Bacillus atrophaeus N-3 and its application in preventing and controlling plant diseases

Abstract

The present invention discloses Bacillus atrophaeus N-3 and its application in preventing and controlling plant diseases. The present invention isolates endophytes from potato plants, and finally screens out an endophyte strain N-3 that can effectively prevent and control plant diseases. The strain N-3 is classified as Bacillus atrophaeus ( Bacillus atrophaeus ), and its microbial preservation number is CGMCC No. 29002. Experiments have shown that Bacillus atrophaeus N‑3 has a good control effect on potato black spot disease, can produce IAA, protease or pectinase, and has the properties of promoting plant growth and improving plant resistance to saline-alkali stress. It has application prospects in preventing and controlling plant diseases, promoting plant growth, producing IAA, protease or pectinase, or improving plant resistance to saline-alkali stress.

Description

Technical Field

[0001] The present invention relates to endophytes isolated from plants, and in particular to Bacillus atrophaeus isolated from potato plants ( Bacillus atrophaeus ) and its application in preventing and controlling plant diseases, belonging to the field of Bacillus isolated from plants and its application. Background Art

[0002] Potato Black Dot is one of the devastating diseases in potato production. The disease is caused by Colletotrichum gloeosporioides ( Colletotrichum coccodes ), the pathogen usually survives in the form of small sclerotia for a long time inside and on the surface of potato tubers, crop debris, soil and air. Colletotrichum can infect various growing parts of the plant, such as leaves, stem base and underground parts, and infect the underground main stem of the plant during the growth period, which can cause the plant to die prematurely; the pathogen infects potato tubers and causes black spots and brown sclerotia on the tubers, which seriously affects the quality of potatoes, and also causes premature death of potatoes in the field, causing cellar rot during storage, causing serious losses to potato yields.

[0003] Traditional prevention and control methods are difficult to effectively control the occurrence of black spot disease during the potato growth period and low-temperature storage period, and the occurrence of potato black spot disease will be more serious in varieties with higher returns and wider planting areas. The use of chemical agents to prevent and control potato black spot disease has a certain prevention and control effect, but it is inconvenient to prevent and control during the low-temperature storage period, and pesticide residues are easy to cause harm to the human body and pollute the environment, which violates the development concept of green agriculture.

[0004] Microorganisms have a good inhibitory effect on fungal diseases. Currently, the use of microbial agents to prevent and control plant diseases is gaining more and more attention. Studies have shown that soil bacteria are a type of microorganism with great development potential. They can not only improve soil structure and promote plant growth, but also inhibit pathogens and improve plant defense capabilities.

[0005] There are many Bacillus atrophaeus ( Bacillus atrophaeus ) were developed and utilized, and it was found that the spores produced by Bacillus atrophaeus have stronger resistance to stress than other Bacillus spores, which makes Bacillus atrophaeus easier to colonize or survive than general biocontrol bacteria, and therefore it has greater potential for development and utilization of biological pesticides. Summary of the invention

[0006] One of the purposes of the present invention is to provide a strain of Bacillus atrophaeus isolated from a plant;

[0007] The second object of the present invention is to provide a microbial preparation or compost containing the atrophic Bacillus;

[0008] The third purpose of the present invention is to apply the Bacillus atrophaeus or the microbial preparation or compost containing the Bacillus atrophaeus to the prevention and treatment of plant diseases, the production of IAA, protease or pectinase, the promotion of plant growth, the improvement of plant resistance to adverse stress or the improvement of soil physical and chemical properties.

[0009] In order to achieve the above object, the main technical solutions adopted by the present invention include:

[0010] One aspect of the present invention is to provide a strain of Bacillus atrophaeus ( Bacillus atrophaeus ) N-3, its microbial preservation number is CGMCC No.29002; its classification name is: Bacillus atrophaeus Bacillus atrophaeus ; The preservation time is: November 14, 2023; The preservation unit is: General Microbiology Center of China Culture Collection Administration; The preservation address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Building 1, Beichen West Road, Chaoyang District, Beijing.

[0011] The colony and bacterial body morphology of the Bacillus atrophaeus described in the present invention are as follows: the colony is nearly circular in shape, has protruding wrinkles in the middle, is sticky when picked up, and is milky white in color.

[0012] The isolated Bacillus atrophaeus N-3 of the present invention has antibacterial ability, such as Colletotrichum sphaeroides, and can play a significant preventive and therapeutic role on potato black spot disease; the Bacillus atrophaeus N-3 can produce IAA, and can promote the growth of plants including potatoes; the Bacillus atrophaeus N-3 can produce protease or pectinase; the Bacillus atrophaeus N-3 has good salt-alkali resistance, can grow normally in a saline-alkali environment, and can improve the physical and chemical properties of the soil.

[0013] Another aspect of the present invention is to apply the Bacillus atrophaeus to plant disease prevention and control, production of IAA, protease or pectinase, promotion of plant growth, improvement of plant resistance to adverse stress or improvement of soil physical and chemical properties.

[0014] In a preferred embodiment of the present invention, the Bacillus atrophaeus N-3 is applied to plant disease prevention and control; wherein the pathogenic bacteria of the plant disease is Colletotrichum sphaeroides; and more preferably, the plant disease is potato black spot disease.

[0015] In a preferred embodiment of the present invention, the Bacillus atrophaeus N-3 is used to produce IAA, protease or pectinase.

[0016] In a preferred specific embodiment of the present invention, the Bacillus atrophaeus N-3 is used to promote plant growth; since the Bacillus atrophaeus N-3 provided by the present invention has the ability to produce IAA, applying the Bacillus atrophaeus N-3, or a microbial preparation or compost prepared from the Bacillus atrophaeus N-3 to the plant growth process can increase the plant height, root length and fresh weight, thereby promoting plant growth.

[0017] In a preferred specific embodiment of the present invention, the Bacillus atrophaeus N-3 is used to improve the resistance of plants to adverse stress; wherein the adverse stress includes salt stress or alkali stress; and the plant is potato.

[0018] In a preferred specific embodiment of the present invention, the Bacillus atrophaeus N-3 is applied to improve the physical and chemical properties of soil; since the Bacillus atrophaeus N-3 provided by the present invention has excellent salt tolerance and alkali tolerance, the microbial preparation or compost prepared by the Bacillus atrophaeus N-3 can be applied to saline-alkali soil to enrich the microbial species in the saline-alkali soil and improve the saline-alkali soil ecological system, thereby effectively improving the physical and chemical properties of the saline-alkali soil, including: regulating the pH value of the soil, increasing the soil porosity or enriching the microbial flora species in the soil, etc.

[0019] Another aspect of the present invention is to provide a microbial preparation or compost, wherein the microbial preparation or compost contains the Bacillus atrophaeus N-3.

[0020] Those skilled in the art can prepare the Bacillus atrophaeus N-3 provided by the present invention into various conventional microbial preparations or composts according to conventional preparation methods of microbial preparations, which are all technical means well known to those skilled in the art; accordingly, various conventional microbial preparations prepared from Bacillus atrophaeus N-3 can be used for plant disease prevention and control, production of IAA, protease or pectinase, promotion of plant growth, improvement of plant resistance to adverse stress or improvement of soil physical and chemical properties, etc.

[0021] The present invention isolates endophytes from potato plants, and finally screens out a strain N-3 that can prevent and control diseases, promote plant growth and improve plant resistance to adverse stress. The strain N-3 is identified by molecular biology combined with morphology and named as Bacillus atrophaeus ( Bacillus atrophaeus ), experiments have shown that the Bacillus atrophaeus N-3 can significantly prevent and treat potato black spot disease, can produce IAA to promote plant growth, can also grow under saline-alkali stress and effectively improve the physical and chemical properties or soil structure of saline-alkali soil. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the colony morphology of Bacillus atrophaeus N-3;

[0023] Figure 2 for Bacillus atrophaeus N-3 against Colletotrichum sphaeroides ( Colletotrichum coccodes ) of the flat plate inhibition effect diagram;

[0024] Figure 3 This is a graph showing the growth of Bacillus atrophaeus N-3 on a protease-producing medium;

[0025] Figure 4 This is a graph showing the growth of Bacillus atrophaeus N-3 on pectinase medium;

[0026] Figure 5 This is a graph showing the growth of Bacillus atrophaeus N-3 under different salt concentrations;

[0027] Figure 6 This is a graph showing the growth of Bacillus atrophaeus N-3 under different alkali concentrations. DETAILED DESCRIPTION

[0028] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, it should be understood that the embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications or replacements all fall within the protection scope of the present invention.

[0029] Example Isolation and Identification of Bacillus atrophaeus N-3

[0030] 1. Strain Isolation

[0031] The potato plant samples were cleaned and dried by dilution and coating method. Several sections of plant samples with a length of about 1 cm were cut and soaked in 75% alcohol for about 2 min. After being taken out and dried, they were soaked in 5% sodium hypochlorite solution for about 2 min. The dried samples were taken out and soaked in 75% alcohol for 1 min again. After that, they were rinsed in sterile water for 2 to 3 times. The potato tissues were dried and ground evenly in a sterilized mortar. 1 mL of the liquid in the mortar was absorbed and evenly applied to the LB solid culture medium plate. The plate was repeated 10 times, and the treated LB plate was inverted and stored in a constant temperature incubator set at 28 °C for 24 to 48 h. The selected endophyte was named N-3 and purified by continuous streaking with LB culture medium. The obtained strain N-3 was stored in a -81 °C ultra-low temperature refrigerator with 70% glycerol.

[0032] 2 Identification of strain N-3

[0033] After the strain N-3 was cultured on LB solid medium at 25℃ for 24 h, the colony morphology was as follows Figure 1 As shown. The colony is nearly round, with a protruding wrinkle in the middle, sticky when picked up, and milky white in color. Pick a single colony of the strain to be identified and culture it in liquid LB medium. After culturing for 3 days, take 1 mL of strain N-3 fermentation broth into a 1.5 mL centrifuge tube and centrifuge at 12000 rpm for 1 min to collect the bacteria. Bacterial DNA was extracted using a bacterial genomic DNA kit purchased from Tiangen Biochemical Technology Co., Ltd. For detailed steps, please refer to the product manual. Using 16S rDNA universal primers and yrA The primers gyrA-F and gyrA-R were used to amplify the 16S rDNA and yrA The gene fragment was amplified by PCR using conventional methods. The PCR product was sequenced after being tested by 1% agarose gel electrophoresis and identified as Bacillus atrophaeus ( Bacillus atrophaeus ).

[0034] Test Example 1 Bacillus atrophaeus N-3 plate confrontation test

[0035] 1 Test method

[0036] The pathogen used in the present invention is Colletotrichum sphaeroides ( Colletotrichum coccodes ), which was preserved and purified by the Institute of Plant Protection, Inner Mongolia Academy of Agricultural and Animal Husbandry Sciences.

[0037] The test was determined by the plate confrontation method. The Colletotrichum sphaeroides was activated and cultured for 5 days. A hole was punched in the colony-rich area with a hole puncher to make a Colletotrichum sphaeroides cake with a diameter of 6 mm. It was inverted in the center of the PDA culture medium plate. At the same time, the isolated single colony of Bacillus atrophaeus N-3 was inoculated 2.5 cm away from the center of the plate. Four points were inoculated per dish. The PDA plate with only the Colletotrichum sphaeroides cake was used as the control. The test was repeated 3 times. The Colletotrichum sphaeroides in the control group was cultured in a 25°C incubator until the hyphae of the Colletotrichum sphaeroides in the control group covered the culture medium. The inhibition zone was observed on the control plate and the diameter of the inhibition zone was measured.

[0038] 2 Test results

[0039] The results of the plate confrontation test of Bacillus atrophaeus N-3 are shown in Tables 1 and Figure 2 As shown, Bacillus atrophaeus N-3 had a significant inhibitory effect on Colletotrichum sphaeroides on the plate, and the average inhibition rate of Bacillus atrophaeus N-3 on Colletotrichum sphaeroides reached 63.48%.

[0040] Table 1 The inhibitory effect of Bacillus atrophaeus N-3 on Colletotrichum sphaeroides

[0041]

[0042] Note: Different lowercase letters in the same column indicate significant differences (P<0.05).

[0043] Test Example 2 Indoor test on the efficacy of Bacillus atrophaeus N-3 against potato black spot

[0044] 1 Test method

[0045] The purified Bacillus atrophaeus N-3 and Colletotrichum sphaeroides were inoculated into LB liquid medium and PDB medium respectively, and cultured in a shaker at 150-180 r / min and 25℃-28℃ for 3 days. The matrix was potted for standby use, and the potato tissue culture seedlings were placed in plug trays for seedling growth. After the seedlings were raised, potato seedlings of the same size were selected and planted in pots. One plant was transplanted into each pot and covered with soil. For the transplanted plants, three treatment groups were set up: CK, F, and N-3+F. F was inoculated with Colletotrichum sphaeroides, N-3+F was inoculated with Bacillus atrophaeus N-3 and Colletotrichum sphaeroides, and CK was not inoculated. The cultured bacterial solution was watered at 20 mL of the original solution per seedling, and CK and F were watered with 20 mL of liquid LB medium respectively. One week after inoculation with Bacillus atrophaeus N-3, the potato seedlings were inoculated with Colletotrichum gloeosporioides using the root wounding method. Each potato tissue culture seedling was inoculated with 50 mL of a 10-fold diluted solution. One week after inoculation, the potato tissue culture seedlings were inoculated again using the above method to cause disease. After growing for 2 months, the root length, root weight, plant height, and fresh weight of the plants were measured to analyze the control effect of Bacillus atrophaeus N-3 on potato black spot disease.

[0046] 2 Test results

[0047] The results of the indoor control efficacy test are shown in Table 2. The root length and fresh weight of the treatment group N-3+F were significantly different from those of the F group, and the incidence of the disease in the N-3+F group was lower than that in the F group. Therefore, Bacillus atrophaeus N-3 has a significant control effect on potato black spot disease, and the indoor control efficacy can reach 61.53%.

[0048] Table 2 Indoor control effect of Bacillus atrophaeus N-3 on potato black spot

[0049]

[0050] Note: Different lowercase letters in the same column indicate significant differences (P<0.05). F is the inoculated anthracis.

[0051] Test Example 3 Test for the IAA production capacity of Bacillus atrophaeus N-3

[0052] 1 Test method

[0053] King's B medium: 20.0 g peptone, 1.5 g potassium dihydrogen phosphate, 1.5 g magnesium sulfate, 10.0 mL glycerol, 0.1 g L-tryptophan, and distilled water to 1 L.

[0054] The strain was inoculated into King's B liquid medium, cultured at 28°C and 150 r / min for 4 days, centrifuged at 8000 rpm for 5 min, and the supernatant was added with Salkowski colorimetric reagent at a ratio of 1:1. The supernatant was placed at 28°C in the dark for 30 min before measuring the OD 530 Using King's B liquid medium without bacteria as control, a standard curve was drawn with IAA (analytical grade) gradient dilutions according to the above method, and the IAA content in the fermentation broth was calculated based on the standard curve.

[0055] 2 Test results

[0056] The test results of the IAA production ability of Bacillus atrophaeus N-3 are shown in Table 3. Bacillus atrophaeus has the ability to produce IAA, and the concentration of IAA produced is determined to be 8.86 mg / L.

[0057] Table 3 IAA production capacity of Bacillus atrophaeus N-3

[0058]

[0059] Test Example 4 Test for the Protease Production Capacity of Bacillus atrophaeus N-3

[0060] 1 Test method

[0061] Protease production medium: 3 g skim milk powder, 3 g agar, 200 mL distilled water, sterilize at 121℃ for 20 min.

[0062] The purified Bacillus atrophaeus N-3 was used to determine the protease production capacity using a protease production medium. The purified Bacillus atrophaeus N-3 was inoculated on the protease production medium using a sterilized toothpick and placed in a 25°C incubator for 3 days. Observe whether apertures are produced and measure the colony diameter and aperture diameter.

[0063] 2 Test results

[0064] The results of the test on the protease production capacity of Bacillus atrophaeus N-3 are shown in Table 4 and Figure 3 As shown, the average colony diameter of Bacillus atrophaeus N-3 in the protease-producing medium is 0.32 cm, but the average aperture diameter is 1.47 cm, which proves that Bacillus atrophaeus N-3 has the ability to produce protease.

[0065] Table 4 Results of the assay of protease production capacity of Bacillus atrophaeus N-3

[0066]

[0067] Test Example 5 Test on the ability of Bacillus atrophaeus N-3 to produce pectinase

[0068] 1 Test method

[0069] Pectinase culture medium: 8 g pectin, 1.4 g ammonium chloride, 2 g potassium dihydrogen phosphate, 6 g dimethyl phosphate, 0.1 g magnesium sulfate, 15 g agar, natural pH, sterilization at 121°C for 30 min.

[0070] The purified Bacillus atrophaeus N-3 was tested for pectinase capacity using pectinase medium. The purified Bacillus atrophaeus N-3 was inoculated on the pectinase medium using a sterilized toothpick and placed in a 25°C incubator for 3 days. Whether an aperture was generated was observed, and the colony diameter and aperture diameter were measured.

[0071] 2 Test results

[0072] The results of the test on the ability of Bacillus atrophaeus N-3 to produce pectinase are shown in Table 5 and Figure 4 As shown, the average colony diameter of Bacillus atrophaeus N-3 on pectinase medium was 0.30 cm, but the average aperture diameter was 1.67 cm, which proved that Bacillus atrophaeus N-3 had the ability to produce pectinase.

[0073] Table 5 Results of the determination of pectinase production capacity of Bacillus atrophaeus N-3

[0074]

[0075] Test Example 6 Salt tolerance test of Bacillus atrophaeus N-3

[0076] 1 Test method

[0077] The salt tolerance of the isolated and purified Bacillus atrophaeus N-3 was determined by the plate method. The activated Bacillus atrophaeus N-3 was inoculated on LB medium with salt concentrations of 9%, 10%, 11%, and 12%, respectively. CK was the control treatment without additional NaCl addition, and the culture was placed in a 25°C incubator for 48-72 hours. The size of the colonies on the salt-tolerant medium was observed, and the diameter of the salt-tolerant colonies was measured. The salt tolerance of Bacillus atrophaeus N-3 was determined according to the numerical value of the colony diameter.

[0078] 2 Test results

[0079] The results of the salt tolerance test of Bacillus atrophaeus N-3 are shown in Table 6 and Figure 5As shown, after the cultivation of Bacillus atrophaeus N-3 in LB culture medium with salt concentrations of 9%, 10%, 11%, and 12%, the colony diameter was measured, and it was concluded that Bacillus atrophaeus N-3 can grow on culture medium with salt concentrations of 9%, 10%, 11%, and 12%.

[0080] Table 6 Determination of salt tolerance of Bacillus atrophaeus N-3

[0081]

[0082] Note: Different lowercase letters in the same column indicate significant differences (P<0.05).

[0083] Test Example 7 Alkali resistance test of Bacillus atrophaeus N-3

[0084] 1 Test method

[0085] The isolated and purified Bacillus atrophaeus N-3 was tested for its alkali resistance using the plate method. The activated Bacillus atrophaeus N-3 was inoculated onto LB medium with pH values ​​of 8, 9, and 10, respectively. CK was the control treatment with pH value of 7, and the cells were cultured in a 25°C incubator for 48 to 72 hours. The size of the colonies on the alkali-resistant medium was observed, and the diameter of the alkali-resistant colonies was measured. The alkali resistance of Bacillus atrophaeus N-3 was determined based on the numerical value of the colony diameter.

[0086] 2 Test results

[0087] Table 7 Alkali resistance determination of Bacillus atrophaeus N-3

[0088]

[0089] Note: Different lowercase letters in the same column indicate significant differences (P<0.05).

[0090] The results of the alkali resistance test of Bacillus atrophaeus N-3 are shown in Table 7 and Figure 6 As shown, after inoculating Bacillus atrophaeus N-3 on an alkali-resistant culture medium and culturing it, the diameter of its colony was measured, and it was concluded that Bacillus atrophaeus N-3 could grow on culture media with pH values ​​of 8, 9, and 10.

Claims

1. A strain of Bacillus atrophaeus ( Bacillus atrophaeus ) N-3, characterized in that, Its microbial preservation number is: CGMCC No.29002.

2. A microbial preparation or compost containing the Bacillus atrophaeus N-3 according to claim 1.

3. Use of the Bacillus atrophaeus N-3 according to claim 1 or the microbial preparation or compost according to claim 2 in preventing and controlling plant diseases; the plant disease is potato black spot disease caused by Colletotrichum sphaeroides.

4. Use of the Bacillus atrophaeus N-3 according to claim 1 or the microbial preparation or compost according to claim 2 in the production of IAA, protease or pectinase.

5. Use of the Bacillus atrophaeus N-3 according to claim 1 or the microbial preparation or compost according to claim 2 in promoting plant growth; wherein: The plant is potato.

6. Use of the Bacillus atrophaeus N-3 according to claim 1 or the microbial preparation or compost according to claim 2 in improving the resistance of potatoes to adverse stress; the resistance to adverse stress is resistance to salt stress or resistance to alkali stress.

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