Procedure for the prevention and/or biological control of wilt caused by ralstonia solanacearum, thr

专利摘要:
Procedure for the prevention and/or biological control of wilt caused by ralstonia solanacearum, through the use of bacteriophages useful for this and compositions thereof. The structural characterization, genomic sequence and activity of three lytic bacteriophages specific for r. Solanacearum are reported. These are podoviruses, which, between 4ºc and 30ºc, have a high stability in aqueous medium in the absence of a host. Due to its high stability, lytic activity, high specificity for r. Solanacearum and the absence of activity against the microbiota associated with the plants to be protected, its use is proposed for the biological control of r. Solanacearum in river courses and irrigation waters, as well as a method for preventing and/or controlling the wilt produced by said bacterium in which at least one of said bacteriophages, or combinations of them, are added to the plants and/or soil in the irrigation water. (Machine-translation by Google Translate, not legally binding)
公开号:ES2592352A1
申请号:ES201530730
申请日:2015-05-26
公开日:2016-11-29
发明作者:Elena GONZÁLEZ BIOSCA；María Milagros López González；María Belén ÁLVAREZ ORTEGA
申请人:Universitat de Valencia；Instituto Valenciano de Investigaciones Agrarias IVIA；
IPC主号:

专利说明:

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In the specific case of the phytopathogenic agent R. solanacearum, whose habitats are host plants and soil, a biological agent that is supplied through water must have biological characteristics that allow it to survive in that environment, which is not the usual nor of the bacterium or its specific bacteriophages. If this survival proves to be prolonged and the bacteriophages maintain their lithic activity on the host after long periods in the water, their applicability in the field is also favored, since they can be directly transported through that natural and simple means such as water, without the need to encapsulate them or add other physical and / or biological supports to protect their viability until the encounter with the target cell. This high survival would also facilitate the preparation of your commercial presentation, which could be in an aqueous medium without requiring refrigeration conditions (or even lower temperatures) for maintenance.
However, it should be borne in mind that bacteriophages are obligate intracellular parasites and, as such, need the host cell for perpetuation. Since they are transported to this cell by different routes, depending on the types of bacteriophages and types of host cells, a survival time in the environment that allows them to contact the host cell is expected. It is known that this time can be variable between different bacteriophages, being necessary to study in each particular case. Thus, for example, there are appreciable variations in the survival of bacteriophages of the same serotype / genotype (Brion et al., 2002) or even among bacteriophages of pathogenic aquatic fish bacteria, whose natural habitat is water (Pereira et al ., 2011). In the latter case, a three month bacteriophage survival in water has been considered as good, accepting that bacteriophages that have greater water survival are good candidates for the control of bacterial diseases of fish in aquatic culture systems (Pereira et al., 2011).
It should be noted that R. solanacearum is a phytopathogenic bacterium whose natural environment is the xylem of plants and often the soil, but not water. Since it is not a native bacterium of aquatic environments, it is not expected a high survival in water of specific bacteriophages. In fact, none of the publications and patents mentioned above describe the viability and lithic activity of specific lytic bacteriophages of the species previously named R. solanacearum in environmental water in the absence of host cells.
And, nevertheless, it would be interesting to have specific lytic bacteriophages against R. solanacearum, particularly showing a broad spectrum of strains of said
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Therefore, the three bacteriophages separately, as well as the combinations thereof, meet the desirable characteristics for biological control agents such as high specificity for the host cell, not posing a risk to the microbiota of water, soil or plants, for being specific against R. solanacearum. Nor do they pose a threat to the health of humans, animals or plants, as they are bacteriophage viruses that only infect bacteria. They are also active in a pH range compatible with the characteristics of different river courses in the Spanish geography, and in a temperature range compatible with their characteristics. This supports the use of the bacteriophages of the present invention, individually or as combinations thereof, and that of the compositions comprising said bacteriophages, for the control of R. solanacearum, either in water of natural water courses such as rivers, streams or streams, natural water reservoirs such as lakes, lagoons, ponds, springs and underground accumulations, artificial water reservoirs such as reservoirs, covered deposits, tanks, ponds (with or without surface cover), wells, irrigation water in general and reservoirs of the same as the natural or artificial reservoirs mentioned above.
In that sense, the field data collected by the present inventors on the natural waters contaminated with R. solanacearum in the different Spanish autonomous communities reveal that in the summer months (which is when the bacteria is detected in water and its use is prohibited for irrigation) the highest daytime temperatures of these waters range between 13ºC and 17ºC, decreasing at night. Thus, for example, in Salamanca and Guadalajara the temperatures of the sampled waters vary between 14ºC and 4ºC. In addition, in the different countries of central and northern Europe with environmental waters 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 its use in natural water courses, in particular in Spain. So is its range of acting pH. However, it is not easy to add bacteriophages to a watercourse in sufficient quantity to achieve effective control of microorganisms in them, especially in the specific place where that water may wish to be used to irrigate, since bacteriophages will be very diluted and they will be transported with the water course along it, so that although their survival time is very high, it is not favored that the bacteriophages contact the host cell in the watercourse section that may be of interest, except that bacteriophages be added to short and / or low flow water courses, such as streams, streams and artificial pipes, especially those that lead to a reservoir where the water will have time
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test temperatures for more than 5 months, and that their survival with lithic activity reaches periods of three years.
Therefore, the bacteriophages of the present invention have a particularly novel feature as their survival for more than 5 months in natural water in the absence of the host cell. This is an appropriate and very advantageous feature for a biological control agent, which must have characteristics that allow it to survive in the environment in which it is intended to apply, which in this case is water.
Thanks to this, it is compatible with the application of the method and the use of the present invention that the composition containing the bacteriophages be maintained during storage and / or use, preferably, at a temperature in the range of 4 ° C and 24 ° C, both included, which can be considered a usual environmental range, although, since the bacteriophages of the invention are active up to 31 ° C, said range can be extended to the range of 4 ° C to 30 ° C, both included, although the latter value is not usual in reservoirs of environmental water. As discussed earlier, conditions where the average water temperature of the reservoir is between 4ºC and 24ºC, both values included, are also considered, given the daily and seasonal ambient temperature fluctuations. This facilitates that the compositions of the present invention can be easily maintained for a long time prior to application, in the form of suspensions in which the bacteriophages are in an aqueous vehicle that 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 when necessary. Therefore, the compositions of the present invention, which may comprise any agronomically acceptable carrier or excipient, may be in liquid form, for example in the form of an aqueous suspension, which can be prepared in water or in an aqueous solution and / or its dilutions . And in this same way they can be used for the control of R. solanacearum and applied with the method of prevention or treatment of the wilt caused by said bacterium, being able therefore to be ready for its direct application from its commercialization and storage form.
The high survival, with maintenance of the lithic activity on the host, of the bacteriophages of the present invention, favors their applicability in the field, since they can be directly transported through water, a natural and simple means, without the need to encapsulate them or to add other physical, chemical and / or biological supports
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CEPA CODE COUNTRY OF ORIGINGUESTYEAR
International strains
NCPPBa 1115 United Kingdom (Ex Egypt)Potato1961
NCPPB 1584 CyprusPotato1963
NCPPB 2505 SwedenPotato1972
NCPPB 2797 SwedenSolanum dulcamara1974
BR 264 United KingdomSolanum dulcamara1993
Bordeaux 11-47 FranceEggplant1994
Nantes 9-46 FranceTomato1994
550 Belgium (Ex Turkey)Potatonineteen ninety five
IPO-1609 HollandPotatonineteen ninety five
Port 448 PortugalPotatonineteen ninety five
W 12 BelgiumPotatonineteen ninety six
WE 4-96 United KingdomRiver waternineteen ninety six
Tom 1 United KingdomTomato1997
Spanish strains
IVIAb-1602.1 Canary IslandsPotatonineteen ninety six
IVIA-2049.53 Canary IslandsGround1999
IVIA-2068.58a Canary IslandsPotato1999
IVIA-2068.61a Canary IslandsPotato1999
IVIA-2093.3.1 Canary IslandsPotato1999
IVIA-2093.5T.1a Canary IslandsPotato1999
IVIA-2128.1b Castilla-LeonPotato1999
IVIA-2128.3a Castilla-LeonPotato1999
IVIA-2167.1a Castilla-LeonRiver water1999
IVIA-2167.2b Castilla-LeonRiver water1999
IVIA-2528.A1-2 Castilla-LeonRiver water2001
IVIA-2528.A3.1 Castilla-LeonRiver water2001
IVIA-2528.54.A2 Castilla-LeonRiver water2001
IVIA-2751.11 EstremaduraRiver water2003
IVIA-2762.1 EstremaduraTomato2003
IVIA-2762.4 EstremaduraTomato2003
IVIA-3090.1 AndalusiaTomato2005
IVIA-3090.5 AndalusiaTomato2005
IVIA-3205.A.22 Castilla la ManchaRiver water2006
IVIA-3243 AndalusiaTomato2006
IVIA-3359.9 Castilla la ManchaRiver water2007
IVIA-3359.10 Castilla la ManchaRiver water2007
aNCPPB: National Collection of Plant Pathogenic Bacteria, United Kingdom. bIVIA: Collection of Bacteria of the Valencian Institute of Agricultural Research, Spain.
NCPPB strains are available in this international collection. The rest of the strains are available in the collection of phytopathogenic bacteria of the IVIA.
The specificity was also tested against other species of phytopathogenic bacteria and various bacterial isolates from river water, to assess the possible impact of isolated bacteriophages on the microbiota of natural water.
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VP4 and, above all, Rhizobium RHEph01, all of them of the type of bacteriophages similar to T7 (T7-like) (Table 4). These regions (corresponding to 5-23% of the complete genome of the bacteriophages vRsoP-WF2, vRsoP-WM2 and vRsoP-WR2) belong to highly conserved regions.
Table 4. Comparison of the genome sequences of the bacteriophages vRsoP-WF2, vRsoP-WM2 and vRsoP-WR2 with genomes of several T7-like bacteriophages.
Sequence compared (SEQ ID NO :) Genome of the virus with the standard sequence of comparison (No. GenBank Access)Coverage*Identity**
one Φ Ralstonia RSB1 (AB597179.1)2%84%
one Φ T7 (NC_001604.1)5%67%
one Φ Rhizobium RHEph01 (JX483873.1)19%68%
one Φ Vibrio VP4 (NC_007149.1)5%70%
2 Φ Ralstonia RSB1 (AB597179.1)fifteen%66%
2 Φ T7 (NC_001604.1)5%67%
2 Φ Rhizobium RHEph01 (JX483873.1)2. 3%68%
2 Φ Vibrio VP4 (NC_007149.1)4%70%
3 Φ Ralstonia RSB1 (AB597179.1)fifteen%66%
3 Φ T7 (NC_001604.1)5%67%
3 Φ Rhizobium RHEph01 (JX483873.1)22%68%
3 Φ Vibrio VP4 (NC_007149.1)2%70%
* Homology between the sequences of the genomes compared, in percentage. ** Nucleotides matching within homology zones of the compared genomes, in percentage.
10 These results reveal that, except in these conserved areas within T7-like bacteriophages, the genomes of the bacteriophages vRsoP-WF2, vRsoP-WM2 and vRsoP-WR2 contain a nucleotide sequence highly divergent from that of the other bacteriophages deposited in the Genbank. Therefore, these high differences in nucleotide sequence guarantee that the bacteriophages vRsoP-WF2, vRsoP-WM2 and
15 vRsoP-WR2 correspond to a new species within the genus of T7-like viruses.
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with this pathogen, as in the prevention and / or control of the disease it causes in the field. This biocontrol capacity is especially important if one considers that there are currently no effective control methods in soil, nor available in water. And, in the present case, as previously discussed and demonstrated in the previous experiments, the biocontrol agents provided by the present invention have the unexpected characteristic of their high survival in water under usual environmental temperature conditions in Spain, which It is an advantage both for its application to the plants through irrigation water and for the control and prevention of the presence of R. solanacearum in it, as well as for the easy and prolonged maintenance of the commercialization forms of the bacteriophages of the invention prior to use. Such maintenance can take place in aqueous medium for a long time without severe losses of lithic activity and would not even require, prior to its direct application to water, the prior dilution of bacteriophages or their mixing with some type of physical or chemical support. that acts as a vehicle to facilitate its interaction with the target bacterium or that guarantees its stability until it is achieved, so that the application to irrigation water or water streams or reservoirs in which it is possible to control possible contamination with R. solanacearum could be, for example, by simple pouring on said water.
Deposit of microorganisms
The bacteriophages vRsoP-WF2, vRsoP-WM2 and vRsoP-WR2, with the ability to lyse R. solanacearum cells have been deposited in the German microbial culture collection Leibniz-Institut DSMZ-Deutsche-Sammlung von Mikro-organism und 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 have been assigned the following access number (Table 5).
Table 5. Bacteriophage deposit data in the German DSMZ collection.
Material Deposit dateAccess number
vRsoP-WF2 April 15, 2015DSM 32039
vRsoP-WM2 April 15, 2015DSM 32040
vRsoP-WR2 April 15, 2015DSM 32041
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权利要求:
Claims (1)
[1]
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JP2005278513A|2004-03-30|2005-10-13|Sanin Kensetsu Kogyo Kk|Bacteriophage having bacteriolytic action on ralstonia solanacearum|

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JP2007252351A|2006-02-24|2007-10-04|Hiroshima Univ|Ralstonia solanacearum infective bacteriophage|

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KR20120055801A|2010-11-24|2012-06-01|동아대학교 산학협력단|Bacteriophage having specific lysis activity for bacterial wilt pathogen in plant|

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JP5812466B2|2011-04-28|2015-11-11|国立大学法人広島大学|Bacterial wilt prevention agent and bacterial wilt prevention method|CN109136194B|2017-06-28|2021-08-24|菲吉乐科生物科技有限公司|Novel solanaceae ralstonia phage and composition and application thereof|

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CN108410825A|2018-04-20|2018-08-17|南京农业大学|A kind of bacteriophage cocktail and its application|

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CN112029789A|2020-08-03|2020-12-04|华南农业大学|Application of phage trp574 gene in reducing resistance of ralstonia solanacearum to phage|

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ES201530730A|ES2592352B2|2015-05-26|2015-05-26|Procedure for the prevention and / or biological control of wilt caused by Ralstonia solanacearum, through the use of bacteriophages useful for it and compositions thereof|ES201530730A| ES2592352B2|2015-05-26|2015-05-26|Procedure for the prevention and / or biological control of wilt caused by Ralstonia solanacearum, through the use of bacteriophages useful for it and compositions thereof|

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PCT/ES2016/070392| WO2016189180A1|2015-05-26|2016-05-24|Method for the prevention and/or the biological control of wilt caused by ralstonia solanacearum, via the use of bacteriophages suitable for this purpose and compositions thereof|

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EP16799402.9A| EP3305892B1|2015-05-26|2016-05-24|Method for the prevention and/or the biological control of wilt caused by ralstonia solanacearum|

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US15/576,798| US10508266B2|2015-05-26|2016-05-24|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|