CN112760229A

CN112760229A - Trichoderma and application thereof

Info

Publication number: CN112760229A
Application number: CN202011610974.3A
Authority: CN
Inventors: 李铭刚; 赵江源; 杨佩文; 周旭峰; 文孟良; 何翔
Original assignee: Yunnan University YNU
Current assignee: Yunnan University YNU
Priority date: 2020-12-30
Filing date: 2020-12-30
Publication date: 2021-05-07
Anticipated expiration: 2040-12-30
Also published as: CN112760229B

Abstract

The invention relates to the field of agricultural microorganisms and discloses trichoderma and application thereof. The preservation number of the trichoderma is CGMCC No. 21023. In addition, the invention also provides application of the trichoderma in regulation and control of plant diseases. The trichoderma of the invention is the dominant strain of high-yield siderophore and can synthesize the secretory hydroxamic acid siderophore. The secondary metabolites of trichoderma of the present invention have bacteriostatic activity against pseudomonas solanacearum (pseudomonas solanacearum), Pectobacterium carotovorum (pebacterium carotovorum) and Ralstonia solanacearum (Ralstonia solanacearum).

Description

Trichoderma and application thereof

Technical Field

The invention relates to the field of agricultural microorganisms, and particularly relates to trichoderma and application thereof.

Background

The use of a siderophore microorganism (and siderophore compounds thereof) for plant protection is expected to be a sustainable plant health management method. By screening high-yield siderophore active strains from nature and investigating the bacteriostatic activity of metabolites thereof, a novel idea can be provided for the development of related microbial biocontrol microbial agents (preparations). The theoretical basis is as follows: iron plays an important role in the growth, development and metabolic processes of microorganisms, but iron mainly exists in an insoluble trivalent stable form in nature. To obtain iron, microorganisms typically secrete iron ions (Fe)³⁺) The process is realized by a siderophore (siderophore), and the microbial community structure in the soil is shaped by the distribution of iron elements among different microorganisms, so that the biological properties of the soil are changed.

How to utilize siderophore produced by non-pathogenic bacteria in nature to inhibit or inhibit the growth of plant pathogenic microorganisms by interfering the normal reproduction and amplification of pathogenic bacteria has become a research hotspot in recent years. For example, Tian F et al screened an antagonistic bacterial strain (accession No. G-229-21T) of the pediatric chlorophenol-type siderophore tobacco Phytophthora parasitica and concluded that: the siderophore compound inhibits the growth of phytopathogens. Solanki MK et al found: rhizobium strains having siderophore activity have stronger inhibitory activity against rice sheath blight disease (Rhizoctonia solani), while the antagonistic activity against siderophore-free strains is weaker. When Dutta S and the like screen the biocontrol bacteria, the strains with stronger control effect are found to have the capability of secreting catecholate type siderophore in general. Ambrosi C et al found that a large amount of carboxylate-type siderophore compound was present in P.fluorescens metabolites; it inhibits the growth of phytopathogens in the soil environment by secreting a range of siderophore materials. The Liangyan R and the like screen PGPR strains with high siderophore activity through CAS detection liquid, and the PGPR strains have bacteriostatic activity on fusarium and the like. Masum MMI et al found that the siderophore-active strain (code K5-3) reduced the number of pathogenic bacteria cells by 73-80% and biofilm formation by 55-65% in studies on bacterial brown streak in rice. In addition, documents report that siderophore microorganisms have absolute advantages for iron ion competition in soil microbial communities, for example, Hedia and the like establish a microbial community model under siderophore intervention based on the siderophore iron chelation principle of microorganisms, draw the conclusion that siderophore producing bacteria have competitive advantages compared with other strains, and regulate the structure and composition of plant pathogenic microbial communities according to the competitive advantages.

At present, plant diseases such as tomato bacterial wilt, potato bacterial wilt and tobacco bacterial wilt mainly occur as a result of mass propagation and amplification of plant pathogenic bacteria. Bacterial strains with high siderophore activity are discovered from healthy soil in the nature, iron competitive factors are actively applied in the soil environment of plant pathogenic bacteria through the intervention of the bacterial strains (and siderophore compounds thereof), and the aim of reducing the occurrence of plant diseases is expected to be achieved by redistributing iron elements among microorganisms, inhibiting the growth of pathogenic microorganisms, regulating the structure of soil microorganisms.

There have been many reports on the bacteriostatic studies of microorganisms belonging to the genus Trichoderma (Trichoderma), and microorganisms belonging to the genus Trichoderma (Trichoderma) have antagonistic activity against plant pathogenic bacteria such as Rhizoctonia solani (Rhizoctonia solani), Botrytis cinerea (Botrytis cinerea), Rhizoctonia fulva (Phellinus noxius), and Fusarium oxysporum (Fusarium oxysporum). At present, no trichoderma has been reported which has both high siderophore activity and good bacteriostatic activity against pseudomonas solanacearum (pseudomonas solanacearum), Pectobacterium carotovorum (pevibacterium carotovorum) and Ralstonia solanacearum (Ralstonia solanacearum).

Disclosure of Invention

The invention aims to overcome the problems in the prior art and provide trichoderma and application thereof, wherein the trichoderma has strong siderophore activity and antagonistic activity, and the antagonistic activity is important related to siderophore thereof.

With the above objects as starting points, the first aspect of the present invention provides a Trichoderma strain (Trichoderma sp.) with the accession number of 2-14F2 and the accession number of CGMCC No. 21023.

The second aspect of the present invention provides a specific method for culturing trichoderma, which provides a material for the research of fermentation metabolites of trichoderma, and the method comprises: inoculating the trichoderma into a culture medium for culturing.

In a third aspect of the present invention, there is provided a method for controlling plant diseases, which comprises applying a preparation containing a metabolite of Trichoderma as described above to plants.

In a fourth aspect of the invention there is provided the use of a Trichoderma reesei as described above in the preparation of a hydroxamic acid type siderophore.

A fifth aspect of the invention provides the use of a trichoderma and/or a metabolite thereof as described above for preventing diseases.

The 2-14F2 strain of the invention can be used as a strain with high siderophore activity and can synthesize the siderophore secreting hydroxamic acid. Therefore, the invention has important application potential in the application of siderophore microorganisms and metabolites thereof in plant health management, and particularly has good control effect on plant bacterial wilt.

Biological preservation

The 2-14F2 bacterial strain is preserved in China general microbiological culture Collection center (address: No. 3 of West Lu No. 1 of the Kyoho, Beijing city, Chaoyang, and the institute of microbiology, national academy of sciences, postal code: 100101) at 23.11.2020, and the preservation unit is abbreviated as CGMCC No. 21023.

Drawings

FIG. 1 shows strain plate culture (PDA and CAS plates) and electron microscope morphology observation (A: strain morphology; B: double-layer chromogenic plate culture; C, D: split spore electron microscope morphology);

FIG. 2 is a phylogenetic tree constructed by the 2-14F2 strain based on ITS gene sequence;

FIG. 3 is the absorbance values of siderophores of 2-14F2 strain at full band;

FIG. 4 shows the results of quantitative determination of the bacteriostatic activity of metabolites of strains 2-14F2 (A: Pseudomonas solanacearum; B: Pseudomonas solanacearum; C: Pectinobacterium carotovorum; D: Ralstonia reyi).

Detailed Description

The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.

In the present invention, without making a reference to the terms herein, the term "siderophore" is used to refer to a small molecule compound having a chelating effect on iron elements, the "siderophore-producing strain" refers to a microorganism capable of efficiently synthesizing siderophores, the "fermentation metabolite" refers to a strain that secretes many metabolites by fermentation, and the "bacteriostatic activity" refers to an activity of inhibiting or retarding growth of phytopathogens.

The preservation number of the trichoderma provided by the invention is CGMCC No. 21023.

The Trichoderma 2-14F2 strain of the present invention is specifically classified into Deuteromycotina, Hyphomycetes, Moniliales, Moniliaceae, Trichoderma. The bacterium has high siderophore activity which is higher than the reported literature data, and the related data are shown in table 1.

Table 1: comparison of siderophore production types and Activities of 2-14F2 Strain

The invention provides a quantitative analysis method for verifying the activity of a strain siderophore, which comprises the following steps: the 2-14F2 strain is inoculated in a culture medium for culture.

According to the present invention, the medium may be a solid medium, which may be a conventional medium capable of being suitable for growth of trichoderma. Preferably the solid medium contains: 400g of potato, 20-40g of glucose, 18-36g of agar, 1-2L of deionized water and pH of 7.0. The color developing agent contains: CAS detection liquid (0.0605g chromium azure S, 1 mmol. L)^-1FeCl₃·6H₂O+10mmol·L^-1HCl, deionized water, etc.). The liquid medium contains: glucose 30.0-60.0 g.L^-1Sodium nitrate 2.0-40.0 g.L^-11.0-2.0 g.L of potassium phosphate trihydrate^-10.5-1.0 g.L of potassium chloride^-1Magnesium sulfate heptahydrate 0.5 g.L^-10.75-1.5 g.L of 8-hydroxyquinoline^-1And 1.0-2.0L of deionized water.

According to the present invention, preferable conditions for the culture of the strain include: the preferred temperature is 25-30 deg.C, and the preferred incubation time is 75-120 h.

The invention provides a specific method for strain expansion and fermentation culture and provides a material for activity research of 2-14F2 strain fermentation metabolites.

According to the present invention, the 2-14F2 strain provided is not particularly required for the conditions of expansion and fermentation culture, but preferably, steamed rice is autoclaved and placed in a tissue culture bottle, and Trichoderma 10 is inoculated per 10g of steamed rice⁷-10⁹CFU (e.g. 10)⁸CFU) and culturing at 25-30 deg.C in dark for 30-60 days.

According to the present invention, the method may further comprise subjecting the culture product to an extraction operation to obtain the hydroxamic acid type siderophore therefrom. The extraction operation may be performed by methanol leaching, i.e., by soaking the culture product with methanol.

The strain provided by the invention is a 2-14F2 strain with high siderophore activity, the metabolite of the strain is effectively extracted through fermentation and expanded culture of the strain, and in order to verify the bacteriostatic function of the metabolite, phytopathogens such as tomato bacterial wilt, potato bacterial wilt, tobacco bacterial wilt and the like are selected for bacteriostatic activity determination.

According to the invention, the 2-14F2 strain with the preservation number of CGMCC No.21023 has the ability of secreting hydroxamic acid type siderophore and inhibiting or hindering the normal growth of a plurality of plant pathogenic bacteria, but preferably has outstanding bacteriostatic activity on pseudomonas solanacearum, and can realize the purpose of preventing and treating plant bacterial wilt.

Accordingly, the present invention also provides a method for controlling plant diseases (or promoting plant growth), characterized in that the method comprises applying a preparation containing a metabolite of trichoderma as described above to plants.

According to the invention, the metabolite may be provided by a culture of the 2-14F2 strain. That is, the metabolite of the 2-14F2 strain can be prepared as described above, specifically, the 2-14F2 strain is inoculated into a rice medium and cultured. The method may further comprise subjecting the culture product to an extraction operation to obtain therefrom the hydroxamic acid type siderophore and the bacteriostatic active substance. According to a preferred embodiment of the present invention, the conditions for the cultivation are as described above and will not be described herein.

According to another preferred mode of the present invention, the culture is performed in a liquid medium, and the conditions of the culture are as described above and will not be described herein.

In the invention, Fe is added into the preparation³⁺The bacteriostatic activity of the strain is obviously reduced, which shows that the activity of the siderophore in the strain is closely related to the bacteriostatic activity. Thus, preferably, the formulation is free of ferric ions, i.e. the method does not comprise the step of applying an agent comprising ferric ions to the plants.

According to the present invention, the plant may be various crops, preferably a plant susceptible to bacterial wilt, more preferably, the plant is selected from at least one of tomato, potato and tobacco.

According to the invention, the metabolite of the strain has outstanding bacteriostatic activity on pathogenic bacteria of tomato bacterial wilt, potato bacterial wilt and tobacco bacterial wilt, therefore, the plant disease is preferably selected from plant diseases caused by at least one of pseudomonas solanacearum, pectobacterium carotovorum and ralstonia solanacearum.

The invention also provides the application of the trichoderma in preparing the hydroxamic acid siderophore.

The invention also provides application of the trichoderma and/or the metabolite thereof in preventing and treating plant diseases (plant bacterial wilt). As described above, the plant disease is preferably selected from plant diseases caused by at least one of Pseudomonas solanacearum, Pectibacterium carotovorum and Ralstonia solanacearum.

The invention also relates to the use of a Trichoderma strain and/or metabolite thereof as described above for inhibiting at least one of Pseudomonas solanacearum, Pectibacterium carotovorum and Ralstonia solanacearum.

The pseudomonas solanacearum, the pectobacter carotovora and the ralstonia solanacearum can be obtained by self-separation and can also be obtained commercially.

The present invention will be described in detail by the following examples. In the following examples, Pseudomonas solanacearum is purchased from Biotechnology Ltd, Baiopabo great, Beijing, under the number bio-51812; the carrot soft rot pathogen (carrot soft rot pectobacterium) is purchased from Beijing Baiohbowei biotechnology, Inc., and numbered bio-02810; ralstonia reyeri was purchased from Beijing Baiohbowei Biotechnology Inc., and numbered bio-103869.

Example 1

This example illustrates the results of morphological and molecular genetic taxonomic identification of the strains according to the invention.

A fungus is separated from original forest soil (100 degrees 49'-101 degrees 53' E, 23 degrees 95'-24 degrees 17' N, elevation 2600m) in national natural reserve of the Lailaishan mountain in Yunnan province, and is preserved in the common microorganism center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No. 21023.

The strain 2-14F2 is cultured on a plate culture medium (300 g of potato, 30g of glucose, 27g of agar, 1.5L of deionized water and pH 7), the colony morphology is round, light green and opaque, and villous hyphae are on the surface of the colony; after the scanning electron microscope is magnified by 21000 times, the conidia are closely spaced and round, the diameter is 1.5um-2.5um, and the surfaces of the conidia are wavy and uneven (see figure 1). Based on morphological observation, the ITS sequencing result of the strain is subjected to similarity search in a Gen Bank database, and is subjected to homology comparison with a standard strain sequence with homology higher than 80%, and the homology with Trichoderma yunnanense (NR 134419) is up to 99.86% (see figure 2), so that the strain is identified as a microorganism of Deuteromycotina, Hyphomycetes, Moniliales, Moniliaceae and Trichoderma.

Example 2

This example illustrates the siderophore capacity of the strains to which the invention relates.

(1) Culture of test tube species

Inoculating 2-14F2 strain on solid slant culture medium, and culturing at 25 deg.C for 80 hr to obtain test tube strain; the formula of the solid culture medium is as follows: 300g of potato, 30g of glucose, 27g of agar, 1.5L of deionized water and pH 7;

(2) solid plate seed culture

The plate culture medium is adopted for culture, and the method comprises the following steps:

preparing an iron-free Chachi solid culture medium: glucose 30.0 g.L^-1Sodium nitrate 2 g.L^-1 Potassium phosphate trihydrate 1 g.L^-10.5 g.L of potassium chloride^-1Magnesium sulfate heptahydrate 0.5 g.L^-10.75 g.L of 8-hydroxyquinoline^-11L of deionized water, and 18g of agar added to the solid.

Double-layer chromogenic medium: the lower layer: 100ml of deionized water, 1.8g of agar and 15ml of CAS solution; and (3) upper layer: iron-free Chachi solid culture medium. The 2-14F2 strain was inoculated on a double-layer plate medium, and the appearance of red color indicated that the strain secreted siderophores. As shown in FIG. 1B, a red halo appeared around the colony, indicating that the strain secretes siderophores.

(3) Quantitative determination of siderophore activity of the strain

Culturing the strain with non-iron Chachien liquid on a constant temperature shaking table (model: HS-200B) at 28 deg.C and 150r/min for 5 days, sucking 3mL of culture solution after culturing, filtering with 0.22 μm sterile filter membrane, adding equal volume of the filtrateCAS detection solution, standing for 1 hr, and measuring OD with full-wavelength microplate reader (MultiskaGO)₆₃₀(As), OD of the liquid medium without inoculated bacteria was measured by the same method₆₃₀As a reference value (denoted as "Ar"). The concentration of the siderophore is expressed by siderophore activity unit (SU), SU ═ Ar-As/Ar]X 100%, the assay was repeated 3 times, and the mean was taken for comparative analysis. As a result, as shown in Table 2, the siderophore productivity of the strain was 87.74% by the cultivation for 5 days. The same method was used to test the siderophore activity of different trichoderma strains, and the results are shown in table 1.

Table 2: quantitative determination of bacterial strain siderophore

	Ar	As	As/Ar	Activity of siderophore%
					2-14F2	4.48	0.55(0.02)	0.12(0.01)	87.74(0.002)

Injecting; the data in the table are the mean (standard deviation) of the results of the soil sample analysis; ar; blank group at OD₆₃₀Absorbance values (reference values); as; treatment group at OD₆₃₀An absorbance value.

Example 3

This example illustrates the identification of the chemical structure type of siderophore compounds produced by the strains of the invention under study.

The identification method comprises the following steps:

identifying hydroxamic acid type siderophores; 1mL of 2% FeCl was added to 1mL of the culture filtrate₃The appearance of a red or purple color in the solution indicates that the test sample contains the ferricarrier substance. When the detection is carried out by an ultraviolet spectrophotometer (EU-2200R), an absorption peak appears between 420 and 450nm, which indicates that the chelating substance is hydroxamic acid type siderophore. As a result, 2-14F2 strain was judged to produce hydroxamic acid type siderophore, as strain culture filtrates showed the highest peaks at 420-450 band after full-band scanning using a spectrophotometer (EU-2200R) (see FIG. 3).

Example 4

This example illustrates the culture of the strain and the extraction of fermentation metabolites according to the invention of the present study.

Selecting a fermentation formula as follows; sterilizing 10g, 25g, 50g, 100g steamed rice under high pressure, and packaging into 100ml, 250 ml, 500ml, 1000ml tissue culture bottles. The strains 2 to 14F2 (. times.10) were inoculated, respectively⁹CFU·ml^-1) The bacterial suspension (2) was cultured in 100. mu.l, 250. mu.l, 500. mu.l, and 1000. mu.l at 28 ℃ in the dark for 45 days. Adding methanol into the fermentation product, soaking at 37 deg.C for 48h (1L/L of fermentation product methanol), dissolving, filtering with filter paper sheet and funnel, bottling, and selecting rotary evaporator (R-210BUCHI), setting temperature at 45 deg.C, and rotating at 700rpm/min until the organic solvent is completely evaporated. The result shows that a solid strain metabolite is obtained, and a raw material is provided for the subsequent antibacterial activity verification.

Example 5

This example illustrates the results of the bacterial wilt inhibition by the metabolites of the strains of the invention of this study.

The implementation method comprises the following steps:

replicate 3 for each strain using 96-well plate a/B/C/D/E/F/G/H rows, 1-12 columns of plate wells (e.g., a1, a2, A3.. a 12; B1, B2.. B12, etc.). Add 100. mu.l of NA liquid medium into the plate hole,then 100 mu L of diluted bacteriostatic solution to be tested (metabolite concentration is 20mg/mL) is respectively added into the first holes of the A/B two rows, blank control is set, 100 mu L of sterile water is added, then the bacteriostatic solution to be tested is fully blown and beaten (at least three times or more) by a pipette to fully and uniformly mix with the liquid culture medium, then 100 mu L of the bacteriostatic solution is sucked and added into the second holes, then the bacteriostatic solution is fully blown and beaten to fully and uniformly mix with the liquid culture medium, the operation is repeated until the last hole is reached, 100 mu L of the bacteriostatic solution is sucked and discarded, and then 100 mu L of bacterial suspension is added into the plate holes. Placing 96-well plate in 37 deg.C constant temperature incubator for 18h, and then labeling with enzyme OD₆₂₅The absorbance was measured. Converted into turbidimetric concentration of Mycoplasma or approximate concentration of microorganisms (. times.10)⁸CFU/ml) to calculate the bactericidal (bacteriostatic) rate. The calculation formula is as follows: the inhibition ratio is "growth control group microorganism concentration (or turbidimetric concentration in my-maiden)" - "inhibition test group microorganism concentration (or turbidimetric concentration in my-maiden)"/"growth control group microorganism concentration (or turbidimetric concentration in my-maiden)" × 100%. The results show that: the metabolite concentration is reduced and the bacteriostatic activity (percentage) is reduced. The bacteriostatic activity of the metabolite aiming at different plant pathogenic bacteria such as pseudomonas solanacearum, pectobacterium carotovorum and ralstonia solanacearum strains is also obviously different. The metabolite concentrations were: 10 mg/ml^-1,5mg·ml^-1,2.5mg·ml^-1,1.25mg·ml^-1,0.625mg·ml^-1,0.3125mg·ml^-1,1.5625mg·ml^-1,0.0781mg·ml^-1,0.039mg·ml^-1,0.019mg·ml^-1,0.0097mg·ml^-1And 0.004 mg/ml^-1At each concentration, the bacteriostatic activity against pseudomonas solanacearum is 93%, 81%, 72%, 67%, 60%, 42%, 23%, 14%, 10%, 9%, 8%, 5%, the bacteriostatic activity against pseudomonas solanacearum is 93%, 80%, 70%, 65%, 60%, 41%, 22%, 13%, 10%, 9%, 5%, 5%, 5%, the bacteriostatic activity against pectobacterium carotovorum is 75%, 72%, 70%, 65%, 60%, 31%, 21%, 17%, 15%, 13%, 10%, 7%, the bacteriostatic activity against pseudomonas solanacearum is 87%, 78%, 73%, 63%, 52%, 32%, 26%, 13%, 10%, 10%, 7%, 4% (see fig. 4).

Example 6

This example illustrates the bacteriostatic effect of the metabolites of the strains of the invention on plant pathogens under iron supplementation.

The implementation method comprises the following steps: adding FeCl of 1mol/ml into metabolite of strain₃Solution (Strain metabolite and FeCl)₃The volume ratio of the solution is 1:1) to prepare a strain metabolite and FeCl with the concentration of 20mg/mL₃The solution was repeated for each strain 3 using 96-well plates, rows a/B/C/D/E/F/G/H, 1-12 columns of wells (e.g., a1, a2, A3.. a 12; B1, B2.. B12, etc.). The minimum inhibitory concentrations (inhibitory rates) against Pseudomonas solanacearum, Pectinobacterium carotovorum and Ralstonia reyi were determined in the same manner as in the test method of example 5. The results show that: adding FeCl of 1mol/ml into metabolite of strain₃After the solution, the bacteriostatic activity is remarkably reduced: the bacteriostatic activity of 2-14F2 strain metabolite against Pseudomonas solanacearum at high concentration (5mg/ml) is about 93%, while FeCl is added₃Then, the antibacterial activity is reduced to 37%; the bacteriostatic activity of 2-14F2 strain metabolite against P.solanacearum at high concentration (5mg/ml) was about 93%, while FeCl was added₃Then, the antibacterial activity is reduced to 36%; aiming at the bacteriostasis rate of the pectobacterium carotovorum under high concentration (5mg/ml) of about 75 percent, FeCl is added₃Then, the bacteriostasis rate is reduced to 29 percent; aiming at the bacterial inhibition rate of ralstonia solanacearum under high concentration (5mg/ml) of 87 percent, FeCl is added₃Then, the bacteriostatic rate decreased to 31% (see fig. 4). This phenomenon indicates that: the siderophore in the metabolite is in important connection with the 2-14F2 strain to play the bacteriostatic role.

Various embodiments of the present invention are described above in detail and result in preferred target pathogens, concentrations, etc., but the present invention is not limited thereto. Various simple modifications can be made to the technical scheme of the invention including the implementation method, technical conception, concentration ratio and the like, and various technical points, characteristics and any other ways can be combined, and the simple modifications and the combinations and the like are considered to be the contents disclosed by the invention and all belong to the protection scope of the invention.

Claims

1. Trichoderma sp (Trichoderma sp.) with preservation number of CGMCC No. 21023.

2. A method of trichoderma culture, comprising: inoculating Trichoderma as defined in claim 1 to a culture medium containing autoclaved steamed rice, and culturing.

3. The method according to claim 2, wherein trichoderma harzianum 10 is inoculated to every 10g of steamed rice⁷-10⁹CFU, conditions of culture including: the temperature is 25-30 deg.C, and the time is 30-60 days.

4. A method for controlling plant diseases, which comprises applying a preparation containing a metabolite of trichoderma as claimed in claim 1 to plants.

5. The method of claim 4, wherein the metabolite is provided by a culture of Trichoderma;

alternatively, the formulation is free of ferric ions.

6. The method according to claim 4 or 5, wherein the plant is selected from at least one of tomato, potato and tobacco;

alternatively, the plant disease is selected from plant diseases caused by at least one of pseudomonas solanacearum (pseudomonas solanacearum), Pectobacterium carotovorum (pebacterium carotovorum) and Ralstonia solanacearum (Ralstonia solanacearum).

7. The method of claim 4 or 5, wherein the metabolite comprises siderophores.

8. Use of a trichoderma as claimed in claim 1 in the preparation of hydroxamic acid type siderophores.

9. Use of a trichoderma and/or its metabolites as claimed in claim 1 for controlling plant diseases.

10. The use according to claim 9, wherein the plant disease is selected from plant diseases caused by at least one of pseudomonas solanacearum, pectobacterium carotovorum, and ralstonia solanacearum.