Application of trichoderma griseagana TR2122 preparation in prevention and treatment of beet root rot

Abstract

The invention discloses an application of trichoderma griseaginii TR2122 in prevention and treatment of beet root rot, a strain of the trichoderma griseaginii TR2122 is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC No.21025, and the strain has strong antagonism and hyperparasitism effects on fusarium oxysporum causing the beet root rot. Meanwhile, the invention further provides a microbial preparation containing the live spores of the strain, the spore content is not lower than 2 * 10 < 8 > CFU / g, and the microbial preparation is preferably wettable powder containing a parent drug, a dispersing agent, a wetting agent and filler. In addition, the invention further discloses a preparation method of the preparation, and the preparation method comprises the steps of fermentation culture, concentration drying and mixing preparation. The strain and the preparation have stable and lasting control effect on beet root rot, are superior to or equivalent to conventional chemical agents, are safe and environment-friendly, and provide an effective solution for beet green control.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microbial pesticides, and particularly relates to application of Trichoderma ghanense TR2122 preparation in prevention and treatment of sugar beet root rot. BACKGROUND

[0002] Sugar beet is an important economic crop in China and plays an important role in sugar industry and agricultural economy. However, sugar beet root rot caused by soil-borne pathogenic fungi such as Fusarium oxysporum and Rhizoctonia solani seriously threatens the yield and quality of sugar beet, often leading to root rot, plant wilting and even large-scale death, and causing significant economic losses. Fusarium spp. Rhizoctonia solani Currently, the prevention and treatment of the disease still mainly relies on seed treatment, soil disinfection or root irrigation with chemical agents such as carbendazim and procymidone. Although chemical control can inhibit the disease in the short term, long-term use may lead to pesticide residues, soil pollution, affect product safety, and be contrary to the development direction of green agriculture. At the same time, the pathogenic fungi gradually increase in drug resistance, and the prevention and treatment effect decreases and the cost increases, which may also destroy the ecological balance of soil microorganisms. Although the disease can be alleviated to some extent through breeding of disease-resistant varieties and reasonable crop rotation, the resistant varieties are easy to be ineffective due to pathogenic variation, and the crop rotation mode is often limited by planting system and economic benefit, and is difficult to be applied in a large scale. Therefore, it is an urgent need of the industry to develop efficient, environmentally friendly and sustainable green prevention and control technology.

[0003] Biological pesticides have the advantages of environmental friendliness and non-induction of drug resistance, and are an important direction of green prevention and control of agricultural diseases. Trichoderma as a representative biocontrol agent can prevent and control soil-borne diseases through various mechanisms such as competition for nutrients and space, secretion of antibacterial substances and induction of plant resistance, and has been widely applied in many crops. However, for sugar beet root rot, especially for the development of efficient, stable and adaptable special biocontrol agents using specific species (such as Trichoderma ghanense), there is still a lack of mature technology and systematic verification, and the existing prevention and treatment methods still have obvious deficiencies in specificity, durability and ecological safety.

[0004] Biological pesticides have the advantages of environmental friendliness and non-induction of drug resistance, and are an important direction of green prevention and control of agricultural diseases. Trichoderma as a representative biocontrol agent can prevent and control soil-borne diseases through various mechanisms such as competition for nutrients and space, secretion of antibacterial substances and induction of plant resistance, and has been widely applied in many crops. However, for sugar beet root rot, especially for the development of efficient, stable and adaptable special biocontrol agents using specific species (such as Trichoderma ghanense), there is still a lack of mature technology and systematic verification, and the existing prevention and treatment methods still have obvious deficiencies in specificity, durability and ecological safety. Trichoderma spp. Trichoderma ghanense Therefore, it is of important practical application value and market prospect to provide a Trichoderma preparation for sugar beet root rot, which has the functions of efficient antibacterial, crop safety, environmental friendliness and stable prevention and treatment effect. SUMMARY

[0005] The purpose of the present application is to provide the application of Trichoderma ghanense TR2122 preparation in prevention and treatment of sugar beet root rot in view of the deficiencies in the prior art.

[0006] To achieve the above-mentioned purpose, the technical solution adopted by the present application is as follows:

[0007] The technical solution adopted by the present application is as follows: ​​The first aspect is to provide an application of Trichoderma ghanense TR2122 in preparing a microbial preparation for preventing and treating sugar beet root rot, wherein the strain TR2122 is preserved in the China General Microbiological Culture Collection Center, with a preservation number of CGMCC No. 21025, a preservation address of No. 3, Beichen West Road, Chaoyang District, Beijing, and a preservation date of December 21, 2020.

[0008] Further, the sugar beet root rot is caused by Fusarium oxysporum. Fusarium oxysporum ).

[0009] The second aspect is to provide a microbial preparation for preventing and treating sugar beet root rot, wherein the preparation comprises live spores of Trichoderma ghanense TR2122 with a preservation number of CGMCC No. 21025.

[0010] Further, the content of the live spores of TR2122 in the preparation is not less than 2×10 8 CFU / g.

[0011] Further, the preparation is a wettable powder, and the components thereof include, in terms of percentage by weight, 8-10% of Trichoderma ghanense TR2122 mother drug, 10-20% of lignin sulfonate, 0.1-0.5% of sodium dodecyl sulfate, 0.1-0.5% of trehalose, 0.1-0.5% of carbon black, and the balance of diatomite and / or talc powder.

[0012] The third aspect is to provide a preparation method of the above microbial preparation, comprising the following steps: S1, fermentation culture: inoculating Trichoderma ghanense TR2122 into a fermentation medium to perform fermentation, so as to obtain a fermentation liquor with a spore concentration of 4×10 8 ~5×10 8 CFU / mL; S2, mother drug preparation: concentrating and drying the fermentation liquor obtained in S1 to prepare a mother drug with a spore content of 20×10 8 ~25×10 8 CFU / g; S3, preparation preparation: mixing the mother drug obtained in S2 with fillers and auxiliaries to prepare the microbial preparation with a spore content of not less than 2×10 8 CFU / g.

[0013] Further, in the drying process of S2, dextrin and sorbitol are added as protective agents.

[0014] The above technical solution is adopted in the present application, and the present application has the following technical effects compared with the prior art: The Ganoderma TR2122 provided by the application is a high-efficiency and specialized biocontrol strain, which shows strong antagonistic activity and hyperparasitism to the main pathogen (Fusarium oxysporum) of sugar beet root rot. Through multi-level verification such as plate confrontation, indoor safety evaluation and field efficacy test, the strain and its preparation have a sustained and stable control effect on sugar beet root rot, and the control effect is better than or equivalent to that of conventional chemical agents, and are safe to crops and friendly to the environment, which provides a new technical support for the green prevention and control of sugar beet root rot, and has a wide application prospect.

[0015] Compared with the Trichoderma strain in the prior art, the core advantage of the Ganoderma TR2122 in the application in preventing and treating sugar beet root rot is that: 1) Excellent environmental adaptability: the strain can grow well in a wide temperature range of 20-40 DEG C, and can still maintain vigorous vitality at high temperature of 30-35 DEG C, which makes it have a competitive advantage in colonization in summer soil environment.

[0016] 2) High reproduction and colonization ability: under suitable conditions (25-30 DEG C), the colony radius can reach 70 mm in 64 hours, and the growth rate is significantly better than that of ordinary strains, which can quickly occupy the rhizosphere niche.

[0017] 3) Specific biocontrol activity: plate confrontation test shows that it has more than 80% inhibition rate on the key pathogen of sugar beet root rot, Fusarium oxysporum, and shows strong hyperparasitism, with obvious mycelial winding and penetration. Fusarium oxysporum

[0018] 4) Crop safety and growth promotion: indoor safety test proves that even at a dose of 1200 g / ha, there is no phytotoxicity to three mainstream sugar beet varieties, and at the recommended dose (600 g / ha), it shows significant growth promotion effect on some varieties. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 The figure shows the winding phenomenon of TR2122 (Trichoderma) mycelium on the mycelium of the pathogen of sugar beet root rot (Fusarium oxysporum) under a microscope.

[0020] Figure 2 The figure shows the hyperparasitism of TR2122 (Trichoderma) on the pathogen of sugar beet root rot (Fusarium oxysporum) under a microscope. DETAILED DESCRIPTION

[0021] The application will be further described below in combination with the drawings and specific examples, but not as a limitation of the application. It should be noted that the examples in the application and the features in the examples can be combined with each other without conflict.

[0022] Example 1 ​The biocontrol strain used in this invention is *Trichoderma kanae* ( Trichoderma ghanense TR2122. This strain was isolated from the rhizosphere soil of tomato plant in Sunqiao, Pudong New Area, Shanghai. Its complete isolation, purification, morphological and molecular biological identification methods, as well as its culture characteristics, have been disclosed in detail in Example 1 and the accompanying drawings of the specification of the applicant's authorized Chinese invention patent CN112680361B (announcement date: July 26, 2022).

[0023] As confirmed by the identification disclosed in the aforementioned patent documents, this strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 21025 and a deposit date of December 21, 2020. Based on this deposited and disclosed strain TR2122, this invention develops new uses for its application in the prevention and control of beet root rot and related formulations.

[0024] Example 2 This embodiment provides a method for preparing a wettable powder (WP) using TR2122 fermented spores as the active ingredient: 1. Fermentation culture After activating the TR2122 strain (CGMCC No. 21025), it was inoculated onto PDA plates and cultured at 25-28℃ for 3-5 days. The spores were washed away with sterile water to prepare a spore suspension, which served as the primary seed culture. The primary seed culture was then transferred to Erlenmeyer flasks containing PDB liquid medium at an inoculation rate of 5-10% (v / v) and cultured at 25-28℃ with shaking at 200-300 rpm for 24-48 hours to obtain the secondary seed culture.

[0025] The secondary seed culture was inoculated into the fermenter at an inoculum rate of 5-10% (v / v) (filling coefficient 60%). The fermentation medium consisted of: peanut powder 25 g / L, glucose 15 g / L, KH₂PO₄ 1.5 g / L, MgSO₄·7H₂O 0.75 g / L, FeSO₄·7H₂O 0.03 g / L, vitamin B1 0.1 mg / L, and natural pH (approximately 5.5-6.5). Fermentation conditions were: temperature 28-30℃, tank pressure 0.04-0.05 MPa, aeration rate 1.5-1.8 vvm, stirring speed 120-150 rpm, and culture for 5-6 days. At the end of fermentation, the spore concentration reached 4 × 10⁻⁶. 8 ~5×10 8 Fermentation broth at CFU / mL.

[0026] 2. Preparation of the parent drug The fermentation broth was concentrated by centrifugation and then spray-dried. Before drying, dextrin (8 wt%) and sorbitol (2 wt%) were added to the concentrated mycelial sludge as protective agents to maximize spore viability. The resulting product had a spore content of 20 × 10⁻⁶. 8 ~25×10 8 CFU / g Trichoderma ganagenii TR2122 mother drug.

[0027] 3. Formulation (wettable powder) Weigh and mix the components according to the following weight percentages: Trichoderma ghana TR2122 technical grade: 9% Lignosulfonate (dispersant): 15% Sodium dodecyl sulfate (wetting agent): 0.3% Trehalose (stabilizer): 0.3% Carbon black (UV protectant): 0.2% Xanthan gum (adhesive): 0.5% Bentonite: 73% Talc (filler): 1.7% After thoroughly mixing, pulverizing, and sieving the above components, a spore content of not less than 2 × 10⁻⁶ is obtained. 8 CFU / g Trichoderma Ghana TR2122 wettable powder (WP).

[0028] Example 3: Plate confrontation test to verify the antagonistic effect of TR2122 on beet root rot. Beetroot rot pathogen (Fusarium oxysporum) with a diameter of 5 mm was inoculated on both sides of the PDA plate. Fusarium oxysporum Two bacterial cultures, TR2122 and TR2122, were used, with a 6cm gap between them. A control group was inoculated with the pathogen alone. Each group was repeated three times. After incubation at 28℃ for 5 days, the colony radius of the pathogen was measured, and the inhibition rate was calculated.

[0029] Inhibition rate (%) = [(Coronavirus radius of control group - Coronavirus radius of treatment group) / Coronavirus radius of control group] × 100% After 48 hours of incubation, TR2122 colonies came into contact with pathogen colonies and gradually covered the pathogens. This was visible under a microscope. Figure 1 , Figure 2 TR2122 hyphae tightly entwine and penetrate the hyphae of pathogenic fungi. After 5 days of culture, TR2122 showed an inhibition rate of over 80% against Fusarium oxysporum, indicating that it has significant antagonistic and reparasitic abilities.

[0030] Example 4 Indoor Safety Test Using sugar beet varieties 'COFCO9128', 'KWS2317', and 'HI1646' as test materials, 200 million spores / gram of Trichoderma ganagenin WP was applied to potted plants at the 3-4 leaf stage via foliar spraying. Three dosages were set: 300, 600, and 1200 g / mu (approximately 667 square meters), with water as a control. The pesticide was applied once every 7 days for 3 consecutive applications. Phytotoxicity was observed 21 days after the last application, plant height was measured, and growth rate was calculated.

[0031] The results are shown in Table 1 below. Under each dosage treatment, no phytotoxicity symptoms were observed in any of the sugar beet varieties, and their growth was normal. Among them, the dosages of 600 g / mu and 1200 g / mu showed a tendency to promote the growth of some varieties.

[0032] Table 1

[0033] Example 5: Potted Plant Efficacy Test Using 'COFCO9128' sugar beet as the experimental material, four treatments were set up: T1 (TR2122 WP, soil treatment at sowing 15 kg / hm) 2 +Drench the roots at the seedling stage: 7.5 kg / hm 2 T2 (pathogen control), T3 (50% carbendazim WP, soil treatment at sowing 1.5 kg / hm²) 2 +Drench the roots with 1.5 kg / hm² during the seedling stage. 2 T4 (water control). Fusarium oxysporum was artificially inoculated, and routine management was implemented. After the onset of disease, the disease index was investigated (experimental data were analyzed using Duncan's multiple range test for significance (p<0.05), and results are expressed as mean ± standard deviation), and the control effect was calculated.

[0034] As shown in Table 2 below, the disease index of treatment T1 (TR2122) was 0.20, which was not significantly different from the 0.40 of treatment T3 (carbendazim) (both marked as b), but both were significantly lower than the disease index of T2 (pathogen control) of 4.80 (marked as a). This indicates that the Trichoderma fungal preparation of the present invention is as effective as the chemical agent carbendazim in controlling the severity of the disease.

[0035] Based on the disease index, the control efficacy of T1 (TR2122) treatment was 95.83%, and that of T3 (carbendazim) treatment was 94.79%. The efficacy values ​​of the two treatments were similar, and there was no statistically significant difference.

[0036] Table 2

[0037] Note: Different upper and lowercase letters indicate significance levels of 0.01 and 0.05, respectively.

[0038] Example 6 Dynamic determination of colonization of Trichoderma ghanense TR2122 in sugar beet rhizosphere soil The TR2122 preparation was inoculated into the soil at a dosage of 15 kg / hm 2 at the sugar beet seedling stage (after emergence), the rapid growth stage of leaf clumps, the sugar content increase stage of tubers, and the sugar accumulation stage, respectively. Soil samples were collected around the rhizosphere of the plants (0-10 cm from the main stem, 5-15 cm deep) at 7 days, 14 days, 21 days, and 28 days after inoculation, and TR2122 was isolated using a selective medium to calculate the colony-forming units (CFU).

[0039] The results are shown in Table 3 below. Trichoderma ghanense TR2122 can effectively colonize the sugar beet rhizosphere and exhibit good adaptability to high temperatures (30-35°C) in Xinjiang summer fields, providing a reliable ecological basis for its sustained biocontrol effect during the entire sugar beet growth period.

[0040] Table 3 Dynamic colonization of Trichoderma ghanense TR2122 in sugar beet rhizosphere soil (CFU / g dry soil)

[0041] Example 7 Field efficacy test (2024) A plot test was conducted in a sugar beet 'COFCO9128' field, with 3 treatments: T1 (TR2122 WP, same as Example 5), T2 (50% carbendazim WP, same as Example 5), and T3 (water control). Each treatment had 4 replicates, and the plot area was 100 m². Disease index and incidence were investigated, and control effect was calculated.

[0042] The results are shown in Table 4 below. The field control effect of T1 treatment on sugar beet root rot was 86.19%, which was significantly higher than that of chemical agent T2 treatment (71.58%), and was safe to crops. This verifies the results of the pot test on the one hand, and on the other hand, its control effect exceeds that of chemical agents in the complex field environment, highlighting the comprehensive advantages of TR2122 strain in realizing sustained control through multiple mechanisms such as competition, hyperparasitism, and induced resistance, rather than the single mode of action of chemical agents.

[0043] Table 4

[0044] Note: Different large and small letters represent significant levels of 0.01 and 0.05, respectively.

[0045] Example 8 Field efficacy test (2025) A large plot test was carried out in a production field, and two treatments were set: T1 (TR2122 WP, used in the same way as in Example 5), T2 (50% carbendazim WP, used in the same way as in Example 5), and the plot area was 667 m². The disease index and incidence were investigated, and the relative control effect was calculated.

[0046] The results are shown in Table 5 below, and the disease index of the TR2122 WP treatment (T1) was significantly lower than that of the carbendazim treatment (T2), and the relative control effect was 31.16%. It can be seen that although there are differences in climate and soil microbial community between years, the preparation of the present application shows stable and significant disease control effect in different years and different scale tests, which proves the reliability and repeatability of the technical scheme.

[0047] Table 5

[0048] The above only describes the preferred embodiments of the present application, and does not limit the implementation and protection scope of the present application. It should be realized by those skilled in the art that any equivalent replacement and obvious changes made according to the content of the present application and the drawings should be included in the protection scope of the present application.

Claims

1. Use of Trichoderma ghanense TR2122 for the preparation of a microbial preparation for the control of beet root rot, characterized in that, The strain TR2122 is preserved in the China General Microbiological Culture Collection Center, with a preservation number of CGMCC No. 21025, a preservation address of No. 3, Xibei Road, Beichen, Chaoyang District, Beijing, and a preservation date of December 21, 2020.

2. Use according to claim 1, characterized in that, The beet root rot is caused by Fusarium oxysporum.

3. A microbial preparation for controlling sugar beet foot and root rot, characterized by, The preparation comprises live spores of the Trichoderma ghanense TR2122 with a preservation number of CGMCC No. 21025.

4. The microbial preparation according to claim 3, characterized in that, The viable spore content of TR2122 in the preparation is not less than 2 x 10 8 CFU / g.

5. The microbial preparation according to claim 3, characterized in that, The preparation is a wettable powder, and the components thereof include, by weight percentage, 8-10% of the Trichoderma ghanense TR2122 mother drug, 10-20% of lignosulfonate, 0.1-0.5% of sodium dodecyl sulfate, 0.1-0.5% of trehalose, 0.1-0.5% of carbon black, and the rest of diatomite and / or talc powder.

6. A method of preparing a microbial preparation as claimed in any one of claims 3-5, characterized in that, The method comprises the following steps: S1, fermentation culture: Trichoderma ghanense TR2122 was inoculated into fermentation medium for fermentation, and a fermentation liquor with a spore concentration of 4 x 105CFU / mL was obtained. 8 8 5 x 105CFU / mL. S2, preparation of the mother drug: the fermentation broth obtained in S1 was concentrated and dried to obtain a mother drug with a spore content of 20 x 10 8 25 x 10 8 CFU / g. S3, preparation of the preparation: the mother drug obtained in S2 is mixed with fillers and adjuvants to obtain the microbial preparation with a spore content of not less than 2×10 8 CFU / g.

7. The method of claim 6, wherein, In the drying process, dextrin and sorbitol are added as protective agents. The strain TR2122 is preserved in the China General Microbiological Culture Collection Center, with a preservation number of CGMCC No. 21025, a preservation address of No. 3, Xibei Road, Beichen, Chaoyang District, Beijing, and a preservation date of December 21, 2020. The beet root rot is caused by Fusarium oxysporum. The preparation comprises live spores of the Trichoderma ghanense TR2122 with a preservation number of CGMCC No. 21025. The preparation is a wettable powder, and the components thereof include, by weight percentage, 8-10% of the Trichoderma ghanense TR2122 mother drug, 10-20% of lignosulfonate, 0.1-0.5% of sodium dodecyl sulfate, 0.1-0.5% of trehalose, 0.1-0.5% of carbon black, and the rest of diatomite and / or talc powder. The method comprises the following steps: In the drying process, dextrin and sorbitol are added as protective agents.

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