Non-pathogenic penicillium ochrochloron and application of non-pathogenic penicillium ochrochloron in biological control and growth promotion

Abstract

The invention belongs to the field of microorganisms, and particularly provides a non-pathogenic penicillium ochrochloron strain which is derived from garlic rhizosphere soil, is classified and identified as penicillium ochrochloron, is preserved in the China General Microbiological Culture Collection Center (CGMCC), and has the preservation number of CGMCC 41151. The strain has broad-spectrum bacteriostatic activity and can antagonize various plant pathogens; the plant growth promoting agent has the growth promoting characteristic, can secrete siderophores and phospholytic enzymes, and improves plant iron and phosphorus nutrition; the composition is safe and free of pathogenicity to crops. Tests show that the P4 strain can relieve plant iron deficiency yellowing and promote crop growth. The invention also provides application of the strain in biological control of plant diseases and plant growth promotion.

Description

Technical Field

[0001] The present invention relates to a strain, specifically to a non - pathogenic Penicillium ochrochloron and its application in biological control and growth promotion. Background Art

[0002] The Penicillium ochrochloron P4 strain is a novel functional strain isolated from the rhizosphere soil of garlic. This strain has the following remarkable characteristics: (1) broad - spectrum antibacterial activity, with the inhibition rate against 19 common plant pathogens reaching 47.82% - 81.57%; (2) high - efficiency growth - promoting ability, capable of secreting siderophores (yield Su = 75.5%) and phosphatase, significantly improving the iron and phosphorus nutritional status of plants; (3) strong environmental adaptability, and its siderophore synthesis can be significantly induced by the sulfur - containing metabolite DADS of garlic; (4) high safety, non - pathogenic to crops such as tomatoes and peppers. Experiments have proved that the P4 strain can not only effectively alleviate iron - deficiency chlorosis in tomatoes (the SPAD value is equivalent to that of chemical iron fertilizers), but also significantly promote the growth of peppers through root irrigation treatment (biomass increased by more than 30%).

[0003] In agricultural production, continuous cropping obstacles and soil - borne diseases of crops are key problems restricting the sustainable development of the industry. Taking Panax notoginseng as an example, its continuous cropping obstacles are mainly manifested as the imbalance of soil microbial communities, the deterioration of physical and chemical properties, and allelopathic autotoxicity, resulting in reduced emergence rate, aggravated diseases, and seriously affecting yield and quality. Similarly, solanaceous crops such as tomatoes and peppers are often invaded by soil - borne diseases such as Fusarium wilt and early blight. Traditional chemical control not only easily causes pathogen resistance, but also causes environmental pollution and agricultural product safety problems.

[0004] Microbial control has become a research hotspot due to its advantages such as environmental friendliness and sustainability. Penicilliumspp., as a widely existing non - pathogenic fungus, has the characteristics of simple cultivation and strong adaptability, showing unique value in the field of biological control. However, existing biocontrol strains generally have problems such as narrow control spectrum or unsatisfactory antagonistic effects, which limit their actual application effects. At the same time, the problem of iron deficiency in plants also seriously affects crop growth. Traditional iron fertilizers have defects such as poor stability and low utilization rate, and there is an urgent need to develop new biological iron - supplement strategies. Summary of the Invention

[0005] Aiming at the problems of narrow control spectrum, poor antagonistic effect and difficult effective improvement of iron deficiency nutrition in plants in the above - mentioned prior art, the present invention provides a new microbial strain with both broad - spectrum biocontrol activity and growth - promoting function and its application to fill the gaps in the prior art, improve the control effect of crop diseases and promote crop growth.

[0006] Specifically, the present invention provides the following technical solutions:

[0007] In the first aspect of the present invention, a non-pathogenic fungal strain, Penicillium ochrochloron P4, isolated from the rhizosphere soil of garlic is provided. The strain was deposited on April 11, 2024 at the China General Microbiological Culture Collection Center (CGMCC), and the deposit number is CGMCC 41151. The strain P4 has the activity of broadly antagonizing a variety of plant pathogenic fungi and can secrete siderophores and phosphatase.

[0008] In the second aspect of the present invention, the application of the strain P4 in controlling plant diseases is provided.

[0009] In the third aspect of the present invention, the application of the strain P4 in promoting plant growth is provided, including being used as a biological fertilizer to supplement iron elements and increase crop yields.

[0010] In the fourth aspect of the present invention, a biological agent containing the strain P4 is provided.

[0011] In the fifth aspect of the present invention, a preparation method of the above biological agent is provided, including culturing the strain P4 and mixing the obtained culture with a carrier to prepare the biological agent.

[0012] In the sixth aspect of the present invention, a method for controlling plant diseases using the strain P4 is provided.

[0013] In the seventh aspect of the present invention, a method for promoting plant growth using the strain P4 is provided.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0015] The Penicillium ochrochloron P4 has both broad-spectrum and highly efficient biocontrol activities and significant growth-promoting functions, and can solve the problems of crop disease control and nutrition improvement with one strain and multiple effects. Using the strain P4 to control diseases can effectively inhibit the growth of a variety of soil-borne plant pathogens, broaden the application scope of biological control and the antagonistic effect is significant; using the strain P4 to supplement iron and promote growth can significantly improve the nutritional status of plants such as iron and phosphorus, relieve the symptoms of iron deficiency chlorosis in crops, promote plant growth and development and increase crop yields. At the same time, the strain P4 is safe and non-toxic to crops, does not cause environmental pollution and pesticide residues, and avoids the drug resistance and food safety hazards brought by chemical pesticides and fertilizers. In addition, the strain has strong adaptability and simple cultivation, and is convenient for large-scale fermentation to prepare biocontrol biological fertilizer preparations for agricultural production. In summary, the present invention provides a new biological control and plant growth-promoting agent that is environmentally friendly, has diverse functions and broad application prospects. Description of the Drawings

[0016] The accompanying drawings are used to provide a further understanding of the present invention and form a part of the specification. They are used together with the embodiments of the present invention to explain the present invention and do not constitute a limitation to the present invention. In the drawings:

[0017] Figure 1 Morphological characteristics of Penicillium ochrochloron P4 strain on PDA medium, where a and b are colony morphologies, and c is the morphology of hyphae and conidia;

[0018] Figure 2 Penicillium ochrochloron P4 strain produces a red halo of siderophore and a transparent halo of phosphatase;

[0019] Figure 3 The iron carrier filtrate produced by P4 strain is mixed with CAS at a ratio of 1:1;

[0020] Figure 4 Confrontation of Penicillium ochrochloron P4 strain with pathogenic fungi on a flat plate;

[0021] Figure 5 Comparison diagrams of new leaves after 7 days of each treatment;

[0022] Figure 6 SPAD values of young leaves after 7 days of each treatment;

[0023] Figure 7 Siderophore production of P4 strain at different DADS concentrations;

[0024] Figure 8 Effect of Penicillium ochrochloron P4 strain on the growth of chili peppers. Detailed implementation mode

[0025] In order to make the technical means, creative features, achieved purposes and effects of the present invention easy to understand, the present invention will be further described below with reference to specific drawings.

[0026] Example 1 Isolation and identification of Penicillium ochrochloron (ochre green penicillium) P4 strain

[0027] 1) Isolation and purification of the strain

[0028] Weigh 10 g of garlic rhizosphere soil and add it to a triangular flask containing 90 mL of sterile water. Shake and culture it on a shaker at 25 °C for 30 min, and then perform 10-fold serial dilutions to 10 -1 、10 -2 . Pipette 0.1 mL of the dilution and spread it evenly on a PDA plate supplemented with streptomycin sulfate (100 ppm). Incubate it upside down in a constant temperature incubator at 28 °C for 1 - 3 d and observe regularly; after suspected colonies grow in the petri dish, immediately pick them and transfer them to a fresh PDA plate for numbering and preservation; use the hyphal tip method for purification to obtain as Figure 1The pure culture shown. The composition of the PDA plate is: 200 g / L of potato, 20 g / L of glucose, and 20 g / L of agar.

[0029] 2) Identification of the strain

[0030] Genomic DNA of the strain after extraction, isolation, and purification was amplified by PCR using the following 2 pairs of primers:

[0031] ① ITS primer pair: Forward primer ITS1: 5’-TCCGTAGGTGAACCTG CGG-3’ (SEQ ID NO.1), reverse primer ITS4: 5’-TCCTCCGCTTATTGATATGC-3’ (SEQ ID NO.2).

[0032] ② Primer pair for β-tubulin: Forward primer BT2a: 5'-GGTAAC CAAATC GGT GCT GCT TTC-3' (SEQ ID NO.3), reverse primer Bt2b: 5'-ACC CTCAGT GTAGTGACC CTT GGC-3' (SEQ ID NO.4)

[0033] The above amplified sequences were aligned in the NCBI database. After BLAST alignment, the similarity with Penicillium ochrochloron was the highest. Combining morphological identification, the taxonomic name was determined to be Penicillium ochrochloron, and the strain was named P4.

[0034] The Penicillium ochrochloron P4 strain was cultured on a glucose potato agar medium (PDA) for 7 days. As Figure 1 shown, the strain could cover a petri dish with a diameter of 9 cm. The spores of Penicillium Q-4 were densely distributed and spread from the middle to the periphery in an oval shape. The growth rate was fast, the surface was smooth, and the colony was villous. The hyphae were septate, the conidiophores were branched and septate, and the conidia were nearly round.

[0035] The preservation information of this Penicillium ochrochloron P4 is as follows: The preservation number is CGMCC No. 41151; the taxonomic name is: Penicillium ochrochloron; the preservation unit is the General Microbiology Center of the China Committee for Culture Collection of Microorganisms; the preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postcode: 100101; the preservation date is April 11, 2024.

[0036] Example 2 Detection of the growth-promoting effect of Penicillium ochrochloron P4 strain

[0037] The following steps are for preparing siderophore and phosphorus-solubilizing detection medium:

[0038] 1) CAS-nutrient double-layer medium: The lower layer is CAS medium (CAS medium is chrome azurol detection medium for detecting the production of siderophores by microorganisms), and the upper layer is PDA medium. When making the medium, use ddH2O, sterilize at 121 °C for 20 minutes, and pour the upper layer medium after the lower layer medium has cooled, and wait for all to cool before use.

[0039] 2) Phosphorus-solubilizing medium: Glucose: 10 g; Ca3(PO4)2: 10 g; MgCl2·6H2O: 5 g; MgSO4·7H2O: 0.25 g; KCl: 0.2 g; (NH4)2SO4: 0.1 g; Agar: 20 g; 1 L ddH2O. Sterilize at 121 °C for 20 minutes, and then pour into petri dishes and wait for cooling before use.

[0040] 3) Quantitative detection of siderophores: ① Fermentation broth preparation: Add P4 bacterial cakes (d = 0.5 cm) to the sterilized PDB at a rate of 5 bacterial cakes / 50 ml. The PDB without added bacterial cakes is used as a control CK. Shake culture at 28 °C and 120 rmp / min for 7 days. Filter the fermentation broth through a 0.22 μm filter membrane and collect the filtrate for use. ② Prepare CAS staining solution: Weigh 0.121 g of CAS (chrome azurol S) and dissolve it in 50 ml of ddH2O, and add 2 ml of 1 mmol / L FeCl3 solution to make solution a. Weigh 0.015 g of CTAB and dissolve it in 8 ml of ddH2O to make solution b. Slowly add solution a to solution b while shaking solution b to make the CAS staining solution. ③ Mix the filtrates of each treatment with CAS at a ratio of 1:1 in a 96-well plate, and detect the OD630 value in an enzyme-linked immunosorbent assay instrument. Calculate the Su value according to the formula Su = (Ar - A) / Ar * 100% (Ar is the OD value of CK, and A is the OD value of the P4 filtrate).

[0041] 4) Result display: If there is a red or yellow halo around the colony on the CAS-nutrient double-layer medium, it indicates that the microorganism can secrete siderophores. If there is a transparent halo ( Figure 2 ) around the colony on the phosphorus-solubilizing medium, it indicates that the microorganism can secrete phosphatase. The amount of siderophores produced by the P4 strain after 7 days of shaking culture is Su = 75.5% ( Figure 3 ).

[0042] Determination of the antagonistic ability of the Penicillium ochrochloron P4 strain described in Example 3 against the pathogenic bacteria of tomato, pepper and Panax notoginseng diseases

[0043] The 19 pathogenic fungi were co-cultured with Penicillium ochrochloron P4 strain by the plate confrontation method. The original fungi include but are not limited to plant pathogenic fungi of the genera Stemphylium, Fusarium, Colletotrichum, Helminthosporium, Alternaria, Fusarium, Peronophythora, Valsa, Cladosporium, Bipolaris, Rhizoctonia, Gaeumannomyces, Cylindrocarpon, such as Fusarium oxysporum f. sp. lycopersici, Alternaria solani, Fusarium verticillioides, Phytophthora capsici, Cladosporium cucumerinum, Peronophythora litchii, Colletotrichum gloeosporioides, Rhizoctonia solani, Gibberella fujikuroi, Bipolaris oryzae, Fusarium graminearum, Gaeumannomyces graminis var. tritici, Exserohilum turcicum, Helminthosporium torulosum (Syd.) Ashby, Didymelia-brvoniaee, Phytophthora parasitica var. Nicotianae, Cylindrocarpon destructans.

[0044] 1) One test pathogen mycelial disc with a diameter of 5 mm was inoculated at the center of the PDA medium, and then four mycelial discs of Penicillium ochrochloron P4 strain were placed around the central mycelial disc, with a distance of about 3 cm between each other. Each treatment was repeated 6 times. The control was inoculated with only the pathogen mycelial disc. It was cultured in a constant temperature incubator at 28 °C. When the control strain covered 2 / 3 of the entire culture dish, the colony radius of the control strain and the colony radius of the pathogen at the confrontation center were measured, and the inhibition rate was calculated.

[0045] Inhibition rate (%) = (control colony radius - confrontation culture colony radius) / control colony radius × 100

[0046] 2) The results are as Figure 4As shown, the Penicillium ochrevicinum P4 strain can significantly inhibit the growth of common crop disease pathogens. Compared with the control group, the growth of 19 pathogens tested was significantly inhibited, with inhibition rates ranging from 47.82% to 81.57%. Among them, the antagonistic inhibition rate against rice seedling disease (Rhizoctonia solani) was the highest, reaching 81.57±4.49%. The Penicillium ochrevicinum P4 strain has broad-spectrum antibacterial activity.

[0047] Table 1 Antagonistic ability of Penicillium ochraceum P4 strain against 19 pathogens

[0048]

[0049]

[0050] Example 4: Penicillium ochregreenii P4 strain alleviates tomato iron deficiency chlorosis detection

[0051] Utilizing Fe3 + environment to explore whether the P4 strain can utilize Fe3 + In order to alleviate the iron deficiency chlorosis of tomatoes, the specific experimental steps are as follows:

[0052] 1) Tomato plants with iron-deficiency etiolation: Prepare Hoagland's iron-deficient nutrient solution in ddH2O as an iron-free treatment, and Hoagland's complete nutrient solution as a control. Place the nutrient solution in tissue culture flasks for later use. Prepare seedling tomato seedlings, clean their roots, and rinse again with ddH2O. Place the treated seedlings in tissue culture flasks, culture in a growing room with uniform light, and observe the etiolation process.

[0053] 2) Preparation of Siderophore-Producing Microbial Fluid: Prepare the purified microbial plate. Use a 0.5 cm diameter punch to punch out bacterial cakes at the edge of the colony. Place the bacterial cakes in sterilized PDB at a density of 30 cakes / L. Then, incubate the plate on a shaker at 120 rpm and shake at 28°C for 7 days.

[0054] 3) Microbial culture iron supplementation experiment

[0055] The hydroponic method was used, and the positive control was set as follows: Hoagland's complete nutrient solution (EDTA-Na2Fe 30mg / L) Fe2+0.071M; Fe3 + Chelated iron positive control: Hoagland's iron-deficient nutrient solution + (EDTA-NaFe 26.15 mg / L) Fe3 +0.071 M; Negative control group: Hoagland's iron-deficient nutrient solution; Microbial negative control: Hoagland's iron-deficient nutrient solution + (FeCl3·6H2O 19.27 mg / L) Fe3+ 0.071 M + Q-9 bacterial solution (Q-9 is a Penicillium strain that does not produce siderophores); Treatment with P4 strain: Hoagland's iron-deficient nutrient solution + (FeCl3·6H2O 19.27 mg / L) Fe3+ 0.071 M + P4 bacterial solution. During the experiment, pay attention to supplementing the basic culture solution, Hoagland's iron-deficient nutrient solution + (FeCl3·6H2O 19.27 mg / L) Fe3 + 。

[0056] 4) Detection of chlorophyll in young tomato leaves:

[0057] On the 5th day, the iron-deficiency chlorotic new leaves changed from yellow to green, and on the 7th day, the SPAD value was measured. The chlorophyll level of fresh young tomato leaves (fully germinated leaves at the top) was measured using a chlorophyll meter. All the young leaves of this treatment were picked as the total sample of this treatment, and 12 leaves were randomly selected from the total sample to measure the SPAD value. Each leaf was measured in 4 replicates, avoiding measuring the main vein, and the average value was taken.

[0058] 5) The results showed that Penicillium ochrochloron P4 strain could significantly alleviate iron-deficiency chlorosis in tomatoes ( Figure 5 ), and its iron-supplementing effect had no significant difference from that of chemical chelated iron fertilizer in naked-eye observation and SPAD value, and significantly alleviated iron-deficiency chlorosis compared with the iron-deficient control and Q-9 treatment ( Figure 6 ).

[0059] Example 5 Garlic sulfur metabolite DADS promotes siderophore production by Penicillium ochrochloron P4 strain

[0060] Use a drug-containing medium to shake-culture microorganisms, then mix the fermentation broth filtrate with CAS at a ratio of 1:1, measure the OD630 value with a 96-well plate, and finally calculate the Su value of each treatment according to the formula. The specific steps are as follows:

[0061] 1) Prepare PDB with four concentrations of DADS at 0 ppm, 4 ppm, 8 ppm, and 40 ppm: Use ddH2O when making the medium, sterilize at 121 °C for 20 minutes, and wait for the medium to cool before use. Add DADS that has passed through a 0.22 μm filter membrane to make its concentration 0 ppm, 4 ppm, 8 ppm, 40 ppm, and 0 ppm as the CK control, with 6 replicates for each treatment.

[0062] 2) Add P4 bacterial cakes (d = 0.5 cm) to the above medium at a rate of 5 bacterial cakes / 50 ml, shake-culture at 28 °C and 120 rmp / min for 7 days, filter the fermentation broth through a 0.22 μm filter membrane, and collect the filtrate for use.

[0063] 3) Prepare the CAS staining solution: Weigh 0.121 g of CAS (Chrome Azurol S) and dissolve it in 50 ml of ddH2O, and add 2 ml of 1 mmol / L FeCl3 solution to make solution a; Weigh 0.015 g of CTAB and dissolve it in 8 ml of ddH2O to make solution b; Slowly add solution a to solution b while shaking solution b to make the CAS staining solution.

[0064] 4) Mix the filtrates of each treatment with CAS at a ratio of 1:1 in a 96-well plate, and detect the OD630 value in an enzyme-linked immunosorbent assay (ELISA) reader. Calculate the Su value according to the formula Su = (Ar - A) / Ar * 100%.

[0065] 5) The results show ( Figure 7 ): The sulfur-containing metabolite DADS in garlic can significantly promote the production of siderophores by strain P4, and there is a strong correlation between the production and the concentration.

[0066] Example 6 Penicillium ochrochloron strain P4 can promote the growth of pepper plants

[0067] A pepper pot experiment was conducted to verify whether Penicillium ochrochloron strain P4 can promote the growth of peppers. The specific experimental operations are as follows:

[0068] Plant four pepper plants in each pepper pot, and set two treatment groups, namely the P4 bacterial liquid treatment group and the blank control group, for the pepper pots with consistent growth, and each treatment has 6 replicates. Punch the purified P4 plate into bacterial cakes with a diameter of 5 mm, inoculate them into the previously prepared sterilized and cooled PDB, and shake culture them on a shaker at 28 °C and 120 rmp / min for 5 days to obtain the P4 bacterial liquid. Irrigate the roots of the P4 bacterial liquid pepper pot treatment group once every week, and irrigate the same amount of water for the blank group, and manage them in the same way at other times. After one month, measure the biomass of the pepper pots in each treatment ( Figure 8 ). Randomly select 6 pepper plants, measure the plant height after washing, then dry them at a low temperature in an oven, and finally weigh the dry weight of the peppers in each treatment.

[0069] The results show that the plant height and biomass of the peppers treated with P4 irrigation are significantly higher than those of the blank treatment, indicating that Penicillium ochrochloron strain P4 can significantly promote the growth of peppers.

[0070] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

1. A non-pathogenic Penicillium ochrochloron strain P4, characterized in that, The strain was deposited on April 11, 2024 at the China General Microbiological Culture Collection Center (CGMCC), and the deposit number is CGMCC 41151.

2. Use of the Penicillium ochrochloron P4 strain according to claim 1 in the control of plant diseases.

3. Use of the Penicillium ochrochloron P4 strain according to claim 1 in promoting plant growth.

4. The use according to claim 3, characterized in that, The promotion of plant growth includes improving plant iron nutrition and increasing crop yield.

5. A biological agent, characterized in that, The preparation contains the Penicillium ochrochloron P4 strain according to claim 1.

6. The preparation method of the biological agent according to claim 5, wherein, The method comprises the following steps: culturing the Penicillium ochrochloron P4 strain according to claim 1 to obtain a strain fermentation product; mixing the fermentation product with a carrier to prepare the biological preparation.

7. A method for controlling plant diseases, characterized in that, Applying an effective amount of the Penicillium ochrochloron P4 strain according to claim 1 to the leaf surface, seeds or growth environment of a plant to inhibit the growth of plant pathogens.

8. A method for promoting plant growth, characterized in that, Applying an effective amount of the Penicillium ochrochloron P4 strain according to claim 1 to the leaf surface, seeds or growth environment of a plant to improve plant iron and phosphorus nutrition and increase crop yield.

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