Application of tobacco stem or tobacco extracting solution in culturing trichoderma harzianum

Abstract

The invention provides a method for preparing a trichoderma harzianum microbial inoculum by using waste tobacco stems or a tobacco extracting solution, and belongs to the technical field of microbial inoculum development. According to the application of the tobacco stems or the tobacco extracting solution in culturing the trichoderma harzianum, through contrast experiments, the chlamydospore production amount of the tobacco stem extracting solution subjected to 140-DEG C near-critical water treatment is superior to that of the tobacco stem extracting solution subjected to contrast and other treatment, and then the waste tobacco extracting solution subjected to 140-DEG C near-critical water treatment is diluted by ten times, so that the chlamydospore production amount of the tobacco stem extracting solution subjected to 140-DEG C near-critical water treatment is reduced; however, the chlamydospore yield of tobacco stems or tobacco extract liquid treated by near-critical water at 180 DEG C and water at 100 DEG C is poor. In a word, although the total amount of spores (the sum of chlamydospores and conidia) of the trichoderma fermented by the tobacco stems or the tobacco extract liquid is less than that of the traditional PDB fermentation, the trichoderma fermented by the tobacco stems or the tobacco extract liquid is more suitable for producing the chlamydospores, tobacco wastes can be fully utilized, and the existing problems in the industry are solved.

Description

Technical Field

[0001] The invention relates to the technical field of bacterial agent development, and in particular to an application of tobacco stems or tobacco extract in culturing Trichoderma harzianum. Background Art

[0002] Tobacco is one of the important cash crops in my country. During its planting and processing, a large amount of waste such as tobacco stalks, unsuitable tobacco leaves and tobacco stems is generated. At present, my country's tobacco waste is restricted by the use method and cost, and there are generally a lot of idle or inefficient use problems, making it difficult to achieve the resource recycling of tobacco waste.

[0003] Trichoderma harzianum is considered to be the most promising biological control factor. Trichoderma harzianum not only has an antagonistic effect on a variety of pathogens, but also promotes the growth of plants. Especially in the process of tobacco growth, it can significantly promote the vigorous growth of tobacco plants, strengthen the root system, and increase tobacco yield. The technical problem to be solved by the present invention is whether the liquid used to treat waste tobacco can be used to cultivate Trichoderma harzianum, thereby realizing resource recycling. Summary of the invention

[0004] The purpose of the present invention is to provide a method for preparing a Trichoderma harzianum agent, which utilizes tobacco stems or tobacco extract to ferment Trichoderma harzianum, thereby realizing resource recycling and laying a foundation for expanding production in the future.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The invention provides an application of tobacco stems or tobacco extract in culturing Trichoderma harzianum.

[0007] Preferably, the tobacco stem or tobacco extract comprises a tobacco stem or tobacco aqueous extract obtained by treating the tobacco stem or tobacco with near-critical water.

[0008] Preferably, the method of near-critical water treatment is to mix tobacco stems or tobacco with water in a weight-to-volume ratio of 1:3-5 g / mL, react for 12-17 minutes at 120-150°C and a pressure of 0.2-0.8 MPa, and then separate the solid and liquid to obtain a water extract after near-critical water treatment of tobacco stems or tobacco.

[0009] The present invention also provides a method for using the tobacco stem or tobacco extract in culturing Trichoderma harzianum, comprising the following steps:

[0010] (1) obtaining a spore suspension of Trichoderma harzianum after culturing;

[0011] (2) inoculating the spore suspension into a fermentation matrix containing tobacco stems or tobacco extract for fermentation to obtain a Trichoderma harzianum agent.

[0012] Preferably, the culture in step (1) is to culture the activated Trichoderma harzianum in PDB medium for 3 to 5 days, and the concentration of the spore suspension is 2 to 4×10 7 cfu / mL.

[0013] Preferably, the tobacco stem or tobacco extract in step (2) is a liquid obtained by diluting the tobacco stem or tobacco aqueous extract after near-critical water treatment by 1 to 10 times.

[0014] Preferably, the spore suspension in step (2) is inoculated at 1-3% of the volume of the tobacco stems or tobacco extract, the pH of the tobacco stems or tobacco extract is 3-5, and fermented at 150-200 r / min and 25-30° C. for 3-7 days.

[0015] The invention also provides a Trichoderma harzianum agent obtained by the application method.

[0016] The invention also provides an application of the Trichoderma harzianum agent in preventing and controlling tobacco pathogenic bacteria.

[0017] The present invention uses tobacco stems or tobacco extracts to prepare Trichoderma harzianum agents. Through comparative experiments, the yield of chlamydospores in tobacco stem extracts treated with 140°C near-critical water is better than that of controls and other treatments, followed by waste tobacco extracts treated with 140°C near-critical water diluted ten times, while the yield of chlamydospores in tobacco stem or tobacco extracts treated with 180°C near-critical water and 100°C water is poor. In short, although the total amount of spores (the sum of chlamydospores and conidia) of tobacco stem or tobacco extract fermentation with Trichoderma is not as good as traditional PDB fermentation, it is more suitable for producing chlamydospores, and can make full use of tobacco waste to solve existing problems in the industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is the number of chlamydospores in each treatment in Comparative Example 1;

[0019] Figure 2 It is the total amount of spores in each treatment in Comparative Example 1. DETAILED DESCRIPTION

[0020] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0021] Example 1

[0022] A method for preparing a Trichoderma harzianum agent comprises the following steps:

[0023] S1: After activation on the plate, Trichoderma harzianum was inoculated into PDB medium and cultured in the dark at 28°C for 4 days to obtain a spore suspension with a concentration of 3×10 7 cfu / mL.

[0024] S2: The preparation process of the liquid after the tobacco stems are treated with near-critical water at 140°C is as follows: tobacco stems and water are placed in a high-temperature and high-pressure reactor at a weight ratio of 1:4, reacted at 140°C for 14 minutes, the reaction pressure is 0.4 MPa, and the solid-liquid separation is carried out after natural cooling. The liquid is the liquid after the tobacco stems are treated with near-critical water at 140°C. The preparation method of the liquid after the tobacco is treated with near-critical water at 160°C and 140°C is the same as that of the tobacco stems.

[0025] S3: The liquid obtained by the treatment in S2 is used as a fermentation substrate, and the spore suspension is inoculated at 1% of the volume of the fermentation substrate, and fermented in a shaking incubator at 28° C. and a rotation speed of 180 r / min for 5 days to obtain a Trichoderma harzianum agent.

[0026] Example 2

[0027] The liquid treated with near-critical water prepared in steps S1 and S2 of Example 2 was diluted 10 times as a fermentation substrate, and the spore suspension was inoculated at 1% of the volume of the fermentation substrate, and fermented in a shaking incubator at 28° C. and a rotation speed of 180 r / min for 5 days to obtain a Trichoderma harzianum agent.

[0028] Comparative Example 1

[0029] Different culture medium treatment groups were set up according to Table 1. The volume of each treatment group was 100 mL. Liquid fermentation was carried out in a conical flask. The laboratory Trichoderma harzianum strain (patent preservation number CGMCC23294) was used as the test Trichoderma. The inoculation amount was 1% of the volume of each culture medium. Under the initial pH 5.0, 25°C, 200r / min conditions, dark culture was carried out for 14 days, and the number of chlamydospores and conidia in each treatment was detected by a hemacytometer.

[0030] Among them, the tobacco stem extract of Example 1 is group T2, the tobacco extract of Example 1 is group T11, the tobacco stem extract diluted 10 times of Example 2 is group T6, and the tobacco extract diluted 10 times of Example 2 is group T14.

[0031] Table 1 Culture medium formula for different treatment groups

[0032]

[0033]

[0034] Each treatment was repeated 3 times, and the results are shown in Table 2 and Figures 1-2 shown.

[0035] Table 2 Number of conidia and chlamydospores in each treatment (×10 8 cfu / ml)

[0036]

[0037]

[0038] Although the fermentation effect of tobacco stem extract is not as good as traditional PDB fermentation, it can make full use of tobacco waste and solve existing problems in the industry.

[0039] Experimental example

[0040] Field application effect

[0041] Field experiments were conducted at the Xuchang campus of Henan Agricultural University to study the effect of the use of Trichoderma harzianum CGMCC23294 strain fermented with waste tobacco or tobacco stem extract in tobacco production. The experiment adopted a random group design, and Trichoderma harzianum agents were added on the basis of conventional fertilization, and 5 treatments were set: CK0 was not applied with agents, CK1 was a Trichoderma agent cultured with PDB, A1 was a Trichoderma agent fermented with the original solution of the extract of tobacco stems treated with near-critical water at 140°C in Example 1, A2 was a Trichoderma agent fermented with the original solution of the extract of tobacco stems treated with near-critical water at 160°C obtained according to the method of Example 1, A3 was a Trichoderma agent fermented with a ten-fold dilution of the waste tobacco extract treated with near-critical water at 140°C in Example 2, and A4 was a Trichoderma agent fermented with a ten-fold dilution of the waste tobacco extract treated with near-critical water at 160°C in Example 2. Each treatment was repeated 3 times, with 100 tobacco plants in each plot, 1500 tobacco plants in total for 5 treatments, 0.55m plant spacing, 1.15m row spacing. Instructions for using the microbial agent: irrigate the roots during transplanting, 2 liters / mu.

[0042] Effects of Trichoderma harzianum inoculations with different fermentation substrates on the control of tobacco black shank in the field

[0043] As shown in Table 3, the incidence and disease index of tobacco treated with Trichoderma harzianum inoculants with different fermentation substrates were significantly reduced compared with the CK0 treatment without the use of inoculants. The relative control effect of Trichoderma harzianum inoculants fermented with traditional PDB medium on tobacco black shank was significantly lower than that of the extracts of discarded tobacco stems and discarded tobacco near-critical water as fermentation substrates. The relative control effects of each treatment in the experimental group were A3>A1>A4>A2>CK1. Although the total amount of Trichoderma harzianum spores in the CK1 treatment was higher than that in other treatments, its relative control effect on black shank was lower. This may be because the tobacco flavonoids and alkaloids contained in the near-critical water extracts of discarded tobacco or tobacco stems improved the control effect of the inoculants on tobacco black shank. In the tobacco or discarded tobacco stem treatment group, the relative control effects of A3 and A1 treatments were significantly higher than those of A4 and A2 treatments, which were the optimal treatments.

[0044] Table 3 Incidence rate and relative control effect of black shank disease in tobacco plants treated with different methods

[0045]

[0046] Effects of Trichoderma harzianum inoculants with different fermentation substrates on economic traits of tobacco

[0047] As shown in Table 4, the yield per hectare, output value, average price and proportion of top-grade tobacco in the tobacco treated with Trichoderma harzianum were all higher than those in the conventional fertilization group. The effects of different fermentation substrates of Trichoderma harzianum on the economic traits of tobacco were different. In terms of yield, A3 and CK1 performed better, followed by A1 and A2; in terms of average price and proportion of top-grade tobacco, A2 and A1 performed better, followed by CK1; in terms of output value, A1 performed better, followed by CK1 and A3. Based on the above analysis, the economic traits of A1 treatment were better than CK1.

[0048] Table 4 Effects of different treatments on economic traits of flue-cured tobacco

[0049]

[0050] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of tobacco stems or tobacco extract in culturing Trichoderma harzianum.

2. The use according to claim 1, characterized in that The tobacco stem or tobacco extract comprises a tobacco stem or tobacco aqueous extract obtained by treating the tobacco stem or tobacco with near-critical water.

3. The use according to claim 2, characterized in that The method for near-critical water treatment is to mix tobacco stems or tobacco with water in a weight ratio of 1:3 to 5, react for 12 to 17 minutes at 120 to 180° C. and a pressure of 0.2 to 0.8 MPa, and then separate the solid and liquid to obtain a water extract after near-critical water treatment of tobacco stems or tobacco.

4. A method for using the tobacco stem or tobacco extract according to any one of claims 1 to 3 in culturing Trichoderma harzianum, characterized in that: The steps include: (1) obtaining a spore suspension of Trichoderma harzianum after culturing; (2) inoculating the spore suspension into a fermentation matrix containing tobacco stems or tobacco extract for fermentation to obtain a Trichoderma harzianum agent.

5. The application method according to claim 4, characterized in that: The culture in step (1) is to culture the activated Trichoderma harzianum in PDB medium for 3 to 5 days, and the concentration of the spore suspension is 2 to 4×10 7 cfu / mL.

6. The application method according to claim 5, characterized in that: The tobacco stem extract in step (2) is a liquid obtained by diluting tobacco stems or tobacco water extract after near-critical water treatment by 1 to 10 times.

7. The application method according to claim 6, characterized in that: The spore suspension in step (2) is inoculated at 1-3% of the volume of the tobacco stems or tobacco extract, the pH of the tobacco stems or tobacco extract is 3-5, and fermented at 150-200 r / min and 25-30° C. for 3-7 days.

8. A Trichoderma harzianum agent obtained by the application method according to any one of claims 4 to 7.

9. Use of the Trichoderma harzianum agent according to claim 8 in preventing and controlling tobacco pathogens.

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