CN113502227B

CN113502227B - Fusarium vine, microbial inoculum and herbicide containing same and application of Fusarium vine

Info

Publication number: CN113502227B
Application number: CN202110638516.9A
Authority: CN
Inventors: 刘敏; 蔡海林; 伍绍龙; 刘天波; 谢鹏飞; 翟争光; 曾维爱; 向世鹏; 赵阿娟; 唐春闺; 戴杏华; 李建勇
Original assignee: Changsha Co Of Hunan Tobacco Co; TOBACCO AGRICULTURAL EXPERIMENT STATION OF CENTRAL-SOUTH CHINA; Hunan Agricultural University
Current assignee: Changsha Co Of Hunan Tobacco Co; TOBACCO AGRICULTURAL EXPERIMENT STATION OF CENTRAL-SOUTH CHINA; Hunan Agricultural University
Priority date: 2021-06-08
Filing date: 2021-06-08
Publication date: 2023-07-07
Anticipated expiration: 2041-06-08
Also published as: CN113502227A

Abstract

The invention relates to the field of biological weed control, and discloses fusarium graminearum, a microbial inoculum and a herbicide containing the fusarium graminearum, and application of the fusarium graminearum and the microbial inoculum and the herbicide. Specifically, the invention provides fusarium graminearum, wherein the preservation number of fusarium graminearum (Fusarium fujikuroi) is CGMCC No.21038. The invention also provides a microbial inoculum and herbicide containing the fusarium graminearum. The fusarium graminearum, the microbial inoculum and the herbicide provided by the invention can be used for preventing and controlling gramineous weeds and broadleaf weeds in agricultural production. The fusarium graminearum has the advantages of strong pathogenicity to weeds, simple components, environment friendliness, simple application and wide weed killing spectrum, and has the potential of further developing into biological herbicides.

Description

Fusarium vine, microbial inoculum and herbicide containing same and application of Fusarium vine

Technical Field

The invention relates to the field of biological weed control, in particular to fusarium graminearum, a microbial inoculum and herbicide containing the fusarium graminearum, and application of the fusarium graminearum and the microbial inoculum and herbicide.

Background

The types of weeds in farmlands in China are complex, and about 60 weeds in 1400 weeds can invade the farmlands, so that the agricultural production in China is seriously affected. Most weeds are developed in root, have strong environment adaptability and strong seed productivity, can be spread rapidly, and increase difficulty for effective prevention and control of weeds. The damage of weeds to a large amount of grains, vegetables, fruit trees and the like is caused each year in China, and the weeds are one of important factors for threatening the safety of farmland output in China.

Weed control is an important link in agricultural production, although weed damage has been controlled to a certain extent with the popularization and application of modern agricultural mechanization and chemical pesticides. Chemical control is the main means for controlling farmland weeds at present, but with the long-term use of chemical herbicides, negative effects such as environmental pollution, agricultural product pesticide residues, rising weed resistance and the like are brought. Compared with chemical herbicide, the microbial herbicide has the characteristics of environmental friendliness, high safety to crops, difficult generation of resistance to weeds and the like. Therefore, the research and development of microbial herbicides have broad prospects.

Disclosure of Invention

The invention aims to overcome the problems in the prior art, provides fusarium graminearum, a microbial inoculum and herbicide containing the fusarium graminearum, and application of the fusarium graminearum and the microbial inoculum and herbicide, can effectively control grassy weeds and broadleaf weeds in fields, and provides a basis for developing novel biological herbicides.

In order to achieve the above purpose, the first aspect of the present invention provides fusarium graminearum, wherein the preservation number of the fusarium graminearum (Fusarium fujikuroi) is CGMCC No.21038.

The second aspect of the invention provides a microbial inoculum comprising the fusarium graminearum.

Preferably, the microbial inoculum contains at least one of living microbial cells, dead microbial cells and fermentation products of the fusarium gracilis, preferably living microbial cells and/or fermentation products.

Preferably, the microbial inoculum is a liquid microbial inoculum and/or a solid microbial inoculum, preferably a liquid microbial inoculum.

The third aspect of the invention provides a herbicide comprising fusarium graminearum and/or a microbial inoculum as described above.

Preferably, the number of conidia of the fusarium graminearum in the herbicide is not less than 10 ⁶ cfu/mL。

Preferably, the preparation method of the herbicide comprises the following steps: and (3) fermenting and culturing fusarium graminearum to obtain a fermentation liquor, and removing mycelia from the fermentation liquor to obtain conidium liquor.

Preferably, the conditions of the fermentation culture at least satisfy: the inoculation amount is 1 multiplied by 10 ² -3×10 ² cfu/mL, 20-35 ℃, 120-200r/min rotational speed and 8-12 days.

The fourth aspect of the invention provides the fusarium graminearum, the microbial inoculum and the application of the herbicide in weed control.

Preferably, the weeds are grassy weeds and/or broadleaf weeds, preferably at least one of gooseberry, huechys, clavicle, aster drillings and alternanthera philoxeroides.

Through the technical scheme, the invention has the beneficial effects that:

the fusarium graminearum provided by the invention is separated from leaves of natural onset of field weeds crabgrass and is derived from natural environment, so that the fusarium graminearum is released into the natural environment without causing any hidden danger in ecology or environment, and is a safe biological control medium; the fusarium graminearum provided by the invention can be used for impregnating and dyeing various grassy weeds and broadleaf weeds such as goosegrass, alopecuroides, clavate grass, aster drillings, alternanthera philoxeroides and the like, has stronger pathogenicity, can play a role in effectively preventing and controlling the grassy weeds and the broadleaf weeds, has the advantages of simple components, environment friendliness, simplicity in application and wider weed killing spectrum, and has the potential of further developing into biological herbicides.

Additional features and advantages of the invention will be set forth in the detailed description which follows.

Preservation of organisms

The strain provided by the invention is fusarium graminearum (Fusarium fujikuroi) and is preserved in China general microbiological culture Collection center (address: north Chen Silu No. 1, 3 of the Korean area of Beijing, national academy of sciences microbiological institute, postal code: 100101) (the preservation unit is abbreviated as CGMCC) in 12 months and 23 days 2020, and the preservation number is CGMCC No.21038.

Drawings

FIG. 1 is a pathogenicity determination of Fusarium gracile (MA 4 strain) isolated in example 1 on crabgrass;

FIG. 2 is the colony morphology of Fusarium tenuis (MA 4 strain) on PDA plates;

FIG. 3 is a conidium morphology of Fusarium tenuifolium (MA 4 strain);

FIG. 4 is a graph showing the effect of conidium fluid of Fusarium vine cartridge (MA 4 strain) on germination of crabgrass seeds.

Detailed Description

The following describes specific embodiments of the present invention in detail. It should be understood that the detailed description and specific examples, while indicating and illustrating the invention, are not intended to limit the invention.

The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.

In a first aspect, the invention provides fusarium graminearum, wherein the preservation number of fusarium graminearum (Fusarium fujikuroi) is CGMCC No.21038.

The fusarium graminearum provided by the invention is separated from naturally-occurring crabgrass leaf tissues. The fusarium bine can be isolated by methods conventional in the art for isolation of new strains, for example, tissue isolation methods can be used.

The tissue separation method specifically may include: rinsing the crabgrass leaves naturally developed in the field twice by using sterile water, cutting the crabgrass leaves into small blocks along the junction of the disease bonds, placing the small tissue blocks into an ethanol-water solution and a sodium hypochlorite solution for surface disinfection respectively, and rinsing the crabgrass leaf tissues subjected to surface disinfection for a plurality of times by using sterile distilled water so as to thoroughly remove disinfectant remained on the surfaces of the tissues; placing the horse Tang Shepian tissue block in a sterilized culture dish, drying in an ultra-clean workbench, placing the dried crab grass leaf tissue on a sterilized PDA flat plate, culturing at constant temperature, picking hypha into a new PDA flat plate by using a sterilized inoculating needle after fungi grow out of the fungus colony, and picking monospore for purification after spore production to obtain a pure culture strain; inoculating the conidium suspension of the pure culture strain back to crabgrass seedlings, generating obvious disease spots after 2-5 days of inoculation, cutting the disease tissues, and carrying out separation culture again to obtain a isolate which is consistent with the original inoculated pathogen, and completing the verification of the Koch rule, wherein the isolate is proved to be crabgrass pathogenic bacteria, thus obtaining the separated strain.

In the invention, the volume fraction of the ethanol in the ethanol-water solution can be 60-80%, and the mass fraction of the sodium hypochlorite in the sodium hypochlorite solution can be 0.5-2%; the media composition of PDA plates may be: 180-220g of potato, 15-25g of glucose, 12-18g of agar powder, 1000mL of distilled water, and sterilizing with natural pH and high-pressure steam at 121 ℃ for 30min for later use; the temperature of constant temperature culture is 25-35 ℃.

The inventor of the invention carries out morphological identification and molecular biological identification on the obtained isolated strain, and the result shows that the strain is fusarium binthini (Fusarium fujikuroi) and is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of 21038 in 12/23 of 2020.

The fusarium graminearum provided by the invention can produce a large number of living thalli and/or fermentation products of fusarium graminearum through fermentation culture. The fermentation culture method is not particularly limited as long as the fusarium graminearum can be propagated in a large amount by the fermentation culture method. For example, fusarium vine canum may be transferred to a plate medium for activation, and then colonies may be picked and transferred to a liquid medium to provide an inoculum size of 1X 10 ² -3×10 ² cfu/mL, and performing shaking culture at 20-35deg.C and rotation speed of 120-200r/min for 8-12 days to obtain fermentation broth. The medium may be a medium conventionally used in the art, for example, a plate medium may be a PDA medium, and a liquid medium may be a PDB medium.

In a second aspect, the invention provides a microbial inoculum, wherein the microbial inoculum comprises fusarium graminearum. In the present invention, the concentration of fusarium graminearum in the microbial inoculum is not particularly limited, and may be specifically selected according to specific conditions.

According to the present invention, the microbial inoculum preferably contains at least one of living microbial cells, dead microbial cells, and fermentation products of fusarium binthini, more preferably living microbial cells and/or fermentation products. The thallus of fusarium graminearum provided by the invention comprises conidium and mycelium, wherein the microbial inoculum can contain the conidium and/or mycelium of fusarium graminearum, and preferably contains the conidium of fusarium graminearum. In the present invention, the term "fermentation product" refers to a metabolite (including intracellular and/or extracellular metabolites) produced by fusarium bine during fermentation or culture.

According to the present invention, the formulation of the microbial inoculum is not particularly limited, and may be prepared into various formulations according to the intended use, and components such as corresponding auxiliary materials (excipients) are added thereto, for example, the microbial inoculum may be a liquid microbial inoculum (for example, a conidium liquid or a dilution thereof) and/or a solid microbial inoculum, and preferably a liquid microbial inoculum. Wherein, which adjuvants are added into the microbial inoculum of which dosage forms are well known to those skilled in the art, and will not be described in detail herein.

In a third aspect, the invention provides a herbicide, wherein the herbicide comprises fusarium graminearum and/or the microbial inoculum.

According to the invention, the herbicide contains at least one of the living bacterial cells, dead bacterial cells and fermentation products of the fusarium graminearum, and can also comprise other weeding active ingredients, wherein the other weeding active ingredients comprise chemical substances and/or biological living bodies. Preferably, the number of conidia of the fusarium graminearum in the herbicide is not less than 10 ⁶ cfu/mL. Further preferably, the herbicide has a conidium number of 10 of fusarium binthini ⁶ -10 ⁸ cfu/mL。

According to the present invention, the method for preparing the herbicide comprises: fermenting and culturing fusarium gracilis to obtain fermentation liquor, and removing mycelium from the fermentation liquor to obtain conidium liquor containing conidium of fusarium gracilis. Illustratively, the fermentation broth is filtered with gauze to remove mycelia; if the concentration of conidium number is too high after mycelium is removed from the fermentation broth, sterile water can be used to dilute the component with conidium number of not less than 10 ⁶ cfu/mL conidium fluid.

The inventor of the invention finds that, although fusarium graminearum provided by the invention is separated from natural onset crabgrass leaves, the fusarium graminearum has certain control effect on common grassy weeds and other broadleaf weeds (such as goosegrass, alopecuroide, banaba, aster drillings, alternanthera philoxeroides and the like) in the field.

In a fourth aspect, the invention also provides the use of the fusarium graminearum, the microbial inoculum and the herbicide for controlling weeds, preferably grassy weeds and/or broadleaf weeds. Illustratively, the grass weeds may be at least one of gooseberry, huechys, and clavate grass, and the broadleaf weeds may be aster drillings and/or alternanthera philoxeroides.

The present invention will be described in detail by examples.

In the following examples, the PDA medium had the following composition: 200g of potato, 20g of glucose, 15g of agar powder, 1000mL of distilled water and natural pH, and sterilizing for 30min at 121 ℃ under high pressure for later use;

the PDB medium comprises the following components: 200g of potato, 20g of glucose, 1000mL of distilled water, natural pH and high-pressure steam sterilization at 121 ℃ for 30min for later use;

in the following examples, the weed infestation rate and fresh weight control effect of fusarium graminearum are obtained by the following calculation formulas:

germination inhibition = (CK germination rate-treated germination rate)/CK germination rate x 100%,

root length inhibition = (CK mean root length-treatment mean root length)/CK mean root length x 100%,

bud length inhibition ratio = (CK mean bud length-treatment mean bud length)/CK mean stem length x 100%,

infection (%) = number of diseased leaves/total number of leaves treated x 100%,

fresh weight control (%) = (control average fresh weight-treated average fresh weight)/control average fresh weight x 100%;

healthy grassy weeds such as crabgrass, gooseberry, alopecuroide, etc., broadleaf weeds such as aster drillings and alternanthera philoxeroides come from a cultivation garden base of Hunan university, and crabgrass seeds are collected from a non-applied empty place of the cultivation garden base of Hunan university, and other raw materials and reagents are all commercially available products.

Example 1

(1) The crabgrass leaves which are naturally ill in the field (crabgrass which is naturally ill in the base of the hogwash agricultural university) are rinsed twice by using sterile water, cut into small pieces along the junction of the disease and the health, and the small pieces of tissue are firstly placed in an ethanol solution with the volume fraction of 70% for surface disinfection for 30s and then placed in a sodium hypochlorite solution with the mass fraction of 1% for surface disinfection for 60s; rinsing the crabgrass leaf tissue after surface disinfection with sterile distilled water for 3-4 times to thoroughly remove disinfectant remained on the surface of the tissue, then placing the crabgrass leaf tissue blocks in a sterilized culture dish, and drying in an ultra-clean workbench;

(2) Placing the dried crabgrass leaf tissue in the step (1) on a sterilized PDA flat plate, placing the sterilized PDA flat plate in a constant temperature incubator at 28 ℃ for culture, picking hypha into a new PDA flat plate by using a sterilized inoculating needle after fungi grow out of the fungus, and picking monospore for purification after spore production to obtain a pure culture strain;

(3) And (3) inoculating the conidium suspension of the pure culture strain obtained in the step (2) back to crabgrass seedlings, wherein obvious lesions appear 3d after inoculation, and as shown in figure 1, cutting the diseased tissue to perform separation culture again, wherein the obtained isolate is consistent with the original inoculated pathogen, and the verification of the Koch rule is completed, so that the isolate is proved to be the crabgrass pathogenic bacteria, and is named as MA4.

Morphological identification and molecular biological identification of crabgrass pathogen MA4

Morphological identification: inoculating the purified MA4 strain to a PDA plate, observing colony morphology of the strain on the PDA plate, wherein the colony morphology of the MA4 strain on the PDA plate is white as shown in figure 2, the mycelium is compact, and observing the conidium morphology of the MA4 strain under an electron microscope after the strain is produced, wherein the conidium of the MA4 strain is elliptical and has a septum and is single-born as shown in figure 3.

Molecular biology identification: extracting DNA of MA4 strain by using fungus genome DNA extraction kit, amplifying ITS (internal transcribed spacer regions), TEF (translation elongation factor-alpha genes) and TUB (beta-tubulin) gene sequences of MA4 strain by using universal primers ITS4/ITS5, EF728F/EF986R and Bt2a/Bt2b respectively, and sequencing, wherein the nucleotide sequence of ITS gene is shown as SEQ ID NO. 1, the nucleotide sequence of TEF gene is shown as SEQ ID NO.2, and the nucleotide sequence of TUB gene is shown as SEQ ID NO. 3; ITS, TEF and TUB gene sequences were submitted to NCBI under GenBank accession numbers MK443269, MK4432671 and MK443268, respectively. BLAST was performed on ITS, TEF and TUB gene sequences in NCBI, and found that the ITS, TEF and TUB genes of MA4 strain were highly homologous to Fusarium bine (Fusarium fujikuroi).

According to morphological identification and molecular biological identification results, the MA4 strain is obtained as fusarium binthini (Fusarium fujikuroi) and is preserved in China general microbiological culture collection center (CGMCC) with a preservation number of 21038 in 12-23 days of 2020.

Example 2

The MA4 strain obtained in example 1 was transferred to a fresh PDA plate for activation, and colonies were picked up and transferred to liquid PDB medium so that the inoculum size was 2X 10 ² Fermenting cfu/mL at 28deg.C and rotational speed of 160r/min for 10 days to obtain fermentation broth, filtering the fermentation broth with four layers of gauze to remove mycelium, diluting with sterile water to obtain a composition with conidium number of 10 ⁸ cfu/mL conidium liquid is used as herbicide.

Example 3

The MA4 strain obtained in example 1 was transferred to a fresh PDA plate for activation, and colonies were picked up and transferred to liquid PDB medium so that the inoculum size was 1X 10 ² Fermenting cfu/mL at 20deg.C and rotation speed of 120r/min for 12 days to obtain fermentation broth, filtering the fermentation broth with four layers of gauze to remove mycelium, diluting with sterile water to obtain a composition with conidium number of 10 ⁶ cfu/mL conidium liquid is used as herbicide.

Example 4

The MA4 strain obtained in example 1 was transferred to a fresh PDA plate for activation, and colonies were picked up and transferred to liquid PDB medium so that the inoculum size was 3X 10 ² cfu/mL, at 35 ℃ and 200r/minFermenting and culturing for 8 days to obtain fermentation broth, filtering the fermentation broth with four layers of gauze to remove mycelium, and diluting with sterile water until the number of conidia is 10 ⁷ cfu/mL conidium liquid is used as herbicide.

Test example 1

The conidium solution obtained in example 2 was subjected to 2-fold gradient dilution to obtain 1/2, 1/4, 1/8, 1/16, 1/32-fold concentration of the conidium solution, respectively, the crabgrass seeds were immersed in the solution and placed in 7 dishes containing two layers of filter paper, each dish containing 100 crabgrass seeds, and treated with 10mL of the conidium solution of the corresponding concentration in the dishes of the treatment group and one blank control group (CK), and after 7 days, 10mL of sterile water was added to the dishes of the blank control group, and the germination percentage, root length and bud length inhibition were calculated, and the results are shown in Table 1.

TABLE 1 Effect of conidium solutions of MA4 strains at different concentrations on crabgrass seeds

From the data in Table 1, it can be seen that the conidium fluid of MA4 strain has strong inhibition effect on germination of crabgrass seeds, and the specific reference is shown in FIG. 4; the undiluted conidium solution has a germination inhibition rate of 94.2% for crabgrass seeds and a root growth and bud growth inhibition rate of 76.5% and 95.9%, respectively. The MA4 has better effect of inhibiting the germination of the crabgrass seeds and the growth of root buds.

Test example 2

Planting crabgrass in a plastic pot, respectively carrying out stem and leaf spraying treatment on the conidium liquid of the MA4 strain prepared in the embodiment 2 in the 3-leaf stage, the 5-leaf stage and the 7-leaf stage of the crabgrass, wherein the symptoms of chlorosis and dead spots begin to appear in the crabgrass after the treatment for 3d, the symptoms of chlorosis and necrosis are obvious in the 5d after the treatment, the overground part of the 8d crabgrass is completely withered after the treatment, and the plant prevention effect reaches 100%; the aerial parts of the crabs are cut and weighed, the non-spray treated crabs are used as a control, the biocontrol effect of the conidium liquid of the MA4 strain on different She Lingma crabs is calculated, the result is shown in a table 2, the fresh weight control effect on different She Lingma crabs is as high as 95.5-97.6%, and the result shows that the MA4 fermentation liquor filtrate has a very strong biocontrol effect on the crabs.

TABLE 2 biocontrol Effect of conidium solution of MA4 Strain on different She Lingma Tang

Test example 3

Crabgrass is planted in the field, when the seedlings of the crabgrass grow to the 4-5 leaf stage, the conidium liquid of the MA4 strain prepared in the example 2 is used for carrying out stem and leaf spraying treatment, and the crabgrass which is not subjected to spraying treatment is used as a control, and the results of the field test are shown in Table 3. As can be seen from Table 3, 28d after the treatment of the conidium liquid of MA4 strain, the plant control effect on crabgrass reaches 95.2%, and the fresh weight control effect reaches 98.4%. The biological test result shows that the conidium liquid of the fusarium graminearum MA4 strain contains substances with strong herbicidal activity to the horse Tang Xingcheng, and has the potential of further research and development.

TABLE 3 MA4 field control of crabgrass

Treatment of	Plant prevention effect	Fresh weight control effect
			21d after treatment	92.2±4.1	--
Post-treatment 28d	95.2±2.3	98.4±1.1

Test example 4

Healthy goosegrass, myrtaria immaturus, lolium clavatum, aster drillings and allium alternifolium are planted in a plastic pot, stem and leaf spray treatment is carried out on the conidium liquid of the MA4 strain prepared in the example 2, the non-spray treated goosegrass is used as a control, and the fresh weight inhibition rate of the conidium liquid of the MA4 strain on various weeds is counted after 14d culture, and the result is shown in a table 4. As can be seen from Table 4, the conidium liquid of MA4 strain has strong infection ability and fresh weight inhibition effect on various gramineous weeds such as goosegrass, alopecuroide and the like, and fresh weight inhibition rates on the goosegrass, the lolium clavatum, aster drillings and alligator alternanthera reach 87.4%, 83.2%, 79.5%, 78.4% and 68.2%, respectively, which shows that the conidium liquid of MA4 strain has excellent prevention effect on horse Tang Juyou, can prevent various gramineous weeds such as goosegrass, alopecuroide and the like, and broadleaf weeds such as aster drillings and alligator alternanthera, and is safe for crops.

TABLE 4 host Range determination of MA4 Strain

Note that: the number of diseased plants is 30% or less of the total treated plants, denoted LS, the number of diseased plants is 30% -70% of the total treated plants, denoted MS, the number of diseased plants is 70% or more, denoted SS, and none of the diseased plants is NS.

The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, a number of simple variants of the technical solution of the invention are possible, including combinations of the individual technical features in any other suitable way, which simple variants and combinations should likewise be regarded as being disclosed by the invention, all falling within the scope of protection of the invention.

SEQUENCE LISTING

<110> Hunan agricultural university, hunan province tobacco public, shaku city, china tobacco middle and south agricultural test station

<120> Fusarium graminearum, microbial inoculum and herbicide containing the same, and applications thereof

<130> 20210608

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Claims

1. Fusarium vine bin is characterized in that the Fusarium vine bin is prepared from the following raw materialsFusarium fujikuroi) The preservation number of the product is CGMCC No.21038.

2. A microbial agent comprising fusarium graminearum of claim 1.

3. The microbial inoculum according to claim 2, wherein the microbial inoculum contains viable cells or conidium fluid of fusarium graminearum.

4. The microbial agent of claim 2, wherein the microbial agent is a liquid microbial agent or a solid microbial agent.

5. The microbial inoculant of claim 4, wherein the microbial inoculant is a liquid microbial inoculant.

6. A herbicide comprising fusarium graminearum of claim 1 and/or a microbial agent of any one of claims 2 to 5.

7. The herbicide according to claim 6, characterized in that the number of conidia of fusarium graminearum in the herbicide is not less than 10 ⁶ cfu/mL。

8. The herbicide according to claim 7, characterized in that the process for the preparation thereof comprises: and (3) fermenting and culturing fusarium graminearum to obtain a fermentation liquor, and removing mycelia from the fermentation liquor to obtain conidium liquor.

9. The herbicide according to claim 8, characterized in that the conditions of the fermentation culture at least satisfy: the inoculation amount is 1 multiplied by 10 ² -3×10 ² cfu/mL, 20-35 ℃, 120-200r/min rotational speed and 8-12 days.

10. Use of fusarium graminearum according to claim 1, a microbial inoculum according to any one of claims 2 to 5, and a herbicide according to any one of claims 6 to 9 for controlling weeds.

11. The use according to claim 10, wherein the weeds are at least one of gooseberry, huechys, clavate, aster drillings and alternanthera.