MXPA00004913A - Biocontrol agents for control of root diseases - Google Patents

Abstract

Fluorescent Pseudomonas spp. are described which are effective for the control of diseases caused by one or more of Rhizoctonia, Gaeumannomyces graminis and Pythium. The subject biocontrol strains have a unique genotype as shown by a characteristic banding pattern, and exhibit root-colonizing ability which is characterized by both higher population density on roots and extended colonizing activity compared to known Gg-suppressive strains. A further property is the ability of a strain to duplicate the level of biocontrol obtained naturally in a take-all decline soil. Methods for isolation and identification of the strains and their use to control diseases caused by Gg are provided. Transgenic fluorescent Pseudomonas spp. which have a locus which encodes for the production of the antibiotic 2,4-diacetylphloroglucinol and have a biosynthetic locus which encodes for the production of the antibiotic phenazine-1-carboxylic acid stably introduced into the genome are also described.

Description

BIOCONTROL AGENTS TO CONTROL ROOT DISEASES Field of the Invention This invention relates to the biocontrol of plant root diseases, caused by one or more Rhizoctonia, Gaeumannomyces graminis and Pythium, using strains of the species Pseudomonas fluorescent (spp.) And particularly strains that have a unique ability to colonize the root of small grain crops and with a biological control activity of putrefaction and root diseases in small grain crops and grass patches on turf. The invention also relates to the isolation and identification of biocontrol strains containing a biosynthetic locus that codes for the production of the antibiotic 2,4-diacetyl-phloroglucinol and which have a unique genotype as can be seen by the banding pattern characteristic, and with strains that additionally have a biosynthetic locus that codes for the production of the antibiotic phenazine-1-carboxylic acid stably introduced into the genome. Also included in this invention are the methods for creating the transgenic strains and the application thereof for the control of root diseases of plants.

Background of the Invention Root diseases, caused by Rhizoctonia, Phythium Gaeumannomyces graminis, cause a major adverse impact on the production of large crops throughout the world, and result in large economic losses due to decreased yields of crops. . Pimple, root disease caused by soil transport fungus, Gaeumannomyces graminis (Ge) (especially the tritici variety (Ggt)), root rot Rhizoctonia, caused by Rhizoctonia solani, and R. oryzae, and Phythium root rot, caused by any of the various serious root diseases of small grain crops, for example, wheat, barley, triticale and rye, throughout the world. Rhizoctonia, a member of the fungi class basidiomycin, causes ^ * rot of the root and stem of most fibrous, food plants and ornamental throughout the world, including small grain crops, turf, asparagus, cañola, corn, beets, tomatoes, potatoes, peas, rice, beans, soybeans, strawberries, zucchini and cotton. Root rot in small grain crops, caused by Rhizoctonia, occurs throughout the Pacific Northwest of the United States, in Australia and South Africa, and potentially in all temperate regions of the world where small grains are grown, especially if they are grown with reduced or no tillage (direct sowing). Rhizoctonia root rot, caused by R. solani AG8, begins as brown, chancroid lesions on the seminal and crown roots that eventually cause ringing that breaks the roots. roots. Plants with roots severed by this disease atrophy and eventually die without forming heads. The disease tends to affect patchy plants and has given rise to other names, such as patch disease, purple patch, crater disease and barley atrophy. Of all small grain crops, barley is especially susceptible to R. solani AG8. Rhizoctonia oryzae infects the embryos of the germinating seeds, preventing the outbreak or limiting the formation of seminal roots to only one or two ™ when in fact healthy seedlings produce five or six seminal roots. These two species of Rhizoctonia, along with Rhizoctonia cerealis and possibly other Rhizoctonia species, are presented as different combinations, depending on the soil, cropping systems, weed management practices, and perhaps other factors not yet identified. The pathogenic complex Pythium spp., Of soil transportation, comprises a group of fungi that is among the most successful of all the microbial colonizers of agricultural soils. It is estimated that almost all the soil : Or that is grown in the world contains spores of at least one, two, three and up to ten species of Pythium. The species Pythium, member of the fungus class oomycetes, like Rhizoctonia, affects almost all food, fibrous and ornamental plants worldwide. Examples of these plants were provided above. The damage caused by Pythium to small grains initiates as infections of the embryo and the consequent poor emergence or acclimatization of the shrub, and continues as a destruction of the fine lateral radicles and the absorbent hairs of the root. Plants that have root rot by Pythium have the appearance of plants that lack fertilizer, since the disease limits the ability of roots to absorb the destruction of plants. fine radicles and absorbent hairs. There are several species of Pythium that have the ability to attack cereals, whether embryos germinated seeds, tips of roots and fine radicles, or all these delicate and normally immature or meristematic tissues. Diseases propagated from small-grain crops and turfgrass are caused by the soil transport fungus Gaeumannomyces graminis (Ge), a member of the ascomyeotin fungi class, and cause significant economic losses due to reduced yields of crops. Pimpanzee, a disease caused by Gaeumannomyces graminis, tritici variety (Gct) occurs in all areas of wheat cultivation in the world and is perhaps the most important root disease of wheat and associated small grains, worldwide. Symptoms of wheat blotch include longitudinal dark lesions on the roots; in severe cases, the whole root turns black with the disease of the fungi that immigrate to the crown of the wheat plant (where the roots of the crown originate) and to the shoots (stems). The plants of severely infected wheat are identified in the field by their white heads that appear when the infection of the crown caused by the fungus interrupts the passage of water to the upper parts of the plant, causing the plant to die prematurely. Losses in yield can be considerable, up to 50% of the potential yield of wheat. There are no resistant extirpators for the :? or wheat and the registered fungicides do not work consistently. In addition, farmers are increasingly encouraged to grow wheat with minimal or no tillage to reduce soil erosion. These practices increase the severity of pietin and other root diseases. Although wheat is especially susceptible to • • fungus of the pietin, many other Gramineae such as barley, rye and triticale can also become infected. Traditionally, pietin has been controlled by combining crop rotation and tillage. These practices reduce the potential of the pathogen inoculum. However, because prolonged rotations are not economically feasible and tillage contributes to soil erosion, the trend in production of cereals is directed towards lower tillage and two or three crops of wheat before an interruption. These two practices aggravate the pietin. There is no known source of genetic resistance in wheat against pietin, and chemical control methods are limited. The need for agriculture to be increasingly sustainable and less dependent on chemical pesticides has required the development of alternative approaches to control pest and other soil-borne diseases. Other Gg fungi, for example, the Gaeumannomyces graminis variety avanea (Gga) infects oats and grasses, and has been found to cause patch patches on grasses such as agrostis. The Gaeumannomyces graminis variety graminis (Ggg) infects certain grasses and is believed to cause rot on the crown pod in rice. ^ All agricultural lands have some degree of antagonism to Ggt and other pathogens that are transmitted through the soil. This is known as "general suppression" (N. Gerlagh, Netherlands Journal of Plant Pathology 74: (Supplement 2) 1-25 97 (1968) or "general antagonism" (D. Hornby, Annual Review of Phytopathology, Annual Reviews Inc., Palo Alto, CA (1983), pp. 65- 85). The general antagonism results from the overall microbial activity in a soil. In addition, a "specific" deletion (biological control) of Ggt, (known as declination of pietin) develops in certain circumstances that overlap with "general suppression" and that result in almost total control of pietin. The declination of pietin (TAD) is a natural biological control of pietin, defined as the spontaneous reduction of the disease and the increase in yield with a prolonged monoculture of small grain crops susceptible to Ggt, such as wheat and barley. TAD ^ was observed for the first time more than 50 years ago and is now recognized as a global phenomenon. The similarity of the TAD throughout the world is remarkable in view of the wide range of soil types, climates and agronomic conditions under which wheat, barley and other small grains are grown. Field studies have clearly indicated that the development of TAD obeys a consistent pattern everywhere, which requires a continuous cultivation of a small grain and the presence of the pneumonia pathogen. Factors such as soil type and harvest history seem to modulate only the degree and speed of development of the TAD. Despite the fact that the pietin eventually declines, most farmers abandon monoculture prematurely because the losses during the interval can be considerable. 15 However, once established, the TAD allows a recovery in yield and persists for as long as monoculture continues. The practical use of the TAD offers the potential of a natural biological control of the pietin. However, to do so, the mechanism (s) responsible for the TAD would need to be identified and applied. However, the investigation to the The date has been descriptive for the most part and the particular occurrence of the TAD has not been fully understood. A similar decline of the foot patch caused by Gga occurs on established turf. The TAD has been studied extensively in an attempt to determine the mechanisms responsible for the natural suppression of pietin. The most theories The common ones that have been exposed to explain this phenomenon include changes in the microbiological condition of the soil, the accumulation of antagonistic bacteria, changes in the pathogenicity and population of the fungus, and the presence of protective fungi (D. Hornby in "Declination of the Pietín: A Paradise for the Theorists ", Soil-borne Plant Pathogens, by B. Schippers and W. Gams, Academic Press, New York (1979), pp. 133-156, and D. Hornby, Annual Review of Phytopathology, Annual Reviews Inc., Palo Alto, CA (1983), pp. 65-85). Hornby reviewed these explanations and concluded that no single mechanism could explain TAD worldwide, and this view has been universally accepted by those working in the area of soil suppressors of diseases. 5 The most widely supported explanation for the TAD is based on the microbial interactions between the pathogen of pietin and the antagonistic specific microorganisms associated with the root (RJ Cook and AD Rovira, So / 7 Biology and Biochemistry 8: 269-273 (1976) ). Various types of evidence support a role for microbial antagonism in the suppression of Ggt. For example, the suppression capacity io can be transferred by incorporating a small amount (1-10% weight / weight) of TAD soil (suppressant) into a TAD floor conductor of the pietin. Likewise, the suppression capacity of a TAD soil is eliminated by pasteurizing the soil with humid heat (60 ° C, 30 minutes), by fumigating the soil with methyl bromide or by cultivating crops that are not host to the soil. pathogen. Studies of the microbial antagonism involved in the TAD have been concentrated in attempts to identify the specific microorganisms antagonistic to the Ggt and to transfer these organisms to the soil in order to reproduce the suppression. A wide variety of microorganisms have been tested given the idea prevalent that the specific strains responsible differ in the different TAD soils. Cook and Rovira, 1876, supra, originally formed the hypothesis that r between antagonistic microorganisms, Pseudomonas spp. Fluorescent has a key role in the TAD. U.S. Patent No. 4,456,684 describes the strains of Pseudomonas that suppress diseases caused by pincushion and others Gg mushrooms, as well as the methods for the selection and application of the strains. Many of the most effective strains produced the antibiotic, 2,4-diacetylphloroglucinol (Phl) (C. Keel et al., Applied and Environmental Microbiology 62: 552-563 (1996)). Phl is a phenolic metabolite with activity against a variety of bacteria, viruses and fungi, including the pathogen of pneumonia (reviewed in L. S. Thomashow and D. M. Weller, In: G. Stacey and N. T. Keen (eds.) Plant-microbe Interactions, Vol. I, Chapman & Hall, Ltd., London, pp. 187-236 (1996). J. M. Raaijmakers et al. (Applied and Environmental Microbiology 63: 881-887 (1997)) report that Pseudomonas spp. Fluorescent Phl producers were present in wheat roots grown in three TAD soils of the State of Washington (USA). In the TAD conductive soils collected in places close to TAD fields, the Pseudomonas spp. Phl producing fluorescents were not detected or detected at densities at least 40 times less than those of the TAD soils. Although the use of microbial biocontrol agents is a great promise as a practical means to control soil transport pathogens, all biocontrol agents published or patented for pest control have the disadvantages of inconsistent performance, are soil-specific, and do not they are able to duplicate the level of control consistently observed in a TAD soil. No microorganism tested to date has demonstrated the disease control capabilities expected from a strain involved in ADT. In this way, it is not surprising that no biocontrol agent has been marketed for pietin. What is required are effective biocontrol agents for the pietin, which duplicate the suppression capacity of a TAD soil, which are effective regardless of the type of soil and have a consistent performance.

Pathogens responsible for foot rot and Rhizoctonia root rot survive as hyphae or mycelium in the tissues of host plants colonized through their parasitic activities. The Pythium species survives in the soil as oospores or sporangia, thick-walled, produced from the nutrients that steal the plant through parasitism. Usually, all three diseases develop simultaneously in the same plants, although a root disease may predominate. Although the Pythium species is ubiquitous in agricultural land harvested for small grains, damage to small grains caused by the Pythium species, for example, reduction in seedling emergence and plant vigor, is greater in soils that they are kept moist, especially if the soils also have a high content of mud, and with pH values below 6.0. If voluntary cereals (plants that develop from seeds spilled or pulled by the harvester on the soil surface) are allowed to grow in the field after harvesting a crop only up to 1 or 2 days before planting the next harvest, and if it is sprayed then with a herbicide like glyphosate, (Round-up®, Monsanto), the weed will be controlled, but root rot by Pythium and root rot by Rhizoctonia will greatly benefit. If wheat is planted directly in the erect stubble of a previous crop of wheat, with the soil wetted by sprinkler irrigation or by allowing the soil to be covered with straw, the three root diseases will be favored. 10 Many diseases of wheat, barley and other crops are controlled by breeding crop varieties with resistance to pathogens. However, this approach has worked mainly for diseases of the leaves and not for diseases of the root of wheat, barley, triticale or rye. The only known source of resistance to foot rot and root rot by Rhizoctonia is found in a very distant diploid relative, Daysapyrum villosum, but so far no use of this source of resistance has been made due to the difficulty of gene transfer at a taxonomically distant distance. Currently, there is no commercial wheat, barley, rye or triticale in the world that has resistance to pestilence, root rot by Rhizoctonia or rot of the root by Pythium. m Methods available for the biological control of fungal pathogens in plants have included bacterial strains of the Pseudomonas species that have a specific activity of the pathogen. U.S. Patent No. 4,456,684 describes the Pseudomonas strains that suppress the disease caused by the pincushion and other fungi Gg. The studies of the microbial antagonism involved in the decline of the pietin, a natural biological control of the pietin, defined as the spontaneous reduction in the disease and an increase in yield with a prolonged monoculture of small grain crops susceptible to Ggt, such as wheat and barley, have concentrated on attempts to identify microorganisms Specific antagonists of the Ggt and transfer these organisms to the soil to reproduce the suppression. Many of the most effective strains produced the antibiotic, 2,4-diacetylphloroglucinol (Phl) (C. Keel et al., Applied and Environmental Microbiology 62: 552-563 (1996)). J. M. Raaijmakers et al. (Applied and Environmental Microbiology 63: 881-887 (1997)) report that Pseudomonas spp. Fluorescent Phl producers were present in wheat roots grown in three TAD soils of the State of Washington (USA). Although the use of microbial biocontrol agents is a great promise as a practical means to control soil transport pathogens, all biocontrol agents published or patented for pest control have the disadvantages of inconsistent performance, are soil-specific, and do not they are able to duplicate the level of control consistently observed in a TAD soil. U.S. Patent No. 4,647,533 reports strains of Pseudomonas that suppress diseases caused by Pythium. Strains of Pseudomonas bacteria inhibiting Rhizoctonia solani or Pythium ultimum have been reported in cotton. (See U.S. Patent No. 5,348,742 to Howell et al.). The Bacillus sp. L324-92 to simultaneously control the species Gaeumannomyces graminis, Rhizoctonia and Pythium (Kim et al., Phytopathology 87: 551-558 (1997)). However, no strain of Pseudomonas is known to be effective in controlling these three pathogens.

Objectives of the Invention We have isolated unique biocontrol agents to control diseases, such as those caused by the soil transport fungus Gaeumannomyces graminis (Gg) in small grain crops and on turfgrass. The invention encompasses unique strains of the spice Pseudomonas fluorescent (spp.) That suppress (inhibit the incidence of or reduce the incidence or severity of) diseases caused by Gg, such as pneumonia, at low doses, and have a much greater capacity to colonize the root in comparison with the capacity of any previous biocontrol agent of Gg. Likewise, the capacity of Root colonization and biocontrol activity of the strains are not affected by the type of soil. The biocontrol agents of the invention provide a biocontrol that is . { • consistently greater than that of all other known biocontrol agents for diseases caused by Gg, such as pneumonia. Additionally, the strains have the unique property of being able to double the level of biocontrol observed in the TAD soil. In a first embodiment of the invention, the biocontrol agents of the invention comprise biologically pure cultures of Pseudomonas strains. spp. fluorescent, containing a biosynthetic locus that codes for the production of the antibiotic 2,4-diacetylchlorchlorucinol, which have a unique genotype as demonstrated by a unique characteristic profile of Amplified Polymorphic DNA Random (RAPD), which present biocontrol activity at dosage levels of to 1000 times lower than the known pneumonic suppressor microorganisms so far, and that they have a much greater capacity for colonization of the root as evidenced by a population density of 10 to 1000 times greater and a prolonged colonization activity. We have found that illustrative strains of the invention have the ability to colonize roots at a population density that averages at least 105 colony formation units per grams of root, including the soil of the rhizosphere, for at least 7 successive growing cycles. This ability to colonize the root is unprecedented.

• ^ In addition, these new strains are not affected by the type of soil. The invention also comprises methods of isolation and identification of these unique strains. A protocol for screening bacteria is presented in FIG. 6, and the sieving method is described in detail below. A further aspect of the invention is the application of the unique strains, or compositions with these strains, for the biocontrol of plant diseases caused by Gaeumannomyces graminis. When used as seed, soil, irrigation treatment in ditches or flood, the unique strains of this The invention has the ability to suppress Gg under field conditions. The application of the strains to the seeds or to the soil showed an unprecedented duplication of the suppression of the pietin, equivalent to the natural decline of the pietin (see Table 1, below), which had never been shown before. Also, because the strains suppress pneumonia at low dosage levels, 5 the strain can be grown and applied at a cost of almost 10 to 1000 times lower than another biocontrol agent of the currently existing pest. Also, because the strains are responsible for the natural TAD, it is expected that they only need to be applied once in a field. All the other biocontrol agents of the pietin require repeated application. In accordance with this invention, one of its objectives is to provide unique strains of Pseudomonas spp. fluorescent that provide a biocontrol of diseases caused by Gg, and that is much greater than that of the known biocontrol strains. It is also an object of the present invention to provide biocontrol strains that have root colonization capacity (higher root population density and prolonged activity) that is greater than that of any other biocontrol agent known to the pest. This property is particularly valuable since it is known that for the suppression of root diseases, such as pietin, increases in root colonization result in a greater and more constant biocontrol activity. Another objective of the invention is to provide biocontrol agents, which are not affected by the type of soil, for the control of diseases caused by Gaeumannomyces graminis in small grains and in lawns. Yet another objective of the invention is to provide methods based on the RAPD analysis to select the strains of the invention, which have a characteristic banding pattern. Another object of the invention is to provide methods for biologically controlling the pincushion in small grain crops and patches in lawns using the strains of the invention and the agricultural compositions that form the strains.

Other objects and advantages of the invention will be apparent from the following description. In a second embodiment of the invention, we have produced biologically pure cultures of transformed strains of the Pseudomonas fluorescent species (spp.) That have a biosynthetic locus that codes for the production of the antibiotic phenazine-1-carboxylic acid stably introduced into the genome, and that suppresses the diseases caused by the Rhizoctonia pathogen of soil transportation. Optionally, the strains suppress diseases caused by Pythium or diseases caused by Gaeumannomyces graminis (Gg), in addition to diseases caused by Rhizoctonia, or have the ability to control the three diseases. The biocontrol agents of the second embodiment are obtained by stably introducing the biosynthetic locus for phenazine-1-carboxylic acid into the genome of a strain of Pseudomonas spp. fluorescent, hereinafter referred to as the strain of the first modality, which contains a biosynthetic locus that codes for the production of the antibiotic 2,4-diacetylchlorchlorucinol, has a unique genotype as evidenced by a unique characteristic profile of Random Amplified Polymorphic DNA (RAPD) , and presents a superior capacity for colonization of the root as discussed below. The screening of transformed strains to select those that have activity against the diseases caused by Rhizoctonia is carried out in bioassays in greenhouses. Optionally, to select transformed strains that are also effective against diseases caused by Gaeumannomyces graminis or Pythium, greenhouse tests can be carried out. An optional step prior to greenhouse classification biosensing is in vitro inhibition, isolation of Rhizoctonia by a transformed strain. Thus, the biocontrol agents of the second modality provide a biocontrol for diseases caused by Rhizoctonia or locally diseases caused by Pythium and by Gaeumannomyces graminis. We have discovered that the illustrative transformed strains of the invention have the ability to suppress root rot by Rhizoctonia at very low doses (102 units of colony formation (CFU) per seed). We have also discovered illustrative strains that additionally have the ability of the strain of the first modality to suppress other root diseases, such as those caused by Gaeumannomyces graminis or Pythium. In this way, the illustrative strains of the invention have activity against the three important root diseases - Rhizoctonia, Pythium and Gaeumannomyces graminis. A further aspect of the invention is the application of strains, or unique compositions of the strains, for the biocontrol of root diseases of plants. When used as seed, soil, irrigation treatment in ditches or flood, the unique strains of this invention have the ability to suppress diseases caused by Rhizoctonia under field conditions, and optionally can suppress diseases caused by Pythium and Gaeumannomyces graminis. Also, the biocontrol agents of the present invention may be used to prepare biocontrol mixtures containing at least one transgenic strain of the invention and including one or more different biocontrol strains, eg, the additional transgenic strains of this invention.; a strain of the first embodiment of the invention, and other biocontrol strains. The invention provides agricultural compositions comprising at least one biocontrol strain of the invention, which compositions are useful for controlling at least one root disease of plants. Other objects and disadvantages of the invention will be apparent from the following description.

Brief Description of the Drawings FIG. 1 is an image showing the banding patterns (RAPD) of strains P Fluorescens Q8rl-96, ML4.9-96, and L5.1-96. Lane 1 shows a 1-kb ladder as a reference. FIGS. 2-4 show the colonization capacity of the strain of the invention P. fluorescens Q8rl-96, compared to the P fluorescens strains M1-96, and Q2-87, (strains not according to the invention) in virgin soil of Quincy, virgin soil of Lind and in virgin soil of Moses Lake, respectively. FIG. 5 shows the colonization capacity of the strain of the invention P. fluorescens Q8rl-96 in virgin soil of Quincy, virgin soil of Lind and virgin soil of Moses Lake. FIG. 6 shows the physical map of the biosynthetic locus of phenazine, linked to the tac promoter. The arrows indicate the genes that encode the enzymes of I biosynthesis of phenazine and the direction of their transcription. The symbol Ptac represents the position and orientation of the tac promoter. FIG. 7 shows the physical map of the plasmid pUTKm:: phz. The diagonals indicate the biosynthetic genes of phenazine. The position and direction of determinant transcription for kanamycin resistance appear as an open box. The small boxes with "x" indicate the position of the ends of terminal Tn5 19-bp.

Detailed Description of the Invention Biocontrol Agents of the Invention. The biocontrol agents of the invention comprise at least one biologically pure strain of Pseudomonas spp. fluorescent. In a first embodiment of the invention, this strain has the following identification characteristics: the strain contains a biosynthetic locus that codes for the production of 2,4-diacetylchlorchlorucinol; it has a unique genotype as evidenced by a characteristic banding pattern described in detail below, and in FIG. 1; It suppresses diseases caused by Gg in oyster crops and in fields grown in fields. It has a root colonization capacity characterized by high population density and prolonged colonization activity, in comparison with known suppressor strains of Gg. (€ i) In the preferred embodiment, this strain has the following characteristics of identification: its biocontrol capacity (capacity to suppress the disease) and the colonization capacity of the root are not affected by the type of soil and / or suppresses Gg in small grain crops equivalent to a level of biocontrol obtained when the small grain crop is grown in a soil in a state of declination of the pietin, that is, it doubles the level of biocontrol observed in a soil with declination of the pietin. Examples of the strains of the first embodiment of the invention are the P fluorescens strains Q8rl-96, ML4.9-96 and L5.1-96. In a second embodiment of the invention, the biocontrol agents comprise at least one biologically pure strain of Pseudomonas spp. fluorescent, which has the following identification characteristics: the strain has a biosynthetic locus that encodes the production of the antibiotic phenazine-1-carboxylic acid stably introduced into its genome; it has the biosynthetic locus of the strain of the first modality and which codes for the production of 2,4-diacetylchloroglucinol; and suppresses diseases caused by the pathogen Rhizoctonia for ground transportation. Optionally, the strain suppresses the diseases caused by Pythium or the diseases caused by Gaeumannomyces graminis (Gg) in addition to the diseases caused by Rhizoctonia, or has a biocontrol activity (ability to suppress the disease) against the three diseases of the root. Examples of the strains of the second embodiment of the invention are the strains P. fluorescens Z30-97; Z32-97; Z33-97 and Z34-97. Characteristic Banded Pattern. Strains of Pseudomonas spp. fluorescent, biologically pure, of the first embodiment of the invention have a characteristic banding pattern. This profile can be identified by a forward. FIG. 1 shows the banding patterns (RAPD) of P fluorescens strains Q8rl-96, ML4.9-96, and L5.1-96, which are illustrative strains of the invention. Lane 1 shows a 1-kb ladder as a reference. As shown in FIG. 1, the bands shared by the strains are: 330 ± 20 bp; 600 bp ± 50 bp; 700 ± 50 bp; 800 bp ± 50 bp; 900 bp ± 50 bp and 1100 bp ± 60 bp. The band at 800 bp ± 50 bp is the most intense. For purposes of the first embodiment, a strain has the characteristic banding profile of the invention if it has bands at 600 bp ± 50 bp; 700 ± 50 bp; 800 bp ± 50 bp; 900 bp ± 50 bp, using the conditions described in Example 1, below. In a preferred embodiment, the strain also has bands at 330 ± 20 bp and 1100 bp ± 60 bp. An optional way to identify if a strain has the banding pattern characteristic of the first mode is to carry out a simultaneous comparison on an agarose gel using the strain P fluorescens Q8rl-96 and the test strain, and visually compare the bands approximately between the base pairs 600 ± 50 and 900 ± 50. If the profile of the test strain conforms to the profile of strain Q8rl-96 in this region, then the test strain has the characteristic banding pattern encompassed by this invention . An alternate way to identify if a transformed strain has the banding pattern characteristic of the first modality is to carry out a simultaneous comparison on an agarose gel using the illustrative strain of the first modality, P fluorescens Qdrl-96, the actual strain of the first embodiment, or an illustrative strain of the invention, such as Z34-97, and the test strain and visually compare the bands approximately between the base pairs 600 ± 50 and 900 ± 50. If the profile of the test strain conforms to the profile of the strain Q8rl-96 illustrative of the first embodiment, the actual parent strain or the illustrative strain of the invention in this region, then the test strain has the characteristic banding pattern that is encompassed by this invention. In the second embodiment of the invention, the transformed strain preferably has the following additional identification characteristics: it has at least four of the characteristic bands of the parent strain or strain of the first embodiment, which codes for the production of 2,4- diacetylchloroglucinol. The transformed strains of the second modality share a banding pattern characteristic of identification with the parent strains or the first modality. This profile can be identified by a Random Amplified Polymorphic DNA (RAPD) analysis using the M13 primer as described in Examples 5 and 11, below. FIG. 1 shows the banding patterns (RAPD) of P fluorescens Q8rl-96, ML4.9-96 and L5.1-96, which are illustrative strains of the first embodiment of the invention. Lane 1 shows a 1-kb ladder as a reference. As indicated in FIG. 1, the bands shared by the illustrative strains of the first embodiment are: 330 ± 20 bp; 600 bp ± 50 bp; 700 ± 50 bp; 800 bp ± 50 bp; 900 bp ± 50 bp and 1100 bp ± 60 bp. The band at 800 bp ± 50 bp is the most intense. Transformed strains Z32-97, Z33-97 and Z34-97 share the following bands with their parent strain P. fluorescens Q8rl-96: 330 ± 20 bp; 600 bp ± 50 bp; 700 ± 50 bp; 800 bp ± 50 bp; 900 bp ± 50 bp and 1100 bp ± 60 bp. The transformed strain Z30-97 shares the following bands with its parent strain Q8rl-96: 600 bp ± 50 bp; 700 ± 50 bp; 800 bp ± 50 bp; 900 bp ± 50 bp. For purposes of this invention, a transformed strain has a characteristic banding profile of the illustrative parent strain if it has bands at 600 bp ± 50 bp; 700 ± 50 bp; 800 bp ± 50 bp; 900 bp ± 50 bp using the conditions described in Example 5 or 11, below. In a preferred embodiment, the transformed strain also has bands at 330 ± 20 bp and 1100 bp ± 60 bp Biocontrol activity. Strains Pseudomonas spp. Biologically pure fluorescents of the first embodiment of this invention have the ability to suppress (inhibit the incidence of or reduce the incidence or severity of) diseases caused by Gg, such as pest, in small grain crops or in lawns. Tables 1, 2 and 4 in the Examples below present the data showing the biocontrol activity. As indicated in Table 2 of Example 3, strain Q8rl-96 reduced the percentage of wheat plants infected with pest in the field by 20%. As shown in Table 4 of Figure 4 the strain at 8rl-96 was found to be almost twice as effective at reducing pneumonia as the P. fluorescens strain Q2-87 (a known suppressor, Phl-producing strain) after of 9 cycles of harvesting wheat. As discussed below, the Q8rl-96 doubled soil biocontrol TAD. Strains of Pseudomonas spp. biologically pure fluorescent bulbs of the invention have a biosynthetic locus that codes for the production of the antibiotic phenazine-1-carboxylic acid stably inserted into the genome and has the ability to suppress (inhibit the incidence of or reduce the incidence or severity of) caused by the Rhizoctonia pathogen of ground transportation. Optionally, strains suppress diseases caused by Pyhtium or diseases caused by Gaeumannomyces graminis in addition to diseases caused by Rhizoctonia, or have the ability to control all three diseases.

Tables 6, 7 and 8 of Example 8 present the data showing the biocontrol activity. As shown in Table 6, the transformed strains Z30-97, Z32-97 and Z34-97 at doses of 102-103 CFU per seed reduce the percentage of wheat roots infected with Rhizoctonia solani AG8 compared to the parent strain Q8rl -96 in a similar dose, P. fíourescens 2-79 (the source of the phenazine-1-carboxylic acid genes) and the two untreated controls. The parent strain Q8rl-96 is equivalent to Z30-97 and Z32-97 in the suppression of Rhizoctonia only when Q8rl-96 is applied in a larger dose. As shown in Table 7, in Example 8, strain Z34-97 at a dose of 102 CFU / seed is as effective in suppressing Pythium as parent strain Q8rl-96 at a dose of 103 CFU / seed. As shown in Table 8, in Example 8, strain Z34-97 provides an important suppression of Gaeumannomyces graminis variety tritici (Ggt). As shown in Tables 4-8, in Example 8, Z34-97 can suppress the three root diseases. Colonization capacity Strains of Pseudomonas spp. Biologically pure fluorescents of the first modality show a unique colonization capacity characterized by (a) greater population density at the roots and known. That is, the strains of the invention have the ability to both colonize and persist in the roots of small grains. As shown in the Examples below, the strains of the invention have the ability to colonize the roots at a population density that averages at least about 105 colony forming units (CFU) / gram of root, including the soil of the associated rhizosphere, for at least 7 successive cultivation cycles. FIG. 2 shows colonization of wheat roots (cv. Panawawa) by strain Q8rl-96 during 9 cycles of wheat in virgin Quincy soil. For cycle 5, the population density of Q8rl-96 was almost 1000 times higher than that of the P fluorescens strains Q2-87 and M1-96. (the strains that do not go according to the invention). The Q8rl-96 also showed significantly higher population densities in virgin Lind and Moses Lake soils in Figures 3 and 4. For purposes of this invention, a strain of the first modality has the characteristic coloriization capacity if it colonizes the roots of wheat at a population density averaging at least about 105 CFU per gram of root, including the associated rhizosphere soil for at least 5 successive growing cycles under the culture conditions described in Example 4, below.

The biologically pure transgenic strains that go according to the second modality have the capacity to colonize the seeds and roots of the plants. As shown in Table 9, in Example 9, the illustrative strains of the invention achieved the same population density as the parent strain, 66 hours and one week after having planted the treated seeds. No affectation due to the type of soil. In a preferred embodiment, a biocontrol strain of the invention has the additional feature that its biocontrol activity and root colonization capacity are not affected by the type of soil. As can be seen in FIG. 5, during the 7 cycles the population densities of the Q8rl-96 were not affected by the type of soil. Duplication of soil biocontrol TAD. A biocontrol strain of the invention is additionally and preferably characterized by having the ability to mimic diseases caused by G in vintage crops, which is equivalent to the level of biocontrol obtained when the small grain crop is grown in a soil in state of declination of pietin (TAD). That is to say, the strain has the capacity to duplicate the level of biocontrol obtained in a soil with declination of the pest. As shown in Table 1, in Example 1, Q8rl-96 applied at a dose of 104 units of colony formation (CFU) / seed and then planted on virgin Quincy soil (conductor) was so effective in suppressing the Pietin as the floor TAD of Quincy. further, Qr8l-96 was as effective in virgin Lind soil (conductor) as in Quincy TAD soil added to 10% w / w in Lind's virgin soil. This duplication of the suppression of pietin, equivalent to the natural decline of pietin, has no precedent. Application of Transgenic Suppressor Strains for the Biocontrol of Diseases of the Root of Plants. Transgenic strains of microorganisms that are in accordance with the second embodiment of the invention are useful for controlling plant root diseases, in particular diseases caused by the soil transport pathogen, Rhizoctonia, and optionally diseases caused by Pythium. , and by Gaeumannomyces graminis. The biocontrol agents of the invention have a particular use for controlling the disease of the root in small grains and in turfgrass. Examples of small grain crops include wheat, barley, rye and triticale. Additionally, the strains can be used to control the root disease of other food, fibrous and ornamental plants, which are susceptible to root diseases caused by soil transport pathogens, Rhizoctonia, Pythium or Gaeumannomyces graminis. These plants include asparagus, sugarcane, corn, beets, tomatoes, potatoes, peas, rice, beans, soybeans, strawberries, zucchini and cotton. To achieve the biocontrol of a specific disease of the root of the plants or diseases in a particular plant, the plant is grown in the presence of an effective suppressive amount of one or more fluorescent Pseudomonas strains of the invention. An effective amount of biocontrol is defined as the incidence or severity of the specific disease of the root or diseases relative to those that occur in a control without treatment. This assumes that factors such as water, fertilizer, soil and air temperatures are not limiting the crop of the specific crop. An effective amount in a particular case can be easily determined by test runs known in the art. The biocontrol is carried out by applying an affective amount of the biocontrol agent to the plant, to a seed of a plant or to the locus of the plant or seed.

For example, the strain can be applied as a seed, soil or irrigation treatment in ditches or flood, or as a flood to grass or soil. Fresh cells or freeze dried cells can be used. The strain can be incorporated into compositions suitable for application to plants where control of root disease is desired. It can be mixed with any suitable carrier, accepted in agriculture or with a suitable carrier accepted in agronomy, that does not interfere with the activity of the strain. Illustrative carriers are water, buffer solution, methylcellulose, peat or vermiculite. When the strain is applied as a suspension or emulsion in a liquid carrier, the suspension or emulsion may optionally contain conventional additives such as surfactants or wetting agents known in the art. The strain of the invention can also be prepared to include other biocontrol strains, including other strains of the second embodiment of this invention, a strain of the first modality, or strains known in the art. The application procedures and the illustrative effective amounts are presented below. The amount that will be within an effective range in a particular example can be determined by experimental tests. For treatment of seeds of small grains or grass or other food plants, fibrous or ornamental, bacteria are added to a suspension containing about 0.5-0.2% methylcellulose to minimize the dehydration of bacteria and promote adherence to the seed.

The suspension is added to the seeds and mixed so that each seed is coated with approximately 102 to 105 CFU per seed. In general, the preferred amount is about 102 to 104 CFU per seed. The treated seeds are dried with air. For soil treatment, the bacteria are suspended in water or in buffer solution and applied to the soil to provide approximately 102 to 105 CFU per gram of soil. For the lawn, the bacteria are suspended in water or in buffer solution and applied to the grass as a bath containing about 102 to 105 CFU per ml. For direct root treatment, the roots are immersed in a bacterial suspension of approximately 102 to 105 CFU per ml of suspension. When the carrier is solid, for example, peat or vermiculite, a typical preparation is about 106 to 109 CFU per gram of carrier. When the carrier is a liquid, a typical preparation is approximately 108 to 1010 CFU per ml of carrier. For a freeze-dried preparation, a typical amount is about 1010 to 1011 CFU per gram of preparation. Freeze-dried preparations may contain additives that are known in the art. Sieving Method. The first embodiment of the invention also encompasses methods for selecting the unique fluorescent Pseudomonas strains of the invention. Using our method, we obtain strains of microorganisms with the characteristics described above. In short, our method includes the steps of: (1) cultivate a small grain crop or turf in successive cycles of cultivation in pest suppressor soil (eg soil in decline state of pietin) to enrich Pseudomonas spp. fluorescent cells containing a biosynthetic locus that codes for the production of 2,4-diacetylchlorchlorucinol (also known as Phl-producing fluorescent Pseudomonas spp.); (2) isolate potentially suppressor fluorescent Pseudomonas bacteria strains; (3) screening the isolated strains to select strains containing a biosynthetic locus that codes for the production of 2,4-diacetylchlorchlorucinol through colony hybridization with a specific DNA probe of 2,4-diacetylchlorchlorucinol to detect Phl producers; and (4) using a RAPD analysis to select the strains having the characteristic banding pattern described above. An optional step can be added after step 3 to confirm the production of Phl. Below is a more detailed description of the selection method of the invention. Step 1. Successive cultivation cycles to enrich the producers of Phl. In this step, a small grain crop or turf is grown in successive cycles in a natural soil suppressor of the pietin for enrichment of 10 Pseudomonas spp. Phl-producing fluorescence as follows: (a) by cultivating seeds from a small grain crop or by cultivating turf in soil in declining state of piedin (TAD soil) in the greenhouse for at least 3 weeks and under conditions that are effective for sustain the growth of said small grain crop or turf to obtain seedlings; (b) collect the soil and roots of the seedlings of the small grain or turf crop grown in the soil and mix them together; and (c) repeating steps (a) to (b) for at least a total of 4 successive cycles wherein the mixture of step (b) is used to grow the seeds in the subsequent cycle. Step 2. Isolation of Pseudomonas spp. fluorescent from the 20 roots produced in cycles in TAD soils. In this step, the potentially suppressor fluorescent Pseudomonas bacteria strains are isolated from the roots and the corresponding rhizosphere soil of the small grain crop or turf cultivated successively in step (1), by cultivating the strains in a seed medium. Pseudomonas for a time and under conditions that are effective for the growth of the Pseudomonas and selecting strains that grow in the medium. Step 3. Hybridization of colony with specific probes to detect Phl. In this step, strains isolated in step 2 are sorted to select a strain containing a biosynthetic locus encoded for the production of 2,4-diacetylchloroglucinol (Phl) by hybridization of a strain colony with a specific probe of 2,4-diacetylfloroglucinol and selecting strains that generate hybrids for probe. The individual colonies selected are stung, striated and striated again until the strain is stable and pure, ie, it is a biologically pure culture. The strain can be stored in glycerol at -80 ° C to keep it stable. Step 3a (optional). Confirmation of Phl producers by PCR. In this optional step, confirmation of the Phl-producing strains is carried out using primers that amplify the sequences within the biosynthetic locus of the Phl, and those strains that provide a positive PCR reaction are selected. Step 4. RAPD analysis to identify Phl producers with a definitive banding pattern. In this step, an analysis of Random Amplified Polymorphic DNA is carried out using primer M13 (Sequence: GGTGGTCAAG) (see Keel et al., Supra). The M13 primer is commercially available from Operon Technologies, Inc., Alameda, California. Strains having bands at 600 bp ± 50 bp are selected; 700 bp ± 50 bp; 800 bp ± 50 bp; 900 bp ± 50 bp. It is preferable that the strains also have bands at 330 bp ± 20 bp and 1100 bp ± 60 bp. Using our method, we obtain biologically pure cultures of the P. fluorescens strains Q8rl-96, L5.1-96 and ML4.9-96 which are examples of the strains of the first modality of this invention. Preparation of Transgenic Strains. Strains of the second embodiment are obtained by recombinant DNA technology wherein the biosynthetic locus for phenazine-1-carboxylic acid is stably introduced into the genome of a fluorescent parent strain of Pseudomonas having the characteristics described in detail below. The genetic locus for the biosynthesis of phenazine-1-carboxylic acid from a bacterial strain, such as a fluorescent Pseudomonas, is modified. This structure is then cloned into a plasmid as a pUT, which contains a mini-Tn5 with cloning sites such as Sfi \ or Not \, and which has the ability to mediate the stable insertion of the biosynthetic phenazine genes into the fluorescent Pseudomonas receptor. The plasmid with the biosynthetic locus of phenazine and the promoter is transported in a strain such as Escherichia coli S17-1 (? Pir). The biosynthetic phenazine genes are stably inserted into the Pseudomonas receptor strain, by pairing with the E. coli strain with the Pseudomonas receptor strain (bacterial conjugation). After the matings, the cultures are suspended in buffer solution and planted on KMB plus kanamycin. Strains that are fluorescent and resistant to kanamycin are putative (transgenic) transformed strains. As presented in Example 5, below, a transposable version of the genetic locus for phenazine-1-carboxylic acid biosynthesis was structured using phenazine biosynthetic genes from the strain of Pseudomonas fluorescens 2-79 (NRRL) B-15132. The biosynthetic locus of phenazine (phz) is located in P fluorescens 2-79 within a DNA fragment Bgl \ l-Xba \ 8,505-bp, and consists of 9 genes designated as phzABCDEFG, phzl and phzR. The complete sequence of the phz locus from P fluorescens 2-79 is listed in the GenBank database with accession number L48616. The phzABCDEFG genes are organized in a single operon and are responsible for the production of phenazine-1-carboxylic acid (PCA) in P fluorescens 2-79. The products of the phzC, phzD and phzE genes share similarities with the shikimic acid and corismic acid metabolism enzymes, and, together with PhzF, are absolutely necessary for the production of PCA. PhzG is similar to the pyridoxamine-5'-phosphate oxidases and is perhaps a cofactor source for the PCA synthesizing enzyme (s). The products of the phzA and phzB genes are highly homologous with each other and may be involved in the stabilization of a putative multi-enzyme complex of PCA synthesizer. The phzABCDEFG genes are located within the Bgl \\ - Xba \ 6.8 kb fragment of the tac promoter control locus, and then cloned into the transfer plasmid described below. The resulting plasmid was used to generate stable chromosomal inserts in Pseudomonas spp. fluorescent that is naturally unable to produce phenazine compounds. To create genetically engineered bacterial strains, the system was based on the transposon characteristics of Tn5 and the transfer properties of plasmid pUT were used (V. de Lorenzo, M. Herrero, U. Jakubzik and KN Timmis, Journal of Bacteriology 172: 6568-6572 (1990)). pUT has an origin of replication of the R6K plasmid that depends on the protein p and is only conserved in bacteria producing the protein p, for example, a strain of Escherichia coli S17-1 (? pi? specially designed. oriT of transfer of plasmid RP4, which results in its transfer of efficient conjugation to the Pseudomonas receptor strains from donor E. cou strains expressing the conjugation functions of RP4 Finally, pUT carries a tnp gene of IS50 that encodes the transposase required for the transposition of the mini-Tn5 elements With the transfer system described above, the phenazine biosynthetic genes are inserted into the chromosome of the Pseudomonas target strains, where they are kept at a low, often natural number , of copies, and should at least theoretically be as stable as other chromosomal genes.After matings, crops are suspended in buffer solution and placed in a plate on KMB plus kanamycin. Strains that are fluorescent and resistant to kanamycin are putative transformed strains. The individual colonies selected are harvested, striated and striated again until the strain is stable and pure, ie, a biologically pure culture. The strain can be stored in glycerol at -80 ° C to keep it stable. Sieving in Greenhouse. The screening of the transformed strains to select those that have activity against the diseases caused by Rhizoctonia, such as the root rot of wheat by Rhizoctonia, is carried out in greenhouse bioassays. The soil is infected with Rhizoctonia inoculum and placed in plastic tubes as described in Example 8, below. The seed of a susceptible crop, such as wheat, is treated with the transformed strain and sown in the soil. Optionally, to select transformed strains that are also effective against the diseases caused by Gaeumannomyces graminis (Gg) or Pythium, greenhouse bioassays are carried out using Gg or Pythium spp. as inoculum, respectively. Optional Sieving In Vitro. An optional step prior to greenhouse classification is the in vitro inhibition of an isolated Rhizoctonia by a transformed strain. The assay includes pairing a strain transformed with the pathogen onto an agar plate and measuring the size of the zone of inhibition. As described in example 6 below, illustrative, biologically pure and transgenic strains showed greater in vitro inhibition of Gaeumannomyces graminis variety tritici, Rhizoctonia solani AG8 and Pythium irregulare than parent strain P fluorescens Q8rl-96. As shown in Table 5, in Example 6, the zones of inhibition of the transgenic strains are significantly greater than that of Q8rl-96 Optionally, the strains suppress diseases caused by Pythium or diseases caused by Gaeumannomyces graminis (Gg), in addition of diseases caused by Rhizoctonia, or have the ability to control all three diseases. Using our method according to the second modality we obtained biologically pure cultures of P fluorescens strains Z30-97; Z32-97; Z33-97, and Z34-97, which are examples of the strains of the invention. These strains were obtained by transforming the parent strain of the first modality, P fluorescens Q8rl-96 to stably introduce the biosynthetic locus for phenazine-1-carboxylic acid into the genome. This is discussed in detail below in Example 5. The parent strain Q8rl-96 normally produces the antibiotic 2,4-diacetylchloroglucinol (Phl) and the introduction of the PCA biosynthetic genes conferred the transforming strains the capacity to produce phenazine. - 1-carboxylic acid in addition to Phl.

As shown in Example 6, below, the addition of the phenazine-1-carboxylic acid biosynthetic genes resulted in transformed strains that are significantly better at suppressing root rot by Rhizoctonia than the parent strain Q8rl-96, or strain 2-79 which is the source of the biosynthetic locus of phenazine-1-carboxylic acid. As shown in the Example, strain Z34-97 was effective in suppressing Rhizoctonia at a dose of 102 to 103 which is a dose 100-1000 times lower than the dose needed for suppression by other Rhizoctonia suppressor strains, including P fluorescens Q8rl-96 and Bacillus sp. L324-92. Characteristics of the Strain. The parent strain example Q8rl-96 shows physiological features and substrate utilization patterns typical of P. fluorescens as described in the Bergey Manual (see Table 10, below). Strain Q8rl-96 also produces the antibiotic 2,4-diacetylphloroglucinol. Transformed strains of the invention, exemplified by Z34-97, share the traits of the illustrative parent strain, except that the transformed strains produce phenazine-1-carboxylic acid in addition to 2,4-diacetylchloroglucinol. Declaration of Deposit. Biologically pure cultures of the strains Q8rl-96, L5.1-96 and ML4.9-96 were deposited on July 8, 1997, according to the terms of the Budapest Treaty, before the Agricultural Research Crops Collection (NRRL), 1815 North University Street, Peoria, Illinois, 61604, and were assigned the accession numbers NRRL B-21806, NRRL B-21807, and NRRL B-21808 respectively. Strains having the identification characteristics of NRRL B-21806, NRRL B-21807, or NRRL B-21808 are included in this invention. For the purposes of this invention, any isolate having the identification characteristics of strains NRRL B-21806, NRRL B-21807, and NRRL B-21808, including subcultures and variants containing a biosynthetic locus encoding the production is included. of the antibiotic 2,4-diacetylphloroglucinol, which has the definitive banding pattern described above, which has the capacity to suppress diseases caused by Gg in small grain crops and turf, and which has the characteristic colonization capacity of the root. The term "variants" is defined herein to include mutants and transformants having the named characteristics. The biologically pure cultures of the P fluorescens strains Z32-97, Z33-97 and Z34-97 were deposited on December 11, 1997 and a biologically pure culture of the P. fluorescens strain Z30-97 was deposited on December 15, 1997. before the Agricultural Research Crops Collection (NRRL), 1815 North University Street, Peoria, Illinois, 61604, and were assigned accession numbers NRRL B-21905, NRRL B-21906, NRRL B-21907 and NRRL B-21908 , respectively. All strains were deposited in accordance with the terms of the Budapest Treaty. Strains having the identification characteristics of NRRL B-21905, NRRL B-21906, NRRL B-21907 and NRRL B-21908 are included in this invention. For the purposes of this invention, any isolate having the identification characteristics of strains NRRL B-21905, NRRL B-21906, NRRL B-21907 and NRRL B-21908 * including its subcultures and variants containing a biosynthetic locus is included. which encodes the production of the antibiotic phenazine-1-carboxylic acid stably introduced into the genome, which suppresses the diseases caused by the Rhizoctonia transport pathogen by land, and which has the 2,4-diacetylchloroglucinol locus of the parent strain. In a preferred embodiment, the isolate also has four bands of the genetic profile of the parent strain, as discussed in detail above. The term "variants" is defined herein to include mutants and transformants having the named characteristics. Cultivation of the Strains of the Invention. Strains of Pseudomonas spp. Fluorescent of the first embodiment of the invention can be cultured in a suitable solid or liquid bacteriological medium. An illustrative medium is King's medium B. The cultivation of the strains is carried out under aerobic conditions at any temperature that is suitable for the culture of the organism, ie from 15 ° C to 30 ° C; The preferred temperature range is from about 24 ° C to 28 ° C. The pH of the nutrient medium is preferably neutral, that is, a pH of 6.7-7.2.

Strains of Pseudomonas spp. fluorescent of the second embodiment of the invention can be cultured in a suitable solid or liquid bacteriological medium. An illustrative medium is King's medium B. The culture of the strains is carried out • under aerobic conditions at any temperature that is suitable for cultivation of the organism, ie from 15 ° C to 30 ° C; The preferred temperature range is from about 24 ° C to 28 ° C. The pH of the nutrient medium is preferably neutral, that is, a pH of 6.7-7.2. Maintenance of Inventory Crops. Each strain must be kept stable, for example, by storing it in glycerol at -80 ° C. io Application of Suppressor Strains of Gg. The microorganism strains of the invention are useful for controlling diseases of small grain or turf crops caused by Gg. Examples of small grain crops are wheat, barley, rye and triticale. The examples of the diseases caused by Gg are the disease of the pneumonia, caused by Ggt which is the most important of the wheat root and that can also infect other Gramineae such as barley, rye and triticale. Other Gg fungi include the Gga that infects the oats and grasses and has been identified as causing patchy patches of turfgrass, such as agrostis; and the Ggg that infects some pastures. To achieve a biocontrol of diseases caused by Gg in crops small grain and on the lawn, the crop or turf is grown in the presence of an effective suppressive amount of one or more fluorescent Pseudomonas strains of the invention. An effective amount of biocontrol is defined as the amount of biocontrol agent that suppresses (inhibits the incidence or reduces the incidence or severity of) diseases caused by Gg, in relation to those that occur in a control without treatment. Optimally, the biologically pure culture is applied to obtain at least 15% less disease in the small grain crop grown in fields, or in the turf, compared to a control without treatment. Biocontrol is carried out by applying an effective amount of biocontrol agent to a plant, to the seed of a plant or to the locus of the plant or of the seed. For example, the strain can be applied as a seed, soil or irrigation treatment in ditches or as a flood to grass or soil. Fresh cells or freeze dried cells can be used. The strain can be incorporated into compositions suitable for application to small grains or turf. It can be mixed with any suitable carrier, accepted in agriculture, or a suitable carrier accepted in agronomy, that does not interfere with the activity of the strain. Illustrative carriers are water, buffer solution, methylcellulose, peat or vermiculite. When the strain is applied as a suspension or emulsion in a liquid carrier, the suspension or emulsion may optionally contain conventional additives such as surfactants or wetting agents known in the art. The strain of the invention can also be prepared to include other strains of biocontrol. The application procedures and the effective illustrative quantities are described below. The amount that will be within an effective range in a particular example can be determined by experimental tests. For treatment of small grain seeds or turfgrass, bacteria are added to a suspension containing about 0.5-2.0% methylcellulose to minimize dehydration of the bacteria and promote adherence to the seed. The suspension is added to the seeds and mixed so that each seed is coated with approximately 102 to 106 CFU per seed. In general, the preferred amount is about 104 to 105 CFU per seed. The treated seeds are dried with air. For soil treatment, the bacteria are suspended in water or in buffer solution and applied to the soil during the first irrigation to provide approximately 102 to 106 CFU per gram of soil. For the lawn, 2 ml of a bath containing about 102 to 106 bacteria per ml is added to a grass plug about 1 cm in diameter. When the carrier is solid, for example, peat or vermiculite, a typical preparation is approximately 107 to 109 CFU per gram of carrier.

When the carrier is liquid, a typical preparation is approximately 108 to 1010 CFU per me of carrier. For a freeze-dried preparation, a typical amount is about 1010 to 1011 CFU per gram of preparation. The freeze-dried preparations may contain additives known in the art. Parent Strains for the Preparation of the Transgenic Strains of the Invention. The parent strains useful for the preparation of the transgenic strains of the invention are those which have the following characteristics: the strain contains a biosynthetic locus which codes for the production of 2,4-diacetylchloroglucinol and which has a unique genotype as demonstrated by a standard of characteristic banding described in detail later and in FIG. 1. This profile can be identified by a RAPD analysis with the M13 primer as described in Example 5. Illustrative parent strains are the P fluorescens strains Q8rl-96, L5.1-96 and ML4.9-96, presented above. For purposes of this invention, a parent strain has the characteristic banding profile if it has bands at 600 bp ± 50 bp; 700 ± 50 bp; 800 bp ± 50 bp; 900 bp ± 50 bp, using the conditions described in Example 5, below. In a preferred embodiment, the parent strain also has bands at 330 ± 20 bp and 1100 bp ± 60 bp. An optional way to identify if a parent strain has the characteristic banding pattern is to perform a simultaneous comparison on an agarose gel using the P fluorescens strain Q8rl-96 and the test strain, and visually compare the bands approximately between pairs of bases 600 ± 50 and 900 ± 50. If the profile of the test strain conforms to the profile of the strain Q8rl-96 in this region, then the test strain has the characteristic banding pattern, and optionally, it also suppresses diseases caused by Gg in small grain crops grown in the field, or in turf, and has a root colonization capacity characterized by higher population density and prolonged colonization activity compared to known Gg suppressor strains . Biocontrol activity of the Parent strain. The biocontrol activity of parent strains Pseudomonas spp. fluorescent, biologically pure, was presented above.

Duplication of soil biocontrol TAD. A parent strain of the invention is also preferably characterized by having the ability to suppress diseases caused by Gg in small grain crops which is equivalent to the level of biocontrol obtained when the small grain crop is grown in a soil that is in a state of declination of the pietin (TAD). That is to say, the strain has the capacity to duplicate the level of biocontrol obtained in a soil with declination of pest. For example, the illustrative parent strain, Q8rl-96, applied at a dose of 104 units of colony formation (CFU) / seed and then sown in a virgin soil (conductor) of Quincy was as effective in suppressing the pincushion as the soil Quincy TAD. In addition, Qr8l-96 was as effective in virgin soil of Lind (conductor) as in Quincy's TAD soil, added to 10% w / w in Lind's virgin soil. This duplication of the suppression of pietin, equivalent to the natural decline of pietin, has no precedent. The method for obtaining parent strains preferably used as starting material for the above processes are discussed above in relation to the biologically pure cultures of P. fluorescens strains Q8rl-96, L5.1-96 and MI4.9-96 which are examples of the father strains. The culture of the parent strains and the maintenance of the stored cultures of the parent strains are carried out as described above with reference to the transgenic strains of this invention.

EXAMPLES The following examples serve only to illustrate the invention and are not intended to limit the scope thereof, which is defined in the claims.

EXAMPLE 1 The following example describes the selection of Pseudomonas spp. fluorescent of the first embodiment of the invention. Summary. Strains of Pseudomonas fluorescens Q8rl-96, L5.1-96 and MI4.9-96 were isolated in 1996 from roots of wheat grown in soils collected from agricultural fields near, respectively, Quincy, Lind and Moses Lake, in Washington (USA), which are in a declining state of pietin (TAD). Q8rl-96 was isolated from the roots of wheat grown in Quincy's TAD soil for 8 successive cycles of 3 weeks each; L5.1-96 of wheat roots grown in Lind's TAD soil for 5 successive cycles of 3 weeks each; ML4.9-96 of wheat roots grown in Moses Lake TAD soil for 4 successive cycles of 3 weeks each. Below is a detailed description of the soils and the isolation and characterization techniques. Soils The three different soils were obtained from agricultural fields in the state of TAD, near Quincy, Lind and Moses Lake in Washington (USA). The three soils are suppressors of the wheat pietín. In 1995, Lind's TAD field had been continuously harvested with wheat for 28 years. In 1980, the TAD fields at Quincy and Moses Lake had been continuously harvested with wheat for 22 years; Between 1980 and 1995 other crops besides wheat were also cultivated. The soils were collected in March 1995 from the top 30 cm of the soil profile, dried with air for 1 week and passed through a 0.5 cm mesh screen. before use. Its physical and chemical properties were determined through the Laboratory of Analytical Sciences of the University of Idaho. Step 1: Successive cycles of wheat cultivation to enrich the producers of Phl. Twelve seeds of wheat were planted in square PVC pots (8 cm high, 7.5 cm wide) with 200 g of natural sifted soil (TAD soil from Quincy, Lind and Moses Lake) and 50 ml of water supplemented with metalaxyl (Novartis , Greensboro, NC) at 2.5 mg mi "1 of active ingredient to control root rot by Pythium Pseudomonas are not affected by this fungicide, a 1 cm layer of soil was spread on top of the seeds. They were grown in an environmental control chamber at 16 ° C with a 12-hour photoperiod.The pots received 50 ml of Hoaglund's solution (macroelements only) diluted (2: 3, vol / vol) twice a week. After 3 to 4 weeks of cultivation, the shoots of the plants were cut at the level of the soil surface, and the soil and the corresponding roots were decanted in a plastic bag and shaken vigorously to be ground and mixed. "was stored for a week at 15 C, is returned to the same pot and then replanted with twelve wheat seeds. This process of cultivation and harvesting of the plant was repeated for at least four and up to eight successive cycles, at which time four plants selected at random from each replica were harvested and root samples were prepared to determine the population size of Pseudomonas spp. . fluorescent antibiotic producer. For each floor, four replicas were used. Step 2: Isolation of Pseudomononas spp. fluorescent of wheat roots produced in cycles in TAD soils. Four plants, selected at random, grown in step 1 were harvested from each replica and loose soil was extracted from the roots, shaking them gently. 1.0 g of roots and the corresponding rhizosphere soil were suspended in 5.0 ml of sterile water and shaken vigorously for 1 minute in a Vortex mixer. The samples were subsequently subjected to sonic processing in an ultrasonic cleaner for 1 minute, and then the serial dilutions of the root wash were plated on King B medium agar [KMB] (Protease Peptone, 20 g; glycerol 10 ml, K2HP04, 1.5 g, MgSO4, 1.5 g, agar, 15 g, H2O, 1000 ml), supplemented with cycloheximide (100 μg mi "1), chloramphenicol (13 μg mi" 1) and ampicillin (40 μg mi "1) [KMB + J.] The plates were incubated at 25 ° C, and the colonies were counted after 48 hours.The colonies of fluorescent Pseudomonas spp. Differed from the non-fluorescent colonies, using UV light (wavelength 266). nm) Step 3: Hybridization of the colony with specific probes to detect Phl producers The number of fluorescent Pseudomonas spp. harboring the genes for Phl was determined by colony hybridization Transfer of bacterial colonies to nylon membranes Hybond-N + (Amersham) was performed by methods is After drying with air, the membrane was baked for 1 hour at 80 ° C in a vacuum oven. To remove bacterial cell debris, the membranes were washed for 1.5 hours at 42 ° C in a solution containing 2 x SSPE (20 mM NaH2P04 [pH 7.4], 0.36 M NaCl, 2 mM EDTA), 0.1% sodium dodecylsulfate (SDS) and pronase (100 μg mi "1), and were washed again for 1 hour at 56 ° C in 2 x SSPE and 0.1% SDS Hybridizations were performed by standard methods.The conditions of high stringency consisted of a prehybridization for 1.5 hours at 65 ° C, annealing for 12 hours at 65 ° C, membrane washing twice each for 5 minutes with 2 x SSC and 0.1% SDS at room temperature, and membrane washing twice each for 30 minutes. minutes with 0.1 x SSC and 0.1% SDS at 65 ° C. The probes were generated from sequences within the biosynthetic locus for 2,4-diacetylchloroglucinol (access no to GenBank U41818) .The probe was developed from sequences within Phl D of Q2-87 by random priming of PCR fragments using e The non-radioactive DIG system (Boehringer Mannheim). The hybridized probes were immunodetected with fragments of anti-digogogenin-AP-Fab and were visualized with the colorimetric substrates nitroblue tetrazolium salt and 5-bromo-chloro-3-indolylphosphate, according to the protocols indicated by the supplier. In order to isolate the Phl producers, the signals on the membrane were aligned with the colonies on the agar plate. Each individual colony that provided a signal on the membrane was harvested, striated and striated again until the strain became stable and pure, ie a biologically pure culture. Each strain was stored in glycerol at -80 ° C to keep it stable. Step 3a (optional): Confirmation of Phl producers by PCR.

This optional step allows false positives from colony hybridization to be removed from the analysis. The bacterial suspensions used by heat used in the PCR analysis were prepared from cultures grown in KMB for 48 hours at 25 ° C. Two bacterial colonies (2 mm in diameter) were suspended in lysis solution of 100 μl (0.05 M NaOH, 0.25% SDS) and incubated for 15 minutes at 100 ° C. The suspension was centrifuged for 1 minute at 12,000 rpm and diluted 50 times in sterile distilled water. Five μl of the diluted suspension was used in each reaction. Primers and PCR analysis. The oligonucleotide primers used in the PCR were developed from sequences within the biosynthetic locus for 2,4-diacetylchloroglucinol (Phl) of P. fluorescens Q2-87 (accession to GenBank U41818). The primers were synthesized by Operon Techn. In. (Alameda, California). The primers Phl2a (Sequence: GAGGACGTCGAAGACCACCA) and Phl2b (Sequence: ACCGCAGCATCGTGTATGAG) were developed from sequences within phID, which predicts a protein of 349 amino acids that is homologous to the chalcone synthase of plants. PCR amplification was carried out in a 25 μl reaction mixture, containing 5 μl of a diluted suspension of heat-lysed cell, 1 x GeneAmp PCR buffer (Perkin Elmer Corp., Norwalk, CT), 200 μM each of dATP, dTTP, dGTP and dCTP (Perkin Elmer), 20 pmol of each primer and 2.0 U of AmpliTaG DNA polymerase; (Perkin Elmer) Each mixture was covered with a drop of mineral oil. The amplifications were performed in a Perkin Elmer 480 Thermal Cycler. The PCR program consisted of an initial denaturation at 94 ° C for 2 minutes, followed by 30 cycles at 94 ° C, for 60 seconds, 67 ° C for 45 seconds and 72 ° C for 60 seconds. Samples (9 μl) of the PCR products were separated on a 1.2% agarose gel in 1 x TBE buffer solution (90 mM Trisborate, 2 mM EDTA (pH 8.3)) at 75 V for 3 hours. The gel was stained with ethidium bromide for 30 minutes, and the PCR products were visualized using a UV transilluminator. Step 4. RAPD analysis to identify Phl producers with a definitive banding pattern. The RAPD analysis (Randomized Amplified Polymorphic DNA) was performed with M13 primer to group the different Phl-producing fluorescent Pseudomonas strains isolated from the roots of the wheat grown in TAD soil of Quincy, Lind and Moses Lake.

DNA amplification was carried out in a 25 μl reaction mixture, containing 5 μl of a diluted suspension of heat-lysed cells, PCR x GeneAmp buffer solution (Perkin Elmer, Corp., Norwalk, CT), 200 μM each of dATP, dTTP, dGTP and dCTP (Perkin Elmer), 80 pmol of primer M13 and 2.0 U of the ft 5 DNA polymerase AmpliTa? r (Perkin Elmer). Each mixture was covered with a drop of mineral oil. The amplifications were performed in a Perkin Elmer 480 Thermal Cycler. The PCR program consisted of an initial denaturation at 94 ° C. for 1 minute 30 seconds, followed by 2 cycles of 94 ° C for 30 seconds, 36 ° C for 30 seconds, 72 ° C for 2 minutes, followed by 40 cycles of 94 ° C for 20 seconds, 36 ° C for 15 seconds, 45 ° C for 15 seconds, 72 ° C for 1 minute 30 seconds, followed by 1 cycle of 72 ° C for 10 minutes. Samples (9 μl) of the PCR products were separated on a 2.5% agarose gel in 1 x TBE buffer solution (90 mM Tris-borate, 2 mM EDTA (pH 8.3)) at 75 V for 5 hours. The gel was stained with ethidium bromide for 60 minutes, and the products PCR was visualized using a UV transilluminator. The biologically pure fluorescent Pseudomonas strains of the invention show a unique banding pattern as demonstrated by the RAPD analysis with M13 primer. FIG. 1 shows the banding patterns (RAPD) of the P fluorescens strains Q8rl-96, ML4.9-96 and 15.1-96. Lane 1 shows a 1-kb ladder as reference. As shown in FIG. 1, the bands shared by the strains of the invention are: 330 ± 20 bp; 600 bp ± 50 bp; 700 ± 50 bp; 800 bp ± 50 bp; 900 bp ± 50 bp and 1100 bp ± 60 bp.

EXAMPLE 2 The following example illustrates the duplication of the TAD using a biocontrol strain of the first embodiment of the invention. The strain P. fluorescens 8rl-96 was isolated and propagated as described in Example 1. Bacteria were suspended in 0.5% methylcellulose and mixed with wheat seed (cv. Penawawa) to provide a dose of 104 30 CFU per seed. The seed used as control received only methylcellulose.

Soils TAD Pullman, Pullman driver, TAD Quincy and Virgen Quincy were modified with 0.5% (weight / weight) of oat grain inoculum (size 0.25-0.5 mm) of the pigeon pathogen. Four treated seeds were planted in square plastic pots (3 cm x 3 cm) containing 50 g of sifted natural earth and moistened with water supplemented with metalaxyl (Novartis, Greensboro, NC) at 2.5 mg / ml of active ingredient to control the Pythium. The plants were grown in an environmental control chamber at 16 ° C with a photoperiod of 12 hours. The pots received diluted solution (2: 3 v / v) of Hoaglund (macroelements only) twice a week. After four weeks of cultivation, the plants were harvested, the root disease was evaluated, and the shoots were weighed and measured. The results are presented below and show that the Q8rl-96 strain introduced in Pullman or Virgin Quincy soil provides the same amount of disease suppression as the TAD Pullman and TAD Quincy soils, and the Quincy TAD soil transferred to Pullman driver. Strain Q8rl-96 did not increase the suppression capacity of the TAD Pullman soil.

Experiment 2 Cond. Pullman not detected x 3.8 to 212 to 142 to Cond. Pullman + TAD Quincy (9: 1) and 4.5 x 105 to 2.1 b 335 b 181 Cond. Pullman + Q8rl-96 4.5 x 107 c 2.5 b 288 b 172 TAD Pullman 1.3 x 106 b 1.9 b 336 b 184 b TAD Pullman + TAD Quincy (9: 1) and 3.5 x 105 a 2.3 b 286 b 177 TAD Pullman + Q8rl -96 3.4 x 107 c 2.3 b 306 b 178 bu Soils Cond. Pullman and Quincy are drivers of the wheat pietin while the complementary TAD soils ^ S3g? SCSg¿ suppressors of the footpad. The Quincy and TAD Quincy virgin soils had been previously cultivated with wheat for six successive periods for four weeks each to activate the microflora. The soil Cond. Pullman and TAD Pullman were obtained directly from fields grown with wheat. All soils were modified with 0.5% (w / w) of an inoculum of wheat from the pest pathogen. Wheat seeds treated with Q8rl-96 received a dose of 104cfu seed'1.

(Z 41 (Table 1, continuation of the legends) v The population sizes of the introduced strain Q8rl-96 or of the Pseudomonas spp. fluorescent producers of natural occurrence, the severity of the disease, fresh weight of the plant, and height of the shoot were determined after four weeks of culture. The average values of six replicas of two plants each were presented. The difference in the treatments in the population densities of Pseudomonas spp. Production of Phl, fresh weight of the plant, from the shoot were determined by analysis of variance followed by the Turkish Student Interval Test. Differences in the severity of the disease were analyzed by the Wilcoxon Interval Sum test. For each experiment and for each parameter, the mean values with different letters indicate a statistically significant difference (a = w The severity of the pietin was classified on a scale of 0-8 (0 = no disease, and 8 = dead plant). x Below the lower limit of detection of 104 cfu g'1 root and Soils Cond. Pullman and TAD Puli were each mixed with Quincy TAD soil in a ratio of 9: 1 (weight / pe ^^ * i ^^ í-á » EXAMPLE 3 The following example describes the biocontrol of pietin in wheat grown in fields. The Q8rl-96 Pseudomonas fluorescens was tested in a commercial field in Almoto, WA. The field used had been harvested with wheat the previous year and was naturally infested with pest. Experimental. Spring wheat (cv. Alpowa) was planted without tillage on winter wheat stubble. The strain P. fluorescens Q8rl-96, isolated and propagated as described in Example 1, in a suspension of 1.5% methylcellulose was applied to Alpowa spring wheat seeds at a dose between 1 x 105 and 1 x 106 CFU per seed. The controls consisted of untreated wheat seed. In a control, called "fumigated" in Table 2, the untreated seed was planted in the soil that had been fumigated with methyl bromide to eliminate the pathogen and to create a "healthy" control. In the other control, called "control", the untreated seed was planted in natural soil to create a control of the disease. The treatment lots were 8 rows (with space of 1 foot) by 25 feet in length. The wheat was harvested 135 days after planting. Results The results are shown in Table 2. As can be seen in the data, strain 8rl-96 reduced the number of infected plants by 20%.

TABLE 2 Treatments Plants Infected with Pietín (% O?) \ Control 69.3A Q8rl-96 48.0B Fumigated 13.3C a The mean values followed by the same letter are not significantly different. a P = 0.1. LSD = 21.1 EXAMPLE 4 The following example demonstrates the superior colonization capacity of the strains of the invention. The strain of the invention, Pseudomonas fluorescens Q8rl-96 was compared with l > P. fluorescens strains M 1-96 and Q2-87 (not according to the invention). The three strains produce the antibiotic 2,4-diacetylchloroglucinol, and show physiological features and substrate utilization patterns typical of P. fluorescens as described in the Bergey Manual (see Table 3, below). However, the strains of the invention exemplified by the Q8rl-96 have a RAPD banding pattern characteristic that the strains that are not of the invention do not have. TABLE 3 Q8rl-96 Q2-87 Gram Reagent - Shape, Cane Cane Fluorescent pigment + + Oxidase + + Arginine dihydrolase + + Fermentation of glucose ß-galactosidase Gelatine hydrolysis + Denitrification + + Use of: D-glucose + + L-arabinose + + Sucrose + + Propionate + + Butyrate Sorbitol + + Adonitol - - D-mannitol + + N-acetyl-D-glucosamine + + Maltose D-gluconate + + Caprate + + Adipate L-malate + + Citrate + + Phenylacetate - In these studies evaluating the colonization capacity, each strain was applied individually in virgin soil of Quincy, virgin soil of Lind and virgin soil of Moses Lake at a dose of 100 CFU (bacterial cells) per gram of soil (size of the used pot 8 cm high and 7.5 cm wide). The wheat (cv.

C > 5 Penawawa) was cultivated in each soil for three weeks, and the plants were harvested and the bacterial populations at the roots were determined by fluted dilution. The soil and the associated roots were decanted in a plastic bag that was stirred to irritate and mix the soil. One week after harvest, each soil was returned to the same pot and wheat was sown again. Thus, each cycle between the plantation lasted 4 weeks. Results FIG. 2 shows the population density (CFU / g root) of the three strains in virgin Quincy soil after 9 cycles. As can be seen in the data, even after 9 cycles, strain Q8rl-96 shows population densities greater than 105 CFU / g root '. In contrast, strains M1-96 and Q2-87 (not in accordance with this invention) dropped to 102 CFU / g root. As can be seen in the data, the colonization capacity of the strain of the invention is superior compared to other biocontrol strains; the strain of the invention colonizes the roots at a significantly higher level and has a colonization activity prolonged. Comparison of biocontrol activity in Virgen de Quincy soil. At the ninth cycle, Ggt inoculum was added to the soil in the form of colonized oat grains. The inoculum was pulverized and hovered, and particles of 0.25 to 0.50 mm in size were mixed with the soil at a concentration of 0.5% (w / w). The pneumonia disease was measured on a scale of 0-8 as described below. The bacteria were compared to a control (check) where no bacteria had been added to the soil. The plants were grown in pots (8 cm high by 7.5 cm wide) for 4 weeks. The data is shown in Table 4. As can be seen in the table, only Q8rl-96 (the strain of the invention) suppressed the disease.

The plants treated with strains that are not of the invention showed the same amount of disease as the control (untreated plants). The superior suppression of the strain of the invention, Q8rl-96, (in the ninth cycle) can be attributed to its greater capacity for colonization of the root and to the persistence of the strain of the invention, and shows how aggressive the strain is as biocontrol agent.

TABLE 4 Strain Severity of the disease Q8rl-96 3.33a 'M1-953 6.42b Q2-873 6.08b Check 6.00b 1It was evaluated on a scale of 0 to 8 (Ownley et al., Phytopathology 82: 178-184 (1992), where: 0 without evident disease, 1 <10% root area with black lesions, 2 10 -25% root area with black lesions, 3> 25% root area with black lesions and one root with lesions at base of stem, 4 = more than one root with lesions at base of stem, 5 all the roots with lesions at the base of the stem, at least one lesion in the lower part of the stem, but without chlorosis in the leaves, 6 many lesions in the stem and first truly chlorotic leaf, 7 all the chlorotic leaves and the plant severely atrophied; 8 dead or almost dead plant.2 Mean values followed by the same letter are not significantly different, P = 0.05 3 Not according to the invention, for comparative purposes only.

FIGS. 3-4 show the population density (CFU / g root) of the three strains in virgin soil of Lind and Moses Lake, respectively, after 7 cycles. As can be seen in the data, even after 7 cycles, the population density of strain Q8rl-96 is greater than 105 CFU / g of root. In contrast, strains M1-96 and Q2-87 (not in accordance with the invention) fell to 102 CFU / g of root. As can be seen in the data, the colonization capacity of the strain of the invention is superior to the other strains; colonizes the roots at a significantly higher level and has prolonged colonization activity. The high colonization results in a higher biocontrol compared to the strains that are not of the invention. FIG. 5 shows the colonization capacity of the strain of the invention, P. fluorescens Q8rl-96 in virgin soil of Quincy, virgin soil of Lind and virgin soil of Moses Lake. As can be seen in the data, the strain of the invention is not affected by the type of soil.

EXAMPLE 5 The following example describes the construction of illustrative, phenazine-producing transgenic fluorescent pseudomonas strains. In all the experiments listed below, standard methods were used for plasmid DNA isolation, restriction enzyme digestion, agarose gel electrophoresis, ligation, and transfusion (Ausubel, FM, R. Brent, RE Kingston, DD Moore, JG Seidman, JA Smith, and K. Struhl (ed.), 1995. Short Protocols in Molecular Biology, J. Wiley and Sons, NY). All enzymes (including restriction endonucleases, T4 DNA ligase, Klenow fragment of DNA polymerase I) and reagents that are necessary to carry out the experiments described below, are commercially available from Life Technologies, Gaithersburg, MD. The QIAEX II Gel Extraction Kit available from QIAGEN, INc, Chatworth, CA, was used to extract DNA from the agarose gels. Strain JM190 Escherichia coli, which was used for all DNA cloning experiments is commercially available from Promega Corporation, Madison, Wl. All strains of bacteria were routinely propagated on LB medium (Bacto tryptone (Difco Laboratories, Detroit, MI), 10 g, Bacto yeast extract (Difco Laboratories, Detroit, MI), 5 g: NaCl, 5 g, H20, 1000 my). Step 1. Cloning of the phz genes under the control of the tac promoter. The 6.8-kb DNA fragment Bg1 \\ - Xba \ containing genes phzA, -B, -C, -D, -E. -F and -G of P. fluorescens 2-79 was inserted into the ßa HI and Xba cloning sites of the cloning vector pALTER-I, which contains a versatile polylinker combined with a strong tac promoter for expression in vivo and in vitro of the cloned genes. The pALTER-x1 cloning vector is commercially available from Promega Corporation, Madison, Wl. After ligation, the DNA sample was transformed into competent E. coli JM109 cells, and the transformants were selected on LB agar modified with 12 μg / ml tetracycline. Several individual colonies were cultured in LB broth with tetracycline and used to purify the plasmid DNA. The insertion of the biosynthetic locus phz was confirmed by digestion with restriction endonucleases. Step 2. Construction of a transposable copy of the biosynthetic locus of phenazine. DNA from pALTER-Ex1 containing phz genes was digested with restriction endonucleases Seal, Xbal and Hindlll that generated the DNA fragments of 6.8-kb, 4.3-kb and 2-kb. The 6.8-kb Hindlll-Xbal DNA fragment containing phz genes linked to the tac promoter (Fig. 6) was separated from the other fragments by gel preparation electrophoresis, purified from the agarose gel, and blunt ended with Klenow fragment. of polymerase I of DNA. This fragment was ligated with DNA from the cloning vector pUC18Sfi (M. Herrero, V. de Lorenzo, K. Timmis, Journal of Bacteriology 172: 6557-6567 (1990)), which was previously cut with the restriction endonuclease EcoRI and was treated with Klenow fragment of DNA polymerase I. The ligation mixture was transformed into E. coli JM109 and those transformed with the inserted phz locus were screened by their white colony color on LB agar supplemented with 40 μg / ml of 5-bromo-4-chloro-3-indolyl. β-D-galactopyranoside (X-gal) and 80 μg / ml ampicillin. Plasmid DNA samples from those transformed with white colonies were screened for the presence of phz genes as described above. The hybrid plasmid pUC18Sf1 containing the phz locus was digested with Sfil restriction endonuclease, and ligated with the pUTKm plasmid (see Herrero et al.) Opened with Sfil. Competent E. coli S17-1 (? Pir) cells were transformed with the ligation mixture and the transformants were selected on LB supplemented with kanamycin and ampicillin, each at 100 μg / ml. Plasmid DNA from the various individual colonies was purified and screened for the presence of phz genes by cleavage with the restriction endonuclease (• EcoRI and Hindlll The genetic map of the resulting plasmid pUTKm:: phz is shown in FIG 6. E. coli S17-1 (? Pir) harboring pUTKm:: phz was used as the donor for conjugation with Pseudomonas Step 3. Conjugation between E. co // S17-1 (? pir) and Pseudomonas spp. Plasmid pUTKm:: phz was mobilized from E. coli S17-1 (? pir) to Pseudomonas fluorescens Q8rl-96 using the mating technique described by Herrero et al. The donor strain E. coli S17-1 (? Pir) harboring pUTKm:: phz was cultured overnight with shaking at 37 ° C in 10 ml of LB broth supplemented with 100 μg / ml of both ampicillin and kanamycin . The rifampin-resistant Q8rl-96 recipient strain was grown separately overnight in 10 ml of broth.

LB without antibiotics. The cultures of the donor and recipient strains were centrifuged separately at 6,000 rpm for 5 minutes and the cells were suspended in 1 ml of fresh LB broth. 30 μl of the cell suspension of the E. coli S17-1 (? Pir) donor strain were placed in a piece (2 x 2 cm) of nitrocellulose membrane placed on a LB plate, and then 20 μl of the The cell suspension of a recipient strain was placed on the donor. The pure nitrocellulose membrane (0.45 micron) is commercially available from Bio-Rad Laboratories, Hercules, CA. The plates with the membranes were incubated at 27 ° C overnight, and then the cells of the membranes were suspended in 1 ml of sterilized water. The cells were made pellets (pellets) by centrifugation at 6,000 rpm for 5 minutes and then suspended in 1 ml of sterilized water. The cell suspension was diluted 10 times, and 100 μl was plated on a minimal M9 medium (Na2HP04 x 7H2O, 21 g, KH2P04 3 g, NaCl, 0.5 g, NH4CI, 1.0 g, 1M CaCl2) 0.1 ml; 1M MgSO, 2 mL; 20% glucose, 20 ml; H20 at 1000 ml), supplemented with 100 μg / ml kanamycin and incubated on-grown on M9 kanamycin plates were transferred to King's medium B plates (Peptone protease (Difco Laboratories, Detroit, MI), 20 g glycerol, 10 ml, K2HP04, 1.5 g, MgSO4 1.5 g, Bacto agar (Difco Laboratories, Detroit, MI) 15 g, H20 at 1000 ml), supplemented with 100 μg / ml kanamycin, and inspected to confirm production of the fluorescent pigment. These sorting clones were then grown overnight in LB broth, and later stored at -80 ° C in 50% glycerol (v / v) for further studies.

EXAMPLE 6 The following example describes an in vitro fungal inhibition assay. Inhibition of the growth of three root pathogens by the Q8rl-96 receptor strains (transformed strains) prepared as described in Example 5, was carried out on potato dextrose agar (PDA) in petri dishes. The method used was similar to that described by Thomashow and Weller, Journal of Bacteriology 170: 3499-3508 (1988), except for the modifications indicated below. 3 μl of LB broth cultures from strains of bacteria from the previous night were placed on the surface of a PDA plate, with two spots per plate on opposite sides and approximately 1 cm away from the edge of the plate. A piece (stopper) of 0.5 cm from the starting edge of cultures of pathogens grown on PDA medium was placed in the center of the plate. The plates were incubated at 28 ° C and were graded by measuring the distance between the edges of the bacterial colony and the fungal mycelium. For the inhibition of Gaeumannomyces graminis, tritici variety, cultures of the bacterial broth were placed on the plates 24 hours after the fungal piece was placed, and the inhibition was scored 5 days after the bacteria were applied. For the inhibition of Rhizoctonia solani AG8, cultures of the bacteria broth were placed on the plates at the same time the fungal piece was placed, and the inhibition was scored 5 days later. For the inhibition of Pythium irregulare, cultures of the bacteria broth were placed on the plates 48 hours before the fungal piece was placed, and inhibition was scored 2 days after the fungus was applied.

Results The results are presented in Table 5. As can be seen in the data, the zones of inhibition of the transgenic strains Z30-97, Z32-97, Z33-97 and Z34-97 were significantly greater than those of the strain Q8rl-96. .

Table 5. In vitro inhibition of Gaeumannomyces graminis, variety tritici, Rhizoctonia solani AG8 and Pythium irregulare by means of the parent strain Pseudomonas fluorescens Q8rl-96 and the transgenic derivatives Z30-97, Z32-97, Z33-97 and Z34-97 on potato dextrose agar.

Size of the Inhibition Zone Ggt strain Rhizoctonia Pythium P. fluorescens Q8rl-96 0.50 D 0.17 B 0.47 B Z30-97 1.55 B 0.55 A 0.80 A Z32-97 1.13 C 0.53 A 0.70 A Z33-97 1.66 AB 0.57 A 0.67 A Z34-97 1.70 A 0.55 A 0.73 A Values means followed by the same letter are not significantly different, P = 0.05.

EXAMPLE 7 The following example describes the greenhouse sieve bioassays. Materials and methods. Seed treatment: Penawawa spring wheat seeds were coated with different doses of bacterial strains grown on KMB (for Pseudomonas sp) or NBY (for Bacillus sp) and suspended in 0.5% methylcellulose. A bacterial suspension was spread evenly over the entire plate and cultured at 27 ° C for 3 days. Three plates were prepared for each strain. The bacterial cells were harvested by scraping them into a capped centrifuge tube and washed twice with 40 ml of sterile distilled water. The bacterial cells were resuspended in 15 ml of 0.5% methylcellulose, and 10-fold dilution series were made with 0.5% methylcellulose. Twenty-five grams of wheat seeds were mixed perfectly with 9 ml of the bacterial suspension - methylcellulose and dried with air overnight. The actual bacterial cell doses in each seed was determined by plate counts by dilution. Greenhouse test on the suppression of the disease. A tube assay (Ownley et al., Phytopathology 82: 178-184 (1992) and Weller et al., Plant Disease 69: 710-713 (1985)) was used to test the transgenic strains in relation to the deletion of the pincushion. (caused by Gaeumannomyces graminis, tritici variety (Ggt)), root rot by Rhizoctonia (caused by Rhizoctonia solani AG8), and root rot by Pythium (caused by a complex of Pythium spp, including Pythium irregulare). The inoculum of each of these three pathogens was prepared by growing each pathogen individually on integral oat grains autoclaved for 4 weeks respectively (see U.S. Patent No. 4,456,684 which is incorporated herein by reference). The inoculum was stored in the refrigerator until it was used. The test used conical plastic tubes (2.5 cm in diameter and 20 cm in length), with holes in the bottom. The tubes were hung on plastic supports. Each tube was capped with cotton balls and then filled in half with fine vermiculite by 13 grams of virgin soil with or without ground oats inoculum. For tests of pest and root rot by Rhizoctonia, the soil was mixed with 1% (w / w) of cultivated inoculum of freshly ground oats kernels (particle size: 0.25-0.50 mm). For tests of root rot by Pythium, the soil was infested with a core of whole oats colonized with Pythium irregulare. Then 10 ml of water was added to each tube for the soil infested with Pythium, and 10 ml of water with metalaxyl (Ridomil 0.075 g / liter, Novartis, Greensboro, NC) for soil infested with Ggt and with Rhizoctonia solani. After irrigation, tubes with soil infested with Pythium irregulare and Rhizoctonia solani were incubated at room temperature for 48 hours before sowing the seeds. The tubes with soil infested with Ggt were seeded immediately after irrigation. Two wheat seeds were planted in each tube, covered by a small amount of fine vermiculite and watered with 3 ml of water for the Pythium test., or were irrigated with metalaxyl for the Ggt and Rhizoctonia tests. Then the tubes were covered with plastic for 3 days to retain soil moisture for germination and were placed in a greenhouse with a 12-hour photoperiod, and a constant of 16 ° C for 4-5 weeks. After emergence of the wheat seedlings (3-4 days), the tubes were irrigated twice a week with 5 ml of Hoagland solution at 1/3 power. All the experiments were carried out in virgin Quincy soil extracted from Quincy, WA., With 5-6 repetitions and 5 tubes for each repetition. The severity of the three root diseases were evaluated after the roots were washed until they were free of the rooting medium. The severity of root rot by Rhizoctonia was evaluated as the percentage of infected roots. The severity of the Pythium infection was assessed by the emergence and height of the seedlings. The severity of the pietin was evaluated on the scale of 0 to 8 developed by Ownley et al., 1992 supra. Statistical analysis. The results were analyzed by SAS statistical software (SAS Institute, Cary, NC). The emergence rate was analyzed after the transformation of the sine arch of the data. The data of proportion of pietin were analyzed by nonparametric statistics. The means of treatment were separated by Fisher's less significant difference (LSD) at P = 0.05. Results The results are shown in Tables 6-10 below. As shown in Table 6, the transformed strains Z30-97, Z32-97, Z33-97 and Z34-97 at doses of 102-103 CFU per seed reduced the percentage of wheat roots infected by Rhizoctonia solani AG-8 in comparison with the parent strain Q8rl-96 at a similar dose, P. fluorescens 2-79 (the phenazine-1-carboxylic acid gene source) and the two untreated controls. The parent strain Q8rl-96 is equivalent to Z30-97 and Z32-97 in the suppression of Rhizoctonia only when Q8rl-96 is applied at a higher dose. As shown in Table 7, below, strain Z34-97 at a dose of 102 CFU / seed is as effective in suppressing Pythium as parent strain Q8rl-96 at a dose of 103 CFU / seed. As shown in Table 8, below, strain Z34-97 provided an important suppression of Gaeumannomyces graminis, tritici variety (Ggt). As shown in Tables 5-8, Z34-97 can suppress the three root diseases.

Table 6. Suppression of wheat root rot by Rhizoctonia (Rhizoctonia solAG-8) by treatment of wild-type seeds and transgenic bacterial biocontrol agents.

Treatment CFU / seed Infected roots (%) 1 MethylcelluloseJ 0 60.7 Å4 No treatment3 0 55.3 ab Pseudomonas fluorescens 2-795 106 48.9 b Pseudomonas fluorescens Q8rl- 103 37.3 c 965.6 Pseudomonas fluorescens Q2-875 106 34.8 cd Pseudomonas fluorescens Q8rl - 104 30.8 c from 965.6 Bacillus spp. L324-925 107 28.1 def Transgenic Z32-97 102 25.1 efg Transgenic Z30-97 102 25.1 efg Transgenic Z34-97 102 21.6 fg Transgenic Z34-97 103 18.8 g 1 The seeds were treated with bacteria suspended in 0.5% methylcellulose to provide the doses indicated by seed. 2 The percentage of roots with typical Rhizoctonia lesions was measured 4 weeks after planting. 3 The seeds treated with methylcellulose were only treated with 0.5% methylcellulose. No untreated seed received any treatment. The mean values followed by the same letter are not significantly different, P = 0.05. 5 They do not agree with the invention. For comparative purposes only. 6 Parent strain.

TABLE 7. Suppression of wheat root rot by Pythium (Pythium irregulare) by treatments of wild-type seeds and transgenic bacterial biocontrol agents.

Treatment CFU / Seed Emergence ^ 1 Methylcellulose J 0 50 d 4 No treatment3, 0 54 cd Pseudomonas fluorescens 2-795 106 63 cd Pseudomonas fluorescens Q8rl- 103 92 ab 965,6 Pseudomonas fluorescens Q2-875 106 50 d Pseudomonas fluorescens Q8rl- 104 71 bed 965.6 Bacillus spp. L324-925 107 92 ab Transgenic Z32-97 102 67 cd Transgenic Z30-97 102 67 cd Transgenic Z34-97 102 96 a Transgenic Z34-97 103 79 abe 1 The seeds were treated with 0.5% methylcellulose bacterial suspension to provide the doses indicated by seed. 2 The number of seedlings that emerged was determined 4 weeks after planting. 3 The seeds treated with methylcellulose were only treated with 0.5% methylcellulose. No untreated seed received any treatment. 4 The mean values followed by the same letter are not significantly different, P = 0.05. 5 They do not agree with the invention. For comparative purposes only. 6 Parent strain.

TABLE 8. Suppression of wheat root rot by pietin (Gaeumannomyces graminis, tritici variety) by treatments of wild-type seeds and transgenic bacterial biocontrol agents.

Treatment CFU / seed1 Proportion of disease2 Methylcellulose3 0 4.2 to 4 No treatment 0 4.2 ab Pseudomonas fluorescens 2-795 106 3.9 Pseudomonas fluorescens Q8rl-965 ß 103 2.9 e Pseudomonas fluorescens Q2-875 10ß 3.7 bed Pseudomonas fluorescens Q8rl-965, ß1010 2.9 e Bacillus spp. L324-925 107 3.5 cd Transgenic Z32-97 102 3.9 ab Transgenic Z30-97 102 3.8 abe Transgenic Z34-97 102 3.5 d Transgenic Z34-97 103 3.4 d 1 The seeds were treated with 0.5% methylcellulose bacterial suspension to provide the doses indicated by seed. 2 Pietin was evaluated on a scale of 0 to 8 (Ownley et al., 1992), where: 0, no evident disease; 1, < 10% area of the root with black lesions; 2, 10-25% root area with black lesions; 3, > 25% root area with black lesions and one root with lesions at base of stem; 4, = more than one root with lesions at the base of the stem; 5, all the roots with lesions at the base of the stem, at least one lesion in the lower part of the stem, but without chlorosis of the leaves; 6, many lesions on the stem and first truly chlorotic leaf; 7, all the chlorotic leaves and the severely stunted plant; 8, Dead or almost dead plant.

The seeds treated with methylcellulose were only treated with 0.5% methylcellulose. No untreated seed received any treatment. The mean values followed by the same letter are not significantly different, P = 0.05. 5 They do not agree with the invention. For comparative purposes only. 6 Parent strain.

EXAMPLE 8 This example describes the colonization of the roots of wheat roots by wild strains and transgenic strains. Colonization of the rhizosphere. Colonization of the rhizosphere of the transgenic strains was characterized using the tube bioassay as described in the previous trial on suppression of the disease. Wheat seeds coated with bacterial cells from the test strains were planted at a dose of 103 CFU / seed. The numbers of CFU present in the seeds were determined at 66 hours and 1 week after planting by dilution plating on KMB medium modified with rifampicin and cyclohexamide at 100 μg / ml each. Results The results are shown in Table 9. As can be seen in the data, the transgenic strains of the invention reached the same population density as the parent strain, 66 hours and one week after planting the treated seeds. TABLE 9 Days after planting Cepa3 66 hr. 7 days (CFU x 107 / seed) (CFU x 107 / g root) __ Pseudomonas fluorescens Q8rl-96 1.4 A "Z32-97 1.1 A 11.7A Z33-97 1.1 A 16.2 A Z34-97 1.1 A 16.0 A Z30- 97 1.5 A 13.2 A a The wheat seeds were coated with a dose of 103 CFU / seed.The mean values followed by the same letter are not significantly different, according to Fisher's least significant difference test (LSD) to P = 0.05.

EXAMPLE 9 The following example describes the physiological features and patterns of substrate utilization. The illustrative parent strain Pseudomonas fluorescens Q8rl-96 was compared to strain Q2-87 P. fluorescens (which also has a locus for the antibiotic 2,4-diacetylchlorchlorucinol). The strains show physiological features and substrate utilization patterns typical of P. fluorescens, as described in the Bergey Manual (see Table 10, below). TABLE 10 Q8rl-96 Q2-87 Gram Reagent - - Shape Rod Cane Fluorescent pigment + + Oxidase + + Arginine dihydrolase + + Fermentation of glucose - - β-galactosidase - - Gelatine hydrolysis - + Denitrification + + Use of: D -glucose + + L-arabinose + + Sucrose + + propionate + + Butyrate - - Sorbitol + + Adonitol - - D-mannitol + + N-acetyl-D-glucosamine + + Maltose - - D-gluconate + + Caprate + + Adipate - - L-malate + + Citrate + + Phenylacetate "*" EXAMPLE 10 This example describes the RAPD analysis to identify the transformed strains with characteristic banding pattern. The RAPD analysis with M13 primer was used to compare the transformed strains with the parent strain Q8rl-96. DNA amplification was carried out in a 25 μl reaction mixture, containing 5 μl of a diluted suspension of heat-lysed cell, 1 x GeneAmp PCR buffer (Perkin Elmer Corp., Norwalk, CT), 200 μM each one of dATP, dTTP, dGTP and dCTP (Perkin Elmer), 80 pmol of primer M13, and 2.0 U of AmpliTao DNA polymerase; (Perkin Elmer) Each mixture was covered with a drop of mineral oil. The amplifications were performed in a Perkin Elmer 480 Thermal Cycler. The PCR program consisted of an initial denaturation at 94 ° C. for 1 minute, 30 seconds, followed by 2 cycles of 94 ° C for 30 seconds, 36 ° C for 30 seconds, 72 ° C for 2 minutes, followed by 40 cycles of 94 ° C for 20 seconds, 36 ° C for 15 minutes seconds, 45 ° C for 15 seconds, 72 ° C for 1 minute, 30 seconds, followed by 1 cycle of 72 ° C for 10 minutes. Samples (9 μl) of the PCR products were separated on an agarose gel in 2.5% buffer solution in 1 x TBE (90 mM Tris-borate, 2 mM EDTA (pH 8.3)) at 75 V for 5 hours. The gel was stained with ethidium bromide for 60 minutes, and the PCR products were visualized using a UV transilluminator. The transformed strains Z32-97, Z33-97, Z34-97 share the following bands with the parent strain P. fluorescens Q8rl-96: 330 ± 20 bp; 600 bp ± 50 bp; 700 ± 50 bp; 800 bp ± 50 bp; 900 bp ± 50 bp and 1100 bp ± 60 bp. The transformed strain Z30-97 shares the following bands with its parent strain Q8rl-96: 6000 bp ± 50 bp; 700 ± 50 bp; 800 bp ± 50 bp; 900 bp ± 50 bp. 1/9 List of Sequences (1) GENERAL INFORMATION: (i) APPLICANTS; The United States of America, as Represented by the Secretary of Agriculture and Washington State University Research Foundation (ii) TITLE OF THE INVENTION: Biocontrol Agents to Control Root Disease. (iii) NUMBER OF SEQUENCES: 10 (iv) ADDRESS FOR CORRESPONDENCE: (A) RECIPIENT: Margaret A. Connor, USDA-ARS (B) STREET: 800 Buchanan Street (C) CITY: Albany (D) STATE: CA (E) ) COUNTRY: USA (F) POSTAL CODE: 94710 (v) COMPUTER LEADABLE FORM: (A) TYPE OF MEDIA: Floppy disk (B), COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) PROGRAM : Patentln Reléase # l.ó, Vers, # 1.30 (vi) INFORMATION ON THE CURRENT APPLICATION: (A) NO. OF APPLICATION: US 08 / 974,938 and 08 / 994,035 (B) F. SUBMITTED: 20-11-1997 and 18-12-1997 (C) CLASSIFICATION: (viii) INFORMATION OF THE EMPLOYEE / AGENT: (A) NAME: Connor , Margaret A (B) REGISTRATION NUMBER: 30043 (C) NO. OF REFERENCE / RECORD: 0027.97 (ix) INFORMATION FOR TELECOMMUNICATIONS: (A) PHONE: (510) 559-6067 (B) TELEFAX: (510) 559-5736 (2) INFORMATION FOR SEQ ID NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 10 base pairs (B) TYPE: nucleic acid (C) CHAIN: simple (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: other nucleic acid (A) DESCRPTION: / desc = "M13 primer" (iii) HYPOTHETICAL: NO 2/9 (iv) ANTI-SENSE: NO (ix) FEATURES: (A) NAME / KEY: mise feature (B) LOCATION: 1. 10 (x) PUBLICATION INFORMATION: (A) AUTHORS: Keel, C. Weller, DM Natsch, A. DeFago, G. Cook, RJ Thomashow, L. S. (B) TITLE: Conservation of the biosynthetic locus of 2,4-Diacetylphloroglucinol between Fluorescent Pseudomonas strains from diverse geographic locations. (C) JOURNAL: Appl. Environ. Microbiol. (D) VOLUME: 62 (E) NUMBER: 2 (F) PAGES: 552-563 (G) DATE: 1996 (K) IMPORTANT RESIDUES IN SEQ ID NO: l: FROM 1 TO 10 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: l: GGTGGTCAAG (2) INFORMATION FOR SEQ ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) CHAIN: simple (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: other nucleic acid (A) DESCRPTION: / desc =, Phl2a "(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (x) INFORMATION FROM THE PUBLICATION: (A) AUTHORS: Raaijmakers, Jos M Weller, David M. Thomashow, Linda S. (B) TITLE: Frequency of Pseudomonas spp. Producers of antibiotic in natural environments (C) JOURNAL: Appl. Environ Microbiol. (D) VOLUME: 63 (E) NUMBER: 3/9 (F) PAGES: 881-887 (G) DATE: March 1997 (K) IMPORTANT RESIDUES IN SEQ ID NO: 2: FROM 1 TO 20 (xi) DESCRIPTION OF: SEQ ID NO: 2: GAGGACGTCG AAGACCACCA (2) INFORMATION FOR SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) CHAIN: simple (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: other nucleic acid (A) DESCRPTION: / desc = "Phl2b" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (X) PUBLICATION INFORMATION: (A) 'AUTHORS: Raaijmakers, Jos M. Weller, David M. Thomashow, Linda S. (B) TITLE: Frequency of Pseudomonas spp. Producers of antibiotic in natural environments. (C) JOURNAL: Appl. Environ. Microbiol. (D) VOLUME: 63 (E) NUMBER: 3 (F) PAGES: 881-887 (G) DATE: March 1997 (xi) DESCRIPTION OF: SEQ ID NO: 3: ACCGCAGCAT CGTGTATGAG (2) INFORMATION FOR SEQ ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 7198 base pairs (B) TYPE: nucleic acid (C) CHAIN: simple (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO 4/9 (vi) ORIGINAL SOURCE: (A) ORGANISM: Pseudomonas fluorescens (ix) CHARACTERISTICS: (A) NAME / KEY : CDS (B) LOCATION: add-on (1810-2859) (C) OTHER INFORMATION: / product "PhID" ft (x) PUBLICATION INFORMATION: (A) AUTHORS: Thomashow, LS Bangera, MG (B) TITLE: U41818 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4: GTCGACCCAG TAAAGGCAGA CAATGCATGG CTCGCTGCTG ATGCTTTGGG CGTGGTAAGG 60 AATCTGCAAC GGGTCCCCGT CAGTCTTGAG CAATCGACCG TTGCCTTTGT AATACCAGGC 120 CTGTTCCGTC TTGGCGGTGC ACAGTGTGAT CTCGCTGACC TGAACAGCCA AGTTGTCATC 180 GGGTTTGAGC GGCGGCCTGT ATTTCGCGCC TGTGGGCGAG AGCGGATAGG GCGTCGATGG 240 GAGGCTCCTG AGGTAGGATT CCCAATCCAT GTTCAGGCGA GCGCAGTGGC TCAGGGCTTC 300 ATAAAGGTTT CCCTGCGGTG ATCGTACTTC CAGGCTTTGC CCGGATTTGA AGTCGAATAC 360 ACCCAGCCCG CGCAGCGGCT GCGGCGAGTC CAATAGCATT ACGTAATGCG TCATCACTTA 420 TCCTCCAGCG TCAAGCGAGC GGGCAGGGGG CCGTAGTCCG CACGGTCATT GAAAACAGGG 80 CTGGCTTCCT TGAGCCGCAG GGACAGCAAC CCGCCCATCA AACTACCAGC CAGTGCCAGA 5 0 AACAGAATGG CGGTAAGCCC CCAGTTCACG GCGATGTACC CGGCGACCAC GGGCGCAACG 600 CCACCGCCCA GGATTTCTCC GCAACCCACC ACCAGGCCCG TAGCGGTGGC CAGTAAACTG 660 GGTGGCACTG ATTCGCTGGT CAGTGGGCCG ACGGTGATGC AGATCAGGCT GAAATTGATG 720 AAAGATAAAA AGAACAGCTG GAGGAACAGT AGCCACGGTA ACGGCGGGGA AATGATGAGC 780 AAGCCGACCA GTAGTGTGCT GATCAGGAAG CAGATGGAAA CGACAGGCTT GCGGCCCAGT 840 TGGTCAGACA AACCGGGAAT GACGAGCTGG CCGAAAAAAC CACCCAGGCC GATCGCGGAG 900 ATGATCATGG CCATGGAGAA ATTGCTCAGG TGCAAGACGT CTGTCAGGTA GCTGGGGAGC 960 AGGGCGCACA GGACGAATTG GCACGTCAGT ATGCATAGCA TCAAGGCAAT GTTGAGGCGC 1020 ACGTTGCCGC TGGACAGGGC TGTTCGCCAT TGGCTGCCGG AGGGTTCTAC GAGCGGCCTT 1080 GGATGGGGCG CCTGGCTCGG TTGGTAGGTT CGATACAGAT ACCAGGCCAC CAGCAGGCCC 1140 GGCAACGAGA TGATGGCGAA CACGGCGCGC CACGATCCGA ACATTTCAAA CAATACGCCC 1200 GCCAGCAGCG GCCCCAGGCA CAGGCCGATG ATGGGAAACA GTGCCTGCTG GATGCCCAGG 1260 TTGAGCCCGC GTCGGCACGG CTGCGAAACT TCATCGGTGA CAATGATGCT GACCGGGGTG 1320 AAGGCGCCTT CGCAGATCCC CATCAAGGCG CGCAGGAGCA CCAGGCCCAT AAGGCTTGAG 1380 5/9 TTGGTGCCCA ATCGCCTGAT AGCAACGCCC ATGAAGAGGG CCGAGCCTCC CCAGGCAAAT 1500 GCCAGGATCG CCGATAACAG GCCCAGGTCC TGATAGTCCA GGGCCAGGTC ATGCATGATC 1560 ACCGGGAACA ACGGCATGAT AATGAATCGA TCAAGTCCTA CCAGCCCGAA GCTCAGCGAC 1620 AAAAGAACGA CCATGCGTCT TTCGTAGCCA CCCCAAGGTC GAGTGGCAAG ATACGTACTC 1680 TCCATGTTCT TCCCCTTCTT TCCTTAGCCC TTTCGACGTT TTCTCGAAAC GGGTGAACGC 1740 TTGTGTTCGA TACTCCTGTA GCCAGGGGCG GATCCGCCCC CGGCTTGGTG CGTGCAATGT 1800 GTTGGTCTGT CAGGCCACCC ACTTGCCCAC GGCCATTTCA GCTGTGAAGC CAGGGCCGAA 1860 GGCTGCCAGC ATGCCGGTCG CTCCATTGGC CGGCCCGCTG TCGAACTGGC GCTTGAGGAC 1920 GTCGAAGACC ACCACGCTGG CAATATTGCC GGCCTCGCTC AAGCTGTCGC GAGACTGCGC 1980 GACCCTGCCA GGTTCCAGAT CGAGCTGCAG CACCAGCTCA TCAAGAATTT TTCGTCCACC 2040 GGTGTGGAAG ATGAAAAAGT CATTTTGAGC GCAATGTTGG TTGAAGGTCT CGAAGTTCAA 2100 TTCCTCCATC ATCGGGGCCA CGTCTTTAAT GGAGTTCATG ACGGCTTTGT CCAGGGTGAA 2160 GTGAAAGCCG CTGTCCTTGA CGTCATATTT AATGTAGTGC TCGCTGTCAG GCAGGAAATA 2220 AGAGCCGGTT TTGGCGATCT TGAATCCCGG CGCCTTATCG TCGGCGCGCA TTACGCAGGC 2280 CGAGACGGCA TCGCCGAACA GCGCTGCGGA TATGAACGCG TGCAACTTGG TGTCCTGTGG 2340 TTGATAGCAG AGTGACGAGA ACTCCAGCGA GACAATAAGG GCGTGGTTGT CTGGAGACAG 2400 GCTGGCAAAG TCGTTGGCTC GATTAATCGC CGCGGCGCCT GCCACGCATC CCAATTGAGC 2460 GATCGGCAAT TGTACGGTCG ACGTTCGCAG TCCCAAGTCA TTGATCAGGT GGGCTGTCAG 2520 CGATGGCATC ATGAACCCGG TGCAAGAGGT AACGGCGACC ATCCGGATGT CGTCCGTGGT 2580 CAAGCCCGCG TTTTCAATGG CCTGGCGCGC GGCGATTGAA GACATGCGGC GAGCCTCTCG 2640 CTCATACACG ATGCTGCGGT GGGTAAAGCC GGTATGCACC GCAAGTTCAT CGATGGGCAA 2700 GACCAGATAC CGTTCATTGA CTTGGGTGTT TTGAATCATC CGTTTAGCCA ATGCCATGCG 2760 CGGATGATCG TCATGCAACT GTTCCAAGTG ATCGATCATC TGTTGTTGGG TAATTTTGTA 2820 ATGCGGGAAA AGCAAGCTGG GTTTGCAAAG AGTAGACATG ACAAGTCCTC GGCTGAAAGC 2880 CAATAAAGAG TAGAAAACCA CGTTTAAGGC AATGGCAAAG CAGGACTCTG AAAAGCAGAA 2940 TCAAACAACG GGCCGGTTGG CCGGAAATAG CGACTGTTGT TATGGATGGC GCGGTATGCA 3000 GCAGTAACTT GTTTGTTATT TCGCCAATAC GAATTTATAA GCGTATTGCC ACGCCAGGTT 3060 GCTTTCCCGA ACGTGCTTGC GAATAACCAT TCGCACTGGT GCTCCAGTCA CGACTTGCCG 3120 GGGATCGACG ACATCGACGA TTTCCGAGGC GATCACCAAG CCATCGTCCA GGCGCACCAT 3180 TGCCATGAAG CGCGGGACGG TTTCGCCATA TCCCATGGCC GCGAGAATGG GGTTTTCAGC 3240 ATGGGCGCTG ACCTGGATCG TGCCGGTGCG TGCGCAGCGA TACGGTTCCA CGTTCAATGA 3300 6/9 GTTGCATGCG CCGCAGACGG TGCGCCGTGG GAAGAAGATT TCTTCGCAAT CCTGGCAGCG 3360 GCTGCCTTCG AGACGATATT TTCCGCCATG TTCGCGCCAT TCGCGCAACA TGCTGGCGGT 3420 GGTCATGCGG TGTATTTGTT CTGGGTAAAG GGACATGCTC GGCTCCTTAA TCGTTGGAAA 3480 GCACAATGAC GCTGTTATGC GCGGCGTAAC CGCCCAAGTT CTGCGAGACG CCAATGCGAG 3540 CGTCCTTGAC TTGGTTGTTG GACTCGCCGC GAAGTTGTCG GAACAGCTCG GTAATGTGCA 3600 GGATGCCGTC GCAACCAGAG GCGTGGCCGC GGCCAATATT GCCGCCATCG GTGTTTAATG 3660 GCAGTTGCCC GTCGAGGGCT ATGCCGCCTT CCAATACAAA GTCGCCTGCC TGGCCTGGAC 3720 CACATACGCC CATGGATTCC ATCTGAATCA ATCCGGCACC CAGCAAGTCG TAGACTTGGG 3780 CCACATCGAT ATCCTTGGCG GTGATGCCGG CTTTTTTGTA GGCGATTTCG GCGCAAGCAA 3840 TGGAGTTGGC GGAAACCGCC ATGCCGACGT CTTTTGGCAG GCCTGGATAT TTCAGGGTCG 3900 GGTTGTGATA GCGCGTCCCG AAATAATGGG ATACGCCGGT ATAGGCACAA CCACGGACGA 3960 ATACCGGTTG GGTCGTGTAG CGGTGCGCCA GGTGTTCGGC GACCAGGATG GCGCAACCGC 4020 TGGCTTCACC CCAGGCCAGC ATCGAGCCAC ATGCTTCGCT GTTCTTGAGG GTTTCAAGGG 4080 ATGGCACCGG CACGCCATAG CGGGTTGCCG TGGGCGTGTT GTGCGCATAG ATGCGCATTT 1 0 GCCGACCAAA CGTTGCCAGG ACATCCGCTT CGCGTCCTGC ATAGCCAAAT TTTTCAAAAT 4200 ATTCGGCGGT TGCGAGGGCA AAGGCGTCGG TGTGCGAAAT GCCCAGGAAA TAATCGTACT 4260 CACATTCGGT ACTGGAGCCG ATGTATTCGG CATAGTTGAA GTGGTCGGTC ATTTTTTTCAA 4320 AGCCACCACA CAGGACGATG TCGTACTCAC CCGAGGCGAC CATCTGATGG GCCATCTGAA 380 AGGAAACCGA GCTGCTGGTG CAGTTGGCAG TGCTCATGAA CGTCGGGGCA GGGCTGATGC 4440 CCAGGGCATC GGAAATAGTC GGGCCCAGGC CGCCGTATTC GGAAATACCT TCACCGTGAT 4500 ATCCATAAGC GACTGCCTGA AGTTCACGGG GATGCATCTT GATGGCGTTG AGCGCCTGAT 4560 AGGCGGACTC GACGATCATC TCCTTGAAGG TTTGACGGAC TCTGGAGCTG CCGGGTTTGG 4620 AAGTATAGGC AGCCGAAACG ATAGCAACGC GTCGTGCGCT CATTGGAAGT GCTCCTTGCT 4680 GGATGGTTGG GAATCAGAGG TAGGCTGTCA GGGCGTAGTC AGGCCGCAAG TATTTGAACT 4740 CGTACTTGAT CGACGTCCCG TAATCCACGT AATACTTGTC TTCCAGCAGC GTGCGCAGCG 4800 CAACGTTGGT CTTTTGGTAG GCTTCGATGG CATCGGTCAC TGTCAACGCA ATCGCATCGC 4860 TGCCCGCACC AAACCCGTAC GACACCAAGA GGATTTTTTC ACCCGGACGC GCTCGGTCCA 4920 GTACGCTCAC CAAGCCCAGC AACGGACTCG CGGCCCCCGC ATCACCGACA CTCTGGGCAT 4980 AAATGCCAGG TTCGATCTGC GCTTTGGTGA AGCCCAGGCC TTTGCCAAGA GAGAAGGGGG 5040 TCGAAACCAG GTTTTGCTGG AATACGACAT AGTCGAAATC GCTGGCCTGT ACATTCATCT 5100 TGGCCATCAA TCCCGACGCA GCACGATGGG TCTGGTCTTC AAGGCCAATG CTGTTCTTGT 5160 CGGAGCCCAG CCCCATTCCT GAGCGAATGT AGCGGTCTCC CTGGGGGCGG ATGTTGTCAG 5220 7/9 CCACATCGGC GGCGCAAGAA AAGCTGGCAT CGAAATGCGC GATCACATTT TCAGTACCCA 5280 ACAACAGTGC GGCGGCTCCC GCTCCGGCGT AGGACTCGGT CAAGTCGCCG GGGGCGGTGT 5340 TGCGGTTGAT CGTATCGGCG CCTATTGCCA GTGCATTGCC GGCCATGCCC GAGGCTACCA 5400 GGGCATAGGC GATCTGCAGG GCGCTGGTGC CTGATTTGCC GGCAAACTGT ACGTCCGCGC 5460 ft AGAAGGCGTC ATAACCGCAG CCGAGCATTT CCAGAATGAC CGCGGCCGAG GCGCGGGAGT 5520 CATATGGGTT GGTGCACGTA CCCAGGTACA GCGCTTCCAG GTCGCAAGAA GGGGCTTTGT 5580 CCAGCGCACG TTGAGCGGCC AGGACACTCA AGGTAATGAC GTCCTCATCG GGTTGGAGTA 5640 CAGCCCTTTC AACGACGCCC AGTTGGTTGG TGACCAGACT CAAGTCTGTG TTTTTCCAGA 5700 CGTGGATCAC GTCTTCCACT TTAAGGCGGC ACACCGGGAT GCCCGCGCCA TAGCTCACAA 5760 TTCCTACTTT ATTCACGTGT ACTTCCTCCA GATTCCTTTC TTCACCTGCC AGCGGATAGC 5820 CGTGACCGAT GCATGAAATA TTTAGAAACT ATCTAACGGT GCCCGCAAAG TGTCGTTGGC 5880 AGTCCTATGC CCGGAAATCG GGCTCCTCAA GGGGGAAAAC TACAGTTCCT TTGAGGGAGA 5940 ACGGGTTTAT TATCCTTCTA TTATTATGTA TGATACGAAA CGTGCCGTAT CGTTAAGGTC 6000 TTGTTAAAAA TTGATGACTA TTTATCGGGT TTCTTCCTAT CTAGTGGCAA GTTCCGCTAT 6060 TGAGGTGTGC AGTTAAGCAG AAÁCTTAGAT CATAAAAACA TACAAAACGA AACGATCCGT 6120 TTCATTGCTT TTCGAGAGAA TCCTATACCT TGCGTCTCTT TTGTCAAGCG CCATATTGGA 6180 GATTTTGAAT TATGGCCCGT AAACCGTCTC GGAGCTCCAT TGGCTCATTG AGGAGCCCAC 6240 ATACGCACAA AGCGATCATC ATCTCCGCTA TAGAAACACT CAAGGAGTGC GGTTATTCAG 6300 GGTTGAGTAT CGAGGCTGTG GCTCGCCGTG CCGGCGCGAG CAAGCCGACC ATCTATCGAT 6360 GGTGGGGTAA CAAGGCGGCT TTGATCGCCG AAGTCTACGA GAGCGAAAGC GAGCAGATTC 6420 GCAAGGAGCC TGATAAAGGA TCCTTCAAGG AGAACCTCAA TTTCCTGCTG CTCAATCTGT 6480 GGAAGGTCTG GAGAGAAACG ATTTGCGGGG AGGCGTTTCG GTGTGTCATC GCTGAAGCCC 6540 AGCTCGACCC CAGTACGCTG CCCAAGCTGA AGGATGAATT CATGGAGCGT CGTCGGGAAT 6600 TGCCGCGAAA GCTGGTGGAA AACGCCATCC AGCAAGGTGA GTTGCCCAAG GACACGTCCC 6660 GTGAGTTGTT GTTGGACATG ATCTTCGGAT TTTGCTGGTA CAGGCTGTTG ACTGAGCAAC 6720 TGGAAGTGGA GGGTGACATC AATGAATTCA CGACGCTTCT GTTGAACGGC GTGTTGCGTA 6780 CGACTTCGGC GGCGGAGTAA GGCGCCGCCG AAGCCTGTTC AAGGGTGAGG ATTGGCCTTA 6840 CGCCGCGCCG CTGAACTGTG CATGAAGGCC AGGCAGGATA CTGGCCAGGT GGTTGAACTC 6900 ACACAGATCA TGCACAGCAA ATTCATAAGC CAGGGTTTCC AGTTCGGCTT CCCCAAACCC 6960 GTTTTCCTTC AACAACTGCG CGGCGCGTTC GGCACCGGGA AAACGCAGCA TCGCTGGGTG 7020 GCTGCCCACC CAGTAACGGC TGGTCAGGTA CAAGCCTTCG GGGCATTCCT TGAACAAGTG 7080 8/9 CACCATGAGC GATATCGGCA CTTGCGGCTG ATCCGCCAGG CTCATCAAGG CGCTGACGCT 7140 GCCGTCTATT TTTGATTCGC GATACAGGTC CGCAGAGAAA CCCAGCTCGC ATGGATCC 7198 (2) INFORMATION FOR SEQ ID NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 350 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: S: Met Ser Thr Leu Cys Lys Pro Ser Leu Leu Phe Pro His Tyr Lys lie 1 5 10 15 Thr Gln Gln Gln Met lie Asp His Leu Glu Gln Leu Hís Asp Asp His 20 25 30 Pro Arg Met Ala Leu Ala Lys Arg Met Met Gln Asn Thr Gln Val Asn 35 40 45 Glu Arg Tyr Leu Val Leu Pro lie Asp Glu Leu Ala Val His Thr Gly 50 t 55 60 Phe Thr His Arg Ser lie Val Tyr Glu Arg Glu Ala Arg Arg Met Ser 65 70 75 80 Ser lie Ala Ala Arg Gln Ala lie Glu Asn Wing Gly Leu Thr Thr Asp 85 90 95 Asp lie Arg Met Val Wing Val Thr Ser Cys Thr Gly Phe Met Met Pro 100 105 110 Ser Leu Thr Wing His Leu lie Asn Asp Leu Gly Leu Arg Thr Ser Thr 115 120 125 Val Gln Leu Pro lie Wing Gln Leu Gly Cys Val Wing Ala Gly Wing Wing 130 135 140 lie Asn Arg Wing Asn Asp Phe Wing Ser Leu Ser Pro Asp Asn His Wing 145 150 155 160 Leu Lie Val Ser Leu Glu Phe Be Ser Leu Cys Tyr Gln Pro Gln Asp 165 170 175 Thr Lys Leu His Wing Phe lie Wing Wing Leu Phe Gly Asp Wing Val 180 185 190 Wing Wing Cys Val Met Arg Wing Asp Asp Lys Wing Pro Gly Phe Lys lie 195 200 205 Wing Lys Thr Gly Ser Tyr Phe Leu Pro Asp Ser Glu His Tyr lie Lys 210 215 220 9/9 Tyr Asp Val Lys Asp Ser Gly Phe His Phe Thr Leu Asp Lys Wing Val 225 230 235 240 Met Asn Ser lie Lys Asp Val Ala Pro Met Met Glu Glu Leu Asn Phe 245 250 255 Glu Thr Phe Asn Gln His Cys Wing Gln Asn Asp Phe Phe He Phe His 260 265 270 Thr Gly Gly Arg Lys He Leu Asp Glu Leu Val Leu Gln Leu Asp Leu 275 280 285 Glu Pro Gly Arg Val Wing Gln Ser Arg Asp Ser Leu Ser Glu Ala Gly 290 295 300 Asn lie Ala Ser Val Val Val Phe Asp Val Leu Lys Arg Gln Phe Asp 305 310 315 320 Ser Gly Pro Wing Asn Gly Wing Thr Gly Met Leu Wing Wing Phe Gly Pro 325 330 335 Gly Phe Thr Ala Glu Met Wing Val Gly Lys Trp Val Wing * 340 345 350

Claims (28)

1. 59 Novelty of the Invention 1. A biologically pure culture of a strain of bacteria Pseudomonas spp. fluorescent that contains a biosynthetic locus that codes for the production of 2,4-diacetylphloroglucinol.
2. 2. The biologically pure culture of claim 1, wherein said Pseudomonas spp. fluorescent has a biosynthetic locus that codes for the production of phenazine-1-carboxylic acid stably introduced into its genome.
3. 3. The biologically pure culture of claim 1 or 2, wherein said Pseudomonas spp. Fluorescent has bands at 600 bp ± 50 bp; 700 ± 50 bp; 800 bp ± 50 bp; 900 bp ± 50 bp, identified by the Random Amplified Polymorphic DNA (RAPD) analysis using the M13 primer.
4. 4. The biologically pure culture of claim 3, which is also characterized as having bands at 330 bp ± 20 bp and 1100 bp ± 60 bp.
5. The biologically pure culture of one of claims 1 to 4, wherein said strain is also characterized as having the ability to suppress diseases caused by the fungus Gaeumannomyces graminis (Gg) in small grain crops grown in fields, and in In the case of turfgrass, said capacity is preferably equivalent to a level of biocontrol obtained when said small grain crop is cultivated in a soil in a declining state of pietin.
6. 6. The biologically pure culture of claim 1 or 2, wherein said strain is also characterized as having the ability to suppress plant root diseases caused by the fungus Rhizoctonia.
7. The biologically pure culture of claim 1 or 2, wherein said strain is also characterized as having the ability to suppress plant root diseases caused by the fungi Gaeumannomyces graminis (Gg) or Pythium.
8. 8. The biologically pure culture of one of claims 1 to 4, wherein said strain is also characterized as having the ability to colonize the roots at a population density averaging at least 105 units of 60 colony formation (CFU) / root gram, including the associated rhizosphere soil, for at least 5 successive growing cycles.
9. 9. The biologically pure culture of claim 8, wherein the colonization capacity of the root of said strain is not affected by the type of soil.
10. The biologically pure culture of claim 1, wherein said fluorescent Pseudomonas strain has the identification characteristics of P fluorescens NRRL B-21806, NRRL B-21807, NRRL B-21808, NRRL B-21905, NRRL B-21906 , NRRL B-21907 or NRRL B-21908.
11. 11. The biologically pure culture of claim 1, further including an agricultural carrier.
12. 12. A method for screening bacteria for the selection of strains having the properties of claim 1 or 3, comprising: (1) successively growing a small grain crop in cycles or a turf in natural soil suppressant to enrich Pseudomonas spp. Phl producing fluorescent, comprising steps e (a) growing seeds of a small grain crop or growing turf in a soil in declining state of piedin (TAD soil) in a greenhouse for at least 3 weeks and under conditions that be effective to support the growth of said small grain crop or turf to obtain seedlings; (b) collecting said crop soil and roots of said seedlings from the small grain or turf crop grown in that soil and mixing them together; (c) repeating steps (a) to (b) for at least a total of 4 successive cycles, wherein the mixture of (b) is used to grow seeds in a subsequent cycle; (2) isolate strains of fluorescent Pseudomonas bacteria potentially suppressing the roots and associated rhizosphere soil of said small grain crop or turf successively grown in step (1), cultivating said strains in a Pseudomonas selector medium for a period and in effective conditions for the growth of Pseudomonas and select the strains that grow in the medium; 61 (3) classifying strains isolated in step (2) to select a strain containing a biosynthetic locus that codes for the production of 2,4-diacetylchloroglucinol (Phl) by hybridizing a colony of said strains with a specific probe of 2. , 4-diacetylchloroglucinol and selecting the strains that hybridize said probe; (4) carrying out a Random Amplified Polymorphic DNA analysis using the M13 primer, and selecting as a Gg suppressor strain those strains having bands at 600 bp ± 50 bp; 700 ± 50 bp; 800 bp ± 50 bp; 900 bp ± 50 bp,
13. 13. The method of claim 12, further including a step between steps (3) and (4), comprising confirming the Phl-producing strains using primers that amplify sequences with the biosynthetic locus of Phl and selecting the strains that provide a positive PCR reaction.
14. 14. A method for controlling a root disease caused by Rhizoctonia, Pythium or Gaeumannomyces graminis (Gg) in plants susceptible to said root disease, which comprises growing said plant in the presence of an effective amount of biocontrol from a culture of a Fluorescent Pseudomonas strain of one of claims 1 to 4.
15. 15. The method of claim 14, wherein said plant is selected from the group consisting of harvesting of small grain, turf or food, fibrous or ornamental plants.
16. The method of claim 14, wherein the soil or furrow for cultivating said small grain crop or turf is treated with an effective amount of biocontrol of the gg of the strain before cultivation.
17. 17. The method of claim 14, wherein the turf is treated with a bacterial treatment solution comprising an effective amount of biocontrol of the Gg of said strain and an appropriate liquid carrier.
18. 18. The method of claim 14, wherein the Pseudomonas strain is a P. fluorescens strain having the identification characteristics of NRRL B-21806, NRRL B-21807, NRRL B-21808, NRRL B-21905, NRRL B -21906, NRRL B-21907 or NRRL B-21908. 62
19. 19. The method of claim 15, wherein said seed has a concentration of about 102 to 106 CFU per seed.
20. The method of claim 17, wherein said treatment solution has a concentration of about 108 to 1010 CFU per ml. of ßl 5 solution.
21. The method of claim 14, wherein the roots of said plant are immersed in a bacterial suspension of 102 to 10d CFU per ml of suspension.
22. 22. An agricultural composition to control a root disease caused by Rhizoctonia, Pythium or Gaeumannomyces graminis (Gg) in plants 10 susceptible to that root disease; the composition comprises a suitable carrier and an effective biocontrol amount of a biologically pure culture of a fluorescent Pseudomonas strain of one of claims 1 to 4.
23. The agricultural composition of claim 22, wherein the carrier is selected from a group consisting of water, buffer solution, methylcellulose, peat 15 and vermiculite.
24. The agricultural composition of claim 22, wherein said strain is at a concentration of about 108 to 1010 CFU per ml of liquid carrier, or 107 to 109 per gram of solid carrier.
25. 25. The agricultural composition of claim 22, wherein said Pseudomonas strain 20 is a P. fluorescens strain having the identification characteristics of NRRL B-21806, NRRL B-21807, NRRL B-21808, NRRL B-21905, NRRL B-21906, NRRL B-21907.
26. 26. A seed of a small grain crop to which an effective amount of biocontrol of a biologically pure culture of any one of claims 1 to 3 is applied.
27. 27. The seed of claim 26 wherein said seed has a Concentration of around 102 to 105 CFU per seed.
28. 28. A method for preparing a transgenic Pseudomonas fluorescent bacterial strain having the properties of claim 1 or 2, which 30 comprises: 63 (1) cloning genes that include a locus of phenazine-1-carboxylic acid under the control of a promoter, and whose locus encodes the production of phenazine-1-carboxylic acid. (2) construction of a transposable copy of said cloned locus of phenazine-1-carboxylic acid in a transfer system having the ability to mediate the stable insertion of said locus of phenazine-1-carboxylic acid within the Pseudomonas receptor strain fluorescent and placing said transfer system within a donor bacterial strain; and (3) by bacterial conjugation, stably inserting said transfer system of the bacterial donor strain into the recipient parent strain of fluorescent Pseudomonas containing a biosynthetic locus that codes for the production of 2,4-diacetylchloroglucinol, and which has bands a 600 bp ± 50 bp; 700 ± 50 bp; 800 bp ± 50 bp; 900 bp ± 50 bp, identified by Random Amplified Polymorphic DNA analysis using the M13 primer, to obtain a transformed strain of Pseudomonas spp. fluorescent that has a biosynthetic locus that codes for the production of phenazine-1-carboxylic acid stably introduced into its genome and that contains a biosynthetic locus that codes for the production of 2,4-diacetylchloroglucinol