An endophytic bacterium JY-1-5R from Aconitum and its application

Abstract

The present invention discloses an endophytic bacterium JY‑1‑5R of Aconitum, which was deposited in Guangdong Provincial Microbiological Collection Center on July 18, 2022, with a deposit number of GDMCC No. 62632. The present invention also discloses the use of the above-mentioned endophytic bacteria JY‑1‑5R of Aconitum in promoting the growth of Aconitum and preventing and treating root rot. The endophytic bacteria JY‑1‑5R of Aconitum in the present invention can secrete indoleacetic acid, and field experiments have confirmed that the bacteria can significantly increase the biomass of Aconitum plants and the yield of lateral root aconites, and can be used as a dedicated growth-promoting bacteria for Aconitum; at the same time, the bacteria has strong stress resistance and has a significant inhibitory effect on the pathogenic bacteria of Aconitum root rot. Therefore, the strain JY-1-5R is applied to the growth of Aconitum plants and the prevention and treatment of root rot in the present invention, which belongs to the category of biological fertilizer and biological control. The endophytic bacteria colonize in the plant body and co-evolve with the host, have no adverse effects on medicinal materials, can significantly improve the yield and quality of Aconitum, do not cause environmental pollution, and have broad application prospects in the green and sustainable development of the Aconitum industry.

Description

Technical Field

[0001] The invention relates to the technical field of biological fertilizers and biocontrol bacteria, and in particular to an endophytic bacterium of Aconitum carmichaelii JY-1-5R and an application thereof. Background Art

[0002] Aconitum carmichaelii Debx is a medicinal plant of the genus Aconitum in the family Ranunculaceae. Its root product is called Fuzi, which is a precious and bulk Chinese medicinal material. Jiangyou, Sichuan is its authentic production area. Fuzi has anti-inflammatory, analgesic, cardiotonic, and anti-arrhythmic pharmacological effects. It is known as the "first product for restoring yang and rescuing adverse conditions" and is the only authentic Chinese medicinal material listed in the "973 Project" of the National Key Basic Research and Development Plan.

[0003] Aconitum requires a large amount of fertilizer during its growth. For example, the base fertilizer at the time of sowing alone requires 15 tons / hectare of organic manure and chemical fertilizers, and chemical fertilizers need to be applied twice during the planting process. In production, Aconitum uses tuberous root asexual propagation. During the planting process of Aconitum, management measures such as root pruning (removing excess lateral roots and retaining only 2-3 lateral roots), topping, and removing lateral buds are often used to improve the yield and quality of Aconitum. The number of root pruning is 1-2 times, but the wounds caused make Aconitum plants more susceptible to infection by pathogens. Among them, root rot is one of the main diseases that restrict the yield and quality of Aconitum. Aconitum root rot mainly occurs in the roots, and the surface gradually expands from water-soaked spots to rotten brown, the cortex rots, and there is a dry rot smell. In humid fields, the stem surface and the ground of the diseased plants are attached with Fusarium oxysporum hyphae and spores; the Aconitum grown at the stem base and the lower part of the mother root are small or rotten, and finally the diseased plants dry up and die. According to statistics, the annual incidence of Aconitum root rot in authentic production areas is around 30-50%. In severe cases, the entire land may even be completely sterile. At present, chemical pesticides are mainly used in the production areas to prevent and control Aconitum root rot. Chemical fertilizers and chemical pesticides are two huge expenses for farmers in the process of growing Aconitum. Coupled with the process of manual root pruning, farmers are less and less motivated to grow Aconitum, and the planting area and yield of Aconitum are declining year by year. In addition, the excessive use of chemical agents will cause environmental pollution, excessive heavy metals, and pathogen resistance, which will seriously restrict the healthy development of the Aconitum industry and is not conducive to the protection of the ecological environment.

[0004] Therefore, finding alternatives to chemical pesticides and fertilizers is an important direction for future development. Among them, biofertilizers and biopesticides with beneficial microorganisms as media are important means to increase the yield of Aconitum and prevent and control diseases. However, there are almost no reports on the use of beneficial microorganisms to increase the yield of Aconitum, and there are also very few biological control measures applied to Aconitum root rot. Plant endophytes colonize in plants and co-evolve with plants. They can promote plant growth, improve plant stress resistance and help plants resist infection by pathogens. They are an important resource library for biofertilizers and biopesticides. At present, there are no reports on biofertilizers and biopesticides using Aconitum endophytes as materials. Therefore, screening for efficient growth-promoting and disease-resistant Aconitum endophytes for Aconitum growth and disease control has important theoretical and application value. Summary of the invention

[0005] The invention aims to solve the problem in the prior art that a large amount of chemical fertilizers and pesticides are used in the cultivation of Aconitum, resulting in serious environmental pollution and restricting the green and sustainable development of the Aconitum industry, and provides an Aconitum endophytic bacterium JY-1-5R and an application thereof.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: an endophytic bacterium JY-1-5R of Aconitum, which was deposited in Guangdong Provincial Microbiological Collection Center on July 18, 2022, with a collection number of GDMCC No.62632.

[0007] Preferably, the 16S rRNA gene sequence of the Aconitum endophytic bacterium JY-1-5R is as shown in SEQ ID No.1.

[0008] The present invention also provides the use of the Aconitum endophytic bacteria JY-1-5R in promoting the growth of Aconitum plants and preventing and controlling root rot.

[0009] Preferably, the Aconitum endophytic bacteria JY-1-5R is used in the form of JY-1-5R bacterial suspension or JY-1-5R fermentation broth.

[0010] Further preferably, the preparation method of the JY-1-5R fermentation broth is as follows: the JY-1-5R strain is inoculated into LB solid culture medium, cultured at 28°C for 3 days, a single colony is picked and inoculated into 1L LB liquid culture medium, and cultured at 28°C and 150rpm / min for one week until the concentration is greater than 1×10 9 CFU / mL, the fermentation broth was obtained.

[0011] The beneficial effects of the present invention are:

[0012] 1) The endophytic bacteria JY-1-5R of Aconitum in the present invention can secrete indoleacetic acid, which can significantly increase the biomass of Aconitum plants and the yield of lateral roots of Aconitum under field conditions, and can be used as a dedicated growth-promoting bacterium to increase the yield of Aconitum; at the same time, the endophytic bacteria JY-1-5R of Aconitum can secrete protein degrading enzymes, glucan degrading enzymes and cellulose degrading enzymes, which have a significant inhibitory effect on the pathogens of Aconitum root rot, and can be used as a biocontrol strain for the prevention and treatment of Aconitum root rot.

[0013] 2) The method of applying strain JY-1-5R to the growth and root rot control of Aconitum in the present invention belongs to the category of biological fertilizer and biological control. The endophytic bacteria colonize in the plant body and will not affect the quality of the medicinal materials, thus avoiding the environmental problems caused by the abuse of chemical fertilizers and pesticides and the problem of decreased quality of Aconitum, and has broad application prospects in the green and sustainable development of the Aconitum industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 is a colony morphology diagram of strain JY-1-5R in Example 1 on LB medium;

[0015] Figure 2 This is a diagram showing the antagonistic effect of strain JY-1-5R in Example 1 on root rot pathogens on a plate, where A and B are the control group and the test group of the plate confrontation test, respectively;

[0016] Figure 3 This is a diagram showing the antagonistic effect of strain JY-1-5R on root rot pathogens on Aconitum carmichaelii slices in Example 1, where A and B are the control group and the test group of the Aconitum carmichaelii slice test, respectively;

[0017] Figure 4 is the phylogenetic tree constructed based on the full-length sequence of the 16S rRNA gene of strain JY-1-5R in Example 2;

[0018] Figure 5 : is the effect diagram of the protein degradation enzyme, glucan degradation enzyme and cellulose degradation enzyme production capacity determination of strain JY-1-5R in Example 3, AC is the effect diagram of JY-1-5R producing degradation circles in protein degradation enzyme medium, glucan degradation enzyme medium and cellulose degradation enzyme medium respectively:

[0019] Figure 6 The control effect of the fermentation liquid of strain JY-1-5R in Example 6 on Aconitum root rot;

[0020] Figure 7 This is the growth-promoting effect of the fermentation liquid of strain JY-1-5R in Example 6 on Aconitum plants.

[0021] The endophytic bacterium JY-1-5R of Aconitum in the present invention is deposited in Guangdong Microbiological Collection Center (GDMCC), the address of which is No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, China. The deposit date is July 18, 2022, the deposit number is GDMCC No. 62632, and the classification name is Bacillus sp. JY-1-5R. DETAILED DESCRIPTION

[0022] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0023] Example 1 Isolation and screening of strain JY-1-5R

[0024] 1. Isolation of endophytic bacteria from Aconitum

[0025] The Aconitum plants in this embodiment were taken from the Aconitum planting area in Yaoyue Village, Qinglian Town, Jiangyou City, Sichuan Province.

[0026] The specific steps include: (1) removing roots (mainly Aconite), stems, and leaves from healthy Aconitum plants, repeatedly rinsing them under tap water, taking care not to cause physical wounds, and absorbing moisture with absorbent paper.

[0027] (2) Soak in 75% ethanol for 1 min in a clean bench, then soak in sterile water for 1 min, then soak in 2% NaClO (effective Cl content) for 5 min, and wash with sterile water 5 times, soaking for 2 min each time and shaking slightly. Take 50 μL of the last washing solution and apply it to LB solid culture medium, culture at 28°C for one week, observe whether there is colony growth to detect the effect of surface disinfection, and select the treatment without any colony formation for subsequent operations.

[0028] (3) After surface disinfection, the sample was crushed with a sterile grinder and inoculated into LB solid medium and cultured in the dark at 28°C. After 3 days, bacterial colonies growing around the sample tissue were selected and purified in LB solid medium. Finally, a milky white, opaque, irregularly shaped bacterial cell was selected and named JY-1-5R (see attached). Figure 1 After three purifications, the purity of the strain was tested and the strain was stored in a -80°C refrigerator with 25% glycerol.

[0029] 2. Plate confrontation test between endophytic bacteria and pathogenic bacteria of Aconitum

[0030] Pick a single colony of strain JY-1-5R and culture it in LB liquid medium until the logarithmic growth phase, adjust the OD 600 = 0.2 for standby; the root rot pathogen cake (diameter 2.0mm) was inoculated in the center of the PDA solid medium, and 10μL (OD 600=0.2), incubated in the dark at 28°C for 3 days, measured the diameter of the pathogen colony, and calculated the inhibition rate (%) = (the diameter of the pathogen colony in the control group - the diameter of the pathogen colony in the test group) / the diameter of the pathogen colony in the control group × 100. The results are shown in the attached Figure 2 As shown, Figure 2 -A is the control group inoculated with pathogens alone; Figure 2 -B is the test group inoculated with the bacterial suspension of strain JY-1-5R and pathogenic bacteria, Figure 2 -A and Figure 2 -B comparison showed that strain JY-1-5R had a significant inhibitory effect on the growth of root rot pathogens on the plate, with the mycelium growth inhibition rate being 53.60%.

[0031] 3. Slice confrontation test of endophytic bacteria and pathogenic bacteria in Aconitum

[0032] The root of Aconitum was cut into 1 cm thick round slices, and the surface of the slices was sterilized.

[0033] Pick a single colony of strain JY-1-5R and culture it in LB liquid medium until the logarithmic growth phase, adjust the OD 600 = 0.2 for standby; 10 μL of the bacterial suspension of strain JY-1-5R was evenly inoculated on the surface of the Aconitum slices, air-dried, and then the root rot pathogen cake (diameter 2.0 mm) was inoculated on the center of the Aconitum slices, and cultured in the dark at 28°C for 3 days, and the diameter of the pathogen colony was recorded, and the antibacterial rate (%) was calculated = (control group pathogen colony diameter - test group pathogen colony diameter) / control group pathogen colony diameter × 100. The results are shown in the attached Figure 3 As shown, Figure 3 -A is the control group inoculated with pathogens alone; Figure 3 -B is the test group inoculated with the bacterial suspension of strain JY-1-5R and pathogenic bacteria, Figure 3 -A and Figure 3 -B comparison showed that JY-1-5R had an inhibitory effect on the growth of root rot pathogens on Aconitum slices, and its inhibition rate on the growth of pathogenic fungi was 27.59%.

[0034] Example 2 Identification of 16S rRNA gene of strain JY-1-5R

[0035] (1) The total DNA of strain JY-1-5R isolated and screened in Example 1 was extracted by GUTC method, and the quality of the DNA was checked by 1% agarose gel electrophoresis.

[0036] (2) 16S rDNA PCR amplification was performed using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACCTTGTTACGACTT-3'). The PCR amplification system was: 2×mix 12.5 μL, 1 μL of 10 μmoL / mL 27F, 1 μL of 10 μmoL / mL 1492R, 1 μL of DNA, and 9.5 μL of ddH2O. PCR amplification conditions: 94°C pre-denaturation for 5 min; 94°C denaturation for 30 s, 50°C annealing for 30 s, 72°C extension for 2 min, 30 cycles; 72°C total extension for 10 min. The amplified product was detected using 1% agarose gel, and the electrophoresis voltage was 80 V for 80 min.

[0037] (3) The PCR product was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The obtained sequence was compared with the NCBI database. The reference strain was selected based on the comparison results, and the phylogenetic tree was constructed using MEGA7.0 software ( Figure 4 ) to clarify its classification status.

[0038] (4) The 16S rRNA gene sequence of strain JY-1-5R is shown in SEQ ID NO: 1. The phylogenetic tree results showed that JY-1-5R and Bacillus toyonensis BCT-7112 were clustered into one branch ( Figure 4 ).

[0039] Example 3 Ability of strain JY-1-5R to produce protein degrading enzymes, glucan degrading enzymes and cellulose degrading enzymes

[0040] Protein degrading enzyme culture medium: 5 g tryptone, 2.5 g yeast powder, 1 g glucose, 7% skim milk, 15 g agar powder, 1000 mL distilled water.

[0041] Glucan degrading enzyme culture medium: K2HPO4 1g, yeast powder 5g, peptone 10g, Congo red 0.4g, dextran 5g, MgSO4·7H2O 0.1g, agar powder 15g, distilled water 1000mL.

[0042] Cellulose degrading enzyme culture medium: sodium carboxymethyl cellulose 10 g, peptone 10 g, NaCl 10 g, yeast powder 5 g, agar powder 15 g, distilled water 1000 mL.

[0043] The strain JY-1-5R isolated and screened in Example 1 was made into a bacterial suspension, and the bacterial suspension was inoculated onto the above three culture media with a bamboo stick. After culturing at 28°C for 5 days, the glucan degrading enzyme culture medium and the cellulose degrading enzyme culture medium were stained with 1g / L Congo red dye, and decolorized with 1mol / L sodium chloride solution after 15 minutes. Whether a degradation zone was generated around the colony was observed, and the diameter of the degradation zone was measured. The results are shown in the attached figure. Figure 5 As shown, Figure 5 AC are the effects of JY-1-5R producing degradation circles in protein-degrading enzyme medium, glucan-degrading enzyme medium and cellulose-degrading enzyme medium respectively; the results show that JY-1-5R can produce protease, glucanase and cellulose-degrading enzyme, and the diameters of its degradation circles are 3.48±0.07cm, 3.80±0.04cm and 3.08±0.02cm, respectively.

[0044] Example 4 Determination of the ability of strain JY-1-5R to secrete indoleacetic acid

[0045] A single colony of endophytic bacteria JY-1-5R was inoculated into LB liquid medium containing L-tryptophan (200 mg / L) and cultured in a constant temperature incubator at 28°C for 48 hours. After taking 1 mL of bacterial suspension and centrifuging it at 10000 rpm / min for 10 minutes, 400 μL of supernatant was taken and added with the same volume of Salkowski colorimetric solution, placed in a cool and ventilated place, dark-treated for 30 minutes, and the absorbance of the mixed solution at a wavelength of 530 nm was measured. 20 mg / L, 40 mg / L, 60 mg / L, 80 mg / L, and 100 mg / L indoleacetic acid standard solutions were prepared, treated in the same way and a standard curve was drawn to calculate the content of indoleacetic acid secreted by strain JY-1-5R. The results showed that the indoleacetic acid secretion of strain JY-1-5R was 3.84±1.23 mg / L.

[0046] Example 5 Tolerance of strain JY-1-5R to temperature, pH, salt concentration and antibiotics

[0047] In this example, the strain JY-1-5R isolated and screened in Example 1 was used as the test strain.

[0048] (1) Temperature: The test strain suspension was inoculated on LB plates and heat-shocked at 4°C, 28°C, 37°C and 65°C for 30 min, then cultured at 28°C incubator for 4 days, and the colony growth was observed and recorded. The results showed that JY-1-5R could grow in LB medium treated at 28°C, 37°C and 65°C.

[0049] (2) pH: The test strain suspension was inoculated into LB medium with pH values ​​of 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, and 11.0, respectively, and cultured at 28°C for 4 days. The growth of the colonies was observed and recorded. The results showed that JY-1-5R could grow in LB medium within the above pH range.

[0050] (3) NaCl concentration: The test strain suspension was inoculated into LB medium with NaCl concentrations of 1%, 2%, 3%, 4%, and 5%, respectively, with NaCl concentration of 0.5% as the control, and cultured at 28°C for 4 days, and the colony growth was observed and recorded. The results showed that JY-1-5R could grow in LB medium with the above 6 NaCl concentrations.

[0051] Example 6 Field application effect of strain JY-1-5R fermentation liquid on Aconitum root rot

[0052] 1. Experimental location: Experimental base of Qingyi Campus of Southwest University of Science and Technology, Mianyang City, Sichuan Province. This is the first time that Aconitum plants have been planted in this experimental site.

[0053] Experimental method: The strain JY-1-5R isolated and screened in Example 1 was inoculated into LB solid medium and cultured at 28°C for 3 days. A single colony was picked and inoculated into LB liquid medium and cultured at 28°C and 150 rpm / min for one week until the bacterial concentration in the solution was greater than 1×10 9 CFU / mL. During the root pruning period of Aconitum (root pruning twice), 10mL of JY-1-5R fermentation liquid was injected into the roots of Aconitum as the experimental group. The control group was not injected with fermentation liquid, and the water and fertilizer management method was the same as the experimental group. Six replicates were set up, with 30 healthy Aconitum plants in each replicate. At harvest, the number of plants infected with Aconitum root rot was counted, and the incidence and biocontrol rate of Aconitum root rot were calculated. Incidence rate = number of infected plants / total number of plants × 100%, biocontrol rate = (incidence rate of control group - incidence rate of experimental group) / incidence rate of control group × 100%. At harvest, the fresh weight of the plant stem, fresh weight of the main root (Aconitum) and fresh weight of the lateral root (commercial Aconitum) were recorded, and the dry weight of the plant stem, dry weight of the main root and dry weight of the lateral root were weighed after drying.

[0054] Test results: Statistics show that the incidence of root rot in the test group was 5.56%, and the incidence of root rot in the control group was 10.67%. The biocontrol rate of JY-1-5R fermented liquid against Aconitum root rot was 47.89% ( Figure 6 In addition, the stem fresh weight, stem dry weight, taproot fresh weight, commercial aconite fresh weight and commercial aconite dry weight of plants inoculated with JY1-5R fermentation liquid increased by 134.22%, 116.21%, 43.59%, 93.74% and 94.82% respectively compared with those of plants not inoculated with bacteria, all reaching significant differences ( Figure 7 ). (Note: * indicates significant difference at p<0.05 level)

[0055] In summary, the Aconitum endophytic bacteria JY-1-5R in the present invention can secrete indoleacetic acid, and field experiments have confirmed that the bacteria can significantly increase the biomass of Aconitum plants and the yield of lateral root aconites. At the same time, the Aconitum endophytic bacteria JY-1-5R can secrete protein degrading enzymes, glucan degrading enzymes and cellulose degrading enzymes, have strong stress resistance, and also have a significant inhibitory effect on Aconitum root rot pathogens, and can reduce the incidence of Aconitum field root rot. Therefore, the above-mentioned Aconitum endophytic bacteria JY-1-5R can be used as a material for Aconitum specific biofertilizers and biopesticides, improve Aconitum yield and reduce the incidence of diseases, and have important theoretical and application value for the green and sustainable development of the Aconitum industry.

[0056] The specification and drawings of the present invention are considered to be illustrative rather than restrictive. On the basis of the present invention, those skilled in the art can make some substitutions and modifications to some of the technical features according to the disclosed technical content without creative labor, all of which are within the protection scope of the present invention.

Claims

1. An endophytic bacterium of Aconitum jy-1-5r, characterized in that The endophytic bacteria JY-1-5R of Aconitum were deposited in Guangdong Provincial Microbiological Collection Center on July 18, 2022, with the deposit number GDMCC No.62632.

2. Use of the endophytic bacteria JY-1-5R of Aconitum as claimed in claim 1 in promoting the growth of Aconitum plants and preventing and controlling Aconitum root rot.

3. The use according to claim 2, characterized in that: The application form of the Aconitum endophytic bacteria JY-1-5R is JY-1-5R bacterial suspension or JY-1-5R fermentation liquid.

4. The use according to claim 3, characterized in that: The preparation method of the JY-1-5R fermentation broth is as follows: the JY-1-5R strain is inoculated into LB solid culture medium, cultured at 28°C for 3 days, a single colony is picked and inoculated into 1L LB liquid culture medium, and cultured at 28°C and 150 rpm / min for one week until the concentration is greater than 1×10 9 CFU / mL, the fermentation broth was obtained.

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