Low-toxicity phytophthora nicotianae JH-11-1 and application thereof in tobacco growth promotion and disease control

Abstract

The invention relates to a low-toxicity phytophthora nicotianae JH-11-1 strain and application of the low-toxicity phytophthora nicotianae JH-11-1 strain in tobacco growth promotion and disease control. The phytophthora nicotianae JH-11-1 has weak pathogenicity and growth-promoting and disease-preventing effects on tobacco, can be developed into a biocontrol bacterial preparation for biological prevention and control of tobacco diseases, has the characteristics of no toxicity, no residue, no pollution, low possibility of generating drug resistance and the like compared with current chemical agents for preventing and controlling the tobacco diseases, and has a good market application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of plant growth-promoting bacteria, and specifically relates to a low-toxic tobacco phytophthora JH-11-1 and application thereof in tobacco growth promotion and disease prevention and control. Background Art

[0002] Tobacco diseases are important limiting factors in tobacco production, seriously affecting tobacco yield and quality. At present, tobacco black shank, bacterial wilt, target spot and other diseases are common diseases in tobacco producing areas. For example, tobacco black shank causes an average annual economic loss of more than 100 million yuan in my country. At present, the application of chemical fungicides is still an effective means of tobacco disease control. The use of broad-spectrum fungicides leads to soil environmental pollution, pathogens develop drug resistance, harm non-target organisms, pesticide residues and a series of other problems, which is not conducive to the promotion of green, environmentally friendly and pollution-free tobacco planting and sustainable development of agriculture. Therefore, it is very necessary to find new, safe and effective measures (such as biological control strategies) to prevent and control tobacco diseases.

[0003] When some pathogenic microorganisms interact with plants, they can induce systemic resistance in plants; pathogens can stimulate plants to produce local resistance and systemic resistance. When they come into contact with plants, plants will produce systemic acquired resistance (systemic acquired resistance) throughout the plant, preventing pathogens from spreading further to other parts of the plant. Plants use receptor kinases (RKs) and receptor-like kinase proteins (RLPs) as pattern recognition receptors (PRRs) to recognize pathogen / microorganism-associated pattern molecules (PAMPs / MAMPs), inducing plants to produce local resistance and systemic disease resistance. Studies have shown that pre-treatment of plants with pathogens can cause changes in their physiological, transcriptional, metabolic and epigenetic levels, activate plant defense mechanisms and improve plant defense capabilities. If the pathogen is encountered again, the plant effectively improves a faster or stronger defense response. This defense response is maintained throughout the plant's growing season and can even be passed on to future generations. Pathogens can induce plant disease resistance and have potential application value. In addition, pathogenic bacteria strains that have lost their pathogenicity grow endogenously in plants, which may improve the disease resistance of plants and promote plant growth, causing plants to produce a series of defense responses, such as producing antibacterial substances and changing the composition of cell walls. These responses may promote plant growth while resisting pathogens. Furthermore, pathogen infection can affect the balance of plant hormones, such as increasing the synthesis of hormones such as cytokinins and gibberellins. These hormones can promote the division and elongation of plant cells, thereby promoting plant growth. Therefore, they can be used as potential strains for the preparation of plant vaccines. Summary of the invention

[0004] Specifically, the present invention provides a low-toxic tobacco Phytophtora nicotianae JH-11-1, which was deposited in the China Center for Type Culture Collection on October 11, 2024, with a deposit number of CCTCC NO: M20242162, and the address of the depository unit is: Wuhan University, Wuhan, China.

[0005] The present invention also provides a bacterial agent or a plant vaccine containing the above-mentioned Phytophtora nicotianae JH-11-1 or its bacterial suspension or its fermentation product.

[0006] Furthermore, the concentration of Phytophtora nicotianae JH-11-1 in the bacterial agent is 0.08 g / ml.

[0007] The present invention also provides the use of the tobacco phytophthora (Phytophtora nicotianae) JH-11-1 or the bacterial agent or plant vaccine in any one of the following A1)-A6):

[0008] A1) Promote tobacco growth;

[0009] A2) preparing a bacterial agent for promoting tobacco growth;

[0010] A3) Improve tobacco disease resistance;

[0011] A4) preparing a bacterial agent that improves the broad-spectrum disease resistance of tobacco;

[0012] A5) Prevention and control of plant diseases;

[0013] A6) Preparation of fungal agents for preventing and controlling various plant diseases

[0014] The plant diseases include tobacco bacterial wilt and tobacco target spot.

[0015] Furthermore, promoting tobacco growth is reflected in increasing tobacco plant height and / or yield, and improving tobacco disease resistance is reflected in promoting high expression of NtPAL and / or NtNPR1.

[0016] The invention also provides a method for promoting tobacco growth and / or increasing tobacco yield, wherein tobacco is treated with the tobacco phytophthora nicotianae JH-11-1 or the bacterial agent, so that the plant height and / or yield of the tobacco plant are increased.

[0017] Furthermore, the concentration of Phytophtora nicotianae JH-11-1 in the bacterial agent is 0.8 g / ml.

[0018] The invention also provides a method for improving tobacco disease resistance or preventing and controlling plant diseases, which comprises treating tobacco with the tobacco phytophthora nicotianae JH-11-1 or the plant vaccine.

[0019] Furthermore, improving tobacco disease resistance is reflected in promoting high expression of NtPAL and / or NtNPR1.

[0020] Furthermore, the plant diseases include tobacco bacterial wilt and tobacco target spot.

[0021] The tobacco phytophthora JH-11-1 described in the present invention has weak pathogenicity and growth-promoting effects on tobacco, and can be developed into a biocontrol fungus preparation for biological control of tobacco diseases. Compared with the current chemical agents for controlling tobacco diseases, it has the characteristics of being non-toxic, residue-free, pollution-free, and not prone to drug resistance, and has good market application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0023] Figure 1 : Colony morphology of Phytophthora nicotianae after 7 days of culture.

[0024] Figure 2 : Growth rate of various tobacco Phytophthora strains.

[0025] Figure 3 :Comparison of pathogenicity of various tobacco Phytophthora strains.

[0026] Figure 4 : The mycelial quantity of Phytophthora nicotianae JH-11-1 cultured on different culture media.

[0027] Figure 5 : Expression levels of tobacco resistance genes NtPAL and NtNPRI at different time periods after treatment with tobacco Phytophthora jH-11-1.

[0028] Figure 6 :Verification of the growth-promoting effect of tobacco phytophthora JH-11-1, Figure A shows the height of tobacco plants, and Figure B shows the tobacco yield. CK: clean water, A: tobacco vaccine, D: 58% mancozeb metalaxyl, E: 10 billion Bacillus subtilis. DETAILED DESCRIPTION

[0029] The following examples are only used to more clearly illustrate the technical scheme of the present invention, and are therefore only used as examples, and cannot limit the scope of protection of the present invention. It should be noted that, unless otherwise stated, the technical terms or scientific terms used in this application should be the usual meanings understood by those skilled in the art to which the present invention belongs. Unless otherwise stated, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise stated, the reagents and materials used in the following examples are commercially available.

[0030] Experimental Example 1: Isolation and purification of tobacco phytophthora and strain identification test

[0031] The tobacco Phytophthora strains isolated in the present invention are all from tobacco black shank diseased plants collected from tobacco bases in Chenzhou City and Xiangxi Prefecture, Hunan Province in 2023. Specifically, tobacco black shank diseased plants are collected from tobacco producing areas and the following operations are performed:

[0032] Use tweezers to remove tobacco discs infected with tobacco phytophthora, then disinfect the surface with 75% alcohol for 30 seconds, wash three times with sterile water and place on sterile filter paper to air dry. Place the washed tobacco discs in an OA culture dish (containing 300μg / mL of cefixime) and culture them in a 28℃ culture room for 3-5 days. When the mycelium grows, pick the mycelium and subculture it in a new OA culture dish. The isolated and purified strains are preserved by two methods: slant preservation and glycerol preservation: pick fresh strain mycelium blocks and place them in 1mL OA slant, culture them in a 28℃ culture room until the mycelium grows all over the slant, and then store them for a long time at 4℃, and save 3 copies for each strain; pick fresh strain mycelium blocks and store them in 25% glycerol, -80℃, and save 3 copies for each strain.

[0033] Extraction of strain DNA: The genomic DNA was extracted using the conventional CTAB method. The specific method is as follows: Use a pick to pick a 4×4 cm2 mycelium block grown for 3 days and place it in a 2 ml sterile centrifuge tube containing steel beads, add 1 ml CTAB extraction solution, placed on a grinder at 50Hz, 300s to break the bacterial blocks, and placed in a 65°C water bath for 10min (inverted 2-3 times during this period); placed a 2ml centrifuge tube in a centrifuge and centrifuged at 12000r / min for 10min, aspirated the supernatant into a new 2ml sterile centrifuge tube, added an equal volume of extraction solution I, and inverted to mix; centrifuged at 12000r / min for 10min, aspirated the supernatant into a new 2ml sterile centrifuge tube, added an equal volume of extraction solution II, and inverted to mix; centrifuged at 12000r / min for 10min, took the supernatant into a new 1.5ml sterile centrifuge tube, added an equal volume of isopropanol, mixed and placed in a -20℃ refrigerator to precipitate for at least 30min; then centrifuged at 12000r / min for 15min, poured out the supernatant, added 700μL 75% anhydrous ethanol, suspended, centrifuged at 12000r / min for 3min, and repeated once. Discard the supernatant, use a pipette to aspirate the remaining liquid, dry it, add 30–100 μL of sterile deionized water to dissolve it, and store it at -20°C.

[0034] PCR amplification using primers ITS1 / ITS4 to amplify the ITS sequence of the strain

[0035] ITS1: 5'-TCCGTAGGTGAACCTGCGG-3'

[0036] ITS4: 5'-TCCTCCGCTTATTGATATGC-3'

[0037] The PCR reaction system (20 μL) is:

[0038]

[0039] PCR amplification procedure:

[0040]

[0041] The PCR products were sent to Tianyi Huiyuan Biotechnology Co., Ltd. (Wuhan) for sequencing.

[0042] The fragment sequencing results were compared by BLAST, and the results showed that the sequence homology of these strains with that of Phytophthora nicotianae was more than 99%.

[0043] Experimental Example 2: Morphological observation and growth rate determination of tobacco phytophthora

[0044] Prepare oatmeal agar medium.

[0045] 1. Weigh 30g of oatmeal, transfer to a glass container, add 1000mL sterile water. Boil on the top of an induction cooker. The oatmeal will then become viscous and the liquid solution will be filtered through a piece of sterile gauze. 2. Pour the filtrate into a 1000mL graduated cylinder and make up to 1000mL with sterile water. 3. Pour the liquid solution into a glass reagent bottle and add 18g agar. Shake well and autoclave at 121℃ for 15min. Let stand at room temperature for 30min. 4. Pour about 20mL of sterilized oatmeal agar medium into each culture dish. Let the culture dishes cool thoroughly at room temperature before mycelial culture.

[0046] Use a 5mm cork puncher to punch out fresh mycelium near the edge of the colony, pick up the mycelium block and place it in the center of a 60mm culture dish containing 10mL OA, and culture it in a 28℃ incubator. Record the colony morphology with a camera at 2d and 4d after inoculation, and measure the diameter of each colony with the cross method.

[0047] The results are as follows Figure 1 , Figure 2 As shown, the mycelial growth of tobacco phytophthora strain JH-11-1 was abnormal, and the growth rate was significantly lower than that of other strains. The strain JH-11-1 was sent to the China Center for Type Culture Collection for preservation, and the strain preservation number is CCTCCNO: M 20242162.

[0048] Experimental Example 3: Determination of the pathogenicity of Phytophthora nicotianae JH-11-1

[0049] 1. Inoculate the mycelium into OA medium and culture it in the dark at 28℃. Prepare a 1cm diameter puncher and toothpick in advance and sterilize them by high pressure at 121℃ for 15min.

[0050] 2. Punch holes on the tobacco black shank pathogen mycelial agar medium to make a circular mycelial mat. 3. Pick up the mycelial mat, place it face down on the oatmeal agar medium, and culture the mycelium at 28°C in the dark for 7 days.

[0051] 3. Use a 1-cm diameter hole puncher to punch holes in the tobacco black shank pathogen agar culture medium to make circular mycelial pads. Use a toothpick to pick up the mycelial pads and inoculate them onto detached leaves. Repeat three times for each strain. After the inoculation is completed, place the inoculated leaves in a 28°C light incubator and observe the disease after four days.

[0052] The results are as follows Figure 3 As shown, the pathogenicity of tobacco Phytophthora strain JH-11-1 was significantly lower than that of other strains, showing weak toxicity.

[0053] Experimental Example 4: Mycelial fermentation experiment of Phytophthora nicotianae JH-11-1

[0054] Effects of different C and N sources on mycelium yield: 9 recipes were designed based on different C and N sources. Magnesium sulfate 1.5 g / L, potassium dihydrogen phosphate 3.0 g / L were added to the recipes, and the pH was natural. The liquid culture medium was 100 mL, the inoculation amount was 14%, and the culture was carried out in a shaker at 120 r / min and 28°C. After 7 days, the results were observed and recorded, and the dry weight of the mycelium was weighed.

[0055] Table 1 Liquid culture formula (g / L)

[0056]

[0057] The results are as follows Figure 4 As shown, JH-11-1 grew better in medium 4 (sucrose + peptone) and medium 7 (glucose + peptone).

[0058] Experimental Example 5: Determination of tobacco-related resistance genes

[0059] The cultured tobacco phytophthora strain JH-11-1 was inoculated into the leaves of live Yunyan 87 according to the method in Experimental Example 3, and the mycelium was placed face down on the tobacco leaves. Samples were taken at 0, 6, 12, 18, 24, 30, and 36 after inoculation, and inoculated at the same time. Four biological replicates were made for each sampling point. Liquid nitrogen was used quickly after sampling. After all the samples at all time points were taken, the total RNA was extracted uniformly using the TRIzol method. The cDNA synthesis used the reverse transcription kit of Beijing Quanshijin Company. Fluorescence quantitative PCR reaction conditions: 95℃ 5min; 95℃ 30s, 60℃ 30s, 72℃ 30s (40cycles).

[0060] like Figure 5 As shown in the figure, after inoculation with tobacco phytophthora strain JH-11-1, the expression levels of phenylalanine ammonia lyase NtPAL-related genes at 12h and 36h were significantly increased compared with 0h. The expression level of NtNPR1-related genes at 12h was significantly higher than that at 0h. This shows that inoculation with tobacco phytophthora strain JH-11-1 can increase the expression of Yunyan 87-related resistance genes, thereby promoting disease resistance in plants.

[0061] Experimental Example 6: Verification of the growth-promoting and disease-resistant effect of tobacco phytophthora JH-11-1

[0062] Test location: Chenzhou, Xiangxi Prefecture

[0063] Test method: 4 treatments were set up, each treatment was repeated 3 times, and there were 12 plots in total. The treatments included clear water control (CK), tobacco vaccine: after optimizing the culture medium according to the conditions in Example 4, the mycelium was cultured, and a mycelium solution with a concentration of 0.08 g / ml was prepared and then sterilized and sealed (A: JH-11-1), chemical agents (D: 58% mancozeb, Sichuan Guoguang), and biological agents (E: 10 billion Bacillus subtilis, purchased from Shaanxi Hengtian Bio-Agriculture Co., Ltd.). The test area of ​​each plot was 30 m2. One mu of test field area was reserved for each point, and the test was arranged in random blocks, with 3-4 ridge protection rows set around the test field.

[0064] Results: 5 points of equidistant sampling were used in the treatment area, and 10 tobacco seedlings were investigated at each point. The effects of different treatment methods on plant disease resistance and yield increase were evaluated by comparing yield and incidence. The incidence rate and disease index base were investigated before application, and the incidence rate and disease index were investigated every 7 days after the control area became ill after application, and the prevention effect was calculated. The tobacco disease investigation standard is based on the Chinese national standard Ren, G., et al. GB / T23222 Grade and Investigation Method of Tobacco Diseases and Insect Pests. China Standard Press, Beijing (2008).

[0065] like Figure 6 As shown in Table 2, compared with the control, the tobacco plant height and yield can be significantly increased after the tobacco vaccine (Group A) is applied. Further investigation of tobacco diseases in the experimental plots was conducted. As shown in Table 2, tobacco black shank did not occur in the experimental plots, while tobacco bacterial wilt and tobacco target spot occurred in all groups. However, the incidence rate and disease grade of the tobacco vaccine group were significantly better than those of the control group and other experimental groups, indicating that tobacco phytophthora JH-11-1 has a good broad-spectrum disease resistance effect.

[0066] Table 2

[0067]

[0068]

[0069] The above specific embodiments describe the implementation of the present invention in detail, but the present invention is not limited to the specific details in the above embodiments. Within the scope of the claims and technical concept of the present invention, the technical solution of the present invention can be modified and changed in many simple ways, and these simple modifications all belong to the protection scope of the present invention.

Claims

1. A strain of low-toxic tobacco Phytophtora nicotianae JH-11-1 was deposited in the China Center for Type Culture Collection on October 11, 2024, with the deposit number CCTCC NO: M 20242162.

2. A bacterial agent or plant vaccine containing the tobacco blight fungus (Phytophtora nicotianae) JH-11-1 or its bacterial suspension or its fermentation product as claimed in claim 1.

3. The bacterial agent according to claim 2, characterized in that: The concentration of Phytophtora nicotianae JH-11-1 in the bacterial agent or plant vaccine is 0.08 g / ml.

4. Use of the tobacco phytophthora (Phytophtora nicotianae) JH-11-1 according to claim 1 or the bacterial agent or plant vaccine according to any one of claims 2 to 3 in any one of the following A1) to A6): A1) Promote tobacco growth; A2) preparing a bacterial agent for promoting tobacco growth; A3) Improve tobacco disease resistance; A4) preparing a bacterial agent that improves the broad-spectrum disease resistance of tobacco; A5) Prevention and control of plant diseases; A6) Preparation of fungal agents for preventing and controlling various plant diseases The plant diseases include tobacco bacterial wilt and tobacco target spot.

5. The use according to claim 4, characterized in that: Promoting tobacco growth is reflected in the increase of tobacco plant height and / or yield, and improving tobacco disease resistance is reflected in the promotion of high expression of NtPAL and / or NtNPR1.

6. A method for promoting tobacco growth and / or increasing tobacco yield, characterized in that: Treating tobacco with the tobacco blight fungus (Phytophtora nicotianae) JH-11-1 described in claim 1 or the bacterial agent described in any one of claims 2-3 increases the plant height and / or yield of the tobacco plants.

7. The method according to claim 6, characterized in that: The concentration of Phytophtoranicotianae JH-11-1 in the bacterial agent is 0.08 g / ml of bacterial suspension.

8. A method for improving tobacco disease resistance or preventing and controlling plant diseases, characterized in that: Tobacco is treated with the tobacco phytophthora nicotianae JH-11-1 according to claim 1 or the plant vaccine according to any one of claims 2-3.

9. The method according to claim 8, characterized in that: Improving tobacco disease resistance is reflected in promoting high expression of NtPAL and / or NtNPR1.

10. The method according to claim 8, characterized in that: The plant diseases include tobacco bacterial wilt and tobacco target spot.

Images