Phytophthora nicotianae JH-11-1 and application thereof in tobacco growth promotion and disease control

Abstract

The present application relates to a low virulence tobacco Phytophthora JH-11-1 and its application in tobacco growth promotion and disease prevention. The tobacco Phytophthora JH-11-1 has weak pathogenicity to tobacco and has growth promotion and disease prevention effects, and can be developed as a biocontrol agent for biological control of tobacco diseases. Compared with the current chemical agents for preventing and treating tobacco diseases, the biocontrol agent has the characteristics of non-toxic, non-residual, non-polluting and not easy to produce drug resistance, and has a good market application prospect.

Description

Technical Field

[0001] This invention belongs to the field of plant growth-promoting bacteria technology, specifically relating to a low-virulence Phytophthora tobaccoii strain JH-11-1 and its application in tobacco growth promotion and disease control. Background Technology

[0002] Tobacco diseases are a significant limiting factor in tobacco production, severely impacting yield and quality. Currently, diseases such as black shank, bacterial wilt, and target spot are common in tobacco-producing areas. For example, black shank causes an average annual economic loss of over 100 million yuan in my country. While chemical fungicides remain an effective means of controlling tobacco diseases, their use leads to soil pollution, pathogen resistance, harm to non-target organisms, and pesticide residues, hindering the promotion of green, environmentally friendly, and pollution-free tobacco cultivation and sustainable agricultural development. Therefore, finding new, safe, and effective measures (such as biological control strategies) to control tobacco diseases is essential.

[0003] When some pathogenic microorganisms interact with plants, they induce systemic resistance. Pathogens can further stimulate plants to develop both local and systemic resistance. Upon contact, plants develop systemic acquired resistance throughout the plant, preventing further spread of the pathogen to other parts. Plants utilize receptor kinases (RKs) and receptor-like kinase proteins (RLPs) as pattern recognition receptors (PRRs) to recognize pathogen / microorganism-associated pattern molecules (PAMPs / MAMPs), inducing both local and systemic resistance. Studies have shown that pathogen pretreatment of plants alters their physiological, transcriptional, metabolic, and epigenetic levels, activating defense mechanisms and enhancing their defensive capabilities. Upon re-encountering the pathogen, plants effectively elevate their defense responses to a faster or stronger degree. This defense response is maintained throughout the growing season and can even be passed on to offspring. Pathogens can induce plant resistance and have potential application value. Furthermore, pathogenic strains that have lost their pathogenicity and grow endophytically in plants may enhance the plant's disease resistance and promote growth, triggering a series of defense responses, such as the production of antimicrobial substances and alterations in cell wall composition. These responses, while defending against pathogens, may also promote plant growth. Moreover, pathogen infection can affect the balance of plant hormones, such as increasing the synthesis of hormones like cytokinins and gibberellins. These hormones promote cell division and elongation, thereby promoting plant growth. Therefore, these strains can serve as potential candidates for preparing plant vaccines. Summary of the Invention

[0004] Specifically, this invention provides a low-virulence strain of Phytophthora nicotianae JH-11-1, which was deposited at the China Center for Type Culture Collection on October 11, 2024, with accession number CCTCC M20242162.

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

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

[0007] The present invention also provides the use of Phytophthora nicotianae JH-11-1 or the fungal agent or plant vaccine in any of the following A1)-A6):

[0008] A1) Promotes tobacco growth;

[0009] A2) Prepare microbial agents that promote tobacco growth;

[0010] A3) Improve tobacco disease resistance;

[0011] A4) Prepare microbial agents to enhance broad-spectrum disease resistance in tobacco;

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

[0013] A6) Preparation of fungicides for the prevention and control of various plant diseases

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

[0015] Furthermore, promoting tobacco growth is manifested in increased tobacco plant height and / or yield, while improving tobacco disease resistance is manifested in the promotion of high expression of NtPAL and / or NtNPR1.

[0016] The present invention also provides a method for promoting tobacco growth and / or increasing tobacco yield by treating tobacco with Phytophthora nicotianae JH-11-1 or the fungal agent, thereby increasing the plant height and / or yield of tobacco plants.

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

[0018] The present invention also provides a method for improving the disease resistance of tobacco or preventing plant diseases by treating tobacco with Phytophthora nicotianae JH-11-1 or the plant vaccine.

[0019] Furthermore, enhancing tobacco disease resistance manifests as promoting high expression of NtPAL and / or NtNPR1.

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

[0021] The *Phytophthora indicum* JH-11-1 described in this invention has weak pathogenicity and growth-promoting effects on tobacco. It can be developed into a biocontrol agent for the biological control of tobacco diseases. Compared with current chemical agents for controlling tobacco diseases, it has the characteristics of being non-toxic, residue-free, pollution-free, and less likely to induce drug resistance, and has good market application prospects. Attached Figure Description

[0022] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 Colony morphology of Phytophthora intoxin cultured for 7 days.

[0024] Figure 2 Growth rate of each strain of Phytophthora indicum.

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

[0026] Figure 4 : Mycelial abundance of Phytophthora tobaccoensis JH-11-1 in different culture media.

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

[0028] Figure 6 Verification of the growth-promoting effect of Phytophthora indicum JH-11-1. Figure A shows tobacco plant height, and Figure B shows tobacco yield. CK: water, A: tobacco vaccine, D: 58% metalaxyl-mancozeb, E: 10 billion Bacillus subtilis. Detailed Implementation

[0029] The following embodiments are only used to more clearly illustrate the technical solutions of the present invention, and are therefore merely examples and should not be used to limit the scope of protection of the present invention. It should be noted that, unless otherwise stated, the technical or scientific terms used in this application should have the ordinary meaning understood by those skilled in the art. Unless specifically stated, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in this technical field. Unless specifically stated, the reagents and materials used in the following embodiments are commercially available.

[0030] Example 1: Isolation and purification of Phytophthora tobaccoii and strain identification test

[0031] The *Phytophthora indicum* strains isolated in this invention were all collected from tobacco black shank diseased plants from tobacco bases in Chenzhou City and Xiangxi Prefecture, Hunan Province in 2023. Specifically, the black shank diseased plants collected from tobacco-producing areas underwent the following procedures:

[0032] Tobacco discs infected with *Phytophthora nicotineae* were carefully handled with tweezers, then surface-sterilized with 75% alcohol for 30 seconds, rinsed three times with sterile water, and air-dried on sterile filter paper. The cleaned tobacco discs were placed in OA (oxygenated oxygen) petri dishes (containing 300 μg / mL cefixime) and incubated statically at 28°C for 3-5 days. Once mycelia had grown, they were transferred to new OA petri dishes for subculture. The isolated and purified strains were preserved using two methods: slant culture and glycerol preservation. Fresh mycelial blocks were placed in 1 mL OA slant and incubated at 28°C until the mycelia covered the slant, then stored at 4°C for long-term preservation; three copies of each strain were also preserved. Alternatively, fresh mycelial blocks were placed in 25% glycerol and stored at -80°C; three copies of each strain were also preserved.

[0033] DNA extraction from the strain: Genomic DNA was extracted using the conventional CTAB method, as follows: A 4 × 4 cm² mycelial block grown for 3 days was picked up with a pick and placed in a 2 ml sterile centrifuge tube containing steel beads. 1 ml of CTAB extraction buffer was added, and the block was broken up at 50 Hz for 300 s using a grinder. The mixture was then incubated in a 65 °C water bath for 10 min (inverting 2–3 times during this period). The 2 ml centrifuge tube was centrifuged at 12000 rpm for 10 min. The supernatant was transferred to a new 2 ml sterile centrifuge tube, and an equal volume of extraction buffer I was added. The mixture was then inverted and mixed. The tube was centrifuged again at 12000 rpm for 10 min, and the supernatant was transferred to a new 2 ml sterile centrifuge tube. An equal volume of extraction buffer II was added, and the mixture was inverted and mixed. The tube was centrifuged again at 12000 rpm for 10 min, and the supernatant was transferred to a new 1.5 ml sterile centrifuge tube. An equal volume of isopropanol was added, and the mixture was incubated at -20 °C. Incubate at °C for at least 30 min to allow precipitation; then centrifuge at 12000 r / min for 15 min, discard the supernatant, add 700 μL of 75% anhydrous ethanol, resuspend, and centrifuge at 12000 r / min for 3 min. Repeat once. Discard the supernatant, aspirate the remaining liquid with a pipette, air dry, dissolve in 30–100 μL of sterile deionized water, and store at -20°C.

[0034] PCR amplification was performed 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 as follows:

[0038]

[0039] PCR amplification procedure:

[0040]

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

[0042] BLAST alignment of the fragment sequencing results showed that these strains had more than 99% homology with Phytophthora tobaccoensis sequences.

[0043] Example 2: Morphological observation and growth rate determination of Phytophthora intoxin

[0044] Prepare oatmeal agar medium.

[0045] 1. Weigh 30 g of rolled oats and transfer them to a glass dish. Add 1000 mL of sterile water. Boil the oats on the top of an induction cooker. Once the oats become viscous, filter the liquid solution through a piece of sterile gauze. 2. Pour the filtrate into a 1000 mL graduated cylinder and bring the volume to 1000 mL with sterile water. 3. Pour the liquid solution into a glass reagent bottle and add 18 g of agar. Shake well and autoclave at 121°C for 15 min. Let stand at room temperature for 30 min. 4. Pour approximately 20 mL of sterilized rolled oat agar medium into each petri dish. Allow the petri dishes to cool completely at room temperature before mycelial culture.

[0046] Fresh hyphae were collected near the edge of the colony using a 5 mm punch. The hyphae were picked up and placed in the center of a 60 mm petri dish containing 10 mL of OA. The dish was incubated at 28°C. The colony morphology was recorded with a camera at 2 days and 4 days after inoculation. The diameter of each colony was measured using the cross-hatching method.

[0047] The results are as follows Figure 1 , Figure 2 As shown, the mycelial growth of Phytophthora tobaccoii strain JH-11-1 is abnormal, and its growth rate is significantly lower than that of other strains. Strain JH-11-1 was deposited at the China Center for Type Culture Collection (CCTCCM) under accession number CCTCCM 20242162.

[0048] Experimental Example 3: Pathogenicity determination of Phytophthora indicum JH-11-1

[0049] 1. Inoculate the mycelium onto OA medium and incubate at 28 ℃ in the dark. Prepare a 1 cm diameter punch and toothpicks beforehand, and autoclave at 121 ℃ for 15 min.

[0050] 2. Make holes in the tobacco black shank mycelial agar medium to form circular mycelial mats. 3. Pick up a mycelial mat, place it with the mycelial side down on oatmeal agar medium, and incubate the mycelium in the dark at 28 ℃ for 7 days.

[0051] 3. Using a 1 cm diameter punch, make holes in the tobacco black shank mycelial agar medium to create circular mycelial pads. Use a toothpick to pick up the mycelial pads and inoculate them onto detached leaves. Perform three replicates for each strain. After inoculation, place the inoculated leaves in a 28 ℃ light incubator for four days and observe the disease development.

[0052] The results are as follows Figure 3As shown, the pathogenicity of Phytophthora tobaccoensis strain JH-11-1 is significantly lower than that of other strains, exhibiting weak virulence.

[0053] Experiment Example 4: Fermentation Experiment of Phytophthora indicum JH-11-1 Mycelium

[0054] Effects of different C and N sources on mycelial yield: Nine formulations were designed based on different C and N sources. All formulations contained 1.5 g / L magnesium sulfate and 3.0 g / L potassium dihydrogen phosphate, with a natural pH. The liquid culture medium was 100 mL, the inoculum size was 14%, and the culture was carried out in a shaker at 120 rpm and 28°C. After 7 days, the mycelial dry weight was observed and recorded.

[0055] Table 1. Liquid culture formulations (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 *Phytophthora indicum* strain JH-11-1 was inoculated onto the leaves of live Yunyan 87 tobacco plants using the method described in Experiment Example 3. The leaves were placed with the mycelial side down. Samples were taken at 0, 6, 12, 18, 24, 30, and 36 days post-inoculation, with simultaneous inoculation and four biological replicates at each sampling point. Samples were rapidly incubated with liquid nitrogen after collection. Total RNA was extracted from all samples using the TRIzol method. cDNA synthesis was performed using a reverse transcription kit from Beijing TransGen Biotech Co., Ltd. Quantitative real-time PCR reaction conditions: 95 °C for 5 min; 95 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s (40 cycles).

[0060] like Figure 5 As shown, after inoculation with *Phytophthora indicum* strain JH-11-1, the expression levels of phenylalanine ammonia-lyase (NtPAL)-related genes were significantly increased at 12 h and 36 h compared to 0 h. Meanwhile, the expression level of NtNPR1-related genes was significantly higher at 12 h than at 0 h. This indicates that inoculation with *Phytophthora indicum* strain JH-11-1 can enhance the expression of resistance genes related to Yunyan 87, thereby promoting disease resistance in the plants.

[0061] Experiment Example 6: Verification of the growth-promoting and disease-resistant effects of Phytophthora indicum JH-11-1

[0062] Test locations: Chenzhou and Xiangxi Prefecture

[0063] Experimental Methods: Four treatments were set up, each repeated three times, for a total of 12 plots. Treatments included a water control (CK), a tobacco vaccine (A: JH-11-1), chemical agents (D: 58% metalaxyl-mancozeb, Sichuan Guoguang), and a biological agent (E: 10 billion Bacillus subtilis, purchased from Shaanxi Hengtian Biological Agriculture Co., Ltd.). Each plot had an experimental area of ​​30 square meters. One mu (approximately 0.067 hectares) was reserved for each experimental plot. The experiment was arranged in a randomized block design, with 3-4 protective rows surrounding the experimental plot.

[0064] Results: Five sampling points were used at equal intervals within the treatment area, with 10 tobacco seedlings surveyed at each point. The effects of different treatments on plant disease resistance and yield were evaluated by comparing yield and disease incidence. Disease incidence and disease index baselines were surveyed before application. After application, disease incidence and disease index were surveyed every 7 days following disease development in the control area, and the control efficacy was calculated. The tobacco disease survey standard was based on the Chinese national standard Ren, G., et al. GB / T 23222 Grade and Investigation Method of Tobacco Diseases and Insect Pests. China Standard Press, Beijing (2008).

[0065] like Figure 6 As shown, compared with the control, the application of the tobacco vaccine (Group A) significantly increased tobacco plant height and yield. Further investigation of tobacco diseases in the experimental plots, as shown in Table 2, revealed that no black shank disease occurred in the experimental plots, while bacterial wilt and target spot disease occurred in all groups. However, the incidence and disease severity in the tobacco vaccine group were significantly better than those in the control group and other experimental groups, indicating that Phytophthora indica JH-11-1 has a good broad-spectrum disease resistance effect.

[0066] Table 2

[0067]

[0068] The above detailed embodiments describe the implementation of the present invention; however, the present invention is not limited to the specific details described in the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. A low-virulence strain of Phytophthora nicotianae JH-11-1 was deposited at the China Center for Type Culture Collection on October 11, 2024, with accession number CCTCC NO: M 20242162.

2. An inoculum containing Phytophthora nicotianae JH-11-1 or its suspension as described in claim 1.

3. The microbial agent according to claim 2, characterized in that: The concentration of Phytophthora icotianae JH-11-1 in the microbial agent is 0.08 g / ml.

4. A plant vaccine containing Phytophthora nicotianae JH-11-1 or a suspension thereof as described in claim 1.

5. The use of Phytophthora nicotianae JH-11-1 as described in claim 1 in any one of the following A1)-A6): A1) Promotes tobacco growth; A2) Prepare microbial agents that promote tobacco growth; A3) Improve tobacco disease resistance; A4) Prepare microbial agents to enhance broad-spectrum disease resistance in tobacco; A5) Prevention and control of plant diseases; A6) Prepare inoculants for the prevention and control of various plant diseases; The plant diseases mentioned are tobacco bacterial wilt and tobacco target spot.

6. The application according to claim 5, characterized in that: Promoting tobacco growth is manifested in increased tobacco plant height and / or yield, while enhancing tobacco disease resistance is manifested in the promotion of high expression of NtPAL and / or NtNPR1.

7. A method for promoting tobacco growth and / or increasing tobacco yield, characterized in that: Tobacco plants are treated with Phytophthora nicotianae JH-11-1 as described in claim 1, or any of the fungal agents described in claims 2-3, or the plant vaccine described in claim 4, resulting in an increase in plant height and / or yield.

8. The method according to claim 7, characterized in that: The concentration of Phytophthora icotianae JH-11-1 in the microbial agent is 0.08 g / ml.

9. A method for improving the disease resistance of tobacco or controlling plant diseases, characterized in that: Tobacco is treated with Phytophthora nicotianae JH-11-1 as described in claim 1, or any of the fungal agents described in claims 2-3, or the plant vaccine described in claim 4; wherein the plant disease is tobacco bacterial wilt or tobacco target spot.

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

Images