CN108002885B

CN108002885B - Trichoderma-validamycin granules and preparation method thereof

Info

Publication number: CN108002885B
Application number: CN201610936252.4A
Authority: CN
Inventors: 陈捷; 吴琼; 余传金; 窦恺; 李雅乾
Original assignee: Shanghai Jiaotong University
Current assignee: Shanghai Jiaotong University
Priority date: 2016-11-01
Filing date: 2016-11-01
Publication date: 2020-12-29
Anticipated expiration: 2036-11-01
Also published as: CN108002885A

Abstract

The invention discloses a trichoderma-validamycin granule and a preparation method thereof in the field of biological control of plants, wherein the granule comprises the following raw materials in parts by weight: 36-45% of trichoderma fermentation liquor, 3-6% of validamycin, 4.5-5.5% of corn flour, 35-38% of diatomite, 11-13% of wheat bran, 0.8-1.2% of zinc sulfate fertilizer, 0.2-0.5% of humic acid and 0.3-0.5% of nitrogen-phosphorus-potassium compound fertilizer. The invention has simple preparation process and low production cost, and is granules taking the trichoderma strains and the validamycin as cores. The trichoderma strain is a wild strain separated from a vegetable field in the Fushan area in Guangdong, and has no ecological threat to microbial flora in the vegetable field; the spore yield is large, the fermentation medium has simple components, the process is easy to optimize, and the preparation cost of the microbial inoculum is low. The two components in the trichoderma-validamycin granules have synergistic effect, the biological control effect is obviously improved compared with that of a single component, and the trichoderma-validamycin granules are particularly suitable for facility vegetable fields with serious fungal diseases caused by primary salinization or continuous cropping obstacles and have high field general use value.

Description

Trichoderma-validamycin granules and preparation method thereof

Technical Field

The invention relates to the technical field of biology, in particular to a trichoderma-validamycin granule and a preparation method thereof, and especially relates to a trichoderma-validamycin granule for preventing and treating corn sheath blight and a preparation method thereof.

Background

As a biological control microorganism widely used internationally, Trichoderma has antagonistic action against at least 18 pathogenic fungi belonging to 29 genera. Currently, trichoderma has been demonstrated to be able to parasitize phytopathogenic fungi from 12 genera, namely Rhizoctonia (Rhizoctonia), Sclerotinia (sclerotiotium), Sclerotinia (Sclerotinia), Helminthosporium (Helminthosporium), Lachnum (Lachnum), Fusarium (Fusarium), Verticillium (Verticillium), hypocotyl (Endothia), Pythium (Pythium), diaporthia (Diaporthe), and nigrospora (Fusarium), among others. Mechanisms of trichoderma biocontrol action are mainly Competition action (Competition) and heavy parasitism (hyperparatism) and induced plant resistance (Inducing plant resistance); in addition, trichoderma has an antibiotic effect (Antibiosis). In most cases, the biocontrol effects of trichoderma are the result of the combined action of the above-mentioned mechanisms.

Validamycin (Validamycin) is a secondary metabolite of streptomyces hygroscopicus (s.hygroscopicus), and the producing bacteria include streptomyces hygroscopicus var citrinum (s.hygroscopicus var. limonenus) IFO12703 and 12704, streptomyces hygroscopicus var. jinggangensis (s.hygroscopicus var. jinggangensis)5008 and the like. As an agricultural antibiotic with strong systemic effect, validamycin can be quickly absorbed by mycelium of rhizoctonia solani and conducted in vivo after contacting with the mycelium of the rhizoctonia solani, and acts on a trehalose energy metabolic system highly dependent on the rhizoctonia solani to interfere and inhibit normal growth and development of cells of the rhizoctonia solani. Validamycin is widely applied in China, the annual output reaches 3000-4000 tons, the validamycin can be used for 6-8 hundred million acres of land, the primary main control object is only rice sheath blight, and the sheath blight of crops such as wheat and corn is expanded later. However, validamycin has some problems at present, and particularly, agricultural products are limited to China, so that the national standards of validamycin residue detection methods are not available in China, and researches on harm to people and livestock, influence on environment and the like are not deep enough.

The trichoderma has obvious competition and heavy parasitism on the corn rhizoctonia solani, and is finally degraded. Validamycin can quickly act on a trehalose energy metabolism system highly dependent on rhizoctonia solani to further inhibit the growth of the rhizoctonia solani, but cannot degrade the rhizoctonia solani. In contrast, the trichoderma has long control period and thorough control effect, but has slight delay; validamycin has quick response but short control period. At present, the prevention and control of the corn sheath blight disease mainly depend on the application of validamycin, but the prevention effect is reduced to some extent due to the fact that the validamycin has been used for many years, and the problem is solved urgently in production because the control effect of the corn sheath blight disease is improved and the use of chemical pesticides is reduced through the synergistic use of trichoderma and validamycin. Aiming at the problems, the invention successfully creates the trichoderma-validamycin granules which take the prevention and the treatment of the corn sheath blight as the main target.

Disclosure of Invention

Aiming at the defects of the prior art, in particular to the problems of the prevention and treatment period and the aging of the domestic corn sheath blight, the invention provides the Trichoderma-validamycin granules and the preparation method thereof, the preparation process is simple, the production cost is low, and the Trichoderma asperellum granules take Trichoderma asperellum (with the number of GDFS1009) and validamycin as the core granules. The trichoderma strain is a wild strain separated from a vegetable field in the Fushan area in Guangdong, and has no ecological threat to microbial flora in the vegetable field; the spore yield is large, the fermentation medium has simple components, the process is easy to optimize, and the preparation cost of the microbial inoculum is low. The two components in the trichoderma-validamycin granules have synergistic effect, the biological control effect is obviously improved compared with that of a single component, and the trichoderma-validamycin granules are particularly suitable for facility vegetable fields with serious fungal diseases caused by primary salinization or continuous cropping obstacles and have high field general use value.

The invention is realized by the following technical scheme:

in a first aspect, the invention provides a trichoderma-validamycin granule, which comprises the following raw materials in parts by weight:

the raw material components and the component contents are recorded through creative screening, and if the content of the raw material is beyond the range limited by the invention, the growth of the rhizoctonia solani and the rhizoctonia solani cannot be obviously inhibited, and the disease attack of pathogenic bacteria cannot be obviously controlled.

Preferably, the granule comprises the following raw materials in parts by weight:

when the granules are prepared according to the raw material proportion, the effect is optimal.

Preferably, the preparation of the trichoderma fermentation broth comprises: inoculating 0.5 wt% of secondary strain into the liquid culture medium, and fermenting at 28 deg.C for 7d until the fermentation liquid turns green, i.e. Trichoderma liquid fermentation liquid; wherein the liquid culture medium comprises 10kg of corn flour, 210kg of water, 766g of monopotassium phosphate, 100g of magnesium sulfate, 0.5g of manganese sulfate, 0.4g of zinc sulfate, 284g of sodium nitrate, 220g of ammonium sulfate and 200g of sodium chloride;

the preparation of the secondary strain comprises the following steps: inoculating Trichoderma into PD, fermenting at 28 deg.C at 180rpm to obtain secondary strain containing a large amount of dispersed flocculent mycelia and dispersed conidium liquid strain.

Preferably, the dosage form of validamycin includes a 5% aqueous solution.

Preferably, the Trichoderma fermentation liquid specifically refers to a fermentation liquid of Trichoderma asperellum GDFS1009 CGMCC NO. 9512.

In a second aspect, the present invention provides a preparation method of the trichoderma-validamycin granules, which comprises the following steps: the materials are prepared according to the weight percentage and are uniformly mixed, extruded and granulated, and dried for 1-2 hours at the temperature of 43-50 ℃ to obtain the trichoderma-validamycin granules.

Preferably, the preparation method further comprises the steps of evaluating the influence of the validamycin on the growth and reproduction of trichoderma, and evaluating the influence of the trichoderma on the absorption and degradation rate of the validamycin.

Preferably, the step of evaluating the influence of validamycin on the growth and reproduction of trichoderma includes the following steps: and (3) taking trichoderma inoculated on a PDA plate containing validamycin as a treatment, taking trichoderma inoculated on a PDA plate without validamycin as a comparison, and counting and comparing the treatment with the comparison on production speed, morphology, spore yield and the like.

Preferably, the step of evaluating the influence of trichoderma on the absorption and degradation rate of validamycin includes:

(1) transferring the trichoderma conidium suspension into a PD culture medium containing validamycin, and culturing at constant temperature to obtain a fermentation liquid;

(2) and filtering the fermentation liquor, collecting supernatant, detecting the content of validamycin by LC-MS (liquid chromatography-mass spectrometry), and taking a PD (PD) culture medium with the initial content of validamycin as a reference.

Preferably, in the step (1), the concentration of validamycin in the PD medium containing validamycin is 500 mug/mL; the concentration of trichoderma conidium in the PD culture medium is 10⁶cfu/mL; the conditions of constant temperature culture are as follows: 28 ℃, 180rpm, 7 d.

Preferably, in step (2), the filtration is performed using a 0.22 μm microporous filter.

Preferably, the mixing uniformly comprises a stirring manner by a stirrer.

Preferably, the granulating comprises using a granulating extruder.

Preferably, the drying comprises using a dryer.

Trichoderma asperellum GDFS1009 CGMCC NO.9512 is preserved in China general microbiological culture Collection center (CGMCC NO.9512), has strong environmental adaptability and rapid growth, and has stronger competitive action compared with rhizoctonia solani which causes corn sheath blight. The strain can produce cell wall degrading enzymes such as chitinase and cellulase, and can inhibit the growth of rhizoctonia by means of heavy parasitic action to degrade the rhizoctonia. In addition, the xylanase secreted by the strain has the functions of inducing plant resistance and increasing plant immunity.

As an agricultural antibiotic with strong systemic property, validamycin can be quickly absorbed and conducted in vivo after being contacted with rhizoctonia mycelium, so that a highly dependent energy metabolism pathway, namely a trehalose pathway, is interfered, the synthesis of glucose is prevented, the growth and development of the glucose are efficiently inhibited, but the glucose cannot be killed or degraded.

Compared with the prior art, the invention has the following beneficial effects:

(1) the results of in vitro bacteriostatic experiments show that the growth of the rhizoctonia solani is reduced by more than 32% and the growth of the rhizoctonia solani is reduced by more than 62% by mixing and adding the trichoderma and the validamycin.

(2) The in vitro leaf experiment result shows that the growth amount of the rhizoctonia solani and the rhizoctonia solani is obviously reduced by mixing and adding the trichoderma and the validamycin A.

(3) By applying the trichoderma-validamycin granules, the morbidity of the corn sheath blight is reduced by 60%, the disease index is reduced by 66%, and the emergence rate and the high grade of the corn are obviously improved.

Drawings

Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:

FIG. 1 shows the effect of different concentrations of validamycin A treatment on Trichoderma asperellum GDFS1009 CGMCC NO. 9512; in the figure, from left to right, 100 mug/mL, 500 mug/mL validamycin A and CK are respectively;

FIG. 2 shows the effect of validamycin A on the competition and the re-parasitic action of Trichoderma asperellum GDFS1009 CGMCC NO. 9512; a, competition effect; b, heavy parasitism;

FIG. 3 shows the effect of validamycin A on the activity of Trichoderma asperellum GDFS1009 CGMCC NO.9512 chitinase;

FIG. 4 shows the effect of validamycin A on the activity of Trichoderma asperellum GDFS1009 CGMCC NO.9512 cellulase;

FIG. 5 shows LC-MS detection of the degradation of validamycin A by Trichoderma asperellum GDFS1009 CGMCC NO. 9512;

wherein, A, 500 mu g/mL validamycin A is fermented in blank PD culture solution for 7 d; b, final concentration 10⁶cfu/mL trichoderma asperellum spore suspension and 100 mug/mL validamycin A are fermented for 7d in PD culture solution;

FIG. 6 is an in vitro experiment of 500. mu.g/mL validamycin A and Trichoderma asperellum GDFS1009 CGMCC NO.9512 in cooperation with Rhizoctonia solani;

FIG. 7 is an in vitro experiment of 500. mu.g/mL validamycin A and Trichoderma asperellum GDFS1009 CGMCC NO.9512 in cooperation with Rhizoctonia solani;

FIG. 8 is an in vitro experiment of 500. mu.g/mL validamycin A synergistically inhibiting Rhizoctonia cerealis with Trichoderma asperellum GDFS1009 CGMCC NO. 9512;

FIG. 9 shows 100. mu.g/mL validamycin A and 10⁶cfu/mL Trichoderma asperellum GDFS1009 CGMCC NO.9512 spore suspension synergistic inhibition of corn rhizoctonia solani in vitro leaf experiments;

FIG. 10 is the experiment of synergistic inhibition of Rhizoctonia solani by validamycin A and Trichoderma asperellum GDFS1009 CGMCC NO.9512 antagonistic factor; a, 100 mu g/mL of validamycin A and 20% of chitinase fermentation liquor; b, 100 mu g/mL of validamycin A and 20% of cellulase fermentation liquor;

FIG. 11 is a graph showing the results of the inoculation test in the adult stage, wherein the left and right sides are respectively the Trichoderma granules and blank control treatment.

Detailed Description

The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications can be made by persons skilled in the art without departing from the spirit of the invention. All falling within the scope of the present invention.

Pathogenic bacteria: rhizoctonia solani comprising: rhizoctonia solani zeae and rhizoctonia solani zea, both of which are stored in the laboratories for the microbiological engineering of the agricultural environment of the college of agriculture and biology of shanghai university, are disclosed in the following documents:

jizhang, Li Bao Du, Biyuping, Zhou Zhuang Yu province corn production variety and part of inbred lines to the identification of sheath blight resistance plant protection, 2010,36(4): 152-154;

the composition and pathogenic type of the rhizoctonia zeae in partial areas of China are analyzed, the plant protection bulletin, 2009,36(4): 289-294.

Corn variety: wang He No. 8 is stored in agricultural environment microbiological engineering laboratories of the colleges of agriculture and biology of Shanghai traffic university.

Validamycin (validamycin a) analytical standards (HPLC grade) were purchased from shanghai kexing biotechnology limited. Natamycin analytical standards (HPLC grade) were purchased from aladin-alatin reagent (shanghai) ltd. 0.22 μm microporous filters were purchased from Millipore, USA. 3, 5-dinitrosalicylic acid (DNS), xylose, sodium carboxymethylcellulose (CMC-Na,800-1200mPa), and Dimethylbenzidine (DAB) were purchased from national pharmaceutical group chemical reagents, Inc. Xylan was purchased from Serva, germany.

Example 1 Effect of Validamycin on Trichoderma growth

1. Effect of different concentrations of validamycin A on Trichoderma asperellum colonies

(1) Culturing trichoderma in an incubator at the constant temperature of 28 ℃ for 3 d;

(2) 5mg and 25mg of validamycin A are dissolved in 5mL of sterile water, filtered by a 0.22 mu m microporous filter and added into 45mL of PDA, after uniform mixing, 100 mu g/mL and 500 mu g/mL of validamycin A plates are prepared, and a sterile water culture medium is used as a reference;

(3) taking the trichoderma fungus cake by a puncher with the diameter of 7mm, inverting and transferring the trichoderma fungus cake to the center of a plate;

(4) culturing in an incubator at 28 ℃ for 3d at constant temperature, measuring the diameter of trichoderma colonies, and observing sporulation. Each experiment was repeated at least 3 times.

Compared with the control, the growth rate, morphology and spore yield of trichoderma asperellum mycelium were not changed in the plates containing validamycin A, and specifically shown in FIG. 1 (100. mu.g/mL, 500. mu.g/mL validamycin A and CK, respectively, from left to right in the figure).

2. Effect of validamycin A on the competitive and parasitizing abilities of Trichoderma asperellum

2.1 Observation of Competition and Reparasitic capabilities

(1) Culturing a PDA culture of trichoderma and corn stalk rot bacteria in an incubator at the constant temperature of 28 ℃ for 3 days;

(2) dissolving 25mg of validamycin A in 5mL of sterile water, filtering by using a 0.22-micron microporous filter, adding into 45mL of PDA, and uniformly mixing to prepare a 500-mu g/mL validamycin A plate; sterile water medium as control;

(3) punching trichoderma and stem rot fungus dishes by a puncher of 7mm, wherein the distance between the two fungus dishes is 4cm, and the two fungus dishes are respectively placed at two ends of a 9cm plate;

(4) culturing at 28 deg.C for 4 days, and observing competitive power;

(5) cutting 2 colonies with a volume of 0.4cm × 0.4cm × 0.1 cm; placing in precooled 2.5% glutaraldehyde, and fixing at 4 ℃ for 12 h;

(6) the ability of Trichoderma to parasitize pathogenic bacteria was observed under an optical microscope (400X).

Results show that the results of confrontation experiments and optical electron microscope observation show that the competition effect and the parasitism capability of the trichoderma asperellum treated by the validamycin A on the corn stalk rot fungi are not different from those of a control (figure 2).

2.2 determination of Activity of enzyme systems associated with the heavy parasitic Effect

2.2.1 preparation of seed bacteria

(1) Transferring trichoderma spores stored at-80 ℃ into a PD culture medium, carrying out constant-temperature shaking culture at 28 ℃ and 180rpm for 2d until mycelia are abundant;

(2) vacuum-pumping and filtering to remove supernatant, and precipitating to obtain seed bacteria;

(3) weighing a plurality of 1g of seed bacteria for transferring an enzyme production induction culture medium;

2.2.2 preparation of mother liquor of validamycin A

300mg of validamycin A is dissolved in 30mL of sterile water and is used for transferring an enzyme production induction culture medium through a 0.22-micron microporous filter;

2.2.3 transfer of chitinase and cellulase Induction Medium, respectively

(1) 1g of seed bacteria is transferred into an enzyme production induction culture medium, 5mL of validamycin A mother solution is added, and sterile water is used as a reference;

(2) carrying out constant temperature shaking culture at 28 ℃ and 180rpm for 4 d;

(3) sampling 1mL each day, centrifuging at 6000rpm for 10min, and taking supernatant for enzyme activity determination.

Chitinase activity assays were repeated at least 3 times per experiment, using t-test to detect differential significance, according to conventional methods.

The results show (FIG. 3) that the Trichoderma asperellum chitinase yield reached plateau at fermentation 3-4 d. The enzyme activity of the trichoderma treated by the validamycin A during fermentation for 4d is as follows: 121.481. + -. 9.114U/mL, controls were: 119.837 + -6.231U/mL, it can be seen that validamycin A has no effect on chitinase production by Trichoderma.

And (3) cellulase activity determination: (1) mixing 100 μ L cellulase solution (cellulase induced culture supernatant as control) and 1.9mL CMC-Na solution (pH 4.8), reacting in water bath at 50 deg.C for 1 hr, and using blank induced culture medium as control; (2) adding 3mL DNS agent, reacting in boiling water bath for 10min, diluting, and determining OD_540nmAnd (4) light absorption value. The glucose standard curve is established according to the light industry standard of the people's republic of China (QB 2583-2003). The amount of enzyme required to release 1mg of glucose per ml of fermentation broth per hour at 50 ℃ and pH 4.8 was defined as one enzyme activity unit. Each experiment was repeated at least 3 times and differential significance was measured using t-test. In the process of fermentation 4d, validamycin A has no influence on the yield of trichoderma asperellum cellulase. The enzyme activity reaches the maximum value after 3d of fermentation, and the enzyme activity after the validamycin A treatment is as follows: 1.717. + -. 0.212U/mL, controls were: 1.723. + -. 0.112U/mL (FIG. 4).

Example 2 absorption and degradation rate of validamycin by Trichoderma

1. Inoculating the conidium suspension of trichoderma into PD culture medium containing 500 mug/mL validamycin A, wherein the final concentration of spores is 10⁶cfu/mL, 28 ℃, 180rpm, culturing for 7 d;

2. filtering with 0.22 microporous filter, collecting supernatant, LC-MS detecting validamycin A content, and using PD culture medium containing 500 μ g/mL validamycin A as reference;

3. the detection device comprises: the Waters Quattro Premier XE triple quadrupole liquid chromatograph-mass spectrometer is provided with UPLC;

4. chromatographic conditions are as follows: the liquid chromatogram is Ultra Performance Liquid Chromatography (UPLC) of Waters company, U.S.A., the chromatographic column is (BEH) C18 column (1.7 μm,2.1 × 100mm), the sample injection amount is 2 μ L, the column temperature is 40 ℃, the temperature of an autosampler is maintained at 4 ℃, the mobile phase A is ultrapure water, the mobile phase B is chromatographic pure acetonitrile, 0.1% (V/V) formic acid is added into the mobile phase, and the flow rate is 0.3 mL/min; eluting by linear gradient, wherein the initial condition is 10% of mobile phase B, the 10% of mobile phase B is maintained for 1min, the 10-90% of mobile phase B is maintained for 1-6 min, the 90% of mobile phase B is balanced for 3min, and the next sample is collected after the 90-10% of mobile phase B is balanced for 0.5 min;

5. mass spectrum conditions: an electrospray ionization (ESI) Source, a positive and negative ion ionization mode, an ion Source temperature (Source temperature) of 150 ℃, a desolvation temperature (desolvation temperature) of 350 ℃, a desolvation nitrogen flow rate (desolvation gas flow) of 500L/h, a cone hole back-flushing nitrogen gas (cone gas flow) of 50L/h, a capillary ionization voltage of the positive and negative ion mode of 3.0kV, a sampling cone hole voltage (sampling cone) of 20eV, an extraction cone hole (extraction cone) of 2eV, and a quadrupole scanning range of 100-1500 m/z;

the LC-MS result shows that the trichoderma asperellum and the validamycin A are fermented together for 7 days, the trichoderma asperellum and the fermentation liquor thereof have very limited degradation rate on the validamycin A, and the degradation rate is only 20%, which indicates that the trichoderma asperellum has no significant influence on the existence of the validamycin A (see figure 5).

Example 3 experiment of synergistic inhibition of Rizoctonia solani by validamycin A and Trichoderma asperellum

3.1 plate experiment

(1) Culturing Trichoderma and Rhizoctonia solani in a PDA culture medium at a constant temperature of 28 deg.C for 3 d;

(2) 25mg of validamycin A was dissolved in 5mL of sterile water, filtered through a 0.22 μm microporous filter, and added to 45mL of PDA, followed by mixing to prepare 500 μ g/mL validamycin A plates. Taking a sterile water culture medium as a control;

(3) after the plate is solidified, punching trichoderma and pathogenic bacteria plates by using a puncher with the diameter of 7mm, respectively placing the rhizoctonia plates on one side of a culture dish plate with the diameter of 9cm, inoculating the trichoderma plate on the other side, and taking independently cultured rhizoctonia solani as a reference, wherein the distance between the two plates is 4 cm;

(4) culturing at 28 deg.C for 4 days, and observing synergistic effect. Each experiment was repeated at least 3 times.

After the culture for 1d, the validamycin A can be efficiently absorbed by the rhizoctonia solani, the bacteriostatic action is very obvious, the diameter of the rhizoctonia solani colony is only 1.8 +/-0.2 cm, and the diameter of the colony of a control group reaches 6.1 +/-0.3 cm. Although the trichoderma asperellum grows rapidly and has obvious bacteriostasis effect, the trichoderma asperellum has obvious difference with the effect of validamycin A, and the diameter of the rhizoctonia solani colony is 4.0 +/-0.2 cm. Further, at this time, the advantage of the synergistic effect of validamycin A and Trichoderma asperellum was not shown, because the colony diameter of Rhizoctonia after synergistic treatment was 1.9. + -. 0.3cm, which was not significantly different from that of validamycin A alone. However, after 4 days of culture, the inhibitory action of validamycin A began to decline or gradually released as the time elapsed, and the colony diameter of Rhizoctonia was increased to 7.2. + -. 0.4cm, which was 9.0. + -. 0.0cm in the control group. However, the biocontrol advantage of Trichoderma is shown, and the colony diameter of Rhizoctonia solani which is effectively inhibited is only 1.8 + -0.2 cm. Moreover, the synergistic disease-resistant effect of validamycin A and trichoderma is also reflected, and the area occupied by rhizoctonia is obviously smaller than that of any single-factor treatment under the sequential action of mechanisms such as antibiotic, competition, heavy parasitism and the like (figure 6). Therefore, the synergistic effect of the validamycin A and the trichoderma can not only realize the advantage complementation of a biological control mechanism, but also make up the deficiency of the mutual action on the aging or period, thereby efficiently and long-term inhibiting the rhizoctonia solani.

3.2 Ex vivo leaf experiment

1. Planting of corn and preparation of in vitro leaves

(1) Planting corn seeds (Wanghe No. 8) in a flowerpot with the caliber of 10cm, culturing 6 seeds in each pot in a phytotron at 25 ℃ until the seven-leaf stage, and illuminating for 10h and darkness for 14h every day;

(2) shearing the 4 th leaf of the healthy 7-leaf stage corn, and placing the 4 th leaf in a plate containing a proper amount of 6-BA (40 mg/mL);

(3) the cake of Rhizoctonia solani was punched out with a punch having a diameter of 0.4cm and placed in the center of the leaf (leaf without cake was used as a control).

2. Treatment of corn in vitro leaves

(1) Adding 100 mu g/mL of validamycin A and 40 mu L of validamycin A to the bacterial cake;

(2)10⁶cfu/mL Trichoderma asperellum GDFS1009 spores, 40 uL added to the fungus cake;

(3)100 μ g/mL ofEvalidamycin A + Trichoderma asperellum GDFS1009 spore/mycelium 10⁶cfu/mL Trichoderma asperellum GDFS1009 spores, 40 uL added to the fungus cake;

(4) adding 40 μ L of sterile water to the cake;

(5) adding 40 mu L of sterile water to the leaves;

(6) after the treatment was completed, the cells were placed in a constant temperature incubator at 28 ℃ with care for moisture retention, and each treatment was repeated at least 3 times.

The results showed that after inoculating Rhizoctonia solani for 12h, the control (CK-1) Rhizoctonia solani grew vigorously, the mycelia appeared pure white, and a large area of the mycelia was infected on the corn leaves. Under the action of a trichoderma single factor, the density and the exuberance degree of the rhizoctonia mycelia are obviously reduced, and the rhizoctonia mycelia infect leaves in a smaller area compared with a control group (CK-1). Under the action of the validamycin A single factor, the shape of the rhizoctonia mycelia is obviously changed, the growth speed is slow, and the rhizoctonia mycelia extend onto corn leaves in a small area. Under the synergistic effect of the validamycin A and the trichoderma, the growth of the rhizoctonia is almost stopped, only weak mycelium exists, and the morphology of the mycelium is obviously changed. Maize leaves (CK-2) that were not inoculated were not damaged at all (FIG. 7).

Example 4 experiment of synergistic inhibition of Rhizoctonia cerealis by validamycin A and Trichoderma asperellum

Dish experiments-validamycin a with trichoderma as in example 3. Ex vivo leaf experiments-validamycin A was the same as for Trichoderma in example 3.

After the culture for 1d, the validamycin A can be efficiently absorbed by the rhizoctonia cerealis, the bacteriostatic action is very obvious, the diameter of the rhizoctonia cerealis colony is only 1.9 +/-0.1 cm, and the diameter of the colony of a control group reaches 5.5 +/-0.2 cm. Although the trichoderma asperellum grows rapidly and has obvious bacteriostasis effect, the trichoderma asperellum has obvious difference with the effect of validamycin A, and the diameter of the rhizoctonia solani colony is 4.5 +/-0.2 cm. Further, at this time, the advantage of the synergistic effect of validamycin A and Trichoderma asperellum was not shown, because the colony diameter of Rhizoctonia after synergistic treatment was 1.9. + -. 0.1cm, which was not significantly different from that of validamycin A alone.

However, the inhibitory effect of validamycin A was gradually released to some extent after 4 days of culture with the lapse of time, and the colony diameter of Rhizoctonia solani was increased to 5.3. + -. 0.2cm, as compared with 9.0. + -. 0.0cm in the control group. However, the biocontrol advantage of Trichoderma is now manifested, and the diameter of the inhibited Rhizoctonia solani colonies is reduced to 3.4. + -. 0.1 cm. Moreover, the synergistic disease-resistant effect of validamycin A and trichoderma is also obviously reflected, and the colony diameter of the rhizoctonia remains only 2.9 +/-0.2 cm under the sequential action of mechanisms such as antibiotic, competition, parasitism and the like, and the occupied area is obviously smaller than that of any single-factor treatment (figure 8). Therefore, the synergistic effect of the validamycin A and the trichoderma can not only realize the advantage complementation of a biological control mechanism, but also make up the deficiency of the mutual action on the aging or period, thereby efficiently and long-term inhibiting the rhizoctonia zeae.

After inoculating the rhizoctonia solani for 12h, the rhizoctonia solani of the control group (CK-1) grows vigorously and is infected on the corn leaves in a large area. Under the action of a trichoderma single factor, the density and the exuberance degree of the rhizoctonia mycelia are obviously reduced, and the rhizoctonia mycelia infect leaves in a smaller area compared with a control group (CK-1). Under the action of the single factor of the validamycin A, the shape of the rhizoctonia mycelia is obviously changed, the growth of the rhizoctonia mycelia is obviously inhibited, and the speed is very slow. Under the synergistic effect of the validamycin A and the trichoderma, the growth of the rhizoctonia is completely finished. Maize leaves (CK-2) that were not inoculated were not damaged at all (FIG. 9).

Dish experiment-validamycin A and trichoderma cell wall degrading enzyme

(1) Culturing the rhizoctonia zeae PDA culture in an incubator at 28 ℃ for 3 d.

(2) 25mg of validamycin A was dissolved in 10mL of sterile water, filtered through a 0.22 μm microporous filter, and added to 40mL of PDA, followed by mixing to prepare a dish containing 100. mu.g/mL of validamycin A.

(3) The two fermentation liquids of 1.2.3.2 and 1.2.4.1 were filtered through a 0.22 μm microporous filter, and added to 40mL of PDA, followed by mixing to prepare a dish containing 20% enzyme solution.

(4) 25mg of validamycin A was dissolved in 10mL of each of the two fermentation solutions 1.2.3.2 and 1.2.4.1, filtered through a 0.22 μm microporous filter, and added to 40mL of PDA, followed by mixing to prepare a dish containing 100. mu.g/mL of validamycin A + 20% enzyme solution.

(5)10mL of sterile water was added to 40mL of PDA, and after mixing, a control plate was prepared.

(6) After the plate is solidified, a 7mm puncher is used for punching a sclerotinia zeae bacterial dish to be placed in the center of the plate, the sclerotinia zeae bacterial dish is cultured at a constant temperature of 28 ℃ until bacterial colonies are contacted, the diameters of the sclerotinia zeae bacterial colonies of the treated group and the control group are respectively measured by adopting a cross method, and the inhibition rate after the culture is respectively calculated. Each experiment was repeated at least 3 times and differential significance was measured using t-test.

The result shows that the diameter of the bacterial colony of the pathogen treated by the validamycin A and the chitinase of the trichoderma is 3.0 +/-0.2 cm after the culture for 4 days; the diameters of the bacterial colonies of the validamycin A and the trichoderma single factor treated germs are respectively 3.7 +/-0.2 cm and 5.9 +/-0.3 cm; in the control, the diameter of the germ colony is 9.0 + -0.1 cm. The disease resistance effect of the synergistic effect is significantly higher than that of the single factor (fig. 10-a). After culturing for 4 days, the diameter of the bacterial colony of the pathogen treated by the validamycin A and the trichoderma cellulase in a synergistic way is 3.5 +/-0.1 cm; the diameters of the bacterial colonies of the validamycin A and the trichoderma single factor treated germs are respectively 4.2 +/-0.2 cm and 7.1 +/-0.2 cm; in the control, the diameter of the germ colony is 9.0 + -0.1 cm. The disease resistance effect of the synergistic effect is significantly higher than that of the single factor (fig. 10-B).

Example 5 preparation of Trichoderma-Validamycin granules

The embodiment provides a preparation method of trichoderma bio-granules, which specifically comprises the following steps:

1. plate strain: inoculating trichoderma asperellum GDFS1009 into a PDA culture medium plate, and performing inverted culture in an incubator at 28 ℃ for 2-3 d;

PDA culture medium: cutting 200g potato into 1cm pieces²Decocting the small blocks with slow fire for 30min, filtering with 4 layers of gauze, and keeping the juice; adding 20g glucose, 20g agar powder and distilled water to constant volume of 1L, and sterilizing with high pressure steam at 121 deg.C for 30 min.

2. Preparing a secondary strain: preparing a secondary fermentation culture solution from 200g of potatoes, 20g of glucose and 1L of water, adjusting the pH value to 6-8, subpackaging in a 250ml triangular flask for 100ml, sterilizing at 121 ℃ for 30min, inoculating a flat strain, rotating the table at 180 revolutions per minute, fermenting at 28 ℃ for 3d, and obtaining a large amount of dispersed flocculent hyphae and dispersed conidium liquid strains.

3. Preparing trichoderma liquid fermentation liquor: the fermentation medium comprises the following components: 10kg of corn flour, 210kg of water, 766g of monopotassium phosphate, 100g of magnesium sulfate, 0.5g of manganese sulfate, 0.4g of zinc sulfate, 284g of sodium nitrate, 220g of ammonium sulfate and 200g of sodium chloride, firstly dissolving inorganic salt in the water, then fully mixing culture materials with the water to prepare a solid fermentation culture medium, filling the solid fermentation culture medium into a 300L fermentation tank, sterilizing for 30-60 min at 121 ℃, inoculating a secondary strain with the weight proportion of 0.5%, controlling the fermentation temperature to be 28 ℃, and fermenting for 7d until the culture materials become green, namely trichoderma liquid fermentation broth;

4. preparation of trichoderma granules: 40% of trichoderma liquid fermentation liquor, 5.2% of corn flour, 37% of diatomite, 12% of wheat bran, 1% of zinc sulfate fertilizer, 0.4% of humic acid, 0.4% of nitrogen-phosphorus-potassium compound fertilizer and 4% of validamycin (5% of water aqua), uniformly stirring by using a stirrer, extruding and granulating by using a granulator, and drying for 1-2 hours at the temperature of 43-50 ℃ by using a dryer to obtain the finished product, namely the trichoderma granule.

The quality standard of the trichoderma-validamycin biological granules prepared by the preparation method is as follows: the particle size is 0.15-0.2 cm, the surface is smooth and hard, the water content is 10-20%, the pH value is 5-7, and the number of live spores is 2-4 multiplied by 10⁸CFU/g, the granules can be completely dissolved in water within 6-10 min, and are suitable for mechanized and chemical fertilizer mixed application.

Examples 6 and 5 the effect of the Trichoderma-validamycin granules on inhibiting Rhizoctonia zeae

6g of trichoderma asperellum GDFS1009 granules are applied to soil holes of each pot before the treatment groups are planted, the normally planted soil is used as blank control, 5 plants are planted in each pot, the base of a potted stem in a large trumpet period is respectively inoculated with pathogenic bacteria (a toothpick carrying rhizoctonia solani is inoculated by a root injury method) in each treatment 5 pots, and the biocontrol effect is counted after the disease is developed.

1. The biocontrol effect of trichoderma asperellum GDFS1009 on corn stem rot is investigated:

the disease rate is the disease rate/the total number of investigated plants multiplied by 100%,

biocontrol effect ═ control diseased plant rate-treated diseased plant rate)/control diseased plant rate ] × 100;

2. the biocontrol effect of trichoderma asperellum GDFS1009 on corn sheath blight is investigated:

disease index [ (number of disease-grade plants × representative number of disease-grade)/total number of plants × representative number of disease-grade at most severe grade) ] × 100% (see table 1),

biocontrol effect is (control disease index-treated disease index)/control disease index x 100%;

TABLE 1

Grade of disease condition	Description of the symptoms
		1	Disease of the 4 th leaf sheath under fruit cluster and the lower leaf sheath
3	The disease of the third leaf sheath under the fruit cluster and the lower leaf sheath
		5	The onset of the 2 nd sheath and the lower sheath under the fruit cluster
7	Disease of the 1 st leaf sheath under fruit cluster and the lower leaf sheath
		9	The disease of ear and sheath of above leaves

TABLE 2

Inoculation test in adult plant stage: as shown in fig. 11, wherein the left and right are trichoderma granules and blank control treatment, respectively; wherein, the morbidity and disease index of the trichoderma granules are respectively 40% and 10, and the blank control groups are respectively 100% and 29. The control effect of the trichoderma granules on corn sheath blight is 65.52 percent respectively. The trichoderma granules have better control effect on corn sheath blight (see table 2 specifically).

At present, the main method for preventing and treating the corn sheath blight caused by rhizoctonia is to directly spray a chemical pesticide, namely validamycin A, on a leaf sheath at the base of a stem. The mechanism of action of validamycin A is mainly to interfere with the energy metabolism of rhizoctonia solani and further inhibit the growth of the rhizoctonia solani, but not to degrade the rhizoctonia solani. The trichoderma can prevent and treat rhizoctonia solani through mechanisms such as competition, heavy parasitic action and the like, and finally, the rhizoctonia solani is degraded. In addition, the resistance of the trichoderma harzianum induced plants to rhizoctonia solani is far more obvious than that of validamycin A, and the trichoderma harzianum can be colonized in plant root systems and stem basal cortex for a long time to form symbiont with plant tissues, so that the infection of the corn rhizoctonia solani is controlled in a lasting manner. Therefore, if validamycin a can act synergistically with trichoderma, the level of control of sheath blight is expected to be significantly increased; meanwhile, the method is also beneficial to realizing the reduction use of antibiotic chemical pesticides and improving the environmental safety. The mode of synergy of validamycin a with trichoderma is essentially as follows: (1) validamycin a, when applied to the base of maize stalks, is rapidly absorbed into the body of rhizoctonia solani, but does not kill it, but rather makes it debilitating. (2) The colonized trichoderma in the plant tissue further competes and re-parasitizes the debilitating pathogen, degrading it. (3) The dual induced resistance effect generated by the validamycin A and the trichoderma can strengthen the resistance response of the corn to the rhizoctonia solani.

In the research, the trichoderma asperellum GDFS1009 is combined with the validamycin A for the first time, and the validamycin A with different concentrations has no obvious influence on the growth and development, the sporulation capacity, the biocontrol function and the like of the trichoderma asperellum. In addition, the Trichoderma asperellum has degradation rate of validamycin A of only 20% in the fermentation process. The method lays a certain theoretical foundation for the synergistic use of the trichoderma asperellum and the validamycin A, and shows that the combination of the trichoderma asperellum and the validamycin A has the characteristic of complementary biocontrol advantages. The conclusion is similar to the only few research results at home and abroad, but the molecular basic research on the affinity interaction of validamycin A and trichoderma at home and abroad is still blank at present.

In conclusion, the trichoderma asperellum can be used for preventing and treating corn sheath blight disease by cooperating with validamycin A. In future, whether other kinds of antagonistic trichoderma can also act with validamycin A in a synergistic manner needs to be explored, and a foundation is laid for developing various composite trichoderma biocontrol agents.

The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes and modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention.

Claims

1. The trichoderma-validamycin granules are characterized by comprising the following raw materials in parts by weight:

the Trichoderma fermentation liquid specifically refers to fermentation liquid of Trichoderma asperellum GDFS1009 CGMCC NO. 9512.

2. The trichoderma-validamycin granules according to claim 1, wherein the granules are prepared from the following raw materials in parts by weight:

3. the trichoderma-validamycin granules according to claim 1 or 2, wherein the preparation of the trichoderma fermentation broth comprises: inoculating 0.5 wt% of secondary strain into liquid culture medium, and fermenting at 28 deg.C for 7d until the fermentation liquid turns green, i.e. Trichoderma fermentation liquid;

wherein the liquid culture medium comprises 10kg of corn flour, 210kg of water, 766g of monopotassium phosphate, 100g of magnesium sulfate, 0.5g of manganese sulfate, 0.4g of zinc sulfate, 284g of sodium nitrate, 220g of ammonium sulfate and 200g of sodium chloride; the preparation of the secondary strain comprises the following steps: inoculating Trichoderma into PD, fermenting at 28 deg.C at 180rpm to obtain secondary strain.

4. The trichoderma-validamycin granules according to claim 1 or 2, wherein the dosage form of validamycin comprises a commercially available 5% aqueous solution.

5. A method for preparing the trichoderma-validamycin granules according to any one of claims 1 to 4, comprising the steps of: preparing the raw materials according to the weight percentage, uniformly mixing, extruding and granulating, and drying at the temperature of 43-50 ℃ for 1-2 hours to obtain the trichoderma-validamycin granules.

6. The method for preparing trichoderma-validamycin granules according to claim 5, further comprising the steps of evaluating the influence of validamycin on the growth and reproduction of trichoderma, and evaluating the influence of trichoderma on the absorption and degradation rate of the trichoderma.

7. The method for preparing trichoderma-validamycin granules according to claim 6, wherein the step of evaluating the influence of validamycin on growth and reproduction of trichoderma specifically comprises the following steps: and (3) taking trichoderma inoculated on a PDA plate containing validamycin as a treatment, taking trichoderma inoculated on a PDA plate without validamycin as a control, and counting and comparing the treatment with the control on production speed, morphology and spore yield.

8. The method for preparing trichoderma-validamycin granules according to claim 6, wherein the step of evaluating the influence of trichoderma on the absorption and degradation rate of validamycin comprises the following steps:

9. The method for preparing trichoderma-validamycin granules according to claim 5, wherein the uniformly mixing comprises stirring by a stirrer; the granulation comprises adopting a granulation extruder; the drying includes using a dryer.