Trichoderma cereus and application thereof

Abstract

The invention discloses a Trichoderma cererum and application thereof, the name of the Trichoderma cererum TR1186A is Trichoderma cererum TR1186A, the preservation unit is China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.21427. The Trichoderma cererum is named as Trichoderma cererum TR1186A. The Trichoderma cererum is named as Trichoderma cererum TR1186A. The trichoderma cereus provided by the invention is an effective biocontrol strain and can antagonize a tobacco black shank strain, and test results of a plate confrontation test, an indoor pharmacodynamic test, an indoor safety test, a field pharmacodynamic test and the like prove that the strain can be developed into a biological pesticide which is environment-friendly, high in safety and good in control effect; particularly, the bacillus subtilis has a good application prospect in biological prevention and control of tobacco black shank.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microbial pesticides, in particular to a Trichoderma cerinum and application thereof. BACKGROUND

[0002] Tobacco is one of the important economic crops in China, and plays an important role in industrial production and national economy. With the development of tobacco industry, tobacco diseases, especially tobacco black shank, have a great destructive power on its yield and quality. Tobacco black shank is mainly caused by infection of Phytophthora nicotianae, and can occur at multiple stages of tobacco growth. From the seedling stage to the adult stage, it can cause harm. At present, the prevention and control of tobacco black shank mainly relies on chemical agents, such as using metalaxyl, hymexazol and other chemical agents for soil treatment, root irrigation or spraying. Chemical control can inhibit the spread of disease in a certain period of time, but long-term and large-scale use of chemical agents can easily cause pollution to soil and tobacco leaves, affect the safety of tobacco products, and also go against the development concept of green agriculture; at the same time, the continuous enhancement of pathogen resistance makes the prevention and control effect of chemical agents decline, the prevention and control cost continues to rise, and it also may destroy the beneficial microbial community in the soil and affect the ecological balance of tobacco field. Although the prevention and control effect can be improved by reasonable rotation, breeding of disease-resistant varieties and other measures, the resistance of some disease-resistant varieties is lost due to the variation of pathogen; the rotation measure is also often limited by planting structure and cost, and it is difficult to be fully and continuously implemented. Therefore, it is urgent to develop more efficient, environmentally friendly and sustainable products for the prevention and control of tobacco black shank.

[0003] Biological pesticides have become the main means of plant disease and pest control and the promotion of green agriculture due to their environmental friendliness and the difficulty of pathogen resistance. Among them, Trichoderma spp. as one of the important members of microbial pesticide products, plays an important role in the prevention and control of various plant diseases. Trichoderma cerinum is a member of the genus Trichoderma. The biocontrol mechanism of Trichoderma spp. includes the following aspects: (1) competition: competing for space and nutrients required for survival of pathogenic bacteria, inhibiting the colonization and expansion of pathogenic bacteria in the rhizosphere environment of tobacco plants; (2) antagonism: producing various antibiotics, hydrolytic enzymes and other secondary metabolites, destroying the cell wall structure of pathogenic bacteria and inhibiting their growth and development; (3) induced resistance: activating the plant's own defense system, enhancing the plant's resistance to pathogenic bacteria and disease resistance.

[0004] Microbial pesticides using Trichoderma spp. as the main active ingredient have been widely used in agricultural production. However, there are few reports on the use of Trichoderma cerinum to control tobacco black shank. SUMMARY

[0005] The application provides a trichoderma cerinum strain and application of the trichoderma cerinum strain in prevention and treatment of tobacco black shank.

[0006] The technical scheme adopted by the application is as follows:

[0007] The first aspect is to provide a trichoderma cerinum strain, the strain is named trichoderma cerinum TR1186A (Trichoderma cerinum TR1186A), the preservation unit is China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 21427.

[0008] The second aspect is to provide a preparation with the trichoderma cerinum strain as an active ingredient.

[0009] The third aspect is to provide a preparation method of the trichoderma cerinum preparation, including the following steps:

[0010] Step one, culturing the trichoderma cerinum strain to obtain trichoderma cerinum fermentation liquor, and the spore content is 4*10 8 -5*10 8 cfu / mL;

[0011] Step two, concentrating and drying the trichoderma cerinum fermentation liquor to obtain trichoderma cerinum mother drug with a spore content of 20*10 8 -25*10 8 cfu / gram;

[0012] Step three, mixing the trichoderma cerinum mother drug with fillers and auxiliaries in proportion to obtain trichoderma cerinum preparation with a spore content of greater than or equal to 2*10 8 cfu / gram.

[0013] Further, the trichoderma cerinum mother drug further includes dry fillers, and the dry fillers include 5-25% of dextrin and 1-5% of glycerol in the total mass of the trichoderma cerinum mother drug.

[0014] Further, the trichoderma cerinum preparation includes 8-10wt% of trichoderma cerinum mother drug, 10-20wt% of water-soluble starch, 0.1-0.5wt% of xanthan gum, 0.1-0.5wt% of sodium dodecyl sulfate, and the rest is bentonite.

[0015] The fourth aspect is to provide application of the trichoderma cerinum strain or the preparation in prevention and treatment of plant pathogenic bacteria; the plant pathogenic bacteria is tobacco black shank bacteria.

[0016] The fifth aspect is to provide application of the trichoderma cerinum strain or the preparation in prevention and treatment of symptoms caused by plant pathogenic bacteria; the plant pathogenic bacteria is tobacco black shank bacteria.

[0017] The sixth aspect is to provide application of the trichoderma cerinum strain or the preparation in prevention and treatment of tobacco black shank.

[0018] Compared with the prior art, the above technical scheme has the following technical effects:

[0019] The trichoderma cerinum provided by the present application is an effective biocontrol strain, which can antagonize the phytophthora parasitica var. nicotianae. The test results of plate confrontation test, indoor efficacy test, indoor safety test, and field efficacy test prove that the strain can be developed into an environmentally friendly, safe, and effective biological pesticide, and has a good application prospect in the biological prevention and control of tobacco black shank. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 It is a plate photo of trichoderma cerinum (colony morphology culture conditions: 28℃ full light culture for 7 days).

[0021] Figure 2 It is a microscope photo of trichoderma cerinum spores.

[0022] Figure 3 It is a microscope photo of trichoderma cerinum conidium. DETAILED DESCRIPTION

[0023] The present application will be further described below in combination with specific examples, but not as a limitation of the present application. It should be noted that the examples in the present application and the features in the examples can be combined with each other without conflict.

[0024] Example 1

[0025] The present application provides a trichoderma cerinum strain, the strain name is trichoderma cerinum TR1186A (Trichoderma cerinum TR1186A), the preservation unit is China General Microbiological Culture Collection Center, the preservation address is No. 3, Beichen West Road, Chaoyang District, Beijing, the preservation number is CGMCC No. 21427, and the preservation date is January 13, 2021.

[0026] The isolation, purification, preservation, and identification of the above strain are as follows:

[0027] 1g of soil sample collected from the rhizosphere of magnolia denudata in Lijiang City, Yunnan Province was weighed in 9ml of sterile water, mixed uniformly on a vortex shaker, and then gradient diluted to 10 -5 1ml was taken and uniformly coated on the surface of the Bengal red culture medium, and after completion, it was placed and cultured at 25℃ for 5-7 days. According to the morphology, color, and size of the colonies screened on the plate, the colonies were picked and transferred to PDA culture medium for purification culture.

[0028] The strain of Trichoderma gamsii was stored in -80°C refrigerator (spores + 20% glycerol) for long-term and in 4°C refrigerator for short-term.

[0029] The biological characteristics of the strain are as follows: the mycelium of the strain spreads over the edge of PDA plate after 3-4 days of culture at 28°C, with a diameter of 9 cm. The initial mycelium is white and sparse, in the form of villi or felt, and then forms a green sporulation zone, with a gray-white edge. The reverse side of the initial PDA plate is white, and the reverse side of the old mycelium is gray-orange after growth. Microscopic observation of the mycelium grown on PDA plate for 2-3 days shows that the conidiophores are short and branched, attached to undifferentiated aerial mycelium, and the conidiogenous structures are also diffuse. The conidia are round or oval, with an average size of 2.3-2.8 um, and are colorless to light green as single, and dark green in groups. The phialides are generally ampoule-shaped, with a size of 3.8-7.6 x 3.0-4.0 um. The conidia are arranged in a regular or irregular pattern on the side of the conidiophores or conidiophore branches, with 3-5 conidia arranged in a false vortex pattern on the partial branches of the conidiophores. The morphological characteristics are consistent with those of Trichoderma gamsii.

[0030] Molecular biological identification of the strain was carried out as follows:

[0031] Fungal universal primers (ITS4: 5'-TCCTCCGCTTATTGATATGC-3', ITS5: 5'-GGAAGGTAAAAGTCAAGG-3', Ef728: 5'-CATCGAGAAGTTCGAGAAGG-3', Tef1aR: 5'-GCCATCCTTGGGAGATACCAGC-3')

[0032] The PCR reaction system was established with a volume of 25 (μl):

[0033] 2x PCR mix 12.5 μl

[0034] 10 mmol F 1.25 μl

[0035] 10 mmol R 1.25 μl

[0036] DNA template 1 μl

[0037] ddH2O 9 μl

[0038] PCR reaction conditions: 95℃ pre-denaturation 2 min; 94℃ denaturation 30 s; 58℃ annealing 30 s, 72℃ extension 1 min for 30 cycles; 72℃ extension 5 min, then stop the reaction. Take 5-10 μl of the amplified product sample solution, add 1-2 μl of 6x loading Dye, mix uniformly, and then detect after electrophoresis on 1.2% agarose gel. Finally, place in a gel imaging system, take a photo by software, and observe the PCR product amplification.

[0039] PCR product sequence sequencing: send the PCR product sample with no difference to Shanghai Shenguo Biotechnology Co., Ltd. for sequencing.

[0040] Sequence alignment and results: the sequencing results are compared for homology in GenBank by using BLAST software, and it is determined that the strain is Trichoderma cerinum.

[0041] Example 2

[0042] The present embodiment provides a preparation method of Trichoderma cerinum preparation, comprising the following steps:

[0043] Step one, first, the laboratory purified Trichoderma cerinum TR1186A is inoculated in a seed flask using PDA medium; then, the laboratory liquid culture (the liquid PD medium is obtained by boiling 200 g of peeled and chopped potatoes in water for 20 minutes, filtering to obtain the filtrate, adding 20 g of glucose, and then adding deionized water to make up to 1000 ml), the culture temperature is 28-30℃, the culture time is 24 h, the inoculation amount is 5-10%, the shaking flask loading coefficient is 200 / 1000 ml, and the rotation speed is 200-300 rpm / min, to obtain the first-stage seed liquid; finally, 1000 liter fermentation tube mass liquid culture (the liquid culture medium is composed of 25 g of soybean cake powder, 15 g of glucose, 1-2 g of potassium dihydrogen phosphate, 0.5-1 g of magnesium sulfate, 0.01-0.05 g of ferrous sulfate, and 0.1 mg of vitamin B1, and then deionized water is added to make up to 1000 ml, and the pH is adjusted to 5.5-6.5). The 1000 liter fermentation tank loading coefficient is 600 / 1000 liters, the inoculation amount is 5-10% of the loading coefficient, the culture temperature is 28-30℃, the sterile air is pressurized in the fermentation tank, the pressure is 0.4-0.5 kg, the air flow is 1500-1800 L / H, the fermentation temperature is 28-30℃, the rotation speed is 120-150 rpm / min, and the culture time is 6 days to obtain the Trichoderma cerinum fermentation liquid.

[0044] Step two, the above Trichoderma cerinum fermentation liquid is concentrated by a centrifugal machine and dried by a spray tower to obtain spores with a quantity of 20×10 8 -25×10 8cfu / g of Trichoderma cerinum mother liquor; the Trichoderma cerinum mother liquor further comprises a dry filler, the dry filler comprising 5-25% of dextrin and 1-5% of glycerol based on the total mass of the Trichoderma cerinum mother liquor; preferably, 8% of dextrin, 2% of glycerol are included;

[0045] Step three, the Trichoderma cerinum mother liquor is mixed with the filler and the adjuvant in proportion, and the spore amount is greater than or equal to 2 x 10 8 cfu / g of Trichoderma cerinum preparation.

[0046] The prepared Trichoderma cerinum preparation comprises 8-10wt% of Trichoderma cerinum mother liquor, 10-20wt% of water-soluble starch, 0.1-0.5wt% of xanthan gum, 0.1-0.5wt% of sodium dodecyl sulfate, and the rest is bentonite.

[0047] Preferably, the Trichoderma cerinum preparation comprises 8wt% of Trichoderma cerinum mother liquor, 77wt% of bentonite, 14wt% of water-soluble starch, 0.5wt% of xanthan gum, and 0.5wt% of sodium dodecyl sulfate.

[0048] Example 3 Plate confrontation test of Trichoderma cerinum TR1186A against the pathogen Phytophthora parasitica var. nicotianae of tobacco black shank disease

[0049] Test pathogen: Phytophthora parasitica var. nicotianae

[0050] Biocontrol agent: Trichoderma cerinum Tr1186A

[0051] Plate confrontation test: a bacterial cake (5mm in diameter) is punched from the edge of a 5-day activated PDA plate of the pathogen Phytophthora parasitica var. nicotianae and placed 1cm from the edge of a PDA plate; at the same time, a bacterial cake (5mm in diameter) is punched from the edge of a 5-day activated PDA plate of Trichoderma cerinum TR1186A and placed 1cm from the edge of the PDA plate on the opposite side; the line connecting the centers of the pathogen and the Trichoderma cerinum passes through the PDA plate in a straight line, and the plates are incubated at 28°C; each treatment has 3 replicates, and the blank control is inoculated with the pathogen alone. After 5 days of incubation, the pathogen colony radius is measured by the cross method, and the inhibition rate is calculated.

[0052] Inhibition rate (%) = (control colony radius - treatment colony radius) / control colony radius * 100%

[0053] Result observation: When the waxwood Trichoderma TR1186A and the pathogen of tobacco black shank Phytophthora parasitica were in confrontation culture on PDA medium plate, the former grew very fast in the early stage on PDA plate, and could contact the mycelium of Phytophthora parasitica after 48h. With the passage of time, it could completely cover the colony of tobacco black shank after 120h. Microscopic observation showed that the mycelium of waxwood Trichoderma completely parasitized on the mycelium of tobacco black shank and degraded the mycelium. The inhibition rate of the plate was more than 80% calculated by measuring the radius, which showed that the strain had good biocontrol potential

[0054] Example 4 Indoor efficacy report of preventing tobacco black shank

[0055] Experimental purpose: To test the control effect of waxwood Trichoderma on tobacco black shank, and to determine the dosage and use method of the agent, so as to provide basis for field test.

[0056] 1. Test agent

[0057] Test agent, 25 billion spores / g waxwood Trichoderma mother drug, Shanghai Wanlihua Biological Technology Co., Ltd.;

[0058] Control agent I, 100 billion spores / g Bacillus subtilis wettable powder, Rushan Hanwei Biological Technology Co., Ltd.;

[0059] Control agent II, 98% emamide, Jiangsu Huifeng Biological Agricultural Co., Ltd.;

[0060] 2. Test method: plate method

[0061] 3. Evaluation method: to evaluate the biological activity of fungicides by virulence baseline and EC 50 , EC 90

[0062] 4. The test results are shown in Table 1 below

[0063] Table 1

[0064]

[0065] ​It can be seen that Trichoderma has good antibacterial activity on tobacco black shank disease. The test agent Trichoderma uses 0.0039 billion spores / gram, the control agent Bacillus subtilis uses 0.0548 billion spores / gram, and the control agent dimethomorph uses 0.9797 mg / L, all of which have 50% effect on preventing and treating tobacco black shank disease; the test agent Trichoderma uses 0.3339 billion spores / gram, the control agent Bacillus subtilis uses 0.6613 billion spores / gram, and the control agent dimethomorph uses 18.3055 mg / L, all of which have 90% effect on preventing and treating tobacco black shank disease. Therefore, the test agent Trichoderma has better effect on preventing and treating tobacco black shank disease than the control agent Bacillus subtilis. Combined with the same effect of the chemical control agent dimethomorph, the test agent Trichoderma has excellent effect on preventing and treating tobacco black shank disease, and the next step can be field efficacy test.

[0066] Example 5: 200 million spores / gram Trichoderma wettability powder indoor safety test on tobacco

[0067] 1. Test crop: tobacco, variety is Daqingjin, Honghuadajinyuan, Qianjinta

[0068] 2. Test agent: 200 million spores / gram Trichoderma wettability powder

[0069] 3. Application method: potting stem and leaf spraying method

[0070] 4. Evaluation method: visual observation and growth rate method

[0071] 5. The test results are shown in Table 2 below

[0072] Table 2

[0073]

[0074]

[0075] It can be seen that under indoor conditions, 200 million spores / gram Trichoderma wettability powder at a dosage of 300-1200 grams / acre is sprayed to treat 3-4 leaf stage Daqingjin, Honghuadajinqin, Honghuadajinyuan and Qianjinta, and the pesticide is applied every 7 days for 3 times. The dosage of 600 grams / acre and 1200 grams / acre has a growth promoting effect on the three varieties of tobacco, and the dosage of 300 grams / acre has a normal growth effect on the three varieties of tobacco.

[0076] Example 6: Field efficacy test of 200 million spores / gram Trichoderma wettability powder on tobacco black shank disease

[0077] 1. Test crop: tobacco, variety, Yunyan 87, tobacco grows well and is in the rosette stage.

[0078] 2. Test agent:

[0079] Test agent, 200 million spores / g Trichoderma virens WP, Shanghai Wanlihua Biotechnology Co., Ltd.

[0080] Control agent, 100 billion spores / g Bacillus subtilis WP, Cangmei, Deqiang Biotechnology Co., Ltd.

[0081] 3. The method of application and the amount of water (seed dressing or soil dressing): stem base spraying method, the amount of water per hectare is 1000 liters.

[0082] 4. The test results are shown in Table 3 below

[0083] Table 3

[0084]

[0085] Note: Different large and small letters represent the difference in significance level of 0.01 and 0.05.

[0086] It can be seen that the control effects of the test agent 200 million spores / g Trichoderma virens WP 3000 g / ha, 3750 g / ha and 4500 g / ha are 67.4%, 72.1% and 75.0% respectively, and the control effect of the control agent 1000 billion spores / g Bacillus subtilis WP 225 g / ha is 68.2%. Among them, the control effect of the test agent 4500 g / ha is the best. It is significantly higher than the control effects of the test agent 3000 g / ha, the control agent 1000 billion spores / g Bacillus subtilis WP 225 g / ha, and the difference reaches 5% significant level. The difference in control effect between other treatments is not significant. Through field observation, under the test dose, the tobacco in each treatment area has no drug damage test, and the agent is not found to have harmful effects on other non-target organisms.

[0087] In summary: the Trichoderma virens provided by the present application is an effective biocontrol strain, which can antagonize the strain of tobacco black shank. And through plate confrontation test, indoor efficacy test, indoor safety test, field efficacy test and other aspects, the good pesticide potential and safety of the strain are confirmed.

[0088] The above only describes the preferred embodiments of the present application, and does not limit the implementation and protection scope of the present application. For those skilled in the art, it should be realized that any equivalent replacement and obvious changes made according to the content of the present application should be included in the protection scope of the present application.

Claims

1. A strain of Trichoderma ceriferm, characterized in that, The strain is named as Trichoderma cerinum TR1186A (Trichoderma cerinum TR1186A), preserved in China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 21427.

2. A formulation characterized in that, The Trichoderma cerinum according to claim 1.

3. A method of preparing a Trichoderma keratinophilic fungal preparation, characterized by, The method comprises the following steps: Step one, culture the Trichoderma ceratii of claim 1 to obtain Trichoderma ceratii fermentation liquor, the spore amount is 4×10 8 -5×10 8 cfu / mL; Step two, the Trichoderma ceratii fermentation broth is concentrated and dried to obtain spores with a quantity of 20 x 10 8 -25 x 10 8 cfu / gram of Trichoderma ceratii mother liquor; Step three, the Trichoderma mother drug is mixed with fillers and adjuvants in proportion to obtain a Trichoderma preparation with a spore content of ≥2×10 8 cfu / gram of Trichoderma harzianum.

4. The production method according to claim 3, characterized by, The Trichoderma cerinum mother drug further comprises a dry filler, wherein the dry filler comprises 5-25% of dextrin and 1-5% of glycerol based on the total mass of the Trichoderma cerinum mother drug.

5. The preparation method according to claim 3, characterized in that, The Trichoderma cerinum preparation comprises 8-10 wt% of the Trichoderma cerinum mother drug, 10-20 wt% of water-soluble starch, 0.1-0.5 wt% of xanthan gum, 0.1-0.5 wt% of sodium dodecyl sulfate, and the rest is bentonite.

6. The Trichoderma cerinum according to claim 1 or the preparation according to claim 2 is applied in the prevention and treatment of plant pathogenic bacteria; the plant pathogenic bacteria is Phytophthora parasitica.

7. The Trichoderma cerinum according to claim 1 or the preparation according to claim 2 is applied in the prevention and treatment of symptoms caused by plant pathogenic bacteria; the plant pathogenic bacteria is Phytophthora parasitica.

8. The Trichoderma cerinum according to claim 1 or the preparation according to claim 2 is applied in the prevention and treatment of tobacco black shank.

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