PL175708B1

Application of streptomyces wyec 108 in fighting against plants pathogens

Abstract

This invention relates to a biocontrol formulation suitable for reducing the susceptibility of plants to fungal phytopathogens. In one aspect of the invention, a newly isolated strain of Streptomyces, Streptomyces WYEC 108, is incorporated into a suitable delivery medium and applied to plant seeds or plant roots.

PL175708B1, drawing sheet 1 — Sheet 1 of 5

Term

Term ended

Expired 29 June 2014, 12.2 years ago.

5 claims: 3 independent, 2 dependent

1
Patent claims Zastrzeżenia patentowe 1. Biologically pure culture of the new microorganism Streptomyces WYEC 108, with identification number ATCC 55445. 1. Biologicznie czysta kultura nowego mikroorganizmu Streptomyces WYEC 108, o numerze identyfikacyjnym ATCC 55445.
2
Composition for the protection of plants against fungal infections, consisting of Streptomyces organisms, characterized in that it contains from 104 to about 100 / g of the new Streptomyces WYEC 108 microorganism and the treatment environment, the treatment environment comprising at least one of the following components:, water, cereal flour, sphagnum, alginate grief, vermiculite and organic and inorganic filler, and possibly a sufficient amount of nitrogen source. 2. Kompozycja do ochrony roślin przed zakażeniami grzybowymi składająca się z organizmów Streptomyces, znamienna tym, że zawiera od 104 do jo^cfu/g nowego mikroorganizmu Streptomyces WYEC 108 oraz środowisko do zabiegów, przy czym środowisko do zabiegów obejmuje co najmniej jeden z następujących składników: piasek, wodę, mąkę zbożową, torfowiec, żal alginianowy, wermikulit i wypełniacz organiczny i nieorganiczny oraz ewentualnie dostateczną ilość źródła azotu. 2. Composition according to claim 2. A process as claimed in claim 2, characterized in that it contains ammonium chloride as a source of nitrogen. 2. Kompozycja według zastrz. 2, znamienna tym, że jako źródło azotu zawiera chlorek amonu.
5
6. A method for improving plant growth, characterized in that a composition comprising a treatment environment and at least one component selected from the group consisting of Streptomyces WYEC 108 cells, Streptomyces WYEC 108 spores and antifungal metabolites produced by Streptomyces WYEC 108 is applied to the seeds or cuttings. 6. Sposób polepszania wzrostu roślin, znamienny tym, że podaje się na nasiona lub sadzonki kompozycję zawierającą środowisko do zabiegów i co najmniej jeden składnik wybrany z grupy obejmującej komórki Streptomyces WYEC 108, zarodniki Streptomyces WYEC 108 i przeciwgrzybowe metabolity wytwarzane przez Streptomyces WYEC 108.

Independent claims3

445 paragraphs in 2 sections, as filed

The subject of the invention is a biologically pure culture of the new microorganism Streptomyces WYEC 108, a composition for protection against fungal infections and a method of improving plant growth. This microorganism is able to inhibit the growth of soil plant pathogens and improve plant growth.

Fungal plant pathogens are the cause of many economic losses in agriculture and horticulture. Many different types of fungal plant pathogens have been described: these pathogens cause plant diseases such as rot, white rot, brown rot and root rot. These diseases can destroy emerging seedlings, reduce plant viability, and reduce crop disadvantage.

To reduce the possibility of fungal infections, plant nurseries can grow seedlings in sterilized or chemically treated soil. However, such treatments also remove beneficial microorganisms from the soil, including microorganisms normally competing with fungi. In such cases, with the accidental introduction of the fungal pathogen, it can grow rapidly and spread the disease widely.

In agricultural conditions, soil infected with fungal pathogens may not be suitable for certain crops. For example, soybean production in Michigan and other states is often severely limited by root rot caused by the Phytopher megasperm fungus (Filinow and Lockwood, 1985). Mushrooms of the genus Pythium are widespread in soils of California, Washington and Idaho. Pythium ultimum is the most common pathogen which is associated with rotting of seedlings both before and after emergence. This species is a serious pathogen for wheat, peas and chickpeas, as well as other crops growing on these soils and soils in other states and countries (Trapero-Casas et al., 1990; Stanghellini and Hancock, 1970; Kraft and

175 708

Burke, 1971; Westerlund et al., 1988). The use of chemicals to combat fungal plant pathogens is often impractical due to the high cost, lack of effectiveness and the emergence of resistant strains of fungi. In addition, the use of chemical fungicides is not recommended for environmental reasons. Therefore, it would be expedient to obtain such a biological agent that will effectively combat fungal infections in plants and at the same time will not be harmful to the environment.

This goal was achieved by isolating a number of actinomycetes that effectively combat fungal plant pathogens. In particular, one of the isolated actinomycin bacteria, referred to as Streptomyces WYEC 108 (also WYEC 108), has shown strong antagonism against a number of plant fungal pathogens, including pathogens that cause plant diseases such as rot, root rot, white rot and brown rot.

The subject of the invention is a biologically pure culture of the new Streptomyces WYEC 108 microorganism with the identification number ATCC 55445.

The invention also relates to a composition for protection against fungal infections consisting of Streptomyces microorganisms, characterized in that it contains from 104 to 10IN cfu / g of the new microorganism Streptomyces WYEC 108 and the treatment environment, the treatment environment comprising at least one of the following components: sand, water, cereal flour, sphagnum, alginate gel, vermiculite, and organic and inorganic filler and possibly a sufficient amount of source nitrogen. Such compositions are useful for reducing plant susceptibility to mycoses and improving the growth of test plants. In a preferred embodiment, such a composition contains ammonium chloride as the nitrogen source. It is also preferred that the composition of the invention contains the spores of Streptomyces WYEC 108 and optionally furthermore the antifungal metabolites produced by Streptomyces WYEC 108.

The invention also relates to a method for improving, growing consisting in administering to a seed or cuttings a composition comprising a treatment environment and at least one component selected from the group consisting of Streptomyces WYEC 108 cells, Streptomyces WYEC 108 spores, and antifungal metabolites produced by Streptomyces WYEC 108.

The invention is illustrated in the drawing, in which Fig. 1 is an electron microscope photograph showing spiral chains (upper) and spore surface (lower) Streptomyces WYEC 108, and Fig. 2 shows pea growing in soil infected with Pythium species. The plants on the right came from seeds treated with Streptomyces WYEC 108. The plants from the left came from untreated seeds.

The present invention describes the isolation of a number of strains of actinomycetes from soil. A number of strains have proven effective in reducing the effects of fungal pathogens on plants, including lettuce, chickpeas and peppers. In particular, the invention relates to the isolation of a new strain called Streptomyces WYEC 108 herein. Strain WYEC 108 exhibits strong antagonism against a wide range of plant fungal pathogens, including rot-causing pathogens, root rot, white rot and brown rot. As such, WYEC 108 is particularly useful as a bio-controlling agent that can be used to protect plants against infection with these plant pathogens. Streptomyces WYEC 108 is also suitable for reducing the susceptibility of plants to fungal infections. Fungal infections in susceptible, untreated plants affect certain growth characteristics of such plants. For example, untreated plants exposed to fungal plant pathogens exhibit significantly lower height, biomass size, and crop yield compared to plants not exposed to fungal pathogens. Plants treated with Streptomyces WYEC 108 according to the method of the invention and subsequently exposed to fungal pathogens show a less significant reduction in height, biomass size and crop yield compared to untreated plants inserted for fungal pathogens. Plants treated with Streptomyces WYEC 108 and exposed to fungal pathogens show better growth characteristics than untreated plants and not infected with pathogens.

175 708

The WYEC 108 strain colonizes plant roots in the presence of competition from root microflora. The WYEC 108 strain has been shown to improve the growth of lettuce growing on steam sterilized soil and paprika growing in the field.

In order to implement the method of the invention, agents for producing vegetative cells or spores of the WYEC 108 strain suitable for inclusion in the treatment environment are also prepared. A composition containing vegetative WYEC 108 cells and spores and a treatment environment has been shown to exhibit high storage stability and is suitable for delivering WYEC 108 strain to plants.

Substances and methodology

Bacterial growth environment

All bacterial growth media were prepared in distilled water and sterilized before autoclaving. All bacterial samples were prepared under standard aseptic laboratory conditions to maintain purity.

YGM environment (yeast extract / glucose / mineral salts) contains 0.6% (w / v) yeast extract (Difco Laboratories, Detroit, Michigan), 1.0% (w / v) glucose, and phosphate salt solution (5, 3 g Na2HPO4, 1.98 g KH2PO4, 0.2 g MgSO4 · 7H2O, 0.2 g NaCl, 0.05 g CaCl2 · 2H2O and 1.0 ml trace elements (Pridham and Gottlieb, 1948) per liter of deionized H2O; pH 7.1 to 7.2). The trace element solution consisted of 0.64 g CuSO4 · 5H20.011 g FeSO4 · 7H20.07.79 g MnCh · 4H20.0.15 g ZnSO4 · 7H2About in 100 ml distilled water.

WYEC (water / yeast extract / agar), modified, according to Reddi and Rao (1971) contained yeast extract (Oxoid, 0.25 g / l) as the only carbon and nitrogen source, and agar (Oxoid, 18.0 g / l). The medium was buffered to pH 7.2-7.4 K2HPO4 (0.5 g / L).

The WYEC medium (water / yeast extract / cellulose / agar) was WYE agar to which a thin layer of agar was added. The agar layer contained 0.25 g / L cellulose (Solka Floc, Sigma Chemical Co) and 18.0 g / L agar in distilled water.

The CYD environment (casein acids / yeast extract / glucose agar) contained casein acids (Difco: 0.5 g / l), yeast extract (Oxoid or Difco: 0.8 g / l), D-glucose (0.4 g / l), K2HPO4 · (2.0 g / l, pH 7.2-7.4) and 18.0 g / l agar in distilled water.

YCED environment (caseaminic acids / yeast extract / glucose / agar); modification according to Reddi and Rao (1971) contained yeast extract (Oxoid, 0.3 g / l), caseamic acids (Difco, 0.3 g / l), D-glucose (0.3 g / l) and agar (Oxoid , 18.0 g / l). The environment was buffered with K2HPO4 (2.0 g / L).

The CYPC environment (cellulose / yeast extract / peptone / compost extract / agar) contained cellulose (Solka Flock, Sigma Chemical Co .; 5.0 g / l), yeast extract (1.0 g / l), peptone (Oxoid, 1 , 0 g / l), phosphate buffer (K2HPO4, 0.75 g / l), agar (18.0 g / l), and compost extract (100 ml / l) replacing 100 ml of distilled water in the environment. It was added directly and not used as a thin agar layer.

The MSSC environment (mineral salts / starch / casein / agar; Turhan, 1981) contained a mineral salt solution composed of NACl (2.0 g / l), MgSo4 · 17H2O (0.05 g / l), CaCO3 (0.02 g / l), FeSO4.18H2O (0.01 g / l), and KNO3 (2.0 g / l), and organic components, including soluble starch (10 g / l) and casein (0.3 g / l) ) and agar (18.0 g / L). The environment was buffered with K2HPO4 (2.0 g / L).

The sporulation environment (ATCC environment # 5) contained yeast extract (1.0 g / l), beef extract (1.0 g / l), tryptose (2.0 g / l), FeSO4 (0.01 g / l) , glucose (10.0 g / l) and agar (15.0 g / l). The environment was adjusted to pH 7.2 before heating in the autoclave. (ATCC Catalog of Bacteria and Bacteriophages - 17th edition).

The CYG medium contained caseamic acid (acid hydrolyzate) (5.0 g / l), yeast extract (5.0 g / l) and glucose) 10.0 g / l) in distilled water, adjusted to pH 7.1-7 2.

The treatment environment (containing sand / cereal flour / water or sphagnum / sand / cereal flour in the proportions given below) was sterilized with steam before use. Sterilization was usually carried out in an autoclave 3 times, each time for 90 minutes.

Collection of grown bacteria

To obtain mycelial growth of Streptomyces WYEC 108, 11 Erlenmyer flasks containing 500 ml of YGM medium (pH 7.1-7.2) were inoculated with 20 ml of the primary culture (prepared as described in Example Π) and incubated with shaking at a rate of 250 rpm at 30 ° C for 3 days. The fungus was centrifuged at 5000 rpm for 10 minutes. Alternatively, the mycelium was harvested, stopping the culture until the mycelium and spores settled at the bottom of the Erlenmeyer flask. The supematant was decanted and the concentrated mycelium and spore suspension was used directly to inoculate the treatment environment.

Cells and spores were also produced by solid culture (e.g. sporulation agar). Mycelia and spores were collected from sporulation agar by scraping the agar surface into distilled water. The spore and mycelium suspension was then mixed directly with the treatment environment.

To create Streptomyces WYEC 108 spores, 2 L Erlenmeyer flasks containing 1200 mL of YGM medium were inoculated with 50 mL of stock culture and incubated with shaking at 250 rpm at 30 ° for 12-18 days. Centrifugation spores at 9,000 rpm for 10 minutes.

Mushroom pathogens

Pythium ultimum PuMXL was obtained from the culture collection of the Department of Microbiology and Plant Protection at Horticulture Research International, Worthing Road, Littlehampton, West Sussex BN 17 6LP, United Kingdom. Mushrooms white rot Phanerochaete chrysosporium and Coriolus versicolor, white rot Paya, Caldariomyces fumago and Gloeophyllum trabeum; soil fungal pathogens Rhizoctonia solani, Fusarium sambucinaum, Geotrichum candidum and Verticillium dahliae were obtained from the culture collection of Professor Don L. Crawford, Department Bacteriology, University of Idaho, Moscow, ID. Pythium irregulare, Phytophthora capsici, Phytophthora cinnamomi, Phytophthora parasitica, Sclerotinia cepivorum and Sclerotinia sclerotiorum were obtained from Dr. Wesley Chun's culture collection, Department of Plant Soil Entomology Science, University of Idaho, Moscow, ID. Fusarium oxysporum was obtained from the culture collection of Dr. Arthur D. Partridge, Department of Forest Resources, University of Idaho, Moscow, ID. All cultures were kept on potato glucose agar or cereal flour agar and grown at 25 ° C. These strains were identified in the preparation as pathogens, but their pathogenicity was not checked.

Soil for bio-tests

For biotests, soil naturally infected with Pythium ultimum was collected from several locations in the Paluose region near Moscow, Idaho. It was harvested from the top 15 cm from half seeded with wheat and peas in the last two seasons. The population of Pythium species was determined as follows: Diluted 1.0 g of air-dried soil in 50 ml of sterilized distilled water by thoroughly mixing in a Vortex tube mixer. A 0.1 ml sample of well mixed dilution was placed as small drops on three-day 2% water agar plates (Stanghellini and Hancock, 1970). Plates were incubated at 25 ° C and periodically read with a weak (x 10) fluorescent microscope to determine the identity and abundance of current Pythium species. Colonies on each plate were checked after 12.48 and 72 hours of incubation before assessing the final population. The identification was based on the morphological characteristics of the mushroom mycelium of the Pythium species under a microscope and the growth pattern on plates with 2% (w / v) water agar. Colonies of pure fungal cultures on 2% (w / v) aqueous agar served as controls for identification (Stanghellini and Hancock, 1970; Stasz et al., 1980).

Soil study revealed that the population densities of P. ultimum and P. irregulare were 354 ± 15 and 194 ± 11 cfu / g, respectively, in air-dried soil during the sowing period (Spring, 1992). The population densities of other Itythium species were 57 ± 9 cfu / g in air-dried soil. P. ultimum and P. irregulare were the predominant species in harvested soil.

Example I. Isolation of actinomycetes strains antagonizing plant fungal pathogens.

Isolation of actinomycetes strains from four root and four adverse plant samples. These strains were tested for usefulness in inhibiting plant fungal pathogens.

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Isolation of actinomycetes strains

Actinomycetes isolates were isolated from 8 different soils by serial dilution and plate spreading. Dilutions (10 '5 up to 10'Twere placed on plates in various agar isolation environments. The compositions of these environments are given in the above Substances and Methodology as cited above. Actinomycetes isolates were determined according to the environment in which they were isolated. For example, WYEC 108 was isolated in a WYEC environment and YcEd 9 was isolated in a yCeD environment. Generally, such environments are poor in organic carbon, which effectively controls the growth of beneficial bacteria and fungi and helps isolate slower-growing actinomycetes. Because WYE and YCED agar were particularly effective isolation environments, this environment was mainly used. The dilution plates were incubated at 25 ° C for 4 to 10 days for the actinomycetes to sporulate, after which the colonies were collected and applied for cleaning to WYE or YCED agar plates. Pure colonies were transferred from these plates to YCED or CYD oblique agar cultures, incubated at 25 ° C or 37 ° C until sporulation and stored at 5 ° C until use. Basic cultures were transferred every 3 to 4 weeks.

Soils (i) Non-root soil

Soil samples (100 to 200 g) were taken from the top 7.5 to 10 cm soil profile from 4 locations in the UK, including soil from a cultivated rose garden (Soil S1) in Rustington, West Sussex; from between wheat fields (Gleb S2) from the Horticulture Research International (HRI) farm in Littlehamton, West Sussex; forest soil (Gleb S6) from the Wynd Cliffhardwood Forest Reserve, South Wales; and pastures (Gleb S7) sometimes grazed by sheep in Hastings Hill, South Downs, West Sussex. These soils were found to be non-root, although they contained the roots of certain plants in varying amounts.

(ii) Soils from the root environment

Soil samples from the root environment from 4 sites were prepared strictly according to the method of Miller et al. (1990). Soil 3 (S3) was associated with the roots of the dandelion (Taracum officinale) in the HRI rose garden in West Sussex, England. Soil (S5) combined with wheat roots and was taken from the same field as S2 soil from a wheat field from a farm (HRI) in Littlehampton, West Sussex. Gleb 4 (S4) was also associated with wheat roots, but on Bignor Hill along South Down Way, West Sussex. Soil 8 (S8) combined with flax roots and was taken from a field adjacent to the S7 sampling site, sometimes grazed sheep in Hastingd Hill, South Downs, West Sussex.

Radiation emitted from soils can be divided into emitted from soils from a non-root environment (S1, S2, S6, and S7) or from a root environment (S3, S4, S5, and S8). All soils were analyzed for water content by drying 3 g (wet weight) samples (three replicates) at 100 ° C for 58 hours and reweighed. The soil pH was determined by thoroughly mixing the soil: slurry water slurry in a 1: 1 ratio allowing solids to deposit for 2 hours, then measuring the pH of the supernatant solution. After collecting the soil, it was stored at 4 ° C until use (24 to 48 hours). A visual examination confirmed that the isolates are strains of actinomycetes, which observation showed that the colonies formed by the strains were typical actinomycete colonies (hard and leathery, with aerial mycelium containing spores).

Determining the useful pH range for growth

Each of the actinomycetes was tested for its ability to grow at pH 5.5 to 8.0. Cultures were point-grafted onto CYD agar plates, buffered to pH 5.5, 6.0, 6.5, 7.0 and 8.0 with a combination of K2HPO4 and KH2PO4 buffers at a concentration of 100 mM. The final pH of each medium was set to the final value just before heating in the autoclave. Cultures were tested for growth after 5 to 7 days of incubation at 25 or 37 ° C. Plates were visually assessed as having poor or no growth (±), some growth (+ or ++) or excellent growth (+++).

As Table I shows, soils from the root environment gave almost twice as much isolates as non-roots. Each isolate was tested for growth on a CYD agar environment in the pH range from 5.5 to 8.0. Only 9 isolates did not grow at pH 6.0, and

175 708 (21%) did not grow at pH 5.5. Of those growing at pH 5.5, depending on the isolate, the growth was from weak to excellent. The isolates' ability to strongly sporulate on CYD agar was also determined visually and microscopically on colonies incubated for 5-10 days.

Table I

<td colspan="5">Selective environment</td>

<td>Soil</td><td>soil pH</td><td>YCED</td><td>WYE</td><td>Together</td>

<td colspan="4">Non-root soils</td><td> 77</td>

<td colspan="4">Root soils</td><td> 140</td>

<td colspan="2">Total isolates</td><td> 114</td><td> 103</td><td> 217</td>

In vitro antagonism test

82 isolates were selected considering their ability to grow well and strongly sporulate on CYD agar.

In vitro plate assay was used to test the ability of isolates to inhibit P. ultimum growth. Each actinomycetes was plated on agar and cereal flour (CMA) plates as a streak culture from one side to the center. The culture was incubated at 25 ° C for about 8 days or until sporulation. A CMA agar block (0.5 cm) containing the actively growing P. ultimum mycelium was placed aseptically in the center of the plate. Incubation continued for 96 hours. After 48 and 96 hours, the plate was examined for P.ultimum growth inhibition.

Inhibition was found when the mycelial growth of P. ultimum towards the actinomycete colonies was retarded or stopped. The results are shown in Table II.

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Table II

<td>Culture</td><td>Source</td><td>Increase for pH</td><td>Antagonism</td><td>watched3</td>

<td></td><td>(soil)</td><td>5.5 (+ or -)</td><td>48 hours</td><td>96 hours</td>

<td colspan="5">Antagonistic (96 hours)</td>

175 708 cont. table II

<td colspan="5">Non-antagonistic (96 hours)</td>

175 708 cont. table II

175 708 cont. table II

P. ultimum inhibition defined as fungal hyphae growth less abundant or slightly delayed in the plaque region towards the side where the actinomycetes grew +++ Very strong inhibition with inhibition zone> 2.0 cm ++ Strong inhibition with inhibition zone> 1.0 cm + Growth virtually retarded with obvious zone of inhibition near the colony ± Weak inhibition of P. ultimum (hyphae of the fungus less abundant or slightly delayed)

- No inhibition

After 96 hours, five isolates (WYEC 108, YCED 9, YWE 91, WYE 90 and YCED 106) showed very strong antagonism against P. ultimum, four isolates (YCED 1, YCED 106, WYE 97 and WYE 98) showed strong antagonism, 10 weak antagonism. The rest of the isolates showed no or very weak antagonism. Cultures clearly inhibiting P. ultimum growth were more or less divided in half from those originating from the root environment and those from the non-root environment.

Seventy isolates that grew at pH 5.5 were also tested for their antagonism in vitro against the white rot Phanerochaete chrysosporium mushroom agar.

175 708 cereal flour (CMA). Thirteen of the isolates showed some degree of antagonism against the white rot fungus, as shown in Table III. The degree of antagonism ranged from strong (+++) to relatively weak (+), given the size of the inhibition zone. Five of the cultures that were antagonistic to P. Chrysosporium (WYEC 108, WYE 78, 2YE 90, YCED 9 and MSSC 2) was further examined for CMA against an additional white rot fungus (coriolus versizolor) and two types of brown rot fungi (placenta and Gloeophyllum trabeum). Four isolates (MSSC 2, YCED 9, WYE 90, WYEC 108) showed very strong antagonism against the above-mentioned white and brown rot fungi. One isolate, WYEC78, showed strong antagonism only against two white rot fungi.

Table III

<td rowspan="2">Culture</td><td rowspan="2">Source (soil)</td><td rowspan="2">Increase for pH 5.5 (+ or -)</td><td colspan="2">Antagonism observed3</td>

<td>48 hours</td><td>96 hours</td>

<td colspan="5">antagonistic</td>

Inhibition of P. chrysosporium defined as fungal hyphae growth less abundant or slightly delayed in the plaque area towards the side where the actinomycetes grew +++ Very strong inhibition with inhibition zone> 2.0 cm

175 708 ++ Strong inhibition with inhibition zone> 1.0 cm + Growth virtually retarded with obvious inhibition zone near colonies ± Weak inhibition P. chrysosporium (hyphae less or slightly delayed growth of hyphae)

- No inhibition b Inhibition of Coriolus versicolor, Paya and Gloeophyllum trabeum pose

P. chrysosporium.

c Inhibition of Postia placenta and Gloeophyllum trabeum outside P. chiysosporium.

In vivo biotest to determine the activity of actinomycete isolates on lettuce seedlings

The biological control test procedure of Lynch et al. (1991, 1992) to test 12 isolates for their effect on germination and growth of lettuce (Latuca sativa).

For control plants, plastic pots with a diameter of 9 cm were filled with soil mix for lettuce and pressed with a Petri dish. Lettuce seed was placed on top, lightly pressed into the soil and covered with a loose mixture. The pots were placed on stands on a layer of water and incubated in the dark at 20 to 22 ° C until a clear germination (about 3 days). They were then transferred to a capillary mat in a greenhouse at 15 to 25 ° C and watered as needed. The number of germinated seedlings in each pot was counted periodically to 18 days.

In the case of plants treated with actinomycetes isolates, a soil mixture inoculated with the spores of the corresponding actinomorph was filled. The soil mix was inoculated with spores from oblique CYD cultures to an average level of 108 up to 109 cfu / g (dry weight) of the mixture. The cfu / g soil mix value was determined by counting viable colonies on CYD agar plates during vaccination. Lettuce seed was sown as before, covered with a small amount of inoculated soil mix and treated identically as controls.

For plants treated with individual actinomycetes and putrefactive Pythium ultimum fungus (strain PuMXL, Lynch et al., 1991), the mix was also inoculated with a fungal pathogen in an amount of about 200 sporangia per gram (dry weight) of the mix. The Lynch et al. Procedure was used. (1991, 1992) to produce sporangia of the pathogen and inoculate soil mix. Sporangia were counted with a hemocytometer, and the pathogenicity of the P. ultimum strain was confirmed prior to use by lettuce passage.

The pots were prepared in repetitions of five times in all treatments. In the greenhouse, the pots were placed in random blocks surrounded by guarding plants, which served to ensure uniformity of conditions and to act as buffers. 18 days after sowing, the plants were harvested, the yield was measured, and weighed (wet and dry masses, above-ground leaves and stem). Dry and wet masses were recorded as total biomass per pot and as average biomass per pot. Values are given as means of five replicates ± standard deviation. Thus, each value was based on 50 seeds planted for each treatment (5 replicates of 10 seeds each). The rise percentage and final yield values were calculated similarly.

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Table IV

<td>Actinomycetes strain</td><td>concentration x105 cfu / g</td><td colspan="2">Healthy number plants</td><td colspan="4">Shoot mass</td>

<td></td><td>dry</td><td colspan="2">for flowerpot</td><td colspan="2">fresh</td><td colspan="2">dry</td>

<td></td><td>compost</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td colspan="2">unvaccinated control</td><td> 9,8</td><td> 3,6</td><td> 1,34</td><td> 0,75</td><td> 0, 074</td><td> 0, 034</td>

P1 P. ultimum: 200 sporangia per g of dry mix

Where:

n = average number of healthy plants per pot from all 5 replicates of 10 plants and Significantly different from the relevant control value at the materiality level

P = 0.05.

Degrees of freedom = 100

In the absence of pathogens (denoted as -P1, in Table IV), no significant differences were found between pots with Actinomycetes grafted plants and controls (without Actinomycetes) in the percentage of seeds giving healthy plants. Germination and growth reached> 98% in all cases. However, the presence of actinomycetes sometimes delayed seed growth by 1 to 3 days (data not shown). Similarly, in the absence of pathogens, there was usually no significant difference between actinomycete vaccinated plants and control shoots in terms of shoot weight, both dry and wet. The exception

175 708 there were 4 WYEC 107 grafted pots in which the presence of actinomycetes significantly improved biomass yield.

In the presence of pathogen (+ P1), the number of plants per pot was on average 3.6 out of 10 in control pots compared to 9.8 out of 10 in pots without pathogen. In the presence of specific actinomycetes in pots inoculated with the pathogen, the number of healthy plants increased significantly.

Seven of the twelve actinomycetes (YCED 85, YCED 64, WYEC107, YCED 106, YCED 71, WYE 88 and wYe 21) significantly increased the yield of healthy plants. Of these YCED 85, YCED 64 and WYE 21 also significantly increased the dry matter yield of plants containing only pathogen-containing control plants. WYE 21 also significantly increased the yield of fresh plants relative to control-containing control plants. One strain that did not significantly improve the number of healthy plants (MSSC 1) significantly increased the yield of fresh and dry mass of plant shoots (Table IV, columns 5 and 7. Thus MSSC 1 may prove useful to protect plants against lower doses of P. ultimum than used here.

Example II Secretion of Streptomyces WYEC 108.

The WYEC 108 strain was identified as a Streptomyces species based on the morphological characteristics of the genus Streptomyces, as defined by Bergey's Manual of Systematic Bacteriology (1986). WYEC 108 is a filamentous bacterium that produces spore chains in antennae mycelium. As described above, Streptomyces WYEC 108 was isolated as one of the actinomycetes strains from soil harvested from eight different locations in the UK. Together with other Streptomyces WYEC 108 radiators, they were isolated by successive dilution and smearing on plate soil from the root environment associated with flax roots from half Hastings Hill, South Downs, West Sussex, England. Dilutions (10 ~5 up to 10'7) this soil was placed on plates on an WYE agar environment. The dilution plates were incubated at 25 ° C for 4 to 10 days to allow colonies to grow and sporulate. Colonies were then picked and smeared on WYEC agar plates for purification. Pure WYEC 108 colonies were transferred from these plates to oblique CYD agar cultures, incubated at 25 ° C for sporulation and stored at 4 ° C until use. Basic cultures were transferred every 3 to 4 weeks.

Identity Streptomyces WYEC 108

As described above, the isolated strains of actinomycetes were tested for their ability to grow well and sporulate strongly on CYD agar. A number of isolates were then tested for their ability to inhibit growth under the conditions of the Pythium ultimatum plant pathogen. Isolates were also tested in vitro for their ability to antagonize white rot fungi Phanerochaete chrysosporium and Coriolus versicolor and brown rot fungi Postia placenta and Gloeophyllum trabeum. As a result of these tests, one of these strains, named Streptomyces WYEC 108, was selected based on its favorable characteristics.

Streptomyces WYEC 108 colonies grown on plates with casein acids / yeast extract / glucose (CYD) were examined under an electron microscope. Samples were prepared as follows:

(1) Streptomyces WYEC 108 colonies on CYD plates were coated with a 1.5% solution of glutaraldehyde in 0.2 M sodium cacodylate buffer and fixed for at least 2 hours;

(2) the colonies were washed thoroughly (2x) with a 0.2 M sodium cacodylate buffer solution by withdrawing the previous fluid with a pipette and replacing it with the buffer solution. Care was taken to ensure that the sample did not dry out;

(3) colonies were removed by collecting agar nuggets with colonies;

(4) the lumps were placed in individual sieve containers and dehydrated with 100% ethanol (2 x) (JT Baker Inc., Phillipsberg, Nj);

(5) the samples were then dried at a critical temperature using a bomb and placed in individual sample rods using colloidal silver conductive paint. They were coated with gold / palladium 60/40 and observed under a scanning electron microscope.

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Figure 1 is a micrograph of a scanning electron microscope showing spiral chains (top) and spore surface (bottom) Streptomyces WYEC 108. The observed spore surface was relatively smooth.

Various physiological characteristics were determined: strain WYEC 108 does not produce melamine or H2S on peptone-yeast-iron agar and peptone-iron agar, respectively (Difco Lab. Detroit, Michigan). The color of the Streptomyces WYEC 108 spore mass on CYD was gray. This strain did not grow at 45 ° C. Streptomyces WYEC 108 may belong to the species Streptomyces lydicus as defined by Bergey's Manual of Determinative Bacteriology (1986). Accordingly, this organism may be referred to as Streptomyces lydicus WYEC 108. Briefly it is referred to as Streptomyces WYEC 108 or simply WYEC 108.

ATCC Access Number

The Streptomyces WYEC 108 was deposited under the terms of the Budapest Pact at the American Type Culture Collection (ATCC), Rockville, MD June 29, 1993. The strain was designated ATCC accession number 55445.

Production of Streptomyces WYEC 108 basic cultures

For short-term use, Streptomyces WYEC 108 was incubated on CYD agar or oblique culture on sporulating agar at 25 ° C until sporulating and allowed to use at 4 ° C. For long-term storage of cultures, 10 ml of spore suspension was prepared from one oblique agar culture or plate in 10 ml of sterile YGM medium. The suspension was inoculated into 250 ml Erlanmayer flasks containing 100 ml YGM (yeast extract / glucose / mineral salts). The flasks were incubated with shaking at 250 rpm for 32-36 hours at 30 ° C to produce a standard seeding mixture.

Samples of the YGN-grown standard grafting mixture were also used to produce glycerin cultures suitable for long-term storage at -70 ° C and for lyophilization.

Example III. In vitro antagonism Streptomyces WYEC 108 against fungal plant pathogens.

The ability of Streptomyces WYEC 108 to inhibit the growth of a number of selected plant fungal pathogens was measured in terms of colony inhibition. Streptomyces WYEC 108 was inoculated on one side up to the center of the cereal flour (CMA) agar plates (Difco Lab., Detroit, Michigan). Inoculated plates were incubated at 25 ° C for about 8-12 days until the cultures sporulated. Seeding was detected by observing the gray mass of antenna mycelium and spores with the naked eye. Seeding was observed under phase contrast microscope (x 1000). A 5 mm CMA agar disk containing the actively growing mycelium of a specific fungal plant pathogen from the anterior edge of the mushroom culture was removed and placed aseptically in the center of the agar plate. Plates were incubated at 25 ° C until the test fungus reached the edge of the control plate not containing Streptomyces WYEC 108. Inhibition of fungal growth was measured by determining the ratio of the radial growth of the fungal plant pathogen under the influence of Streptomyces WYEC 108 to the growth of the pathogen alone in the control plate. Percent inhibition was recorded after 48, 96 and 192 hours of incubation depending on the pathogenic fungus. The bioassay was repeated on five plates, inhibition was measured separately and recorded as mean ± standard deviation.

The in vitro test results are given in Table V. These data show that Streptomyces WYEC 108 shows very strong antagonism against a wide spectrum of fungal plant pathogens, including rot (Pythium ultimum), root rot (Pythium ultimum, Rhizoctonia solani, Fusarium solani and Phytophthora cinnamomi) , white rot (Phanerochaete chrysosporium and Coriolus versicolor), brown rot (Postia placenta and Gloeophyllum trabeum) and leaf and stem rot (Sclerotinia species).

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Table V

<td rowspan="3">Mushroom pathogens</td><td colspan="2">% inhibition ± standard deviation3</td>

<td>observed the</td><td>antagonisms0</td>

<td>48 hours</td><td>96 hours</td>

<td>Pythium irregulare</td><td> 700±0,0</td><td> 100±0, 0</td>

<td>Pythium ultimum</td><td> 100±0,0</td><td> 100±0,0</td>

<td>Rhizoctonia solani</td><td> 700±0,0</td><td> 84±0,0</td>

<td>Fusarium oxysporum</td><td>2β ± 2.5</td><td> 26±3, 6</td>

<td>Fusarium sambucinetum</td><td> 44±2,4</td><td> 35±2,4</td>

<td>Fusarium solani</td><td> 36±2,5</td><td> 19±2,5</td>

<td>Phytophthora capsici</td><td> 700±0,0</td><td> 100±0,0</td>

<td>Phytophthora cinnamomi</td><td> 100±0,0</td><td> 100±0,0</td>

<td>Phytophthora parasitica</td><td> 700±0,0</td><td> 100±0,0</td>

<td>Selerotinia cepivorum</td><td> 100±0,0</td><td> 95±1,5</td>

<td>Selerotinia selerotiorum</td><td> 700±0,0</td><td> 100±0,0</td>

<td>Phanerochaete chrysosporium</td><td> 700±0,0</td><td> 100±0,0</td>

<td>Coriolus versicolor</td><td> 100±0,0</td><td> 100±0,0</td>

<td>Placenta post</td><td> 100±0,0C</td><td>100 ± 0,0d</td>

<td>Caldariomyces fumago</td><td> 700±0, 0C</td><td> 100 + 0,0d</td>

<td>Gloeophyllum trabeum</td><td> 100±0,0C</td><td> 1000,0d</td>

<td>Geotrichum candidum</td><td> 47±2,1</td><td> 45±2,1</td>

<td>Verticillium dahliae</td><td>73 ± 2,0C</td><td>59 ± 2,0D</td>

a Values based on averages of individual values of five replicates on plates. Individual values were determined separately by measuring the mycelium growth on each plate.

Inhibition of fungal pathogens defined as fungal hypha growth under the influence of Streptomyces WYEC 108 relative to growth on CMA control plates.

cdi% inhibition after 96 and 192 hours, respectively.

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Example IV Use of Streptomyces WYEC 108 for seed treatments.

The effectiveness of Streptomyces WYEC 108 cells in protecting plants against plant pathogens was determined using the WYEC 108 strain on non-germinated chickpea seeds and sowing seeds in soil infected with fungal pathogens of P. ultimatum and P. irregulare. The extracellular metabolites produced by the WYEC 108 strain were extracted from the culture by ether extraction.

The effect of these metabolites on fungal infection of emerging chick peas was also determined.

Growth of Streptomyces WYEC 108

To grow cells of the WYEC 108 cells, a 1 L Erlenmeyer flask containing 500 ml YGM (pH 7.1-7.2) was inoculated with 20 ml stock culture and incubated with shaking at 250 rpm at 30 ° C for 3 days to produce cell mass. To produce anti-fungal metabolites, a 1 L Erlenmeyer flask containing 500 ml CYD (pH 7.1-7.2) was inoculated with 20 ml stock culture and incubated with shaking at 250 rpm at 30 ° C for 7 days.

Seed treatment using Streptomyces WYEC 108 and antifungal metabolites.

The mycelial suspension Streptomyces WYEC 108 was collected by centrifugation at 5000 rpm for 10 minutes from a three-day 500 ml culture with yGm. The collected mycelia were suspended in 200-300 ml sterilized 3% (w / v) sodium alginate solution for culture density 1.0-1.2x104 cfu / ml. Pea seeds were added to the well-mixed cell suspension and alginate and the seeds were transferred one into a sterilized 0.25 M CaCi ™ solution in distilled water. Seeds were used in the biological control test described below.

Antifungal metabolites produced by Streptomyces WYEC 108 were obtained in purified form as follows. A seven day culture of 500 ml was filtered to remove cells and then extracted with 150 ml of ether using an extraction funnel. The ether was removed by evaporation under reduced pressure and the resulting extracts were dissolved in 1.5 ml of distilled water. The solution was sterilized by filtration through a 0.45 gm filter and added to 10 ml of a 3% (w / v) sodium alginate solution. Purified anti-fungal metabolites can be used to protect plants against fungal infections. Preferably, the antifungal metabolites will be purified in such a way that they are completely free of WYEC 108 cells. However, the composition of WYEC 108 cells and / or spores together with antifungal metabolites is also considered effective against plant fungal pathogens. The antifungal suspension metabolite-alginate was applied as described to chickpea seeds as described in the biological control test.

In vivo biological control test

Soil naturally infected with P.ultimum and P. irregulare is described in Substances and methodology. This soil was used in in vivo biological control tests. The soil pH was determined to be 5.6 by thoroughly mixing the soil / water slurry suspension (1: 1), allowing the parts to settle for 2 hours and testing the pH of the supernatant solution. The soil was ground, mixed thoroughly and placed in seedling pots (10 cm deep and 10 cm in diameter).

The in vivo biological control test was conducted judging in uninfected soil non-germinated pea seeds treated with Streptomyces WYEC 108 or antifungal metabolites.

Untreated seeds placed in the same soil were used as controls. The procedure included the following steps:

1) One cm sphagnum was placed at the bottom of each pot to protect against soil loss while aerating and draining;

2) The pots were filled with infected soil;

3) The soil was watered for saturation from the bottom side. After saturating the soil surface, the untreated and treated pea seeds were placed in the soil and covered with 1.5-2.0

175 708 cm of the same soil. The added soil was allowed to wet in a capillary way, from the side located below the moist soil. Seeds were planted in each of three identical pots. No fertilizer was added to the soil. To minimize drying and crust formation, the pots are covered with clean material until the seedling emerges. After the cuttings emerged, the top of the pots was sprayed as needed. The experiments were carried out in a greenhouse at 15-30 ° C with a lighting cycle of 12 hours a day and 12 nights (16,000 lux).

Periodic seedlings were counted periodically and the final count was made after 20 days. These data were means from each type of surgery. The ability of Streptomyces WYEC 108 to act as a biological control agent was evaluated based on the total number of ascents, plant heights, and fresh plant weights, compared to control plants growing from seeds not treated with a biological control agent. The results of the biological control test are shown in Table VI.

Table VI

<td rowspan="2">intervention</td><td colspan="2">Rotting (%)</td><td rowspan="2">Germination (%)</td><td rowspan="2">Height (Cm)</td><td rowspan="2">Fresh Mass (G / plant)</td>

<td>In front of emergence</td><td>After rising</td>

<td>control</td><td> 86, 7</td><td> 6, 6</td><td> 6,7</td><td> 4,3X</td><td> 0, 34x</td>

<td>Streptomyces</td><td> 36, 7</td><td>ABOUT about</td><td> 63, 3</td><td> 11, 3</td><td> 1,05</td>

<td>3% alginated</td><td> 83, 3</td><td> 10, 0</td><td> 6,7</td><td> 4,1x</td><td> 0,32x</td>

<td>przeciwgrzy-</td><td> 63, 3</td><td> 3,3</td><td> 33,3</td><td> 8,9</td><td> 0, 66</td>

<td>bowe metabo-</td><td></td><td></td><td></td><td></td><td></td>

<td>solidc</td><td></td><td></td><td></td><td></td><td></td>

c in 3% alginate as a coating on seeds d does not contain WYEC 108 x so marked means in the column were not significant.

Both Streptomyces WYEC 108 cells and the antifungal metabolites formed in these cells reduced the intensity of Pythium rotting pea.

Plants showed spontaneous growth when seeds were coated with Streptomyces WYEC 108 cells. The height and weight of fresh plants growing from control (untreated) chickpea seeds were significantly reduced compared to plants germinating from seeds coated with Streptomyces WYEC 108 cells. The emergence of untreated chickpea seeds was very much reduced (6.7% rise) due to root rot and decay before emergence caused by P. ultimum when seeds were planted in soil naturally infected with P. ultimum and P. irregulare. In contrast, seed emergence of Streptomyces WYEC 108 treated seed was 63.3%. Seeds treated with alginate only did not show an increase in the number of emergence. Symptoms typical of Pythium root rot, including hair loss and root discoloration, were seen on harvested roots

175 708 chickpea from control seeds, but these symptoms did not occur in plants grown from seeds treated with Streptomyces WYEC 108 cells. In control tests, chick pea damage was mainly in the form of seed spoilage and rotting before emergence. Pea seedlings that emerged and grew were stunted and their roots strongly infected with P. ultimum. Figure 2 shows a comparison of chick peas from a biological control test. The control plant, shown on the left, germinating from untreated seed, shows a far-reaching root infection and no secondary roots and root hairs, and the plant emerging from the seed coated with Streptomyces WYEC 108 shown on the right shows good growth and normally formed secondary roots and root hairs.

The emergence of chick pea seeds treated with the antifungal metabolite in the form of an ether soluble metabolite was more intense (33.3%) than control seeds (6.7%), but lower than seeds treated with Streptomyces WYEC 108 cells (63.3%). Emerging plants from the seed treated with the antifungal metabolite showed vigorous growth, had longer roots and higher root hair density compared to control plants.

Example V. Effect of Streptomyces WYEC 108 on root infection and seed rot caused by Pythium ultimum.

Pea seedlings, as described in example IV, in soil naturally infected with Pythium ultimum, with or without pretreatment with WYEC 108 was examined to determine the effect of WYEC 108 on P. ultimum infection.

P. ultimum root infection was examined on control (treated) seedlings harvested after 20 days of growth. P. ultimum causing root rot was also isolated from damaged roots of control plants. Secretion of P. ultimum was carried out by rinsing the soil from the chickpea roots (rootlets and root hairs) first with tap water and then washing twice with sterile distilled water. Discolored and rotten roots were cut with a razor under aseptic conditions and placed on 3 day old plates with 2% water agar. Plates were incubated for 24-48 hours at room temperature and observed under phase contrast microscope (x 40). P. ultimum grown from infected pea roots was grown on 2% water agar plates and identified as described above (Ingram and Cook, 1990).

Root rot caused by P. ultimum was studied on damaged control seeds harvested after 20 days. Some rotten seeds were placed under aseptic conditions on 3-day 2% water agar plates with a sterilized toothpick and incubated for 24-28 hours at room temperature, then examined as described above.

Pythium has been observed to infect the roots of untreated pea seeds growing in naturally infected soil. P. ultimum was the predominant species isolated from rotten seeds and roots. P. irregulare was the least observed species.

Root colonization by Streptomyces WYEC 108 was studied on the roots of 20-day-old chick pea plants germinating from seeds treated with Streptomyces WYEC 108 cells, suspended in alginate described in Example II. Plants were removed from the pots and gently washed with tap water to remove root soil. They were then washed with sterile distilled water. Root samples were prepared for microscopy by placing part of the root on a slide, adding a drop of methylene blue, and then covering with a lid slide. Prepared samples were observed under phase contrast microscope (x 1000).

Streptomyces WYEC 108 present on the seeds come into contact with the growing roots. During their elongation, Streptomyces moves along elongated hairs and cones. It was observed that Streptomyces WYEC 108 widely colonized the primary root, secondary roots, root hairs and cones. Plants growing from Streptomyces WYEC 108 coated seeds were healthier, had longer roots and root hairs and were more dense than those on control plants growing from uncoated WYEC 108 seeds. This difference was clearly related to root colonization by the control agent. The roots colonized by this agent showed no symptoms of root disease. Streptomyces WYEC 108 perfectly colonized the root in the presence of competition from native root microflora.

Anti-fungal activity

In addition to the ability of Streptomyces WYEC 108 to colonize plant roots and produce antifungal metabolites, WYEC 108 has been observed to dissolve cell walls and fungal oospores. Using scanning electron microscopy, Streptomyces WYEC 108 mycelium has been shown to colonize the surface of a fungal or oospore strand, including a fungal strand or a Pythium ultimum oospore. Colonized fungal or oospore strands are degraded by Streptomyces WYEC 108, most likely due to the secretion of extracellular enzymes by WYEC 108, such as chitinases and cellulases. Streptomyces WYEC 108 has been shown to produce both of these enzymes. It is possible that Streptomyces WYEC 108 also produces other extracellular degradation enzymes.

Example VI. Placing Streptomyces WYEC 108 in a treatment environment.

A composition suitable for long-term storage containing viable spores Streptomyces WYEC 108 and for use in agrotechnical treatments was prepared as follows.

One liter Erlenmeyer flask containing 500 ml YGM (pH 7.0-7.1) was inoculated with 20 ml stock culture and incubated with shaking at 250 rpm at 30 ° C for 3 days. After incubation, the culture was harvested by centrifugation at 5000 rpm for 10 minutes. The collected material was suspended in 1600 ml of 10% YGM and suspended with 8 g of sterilized NH4Cl dissolved in 400 ml of distilled water. Two liters of the mixture of cells and NHaCl were inoculated in a plastic container containing 4 kg of sterilized treatment environment consisting of a sand-water-cereal flour mixture in a ratio of 9-2-1 (by weight). The treatment environment was sterilized twice (3 hours at 121 ° C) before incubating the culture. The mixture was incubated for 10-14 days at 25 ° C to maximize the number of spores in the mixture. Streptomyces WYEC 108 produced spores during a 10-14 day incubation, resulting in increased cfu / g treatment environment (to an average of 108 to 109 cfu / g in the environment (dry matter)). The mixture was then stored at 4 ° C before use.

Alternatively, instead of the YGM environment, cells and spores can be produced in the CYG environment. As an alternative to centrifuging the culture flask, it can also be set aside to settle the mycelium and bacterial spores. The pure supernatant is then decanted and the concentrated mycelium / spores suspension inoculated directly into the treatment environment. When this collection method is used, it is not necessary to add NH4Cl to the environment because the bacterial growth environment (YGM or CYG) is a suitable source of nitrogen.

Example VII. Effect of Streptomyces WYEC 108 on emergence and weight of fresh lettuce seedlings.

To determine the effect of Streptomyces WYEC 108 on lettuce growth, lettuce seeds were grown either in a treatment environment containing Streptomyces WYEC 108 as in Example VI above, or in steam sterilized soil. A total of 30 seeds were planted in each of the growth environments (one seed per 4 x 13.5 cm pot) and emergence noted after 21 days. The mass of fresh plants was determined 35 days after harvesting from above the soil surface. The weight of fresh plants was determined as the mean value. The results are shown in the table below.

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Tabe 1 and VII

<td rowspan="2">intervention</td><td colspan="3">Number of emerging plants</td><td rowspan="2">emergence (%)</td><td rowspan="2">Fresh Mass ZL. )</td>

<td>sterile soil + seeds</td><td> 13</td><td> 17</td><td> 21</td><td> (21/30) 70</td><td> 0, 81</td>

<td>Environment + WYEC 108 + seeds</td><td> 23</td><td> 27</td><td> 29</td><td> (29/30) 97</td><td> 1,41</td>

The results indicate that the treatment of Streptomyces WYEC 108 lettuce seeds improves both the number of emergence and the mass of fresh plants.

Example VIII. The use of Streptomyces WYEC 108 in a field treatment environment.

An in vivo biological control test was performed to determine the efficacy of Streptomyces WYEC 108 as a biological control agent when introduced into the treatment environment described above.

Streptomyces WYEC 108 was prepared and included in the treatment environment as described in Example VII above, using Streptomyces WYEC 108 grown for 3 days in a YGM environment. The initial population of Streptomyces WYEC 108 was approximately 1.0-1.2 x 105 cfu / g soil after calculation on CYD plates immediately before planting seedlings in pots. The treated soil was used to fill pots (4 x 13.5 cm).

Control plants were grown in pots containing soil sterilized with steam (at 100 ° C for 60 minutes).

Pepper seedlings (green hot peppers) were planted in pots with steam sterilized soil or with a mixture of steam sterilized soil inoculated with a treatment environment containing Streptomyces WYEC 108. After 6 weeks of growing in the greenhouse, the plants were moved to the field. In some cases, before transferring 100 g of treatment environment, containing 450 ± 17 cfu / g Phytophthora parasitica, was added to the seedling hole.

Plant heights were determined 55 days after transfer and recorded as means. Plant biomass was determined by collecting and measuring the weight of fresh plants 110 days after transfer. During this time, the number of pepper fruits formed on each plant and their weight was recorded as the average value. The results of the field test are given in Table VIII.

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T abe 1 a νίΠ

<td>intervention</td><td>Plant height (cm)</td><td>Fresh weight bimasy (G / plant))</td><td>% peppers on plant</td><td>Performance peppers fresh (G / plant)</td>

<td>^ Control untreated</td><td>22,4k7</td><td>274, Lkv</td><td>19, lk ^</td><td>158,8ks</td>

<td>cWYEC 108</td><td>29 lm</td><td> 353,3m</td><td>36, 5m</td><td>279,8m</td>

<td>^ untreated + P. para- ^ Sitic</td><td>20,3k</td><td>238.5k 13.8k</td><td>13,8k</td><td>151.2k</td>

<td>cWYEC 108 + P. para- ^ Sitic</td><td>28,6m</td><td>266.6k 22.7k</td><td> 22,7</td><td>221,6k</td>

b Pepper plants growing in pots (4 cm x 13.5 cm) containing only soil sterilized with steam.

c Pepper plants growing in pots containing a mixture of steam sterilized soil and the environment for treatment with Streptomyces WYEC 108.

100 g treatment environment with Phytophthora parasitica was introduced into the opening prior to transfer (450 ± 17 cfu / g treatment environment) s The means in the column before the same letter are not significantly different at the significance level P = 0.05.

As Table VIII shows, the treatment of WYEC 108 paprika seedlings in the absence of P. parasitica gave a statistically significant increase in plant height, biomass, paprika fruit and paprika yield compared to control plants not receiving WYEC 108. Comparison of row 3 and 4 of table V shows that strain WYEC 108 protected peppers from the harmful effects of P. parasitica. In addition, there was a significant improvement in the growth of plants treated with WYEC 108 in the absence of P. parasitica compared to untreated plants and without P. parasitica.

In addition to the experiments described in this example, WYEC 108 has proved effective in protecting a number of plants from fungal infections. Test plants include the plants listed in Table IX below.

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Table IX

Crops protected against mycosis by WYEC 108 Plants tested with wYEc 108and

<td>Plant</td><td>Field test (+/-)</td><td>Greenhouse test / Labware. (+/-)</td>

<td>lettuce</td><td> —</td><td> +</td>

<td>chick-pea</td><td> —</td><td> +</td>

<td>green peas</td><td></td><td> +</td>

<td>pepper</td><td> +</td><td> —</td>

<td>Cotton</td><td> +</td><td> +</td>

<td>grass</td><td> +</td><td> +</td>

<td>onion</td><td> +</td><td> +</td>

<td>potato</td><td> +</td><td> +</td>

and Tests were carried out either in the greenhouse or in the field, or in both places. The tests compared plants treated with WYEC 108 with untreated control and / or treated with WYEC 108 and the specific fungal pathogen (Pythium, Aphanomyces, Rhizoctonia, Fusarium, Phytophthora or Phytomatotrichum). In each case, WYEC 108 protected plants against fungal infections.

Example IX. Production of Streptomyces WYEC 108 spores in a liquid environment.

Biological control measures must be able to survive for a longer period of time to meet the requirements of transport and farming times. The use of WYEC 108 strain spores, rather than vegetative cells, in specific compositions for biological control extends the shelf life of the composition, since the spores remain viable under harsh conditions and for a longer period of time.

Typically, Streptomyces spores are produced in permanent environments. However, as noted below, the following method has proven suitable for producing spores in a liquid culture.

2 L Erlenmeyer flasks containing 1200 mL of YGM medium (pH 6.5) were inoculated with 50 mL of stock culture (prepared as in Example 2) and incubated with shaking at 250 rpm at 30 ° C for 12-18 days. Spore production in culture was observed under a phase contrast microscope (x 1000, methylene blue). The spores were centrifuged at 9,000 rpm for 10 minutes.

Then the spores were suspended in 1600 ml of 10% liquid YGM and 400 ml sterilized solution containing 8 g NH4Cl in distilled water was added (up to a final density of 1.0-1.2x10 spores7 cfu / ml). The mixture was directly inoculated with 4 kg of sterilized treatment environment consisting of a sand-water-cereal flour mixture in a ratio of 9-2-1 (by weight). The treatment medium was sterilized twice in an autoclave (3 hours at 121 ° C) before inoculation with spores.

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Production of spores directly in the liquid culture as described allows avoiding further incubation of the mixture. The treatment environment containing the spores is stored at 4 ° C before use.

The spores produced in the liquid culture, as described, were tested for viability after 4 months storage at 4 ° C. One ml of spore suspension was inoculated into flasks containing 100 ml of liquid sterilized 10% YGM (pH 6.5) and incubated with shaking at 250 rpm at 30 ° C. Spore production in culture was observed under a phase contrast microscope (x 1000, methylene blue). The spores germinated after about 8 days. A simple observational test showed no loss of viability after this storage period.

Streptomyces WYEC 108 connected to the treatment environment (sand-water-cereal flour 9-2-1) was tested for viability as follows.

A 1.0 g sample of the Streptomyces WYEC 108 treatment medium was serially diluted and applied to CYD agar plates. Plates were incubated at 25 ° C until colonies. Average level 10 recorded8 up to 109 cfu / g treatment environment (dry matter) for samples stored 30 days.

Alternatively, the spores from the sporulating agar plates were suspended in 10-20 ml of sterile distilled water or YGM broth and mixed with 10-100 g of treatment medium to obtain the number of viable spores 1012 up to 1014 cfu / g environment. The mixture was air dried, mixed thoroughly and stored at 4 ° C before use. The composition is a concentrated product that can be diluted with the addition of treatment environment to any level of final life cfu / g.

Example X. Stability of an alginate gel composition.

Mycelial Streptomyces WYEC 108 was collected by centrifugation at 5000 rpm for 10 minutes with 500 ml of a 3-day liquid YGM culture. Harvested mycelia were suspended in 125 ml of 10% YGM and 125 ml of sterilized 5% (w / v) sodium alginate solution was added to a culture density of 1.0-1.2x104 cfu / ml. Alginate granules containing the mycelium Streptomyces WYEC 108 were formed by adding the cell-alginate suspension drop by drop to a sterilized solution of 0.25 M CaCl2 in distilled water.

To determine the viability of the alginate granules formed by this method, they were spread on a sterilized plastic Petri dish (10 cm x 10 cm) and dried for an hour in a laminar stream of sterile air under the hood. Granulated Streptomyces WYEC 108 spore easily after storage at 25 ° C for 6 to 8 months (up to an average of 108 up to 109 cfu / g dry alginate beads). These spores germinated easily when incubated in sterilized water at 25 ° C. Germination of spores was observed under a phase contrast microscope (x 1000, methylene blue).

Example XI. Favorable environmental compositions for treatment with Streptomyces WYEC 108.

After becoming familiar with the above methods of isolating actinomycosis strains, testing the strains for suitability as biological control agents, methods for producing biological control agents in the mycelium form and as spores, appropriate treatment environments and in a preferred Streptomyces WYEC 108 variant, it will be apparent to those skilled in the art that the present invention can be modified in many ways.

The following are examples of alternative variations of the invention with a description of particularly preferred variants.

Optimal breeding conditions

Optimal growth conditions for the WYEC 108 strain include temperatures between 20 ° C and 30 ° C, a pH of 5.5 to 7.5, and a stirring speed at fermentation of 200 to 300 rpm. Streptomyces WYEC 1-8 typically reaches a maximum cell mass of about 5.3 g (dry) biomass / L liquid YGM medium at 30 ° C, pH 6.5 and shaking at 200 rpm for 72 hours (end of logarithmic phase). The doubling of the time in the logarithmic phase is about 10 hours. Incubation for 72 hours can be significantly shortened by using a higher concentration of cell vaccine in the logarithmic phase.

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Alternatively, the spores may be formed on a solid agar medium such as sporulating agar. Spores can be collected directly by scraping into a suitable liquid such as 10% YGM and directly introduced into the environment for surgery. This approach avoids the stage of culture growth in the liquid and thus shortens the production process.

Beneficial and alternative environments for surgery

Streptomyces WYEC 108 can be incorporated into the treatment environment for horticultural and agricultural applications. Example VI described one treatment composition containing sand-water-cereal flour in a ratio of 9-2-1 (by weight). Those skilled in the art will understand that the composition of the treatment environment will be dictated by the specific use of the biological control agent. Eg. various organic and inorganic fillers, such as clay, vermiculite, wheat bran, corncobs or chitin, can be added to the treatment environment. The ratio of environmental components to treatment will be determined by the desired texture and physical properties. For example, properties such as the ability to hold water, light weight to facilitate handling and transport, porosity leaving room for mycelium and plant root growth may be important.

Alternatively, vegetative mycelium or spores of Streptomyces WYEC 108 may be added to the alginate suspension to produce an alginate entrapped granulate strain. Methods for making alginate granules are known to those skilled in the art and are described more fully in U.S. Patent No. 4,665,812 to Lewis et al. Other ingredients, such as fertilizer, can be added to the granules.

In a preferred embodiment, the inventors have found that the treatment environment consisting of sphagnum-sand and cereal flour in a weight ratio of 1: 3.5: 1 is particularly useful. This ratio gives the right density and water holding capacity for the product for agricultural and horticultural applications. However, as previously stated, other ratios of these ingredients and other ingredients are also acceptable in the treatment environment. Eg. an effective alternative treatment environment consists of sphagnum (620 g) - sand (3380 g) - cereal flour (270 g) chitin (10 g).

In one variation of about 1.61 harvested broth from the culture (log phase cells, e.g. after about 72 hours of culture) containing Streptomyces WYEC 108 mycelium grown on YGM medium described above supplemented with 400 ml sterile NH4G solution (containing 8 g NH4G distilled water) inoculated in a plastic container containing 4 kg of sterilized treatment environment consisting of a mixture of sphagnum-sand-cereal flour. The treatment environment is sterilized twice in an autoclave (3 hours at 121 ° C) before inoculation with Streptomyces WYEC 108. Inoculated containers are incubated at 30 ° C for 10 to 14 days to maximize spore production. The containers are stored at 4 ° C before use.

The use of NH4Cl in the treatment environment gives a nitrogen source for the germinating spores of Streptomyces WYEC 10. It will be obvious to the skilled person that other sources of nitrogen besides NH4CL can be used for this purpose, e.g. 10% YGM) before adding to the environment for treatments, the addition of a nitrogen source is unnecessary. In preferred embodiments of the invention, the treatment environment contains a sufficient amount of nitrogen source. One of ordinary skill in the art will understand that the determination of a sufficient amount of nitrogen source may be clarified by determining the effect on the germination frequency of reducing or increasing the content of a given nitrogen source, or the effect of changing the nitrogen source. A sufficient amount of nitrogen source is the amount of a given nitrogen source that facilitates the germination of Streptomyces WYEC 108 spores.

In an alternative embodiment described in Example 11, Streptomyces WYEC 108 spores are produced in a liquid medium and the safety is added to the beneficial treatment environment, which is then stored at 4 ° C.

In a preferred embodiment of the invention, Streptomyces WYEC 108 is added to the treatment environment at a final concentration of Streptomyces WYEC 108 of at least 1x105 cfu / g. In a more preferred embodiment, the final concentration of Streptomyces WYEC 108 is from 1x1) 5 to 1x 108 cfu / g.

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Example XII. A detailed description of the composition of the treatment environment containing Streptomyces WYEC 108.

A preferred treatment environment composition containing Streptomyces WYEC 108 is prepared on a large scale by the procedure described below. All procedures described are carried out using standard aseptic techniques (e.g., ultraviolet sterilized laminar flow chamber) to ensure sterility until the package is opened by the user.

Cell production

1) Suspend spores from an oblique CYD Streptomyces WYEC 108 culture in 10 ml sterile YGM or CYG broth (pH 6.5). This vaccination suspension is used to inoculate cultures in flasks.

2) Inoculate 6 flasks containing 100 ml of YGM (pH 6.5). Use 10 ml spore suspension per flask. After inoculation, the shake flasks are incubated at 200 rpm at 30 ° C for approximately 36 hours.

3) Inoculate 6 2.0 L flasks containing 1.1 L YGM broth (pH 6.5) mycelium vaccine prepared above (100 ml per flask). After inoculation, the shake flasks are incubated at 200 rpm at 30 ° C for approximately 24 to 48 hours or longer (up to 4 days). This is a fermenter vaccine.

Fermentation

Approximately 7.2 L of the basic culture obtained by the method described above was inoculated with a fermenter containing 401 sterile YGM broth (pH 6.5) (= 15% by volume of vaccine, the approach is to use high density cell suspension as practical). The fermenter works with stirring (200 rpm) at 30 ° C for about 72 hours (almost to the end of the logarithmic phase).

Harvesting after fermentation

1) Post-fermentation broth containing WYEC 108 cells (after about 72 hours of incubation) are collected aseptically into sterile 20L plastic bottles.

2) Sterile NH4Cl solution is added to the culture broth containing WYEC 108 cells (16 g NH4Cl dissolved in 800 ml distilled water per 3.21 harvested culture broth, pre-sterilized in an autoclave). The resulting volume of cell suspension containing NH1Cl is mixed thoroughly by shaking the bottles before inoculating the previously prepared treatment environment.

Preparation of the environment for treatments

1) Each component of the treatment environment is measured out separately and added to a large sterilizable vessel. The combined mixture is referred to as the treatment environment. It consists of sphagnum, sand and cereal flour (540 g: 2700 g: 540 g; 1: 5: 1 by weight).

2) The treatment environment is mixed thoroughly and covered with hard aluminum foil or cotton wool and sterilized twice (90 minutes at 121 ° C with 12 hours between sterilizations).

3) The treatment environment is cooled to room temperature after the second sterilization and before inoculation with harvested broth containing strain WYEC 108 and the solution of NHqCl (prepared as above).

Introduction of Streptomyces WYEC 108 into the treatment environment to create a composition of sphagnum, sand, water, cereal flour and NH4CL

1) Approximately 0.5 L of harvested broth containing strain WYEC 108 and NHCl solution (prepared above) are placed in exactly as many as needed, sterilized plastic containers containing 3.78 kg of treatment environment.

2) Inoculated containers are incubated at 30 ° C for 10-14 days (up to 20 days of incubation may be optimal), then stored at 4 ° C before use (the composition is stable for months).

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Manipulation and transport

1) The complete composition containing Streptomyces WYEC 108 is aseptically transferred to sterile three-layer plastic bags using a small sterilized scoop or other tool, preferably under the laminar flow hood with sterilized UV.

2) The filled bags are then tied up and placed in movable boxes with a capacity of 0.042 m3 for transport. Each box is closed with a strong tape.

Example XIII. Incorporation of compositions containing Streptomyces WYEC 108 into seedbeds.

The composition containing the biological control agent Streptomyces WYEC 108 and the treatment environment described in Example 12 are mixed with seedbed or pot soil to a final concentration of Streptomyces> 1, ^ - 1.2x105 cfu / g soil or more). The sowing procedure is as follows.

1) Approximately 1.0 cm sphagnum is placed at the bottom of each pot (or seedbed) to protect against soil loss (potting mixture) with simultaneous aeration and drainage;

2) Pots are filled with agricultural (seedling or potted) soil to about 3.0 cm below the edge of the pot (seedbed). Pots (seedlings) are watered until saturation.

3) About 1.5 cm of the composition containing Streptomyces WYEC 108 and the treatment environment is added to the top of each pot (seedbed). If desired, the composition is premixed with seedling or potting soil to increase volume and bring to cfu / g. However, optimal performance requires at least 105 cfu / g in the final mixture.

4) Seeds are placed on the surface of pots or seedling and covered with an additional layer of about 1.5 cm seedling or potting soil.

5) A small amount of water is added to moisten the soil and seeds.

6) To minimize drying and crust formation, the pots are usually covered with clean black plastic until seedlings rise (this may not be necessary when controlling humidity).

7) The top of the pots (seedbed) was sprayed as needed with water after the seedlings emerged.

Literature

ATCC Catalog ofBacteria and Bacteriophages, edition 17,1989. American Type Culture Collection, Rockville, MD.

Locci, R. 1989. Streptomyces end Related Genera in Bergey 'Manual of Systemaric Bacteriology; Williams and Wilkens, Baltimore, MD. 4: 2451-2492.

Filnow, AB and JL Lockwood. 1985. Evaluation of several actinomycetes and the fungus Hypochytrium catenoides as biocontrol agents of Phytophthora root rot of soybean. Plant Disease 69: 1033-1036.

Ingram, DM and RJ Cook. 1990. Pathogenicity of four Pythium species to wheat, barley, peas, and lentils. Plant Pathology 39: 110-117.

Kraft, JM and DW Burke. 1971. Pythium ultimum as a pathogen of beans and peas in Washington. Plant Dis. Rep. 55: 1056-1060.

Lynch, JM, RD Lumsden, PT Atkey, and MA Ousley. 1992. Prospects for control of Pythium damping-off of lettuce with Trichoderma, Gliocladium, and Enterobacter spp. Biol. Fertil. Soils 12: 95-99.

Lynch, JM, KL Wilson, MA Ousley, and JM Whipps. 1991. Response of lettuce to Trichoderma treatment. Lett. Appl. Microbiol. 12: 59-61.

Miller, JJE Liljeroth, G. Henken, and JA van Veen, 1990. Fluctuations in the fluorescent pseudomonad and actinomycete populations of rhizosphere and rhizoplane during the growth of spring wheat. Can. J. Microbiol. 36: 254-258.

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Pridham, TG and D. Gottlieb. 1948. The utilization ofcarbon compounds by some actinomycetales as an aidfor species determination. J. Bacteriol. 56: 107-114.

Reddi, GS, and AS Rao 1971. Antagonism of soil actinomycetes do some soil borne plant pathogenic fungi. Indian Phytopathol. 24: 649-657.

Stanghellini, ME and JG Hancock. 1970. A Quantitative Method for the Isolation of Pythium ultimumfrom Soil. Phytopathology. 60: 551-552.

Stasz, TE, GE Harman and GA Marx. 1980. Time and site of infection of resistant and susceptible germinating pea seeds by Pythium ultimum. Phytopathology. 70: 730-733.

Trapero-Casas, A., WJ Kaiser and DM Ingram. 1990. Control of Pythium seed rot and preemergence damping-off of chickpea in the US pacific northwest and Spain. Plant Dis. 74: 563-569.

Westerlund, FV, Jr., RN Campbell and KA Kimble. 1974. Fungal root rots and wilt of chickpea in California. Phytopathology 64: □ 32-436.

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FIG. 1

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Document	Office	Kind	Date
8544893	United States of America	A	1993-06-30
8544893	United States of America	A	1993-06-30
9407373	United States of America	W	1994-06-29
9407373	United States of America	W	1994-06-29
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US19930085448	–	–	–
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WO9501099A1	World Intellectual Property Organization (WIPO)	A1	1995-01-12
AU7253194A	Australia	A	1995-01-24
US5403584A	United States of America	A	1995-04-04
PL312385A1	Poland	A1	1996-04-15
EP0706321A1	European Patent Office (EPO)	A1	1996-04-17
US5527526A	United States of America	A	1996-06-18
CN1126424A	China	A	1996-07-10
KR960703318A	Republic of Korea	A	1996-08-17
CA2216794A1	Canada	A1	1996-10-03
WO9629874A1	World Intellectual Property Organization (WIPO)	A1	1996-10-03
AU5382596A	Australia	A	1996-10-16
JPH09500268A	Japan	A	1997-01-14
PH30701A	Philippines	A	1997-09-16
NZ269070A	New Zealand	A	1997-09-22
MX9707514A	Mexico	A	1997-11-29
AU684809B2	Australia	B2	1998-01-08
EP0817567A1	European Patent Office (EPO)	A1	1998-01-14
SG49012A1	Singapore	A1	1998-05-18
PL175708B1This record	Poland	B1	1999-01-29
RU2127521C1	Russian Federation	C1	1999-03-20
EP0706321B1	European Patent Office (EPO)	B1	1999-04-07
AT178457T	Austria	T	1999-04-15
ATE178457T1	Austria	T1	1999-04-15
DE69417750D1	Germany	D1	1999-05-12
CA2166096C	Canada	C	1999-06-08
MY111006A	Malaysia	A	1999-07-31
ES2132416T3	Spain	T3	1999-08-16
DK0706321T3	Denmark	T3	1999-10-18
US5968503A	United States of America	A	1999-10-19
GR3030695T3	Greece	T3	1999-11-30
DE69417750T2	Germany	T2	1999-12-23
CA2216794C	Canada	C	2001-09-25
CN1113604C	China	C	2003-07-09
KR100331125B1	Republic of Korea	B1	2005-01-13
JP3612071B2	Japan	B2	2005-01-19

Application of streptomyces wyec 108 in fighting against plants pathogens

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