Method for promoting growth or resisting diseases of oilseed rapes

Abstract

The invention discloses a method for promoting growth or resisting diseases of oilseed rape. The research finds that any one or a combination of more of bacillus subtilis B.subtilis 168, trichoderma harzianum T.harzianum 25852 and trichoderma harzianum T.harzianum 52623 can effectively promote the growth of rape seedlings, and the growth mainly shows that the plant stem diameter, the plant height and the increase of the biomass of the overground part and the underground part. Wherein the trichoderma harzianum T.harzianum 25852, and the combination of the bacillus subtilis B.subtillis 168 and the trichoderma harzianum T.harzianum 25852 also have a relatively good sclerotinia rot prevention and treatment effect. The microorganism provided by the invention has the characteristics of high efficiency, no pollution, difficult generation of drug resistance and the like in the aspect of oilseed rape production, has great application potential, and has important significance for promoting the improvement of oil productivity and green low-carbon efficient production in China.

Description

Technical Field

[0001] The embodiments of the present invention relate to the field of agricultural technology, and in particular to a method for promoting growth or resisting disease of rapeseed. Background Art

[0002] Rapeseed is the main oil crop with the greatest development potential in my country. Rapeseed oil is the largest source of vegetable oil in my country. Its high and stable yield is crucial to ensuring the security of the country's edible oil supply. At present, applying sufficient basal fertilizer before planting rapeseed is an important measure to promote the growth of rapeseed. In different growth stages of rapeseed, topdressing fertilizers can be applied to meet the growth needs of crops to the greatest extent, so as to achieve the goal of stable and high yield of rapeseed. Rapeseed requires a large amount of fertilizer. Long-term and large-scale use of fertilizers will lead to the destruction of soil structure, which not only damages the ecological environment, but also reduces economic benefits.

[0003] At present, the main method for controlling sclerotinia rot in rapeseed is to use pesticides during the flowering period of rapeseed, such as spraying carbendazim, sclerotinia chlorantraniliprole, etc. The use of pesticides is prone to drug resistance. In addition, the bacteria in seeds and the sclerotia remaining in the soil are the main sources of sclerotinia rot infection in the next year. The effect of pesticides on pathogens in the soil is limited, so the control effect of rapeseed diseases and pests is not ideal.

[0004] Existing studies have found that bacteria, fungi and other microorganisms play an important role in soil ecosystems and plant growth and development. They promote crop growth and development by increasing nutrient utilization, improving soil conditions and antagonizing crop pathogens, thereby increasing crop yields.

[0005] However, there are no highly functional microbial products currently available on the market for use in rapeseed production. In view of this, providing a beneficial microorganism that can effectively promote rapeseed growth and prevent and control rapeseed sclerotinia disease is a technical problem that needs to be solved urgently. Summary of the invention

[0006] To this end, an embodiment of the present invention provides a method for promoting the growth or resisting diseases of rapeseed. The inventors have conducted extensive research on existing microorganisms, screened out microorganisms that can promote the growth of rapeseed and have a good inhibitory effect on Sclerotinia sclerotiorum, and developed them into microbial agents or fertilizers for application in rapeseed production, which has significant social and ecological benefits and is of great significance to promoting the increase of my country's oil production capacity and green, low-carbon and efficient production.

[0007] In order to achieve the above purpose, the embodiment of the present invention provides the following technical solutions:

[0008] According to a first aspect of an embodiment of the present invention, the present invention provides use of at least one of Bacillus subtilis 168, Trichoderma harzianum 25852 and Trichoderma harzianum 52623 in rapeseed production.

[0009] Furthermore, the application includes promoting rapeseed growth and preventing and controlling rapeseed sclerotinia disease.

[0010] According to a second aspect of an embodiment of the present invention, the present invention provides a microbial agent for rapeseed production, wherein the microbial agent includes at least one of Bacillus subtilis 168, Trichoderma harzianum 25852 and Trichoderma harzianum 52623.

[0011] Furthermore, the microbial agent includes at least one of a bacterial liquid of Bacillus subtilis 168, a conidia liquid of Trichoderma harzianum 25852, and a conidia liquid of Trichoderma harzianum 52623.

[0012] According to a third aspect of an embodiment of the present invention, the present invention provides a microbial fertilizer for rapeseed production, wherein the microbial fertilizer comprises the microbial agent as described above.

[0013] According to a fourth aspect of an embodiment of the present invention, the present invention provides a method for producing rapeseed, using the microbial agent as described above, or the microbial fertilizer as described above to treat rapeseed.

[0014] Furthermore, the rapeseed is in the seedling stage.

[0015] The embodiments of the present invention have the following advantages:

[0016] The present invention has found that any one or more combinations of Bacillus subtilis 168, Trichoderma harzianum 25852 and Trichoderma harzianum 52623 can effectively promote the growth of rapeseed seedlings, which is mainly manifested in the increase of plant stem thickness, plant height, aboveground and underground biomass. Among them, Trichoderma harzianum 25852, and the combination of Bacillus subtilis 168 + Trichoderma harzianum 25852, also have a better effect on the prevention and treatment of sclerotinia. The microorganism of the present invention has the characteristics of high efficiency, no pollution, and not easy to produce drug resistance in rapeseed production. At the same time, it is easy to use, has significant benefits, has great application potential, and is of great significance to promoting the improvement of oilseed production capacity and green, low-carbon and efficient production in my country. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the implementation methods of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the implementation methods or the description of the prior art. Obviously, the drawings in the following description are only exemplary, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0018] Figure 1 The invention discloses the growth-promoting effect of Trichoderma harzianum and Bacillus subtilis and their combination on rapeseed seedlings.

[0019] Figure 2 This is a plate confrontation growth experiment between Sclerotinia sclerotiorum 1980 strain and Trichoderma harzianum strains T.harzianum 25852 and T.harzianum52623.

[0020] Figure 3 The rapeseed plants treated with B and AB showed resistance to infection by Sclerotinia sclerotiorum 1980 strain.

[0021] Figure 4 The lesion statistics of rapeseed plants treated with B and AB infected by Sclerotinia sclerotiorum 1980 strain. DETAILED DESCRIPTION

[0022] The following is a description of the implementation of the present invention by specific embodiments. People familiar with the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Obviously, the described embodiments are 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 are within the scope of protection of the present invention.

[0023] Bacillus subtilis 168 was obtained from Guangdong Microbiological Culture Collection Center.

[0024] Trichoderma harzianum 25852 and Trichoderma harzianum 52623 were obtained from Beijing Bio-Bowei Biotechnology Co., Ltd.

[0025] The sclerotinia pathogen Sclerotinia sclerotiorum strain 1980 was provided by the Oil Crops Research Institute, Chinese Academy of Agricultural Sciences.

[0026] The rapeseed variety Double 11 was provided by the Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences.

[0027] Example 1 Growth-promoting effect of Bacillus subtilis, Trichoderma and their combination on rapeseed seedlings

[0028] 1.1 Test materials

[0029] Bacillus subtilis (B.subtilis 168), Trichoderma strains (Trichoderma harzianum 25852, Trichoderma harzianum 52623), rapeseed variety Zhongshuang 11.

[0030] 1.2 Test methods

[0031] (1) Preparation of bacterial solution

[0032] Preparation of Bacillus subtilis culture solution

[0033] The Bacillus subtilis strain was inoculated into LB solid medium and cultured at 37°C. After 12 h, a single colony was picked and transferred to LB liquid medium and cultured at 37°C until OD 600 About 0.8, for spare use.

[0034] Preparation of Trichoderma harzianum conidia solution

[0035] The Trichoderma strain was inoculated into PDA solid medium and cultured at 28°C until complete sporulation. The plate was washed with sterile water to collect the spores to obtain conidia. The concentration of the spore suspension was adjusted to 10 using a hemocytometer under a microscope. 6 CFU / mL, for future use.

[0036] (2) Seedling cultivation and inoculation

[0037] Select plump rapeseed grains, disinfect them with 1% sodium hypochlorite for 15 minutes, then rinse them with sterile double distilled water for 4 times, place the seeds in a sterile culture dish covered with two layers of moist filter paper, put them in a 24°C incubator away from light for germination for 24-48 hours, then sow the rapeseed seeds with white buds into seedling pots, water them regularly every day, and set 4 seedlings in each pot.

[0038] A total of eight treatments were set up in the experiment, namely: sterile culture medium (CK), Bacillus subtilis 168 (A), Trichoderma harzianum 25852 (B), Trichoderma harzianum 52623 (C), Bacillus subtilis 168 + Trichoderma harzianum 25852 (AB), Bacillus subtilis 168 + Trichoderma harzianum 52623 (AC), Trichoderma harzianum 25852 + Trichoderma harzianum 52623 (BC), Bacillus subtilis 168 + Trichoderma harzianum 25852 + Trichoderma harzianum 52623 (ABC). The seedlings were inoculated with roots, and the suspension containing Bacillus subtilis and Trichoderma harzianum conidia was inoculated into 1-week-old rapeseed seedlings alone and in combination (mixed in equal volumes). Each pot was inoculated with 10 ml of the bacterial solution, and inoculated once every other week for a total of 3 times. Each treatment was repeated 4 times, and a control group was set up. The rapeseed seedlings in the control group were inoculated with the same amount of sterilized culture medium. After 30 days of cultivation in the greenhouse, the whole plant was dug out, the stem diameter and plant height of the plant were measured, and the fresh weight and dry weight of the aboveground and underground parts were determined.

[0039] 1.3 Test results

[0040] Inoculation of the above-mentioned microorganisms has a significant growth-promoting effect on rapeseed, mainly manifested in increasing the stem diameter, plant height, and aboveground and underground biomass of rapeseed plants ( Figure 1 , Table 1). Among them, treatments A, B, C, and AC had better growth-promoting effects on rapeseed.

[0041] Table 1 Growth-promoting effects of Trichoderma harzianum, Bacillus subtilis and their combination on rapeseed seedlings

[0042]

[0043] Example 2: Control effect of Bacillus subtilis and Trichoderma and their combination on rapeseed sclerotinia

[0044] 2.1 Test materials

[0045] Bacillus subtilis (B.subtilis 168), Trichoderma strains (Trichoderma harzianum 25852, Trichoderma harzianum 52623), Sclerotinia sclerotiorum pathogen strain 1980, rapeseed variety Zhongshuang 11.

[0046] 2.2 Test methods

[0047] (1) Strain activation

[0048] The Trichoderma strain and the Sclerotinia sclerotiorum strain were inoculated into PDA solid culture medium, and the Bacillus subtilis strain was inoculated into LB solid culture medium for later use.

[0049] (2) Confrontation experiment between Trichoderma strains and Sclerotinia sclerotiorum: Mycelial blocks were punched out from the colony edges of the activated Trichoderma harzianum strain and Sclerotinia sclerotiorum strain 1980 using a 5 mm borer, and inoculated on PDA plates for confrontation culture. Three replicates were set for each treatment, and the plates with only Sclerotinia sclerotiorum mycelial blocks were used as controls and cultured in an incubator at 28°C. The colony growth radius of Trichoderma and Sclerotinia sclerotiorum was observed, and whether the Trichoderma colonies covered the pathogen colonies.

[0050] (3) Detached leaf protection efficacy determination: The experiment was set up with eight treatments, namely: sterile water (CK), Bacillus subtilis 168 (A), Trichoderma harzianum 25852 (B), Trichoderma harzianum 52623 (C), Bacillus subtilis 168 + Trichoderma harzianum 25852 (AB), Bacillus subtilis 168 + Trichoderma harzianum 52623 (AC), Trichoderma harzianum 25852 + Trichoderma harzianum 52623 (BC), Bacillus subtilis 168 + Trichoderma harzianum 25852 + Trichoderma harzianum 52623 (ABC). The preparation of each group of bacterial liquid was the same as in Example 1.

[0051] Pick the rape leaves grown for 30 days, disinfect them with 75% alcohol, and rinse them with sterile water; spray the bacterial solution of each group on the surface of the rape leaves, 3 leaves for each treatment, 3 times, and spray sterile water for the blank control. After 24 hours, inoculate the sclerotinia cake (about 5mm in diameter) in the center of the leaves, evenly place the leaves in a plastic box saturated with double-layer gauze and water, seal them with sealing film, and then place them in a 24℃ incubator (14h / 10h) for cultivation, add water regularly to maintain the humidity in the plastic box, and measure the diameter of the lesions on each treated leaf after the blank control shows obvious disease.

[0052] 2.3 Experimental Results

[0053] The confrontation culture of Trichoderma harzianum and Sclerotinia sclerotiorum revealed that when the two strains were not in contact, the growth of Sclerotinia sclerotiorum 1980 was consistent with the mycelial growth of the control strain, and its growth was not inhibited. After the two strains were in contact for 7 days, the growth of Sclerotinia sclerotiorum 1980 was inhibited and eventually covered by Trichoderma spores. The inhibitory effect of Trichoderma harzianum 25852 on Sclerotinia sclerotiorum 1980 was more obvious, and Sclerotinia sclerotiorum 1980 basically did not produce sclerotia ( Figure 2 ).

[0054] The results of the in vitro leaf protection test are as follows: the average diameter of the lesions of the blank control CK is 13mm, the diameter of the lesions of the leaves treated with A, C, AC, BC, and ABC is not significantly different from that of the CK treatment, the diameter of the lesions of the leaves treated with B is 6.7mm, and the diameter of the lesions of the leaves treated with AB is 6.3mm. The results showed that B and AB can effectively inhibit the spread of lesions. The results showed that Trichoderma harzianum 25852 and the combination of Bacillus subtilis 168 + Trichoderma harzianum 25852 can effectively inhibit the infection of Sclerotinia sclerotiorum on rapeseed leaves, and have a better control effect on rapeseed sclerotinia sclerotiorum ( Figure 3 and Figure 4 ).

[0055] Although the present invention has been described in detail above by general description and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

Claims

1. Use of at least one of Bacillus subtilis 168, Trichoderma harzianum 25852 and Trichoderma harzianum 52623 in rapeseed production.

2. The use according to claim 1, characterized in that: The application includes promoting rapeseed growth and preventing and controlling rapeseed sclerotinia disease.

3. A microbial agent for rapeseed production, characterized in that: The microbial agent includes at least one of Bacillus subtilis 168, Trichoderma harzianum 25852 and Trichoderma harzianum 52623.

4. The microbial agent for rapeseed production according to claim 3, characterized in that: The microbial agent comprises at least one of a bacterial liquid of Bacillus subtilis 168, a conidia liquid of Trichoderma harzianum 25852 and a conidia liquid of Trichoderma harzianum 52623.

5. A microbial fertilizer for rapeseed production, characterized in that: The microbial fertilizer comprises the microbial agent as described in claim 3 or 4.

6. A method for producing rapeseed, characterized in that: The rapeseed is treated using the microbial agent described in claim 3 or 4, or the microbial fertilizer described in claim 5.

7. The rapeseed production method according to claim 6, characterized in that: The rapeseed is in the seedling stage.

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