Microbial agent for inhibiting root rot of beet and application of microbial agent

Abstract

The invention discloses a microbial inoculum for inhibiting beet root rot and application of the microbial inoculum, the microbial inoculum is obtained by fermenting brevibacterium alimentarius, the microbial inoculum has the remarkable characteristics of saline-alkaline tolerance, high viable count and the like, and the yield loss caused by diseases can be reduced by effectively preventing and treating the beet root rot, so that the yield and quality of crops are improved. After the fungicide is used for treatment, in normal soil, the seed germination rate and morbidity are different from those of a control group. Particularly, in disease nursery soil, the germination rate of the seeds can be remarkably increased and the morbidity can be reduced by treating the seeds with the bacterial liquid. The microbial agent provided by the invention not only reduces the investment cost of chemical pesticides, but also can improve the yield and quality of crops, and brings higher economic benefits. In addition, the biological agent is environment-friendly, is beneficial to maintaining the health and stability of a soil ecosystem, and promotes the sustainable development of agriculture.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and particularly relates to a bacterial agent applied in agricultural production, and more particularly to a bacterial agent for inhibiting beet root rot and the technical field of its application. Background Art

[0002] Beet root rot is one of the root diseases with the widest range of damage and the greatest degree of damage in current beet production. So far, there is still a lack of effective chemical agents to completely control it. Due to the inability to predict and the lack of effective agents (Qiao Zhiwen, 2011), at present, agricultural control, biological control, and disease-resistant varieties are mainly relied on to reduce the occurrence degree of the disease. The annual increase in the incidence of root rot seriously threatens the safe supply of beet raw materials. Beet root rot occurs in all major beet-growing countries in the world. In recent years, there have been frequent occurrences of complete crop failures in some planting plots in the Yili sugar area and the Ta'er Basin sugar area due to the occurrence of root rot. The pathogenic populations identified from the isolates of beet root rot samples in Xinjiang are mainly Fusarium, Rhizoctonia, and Pythium, accounting for 68.16%, 15.37%, and 11.02% of the isolates respectively. These pathogenic bacteria are distributed in all major production areas of beets in Xinjiang without obvious regional characteristics (Wang Wenjun, 2011). When root rot occurs, it generally causes a 10% - 40% reduction in root tuber yield, and when the disease is severe, it can reach more than 60%, and even complete crop failure (Xue Jin, 2006). In the prevention and control of root rot, large doses of chemical pesticides are often used for root irrigation in production, which will not only affect the quality of beets, but also cause chemical residues in the soil, and the control effect cannot last. However, the use of biological bacterial agents for prevention and control avoids the occurrence of the above phenomena.

[0003] The invention patent "Application of a Microbial Bacterial Agent in Beet Root Rot" with the application number 202110038529.2 provides the product "Jiufeng" using "Purpureocillium lilacinum". For beets that have been continuously cropped for 4 years, deep autumn plowing and basal fertilization are carried out, which has a certain inhibitory effect on beet root rot and can greatly increase the yield per mu and sugar content of beets. The invention patent with the application number 201510455413.3, Bacillus cereus ( Bacillus cereus ) LHTYBA, whose preservation number in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms is CGMCC No. 7566. The provided Bacillus cereus ( Bacillus cereus ) LHTYBA can colonize in the rhizosphere of crops and has a significant effect on increasing the yield of beet production.

[0004] How to further enrich the relevant microbial inoculants for controlling beet root rot in soils suitable for long-term beet cultivation with a large content of pathogenic bacteria, and provide a green and environmentally friendly control method for the beet planting industry, which can replace some chemical pesticides. This not only reduces the environmental pollution caused by chemical pesticides, but also reduces the potential risks of pesticide residues to human health. Summary of the Invention

[0005] Regarding the problem that there are few reports on the use of relevant inoculants of Brevibacterium linens ( Brachybacterium alimentarium ) in inhibiting beet root rot in the prior art, the present invention provides an inoculant for inhibiting beet root rot and a preparation method thereof. The present invention also provides the application of the inoculant for inhibiting beet root rot in the control of beet root rot. By implementing the technical solution of the present invention, the germination rate of seeds can be significantly increased and the incidence rate can be reduced. It can promote the effective development of the beet planting industry.

[0006] To achieve the above object, the present invention adopts the following technical solutions: The present invention provides an inoculant for inhibiting beet root rot, and the active ingredient of the inoculant is composed of a resuspension obtained by fermenting Brevibacterium linens ( Brachybacterium alimentarium ).

[0007] The optical density OD 600 of the resuspension of the inoculant is 0.02.

[0008] Furthermore, the present invention also provides a preparation method of an inoculant for inhibiting beet root rot, which is specifically prepared by the following steps: (1) Inoculate the strain of Brevibacterium linens ( Brachybacterium alimentarium ) into TSA medium containing 5% NaCl, and incubate at 30 °C for 3 - 15 days for standby; (2) Inoculate the strain of Brevibacterium linens ( Brachybacterium alimentarium ) in step (1) into TSA containing 2% NaCl and culture until the exponential growth phase, and centrifuge to collect cells; (3) Wash the product in step (2) once with 1×PBS, resuspend it in 10 mM MgCl2 solution, and dilute the optical density (OD 600 ) of the cell solution to OD 600 to 0.02.

[0009] Furthermore, the present invention also provides the application of an inoculant for inhibiting beet root rot or the preparation method of the inoculant in any one of the following (1) - (3): (1) An inoculant for controlling beet root rot; (2) An inoculant for reducing the root incidence rate of beets planted in the area where beet root rot occurs; (3)A bacterial agent for increasing the seed germination rate of sugar beets planted in the area affected by sugar beet root rot.

[0010] Furthermore, the present invention also provides a biological organic fertilizer prepared by the bacterial agent for inhibiting sugar beet root rot or the preparation method of the bacterial agent.

[0011] Furthermore, the applicable sugar beet varieties for the above application are variety ST13092 or variety SD13829.

[0012] In the above application, when applying the bacterial agent for inhibiting sugar beet root rot, clear water and the bacterial liquid are used. 20 milliliters of the bacterial liquid is added to each liter of clear water for watering, and then the flowerpot is covered with a plastic film, and watering is carried out every 7 days later.

[0013] Compared with the prior art, the present invention has the following characteristics: (1)The bacterial agent for inhibiting sugar beet root rot provided by the present invention uses a fermentation resuspension of Brevibacterium alimentarium ( Brachybacterium alimentarium ), and the bacterial agent has remarkable characteristics such as salt and alkali tolerance and a high viable bacteria count.

[0014] (2)During the seed germination period when the provided bacterial agent is most sensitive to environmental stress, the microorganisms in the biological bacterial agent can help the seeds germinate better under adverse environmental conditions by improving the stress resistance of plants. By effectively preventing and controlling sugar beet root rot, the yield reduction loss caused by diseases can be reduced, thereby increasing the yield and quality of crops. After treatment with the bacterial agent, for variety ST13092 in normal soil, there is no significant difference in the seed germination rate and incidence rate compared with the control group, but in the diseased nursery soil, treatment with the bacterial liquid can significantly increase the seed germination rate and reduce the incidence rate. For variety SD13829, whether in normal soil or diseased nursery soil, the seed germination rate is significantly increased after treatment with the bacterial agent, and at the same time the incidence rate is significantly reduced.

[0015] (3)Using the bacterial agent provided by the present invention not only reduces the input cost of chemical pesticides, but also can increase the yield and quality of crops, bringing higher economic benefits. In addition, the biological bacterial agent is environmentally friendly, helps to maintain the health and stability of the soil ecosystem, and promotes the sustainable development of agriculture. Description of the Drawings

[0016] Figure 1 It is the observation result of the colony morphology of strain culture.

[0017] Figure 2 It is the test result diagram of variety SD13092 sugar beet.

[0018] Among them, all "variety SD13092 sugar beet" are abbreviated as "SD", and the treatment group using the bacterial agent provided by the present invention is marked as "treatment with bacterial liquid No. 10".

[0019] Figure A shows the pot control diagrams of the control group of "SD" in normal soil and the treatment group of "Bacterial solution No. 10"; Figure B shows the pot control diagrams of the control group of "SD" in continuous cropping disease soil and the treatment group of "Bacterial solution No. 10"; Figure C shows the comparison diagram of the germination rate of sugar beet seeds in the control group of "SD" in normal soil and the treatment group of "Bacterial solution No. 10"; Figure D shows the comparison diagram of the germination rate of sugar beet seeds in the control group of "SD" in continuous cropping disease soil and the treatment group of "Bacterial solution No. 10"; Figure E shows the comparison diagram of the root incidence rate of sugar beet in the control group of "SD" in normal soil and the treatment group of "Bacterial solution No. 10"; Figure F shows the comparison diagram of the root incidence rate of sugar beet in the control group of "SD" in continuous cropping disease soil and the treatment group of "Bacterial solution No. 10".

[0020] Figure 3 It is the test result diagram of sugar beet variety ST13092.

[0021] Figure A shows the pot control diagrams of the control group of "ST" in normal soil and the treatment group of "Bacterial solution No. 10"; Figure B shows the pot control diagrams of the control group of "ST" in continuous cropping disease soil and the treatment group of "Bacterial solution No. 10"; Figure C shows the comparison diagram of the germination rate of sugar beet seeds in the control group of "ST" in normal soil and the treatment group of "Bacterial solution No. 10"; Figure D shows the comparison diagram of the germination rate of sugar beet seeds in the control group of "ST" in continuous cropping disease soil and the treatment group of "Bacterial solution No. 10"; Figure E shows the comparison diagram of the root incidence rate of sugar beet in the control group of "ST" in normal soil and the treatment group of "Bacterial solution No. 10"; Figure F shows the comparison diagram of the root incidence rate of sugar beet in the control group of "ST" in continuous cropping disease soil and the treatment group of "Bacterial solution No. 10". Detailed implementation mode

[0022] The following examples are used to further illustrate the content of the present invention, but should not be construed as a limitation of the present invention. Without departing from the spirit and essence of the present invention, any modification or replacement of the methods, steps or conditions of the present invention belongs to the scope of the present invention.

[0023] The Brevibacterium linens used in the present invention ( Brachybacterium alimentarium ) is a harmless and safe strain, which is obtained by purchasing from Beijing BioWin Biotechnology Co., Ltd., and the general public can also purchase it through other public channels such as Beijing BioWin Biotechnology Co., Ltd.

[0024] The Purpureocillium lilacinum used in this application ( Paecilomyces lilacinus ) is obtained by purchasing from Beijing BioWin Biotechnology Co., Ltd., and the general public can also purchase it through other public channels such as Beijing BioWin Biotechnology Co., Ltd.

[0025] In the present invention, in the following embodiments, "Brevibacterium alimentarium ( Brachybacterium alimentarium )" is abbreviated as "Brevibacterium alimentarium"; The sugar beet variety SD13092 is abbreviated as the "SD" variety; the sugar beet variety ST13092 is abbreviated as the "ST" variety.

[0026] In the present invention, the sugar beet varieties SD13092 and ST13092 adopted are both diploid single-embryo male sterile hybrids introduced from abroad, selected by Strube Limited, standard varieties, blue pelletized commercial seeds, donated by the Institute of Industrial Crops of the Chinese Academy of Agricultural Sciences, and the general public can purchase and obtain them through the Institute of Industrial Crops of the Chinese Academy of Agricultural Sciences or the Beijing Representative Office of Strube Limited.

[0027] In the present invention, the test soil is taken from the diseased plot and the field of the Manas sugar beet breeding base. The diseased plot is a mixed infection diseased plot of rhizomania and root rot, and sugar beets have been continuously planted for 25 years, and the soil contains a large number of pathogenic bacteria of root rot. The field soil is normal rotation farmland soil.

[0028] In the present invention, the broth soy peptone agar medium TSA, ADF liquid medium, and ADF medium adopted are conventional media in the technical field.

[0029] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. Unless otherwise specified, the materials, reagents, etc. used in the following examples can all be obtained from commercial channels.

[0030] Example 1: A bactericide for inhibiting sugar beet root rot The present invention provides a bactericide for inhibiting sugar beet root rot, and the active ingredient of the bactericide is composed of a resuspension obtained by fermenting Brevibacterium alimentarium ( Brachybacterium alimentarium )

[0031] The optical density OD of the resuspension of the bactericide 600 is 0.02.

[0032] Furthermore, the present invention also provides a preparation method of a bactericide for inhibiting sugar beet root rot, which is specifically prepared by the following steps: (1) Inoculate the strain of Brevibacterium alimentarium ( Brachybacterium alimentarium ) into TSA medium containing 5% NaCl, and incubate at 30 °C for 3 - 15 days for standby; (2) Inoculate the strain of Brevibacterium alimentarium ( Brachybacterium alimentarium ) in step (1) into TSA containing 2% NaCl and culture until the exponential growth phase, and centrifuge to collect cells; (3) Wash the product in step (2) once with 1×PBS and resuspend it in 10 mM MgCl2 solution. Dilute the optical density (OD 600 ) of the cell solution to OD 600 of 0.02.

[0033] Example 2: Hemolytic assay Spot the Brevibacterium linens strain onto the hemolytic medium and culture it at 30 °C for 3 - 7 days. A 1 - 2 mm translucent hemolytic zone can be observed around the colony, indicating that the strain has hemolytic activity. The assay results show that the selected Brevibacterium linens strain does not have a hemolytic reaction, indicating that the selected five strains are biosafe strains and can be used for the construction of synthetic bacterial agents in the future. The results are shown in the appendix Figure 1 as shown.

[0034] Example 3: Identification of the ability to produce 1 - aminocyclopropane - 1 - carboxylic acid deaminase (ACC) Inoculate the Brevibacterium linens strain into a liquid nitrogen - free medium and culture it with shaking at 30 °C and 200 rpm for 24 h. Pipette 0.1 mL of the bacterial solution and inoculate it into 5 mL of DF liquid medium. After culturing with shaking for 24 h, pipette 0.1 mL of the above culture solution and transfer it to ADF liquid medium. After culturing with shaking for 48 h, repeat the transfer to ADF medium. Strains that can grow are determined to be ACC deaminase - positive strains.

[0035] Example 4: Detection of the usage effect Based on the descriptions in the above Examples 1 to 3, using the potted method, mix the soil from the diseased nursery, field soil, and substrate in a ratio of 1:1, mix them evenly and put them into small flower pots. Evenly sow two varieties, SD and ST, on the soil surface in the flower pots, with 80 seeds sown in each pot. After covering the soil, water them. Set a control group (CK) and a treatment group for each variety. The control group is watered with clear water, and the treatment group is watered with clear water plus bacterial solution (add 20 mL of bacterial solution to each liter of clear water). Then cover the flower pots with plastic film and water them once every 7 days according to the method of the first watering, and observe the emergence, growth, and occurrence of root rot during the seedling stage.

[0036] After culturing for 7 - 30 days, observe and count the results. After assisting with the "No. 10 bacterial solution" (i.e., the bacterial agent provided by the present invention), for the ST variety in normal soil, the seed germination rate and disease incidence are not significantly different from those of the control group. However, in the diseased nursery soil, using the bacterial solution treatment can significantly improve the seed germination rate and reduce the disease incidence. For the SD variety, whether in normal soil or diseased nursery soil, the seed germination rate is significantly increased after treatment with the No. 10 bacterial solution, and the disease incidence is significantly reduced at the same time.

[0037] Comparative Example 1: Comparison of the control effects of different bacterial agents Based on the bacterial agent for inhibiting beet root rot constructed and obtained in the examples, and the method for applying the bacterial agent in Example 4, compare the application effects of the bacterial agent for inhibiting beet root rot constructed in Example 3 under different concentrations, and the comparison of the application effects of different concentrations of "Purpureocillium lilacinum". The treatments for each group are as follows: Group of Example 1: Inoculate the Brevibacterium alimentarium strain in TSA medium containing 5% NaCl, and incubate it at 30 °C for 3 - 15 days for standby; inoculate the Brevibacterium alimentarium strain in TSA containing 2% NaCl and culture it until the exponential growth phase, centrifuge to collect cells; wash once with 1×PBS, resuspend in 10 mM MgCl2 solution, and dilute the optical density (OD 600 ) to OD 600 of 0.01.

[0038] Group of Example 2: Inoculate the Brevibacterium alimentarium strain in TSA medium containing 5% NaCl, and incubate it at 30 °C for 3 - 15 days for standby; inoculate the Brevibacterium alimentarium strain in TSA containing 2% NaCl and culture it until the exponential growth phase, centrifuge to collect cells; wash once with 1×PBS, resuspend in 10 mM MgCl2 solution, and dilute the optical density (OD 600 ) to OD 600 of 0.02.

[0039] Group of Example 3: Inoculate the Brevibacterium alimentarium strain in TSA medium containing 5% NaCl, and incubate it at 30 °C for 3 - 15 days for standby; inoculate the Brevibacterium alimentarium strain in TSA containing 2% NaCl and culture it until the exponential growth phase, centrifuge to collect cells; wash once with 1×PBS, resuspend in 10 mM MgCl2 solution, and dilute the optical density (OD 600 ) to OD 600 of 0.03.

[0040] Group of Comparative Example 1: Inoculate the purchased Purpureocillium lilacinum in PDA medium, and culture it at 25 °C - 30 °C until the exponential growth phase, centrifuge to collect cells; wash once with 1×PBS, resuspend in 10 mM MgCl2 solution, and dilute the optical density (OD 600 ) to OD 600 of 0.01.

[0041] Group of Comparative Example 2: Inoculate the purchased Purpureocillium lilacinum in PDA medium, and culture it at 25 °C - 30 °C until the exponential growth phase, centrifuge to collect cells; wash once with 1×PBS, resuspend in 10 mM MgCl2 solution, and dilute the optical density (OD 600 ) to OD 600 of 0.02.

[0042] Control group three: The purchased Purpureocillium lilacinum was inoculated into PDA medium and cultured at 25°C - 30°C until the exponential growth phase, and the cells were collected by centrifugation; washed once with 1×PBS and resuspended in 10 mM MgCl2 solution. The optical density (OD 600 ) of the cell solution was diluted to OD 600 of 0.03.

[0043] Using the potting method, the soil in the disease nursery, the field soil and the substrate were mixed in a ratio of 1:1, and after mixing evenly, they were put into small flower pots. Two varieties, SD and ST, were evenly sown on the soil surface in the flower pots, with 80 seeds sown in each pot. After covering the soil, watering was carried out. For each variety, a control group (CK) and a treatment group were set up. The control group was watered with clean water, and the treatment group was watered with clean water plus bacterial solution (20 ml of bacterial solution was added to each liter of clean water). Then, the flower pots were covered with plastic film, and watering was carried out once every 7 days according to the method of the first watering. The emergence, growth and occurrence of root rot disease in the seedling stage were observed. After culturing for 7 - 30 days, the results were observed and statistically analyzed. The statistical results are shown in Table 1.

[0044] Table 1: Grouping treatment conditions, beet germination rate and beet root incidence

[0045] By comparing the application effects of the above different strains, it can be seen that in the application results of the bacterial agent provided by the present invention in the two varieties, the beet seed germination rate and the beet root incidence are at relatively good levels. Compared with the Purpureocillium lilacinum bacterial agent provided in the control group, the beet seed germination rate is lower and the beet root incidence is higher. It can be seen that the bacterial agent provided in this application has a good beet planting effect on the stubborn pathogen-containing soil of "the mixed infection disease nursery of rhizomania and root rot, where sugar beets have been continuously planted for 25 years and a large amount of root rot pathogenic bacteria are contained in the soil".

[0046] It can be seen that for the seed germination period when the bacterial agent provided by the present invention is most sensitive to environmental stress, the microorganisms in the biological bacterial agent can help the seeds germinate better under adverse environmental conditions by improving the stress resistance of plants. By effectively preventing sugar beet root rot disease, the yield reduction loss caused by the disease can be reduced, thereby increasing the yield and quality of crops. Using the bacterial agent provided by the present invention not only reduces the input cost of chemical pesticides, but also can increase the yield and quality of crops, bringing higher economic benefits. In addition, the biological bacterial agent is environmentally friendly, helps to maintain the health and stability of the soil ecosystem, and promotes the sustainable development of agriculture.

[0047] The above embodiments are merely examples for clearly illustrating the present invention, rather than limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all the implementation manners here. And the obvious changes or modifications derived therefrom still fall within the protection scope of the present invention.

Claims

1. A bacterial agent for inhibiting beet root rot, characterized in that: The active ingredient of the bacterial agent is Brevibacterium spp. Brachybacterium alimentarium ) The composition of the resuspension obtained by fermentation.

2. A bacterial agent for inhibiting beet root rot according to claim 1, characterized in that: The optical density OD600 of the bacterial agent resuspension is 0.

02.

3. A method for preparing a bacterial agent for inhibiting beet root rot according to claim 1 or claim 2, characterized in that: The specific preparation steps are as follows: (1) Add food Brevibacterium ( Brachybacterium alimentarium )The strain was inoculated into TSA medium containing 5% NaCl and cultured at 30°C for 3-15 days for later use; (2) Add the food bacteria ( Brachybacterium alimentarium ) The strain was inoculated in TSA containing 2% NaCl and cultured until the exponential growth phase, and the cells were collected by centrifugation; (3) Wash the product from step (2) once with 1× PBS, resuspend in 10 mM MgCl2 solution, and measure the optical density (OD 600 ) was diluted to OD 600 is 0.

02.

4. Use of the bacterial agent for inhibiting beet root rot according to claim 1 or claim 2 or the method for preparing the bacterial agent according to claim 3 in any one of the following (1) to (3): (1) Bacterial agents for the prevention and treatment of beet root rot; (2) A microbial agent used to reduce the incidence of root rot in sugar beets grown in areas affected by sugar beet root rot; (3) A microbial agent used to increase the germination rate of sugar beets grown in areas affected by beet root rot.

5. A biological organic fertilizer containing the bacterial agent for inhibiting beet root rot according to claim 1 or claim 2 or the method for preparing the bacterial agent according to claim 3.

6. The use according to claim 4, characterized in that The applicable sugar beet variety is ST13092 variety or SD13829 variety.

7. The use according to claim 4, characterized in that In the application, clean water is added with bacterial agent, 20 ml of bacterial solution is added to each liter of clean water for watering, and then the flower pot is covered with ground film, and watered once every 7 days in the later stage.

Images