy and molecular characterization by DNA restriction analysis have shown that the virions belong to the Podoviridae family (specifically, the genus of T7-like viruses), a family of which so far only one bacteriophage has been described with lytic activity against the species formerly known as Ralstonia solanacearum, i.e. the bacteriophage RSB1, described by Fujiwara et al (Fujiwara et al., 2011) and which has a larger genome than the bacteriophages of the present invention, whose genome does not exceed 41,000 base pairs (bp) in any of the three cases (see Table 2), while the RSB1 genome has a size of 43,077 bp. Furthermore, the three inventions of other authors mentioned in the present application, relating to the use of bacteriophages to control R. solanacearum refer to the species formerly known as Ralstonia solanacearum, and, where information is available (patent documents, scientific publications, etc.), it can be confirmed that the strains dealt with are mostly reclassified as the new species R. pseudosolanacearum.

[0080] Therefore, the bacteriophages of the present invention do not belong to one of the most common families of bacteriophages lytic for the species formerly known as R. solanacearum, i.e. Myoviridae, but rather to a different family. In addition, they appear to be part of the same species, different from other species of T7-like viruses described so far. The discovery of viruses belonging to a new species of bacteriophages which attack R. solanacearum is an unexpected event.

[0081] The genome of any of the three bacteriophages of the present invention appears to have recognition targets for PstI restriction enzyme, which does not result in digestion of them, which represents a difference with lytic bacteriophages of Japanese Patent JP4532959-B2, wherein they are digested by the enzyme.

[0082] Thus, in the present invention are provided first data to identify the three bacteriophages of the present invention and distinguish them from any known bacteriophage, such as the family to which they belong, genus, the assignment of all of them to a single species, the sequence of the genome and distinctive restriction profile, obtained with several enzymes after digestion of the genome (see Examples 1 and 2, and FIGS. 3, 4, 5 and 6). The isolation method used and a source of each is further described. Additionally, for clear definition , the deposit number issued by the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikro-Organismen and Zellkulturen GmbH is provided, as authority for international deposit under the Budapest Treaty for each of the bacteriophages.

[0083] In addition to the structural characterization (morphological characterization of the viral particles and genomic characterization), in the present application the functional characterization (physiological and lytic) of the isolated bacteriophage is also described. As noted previously, one of the important characteristics for a biocontrol agent to be effective is to be shown to act on a wide range of strains. As shown hereinafter in the Examples section of the present application, the data previously described by Alvarez et al (Alvarez et al., 2006a, Alvarez et al., 2006b) were confirmed for a bacteriophage that was known to have been isolated from a watercourse in Spain that had not been specifically identified and for which insufficient structural data had been provided to ascribe them to a family and, much less, to a genus and specific species. In particular, the lytic capacity was confirmed for 30 strains that, at the time, were all considered to belong to the same species, i.e. the species formerly known as R. solanacearum, the phylotype of which was unknown, and the pH and temperature ranges in which it showed activity: between 14 C. and 31 C., and a pH range of 6.5 to 8.2. These data correspond to the bacteriophage referred to in this application as vRsoP-WF2. It has been found that the other two bacteriophages of the present invention, vRsoP-WM2 and vRsoP-WR2 exhibit lytic capacity for the same strains of Ralstonia solanacearum, are active in the same ranges of pH and temperature, thus expanding the aforementioned characterization to the three bacteriophages of the present invention. The same results were obtained with mixtures of two of the bacteriophages (vRsoP-WF2 with vRsoP-WM2, vRsoP-WF2 with vRsoP-WR2, or WM2-vRsoP with vRsoP-WR2) or the combination of all three (vRsoP-WF2, vRsoP-WM2 and vRsoP-WR2). Thus, mixtures comprising combinations of these bacteriophages as well as the compositions comprising at least one of the bacteriophages of the present invention are a possible embodiment of the compositions of the present invention.

[0084] The specificity for Ralstonia solanacearum was also confirmed, no lytic activity being observed with bacteria isolated from river water with which tests were conducted, or with strains of other species of pathogenic plant bacteria. Again, these data are valid both for the three bacteriophages of the present invention and for combinations.

[0085] Therefore, the three bacteriophages separately, as well as combinations, fulfil the desirable characteristics for biological control agents such as high specificity by the host cell, and not posing a risk to the microbiota of water, soil or plants; being specific against Ralstonia solanacearum. Nor they pose a threat to the health of humans, animals or plants, being bacteriophages viruses that only infect bacteria. They are active also in a pH range compatible with the characteristics of different watercourses of Spain, and in a range compatible with the characteristics temperatures. This supports the use of bacteriophages of the present invention, individually or as combinations, and of compositions comprising such bacteriophages, to control R. solanacearum, whether in water from natural watercourses such as rivers, streams or creeks, natural reservoirs of water such as lakes, lagoons, ponds, springs and underground accumulations, artificial water reservoirs and dams, covered storage vessels, tanks, ponds (with or without surface covers), wells, irrigation water in general, or reservoirs of irrigation water as well as the aforementioned natural or artificial reservoirs.

[0086] In that sense, the field data collected by the present inventors on natural waters contaminated with R. solanacearum in different Spanish autonomous communities reveal that in the summer months (when the bacteria in water is detected and prohibits its use for irrigation) the highest daytime temperatures of these waters are between 13 C. and 17 C. and decrease at night. For example, in Salamanca and Guadalajara sampled water temperatures vary between 14 C. and 4 C. In addition, in countries in central and northern Europe with environmental water contaminated with R. solanacearum temperatures are lower in the summer months. Therefore, the range of activity observed for the bacteriophages of the present invention is compatible with use in natural watercourses, particularly in Spain. So is the pH range of action. However, it is not easy to add bacteriophages to a river watercourse in sufficient amount to achieve effective control of microorganisms therein, especially in the particular place where this water will be drawn for irrigation, as bacteriophages will be very diluted and will be carried along watercourse so that although their survival time is very high, such use does not favour bacteriophages to come into contact with the host cell in the section of watercourse that may be of interest, unless bacteriophages are used in short watercourses and/or watercourses with reduced flow, such as rivulets, brooks and artificial pipes, especially those leading to a reservoir where the water will stay for a longer time. Therefore, it is preferred that use should take place in a natural or artificial water reservoir, such as lakes, lagoons, ponds, streams, reservoirs, covered vessels, tanks, ponds (with or without surface covering) or wells. In them, if contamination with R. solanacearum is suspected, it is easier to estimate the extent of such contamination and determine the amount or concentration of bacteriophages to add depending thereon. In any case, it is preferred that the water in the reservoir should be maintained at a temperature in the temperature range of 4 C. and 30 C. inclusive, interval in which the bacteriophages of the present invention survive prolonged periods, keeping their lytic activity, both alone and as part of compositions containing at least one of them. This temperature range also comprises the ambient temperatures of survival and/or multiplication of the pathogen Ralstonia solanacearum, i.e. the environmental range of 4 C. and 24 C., so that the lytic activity of the bacteriophages of the present invention is effective at the temperatures that such bacteria presents a real threat of development of disease in crops. Since temperatures approaching 30 C. are not common in reservoirs of environmental water, and taking into account fluctuations in daily and seasonal environmental temperature, conditions where the average water temperature in the reservoir is between 4 C. and 24 C. inclusive are preferred.

[0087] It should be noted that the tests described in Example 4 of the present application confirm the applicability of bacteriophages of the present invention via irrigation water and the usefulness in reducing damage caused by R. solanacearum wilting in plants. That is why the present invention also provides a method for preventing or controlling wilt caused by Ralstonia solanacearum in a plant, comprising the steps of adding to the water be used to water the plant a composition comprising at least one of the bacteriophages of the present invention, or combinations, and watering the plant with the aforementioned treated water.

[0088] The proposal of pre-treatment of irrigation water before use for irrigation is an option not considered in previous studies of Japanese authors discussed hereinbefore and is an option of great interest, since the main crops affected by R. solanacearum are irrigated crops. Thus, although it is compatible with the invention that plant cultivation may be carried out in any of the conditions under which cultivation is possible, a possible embodiment of the invention which may be very important use on plants growing in a field, in a nursery, in a greenhouse or any other type of substrate, or hydroponics, where it can be easy to plan and implement the method of the invention within the irrigation system and also do so in ways that benefit many plants simultaneously. Moreover, but perfectly compatible with the aforementioned embodiment, within the possible application to any crop of a species susceptible and/or tolerant to R. solanacearum, one embodiment of the invention of great interest is that in which the plant is a species belonging to the family of the Solanaceae (Solanaceae family) and in particular one in which the plant is selected from among tomatoes (Solanum lycopersicum), potatoes (the two crops most frequently affected) (Solanum tuberosum), sweet peppers (Capsicum annuum) or aubergines (Solanum melongena). The application of the method of the invention is perfectly compatible whether the plant is in a growing area dedicated to plants of a single species, or growing areas where there are plants of different species, usually with specific sections for each, as is the case of traditional orchards, usually with an irrigation system common to them all and a common irrigation water reservoir. The characteristics of the bacteriophages of the present invention allow for individual application (plant by plant), as is the case with the applications proposed by Japanese authors and with other biocontrol agents is not necessary, to be unnecessary. Irrigation can be performed by any known system, such as traditional systems of partial or total flooding, drip irrigation, subsurface irrigation via perforated pipes, by exudation via porous pipes, or spray irrigation.

[0089] As discussed above, it is desirable that, prior to irrigation, the water which the composition with one or more bacteriophages of the present invention will be added to should be maintained at a temperature in the range of 4 C. and 24 C. which can be considered a usual environmental range, although, as bacteriophages of the invention are active up to a temperature of 31 C., this range can be extended to the range of 4 C. and 30 C. inclusive, although the latter value is unusual in environmental water reservoirs. As discussed above, conditions where the average water temperature in the reservoir is between 4 C. and 24 C. inclusive are considered suitable, given the daily and seasonal fluctuations of ambient temperature.

[0090] It is also desirable that the water pH should be in the range of 6.5 to 9.0 (both inclusive) to favour the lytic activity of the bacteriophage of the present invention.

[0091] For the same reasons discussed above, it is preferable that, prior to irrigation of the plant with water, irrigation water should stay in a reservoir, natural or artificial, from the time when the composition comprising one or more bacteriophages of the present invention is added; this approach is consistent with the addition of the bacteriophages when the water is not necessarily in such reservoir, but it is a watercourse that feeds or pours into the reservoir, especially when it is a pipe or a natural watercourse with a low flow rate that flows off of a natural watercourse with a high flow rate or a large reservoir, natural or artificial, such as a lake or a dam reservoir.

[0092] As for the reservoir itself, can be a storage vessel with or without surface coverage, including tank-type or pond-type; it can also be a natural accumulations of water, such as those that occur in the upwelling of certain springs, or artificial, natural or semi-natural wells as those formed in certain natural cavities, which are accessed by man at a later time.

[0093] On the other hand, it is important to note, as previously commented that one of the key points that determine the effectiveness of bacteriophages as biocontrol agents is their survival in the environmental conditions in which they are intended to be applied. In general, survival of the bacteriophages outside the host, as discussed previously, is extremely variable and depends on the particular nature of each bacteriophage, being strongly influenced by the surrounding environment, by conditions such as pH of the medium, temperature or sunlight (Iriarte et al., 2007). Because sunlight is a factor that often adversely affects the survival of bacteriophages under natural conditions, in considering the use in surface water, it is important for them to survive adequately in the presence of this factor. In this regard, the bacteriophages of the invention were isolated from different Spanish watercourses exposed to different levels of sunlight, unlike Japanese bacteriophages isolated from soil and plant material. Moreover, as bacteriophages of the invention were isolated from water samples in which host cells were present, the unexpected survival in water in the absence of host cells was unknown.

[0094] For all these reasons, survival of the bacteriophages isolated by the present inventors is considered an important factor, nearly a crucial feature that is an important advantage for use as a biocontrol agent in water. As described below in Example 3, the aforementioned survival was tested in the absence of the host cell in natural water from two Spanish rivers (the Tormes River in Salamanca and the Turia River in Valencia) of different chemical composition and pH, and at different temperatures. These two types of water show substantial differences in the main physico-chemical parameters referenced in the composition: specifically with the water of the Turia River values were comparatively 100 times higher in Mn, 10 times higher in Fe, between 5 and 10 times higher in chlorides and triple in nitrates; with water from the Tormes River values being approximately 4 times higher in phosphate; the average values of pH were around 8.13 in the water from the Turia River and 7.36 in the water from the Tormes river. Temperature ranges of water ranged from 3.5 C. to 20.9 C. for the Tormes River and from 11.5 C. to 22.0 C. for the water from the Turia River, i.e. temperature ranges for both environmental waters are within the temperature values used for testing survival of the bacteriophages, which were: 4 C., 14 C. and 24 C., and the pH values were 7.2 for water from the Tormes River and 8.1 for the water from the Turia River. Of these rivers, the Tormes River is contaminated with R. solanacearum and the use of the water is prohibited for irrigation, while contamination has not been observed in the Turia River so far. For tests of the present invention, the aforementioned natural water was filtered through a 0.22 n filter and sterilized, so that survival tests were performed in the absence of the host. As demonstrated in Example 3, the three bacteriophages of the present invention were active and at high levels of lytic activity for more than 5 months, longer than three months considered good survival period for bacteriophages of aquatic bacteria that affected fish and which are therefore in their natural environment (Pereira et al., 2011). This high survival was observed at the three temperatures tested (4 C., 14 C. and 24 C.), which were intended to cover the environmental range of interest for use in watercourses and natural reservoirs of water, artificial reservoirs and irrigation water. Subsequently, as mentioned in Example 3, the test was continued, and it was found that, after 3 years in natural water, they remain active. This long period of survival with maintenance of lytic activity is unexpected and surprising, particularly for a lytic bacteriophage of R. solanacearum because it is not a native bacteria from aquatic environments, but rather its natural environment is the xylem of plants and often the ground, and it was not expected that this bacteriophages specific of such bacteria would present a high survival rate in water outside the host. In fact, in the studies of Fujiwara et al (Fujiwara et al., 2011), for example, stability was monitored only for 15 days, in which clear differences were observed among the stability of the three bacteriophages tested, more pronounced in a buffer than in the presence of soil, with marked differences observed in the survivability of the two bacteriophages of the Myoviridae family, RSL1 and RSA1.

[0095] As discussed previously, survival outside the host varies greatly between different bacteriophages, even among those belonging to the same serotype/genotype (Brion et al., 2002) or even among those who share a common natural habitat such as water (Pereira et al., 2011), habitat in which survival of aquatic bacteriophages of at least three months previously was considered a suitable characteristic for selecting good candidates for the control of bacterial fish diseases transmitted via water. Thus, the survival of the bacteriophages isolated by the present inventors was not predictable at all, especially considering that not even the family to which they belong was known and a high survival rate in water was not expected, since the usual habitat of its host are plants and soil, not water.

[0096] Therefore, the survival results in the absence of the host are noteworthy of the bacteriophages of the invention obtained at 24 C. because the previous results of the present inventors in survival studies of R. solanacearum in which lytic activity of other bacteriophages was tested in the presence of the aforementioned host bacteria indicated that the temperature of 24 C. promotes the rate of disappearance of the pathogen with respect to a temperature of 14 C. (Alvarez et al., 2007). However, at a temperature of 14 C., the bacteriophages of the invention maintain lytic activity on the host in natural water even after three years of the absence, and it has been observed that, in conditions similar to natural conditions, studies by the present inventors with other bacteriophages of this pathogen, lysis also causes a significant reduction of the populations of this bacterium (Alvarez et al., 2007). In addition, 14 C. is a temperature closer to those recorded in most aquatic habitats where the pathogen has been detected in Spain and other European countries.

[0097] Moreover, it is noteworthy that it is common conserve biocontrol agents, prior to their use, at low temperatures, preferably at 4 C., but they can also be stored at 14 C., as well as 24 C. Therefore, it is noteworthy that the bacteriophages of the present invention remain active and at high levels at all three test temperatures for more than 5 months and the survival with lytic activity can be as long as a period of three years.

[0098] Therefore, a particularly novel feature of the bacteriophages of the present invention is the survival for more than 5 months in natural water in the absence of the host cell. This is an adequate and very advantageous feature for a biological control agent, which must have features that enable it to survive in the medium in which it is intended to be applied, in this case, water.

[0099] As a result, it is compatible with the use of the method and use of the present invention that the composition containing bacteriophages should be maintained during storage and/or use, preferably at a temperature in the range from 4 C. to 24 C., inclusive, which can be considered a regular environmental range, however, since bacteriophages of the invention are active up to 31 C., this range may extend to a range from 4 C. to 30 C. inclusive, despite the latter value not being usual in environmental water reservoirs. As discussed above, an average temperature of the water in the reservoir from 4 C. to 24 C. inclusive, given the daily and seasonal fluctuations of ambient temperature, is also considered to be a suitable condition. This enables the compositions of the present invention to be easily preserved for a long time prior to their use in the form of suspensions in which the bacteriophages are in an aqueous vehicle which can be water (environmental, natural, distilled, previously sterilized, or subjected to another usual treatment for aqueous vehicles) or an aqueous solution (such as sterile saline, phosphate buffered saline, etc.) and ready to use and apply directly where needed. Therefore, the compositions of the present invention may comprise any carrier or excipient agronomically acceptable, and may be in liquid form, e.g. as an aqueous suspension, which can be prepared in water or in an aqueous solution and/or dilutions. In this way, they can be used to control R. solanacearum and can be applied with the method of prevention or treatment of wilt caused by the aforementioned bacteria and can be therefore ready for direct use from the stored and marketed form.

[0100] The high survival rate, with maintained lytic activity on the host, of the bacteriophages of the present invention, favours the use in the field because they can be transferred directly via water, a natural and simple way, without encapsulating or adding other physical, chemical and/or biological mediums to protect their viability until coming in contact with the target cell. This facilitates the production process, lowers costs and eliminates the need for complex formulations for implementation as well as the addition of chemicals to the environment. Thus, the high survival rate of the bacteriophages in water in the absence of the target cell reduces the implementation costs by decreasing the number of uses required over time, and increases long-term product efficiency in the agricultural systems where they are intended to be applied, and can thus more effectively prevent outbreaks of disease caused by R. solanacearum. All this leads to a product with more added value for farmers and nursery keepers, the major potential consumers of the aforementioned product, i.e. the bacteriophages of the present invention and/or compositions comprising them.

[0101] Moreover, this high survival in natural water while in the extracellular state facilitates combination with other control strategies (chemical and/or physical, and even biological) for the same plant pathogen or others, which may be an additional optional step of the method of the present invention. The method of the present invention is also compatible with the use of copper compounds, antibiotics and/or soil fumigants, whose application to the soil where plant is growing can also be considered an additional optional step of the method of the present invention.

[0102] The present invention is also compatible with additional use not only an agent of chemical or physical control, but rather as one or more additional biological control agents other than any of the bacteriophages of the present invention (other microorganisms such as bacteria, fungi and other bacteriophages, etc.). One possibility is any of the lytic or lysogenic bacteriophages previously known, which have activity against the aforementioned bacteria. For use, the additional agent may be further comprised in a composition of the present invention, or may be applied separately.

[0103] Although the preferred form of the compositions of the present invention is the liquid form, in aqueous medium, especially when applied to water to control R. solanacearum and/or preventing or reducing bacterial wilt caused by the aforementioned bacteria in plants which are to be irrigated with the aforementioned water, other forms of the composition are also compatible with the invention, especially those known to those skilled in the art for the conservation of bacteriophages, such as in a lyophilized form (which facilitates preservation at room temperature) or as a refrigerated and/or frozen aqueous suspension, preferably from 4 C. to 20 C., and even lower temperatures, such as 20 C. to 80 C.

[0104] As already mentioned, the compositions of the present invention may contain one of three bacteriophages whose isolation and morphological and genomic characterization is described in the present application (vRsoP-WF2, vRsoP-WM2 or vRsoP-WR2), or combinations (vRsoP-WF2 and vRsoP-WM2, vRsoP-WF2 and vRsoP-WR2, vRsoP-WM2 and vRsoP-WR2, or vRsoP-WF2, vRsoP-WM2 and vRsoP-WR2). The tests performed and described in Example 4 suggest that combinations, either two of or all three of the bacteriophages of the present invention are more effective than the use of the bacteriophages separately, so they may be a good choice for use against Ralstonia solanacearum in water to be treated, and particularly in water that is to be used for irrigation in order to prevent or reduce wilt caused by this bacteria. In compositions with combinations of several bacteriophages, each may be at the same concentration as in Example 4 of the present application, but different concentrations are also compatible with the invention.

[0105] Moreover, an advantage to consider of using the combination of two or more bacteriophages of the present invention is that mixtures prevent the appearance of strains of R. solanacearum that are resistant to the lytic action of any one of them.

[0106] Regarding the total concentration of bacteriophages in the compositions of the present invention, there are no limitations except those imposed for chemical reasons, resulting in the suspension being saturated and bacteriophages precipitating or settling. However, in practice, this is highly unlikely. One option is for the total concentration of bacteriophages of the invention to range between 10.sup.5 and 10.sup.9 plaque forming units per millilitre (PFU/ml), which are concentrations that have been tested in the Examples section of the present application and which can also be a suggested range of concentrations in order to choose the final concentration of bacteriophages desired to be present in the irrigation water. However, concentrations may be higher or lower than those included in that range, with concentrations of 10.sup.3 PFU/ml being able to be maintained and/or used as in section 4.1 of Example 4, or even lower, as the present inventors have obtained lysis data in liquid medium with bacteriophages of the present invention at concentrations of about 10.sup.2 PFU/ml. Thus, the range 10.sup.2 to 10.sup.9 PFU/ml or 10.sup.3 to 10.sup.9 PFU/mL are also possible concentration ranges of the compositions of the present invention or the conditions of action of the bacteriophages of the present invention, as well as other upper or lower limits, since the bacteriophages multiply inside the bacteria.

[0107] As far as the different bacteriophages of the present invention, under the envisaged use and/or maintenance conditions, survival data may lead to a preference for vRsoP-WM2, while macrotests carried out on plants described in Example 4, specifically on tomato plants, can lead to a preference for vRsoP-WR2, because in such tests a greater reduction in bacterial wilt was observed from applying this bacteriophage individually with respect to the other two bacteriophages of the present invention.

[0108] The invention will now be explained in more detail by the Examples and Figures described hereinafter.

EXAMPLES

Example 1

Origin and Isolation of the Bacteriophages

[0109] Lytic bacteriophages against R. solanacearum were isolated from several rivers of Castilla-Leon, Extremadura and Andalusia, in the vicinity of fields affected by bacterial wilt. A selection of these bacteriophages was purified and their lytic activity was tested in the laboratory against R. solanacearum, as shown in FIG. 1.

[0110] Among them, three bacteriophages (vRsoP-WF2, vRsoP-WM2 and vRsoP-WR2), from different origins, were chosen for further characterization.

[0111] VRsoP-WF2: isolated from the Tormes River in the vicinity of Salamanca.

[0112] VRsoP-WM2: isolated from the Cayo River in the province of Badajoz.

[0113] VRsoP-WR2: isolated from the Yator River in the area of the Alpujarras, in the province of Granada.

[0114] The three bacteriophages were purified via successive plaque passages in general LPGA medium (yeast extract [5g]-peptone [5 g]-glucose [10 g]-agar [20 g], dissolved in distilled water [1 litre]; the glucose is sterilized by filtration and subsequently added to the rest of the medium sterilized by autoclaving) with host cells of a standard strain of R. solanacearum (IVIA 1602.1 strain, deposited at the French Collection of Plant-associated Bacteria [CFBP] under CFBP number 4944 and at the DSMZ free access collection under the number DSM 100387). This method is also preferred for amplification of any of the aforementioned bacteriophages of the present invention on a solid medium.

[0115] Once purified, the lytic activity, pH and temperature range, and range of hosts of them were characterized as described hereinafter.

1.1. Temperature Range.

[0116] The lytic activity observed of the characterized bacteriophages against the selected strain of R. solanacearum (IVIA-1602.1) is observed between 14 C. and 31 C. in all three cases.

[0117] Within this range, the routine incubation temperature of the bacteriophages to multiply in the host, both on solid medium and in liquid medium, under laboratory conditions, is between 28-30 C., because they are the values that are considered optimal for the growth of R. solanacearum in these conditions.

[0118] Mixtures of two (vRsoP-WF2-vRsoP-WM2, vRsoP-WF2-vRsoP-WR2, vRsoP-WM2-vRsoP-WR2) or all three bacteriophages (vRsoP-WF2-vRsoP-WM2-vRsoP-WR2) were also tested. Each of the four mixtures of bacteriophages also showed activity in the same temperature range.

1.2. pH Range.

[0119] The lytic activity observed of the characterized bacteriophages against the selected strain of R. solanacearum (IVIA-1602.1) tested in different irrigation water, river water, canal water and lake water, was positive in all of the aforementioned types of water at pHs ranging between 6.5 and 9.0. Similarly, the mixtures of bacteriophages showed lytic activity in the same irrigation water and, thus, within the same pH range. The minimum and maximum pH values for the lytic activity of the bacteriophages have not yet been determined.

1.3. Salinity

[0120] Since it has been reported that R. solanacearum can grow in the presence of NaCl at concentrations of 1%, and even sometimes up to 2%, the lytic activity of bacteriophages in brackish water of different origins was tested, with salt concentrations of about 1.5%. Lytic activity was observed with all three bacteriophages. The four mixtures of bacteriophages of the invention mentioned in point 1.1 also showed lytic activity in the tested salinity conditions.

1.4. Visible Light

[0121] Since visible light can sometimes affect the lytic activity of bacteriophages, their activity was tested in conditions of light and darkness. It was observed that after 48 hours of uninterrupted exposure to intense light (approximately 15,000 lux), the lytic activity was similar to that observed in dark conditions, so the presence of light does not affect the lytic activity of the bacteriophages of the invention. Similarly, the different mixtures of tested bacteriophages had similar lytic activity both in the presence of light and in darkness.

1.5. Aeration

[0122] Since R. solanacearum is an aerobic bacteria and typically grows in liquid medium with stirring (aeration), the effect of the absence of aeration on the lytic activity was determined, as in field conditions aeration is not always assured (by example in tank storage). Activity was observed both in the presence and absence of stirring, of the same magnitude, being faster with aeration. Similarly, the mixtures of the bacteriophages showed lytic activity both with and without aeration.

1.6. Specificity

[0123] Specificity was tested in Petri dishes against bacterial lawns of R. solanacearum strains in the LPGA general culture medium, on which two drops of a suspension of each of the three bacteriophages (FIG. 2) were poured. The lytic activity was visualized by the appearance of areas of clearance formed by lysed bacteria in the bacterial lawn where drops of the suspensions with bacteriophages were placed (FIG. 2, corresponding to the test on R. solanacearum strain IVIA-1602.1).

[0124] According to experimental data, the lytic activity of the characterized bacteriophages was positive for 35 strains of R. solanacearum of different origins, hosts and years of isolation (Table 1). Among the aforementioned, 13 are international in scope and/or standard. The remaining are all strains isolated in Spain, belonging to the collection of the Valencian Institute of Agricultural Research (IVIA).

TABLE-US-00001 TABLE 1 R. solanacearum strains sensitive to the lytic action of the three bacteriophages of the present invention. STRAIN CODE COUNTRY OF ORIGIN HOST YEAR International Strains NCPPB.sup.a 1115 United Kingdom Potato 1961 (Ex Egypt) NCPPB 1584 Cyprus Potato 1963 NCPPB 2505 Sweden Potato 1972 NCPPB 2797 Sweden Solanum dulcamara 1974 BR 264 United Kingdom Solanum dulcamara 1993 Bordeaux 11-47 France Aubergine 1994 Nantes 9-46 France Tomato 1994 550 Belgium (Ex Turkey) Potato 1995 IPO-1609 Netherlands Potato 1995 Port 448 Portugal Potato 1995 W 12 Belgium Potato 1996 WE 4-96 United Kingdom River Water 1996 Tom 1 United Kingdom Tomato 1997 Strains from Spain IVIA.sup.b-1602.1 Canary Islands Potato 1996 IVIA-2049.53 Canary Islands Soil 1999 IVIA-2068.58a Canary Islands Potato 1999 IVIA-2068.61a Canary Islands Potato 1999 IVIA-2093.3.1 Canary Islands Potato 1999 IVIA-2093.5T.1a Canary Islands Potato 1999 IVIA-2128.1b Castile-Leon Potato 1999 IVIA-2128.3a Castile-Leon Potato 1999 IVIA-2167.1a Castile-Leon River Water 1999 IVIA-2167.2b Castile-Leon River Water 1999 IVIA-2528.A.sub.1-2 Castile-Leon River Water 2001 IVIA-2528.A.sub.3.1 Castile-Leon River Water 2001 IVIA-2528.54.A.sub.2 Castile-Leon River Water 2001 IVIA-2751.11 Extremadura River Water 2003 IVIA-2762.1 Extremadura Tomato 2003 IVIA-2762.4 Extremadura Tomato 2003 IVIA-3090.1 Andalusia Tomato 2005 IVIA-3090.5 Andalusia Tomato 2005 IVIA-3205.A.22 Castile-La Mancha River Water 2006 IVIA-3243 Andalusia Tomato 2006 IVIA-3359.9 Castile-La Mancha River Water 2007 IVIA-3359.10 Castile-La Mancha River Water 2007 .sup.aNCPPB: National Collection of Plant Pathogenic Bacteria, United Kingdom. .sup.bIVIA: Bacteria Collection of the Valencian Institute of Agricultural Research, Spain.

[0125] Strains from the NCPPB are available in this international collection. The other strains are available in the IVIA collection of phytopathogenic bacteria.

[0126] Specificity was also tested against other species of plant pathogenic bacteria and various bacterial isolates of river water to assess the possible impact of the isolated bacteriophages on the microbiota of natural water.

[0127] The lytic activity was negative for the 14 bacterial isolates of river water in the tests, which were selected from various water samples and presented different colonial morphologies from each other and with respect to the host. The activity was also negative for the 11 tested phytopathogenic bacteria strains belonging to other genera, demonstrating the specificity of the selected bacteriophages against Ralstonia solanacearum. The same results were obtained with the four possible mixtures of the aforementioned bacteriophages.

Example 2

Structural Characterization: Morphological and Molecular Characterization.

2.1. Morphological Characterization.

[0128] A study of the morphology of the selected bacteriophages was carried out via transmission electron microscopy of the viral particles after negative staining with phosphotungstic acid. It is observed that they present the characteristic morphology of the Podoviridae family: polygonal, non-enveloped heads 40 to 60 nm in diameter and short tails (FIG. 3). The bacteriophages of this family are also characterized by a genome of double-stranded DNA, a fact which was confirmed in the tests described below.

2.2. Molecular Characterization.

2.2.1. Extraction of the DNA of the Three Bacteriophages.

[0129] Concentrated capsid suspensions were obtained from the three types of bacteriophages from the corresponding bacterial lysates (filtered and treated with DNAse and RNAse to degrade the bacterial nucleic acids), by polyethylene glycol capsid precipitation protocol. After treatment of the aforementioned capsids with proteinase K, extraction of genomic DNA was performed after the addition of phenol, chloroform and isoamyl alcohol. After confirming that concentration and purity were adequate, the obtained DNAs were analysed by electrophoresis in agarose gel to verify the integrity as a preliminary step to the restriction analysis (see section 2.2.2) and purification for subsequent sequencing (see section 2.2.3).

2.2.2. Restriction Analysis of the Genomes of the Three Bacteriophages.

[0130] From the obtained genomic DNAs of the three bacteriophages, restriction analysis was carried out with various restriction enzymes, chosen to give a banding pattern belonging to T7 genus bacteriophages of the Podoviridae family. These enzymes were KpnI, ScaI, SpeI and XmnI. PstI was also tested because it is an enzyme used to cut the genome of bacteriophages of the species formerly known as R. solanacearum described in Japanese Patent JP4532959-B2 (publication number JP2005278513).

[0131] As shown in FIG. 4, the profile bands obtained with these five restriction enzymes is apparently the same for the three bacteriophages: complete digestion with XmnI and partial digestion with KpnI, ScaI and SpeI was observed, while no PstI digestion was appreciable. These results indicate the genetic proximity of the three bacteriophages to each other, and the difference from the bacteriophages of Japanese patent application JP4532959-B2, whose genomes themselves are cut by PstI.

2.2.3. Mass Sequencing of Genomic DNAs of the Three Bacteriophages and Bioinformatic Analysis.

[0132] From the resulting genomic DNA belonging to each of the three bacteriophages, we proceeded to the massive sequencing of the nucleotide bases and subsequent bioinformatic analysis and complete annotation of the genomic sequences found (SEQ ID NO: 1, corresponding to vRsoP-WF2; SEQ ID NO: 2, corresponding to vRsoP-WM2 and SEQ ID NO: 3, corresponding to vRsoP-WR2). This part was entrusted to the Valgenetics, S.L. (University of Valencia Science Park, Valencia, Spain).

[0133] The main findings were as follows:

[0134] The assembly of sequences obtained by massive sequencing yielded final sequences assembled with 100% fidelity, whose sizes are shown in Table 2.

TABLE-US-00002 TABLE 2 Size of the Genomic Sequences Obtained for Each Bacteriophage. SEQ ID NO: Bacteriophage Number of Base Pairs (bp) 1 vRsoP-WF2 40,409 2 vRsoP-WM2 40,861 3 vRsoP-WR2 40,408

[0135] The results indicated that each of the majority sequences included in the samples of SEQ ID NO: 1 (corresponding to vRsoP-WF2), SEQ ID NO: 2 (corresponding to vRsoP-WM2) and SEQ ID NO: 3 (corresponding to vRsoP-WR2) is readily identifiable as a complete genome of a bacteriophage belonging to the genus of T7-like viruses, which is the type species of enterobacteria bacteriophage known as T7 (enterobacteria phage T7), which belongs to the Podoviridae family.

[0136] Comparing the genomes of the three bacteriophages, it was showed that they were 99% identical over the whole of the genome. However, analysis of these sequences made obvious the presence of small genomic differences such as mutations, insertions and deletions distributed throughout the genome. These differences are higher in the sequence of SEQ ID NO: 2 (corresponding to vRsoP-WM2) than in the sequences of SEQ ID NO: 1 (corresponding to vRsoP-WF2) and SEQ ID NO: 3 (corresponding to vRsoP-WR2), which are almost identical. Thus, the sequence of SEQ ID NO: 2 contains an insertion of 468 nucleotides compared to the sequences of SEQ ID NO: 1 and SEQ ID NO: 3. FIG. 5 shows the sequence alignment extract corresponding to the area of the insertion. Small differences found in the nucleotide sequences indicated that bacteriophages vRsoP-WF2, vRsoP-WM2 and vRsoP-WR2 are different bacteriophages of the same viral species (Table 3).

TABLE-US-00003 TABLE 3 Comparison of the Sequences of the Genomes of Bacteriophages yRsoP-WF2, yRsoP-WM2 and yRsoP-WR2 Compared Pattern Sequence Sequence of the Comparison (SEQ ID NO:) (SEQ ID NO:) Coverage* Identity** 1 2 98% 99% 1 3 100% 99% 2 3 99% 99% *Homology between sequences of compared genomes, expressed as a percentage. **Nucleotides matching within areas of homology of the genomes compared, expressed as a percentage.

[0137] Additionally, using the BlastN and Blast2Seq analyses carried out with the tools accessible to the public via the website of the National Center for Biotechnology Information of the USA (http://www.ncbi.nlm.nih.gov/), it was found that the genomes of bacteriophages vRsoP-WF2, vRsoP-WM2 and vRsoP-WR2 exhibit some regions with high identity (about 70%) with bacteriophages: Ralstonia RSB1, Vibrio VP4 and, especially, Rhizobium RHEph01, all of them being T7-like bacteriophages (Table 4). These regions (corresponding to 5-23% of the entire genome of bacteriophages vRsoP-WF2, vRsoP-WM2 and vRsoP-WR2) belong to highly conserved regions.

TABLE-US-00004 TABLE 4 Comparison of the Sequences of the Genomes of Bacteriophages vRsoP-WF2, vRsoP-WM2 and vRsoP-WR2 with Several T7-like Bacteriophage Genomes. Compared Sequence Genome of the Virus Pattern (SEQ Sequence of the Comparison Coverage* ID NO:) (GenBank Access Number) Identity** 1 Ralstonia RSB1 (AB597179.1) 2% 84% 1 T7 (NC_001604.1) 5% 67% 1 Rhizobium RHEph01 19% 68% (JX483873.1) 1 Vibrio VP4 (NC_007149.1) 5% 70% 2 Ralstonia RSB1 (AB597179.1) 15% 66% 2 T7 (NC_001604.1) 5% 67% 2 Rhizobium RHEph01 23% 68% (JX483873.1) 2 Vibrio VP4 (NC_007149.1) 4% 70% 3 Ralstonia RSB1 (AB597179.1) 15% 66% 3 T7 (NC_001604.1) 5% 67% 3 Rhizobium RHEph01 22% 68% (JX483873.1) 3 Vibrio VP4 (NC_007149.1) 2% 70% *Homology between sequences of compared genomes, expressed as a percentage. **Nucleotides matching within areas of homology of the genomes compared, expressed as a percentage.

[0138] These results reveal that, except in these conserved regions within the T7-like bacteriophages, the genomes of bacteriophages vRsoP-WF2, vRsoP-WM2 and vRsoP-WR2 contain a highly divergent nucleotide sequence with respect to other bacteriophages deposited in GenBank. Therefore, these high differences in nucleotide sequence guarantee that bacteriophages vRsoP-WF2, vRsoP-WM2 and vRsoP-WR2 correspond to a new species within the genus of T7-like viruses.

[0139] Moreover, the identification of open reading frames (ORF) and characteristic features of the bacteriophages revealed that the sequences of the genomes of bacteriophages vRsoP-WF2, vRsoP-WM2 and vRsoP-WR2 have genomic organization and ORF expression that is mutually similar, and similar to T7-like bacteriophages in part of the genome (FIG. 6).

[0140] In summary, the three bacteriophages of the present invention are three isolates of the same viral species, being a new species classified as belonging to the genus T7 of the Podoviridae family, with organization very similar but distinct from the T7 bacteriophages deposited in Gen Bank (FIG. 6). The novel bacteriophages have different sequences to T7 bacteriophages, only resembling in some highly conserved areas, such as those related to replication and encapsidation.

Example 3

Survival of the Three Bacteriophages in Natural River Water

[0141] The survival of the three selected bacteriophages was tested in two different types of river water: Tormes, from Salamanca, and Turia, from Valencia, both in Spain. These two types of water show substantial differences in the main physico-chemical parameters analysed of the composition: specifically with the water of the Turia River values were comparatively 100 times higher for Mn, 10 times higher for Fe, between 5 and 10 times higher for chlorides and triple for nitrates; with water from the Tormes River, values were approximately 4 times higher in phosphate; the average values of pH were around 8.13 in the water from the Turia River and 7.36 in the water from the Tormes river. Temperature ranges of water ranged from 3.5 C. to 20.9 C. for the Tormes River and from 11.5 C. to 22.0 C. for the water from the Turia River, i.e. temperature ranges for both types of environmental water are within the temperature values used for testing survival of the bacteriophages, which were: 4 C., 14 C. and 24 C., and the pH values were 7.2 for water from the Tormes River and 8.1 for the water from the Turia River.

[0142] Surprisingly, it was observed that all the bacteriophages maintained their lytic activity against R. solanacearum after more than five months under these conditions, in the absence of the host since, prior to inoculation, the water had been filtered through a 0.22 m filter and autoclaved.

[0143] FIG. 7 shows graphics for the evolution of plaque forming units per millilitre (PFU/ml) of the bacteriophages in both the water from the Tormes River (panel A) and from the Turia River (panel B), in samples incubated at 14 C. It is observed that PFU/ml are maintained in the absence of Ralstonia solanacearum. The survival curves of the three bacteriophages samples kept at 4 C. and 24 C. were similar.

[0144] Thus, it is noteworthy that the three bacteriophages are active and have high lytic activity at all three temperatures tested for more than 5 months.

[0145] In addition, survival and maintenance of the lytic activity of all bacteriophages were confirmed at 4 C. and 14 C. for a period of time as long as three years. This result is important for conservation within this temperature range when such long storage periods are required.

Example 4

Biocontrol of Bacterial Wilt Caused by R. solanacearum

4.1. Ability to Control Bacterial Populations in Natural River Water.

[0146] Since the three bacteriophages of the invention had similar lytic activity at the different tested temperatures and pH values in natural water, initially one of them was chosen as a model (bacteriophage vRsoP-WF2) to perform biocontrol tests of bacterial wilt caused by R. solanacearum.

[0147] A bacteria-bacteriophage coinoculation test was carried out in sterile river water, in a closed system controlled in the laboratory, for simultaneous quantification of the population levels of both microorganisms over time. To do this, the bacteria was inoculated at a concentration of 10.sup.6 of colony forming units per millilitre (CFU/ml) in the liquid medium (sterile river water) and the bacteriophage was added at a concentration of 10.sup.3 plaque forming units per millilitre (PFU/ml). As shown in FIG. 8, it was confirmed that populations of the inoculated bacteria (reference strain IVIA-1602.1 of R. solanacearum) descended substantially in a few hours due to the lytic activity of the inoculated bacteriophages (bacteriophage vRsoP-WF2), the aforementioned pathogenic bacteria virtually disappearing after about 10 hours.

4.2. Tests of Biocontrol of Bacterial Wilt in Host Plants: Bacteriophage vRsoP-WF2.

[0148] The ability of the river water bacteriophage vRsoP-WF2 for biocontrol of the disease caused by R. solanacearum was tested in two independent experiments, watering plants of a susceptible host (tomato plants) with a concentration of the standard bacterial strain IVIA 1602.1 (10.sup.5 CFU/ml) and two different concentrations of the aforementioned bacteriophage (10.sup.6 and 10.sup.9 PFU/ml), and the decimal dilutions (10.sup.5 and 10.sup.3 PFU/ml, respectively), in conditions of optimum temperature and humidity for the development of the disease. The experimental procedure is shown in FIG. 9.

[0149] The results of both experiences are shown in FIG. 10. Overall, the disease incidence decreased to 0-5% in plants irrigated with the pathogen and the bacteriophage, in independent experiments, while in the controls without bacteriophages wilt incidence was 25-50%.

4.3. Tests of Biocontrol of Bacterial Wilt in Host Plants: vRsoP-WF2, vRsoP-WM2, vRsoP-WR2, and their Combinations.

[0150] Similar to the experiments of biocontrol in plants carried out with the bacteriophage vRsoP-WF2 described in section 4.2, a macrotest was carried out, which could simultaneously study the ability for biocontrol of each of the three bacteriophages vRsoP-WF2, vRsoP-WM2 and vRsoP-WR2, separately as well as in combinations of two and a mixture of all three. This is considered a macroassay, since it is performed with a large number of plants requiring sufficient room for incubation and people qualified for carrying it out. In all cases, tomato plants, which are a host susceptible to the pathogen, were tested, wherein approximately 35 plants per experimental condition were inoculated, which is approximately 315 plants. Inoculated plants were kept in a climatic chamber of suitable size, in day/night cycles of 16 hours of light at 26 C. and 8 hours dark at 22 C. and a humidity of about 70%, in conditions of biological containment, in a BSL3 laboratory. In the cited macroassay, the concentration of R. solanacearum (strain IVIA 1602.1) in irrigation water was 10.sup.5 CFU/ml, while the total concentration of bacteriophages was 10.sup.7 PFU/ml in all the tested experimental conditions.

[0151] The graph of FIG. 11 shows the results obtained. These results indicate that:

[0152] The bacteriophage vRsoP-WR2 is the most effective of the three, resulting in a greater decrease of bacterial wilt when added to irrigation water at the same concentration as the other two bacteriophages.

[0153] Any mixture of the bacteriophages (either binary combinations, or the combination including the three) are more effective than the separate bacteriophages.

[0154] All these experiments demonstrate the lytic potential of the bacteriophages of the present invention, isolated at different places in Spain, for the biocontrol of R. solanacearum and, therefore, the applicability of the aforementioned lytic activity in both the treatment of environmental water for agricultural use that has been contaminated with the aforementioned pathogen, or other uses, such as in the prevention and/or control of the disease caused in the field. This biocontrol ability is especially important, since it is considered that there is currently no effective control methods available via soil or water. And, in this case, as previously discussed and demonstrated in the aforementioned experiments, the biocontrol agents provided by the present invention have the unexpected feature of a high survival rate in water under normal environmental temperatures in Spain, which it is an advantage for use on plants via irrigation water and for the control and prevention of the presence of R. solanacearum therein, and for easy and prolonged maintenance of the marketed forms of the bacteriophages of the invention prior to use. Such maintenance may take place in an aqueous medium for a long time without severe loss of lytic activity and not even require, prior to the application to water, pre-dilution of the bacteriophages or their mixtures with any kind of physical or chemical vehicle to facilitate the interaction with the target bacteria or to ensure the stability before coming into contact with it, so that applying bacteriophages to irrigation water, water streams or water reservoirs can be simply by pouring them into R. solanacearum contaminated water to be controlled.

Deposit of Microorganisms

[0155] The bacteriophages vRsoP-WF2, vRsoP-WM2 and vRsoP-WR2, with ability to lyse R. solanacearum cells, have been deposited in the German Collection of microbial cultures Leibniz-Institut DSMZ-Deutsche Sammlung von Mikro-organismen and Zellkulturen GmbH, Inhoffenstrasse 7B, 38124 Braunschweig, Germany, following the rules of the Budapest Treaty for the deposit of microorganisms for patent purposes, on the following dates and assigned the following access numbers (Table 5).

TABLE-US-00005 TABLE 5 Data on the Deposit of Bacteriophages in the DSMZ German Collection. Material Deposit Date Access Number vRsoP-WF2 15 Apr. 2015 DSM 32039 vRsoP-WM2 15 Apr. 2015 DSM 32040 vRsoP-WR2 15 Apr. 2015 DSM 32041

REFERENCES

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