Biocontrol agents for take-all

This application is a continuation of application Ser. No. 08/974,938, filed Nov. 20, 1997, now abandoned which is incorporated herein by reference.

1. Field of the Invention

The present invention relates to biocontrol of diseases caused by the soil-borne fungus Gaeumannomyces graminis . In particular, the invention relates to strains of fluorescent Pseudomonas species (spp.) which have unique root-colonizing ability for small grain crops and biocontrol activity for diseases caused by Gaeumannomyces graminis in small grain crops and take-all patch in turf grass. The invention further relates to isolation and identification of the unique strains, and application thereof to control plant diseases caused by Gaeumannomyces graminis.

2. Description of the Art

Widespread diseases of small grain crops and turf grass are caused by the soil-borne fungus Gaeumannomyces graminis ( Gg ), and result in significant economic losses due to reductions in crop yield. Take-all, a disease caused by Gaeumannomyces graminis var. tritici ( Ggt ) occurs in all wheat-growing regions of the world and is the most important root disease of wheat. Symptoms of wheat take-all include dark longitudinal lesions on roots; in severe cases, the entire root may become blackened with disease with the fungus migrating to the crown of the wheat plant (where the crown roots originate) and the tillers (stems). Severely infected wheat plants are identified in the field by their white heads which result when infection of the crown by the fungus cuts off water transport to upper plant parts causing the plant to die prematurely. Yield losses can be considerable up to 50% of the potential wheat yield. There are no resistant wheat cultivars and registered fungicides perform inconsistently. Further, growers are being increasingly challenged to grow wheat with minimum or no tillage to reduce soil erosion. These practices increase the severity of take-all and other root diseases. Although wheat is particularly susceptible to the take-all fungus, many other Gramineae such as barley, rye, and triticale can also be infected.

Traditionally, take-all has been controlled by a combination of crop rotation and tillage, practices which reduce the inoculum potential of the pathogen. However, because long rotations are often not economically feasible and tillage contributes to soil erosion, the trend in cereal production is toward less tillage and two or three wheat crops before a break. Both of these practices exacerbate take-all. There is no known source of genetic resistance in wheat against take-all, and methods of chemical control are limited. The need for agriculture to become more sustainable and less dependent on chemical pesticides has necessitated the development of alternative approaches to control take-all and other soil-borne diseases.

Other Gg fungi, for example, Gaeumannomyces graminis var. avenae ( Gga ) infects oats and grasses and have been identified as causing take-all patch in turf grasses such as bent grass. Gaeumannomyces graminis var. graminis ( Ggg ) infects some grasses and has been suggested as causing crown sheath rot in rice.

All agricultural soils show some degree of antagonism to Ggt and other soil-borne pathogens. This has been referred to as “general suppression” (N. Gerlagh, Netherlands Journal of Plant Pathology 74:(Suppl. 2) 1-97 (1968) or “general antagonism” (D. Hornby, Annual Review of Phytopathology , Annual Reviews Inc. Palo Alto, Calif. (1983), pp. 65-85). General antagonism results from the overall microbial activity in a soil. In addition, a “specific” suppression (biological control) of Ggt (known as take-all decline) develops in certain circumstances which is superimposed over “general suppression” and which results in a nearly complete control of take-all. Take-all decline (TAD) is a natural biological control of take-all, defined as the spontaneous reduction in disease and the increase in yield with extended monoculture of Ggt -susceptible small grain crops such as wheat and barley. TAD was first observed more than 50 years ago and is now recognized as a worldwide phenomenon. The similarity of TAD throughout the world is remarkable in view of the broad range of soil types, climates, and agronomic conditions under which wheat, barley, and other small grains are cultivated. Field studies have clearly indicated that the development of TAD follows a consistent pattern everywhere, requiring the continuous cultivation of a small grain and the presence of the take-all pathogen. Factors such as soil type and previous cropping history only seem to modulate the extent and speed of development of TAD. Despite the fact that take-all eventually declines, most growers abandon monculture prematurely because interim losses can be considerable. Once established, however, TAD permits a recovery in yield and persists as long as monoculture continues. Practical exploitation of TAD offers the potential as a natural biological control of take-all. However, to do this, the responsible mechanism(s) for TAD would need to be identified and applied. However, research to date has been mostly descriptive and no particular occurrence of TAD is yet fully understood. A similar decline of take-all patch caused by Gga occurs in established turf.

TAD has been extensively studied in an attempt to determine the mechanisms responsible for natural take-all suppression. The most common theories put forward to explain this phenomenon include changes in the microbiological status of the soil, build up of antagonistic bacteria, changes in the pathogenicity and population of the fungus, and presence of protective fungi (D. Hornby in “Take-All Decline: A Theorists's Paradise,” Soil - borne Plant Pathogens , Ed. 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, Calif. (1983), pp. 65-85). Homby 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 field of disease suppressive soils.

The most widely held explanation for TAD is based on microbial interactions between the take-all pathogen and specific antagonistic root-associated microorganisms (R. J. Cook and A. D. Rovira, Soil Biology and Biochemistry 8: 269-273 (1976)). Several types of evidence support a role for microbial antagonism in the suppression of Ggt . For example, suppressiveness can be transferred by incorporation of a small amount (1-10% w/w) of a TAD (suppressive) soil into a take-all conducive soil. Furthermore, the suppressiveness of a TAD soil is eliminated by pasteurization of the soil with moist heat (60° C., 30 min.), by soil fumigation with methyl bromide or by growing crops which are non-hosts of the pathogen.

Studies of the microbial antagonism involved in TAD have focused on attempts to identify specific Ggt -antagonistic microorganisms and to transfer these organisms to soil to reproduce suppression. A wide variety of microorganisms have been tested given the prevailing idea that the specific strains responsible differ among TAD soils. Cook and Rovira, 1976, supra, originally hypothesized that among the antagonistic microorganisms the fluorescent Pseudomonas spp. have a key role in TAD. U.S. Pat. No. 4,456,684 describes Pseudomonas strains which suppress diseases caused by take-all and other Gg fungi and methods for 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 take-all pathogen (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 Phl-producing fluorescent Pseudomonas spp. were present on roots of wheat grown in three TAD soils from Washington State (USA). In take-all conducive soils collected from sites near the TAD fields, Phl-producing fluorescent Pseudomonas. spp. were not detected or were detected at densities at least 40-fold lower than those in the TAD soils. Although use of microbial biocontrol agents holds great promise as a practical means to control soilborne pathogens, all published or patented biocontrol agents for take-all have the disadvantages of performing inconsistency, being soil-specific, and being unable to duplicate the level of control consistently observed in a TAD soil. No microorganism tested to date has demonstrated the disease control abilities expected of a strain involved in TAD. Thus, it is not surprising that no biocontrol agent for take-all has been commercialized. What is needed are effective biocontrol agents for take-all which duplicate the suppressiveness of a TAD soil and are effective independent of soil type, and perform consistently.

We have discovered unique biocontrol agents for control of the diseases caused by the soil-borne fungus Gaeumannomyces graminis ( Gg ) in small grain crops and turf grass. The invention encompasses unique strains of fluorescent Pseudomonas species (spp.) which suppress (inhibit the incidence of or reduce the incidence or severity of) diseases caused by Gg , such as take-all, at low doses, and have root-colonizing ability which is greater than any previous biocontrol agent of Gg . In addition, the root colonization ability and biocontrol activity of the strains are not affected by soil type.

The biocontrol agents of the invention provide biocontrol which is consistently greater than that of all known biocontrol agents for diseases caused by Gg , such as take-all. Additionally, the strains have the further unique property of being able to duplicate the level of biocontrol observed in a TAD soil.

The biocontrol agents of the invention comprise biologically pure cultures of strains of fluorescent Pseudomonas spp. which contain a biosynthetic locus which encodes for the production of the antibiotic 2,4-diacetylphloroglucinol, which have a unique genotype as shown by a characteristic unique Random Amplified Polymorphic DNA (RAPD) profile, exhibit biocontrol activity at dose levels 10 to 1000 times lower than take-all-suppressive microorganisms known heretofore, and exhibit superior root colonizing ability as demonstrated by 10 to 1000-fold higher population density and extended colonizing activity. We have found that exemplary strains of the invention have the ability to colonize roots at a population density averaging at least 10 5 colony forming units per gram of root, including the rhizosphere soil, for at least 7 successive growth cycles. Such root-colonizing ability is unprecedented. Further, the novel strains are not affected by soil type.

The invention further comprises methods of isolation and identification of these unique strains. A protocol for screening of bacteria is shown in FIG. 2, and the screening method is described in detail, below.

A further aspect of the invention is application of the unique strains or compositions comprising the strains for biocontrol of plant diseases caused by Gaeumannomyces graminis . When used as a seed, soil, furrow treatment or drench, the unique strains of the invention have the ability to suppress Gg under field conditions. Application of the strains to seed or soil showed the unprecedented duplication of suppression of take-all equivalent to natural take-all decline (see Table 1, below), which has never been shown before. Further, because the strains suppress take-all at low dose levels, the strain can be grown and applied at a cost of nearly 10 to 1000-fold less than other currently existing biocontrol agent of take-all. In addition, because the strains are responsible for natural TAD, it is envisioned that they need only be applied only once in a field. All other biocontrol agents of take-all require repeated application.

In accordance with this discovery, it is an object of the invention to provide unique strains of fluorescent Pseudomonas spp. which provide biocontrol of the diseases caused by Gg which is greater than known biocontrol strains.

It is also an object of the invention to provide biocontrol strains that have root colonizing ability (higher root population density and extended activity) which is greater than any known biocontrol agent for take-all. This property is particularly valuable because it is known that for suppression of root diseases such as take-all, increases in root colonization result in greater and more consistent biocontrol activity.

Another object of the invention is the provision of biocontrol agents for control of diseases caused by Gaeumannomyces graminis in small grains and turf grass which are not affected by soil type.

It is a further object of the invention to provide methods based on RAPD analysis for selecting the strains of the invention having a characteristic banding pattern.

A still further object of the invention is the provision of methods for biologically controlling take-all in small grain crops and patch in turf grass using the strains of the invention and agricultural compositions which incorporate the strains.

Other objects and advantages of the invention will become readily apparent from the ensuing description.

FIG. 1 is an image which shows the banding patterns (RAPD) of P. fluorescens strains Q8r1-96, ML4.9-96, and L5.1-96. Lane 1 shows a 1-kb ladder as a reference.

FIG. 2 shows a protocol for isolation and identification of the unique fluorescent Pseudomonas spp. of the invention from take-all suppressive soils.

FIGS. 3-5 show the colonizing ability of the invention strain P. fluorescens Q8r1-96 compared to P. fluorescens strains M1-96 and Q2-87, (strains not in accordance with the invention) in Quincy virgin soil, Lind virgin soil, and Moses Lake virgin soil, respectively.

FIG. 6 shows the colonizing ability of the invention strain P. fluorescens Q8r1-96 in Quincy virgin soil, Lind virgin soil, and Moses Lake virgin soil.

Biocontrol Agents of the Invention. The biocontrol agents of the invention comprise at least one biologically pure strain of fluorescent Pseudomonas spp. which has the following identifying characteristics: the strain contains a biosynthetic locus which encodes for the production of 2,4-diacetylphloroglucinol; it has a unique genotype as shown by a characteristic banding pattern described in detail, below, and in FIG. 1; it suppresses diseases caused by Gg in field-grown small grain crops or turf grass; and it exhibits root-colonizing ability which is characterized by both higher population density and extended colonizing activity compared to known Gg -suppressive strains.

Preferably, the strain has the following additional identifying characteristics: it's biocontrol activity (disease suppression ability) and root colonization ability are not affected by soil type and/or it 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 take-all decline, that is, it duplicates the level of biocontrol observed in a take-all decline soil.

Exemplary of the strains of the invention are P. fluorescens strains Q8r1-96, ML4.9-96, and L5.1-96.

Characteristic Banding Pattern. The biologically pure fluorescent Pseudomonas spp. strains of the invention have an identifying characteristic banding pattern. This profile can be identified by RAPD-analysis with primer M13 as described in Example 1, below. FIG. 1 shows the banding patterns (RAPD) of P. fluorescens strains Q8r1-96, ML4.9-96, and L5.1-96, which are exemplary 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 this invention, 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 alternative way to identify if a strain has the characteristic banding pattern of the invention is to carry out a side-by-side comparison in an agarose gel using P. fluorescens strain Q8r1-96 and the test strain and visually comparing the bands between about 600±50 and 900±50 base pairs. If the profile of the test strain matches the profile of strain Q8r1-96 in this region, then the test strain has the characteristic banding pattern encompassed by this invention.

Biocontrol activity. The biologically pure fluorescent Pseudomonas spp. strains of the invention have the ability to suppress (inhibit the incidence of or reduce the incidence or severity of) diseases caused by Gg , such as take-all, in small grain crops or turf grass. Tables 1, 2, and 4 in the Examples, below, present data showing biocontrol activity. As shown in Table 2 in Example 3, below, strain Q8r1-96 reduced the percentage of wheat plants infected with take-all in the field by 20%. As shown in Table 4 in Example 4, below, strain Q8r1-96 was shown to be nearly twice as effective at reducing take-all as P. fluorescens strain Q2-87 (a known suppressive Phl-producing strain) after 9 cycles of cropping wheat. As discussed below, Q8r1-96 duplicated the biocontrol of TAD soil.

Colonizing ability. The biologically pure fluorescent Pseudomonas spp. strains of the invention show a unique colonizing ability which is characterized by both (a) higher population density on the roots and (b) extended colonizing activity compared to known suppressive strains. That is, the strains of the invention have the ability to both colonize and persist on the roots of small grains. As shown in the Examples, below, strains of the invention have the ability to colonize roots at a population density averaging at least about 10 5 colony forming units (CFU)/gram of root, including the associated rhizosphere soil, for at least 7 successive growth cycles. FIG. 3 shows colonization of wheat (cv Penawawa) roots by Q8r1-96 during 9 cycles of wheat in Quincy virgin soil. By cycle 5, the population density of Q8r1-96 was nearly 1000-fold greater than that of P. fluorescens strains Q2-87 and M1-96 (strains not in accordance with the invention). Q8r1-96 also showed significantly greater population densities in the Land and Moses lake virgin soils in FIGS. 4 and 5.

For purposes of this invention, a strain has the characteristic colonizing ability of the invention if it colonizes roots of wheat at a population density averaging at least about 10 5 CFU per gram of root including associated rhizosphere soil for at least 5 successive growth cycles under growth conditions described in Example 4, below.

Unaffected by Soil Type. In a preferred embodiment, a biocontrol strain of the invention has the further characteristic that its biocontrol activity and root colonization ability are not affected by soil type. As can be seen in FIG. 6, during 7 cycles, the populations densities of Q8r1-96 were not affected by soil type.

Duplication of Biocontrol of TAD Soil. A biocontrol strain of the invention is preferably further characterized by having the ability to suppress diseases caused by Gg in small grain crops that is equivalent to a level of biocontrol obtained when the small grain crop is grown in a soil in a state of take-all decline (TAD). That is, the strain has the ability to duplicate the level of biocontrol obtained in a take-all decline soil. As shown in Table 1 in Example 1, below, Q8r1-96 applied at a dose of 10 4 colony forming units (CFU)/seed and then sown in Quincy virgin (conducive) soil was as effective at suppressing take-all as the Quincy TAD soil. In addition, Qr81-96 was as effective in Lind virgin (conducive) soil as Quincy TAD soil added at 10% w/w into the Lind virgin soil. This duplication of suppression of take-all equivalent to natural take-all decline is unprecedented.

Screening Method. The invention also encompasses methods for selecting the unique fluorescent Pseudomonas strains of the invention. Using our method, microorganism strains having the above-described characteristics are obtained, In brief, our method includes the steps of: (1) cultivating a small grain crop or turf grass in successive growth cycles in take-all suppressive soil (e.g., soil in a state of take-all decline) to enrich for fluorescent Pseudomonas spp. which contain a biosynthetic locus which encodes for the production of 2,4-diacetylphloroglucinol (also referred to as Phl-producing fluorescent Pseudomonas spp.); (2) isolating strains of potentially-suppressive fluorescent Pseudomonas bacteria; (3) screening the isolated strains to select strains which contain a biosynthetic locus which encodes for the production of 2,4-diacetylphloroglucinol by colony hybridization with a 2,4-diacetylphloroglucinol-specific DNA probe to detect Phl-producers; and (4) using RAPD analysis to select those strains having the characteristic banding pattern, described above. An optional step can be inserted after step 3 to confirm Phl production. FIG. 2 shows the screening protocol using wheat.

The following is a more detailed description of the selection method of the invention.

In this step, a small grain crop or turf grass is cultivated in successive growth cycles in natural take-all suppressive soil to enrich for Phl-producing fluorescent Pseudomonas spp. as follows: (a) growing seeds of a small grain crop or growing turf grass in a soil in a state of take-all decline (TAD soil) in the greenhouse for at least 3 weeks and under conditions effective to support growth of said small grain crop or turf grass to obtain seedlings; (b) collecting the soil and roots of the small grain crop or turf grass seedlings grown in the soil and mixing them together; and (c) repeating steps (a) through (b) for at least a total of 4 successive cycles, wherein the mixture of step (b) is used to grow the seeds in the succeeding cycle.

In this step, strains of potentially-suppressive fluorescent Pseudomonas bacteria are isolated from the roots and associated rhizosphere soil of the small grain crop or turf grass successively cultivated in step (1) by growing the strains on a Pseudomonas-selective medium for a time and under conditions effective for growth of Pseudomonads and selecting strains which grow on the medium.

Step 3. Colony hybridization with specific probes to detect Phl producers. In this step, strains isolated in step 2 are screened to select a strain which contains a biosynthetic locus which encodes for the production of 2,4-diacetylphloroglucinol (Phl) by hybridizing a colony of the strains with a 2,4-diacetylphloroglucinol-specific probe and selecting strains that hybridize to the probe. The selected individual colonies are picked, streaked, and restreaked until the strain is stable and pure, that is, it is a biologically pure culture. The strain can be stored in glycerol at −80° C. to keep it stable.

In this optional step, confirmation of Phl producing strains is carried out using primers which amplify sequences within the Phl biosynthetic locus, and those strains that give a positive PCR reaction are selected.

In this step, Random Amplified Polymorphic DNA analysis is carried out using primer M13 (Sequence: GGTGGTCAAG) (see Keel et al., supra). Primer M13 is available commercially from Operon Technologies Inc., Alameda, Calif. Strains which have bands at 600 bp±50 bp; 700±50 bp; 800 bp±50 bp; 900 bp±50 bp are selected. It is preferred that the strains also have bands at 330±20 bp and 1100 bp±60 bp.

Using our method we obtained biologically pure cultures of P. fluorescens strains Q8r1-96, L5.1-96, and ML4.9-96 which are exemplary of the strains of the invention.

Statement of Deposit. Biologically pure cultures of strains Q8r1-96, L5.1-96, and ML4.9-96 were deposited Jul. 8, 1997 under terms of the Budapest Treaty in the Agricultural Research Culture Collection (NRRL), 1815 North University Street, Peoria, Ill. 61604, and have been assigned the accession numbers NRRL B-21806, NRRL B-21807, and NRRL B-21808, respectively. Strains having the identifying characteristics of NRRL B-21806, NRRL B-21807, or NRRL B-21808 are encompassed by this invention. For the purpose of this invention, any isolate having the identifying characteristics of strains NRRL B-21806, NRRL B-21807, or NRRL B-21808, including subcultures and variants thereof which contain a biosynthetic locus which encodes for the production of the antibiotic 2,4-diacetylphloroglucinol and have the definitive banding pattern described above, and which retain the ability to suppress diseases caused by Gg in small grain crops or turf grass and retain the characteristic root-colonizing ability, are included. The term variants is defined herein to include transformants and mutants which having the aforenamed characteristics.

Growth of the Strains of the Invention. The fluorescent Pseudomonas spp. strains of the invention can be grown on any suitable solid or liquid bacteriological medium. An exemplary medium is King's medium B. Growth of the strains are effected under aerobic conditions at any temperature satisfactory for growth of the organism, i.e., from about 15° C. to 30° C.; the preferred temperature range is about 24° C. to 28° C. The pH of the nutrient medium is preferably about neutral, i.e., pH 6.7-7.2.

Maintenance of Stock Cultures. Each strain is maintained to keep it stable, such as by storing in glycerol at −80° C.

Application of Gg -Suppressive Strains. The microorganism strains of the invention are useful in controlling diseases of small grain crops or turf grass caused by Gg . Examples of small grain crops are wheat, barley, rye, and triticale. Examples of diseases caused by Gg are take-all disease caused by Ggt which is the most important root disease of wheat and which can also infect other Gramineae such as barley, rye, and triticale. Other Gg fungi include Gga which infects oats and grasses and which has been identified as causing take-all patch in turf grasses such as bent grass; and Ggg which infects some grasses.

To achieve biocontrol of diseases caused by Gg in small grain crops and turf grass, the crop or grass is grown in the presence of an effective suppressive amount of one or more fluorescent Pseudomonas strains of the invention. An effective biocontrol amount is defined as that quantity of biocontrol agent which suppresses (inhibits the incidence or reduces the incidence or severity of) diseases caused by Gg relative to that occurring in an untreated control. Optimally, the biologically pure culture is applied to obtain at least 15% less disease in the field grown small grain crop or turf grass compared to an untreated control. Biocontrol is carried out by applying an effective amount of the biocontrol agent to a 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 furrow treatment or as a drench to turf or soil. Fresh cells or freeze-dried cells may be used.

The strain may be incorporated into compositions suitable for application to small grains or turf grass. It can be mixed with any agriculturally acceptable carrier or suitable agronomically acceptable carrier which does not interfere with the activity of the strain. Exemplary carriers are water, buffer, methylcellulose, ground peat or vermiculite. Where 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 as known in the art. The strain of the invention can also be formulated to include other biocontrol strains.

Exemplary application procedures and effective amounts are described below. The amount that will be within an effective range in a particular instance can be determined by experimental tests.

For seed treatment of small grain or turf, bacteria are added to a suspension containing 0.5-2.0% methylcellulose to minimize desiccation 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 about 10 2 to 10 6 CFU per seed. In general, the preferred amount is about 10 4 to 10 6 CFU per seed. Treated seeds are air dried.

For soil treatment, bacteria are suspended in water or buffer and applied during the first watering of the soil to give about 10 2 to 10 6 CFU per gram of soil. For turf, 2 ml of a drench containing about 10 2 to 10 6 bacteria per ml is added to about a 1-cm diameter plug of grass.

Where the carrier is a solid, e.g., peat or vermiculite, a typical formulation is about 10 7 to 10 9 CFU per gram of carrier. Where the carrier is a liquid, a typical formulation is about 10 8 to 10 10 CFU per ml of carrier. For a freeze-dried formulation, a typical amount is about 10 10 to 10 11 CFU per gram formulation. Freeze dried formulations may contain additives as known in the art.

The following examples are intended only to further illustrate the invention and are not intended to limit the scope of the invention which is defined by the claims.

The following example describes the selection of fluorescent Pseudomonas spp. of the invention.

Overview. Pseudomonas fluorescens strains Q8r1-96, L5.1-96, and ML4.9-96 were isolated in 1996 from roots of wheat grown in soils collected from agricultural fields near, respectively, Quincy, Lind, and Moses Lake, Wash. (USA) that are in the state of take-all decline (TAD). Q8r1-96 was isolated from roots of wheat grown in Quincy TAD soil for 8 successive cycles of 3 weeks each; L5.1-96 from roots of wheat grown in Lind TAD soil for 5 successive cycles of 3 weeks each; ML4.9-96 from roots of wheat grown in Moses Lake TAD soil for 4 successive cycles of 3 weeks each. The protocol for isolation of these strains is described in FIG. 2 .

The following 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, Wash. (USA). All three soils are suppressive to take-all of wheat. In 1995, the Lind TAD field had been cropped continuously to wheat for 28 years. In 1980, the TAD fields at Quincy and Moses Lake had been cropped continuously to wheat for 22 years; between 1980 and 1995 other crops besides wheat also were grown. The soils were collected in March 1995 from the upper 30 cm of the soil profile, air-dried for 1 week and passed through a 0.5-cm mesh screen prior to use. Their physical and chemical properties were determined by the Analytical Sciences Laboratory, University of Id.

Twelve wheat seeds were sown in square PVC pots (8 cm high, 7.5 cm wide) containing 200 g of sieved natural soil (Quincy, Lind, or Moses Lake TAD soil) and 50 ml of water supplemented with metalaxyl (Novartis, Greensboro, N.C.) at 2.5 mg ml −1 active ingredient to control Pythium root rot. Pseudomonads are not affected by this fungicide. A 1-cm layer of soil was spread on top of the seeds. Plants were grown in a controlled-environment chamber at 16° C. with a 12-hour photoperiod. Pots received 50 ml of dilute (2:3, vol/vol) Hoaglund's solution (macro-elements only) twice a week. After 3 to 4 weeks of growth, the shoots of the plants were excised at the soil surface, and the soil and associated root system was decanted into a plastic bag and shaken vigorously to aerate and mix. This ‘cultivated’ soil was stored for 1 week at 15° C., returned to the same pot, and then replanted with twelve wheat seeds. This process of plant growth and harvesting was repeated for at least four and up to eight successive cycles, at which time four randomly selected plants were harvested from each replicate and root samples were prepared to determine the population size of antibiotic-producing fluorescent Pseudomonas spp. For each soil, four replicates were used.

Step 2: Isolation of fluorescent Pseudomonas spp. from roots of wheat cycled in TAD soils.

Four randomly selected plants grown in step 1 were harvested from each replicate, and loosely adhering soil was removed from the roots by gently shaking. 1.0 g of roots and associated rhizosphere soil was suspended in 5.0 ml of sterile water and shaken vigorously for 1 minute on a Vortex mixer. The samples were subsequently sonicated in a ultrasonic cleaner for 1 minute, and then serial dilutions of the root wash were plated onto King's medium B [KMB] agar (Proteose peptone, 20 g; glycerol, 10 ml; K 2 HPO 4 , 1.5 g; MgSO 4 , 1.5 g; agar, 15 g; H 2 O, 1000 ml) supplemented with cycloheximide (100 μg ml −1 ), chloramphenicol (13 μg ml −1 ) and ampicillin (40 μg ml −1 ) [KMB + ]. Plates were incubated at 25° C., and colonies were enumerated after 48 hours. Colonies of fluorescent Pseudomonas spp. were differentiated from non-fluorescent colonies under UV light (wavelength 366 nm).

The number of fluorescent Pseudomonas spp. that harbor the genes for Phl was determined by colony hybridization. Transfer of bacterial colonies to Hybond-N + nylon membranes (Amersham) was performed by standard methods. After air drying, the membranes were baked for 1 hour at 80° C. in a vacuum oven. To remove bacterial cell debris, membranes were washed for 1.5 hours at 42° C. in a solution containing 2×SSPE (20 mM NaH 2 PO 4 [pH 7.4], 0.36 M NaCl, 2 mM EDTA), 0.1% sodium dodecyl sulfate (SDS) and pronase (100 μg ml −1 ) and washed again for 1 hour at 56° C. in 2×SSPE and 0.1% SDS. Hybridizations were performed by standard methods. High stringency conditions consisted of prehybridization for 1.5 hour at 65° C., hybridization for 12 hours at 65° C., membrane washing twice each for 5 minutes with 2×SSC and 0.1% SDS at room temperature, and membrane washing twice each for 30 minutes with 0.1×SSC and 0.1% SDS at 65° C.

Probes were generated from sequences within, the biosynthetic locus for 2,4-diacetylphloroglucinol (GenBank accession no. U41818). The probe was developed from sequences within Phl D of Q2-87 by random primed labeling of PCR fragments using the nonradioactive DIG system (Boehringer Mannheim). The hybridized probes were immunodetected with anti-digoxigenin-AP-Fab fragments and were visualized with the colorometric substrates nitroblue tetrazolium salt and 5-bromo-4-chloro-3-indolylphosphate, according to protocols provided by the supplier.

In order to isolate Phl producers, the signals on the membrane were aligned with the colonies on the agar plate. Each individual colony which gave a signal on the membrane was picked, streaked, and restreaked until the strain was stable and pure, that is, it is 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 the colony hybridization to be eliminated from analysis.

Heat-lysed bacterial suspensions used in PCR analysis were prepared from cultures grown on KMB for 48 hours at 25° C. Two bacterial colonies (2 mm diam) were suspended in 100 μl lysis solution (0.05 M NaOH, 0.25% SDS) and incubated for 15 min at 100° C. The suspension was centrifuged for 1 min at 12,000 rpm and diluted 50-fold in sterile distilled water. Five μl of the diluted suspension were 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-diacetylphloroglucinol (Phl) of P. fluorescens Q2-87 (GenBank accession no. U41818). Primers were synthesized by Operon Techn. Inc. (Alameda, Calif.). Primers Phl2a (Sequence: GAGGACGTCGAAGACCACCA) and Phl2b (Sequence: ACCGCAGCATCGTGTATGAG) were developed from sequences within phlD, which predicts a protein of 349 amino acids that is homologous to chalcone synthase from plants.

PCR amplification was carried out in a 25-μl reaction mixture, which contained 5 μl of a diluted heat-lysed cell suspension, 1×GeneAmp PCR buffer (Perlin Elmer Corp., Norwalk, Conn.), 200 μM each of dATP, dTTP, dGTP, and dCTP (Perkin Elmer), 20 pmole of each primer, and 2.0 U of AmpliTaq DNA polymerase (Perkin Elmer). Each mixture was covered with one drop of mineral oil. Amplifications were performed in a Perkin Elmer Thermal Cycler 480. The PCR program consisted of an initial denaturation at 94° C. for 2 min followed by 30 cycles of 94° C. for 60 s, 67° C. for 45 s, and 72° C. for 60 s. Samples (9 μl) of the PCR products were separated on a 1.2% agarose gel in 1×TBE buffer (90 mM Tris-borate, 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 the definitive banding pattern. RAPD-analysis Random Amplified Polymorphic DNA) with primer M13 was performed for clustering the different Phl-producing fluorescent Pseudomonas strains isolated from roots of wheat grown in Quincy, Lind and Moses Lake TAD soil.

Amplification of the DNA was carried out in a 25-μl reaction mixture, which contained 5 μl of a diluted heat-lysed cell suspension, 1×GeneAmp PCR buffer (Perkin Elmer Corp., Norwalk, Conn.), 200 μM each of dATP, dTTP, dGTP, and dCTP (Perkin Elmer), 80 pmole of primer M13, and 2.0 U of AmpliTaq DNA polymerase (Perkin Elmer). Each mixture was covered with one drop of mineral oil. Amplifications were performed in a Perkin Elmer Thermal Cycler 480. The PCR program consisted of an initial denaturation at 94° C. for 1 min 30 sec followed by 2 cycles of 94° C. for 30 sec, 36° C. for 30 sec, 72° C. for 2 min, followed by 40 cycles of 94° C. for 20 sec, 36° C. for 15 sec, 45° C. for 15 sec, 72° C. for 1 min 30 sec, followed by 1 cycle of 72° C. for 10 min. Samples (9 μl) of the PCR products were separated on a 2.5% agarose gel in 1×TBE buffer (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 biologically pure fluorescent Pseudomonas strains of the invention show a unique banding pattern as demonstrated by RAPD-analysis with primer M13. FIG. 1 shows the banding patterns (RAPD) of P. fluorescens strains Q8r1-96, ML4.9-96, and 15.1-96. Lane 1 shows a 1-kb ladder as a 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.

(1) GENERAL INFORMATION:

(iii) NUMBER OF SEQUENCES: 5

(2) INFORMATION FOR SEQ ID NO:1:

(A) LENGTH: 10 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = “M13 primer”

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1..10

(A) AUTHORS: Keel, C

Weller, D M

Natsch, A

DeFago, G

Cook, R J

Thomashow, L S

(B) TITLE: Conservation of the

2,4-Diacetylphloroglucinol Biosynthesis Locus

among Fluorescent Pseudomonas Strains from Diverse

Geographic Locations

(C) JOURNAL: Appl. Environ. Microbiol.

(D) VOLUME: 62

(E) ISSUE: 2

(F) PAGES: 552-563

(G) DATE: 1996

(K) RELEVANT RESIDUES IN SEQ ID NO:1: FROM 1 TO 10

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

GGTGGTCAAG 10

(2) INFORMATION FOR SEQ ID NO:2:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = “Phl2a”

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(A) AUTHORS: Raaijmakers, Jos M

Weller, David M

Thomashow, Linda S

(B) TITLE: Frequency of Antibiotic-Producing Pseudomonas

spp. in Natural Environments

(C) JOURNAL: Appl. Environ. Microbiol.

(D) VOLUME: 63

(E) ISSUE: 3

(F) PAGES: 881-887

(G) DATE: March-1997

(K) RELEVANT RESIDUES IN SEQ ID NO:2: FROM 1 TO 20

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

GAGGACGTCG AAGACCACCA 20

(2) INFORMATION FOR SEQ ID NO:3:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = “Phl2b”

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(A) AUTHORS: Raaijmakers, Jos M

Weller, David M

Thomashow, Linda S

(B) TITLE: Frequency of Antibiotic-Producing Pseudomonas

spp. in Natural Environments

(C) JOURNAL: Appl. Environ. Microbiol.

(D) VOLUME: 63

(E) ISSUE: 3

(F) PAGES: 881-887

(G) DATE: March-1997

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

ACCGCAGCAT CGTGTATGAG 20

(2) INFORMATION FOR SEQ ID NO:4:

(A) LENGTH: 7198 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(A) ORGANISM: Pseudomonas fluorescens

(ix) FEATURE:

(B) LOCATION: complement (1810..2859)

(D) OTHER INFORMATION: /product= “PhlD”

(A) AUTHORS: Thomashow, L S

Bangera, M G

(B) TITLE: U41818

(xi) SEQUENCE DESCRIPTION: 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 480

CTGGCTTCCT TGAGCCGCAG GGACAGCAAC CCGCCCATCA AACTACCAGC CAGTGCCAGA 540

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

ATCAACGCAG ATGCGCCGGC CAGGAGCGAT ACCAAGGTAA TCGAAAGCAC CAGCAGTTGC 1440

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

GTTGCATGCG CCGCAGACGG TGCGCCGTGG GAAGAAGATT TCTTCGCAAT CCTGGCAGCG 3360

GCTGCCTTCG AGACGATATT TTCCGCCATG TTCGCGCCAT TCGCGCAACA TGCTGGCGGT 3420

GGTCATGCGG TGTATTTGTT CTGGGTAAAG GGACATGGTC 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 4140

GCCGACCAAA CGTTGCCAGG ACATCCGCTT CGCGTCCTGC ATAGCCAAAT TTTTCAAAAT 4200

ATTCGGCGGT TGCGAGGGCA AAGGCGTCGG TGTGCGAAAT GCCCAGGAAA TAATCGTACT 4260

CACATTCGGT ACTGGAGCCG ATGTATTCGG CATAGTTGAA GTGGTCGGTC ATTTTTTCAA 4320

AGCCACCACA CAGGACGATG TCGTACTCAC CCGAGGCGAC CATCTGATGG GCCATCTGAA 4380

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

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

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 AAACTTAGAT 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

CACCATGAGC GATATCGGCA CTTGCGGCTG ATCCGCCAGG CTCATCAAGG CGCTGACGCT 7140

GCCGTCTATT TTTGATTCGC GATACAGGTC CGCAGAGAAA CCCAGCTCGC ATGGATCC 7198

(2) INFORMATION FOR SEQ ID NO:5:

(A) LENGTH: 349 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

Met Ser Thr Leu Cys Lys Pro Ser Leu Leu Phe Pro His Tyr Lys Ile

1 5 10 15

Thr Gln Gln Gln Met Ile Asp His Leu Glu Gln Leu His Asp Asp His

20 25 30

Pro Arg Met Ala Leu Ala Lys Arg Met Ile Gln Asn Thr Gln Val Asn

35 40 45

Glu Arg Tyr Leu Val Leu Pro Ile Asp Glu Leu Ala Val His Thr Gly

50 55 60

Phe Thr His Arg Ser Ile Val Tyr Glu Arg Glu Ala Arg Arg Met Ser

65 70 75 80

Ser Ile Ala Ala Arg Gln Ala Ile Glu Asn Ala Gly Leu Thr Thr Asp

85 90 95

Asp Ile Arg Met Val Ala Val Thr Ser Cys Thr Gly Phe Met Met Pro

100 105 110

Ser Leu Thr Ala His Leu Ile Asn Asp Leu Gly Leu Arg Thr Ser Thr

115 120 125

Val Gln Leu Pro Ile Ala Gln Leu Gly Cys Val Ala Gly Ala Ala Ala

130 135 140

Ile Asn Arg Ala Asn Asp Phe Ala Ser Leu Ser Pro Asp Asn His Ala

145 150 155 160

Leu Ile Val Ser Leu Glu Phe Ser Ser Leu Cys Tyr Gln Pro Gln Asp

165 170 175

Thr Lys Leu His Ala Phe Ile Ser Ala Ala Leu Phe Gly Asp Ala Val

180 185 190

Ser Ala Cys Val Met Arg Ala Asp Asp Lys Ala Pro Gly Phe Lys Ile

195 200 205

Ala Lys Thr Gly Ser Tyr Phe Leu Pro Asp Ser Glu His Tyr Ile Lys

210 215 220

Tyr Asp Val Lys Asp Ser Gly Phe His Phe Thr Leu Asp Lys Ala Val

225 230 235 240

Met Asn Ser Ile Lys Asp Val Ala Pro Met Met Glu Glu Leu Asn Phe

245 250 255

Glu Thr Phe Asn Gln His Cys Ala Gln Asn Asp Phe Phe Ile Phe His

260 265 270

Thr Gly Gly Arg Lys Ile Leu Asp Glu Leu Val Leu Gln Leu Asp Leu

275 280 285

Glu Pro Gly Arg Val Ala Gln Ser Arg Asp Ser Leu Ser Glu Ala Gly

290 295 300

Asn Ile Ala Ser Val Val Val Phe Asp Val Leu Lys Arg Gln Phe Asp

305 310 315 320

Ser Gly Pro Ala Asn Gly Ala Thr Gly Met Leu Ala Ala Phe Gly Pro

325 330 335

Gly Phe Thr Ala Glu Met Ala Val Gly Lys Trp Val Ala

340 345