Composite trichoderma fungicide for preventing and treating ginger root rot as well as preparation method and application of composite trichoderma fungicide

Abstract

The invention relates to the technical field of microorganisms, in particular to a composite trichoderma agent for preventing and treating ginger root rot as well as a preparation method and application of the composite trichoderma agent. The preparation method comprises the following steps: adsorbing a composite spore suspension containing trichoderma atroviride HB20111 spores and trichoderma harzianum TW20321 spores by using a carrier to form a solid fungicide, then uniformly stirring and mixing the solid fungicide with carboxymethyl cellulose sodium and sodium alginate, and drying until the moisture content is less than or equal to 10% to prepare the composite trichoderma fungicide. The composite trichoderma agent disclosed by the invention has a strong inhibition effect on ginger root rot pathogenic bacteria, namely fusarium oxysporum, ginger specialization, so that the effect of preventing and treating the ginger root rot is achieved; in addition, the two strains of trichoderma have a rhizosphere growth promoting effect, so that the micro-ecological environment of ginger planting soil can be improved, the soil structure and physicochemical properties can be improved, and the biological yield and quality of ginger can be improved.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a composite trichoderma agent for preventing and treating ginger root rot, and a preparation method and application thereof. Background Art

[0002] Big ginger ( Zingiber officinale Rosc.), belonging to the genus Zingiberaceae, is a perennial monocotyledonous herb. The rhizome of ginger is large and can be used for food and medicine. It contains gingerol, gingerolone, shogaol, gingerol, starch, pentosan, protein, cellulose, pigment, multiple vitamins and trace mineral elements. Ginger is warm in nature and has a special spicy taste. It is a common spicy seasoning vegetable and is known as the "ancestor of vegetables". In addition to being used directly as a seasoning, ginger can also be processed into ginger juice, ginger slices, ginger powder, ginger wine, ginger oil, etc. It can also be salted, candied, and pickled to make a variety of foods.

[0003] With the continuous expansion of planting area, more and more diseases are harming ginger, such as Fusarium oxysporum, ginger-specific type ( Fusarium oxysporum f. sp. Zingiberi , Foz) occurs frequently in ginger producing areas. Ginger root rot is a soil-borne fungal disease. The pathogen can spread in the soil through agricultural tools, ginger seeds, and soil irrigation water, or through diseased residues when ginger is planted. The initial point of occurrence of ginger root rot is the underground rhizome. The pathogen reproduces and secretes toxins in the vascular bundles of the roots and stems, destroying the vascular function and affecting the growth of ginger. After infection, the vascular bundles of the roots and stems of ginger will turn brown to dark brown, the base of the stem will wither and wilt, and the whole plant will die.

[0004] At present, the prevention and control methods of ginger root rot include agricultural prevention, physical prevention, chemical prevention and biological prevention. Agricultural prevention mainly includes crop rotation, intercropping, detoxification, etc. The large-scale application of these technologies will increase labor intensity and cost. Physical prevention mainly uses solar radiation sterilization, etc. These technologies have problems such as incomplete sterilization and easy to be affected by external conditions. Chemical prevention is currently the most commonly used method for the prevention and control of ginger root rot, mainly including chemical fumigation soil disinfection and chemical pesticide spraying. Commonly used chemical pesticides for fumigation include chloropicrin, dazomethane, and pyraclostrobin, and chemical pesticides for spraying include thiazolyl, carbendazim, metalaxyl, and methyl thiophanate, etc. However, there are generally vicious cycle problems such as strong drug resistance of pathogens, reduced prevention and control effects year by year, imbalance of the proportion of beneficial bacteria in the soil, and serious soil environmental pollution. Biological control is the use of beneficial bacteria with biological control functions to prevent and control ginger root rot, which can reduce chemical residues and improve food safety, but the adaptability of a single strain is often limited. Summary of the invention

[0005] In view of the technical problem that the adaptability of the strains existing in the prevention and control of ginger root rot is limited when a single strain is used, the present invention provides a composite Trichoderma agent for preventing and controlling ginger root rot, and a preparation method and application thereof. The composite Trichoderma agent shows a strong inhibitory effect on the ginger-specific type of Fusarium oxysporum, the pathogen of ginger root rot, thereby achieving the effect of preventing and controlling ginger root rot; in addition, both Trichoderma strains have a rhizosphere growth-promoting effect, which can improve the microecological environment of ginger planting soil, improve soil structure and physical and chemical properties, and increase the biological yield and quality of ginger.

[0006] In a first aspect, the present invention provides a method for preparing a composite Trichoderma agent for preventing and treating ginger root rot, comprising the following steps: Use carrier to adsorb dark green Trichoderma ( Trichoderma atroviride )HB20111 spores and Trichoderma harzianum ( Trichoderma harzianum ) a composite spore suspension of TW20321 spores to form a solid bacterial agent, which is then mixed with sodium hydroxymethyl cellulose and sodium alginate and dried to a moisture content of ≤10% to obtain a composite Trichoderma agent; Among them, Trichoderma viridis HB20111 has been disclosed in the Chinese invention patent CN110915822B, with a deposit date of December 5, 2018, and a depository unit named China General Microbiological Culture Collection Center, with a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and a deposit number of CGMCC No. 16963, and a classification name of Trichoderma viridis Trichoderma atroviride ; Trichoderma harzianum TW20321 was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on November 26, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No.41641 and the classification name is Trichoderma harzianum Trichoderma harzianum .

[0007] Furthermore, the adsorption carrier is selected from diatomaceous earth, peat soil or medical stone.

[0008] Furthermore, based on the mass of the solid bacterial agent, the dosage of sodium hydroxymethyl cellulose and sodium alginate is 3g / 100g respectively.

[0009] Furthermore, the bacterial count in the composite Trichoderma agent was 2.0×10 8 cfu / g or above.

[0010] Further, the composite spore suspension is obtained by the following method: The spore suspension of Trichoderma harzianum TW20321 and the fermentation medium are uniformly mixed and cultured together. After the culture is completed, the culture system is immersed in water and stirred to disperse the conidia of Trichoderma harzianum TW20321 and the conidia of Trichoderma harzianum HB20111 in the water. The filtrate is filtered to obtain a composite spore suspension.

[0011] In a second aspect, the present invention provides a composite Trichoderma agent prepared by the above preparation method.

[0012] In a third aspect, the present invention further provides a use of the above-mentioned composite Trichoderma agent in preventing and treating ginger root rot, wherein the pathogen of ginger root rot is Fusarium oxysporum ginger-specific type.

[0013] Furthermore, the composite Trichoderma agent also has a growth-promoting effect on ginger.

[0014] Furthermore, the application method is: ① evenly apply it into the soil by mixing it with soil, and plant ginger sprouts, ② mix it with nutrient medium as a ginger seedling cultivation medium, or ③ dilute it with water and spray it on the leaves of ginger.

[0015] The beneficial effects of the present invention are: The two Trichoderma strains used in the present invention, Trichoderma harzianum TW20321 and Trichoderma harzianum, are both isolated from the soil and can produce fungal cell wall degrading enzymes including protease, cellulase and β-1,3-glucanase, as well as growth-promoting substances such as indole-3-acetic acid (IAA) and siderophore. Both have rhizosphere growth-promoting effects and can colonize and occupy positions at the roots of crops, inside plants or in soil ecology, thereby improving the microecological environment of ginger planting soil, improving soil structure and physical and chemical properties, and increasing the biological yield and quality of ginger.

[0016] The composite Trichoderma agent provided by the invention has cost-saving preparation process, stable fermentation method, simple usage, and is environmentally friendly. Compared with a single biocontrol strain, it is not easy to produce drug resistance in preventing and controlling ginger root rot, has significant effect, and has high application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0018] Figure 1These are photos of the antibacterial activity of Trichoderma harzianum TW20321 against Fusarium oxysporum Foz on PDA plates, where A is the antibacterial result of Trichoderma harzianum TW20321, B is the antibacterial result of Trichoderma harzianum TW20321, and C is the blank control inoculated with only Fusarium oxysporum Foz.

[0019] Figure 2 These are photos of plate tests on the ability of dark green Trichoderma HB20111 and Trichoderma harzianum TW20321 to produce siderophore, protease, cellulase and β-1,3-glucanase, where A is the result of siderophore production, B is the result of protease production, C is the result of cellulase production, and D is the result of β-1,3-glucanase production. DETAILED DESCRIPTION

[0020] In order to enable those skilled in the art to better understand the technical solutions in the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0021] The dark green Trichoderma HB20111 used in the present invention has been disclosed in Chinese invention patent CN110915822B, and the deposit information is as follows: Deposit date: December 5, 2018; Name of depository: General Microbiology Center of China Microbiological Culture Collection Administration; Storage address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; Deposit number: CGMCC No.16963; Classification name: Dark green Trichoderma Trichoderma atroviride .

[0022] The Trichoderma harzianum TW20321 used in the present invention was collected from Dongying City, Shandong Province in December 2015, and the ITS sequence (sequence 1) is as follows: .

[0023] The deposit information of Trichoderma harzianum TW20321 is as follows: Deposit date: November 26, 2024; Name of depository: General Microbiology Center of China Microbiological Culture Collection Administration; Storage address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; Deposit number: CGMCC No.41641; Classification name: Trichoderma harzianum Trichoderma harzianum .

[0024] Example 1 Preparation of composite Trichoderma agent (1) Strain activation: dark green Trichoderma T. atroviride HB20111 and Trichoderma harzianum T. harzianumTW20321 was inoculated into PDA medium (20% peeled potatoes, 2% glucose, 1.5% agar powder, 1 L distilled water, natural pH) and placed in a constant temperature incubator at 28°C for activation culture for 5 to 7 days.

[0025] To verify the inhibitory activity of Trichoderma HB20111 and Trichoderma harzianum TW20321 against Fusarium oxysporum ginger-specific Foz, two Trichoderma strains and pathogenic bacteria with a diameter of 5 mm were inoculated at both ends of the PDA plate, and the PDA plate inoculated with Foz alone was used as a blank control. The plates were cultured at 28°C for 5 days to observe the antagonistic inhibition of the two Trichoderma strains on Foz. Figure 1 As shown, dark green Trichoderma HB20111 and Trichoderma harzianum TW20321 in culture dishes A and B significantly inhibited the growth of Foz and covered most of its colonies, indicating that the two have significant inhibitory activity against the ginger-specific type of Fusarium oxysporum.

[0026] Both Trichoderma harzianum HB20111 and Trichoderma harzianum TW20321 were isolated from soil and were able to produce fungal cell wall degrading enzymes including proteases, cellulases and β-1,3-glucanases, as well as growth-promoting substances such as indole-3-acetic acid (IAA) and siderophores. The rhizosphere growth-promoting functions of the two strains were qualitatively analyzed. The specific methods are as follows: Prepare each activity test medium plate, inoculate two 5mm diameter Trichoderma plugs in the center of the plate, culture at 28℃ for 5-7 days, and record the diameter of the hydrolysis zone. Figure 2 As shown, Trichoderma aureum HB20111 has stronger siderophore, glucanase and protease activities, while Trichoderma harzianum TW20321 has stronger siderophore, cellulase and protease activities.

[0027] (2) Seed solution preparation: Scrape the spores of Trichoderma harzianum TW20321 and Trichoderma harzianum HB20111 with sterile water and dilute them to 1.0×10 8 cfu / mL as seed solution.

[0028] (3) Solid culture: The two seed solutions obtained in step (2) were inoculated into a fermentation medium (1 wt % corn flour, 42 wt % bran, 5 wt % rice husk, and the balance was water) at an inoculum rate of 5% (v / v), mixed evenly, and cultured together for 120 h.

[0029] (4) Spore suspension collection: The fermented culture system and water were immersed in water at a mass ratio of 1:1, stirred to disperse the conidia in the culture system into the water, filtered to remove the fermentation medium raw materials, and obtained a composite spore suspension. The concentration was adjusted to 5.0×10 9 cfu / mL.

[0030] (5) Preparation of composite Trichoderma inoculant: diatomaceous earth, peat soil, and medical stone were used as adsorption carriers to adsorb the composite spore suspension to form a solid inoculant. 3 wt% sodium carboxymethyl cellulose and 3 wt% sodium alginate were added and mixed with a blender. The bacterial count was adjusted to 2.0 × 10 8 cfu / g, dried at 50℃ in a fluidized bed to a moisture content below 10%, namely, the composite Trichoderma agent adsorbed by diatomaceous earth, the composite Trichoderma agent adsorbed by peat soil and the composite Trichoderma agent adsorbed by medical stone. The dosage forms of the three composite Trichoderma agents are all wettable powders.

[0031] Example 2 Field application demonstration of composite Trichoderma agent Experimental location: Zhaili Town, Laiwu District, Jinan City, Shandong Province, a ginger-growing area with a high incidence of ginger root rot in the early stage, with an experimental area of ​​100 square meters.

[0032] Test crop: Laiwu ginger.

[0033] The experiment was divided into three treatment groups: blank control group, carbendazim treatment group (positive control) and co-culture bacterial agent treatment group.

[0034] The blank control group did not receive any pretreatment.

[0035] Carbendazim-treated group: During the ginger sowing period, 50% carbendazim wettable powder was mixed with soil at a dosage of 2kg / mu and applied to plant ginger seed buds.

[0036] The composite Trichoderma agent treatment group: During the ginger sowing period, the composite Trichoderma agent adsorbed with medical stone prepared in Example 1 was evenly applied to the soil at a dosage of 5 kg / mu by mixing with soil, and ginger seed sprouts were planted.

[0037] The planting and management of ginger in the three treatment groups were consistent with local cultivation conditions. 180 days after planting, the field plot prevention and growth-promoting effects of the composite Trichoderma agent on ginger root rot were investigated and tested.

[0038] The root rot of ginger was investigated plant by plant according to the 7-level classification method. The classification standards for the severity of the disease are as follows: Level 0: The whole plant is disease-free; Level 1: The base of the stem is slightly discolored, or less than 1 / 4 of the leaves are slightly faded or wilted; Level 3: The base of the stem is obviously discolored, or 1 / 4 to 1 / 2 of the leaves are slightly wilted; Level 5: Browning at the base of the stem covering 1 / 2 of the entire stem, or more than 2 / 3 of the leaves withering; Level 7: All diseased plants wilt or die.

[0039] The disease index and disease prevention effect are calculated according to the following formula: ; .

[0040] The field plot control results of ginger root rot are shown in Table 1. The application of compound Trichoderma can effectively reduce the incidence of ginger root rot, and its control effect on ginger root rot reached 68.90%, which is significantly higher than the 63.88% of chemical pesticide carbendazim. The application of compound Trichoderma can also significantly improve the growth indicators of ginger, and the plant height and single plant yield are significantly different from the blank control ( p <0.05), and the promotion effect on ginger plant height and single plant yield increased by 19.31% and 27.62% respectively compared with the control group; there was no significant difference with the effect of carbendazim treatment (18.83% and 24.76% respectively). This shows that the application of composite Trichoderma agent can not only effectively prevent and control ginger root rot, but also play a significant role in promoting ginger yield.

[0041] Table 1 Statistics of ginger field plot test

[0042] In other embodiments, the composite Trichoderma agent of the present invention can also be added to a nutrient medium at a ratio of 10-20 g / L and used as a cultivation medium for ginger seedlings; or diluted with water to 10-100 times and then sprayed on the leaves of ginger.

[0043] Although the present invention has been described in detail with reference to the accompanying drawings and in combination with the preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and essence of the present invention, a person of ordinary skill in the art may make various equivalent modifications or substitutions to the embodiments of the present invention, and these modifications or substitutions shall be within the scope of the present invention. Any person of ordinary skill in the art may easily think of changes or substitutions within the technical scope disclosed by the present invention, and these shall be within the scope of protection of the present invention.

Claims

1. A method for preparing a composite trichoderma agent for preventing and treating ginger root rot, characterized in that: The steps include: Use carrier to adsorb dark green Trichoderma ( Trichoderma atroviride )HB20111 spores and Trichoderma harzianum ( Trichoderma harzianum ) a composite spore suspension of TW20321 spores to form a solid bacterial agent, which is then mixed with sodium hydroxymethyl cellulose and sodium alginate and dried to a moisture content of ≤10% to obtain a composite Trichoderma agent; Among them, Trichoderma viridis HB20111 was deposited in the General Microbiological Center of China Microbiological Culture Collection Administration on December 5, 2018. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No. 16963, and the classification name is Trichoderma viridis Trichoderma atroviride ; Trichoderma harzianum TW20321 was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on November 26, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No.41641 and the classification name is Trichoderma harzianum Trichoderma harzianum .

2. The preparation method according to claim 1, characterized in that The adsorption carrier is selected from diatomaceous earth, peat soil or medical stone.

3. The preparation method according to claim 1, characterized in that: Calculated based on the mass of solid bacterial agent, the dosage of sodium hydroxymethyl cellulose and sodium alginate is 3g / 100g respectively.

4. The preparation method according to claim 1, characterized in that: The bacterial count in the composite Trichoderma agent was 2.0×10 8 cfu / g or above.

5. The preparation method according to claim 1, characterized in that: The composite spore suspension is obtained by the following method: The spore suspension of Trichoderma harzianum TW20321 and the fermentation medium are uniformly mixed and cultured together. After the culture is completed, the culture system is immersed in water and stirred to disperse the conidia of Trichoderma harzianum TW20321 and the conidia of Trichoderma harzianum HB20111 in the water. The filtrate is filtered to obtain a composite spore suspension.

6. A composite Trichoderma agent prepared by the preparation method according to any one of claims 1 to 5.

7. Use of the composite Trichoderma agent as claimed in claim 6 in preventing and treating root rot of ginger, characterized in that: The pathogen of ginger root rot is Fusarium oxysporum, a special strain of ginger ( Fusarium oxysporum f. sp. Zingiberi ).

8. The use according to claim 7, characterized in that The compound Trichoderma agent also has a growth-promoting effect on ginger.

9. The use according to claim 7, characterized in that The application method is: ① Apply it evenly into the soil by mixing it with soil and planting ginger sprouts, ② Mix it with nutrient medium as a ginger seedling cultivation medium, or ③ Dilute it with water and spray it on the leaves of ginger.

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