Streptomyces Venezueli for biological control of plant diseases

Abstract

The present invention relates to the use of at least one strain of Streptomyces Venezuralensis, in particular a strain of Streptomyces Venezuralensis DSM 33887, as a botanical drug, in particular as a fungicide or a biological fungicide. Another object of the invention is a strain of Streptomyces Venezuelensis, referred to as DSM 33887, as well as compositions and uses thereof. The present invention relates to a method of controlling plant pathogens and a method of preventing, controlling and treating plant diseases caused by plant pathogens, comprising the use of a Streptomyces Venezueli strain, preferably a DSM 33887 strain.

Description

Technical Field

[0001] The present invention relates to the use of at least one strain of Streptomyces venezuelae as a phytomedicine and a method for controlling plant pathogens to prevent, control and treat plant diseases caused by them. The present invention relates to the strain of Streptomyces venezuelae DSM 33887, compositions and uses thereof. Background Art

[0002] Agriculture occupies more than one third of the land surface (FAOSTAT, 2016). The main component of agricultural production systems is seeds, both for perpetuating genotypes and as a component required for food production. Therefore, seed quality plays an important role in agriculture: characteristics such as health, germination performance, vigor and purity must be guaranteed at all times. Seed treatment and coating are widespread practices to guarantee the phytosanitary quality of seeds and avoid that the seeds themselves become vectors of pathogens and, therefore, diseases.

[0003] In the past, phytomedicines have been used extensively to guarantee high phytosanitary standards for seeds, however, the need to guarantee greater sustainability and safety of agricultural systems and the need to be more respectful of the environment have led to increasing restrictions on the use of synthetic phytomedicines that are known to be harmful to humans and the environment.

[0004] In recent years, the European Commission has gradually banned the use of most active synthetic substances traditionally used for seed coating - mainly fungicides. As a result, phytomedicines valid for this use to date are no longer on the phytosanitary lists of authorised plant protection products, with the exception of the active substance fludioxonil, which is authorised on cereal seeds but not on horticultural crops, unless a regional procedure is used (i.e. an exemption authorisation with an extended scope of use in response to a phytosanitary emergency).

[0005] Therefore, the need to find alternative active substances or microorganisms with antibacterial and antifungal activity has increased over the past few years. In recent years, several microorganisms have been authorized for use as active substances in plant protection. For example, Bacillus thuringiensis for the management of certain insect pests; Bacillus amyloliquefaciens for the management of several bacterial diseases, gray mold (Botrytis cinerea) and brown rot; and Ampelomyces quisqualis for the management of powdery mildew in grapes. None of the registered microorganisms is specifically for seed coating, but all of them should be applied to the leaves of the relevant crop.

[0006] In view of the above, there is still a need to find alternative microorganisms that are effective in biologically controlling plant diseases caused by plant pathogens (microorganisms), especially fungi, preferably suitable for seed coating, and are able to combat infections caused by pathogenic microorganisms, especially fungi, which live in the soil and are harmful to plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1 Shown are the percent inhibition of fungal growth (IG%) of the following strains against phytopathogenic fungi on potato dextrose agar (PDA) medium: A) Streptomyces sp. DLS1568; B) Streptomyces sp. SA51; C) Pseudomonas sp. PT 65 and D) Agrobacterium sp. AR 39. The bar graph represents the standard deviation of the mean associated with the percent inhibition of fungal growth (± SD).

[0008] Figure 2 Siderophore production of Streptomyces sp. DLS1568 and Streptomyces sp. SA51 strains and a positive control (1 M catechol) are shown. Mean values ​​marked with different letters indicate significant differences among the three samples according to statistical analysis (one-way ANOVA, Tukey's test; p < 0.001). The bar graph represents the standard deviation of the mean (± SD).

[0009] Figure 3 Phosphate solubilization activity of Streptomyces sp. DLS1568 and Streptomyces sp. SA51 and negative control are shown. Mean values ​​marked with different letters indicate significant differences among the three samples according to statistical analysis (one-way ANOVA, Tukey's test, p<0.001). Bar graphs represent standard deviation values ​​(± SD) of the mean.

[0010] Figure 4Shown are the germination results expressed as percentage values ​​(%) of seed samples belonging to: A) onion, B) fennel, treated with Streptomyces sp. DLS1568 and Streptomyces sp. SA51, respectively, and negative control (uncoated). According to the statistical analysis (one-way ANOVA, Tukey's test, p>0.05), the mean values ​​marked with the same letters indicate no significant difference between the two treatment groups. According to the statistical analysis results (one-way ANOVA, Tukey's test, p<0.001), the mean values ​​marked with different letters indicate significant differences between the two treatment groups. The bar graph represents the standard deviation value (± SD) of the mean.

[0011] Figure 5 Shown are the disease index values ​​of seedlings emerging from seeds coated with Streptomyces species DLS1568, Streptomyces species SA51 and uncoated seeds (control) after experimental inoculation with the plant pathogenic fungus F. oxysporum f.sp. raphani (Fus Ruc 9 strain). Data are reported as mean ± SD of disease index. Means marked with the same letter indicate no significant differences between treatment groups (one-way ANOVA, Tukey's test, p>0.05). Means marked with different letters indicate significant differences between treatment groups (one-way ANOVA, Tukey's test, p<0.001). Bar graphs represent standard deviation values ​​(± SD) of the mean.

[0012] Figure 6 Shown are categories associated with different relative phenological parameters such as A) plant height (cm); B) number of leaves, evaluated during agronomic trials of onion variety Elenka in experimental fields during the planting season. Data are reported as standard deviations (± SD) of the means of the relative recorded values ​​(i.e., plant height (cm) and number of leaves). Mean values ​​marked with the same letter indicate no significant differences between treatment groups. Mean values ​​marked with different letters indicate significant differences between treatment groups (one-way ANOVA, Tukey's test, p < 0.001). The bar graph represents the standard deviation values ​​of the mean (± SD).

[0013] Figure 7Shown are the mean values ​​of the vigor data recorded in agronomic trials of fennel variety Crono during the planting season: A) second survey; and B) third survey. Data are reported as mean ± SD. Mean values ​​marked with the same letter indicate no significant difference between the treatment groups (one-way ANOVA, Tukey's test, p>0.05). Mean values ​​marked with different letters indicate significant differences between the treatment groups (one-way ANOVA, Tukey's test, p<0.001). The bar graph represents the standard deviation of the mean (± SD). Summary of the invention

[0014] The object of the present invention is the use of at least one strain of Streptomyces venezuelae as a phytomedicine or phytosanitary product, in particular as a fungicide or bio-fungicide, and the use of said strain for triggering the immune defence of plants.

[0015] Another object of the invention is a phytosanitary treatment method for controlling plant pathogens or eradicating plant pathogens, comprising applying a formulation comprising a strain of Streptomyces venezuelae on seeds, plants or soil; another phytosanitary treatment method for preventing and / or treating plant diseases caused by plant pathogens, comprising applying a formulation comprising a strain of Streptomyces venezuelae on seeds, plants or soil. Another object of the invention is a phytosanitary treatment method for triggering plant immune defenses, comprising applying a formulation comprising a strain of Streptomyces venezuelae on seeds, plants or soil.

[0016] Furthermore, the present invention relates to a formulation comprising a strain of Streptomyces venezuelae as active ingredient and at least one acceptable excipient (co-adjutant and / or co-formulation agent) and / or carrier.

[0017] Another object of the present invention is the Streptomyces venezuelae strain known as DLS1568, which has been deposited at the Leibniz-Institute DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH under the deposit number DSM 33887. DETAILED DESCRIPTION

[0018] The present invention relates to the use of at least one strain of Streptomyces venezuelae, in particular the strain Streptomyces venezuelae DSM 33887, as a phytomedicine or phytosanitary product which can be used for biomanagement, ie controlling or eradicating plant pathogens, in particular fungi, which cause plant diseases.

[0019] Surprisingly, it was found that microorganisms belonging to the genus Streptomyces venezuelae, in particular the strain Streptomyces venezuelae DSM 33887, are able to control or eradicate a variety of plant pathogens that are taxonomically different from one another.

[0020] According to the invention, the term "control of phytopathogens" refers to biological control activity, ie the biological control activity of microbial agents, in particular fungal pathogens, which are directly antagonistic to plant harmful microorganisms, in particular fungal pathogens, which are cultivated plants, in particular those whose seeds represent the vegetative reproductive organs.

[0021] Streptomyces venezuelae is a Gram-positive bacterium belonging to the order Streptomyces, family Streptomycetes, genus Streptomyces, isolated from the microbiota associated with rice (Oryza sativa L.) seeds. Streptomyces venezuelae is an aerobic, filamentous, and spore-forming bacterium.

[0022] Another object of the present invention is the Venezuelan Streptomyces strain known as DLS1568, which has been deposited on June 1, 2021 at the Leibniz-Institute DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH under the Budapest Treaty with the deposit number "DSM 33887".

[0023] According to a preferred aspect, at least one strain of Streptomyces venezuelae, in particular the strain Streptomyces venezuelae DSM 33887, is used as a fungicide or biofungicide for controlling or eradicating plant pathogens, in particular fungi, that affect and damage agricultural target plants.

[0024] The pathogenic fungus is preferably selected from: Fusarium oxysporum f.sp.Cepae (FOC), F.oxysporum f.sp.raphani (FOR), F.oxysporum f.sp.lactucae (FOL), Sclerotinia sp., Pythium sp., Stemphylium botryosum, and Colletotrichum dematium.

[0025] The present invention relates to a formulation comprising as active ingredient a strain of Streptomyces venezuelae, preferably Streptomyces venezuelae DSM 33887, at least one acceptable excipient and / or co-formulator and / or carrier, ie compatible with the biology, activity and efficacy of Streptomyces.

[0026] The composition can be formulated according to conventional methods and is preferably used in liquid form, for example in the form of a dispersion or suspension of the microorganism, or in the form of a powder preparation of the same microorganism.

[0027] Preferably, the excipient, particularly in formulations applied to seeds, is Bio-Friendly 1 (binder / coating) SEEDWORX TM [Centor Europe, Andijk, The Netherlands], which does not impair microbial viability, seed germination and seed development.

[0028] Streptomyces venezuelae, preferably Streptomyces venezuelae DSM 33887 strain, may be present in the formulation in a weight ratio of 5% to 75%, preferably 20% to 40%, even more preferably equal to 30%, of the total weight (w / w) of the composition. Preferably, Streptomyces venezuelae, preferably Streptomyces venezuelae DSM 33887 strain, may be present in the formulation in a weight ratio of 10 7 Up to 10 9 The amount present in the composition is preferably equal to 10 for 1 g of product. 8 CFU (colony forming units).

[0029] Another object of the present invention is the use of a strain of Streptomyces venezuelae, preferably the strain Streptomyces venezuelae DSM 33887, or a preparation thereof, for preventing and / or treating plant diseases caused by phytopathogens, in particular fungi, which preferably affect crops at the collar and canopy of the plants.

[0030] An object of the present invention is a phytosanitary treatment method for controlling plant pathogens that cause plant diseases or a method for preventing and / or treating plant diseases caused by plant pathogens, especially fungi, which comprises applying a preparation comprising a strain of Streptomyces venezuelae, preferably a strain of Streptomyces venezuelae DSM 33887, on seeds, plants or soil.

[0031] According to a preferred aspect, application comprises seed coating, for example by dry mixing or admixture with a liquid composition prior to sowing.

[0032] According to another preferred aspect, application comprises spraying the seedlings or, as an alternative, mixing the solution as an aqueous solution with the soil substrate or with the cultivated plants before sowing, or performing a soil treatment immediately after transplanting.

[0033] According to another preferred aspect, applying comprises dipping the plantlet roots or rooted bulbs into an aqueous suspension comprising Streptomyces venezuelae, preferably the DSM 33887 strain, before transplanting.

[0034] In addition, application may include treatment during the cultivation stage of crops in the form of foliar sprays to combat pathogenic microorganisms, in particular fungal diseases, which cause diseases of leaves and other above-ground parts of plants.

[0035] Furthermore, in preparation for treatment with Streptomyces venezuelae, preferably strain DSM 33887, the seeds can be treated by ozonation, preferably at 20 ppm ozone for 45 minutes, to significantly reduce the microbial load associated with the seeds, thereby both reducing the seed microbial population that may compete with the same Streptomyces and stimulating seedling germination and development.

[0036] The experimental analysis conducted showed that the main mechanisms of action of the antagonistic microorganism Streptomyces venezuelae, especially the strain DSM 33887, in the biological control of plant pathogens include:

[0037] 1) Pathogens directly parasitize through physical attack and secretion of lytic enzymes;

[0038] 2) competition for nutrients required for pathogen development or its ability to colonize a habitat;

[0039] 3) Produce secondary metabolites with toxic and inhibitory activity against plant pathogens (antimicrobial).

[0040] Furthermore, the experimental analyses conducted have shown that the microorganisms of Streptomyces venezuelae, in particular the strain DSM 33887, are internalized in plant tissues and that the microorganisms subsequently colonize the plant, promoting plant growth and possibly stimulating the endogenous immune defenses of the plant itself. During the endophytic phase, the microbial colony rapidly multiplies, colonizing first the roots of the host plant and then the aerial parts, such as the stems and leaves, occupying important ecological niches at the expense of possible colonization by pathogenic microorganisms, thus losing their invasiveness and pathogenicity efficiency. In addition, the microorganisms stimulate the development of the host plant, since during the process of rhizosphere colonization, growth-promoting enzymes and metabolites are produced, which stimulate the development of the roots and aerial parts, as well as the immune defenses of the host plant.

[0041] From the studies conducted, it is evident that Streptomyces is able to induce the expression of genes in planta that are associated with systemic resistance responses against pathogens, indicating that plant immune responses occur when plants are stimulated through contact with Streptomyces.

[0042] Another object of the present invention is the use of at least one strain of Streptomyces venezuelae, preferably the strain Streptomyces venezuelae DSM 33887, for enhancing the immune defense of plants.

[0043] Another aspect of the invention is a phytosanitary treatment method for enhancing the immune defense of a plant, comprising applying a composition comprising a strain of Streptomyces venezuelae on seeds, plants or soil.

[0044] The following examples further illustrate the present invention.

[0045] Example

[0046] Formulation Examples

[0047] Example 1

[0048] A formulation for seed tanning was prepared in which Streptomyces venezuelae DSM 33887 was applied at a final concentration of 10% per kg of seeds. 9 UFC ml -1 20 ml of deionized water and add the co-formulation Bio Friendly 1 (binder / coating) SEEDWORX in a 1:1 ratio TM [Centor Europe, Andijk, The Netherlands].

[0049] Experimental example

[0050] The following experimental examples are concerned with evaluating the efficacy of the composition of the invention of Example 1 in controlling plant pathogens and in preventing, controlling and treating plant diseases caused by plant pathogens by using Streptomyces venezuelae through in vitro tests, under controlled environments and through field trials.

[0051] Materials and methods

[0052] Microbial cultures

[0053] The bacterial strains used in this study were from the collection of the Laboratorio di Patologia Vegetale, Department of Life Sciences, University of Modena and Reggio Emilia (Table 1).

[0054] Table 1 – List of antagonistic bacterial strains used in this study

[0055]

[0056] The plant pathogenic fungi used in this study are the most common agents causing soil diseases affecting horticultural crops (Table 2).

[0057] Table 2 – List of plant pathogenic fungi used in this study

[0058] Plant pathogenic fungi Identification code Isolated plant matrix Fusarium oxysporum 1585 onion Fusarium oxysporum Fus Ruc 9A Wild Arugula Sclerotinia species 1584 Spatholobus species 1773 onion Colletotrichum fasciculata 1772 spinach Pythium species 1583 onion

[0059] Plant materials

[0060] Four different horticultural crops were used during the study period (Table 3).

[0061] Table 3 – List of horticultural species used in this study

[0062]

[0063] Example 2 - In vitro antagonism assay

[0064] The double plate culture method was used to study microbial antagonism. Each bacterial culture of Table 1 was cultured at 27±2°C in nutrient liquid broth (NSB = nutrient broth 8.0 gr l -1 , sucrose 50gr l -1 ) for 24 h, then a drop (20 μl) of bacterial suspension was recovered from the growing culture and smeared on one side of a Petri dish containing potato dextrose agar (PDA = potato extract 4 g l) 24 h before inoculation with fungi, 1 cm from the edge. -1 , Glucose 20g l -1 , agar 15g l -1 ). A disk (3 mm) of fungal mycelium grown on PDA for five days was then placed on the opposite side of each plate (1 cm from the edge) in order to evaluate the antagonistic effect of the bacterial inoculum. Plates containing the same nutrient matrix but without the addition of antagonistic bacteria were used as controls. All plates were then incubated at 27 ± 2 °C for seven days. Fungal growth inhibition (IG) was evaluated as a percentage and calculated as the average value of the diameter of the fungal mycelium formed around the bacterial colony area according to the following formula (Wang et al., 2011) (three experimental replicates were performed for each isolate):

[0065]

[0066] (C = diameter of positive control, S = diameter of sample)

[0067] result

[0068] The antagonistic activity of four isolated microorganisms: Streptomyces species DLS1568, Streptomyces species SA51, Agrobacterium species AR 39 and Pseudomonas species PT 65 against seven plant pathogenic fungi: Fusarium oxysporum race 1, Fusarium oxysporum race 2, Sclerotinia species, Stolonifera species, Colletotrichum fasciculata and Pythium species were evaluated in vitro.

[0069] exist Figure 1 The IG (%) values ​​obtained for each microorganism are reported in the histogram.

[0070] All four microorganisms showed inhibitory activity against the growth of phytopathogenic fungi. However, Streptomyces sp. DLS1568 was particularly effective, showing IG (%) higher than 55% in all seven phytopathogenic fungi tested in vitro ( Figure 1 ).

[0071] Streptomyces sp. DLS1568 also showed the highest inhibitory activity against Fusarium oxysporum race 1 and Fusarium oxysporum race 2, with IG (%) as high as 60% and 65%, respectively.

[0072] Pseudomonas sp. PT 65 and Agrobacterium sp. AR 39 showed lower inhibitory activity (IG less than 50%) against the above seven plant pathogenic fungi.

[0073] Using the double plate culture assay, Streptomyces sp. DLS1568 and Streptomyces sp. SA51 showed IGs higher than 50% against all seven plant pathogenic fungi.

[0074] Example 3 - Plant growth promotion mechanism

[0075] Two plant growth-promoting mechanisms, such as siderophore production and phosphate solubilizing activity, of Streptomyces sp. DLS1568 and Streptomyces sp. SA51 strains were validated by in vitro assays on agar-based plates.

[0076] Siderophore production

[0077] To evaluate the production of siderophores, a modification of the method of Schwyn and Neilands (1987) was used. Both Streptomyces were cultured in a liquid medium “International Streptomyces Project-2” (ISP-2, yeast extract 4.0 g -1 Malt extract 10.0gl -1 , glucose 4.0gl -1) in a flask at 26±2°C for 48 hours. The Streptomyces microbial suspension was then centrifuged at 6000 rpm for 20 minutes, and the resulting precipitate was preliminarily washed and then resuspended in sterile distilled water; a portion of the suspension was inoculated in agar CAS-LB (agar base LB with Chrome Azurol S added) prepared according to Schwyn and Neilands (1997). Sterile distilled water was used as a negative control, and 1M catechol solution (Lakshmanan et al., 2015) was used as a positive control. The plates were then incubated at 28±2°C for 5 days, and the formation of yellow-orange halos, which indicates the production of siderophores, was measured. Three experimental replicates were performed for each strain. The test data were subjected to statistical analysis of variance (one-way ANOVA). The results were compared with Tukey's HDS post hoc test.

[0078] result

[0079] Microorganisms may be characterized by the production and secretion of siderophores suitable for sequestering and metabolizing iron present in the rhizosphere. Two isolated strains: Streptomyces sp. DLS1568 and Streptomyces sp. SA51 showed the ability to produce siderophores, as highlighted by the color change of the blue CAS matrix from turquoise to orange.

[0080] Differences in siderophore production were assessed by measuring the diameter of the orange halo. Streptomyces species DLS1568 showed higher siderophore production, with a recorded mean halo diameter of 43 mm (SD = 5.00), compared to a mean halo diameter of 11 mm (SD = 5.29) for Streptomyces species SA51. Statistical analysis showed that the mean diameter values ​​(mm) of the yellow halo of Streptomyces species DLS1568, Streptomyces species SA51 were significantly different from the positive control (1 M catechol) (p < 0.001) ( Figure 2 ).

[0081] Solubilization of phosphate

[0082] The phosphate solubilization capacity of Streptomyces species DLS1568 and Streptomyces species SA51 strains was studied using a modified Lucas et al. (2014) protocol. A drop of Streptomyces bacterial suspension (20 μl) was placed in the center of a petri dish with an agar matrix. A drop of sterile distilled water (20 μl) was used as a negative control. After 14 days of cultivation, the phosphate solubilization capacity was evaluated by evaluating the presence and diameter measurement of lighter colored areas (so-called halos) around the colonies of Streptomyces species strains. Three technical replicates were performed for each strain. The recorded data were subjected to statistical analysis of variance (one-way ANOVA). The results were compared with Tukey's HDS post hoc test.

[0083] result

[0084] The ability of Streptomyces species DLS1568 and Streptomyces species SA51 strains to metabolize phosphate was determined by evaluating their ability to extracellularly solubilize tricalcium phosphate precipitated with glucose as the sole carbon source. Streptomyces species DLS1568 strain showed a higher and statistically significant mineral phosphate solubilization activity (p < 0.001), with a mean value of 24.33 mm (SD = 3.05) for the light halo compared to a mean value of 9.00 mm (SD = 2.00) for Streptomyces species SA51 strain ( Figure 3 ).

[0085] Example 4 - Observation of EGFP-Streptomyces sp. DLS 1568 using confocal laser scanning microscopy (CLSM) Endophytic colonization in plants

[0086] Onion, fennel, wild rocket and lettuce seeds were used for microbial seed coating, where the EGFP-Streptomyces sp. DLS1568 mutant strain was transformed with GFP protein (green fluorescent protein) so that it could be tracked during internalization and colonization in plants. Each seed sample (n = 1 g of seeds) was surface sterilized according to the method of Fatmi et al. (1991). Briefly, seeds were treated in 2% sodium hypochlorite (NaOCl) for 20 minutes at 25°C on a rotary shaker at a speed of 250 rpm (Innova, New Brunswick Scientific, Edison, NJ), and then rinsed under tap water for three minutes.

[0087] The Streptomyces bacterial suspension used for seed coating was grown in liquid medium ISP-2 at 27 ± 2 ° C for 24 hours. The suspension was then centrifuged at 6000 rpm for 20 minutes. The supernatant was discarded and the pellet was resuspended in 20 ml of sterile distilled water. The bacterial concentration (optical density at 600 nm = 0.3; about 1 × 10 8 CFU ml -1 ) and calibrated to 10 8 CFU ml -1 final concentration.

[0088] According to Kaufman (1991), seed samples were treated with sterile distilled water (used as negative control) and the above bacterial suspension. Briefly, each seed sample was incubated on a rotary shaker at 250 rpm at 25°C for 20 minutes (Innova, New Brunswick Scientific, Edison, NJ), air-dried at room temperature for 24 hours, and then stored at 4°C until sowing.

[0089] Seedlings grown 20 days after sowing were evaluated for internalization of Streptomyces sp. DLS1568 strain. For each sample, roots, stems and leaves (approximately 10 mm) were carefully dissected with a sterile scalpel and placed on a slide for observation by confocal microscopy (CLSM) Leica TCS SP8 (Leica Microsystems, Mannheim, Germany) equipped with a solid-state laser capable of exciting the fluorescence of the mutant EGFP-GFP protein of Streptomyces sp. DLS1568 strain, thus allowing verification of the presence and possible colonization of plant tissues. Laser scanning microscopy analysis was performed at CIGS (CentroInterdipartimentale Grandi Strumenti, University of Modena and Reggio Emilia, Italy).

[0090] result

[0091] The internalization of Streptomyces sp. DLS1568 was investigated using confocal laser scanning microscopy (CLSM) using different tissues of seedlings developed 3 weeks after sowing, such as roots and leaves of onion, fennel, wild rocket and lettuce. Endophytic colonization of plant tissues by EGFP-Streptomyces sp. DLS1568 was observed in 99% of tissue sections of all seedling samples. The extensive colonization of EGFP-Streptomyces sp. DLS1568 demonstrated the ability of this microorganism to interact with the host from the early stages of germination from seeds and during root and leaf development. Cells of the mutant EGFP-Streptomyces sp. DLS1568 showed uniform distribution in all the various plant tissues. These results confirm that Streptomyces sp. DLS1568 can internalize and colonize plant tissues, highlighting the potential for long-term interactions with all four different crops evaluated in this study.

[0092] Example 5 - Germination Test

[0093] Onion, fennel, wild rocket and lettuce seed samples were coated with Streptomyces species DLS1568 (treatment specifications see Example 2) and experimentally treated with ozonation to determine their germination performance together with the corresponding uncoated controls. Four replicates were performed for each seed sample (n = 100 seeds) of each plant species according to the ISTA protocol (International Seed Testing Association, 2018). The seed samples were placed on germination test strips (Sartorius, DE), moistened and subsequently placed in plastic boxes. The plastic boxes were then placed in a germination room at an average temperature of 21±2°C and daylight for 16 hours. After the incubation period, the number of germinated seeds in each box was counted and the germination percentage of the total tested seeds was calculated. According to the Seedling Evaluation Manual (International Seed Testing Association, 2018), seeds are considered to have germinated when the seedlings and all their basic structures (such as stems, epicotyls, cotyledons, hypocotyls and roots) are well developed, complete, well-proportioned and healthy. The germination test results are expressed as a percentage, taking into account the number of normal, abnormal seedlings, hard seeds, fresh seeds and dead seeds according to the following formula. Four technical replicates were performed for each sample.

[0094]

[0095] Where n represents the number of germinated seeds and N represents the total number of seeds used for testing.

[0096] Seed coating was performed with Streptomyces sp. SA51 by the method previously described in Example 2 and by using Bio-Friendly 1 (binder / coating) SEEDWORX TMSeed samples of onion variety Elenka and fennel variety Crono obtained from [Centor Europe, Andijk, The Netherlands] were used as commercial controls. The recorded data were subjected to statistical analysis of variance (one-way ANOVA). The results were compared with Tukey's HDS post hoc test.

[0097] result

[0098] Germination data obtained for seeds coated with Streptomyces sp. DLS1568 and Streptomyces sp. SA51 showed improved germination performance compared to the uncoated control. Figure 4 A), germination rate was recorded for uncoated seeds (negative control) of 65%, whereas for both treatments with Streptomyces sp. DLS1568 and Streptomyces sp. SA51, germination rate was significantly increased up to 20% (p<0.05). Similar results were obtained in fennel germination tests. Figure 4 B shows that for the treatments with Streptomyces species DLS1568 and Streptomyces species SA51, germination was increased by 30% compared to the uncoated control. In addition, Streptomyces species DLS1568 had higher germination values ​​than Streptomyces species SA51 in both tests (i.e., onion and fennel), even though there were no non-significant differences (p>0.05).

[0099] Example 6 - In planta experiments under controlled conditions to evaluate the microbial coating of wild rocket as a Effectiveness of biological control operations for tracheofusarium disease (FOR)

[0100] According to the experimental protocol of Gilardi et al. (2007), wild rocket variety Adagio, susceptible to infection by Fusarium oxysporum race radish (FOR), was experimentally analyzed to evaluate the effectiveness of Streptomyces sp. DLS1568 as a biological control agent applied by seed coating against the plant pathogenic fungus FOR (Fus Ruc 9A strain). The experimental analysis was performed under controlled conditions (phytotron) on samples of wild rocket variety Adagio seeds treated with Streptomyces sp. DLS1568 and Streptomyces sp. SA51 (commercial control), following the protocol described in Example 2. Samples of uncoated seeds were used as controls.

[0101] To prepare the fungal inoculum, 15 ml of sterile distilled water was added to the corresponding PDA plate to rehydrate the fungal conidia, thereby preparing the conidial suspension of FOR, in which the plant pathogenic fungi had been grown in the plate for 7 days. Afterwards, the conidial suspension was filtered through gauze. The conidial concentration was then determined by using a hemocytometer and calibrated to 10 with deionized water. 7CFU ml -1 The final concentration was then used as inoculum in all experiments.

[0102] The seeds were then sown in polystyrene trays for a total of 104 seeds per track. Twenty days after sowing, the roots of the seedlings were cut into 5 cm lengths and placed in a calibrated conidia suspension of the fungal plant pathogen strain FOR (Fus Ruc 9A) for 20 minutes as described above. Twenty-five treated seedlings from each track were transplanted to new trays. Each tray (n=25) was considered an experimental replicate, with a total of 4 replicates per treatment. The disease severity index values ​​(severity) are reported in Table 4. The recorded disease severity data were subjected to statistical analysis of variance (one-way ANOVA). The results were compared with Tukey's HDS post hoc test.

[0103] Table 4 - Plant pathology scale showing disease severity index with values ​​ranging from 0 (healthy plants) to 100 (dead plants)

[0104]

[0105] result

[0106] The experimental analysis conducted under controlled conditions (climate chamber) showed that the mean disease severity index of plantlets developed from seeds coated with Streptomyces sp. DLS1568 and Streptomyces sp. SA51, caused by infection with the pathogenic strain FOR (Fus Ruc 9A), was significantly lower (p<0.05) than that of uncoated seed samples (control). For uncoated wild rocket (control), the mean disease severity index recorded was 36.25%. Seed coating treatments with isolated strains Streptomyces sp. DLS1568 and Streptomyces sp. SA 51 had a significant protective effect against Fusarium wilt pathogen (p<0.05). The highest biocontrol effect / efficacy was recorded for seeds coated with the isolate Streptomyces sp. DLS1568, showing a relative mean disease severity index of 16.50%, even though it was not significantly different (p>0.05) from seeds coated with Streptomyces sp. SA 51, which showed a mean disease severity index of 20.75% ( Figure 5 ).

[0107] Example 7 - Field trials of onion variety Elenka to evaluate its resistance to natural Fusarium spp. Germination performance and biocontrol activity of Fusarium spp. under inoculation stress (FC2020)

[0108] In this experiment, the onion variety Elenka was tested. Table 5 lists the corresponding tanned and ozonated seed treatments. The test experimental design was organized in the form of randomized blocks for four treatment types, each in duplicate. In the extended tests carried out in experimental fields with high natural pressure of Fusarium inoculum, the dimensions of the experimental plots were as follows: 1.8 m (width) × 25 m (length), with four sowing rows per plot. Sowing was carried out with a mechanical seeder (mod. SPX 2015 - Bassi Seminatrici, IT) at a density of 600,000 onion seeds per hectare. Surveys were carried out over a length of 20 meters in the middle part of each corresponding plot. The recorded disease severity data were subjected to statistical analysis of variance (one-way ANOVA). The results were compared with Tukey's HDS post hoc test.

[0109] During the preparation phase before sowing, the fields were not treated with herbicides or fungicides. During the experimental tests, which were carried out in accordance with good agronomic practices, no infection with harmful bacteria and insects that could damage the cultures during the experimental period was observed. Weed control was carried out according to the disciplines described in the Cornell Vegetable Management Guide and Recommendations (Reifers et al., 2019).

[0110] During the cultivation season, samples of symptomatic materials such as onion roots and bulbs were collected to isolate Fusarium-like fungal colonies, which were subsequently identified and confirmed as pathogens by preliminary analysis of conidia under a microscope and then molecular analysis. According to White et al., 1990, molecular identification was performed by polymerase chain reaction (PCR) amplification of the conserved region ITS. The amplified product was then purified and sequenced at Bio-Fab Research srl (Rome, IT). The nucleotide sequence of the conserved region ITS (Internal Transcription Spaces) of ribosomal DNA was then analyzed by the sequence similarity study program in the international DNA database BLAST (Basic Local Alignment Search Tool; / / www.ncbi.nlm.nih.gov / BLAST).

[0111] Table 5 – List of onion seed treatments investigated during the Cesena (FC) field trials

[0112]

[0113] Note: * Bacterial suspension concentration: 1×10 9 CFU ml -1

[0114] result

[0115] In the experimental tests carried out during the first survey period (18 days after sowing), the number of seedlings was evaluated according to the above procedure. Seedlings produced from seeds treated with Streptomyces species DLS1568 (210.75; SD = 36.71) and ozone (T3) (214.63; SD = 53.4) showed an increase of 15% and 17% in germination, respectively, compared to the control (182.50; SD = 26.22). The germination rate of such seedlings (produced from seeds treated with Streptomyces species DLS1568 and ozone (T3)) increased by 20% and 22%, respectively, compared to the fludioxonil treatment (175.63; SD = 32.70). For such treatments (i.e. Streptomyces species DLS1568 and ozone (T3)), the germination rate increased by 24% and 26%, respectively, compared to the treatment with Streptomyces species SA51 (169.13; SD = 25.82). However, statistical analysis of variance (one-way ANOVA, Tukey's test) did not show significant differences (p>0.05).

[0116] During the second investigation period (42 days after sowing), the number of plants emerging from seeds treated with ozone (207.88; SD = 35.62) was significantly higher (p < 0.05) by 41% and 51% relative to treatments with fludioxonil (147.00; SD = 41.52) and Streptomyces sp. SA51 (137.71; DS = 18.25). In addition, the number of plants emerging from seeds treated with Streptomyces sp. DLS1568 (198.13; SD = 43.53) showed a significant increase of 44% (p < 0.05) and 34% (p < 0.05) relative to treatments with Streptomyces sp. SA51 and fludioxonil, respectively.

[0117] In the third survey (70 days after sowing), the germination performance trends were consistent with the first two surveys, especially the germination performance of the above treatments was better. Finally, in the fourth survey (97 days after sowing), the data related to the number of plants emerged showed that the number of plants emerged in the two treatments of Streptomyces species DLS1568 (137.40; SD = 16.41) and ozone (119.17; SD = 19.24) was greater than that in the treatments of fludioxonil (72.75; SD = 8.10) and Streptomyces species SA51 (87.17; SD = 10.57). In particular, the number of plants grown from seeds treated with Streptomyces species DLS1568 (198.13; Sd = 43.53) was significantly higher than that of plants treated with Streptomyces species SA51 and fludioxonil by 57% (p < 0.05) and 72% (p < 0.05), respectively.

[0118] By analyzing symptomatic samples of onion plants collected from field trials, the presence of a high natural inoculation pressure was confirmed by isolating and identifying Fusarium oxysporum, which was identified by morphological examination of the fungal isolates (colony characteristics and observation of fruiting bodies, phialides, chlamydospores, etc. under a microscope). By using BLAST to compare sequences with the National Center for Biotechnology Information (NCBI) database, it was found that the sequence similarity of the internal transcribed spacer (ITS) region of the conserved ribosomal region was 98.50% and 100%.

[0119] The higher number of seedlings, germination, and developing plants recorded in the four surveys, especially compared with the fungicide treatment with fludioxonil, highlights the biocontrol activity of Streptomyces sp. DLS1568 against the plant pathogenic fungus Fusarium oxysporum by seed coating.

[0120] Example 8 - Field trials of onion variety Elenka to evaluate its agronomic characteristics (phenological development) (FC2020)

[0121] For description of the analysis, experimental design, agronomic practices, see Example 7. Before sowing, the fields were not treated with herbicides or fungicides. During the experimental field trials conducted according to the best agronomic practices, no infection with harmful bacteria and insects that caused damage to the cultures during the experiments was observed. Weeds were managed according to the disciplines described in the Cornell Vegetable Management Guide and Recommendations (Reifers et al., 2019).

[0122] The recorded data were subjected to statistical analysis of variance (one-way ANOVA). The results were compared with Tukey's HDS post hoc test.

[0123] result

[0124] like Figure 6 As shown in A, in the plant height assessment (second survey), Streptomyces DLS1568 treatment showed a significant increase in plant height (12.23 cm; SD = 0.9) compared to fludioxonil (10.11 cm; SD = 0.7; p < 0.05). The number of leaves on the plants in the five tracks was assessed (third survey), and the values ​​recorded did not indicate significant differences between the tracks (p > 0.05), although the highest values ​​were recorded in the tracks of seeds treated with Streptomyces DLS1568 (5.23; SD = 0.2) and ozone (4.69; SD = 0.6). Figure 6 B).

[0125] Example 9 - Field trials using the onion variety Fundador to evaluate the presence of Fusarium spp. in experimental fields (FC2020) Germination performance and biological control under natural inoculation pressure

[0126] For experimental description, experimental design, agronomic practices, see Example 7. In this experiment, onion variety Fundador was used and the seed treatments listed in Table 5 were applied. The recorded data were subjected to statistical analysis of variance (one-way ANOVA). The results were compared with Tukey's HDS post hoc test.

[0127] result

[0128] During the first survey period (18 days after sowing), the number of plants emerging from seeds treated with Streptomyces sp. DLS1568 (212.87; SD = 33.99) was significantly higher (p < 0.05) by 20%, 51% and 15% compared to the control (178.12; SD = 30.92), Streptomyces sp. SA51 (141.12; SD = 35.71) and ozone treatment (185.87; SD = 33.24). There was no significant difference (p > 0.05) between Streptomyces sp. DLS 1568 and fludioxonil treatments (210.12; SD = 57.65). In the fourth survey, the number of plants emerging from seeds treated with Streptomyces sp. DLS1568 (154.57; SD = 16.38) was significantly higher (p < 0.05) than from the treatment with Streptomyces sp. SA51 (103.50; SD = 35.61). From the isolation and identification of symptomatic plant material, the presence and identity of the plant pathogen Fusarium oxysporum fungal isolates were confirmed, confirming the high pressure of the natural inoculum (see Example 7).

[0129] Increased values ​​were recorded in the number of seedlings, germination and developing plants from seeds coated with Streptomyces sp. DLS1568 in the four surveys compared to Streptomyces sp. SA 51. Moreover, these values ​​were consistent with the results of sowing fungicide treatments with fludioxonil, highlighting the biocontrol effect of Streptomyces sp. DLS1568 against the pathogenic fungus Fusarium oxysporum by seed coating.

[0130] Example 10 - Commercial field trials of fennel variety Crono to evaluate its emergence and germination performance (AQ2020)

[0131] In this experiment, fennel variety Crono was used to study the effect of applying Streptomyces species DLS1568 as a microbial coating agent on crop emergence and germination. The corresponding seed treatments are listed in Table 6.

[0132] The test experimental design was organized in the form of randomized blocks for 4 treatment types, each type in duplicate (replication). In the experiments conducted in plots, the dimensions of the experimental plots were as follows: 1.5 m (width) × 100 m (length), each plot had four sowing rows. Sowing was carried out with a mechanical seeder. During the experimental field trials conducted according to the best agronomic practices, no infection with harmful bacteria and insects that caused damage to the cultures during the experiments was observed. Weeds were controlled according to the integrated weed management on the farm.

[0133] The first survey was conducted 20 days after sowing to evaluate the total length of each plot. The second survey was conducted 32 days after sowing the seeds, and the third and final survey was conducted one month after the second survey. The recorded data were subjected to statistical analysis of variance (one-way ANOVA). The results were compared with Tukey's HDS post hoc test.

[0134] Table 6 – List of seed treatments applied to fennel variety Crono during field trials (AQ)

[0135]

[0136] Note: * Bacterial suspension concentration: 1×10 9 CFU ml-1

[0137] result

[0138] All the seedling and germination values ​​recorded indicated that the germination performance and number of healthy plantlets were increased in the Streptomyces sp. DLS 1568 treatment compared to the control and fludioxonil treatments (Table 7). In the first investigation of the number of seedlings, the mean value of the Streptomyces sp. DLS 1568 treatment was higher, 365.25 (SD = 26.79), but not significantly higher than the control (339.50; SD = 9.33) and fludioxonil (339.50; SD = 45.35) related tracks. In the second survey (after the agronomic practice of artificial thinning of plantlets, usually applied by agricultural operators to weak / abnormal plantlets, leaving an average distance of 20-25 cm on the rows), the data collected and statistically analyzed showed that the number of plantlets in seeds treated with Streptomyces sp. DLS1568 (227.94; SD=8.31) was significantly increased (p<0.05) compared to the control (194.40; SD=14.85). Although the values ​​of tracks treated with fludioxonil (217.30; SD=3.89) were lower than those of tracks treated with Streptomyces sp. DLS1568, there was no significant difference (p>0.05). In the third survey, the data collected and statistically analyzed showed that the number of plants treated with Streptomyces sp. DLS1568 was significantly increased (p<0.05) with a mean of 212.50 (SD=16.26) compared to the control track (187.00; SD=7.07) and fludioxonil (198.62; SD=0.88) (Table 7).

[0139] Table 7 - Number of seedlings of fennel variety Crono recorded during the extended plot field trial (AQ2020). Means with the same letters indicate no significant difference between treatment groups (one-way ANOVA, Tukey's test, p>0.05). Means with different letters indicate significant difference between treatment groups (one-way ANOVA, Tukey's test, p<0.05). Bars represent standard deviation of the mean (±SD).

[0140]

[0141] Example 11 - Field trials using fennel variety Crono to assess emergence, germination and vigor of plots (AQ2021)

[0142] See Example 10 for field trial description, experimental design, and agronomic practices applied.

[0143] For this experiment, sowing was carried out with a mechanical seeder. The first survey was carried out 14 days after sowing, evaluating the entire length of each plot: 1.5 m (width) × 100 m (length), each plot having four sowing rows. The second survey was carried out one month after sowing, and the third and final survey was carried out two months after sowing, evaluating the number and vitality of the emerged plants according to the evaluation criteria described in Table 8. The recorded data were subjected to statistical analysis of variance (one-way ANOVA). The results were compared with Tukey's HDS post hoc test.

[0144] Table 8 – Vigor Assessment Scale for Commercial Field Trials of Fennel Variety Crono (AQ2021)

[0145]

[0146] The emergence data of tracks treated with seed tanning with Streptomyces sp. DLS1568 showed an improvement in germination performance and development of healthy plantlets compared to the control and fludioxonil treatments (Table 9). In the first survey, the mean recorded for tracks treated with Streptomyces sp. DLS1568 coating in terms of number of seedlings emerged showed a higher, but not significantly (p>0.05) mean of 335.13 (SD=6.89) compared to the coated treatments associated with the uncoated control (313.00; SD=4.60) and fludioxonil (323.88; SD=6.19). In the second survey, to assess the emergence of plants (in agronomic practice, manual thinning of weak / abnormal plantlets is usually applied by agricultural operators to weak / abnormal plantlets), data collected and statistically analyzed showed that the number of plantlets in seeds treated with Streptomyces sp. DLS1568 (209.13; SD = 8.31) was significantly increased compared to the control group (180.75; SD = 14.85) (p < 0.05). There was no significant difference (p > 0.05) between the number of existing plantlets in seeds coated with Streptomyces sp. DLS1568 and fludioxonil (205.00; SD = 3.89). In the third survey, the data collected and statistically analyzed showed that the number of plants treated with Streptomyces sp. DLS1568 (194.50; SD = 12.24) was significantly increased (p < 0.05) relative to the number of plants treated with uncoated controls (173.00; SD = 8.05) and those treated with fludioxonil coating (186.63; SD = 1.98).

[0147] Table 9 - Number of emerged plants of fennel variety Crono detected in the extended plot field trial (AQ2021). Mean values ​​with the same letters indicate no significant difference between treatment groups (one-way ANOVA, Tukey's test, p>0.05). Mean values ​​with different letters indicate significant difference between treatment groups (one-way ANOVA, Tukey's test, p<0.05). The bar graph represents the standard deviation of the mean (±SD).

[0148]

[0149] In the second survey, the vigor of the plantlets was evaluated and the data recorded showed that the mean vigor index of plants germinated from seeds treated with Streptomyces sp. DLS1568 was significantly increased (p<0.05) with a mean value of 2.69 (SD=0.20) compared to plants treated with fludioxonil coating (2.49; SD=0.27) and uncoated controls (2.34; SD=0.22) ( Figure 7 A). Even in the third survey, the data collected and statistically analyzed showed that the average vigor index of plants germinated from seeds treated with Streptomyces sp. DLS1568 was significantly increased (p<0.05), with a mean of 3.09 (SD=0.21), while the mean of the uncoated control was 2.71 (SD=0.19) ( Figure 7 B) No significant difference was found between the tanning treatment with Streptomyces sp. DLS1568 and the tanning treatment with fludioxonil (3.01; SD = 0.15).

[0150] Example 12 - In planta detection of Streptomyces sp. DLS 1568

[0151] As reported in Examples 7, 8, 9, 10 and 11, during field trials on onion and fennel crops, plant samples corresponding to seeds treated with Streptomyces sp. DLS1568 were collected at different phenological stages (i.e., seedlings, plantlets and plants) to detect and confirm the presence and colonization of the microorganism in plant tissues. Plants germinated from uncoated seeds were used as negative controls. Plant material was collected and used Plant Mini Kit (Qiagen, Hilden, Germany) was used for DNA extraction. Then, 2 μl DNA aliquots were subjected to PCR molecular tests (polymerase chain reaction) using specific primers for the 16S rRNA gene of Streptomyces: StrepB (forward) (5'-ACAAGCCCTGGAAACGGGT-3') (SEQ ID N.1) and StrepF (reverse) (5'-ACGTGTGCAGCCCAAGACA-3') (SEQ ID N.2), according to the experimental protocol reported by Rintala et al. (2001). Gene amplification (PCR) was performed in a 25 μl amplification reaction in a thermal cycler (Applied Biosystems 9700). After amplification, samples were analyzed by electrophoresis in 2% agarose gels, stained with ethidium bromide for 30 minutes, and images were stored in BioDoc Analysis (Biometra, Göttingen, Germany).

[0152] result

[0153] 93% and 89% of onion and fennel plant samples, respectively, showed positive results for specific gene amplification with the primer pair of rRNA 16S StrepB / StrepF gene, generating a specific fragment of 507 bp. Thus, it was confirmed that Streptomyces sp. DLS1568 was able to colonize onion and fennel plant tissues by seed coating treatment with this microorganism. No specific amplification was recorded for the uncoated control.

Claims

1. Use of at least one strain of Streptomyces venezuelae as a phytomedicine or phytosanitary product.

2. The method according to claim 1, wherein the strain is Streptomyces venezuelae DSM 33887.

3. Use according to claim 1 or 2 as a fungicide or biofungicide.

4. The method according to claim 1 for controlling or eliminating plant pathogens.

5. The use according to claim 1 or 2 for preventing and / or treating plant diseases caused by plant pathogens.

6. The use according to claim 4 or 5, wherein the plant pathogen is a pathogenic fungus causing plant diseases.

7. The use according to claims 3-6, wherein the fungus is selected from: Fusarium oxysporum f.sp.cepae (FOC), Fusarium oxysporum f.sp.raphani (FOR), Fusarium oxysporum f.sp.lactucae (FOL), Sclerotinia species (Sclerotinias p.), Pythium species (Pythium sp.), Stemphylium botryosum, Colletotrichum dematium.

8. A phytosanitary treatment method for controlling or eradicating plant pathogens, comprising applying a composition comprising a strain of Streptomyces venezuelae to seeds, plants or soil.

9. A phytosanitary treatment method for preventing and / or treating plant diseases caused by plant pathogens, comprising applying a composition comprising a strain of Streptomyces venezuelae to seeds, plants or soil.

10. A method of plant quarantine treatment for enhancing the immune defence of a plant, comprising applying a composition comprising a strain of Streptomyces venezuelae to seeds, plants or soil.

11. The method according to claims 8-10, wherein the strain is Streptomyces venezuelae DSM 33887.

12. A method according to claims 8-11, wherein applying comprises seed coating.

13. A method according to claims 8 to 11, wherein application comprises spraying the seedlings, or alternatively, mixing with the culture medium or cultivated plants as an aqueous solution before sowing, or as a soil treatment immediately after transplanting.

14. A method according to claims 8-11, wherein applying comprises dipping the plantlet roots or rooted bulbs in the aqueous suspension prior to transplanting.

15. The method according to claims 8 to 11, wherein applying comprises treating as a foliar spray during the cultivation phase.

16. The method according to claims 8-15, wherein applying further comprises seed ozonation.

17. A formulation comprising a strain of Streptomyces venezuelae as an active ingredient, and at least one acceptable co-adjuvant and / or co-formulator and / or carrier.

18. The preparation according to claim 7, wherein the strain is Streptomyces venezuelae DSM 33887.

19. The formulation according to claim 7 or 8, wherein the co-formulator is Bio-Friendly 1.

20. Formulation according to claims 7-9, wherein the Streptomyces venezuelae strain or Streptomyces venezuelae DSM 33887 is present in the composition in a weight ratio ranging from 5% to 75%, preferably from 20% to 40%, even more preferably equal to 30% of the total weight of the composition (w / w).

21. The preparation according to claim 20, wherein the Streptomyces venezuelae strain or Streptomyces venezuelae DSM 33887 is present in an amount of 10 7 Up to 10 9 , preferably equal to 10 8 The amount of UFC / 1 g of the composition is present in the composition.

22. A Streptomyces venezuelae strain known as DLS1568, which has been deposited at the Leibniz-Institute DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH under the accession number DSM 33887.

23. Use of at least one strain of Streptomyces venezuelae for enhancing the immune defence of a plant.

24. Use according to claim 23, wherein the strain is Streptomyces venezuelae DSM 33887.

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