Application of rhizopus microsporus var. Microsporus var. Microsporus var. Microsporus var. Microsporus var.

Abstract

The invention provides application of rhizopus microsporus var. Microsporus var. Microsporus var. Microsporus var. Microsporus var. Microsporus var. Microsporus var. Microsporus var. Microsporus It is found for the first time that the rhizopus microsporus var. Microsporus var. Microsporus var. Microsporus var. Microsporus var. Microsporus var. Microsporus var. Microsporus var. Microsporus var. Microsporus var. Microsporus var. Microsporus var. Microsporus var. Microsporus var. Microsporus var. Microsporus var. Microsporus var. Microsporus var. Moreover, the strain has the capabilities of producing cellulase, fixing nitrogen, decomposing inorganic phosphorus and producing siderophores, and pot experiments show that the strain can promote the growth of peppers. The invention provides a new way for biological prevention and control of pepper phytophthora blight, and has positive significance for reducing the use of pesticides and improving the yield and quality of pepper.

Description

Technical Field

[0001] This invention relates to the field of plant disease control technology, specifically to the application of a Microsporum hua variety in controlling pepper blight and promoting pepper growth. Background Technology

[0002] Phytophthora blight is a disease caused by Phytophthora capsici (Phitophthora caps Phytophthora capsici) This soil-borne disease is widespread in chili-growing regions around the world, causing significant economic losses to chili production. It can occur at any stage of chili growth and is primarily spread through soil, irrigation water, and farm implements. Once infected, it spreads rapidly, causing water-soaked lesions on leaves, stems, and fruits, eventually leading to rot and wilting, severely impacting the yield and quality of chilies.

[0003] Currently, the control of pepper blight mainly relies on chemical pesticides and the breeding of disease-resistant varieties. However, the long-term use of chemical pesticides leads to pesticide residues in the soil and environmental pollution, and also enhances the resistance of pathogens, reducing the effectiveness of control. Furthermore, the breeding of disease-resistant pepper varieties is hampered by a lack of suitable materials; many varieties are only moderately resistant or tolerant, with very few highly resistant varieties, and therefore cannot effectively control pepper blight. Biological control, with its advantages of effectiveness, safety, low toxicity, low residue, and no development of resistance, is receiving increasing attention and has become a hot topic in research on the control of various plant diseases, showing broad development prospects.

[0004] Biological control mainly involves using antagonistic microorganisms, plant extracts, and biological agents to inhibit the growth and reproduction of pathogens. Antagonistic microorganisms primarily include bacteria, fungi, and actinomycetes. Endophytic fungi are fungi that live within plant tissues but do not cause obvious plant diseases; they form a symbiotic relationship with plants, enhancing plant resistance, promoting plant growth, and controlling various plant diseases. In recent years, an increasing number of endophytic fungi have been found to possess significant biocontrol potential, such as inhibiting pathogen growth by producing active substances like antibiotics, chitinases, and glucanases. The application of endophytic fungi in the control of pepper blight has also gradually attracted attention; therefore, it is necessary to screen and develop new plant endophytic fungi for the control of this disease to reduce pesticide use and improve pepper yield and quality. Summary of the Invention

[0005] In view of this, the present invention provides an endophytic fungus and method for the prevention and control of pepper blight, aiming to provide a safe and effective new biological control approach for the prevention and control of pepper blight.

[0006] In a first aspect, the present invention provides a method for inhibiting the growth of *Phytophthora capsici*, which uses *Microsporum hua* var. *hua* (… Rhizopus microsporus var. chinensis GZ1F2 is used as an inhibitor.

[0007] Furthermore, in the above method, a fermentation broth of *Microsporum hua* var. *hua* GZ1F2 is first prepared, and then the fermentation broth is used to inhibit the growth of *Phytophthora capsici*. In some embodiments of the present invention, the fermentation broth is prepared as follows: mycelial blocks of *Microsporum hua* var. *hua* GZ1F2 are inoculated into PDA liquid culture medium and cultured (28°C, 180 rpm for 72 h), the mycelia are removed with gauze, and then the culture medium is filtered through a 22 μm microporous membrane to obtain the fermentation broth.

[0008] Experimental data show that the fermentation broth of *Microsporum hua* var. GZ1F2 has at least the following effects on *Phytophthora capsici*: a) Inhibits the growth of *Phytophthora capsici* mycelium; b) Inhibits the formation of zoosporangia of Phytophthora capsici; c) Inhibit the motility of zoospores of Phytophthora capsici; d) Inhibit the germination of dormant spores of Phytophthora capsici.

[0009] Secondly, this invention provides the application of *Microsporum hua* variate GZ1F2 in the control of pepper blight and the promotion of pepper growth. *Microsporum hua* variate GZ1F2 not only significantly inhibits the growth and development of *Phytophthora capsici* in vitro, but pot experiments also show that it can effectively control pepper blight and promote pepper growth.

[0010] Furthermore, in the above application, the chili pepper is treated with root drenching of the microsporum rhizogenes var. GZ1F2. In some embodiments of the present invention, the root drenching treatment is performed by preparing a spore suspension of microsporum rhizogenes var. GZ1F2 and then using the spore suspension to drench the chili pepper roots. In some embodiments of the present invention, the root drenching treatment is carried out at the 4-leaf-1-heart stage of the chili pepper.

[0011] Thirdly, the present invention provides a microbial preparation for preventing and / or promoting the growth of peppers, comprising *Microsporum huahua* var. GZ1F2 and / or its spore suspension and / or its fermentation broth. The microbial preparation may be any one of a dry powder, a wettable powder, or a liquid formulation.

[0012] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention marks the first discovery that the *Microsporum hua* varietal *Phytophthora capsici* has a significant inhibitory effect on this fungus. Furthermore, applying a suspension of its spores to the roots of chili seedlings significantly enhances the chili's resistance to the disease, achieving a control efficacy of 68.42%. In addition, *Microsporum hua* varietal *Phytophthora capsici* GZ1F2 possesses the ability to produce cellulase, fix nitrogen, decompose inorganic phosphorus, and produce siderophores, which facilitates the absorption of nutrients from the soil by chili peppers, thereby promoting their growth. This invention provides a novel approach for the biological control of *Phytophthora capsici* in chili peppers, and has positive implications for reducing pesticide use and improving chili yield and quality. Attached Figure Description

[0013] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the present invention will be briefly described below. Obviously, the drawings described below are merely some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0014] Figure 1 The image shows the inhibitory effect of Microsporum vulgare GZ1F2 on Phytophthora capsici in Example 1. The left image is the control group, and the right image is the Microsporum vulgare GZ1F2 treatment group. Figure 2 The image shows the inhibitory effect of the fermentation broth of Microsporum vulga GZ1F2 on the mycelial morphology of Phytophthora capsici in Example 2. The left image is the control group, and the right image is the fermentation broth treatment group of Microsporum vulga GZ1F2. Figure 3 The graph shows the inhibitory effect of the fermentation broth of Microsporum vulga GZ1F2 on the zoosporangia of Phytophthora capsici in Example 2. The left graph is the control group, and the right graph is the fermentation broth treatment group of Microsporum vulga GZ1F2. Figure 4 The image shows the inhibitory effect of the fermentation broth of Microsporum vulga GZ1F2 on the germination of dormant spores of Phytophthora capsici in Example 2. The left image is the control group, and the right image is the fermentation broth treatment group of Microsporum vulga GZ1F2. Figure 5 The graph shows the results of the growth-promoting ability test of Microsporum vulga GZ1F2 in Example 4. From left to right, the results are the test results of cellulase, nitrogen fixation, inorganic phosphorus decomposition ability, and iron carrier production ability. Figure 6 The image shows the effect of Microsporum vulga GZ1F2 on promoting the growth of peppers in Example 5. The left image is the Microsporum vulga GZ1F2 treatment group, and the right image is the control group. Detailed Implementation

[0015] The technical solution of the present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0016] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art; the terminology used herein is for the purpose of describing particular implementations only and is not intended to limit the invention.

[0017] Microsporum var. hua, GZ1F2, is an endophytic fungus isolated from tobacco leaves. It has been deposited at the Guangdong Provincial Microbial Culture Collection Center, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No. 65361. Current research indicates that it can form a symbiotic relationship with tobacco and is used to control tobacco bacterial wilt. However, there are currently no reports of this fungus colonizing other plants, forming symbiotic relationships with them, or controlling other plant diseases.

[0018] To enrich the biological control methods for pepper blight, this invention has surprisingly discovered that the microsporidis var. *hua* GZ1F2 can also be used on peppers, playing a positive role in controlling pepper blight and promoting pepper growth. Data from the examples show that the microsporidis var. *hua* GZ1F2 has a comprehensive inhibitory effect on the growth and development of *Phytophthora capsici*, and that applying it as spores to the roots of pepper seedlings significantly improves the pepper's resistance to blight.

[0019] The following are some specific embodiments. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0020] The specific composition of each culture medium used in the following examples is as follows: Potato glucose broth (PDA): 200g peeled potatoes, 20g glucose and 18g agar powder, bring the volume to 1000mL with deionized water, sterilize at 121℃ for 20min; PDA liquid broth does not contain agar powder. V8 liquid culture medium: Add 1g of calcium carbonate to 100mL of American Kingston V8 original vegetable juice, stir until completely dissolved, 7000rpm for 10min to obtain clear V8 fruit and vegetable juice, add deionized water to 1000mL, and sterilize at 121℃ for 20min.

[0021] Example 1 This example uses the plate confrontation method to determine the inhibitory effect of Microsporum vulgare GZ1F2 on Phytophthora capsici. The specific experiment is as follows: Microsporum var. *Hydroxella* GZ1F2 and *Phytophthora capsici* were activated on PDA medium for 5 days. 5mm diameter mycelial blocks were punched and inoculated onto the left and right sides of a PDA plate, approximately 5cm apart and 2cm from the edge of the plate. The line connecting the centers of the mycelial blocks passed through the center of the plate. After inoculation, the PDA plates were inverted and incubated at 28℃ for 5 days. The growth radius of *Phytophthora capsici* colonies pointing towards *Microsporum var. *Hydroxella* GZ1F2 was measured (measured from the center of the mycelial block), and the inhibition rate was calculated. Inhibition rate (%) = (control group colony diameter - treatment group colony diameter) / (control group colony diameter - mycelial block diameter) × 100. A blank medium was used as a control, and the experiment was repeated three times.

[0022] The results of the flat plate confrontation experiment are as follows Figure 1 As shown, the microsporidis var. GZ1F2 exhibited very good inhibitory effect on Phytophthora capsici, with an inhibition rate of 93.28%.

[0023] Example 2 This study investigated the effects of Microsporum var. hua (GZ1F2) on the mycelium, zoosporangia, and zoospores of Phytophthora capsici. The experimental procedure is as follows: (1) Preparation of fermentation broth of Microsporum vulga GZ1F2.

[0024] The mycelial blocks of Microsporum var. GZ1F2 were transferred into a shake flask containing 150 mL of PDA liquid medium and cultured on a shaker at 28 °C and 180 rpm for 72 h. The mycelium was filtered with gauze and then the culture medium was filtered through a 22 μm microporous membrane to obtain the fermentation broth of Microsporum var. GZ1F2.

[0025] (2) Effect of fermentation broth of Microsporum vulga GZ1F2 on the mycelial morphology of Phytophthora capsici.

[0026] Mycelial blocks of *Phytophthora capsici* were transferred into petri dishes, and 10 mL of V8 liquid medium was added. Simultaneously, 5 mL of fermentation broth from *Microsporum hua* var. *hua* GZ1F2 was added. A control group was prepared by adding 5 mL of PDA liquid medium. The petri dishes were incubated in the dark at 28°C for 3 days. A small amount of mycelium was then collected and prepared as a slide for microscopic observation of mycelial morphology.

[0027] The results are as follows Figure 2 As shown, in the control group, the hyphae of *Phytophthora capsici* were slender, septate, smooth, and contained homogeneous contents. After treatment with the fermentation broth of *Microsporum hua* var. GZ1F2, the contents of the *Phytophthora capsici* hyphae agglomerated into clumps, resembling beads, and exhibited deformed morphology. These results indicate that the fermentation broth of *Microsporum hua* var. GZ1F2 has a strong inhibitory effect on the hyphae of *Phytophthora capsici*.

[0028] (3) Effect of fermentation broth of Rhizopus var. GZ1F2 on the formation of zoosporangia of Phytophthora capsici.

[0029] Transfer the mycelial blocks of *Phytophthora capsici* into petri dishes, pour in 10 mL of V8 liquid medium, and incubate in the dark at 28°C for 3 days. Discard the culture medium, add 10 mL of sterile water to re-submerge the mycelial clusters, and continue incubation. Change the water every 12 hours for a total of 3 times. Each time the sterile water is changed, add 5 mL of *Microsporum hua* var. *GZ1F2* fermentation broth, with 5 mL of sterile water added as a control.

[0030] After water replacement, 10 mL of sterile water and 5 mL of *Microsporum hua* var. *hua* GZ1F2 fermentation broth were added to the petri dish. The dish was incubated in the dark at 28°C for 1 hour, then placed in a 4°C refrigerator for 15 minutes and a 28°C incubator for 30 minutes to stimulate the release of zoospores from the sporangia. The formation of *Phytophthora capsici* sporangia and the release of zoospores were observed under a microscope and counted.

[0031] The results are as follows Figure 3 As shown, in the control group, *Phytophthora capsici* produced a large number of zoosporangia after treatment, while *Phytophthora capsici* did not produce zoosporangia after treatment with fermentation broth of *Microsporum hua* var. GZ1F2. This result indicates that the fermentation broth of *Microsporum hua* var. GZ1F2 has a significant inhibitory effect on zoosporangia production in *Phytophthora capsici*.

[0032] (4) Effects of fermentation broth of Microsporum vulga GZ1F2 on the motility of zoospores and the germination of rest spores of Phytophthora capsici.

[0033] Following the method in step (3), a suspension of zoospores of *Phytophthora capsici* was obtained. 0.5 mL of fermentation broth of *Microsporum hua* var. GZ1F2 was added to 1 mL of the zoospore suspension. 0.5 mL of PDA liquid culture medium was added as a control. After mixing, the movement of the zoospores was immediately observed under a microscope and recorded. Microscopic observation of the zoospore motility of *Phytophthora capsici* revealed that after treatment with the fermentation broth of *Microsporum hua* var. GZ1F2, the zoospores of *Phytophthora capsici* quickly stopped motility and formed resting spores, while the zoospores in the control group still motility for a period of time before forming resting spores.

[0034] Take another 3 mL of zoospore suspension of *Phytophthora capsici* and place it in a 10 mL centrifuge tube. Vortex the tube to remove the flagella from the zoospores, resulting in resting spores. Add 1.5 mL of fermentation broth of *Microsporum hua* var. *hua* GZ1F2, using 1.5 mL of LPDA liquid medium as a control. Incubate at 28 °C. After 2 hours, observe the germination of the resting spores under a microscope. The results are as follows: Figure 4As shown, in the control group, the zoospores of *Phytophthora capsici* germinated normally after becoming resting spores. However, after treatment with the fermentation broth of *Microsporum hua* var. GZ1F2, the resting spores of *Phytophthora capsici* ruptured, releasing their contents and failing to germinate. This result indicates that the fermentation broth of *Microsporum hua* var. GZ1F2 significantly inhibited the motility of *Phytophthora capsici* zoospores and suppressed the germination of resting spores.

[0035] Example 3 This example demonstrates the control effect of Microsporum vulgare GZ1F2 on pepper blight through a pot experiment, as detailed below: Microsporum var. var. GZ1F2 was cultured on PDA medium for 5 days. After microscopic observation of sporulation, sterile water was added, spores were scraped off, filtered, and diluted to a spore concentration of 1.0 × 10⁻⁶. 6 1 / mL, for use in inoculating peppers.

[0036] Chili seeds were soaked in 75% alcohol for 30 seconds to disinfect their surface, and then washed five times with sterile water. Two layers of sterile, moistened filter paper were placed in a sterile petri dish, and the treated seeds were evenly distributed on the filter paper. The dish was then placed in a dark environment at 25°C for 4 days. After germination, the seeds were transplanted into flowerpots and cultured in a greenhouse at 25°C until they reached the 4-leaf, 1-heart stage. A suspension of Microsporum hua var. GZ1F2 spores (1×10⁻⁶) was then used for root irrigation. 6 Spores / mL, 10mL per plant (control group with equal volume of sterile water), when 6-7 true leaves, drench the roots with a suspension of zoospores of Phytophthora capsici (1×10). 3 (1 zoospore / ml, 3ml per plant), observe and record the disease incidence of peppers at 0, 1, 3, 5, and 7 days after inoculation. Each treatment consisted of 30 plants, replicated 3 times.

[0037] Incidence rate = Number of diseased plants / Total number of plants. Disease index = (∑(Disease level × Number of plants at that level) / (Highest disease level × Total number of plants)) × 100, disease levels are shown in Table 1. Relative control efficacy = (Disease index of control group - Disease index of experimental group) / Disease index of control group.

[0038] Table 1. Grading Table for Phytophthora blight in chili peppers

[0039] Table 2. Control results of Microsporum vulgare GZ1F2 against Phytophthora blight in pepper.

[0040] As shown in Table 2, the microsporum rhizogenes var. GZ1F2 significantly reduced the incidence and disease index of pepper blight. On day 7 after inoculation with Phytophthora capsici, the disease index of pepper blight in the treatment group was 4.00, while that in the control group was 12.67, which was significantly higher than that in the treatment group. The calculated control efficacy of microsporum rhizogenes var. GZ1F2 against pepper blight was 68.42%, indicating that it has good application potential.

[0041] Example 4 This example tested the in vitro life-promoting activity of Microsporum vulga GZ1F2. The experimental procedure is as follows: Microsporum spp. var. GZ1F2 was inoculated onto cellulase detection medium, inorganic phosphorus bacteria medium, Ashby's medium, and CAS detection medium (the inorganic phosphorus bacteria medium, Ashby's medium, and CAS detection medium were purchased from Haibo Biotechnology Co., Ltd., and the cellulase detection medium was purchased from Beijing Coollife Technology Co., Ltd.). The cultures were incubated at 28℃ for 5 days, repeated three times, and the results were observed. If Microsporum spp. var. GZ1F2 produced a clear zone on the cellulase detection plate, it indicated that the strain produced cellulase; if it grew on the CAS detection plate and produced a yellow ring, it indicated that the strain had the ability to produce siderophores; if it could grow on Ashby's nitrogen-free medium, it indicated that the strain had the ability to fix nitrogen; and if it produced a clear zone on the inorganic phosphorus plate, it indicated that the strain had the ability to decompose inorganic phosphorus.

[0042] The results are as follows Figure 5 As shown, this strain produces a clear zone on cellulase detection plates, can grow on Assumption nitrogen-free medium, produces a clear zone on inorganic phosphorus plates, and produces a yellow ring on CAS detection plates. This indicates that the strain has the ability to produce cellulase, fix nitrogen, decompose inorganic phosphorus, and produce siderophores, and can play a positive role in the absorption of nutrients from the soil by plants.

[0043] Example 5 This example demonstrates the growth-promoting effect of Microsporum vulgare GZ1F2 on pepper through a pot experiment, as detailed below: Microsporum var. var. GZ1F2 was cultured on PDA medium for 5 days. After microscopic observation of sporulation, sterile water was added, spores were scraped off, filtered, and diluted to a spore concentration of 1.0 × 10⁻⁶. 6 1 / mL, for use in inoculating peppers.

[0044] Soak chili seeds in 75% alcohol for 30 seconds to disinfect their surface, then wash them five times with sterile water. Line a sterile petri dish with two layers of sterile, moistened filter paper, and evenly place the treated seeds on the filter paper. Incubate in a dark environment at 25°C for four days. After germination, transplant the seeds into flowerpots and cultivate them in a greenhouse at 25°C until they have four leaves and one bud. Then, drench the roots with a suspension of Microsporum vinifera var. GZ1F2 spores (1×10⁻⁶). 6 Spores / mL, 10mL per plant (control group with an equal volume of sterile water). Each treatment consisted of 30 plants, replicated 3 times. After 30 days, the growth of the peppers was observed, and the dry and fresh weights of the above-ground and underground parts were measured.

[0045] The results are as follows Figure 6 As shown in Table 3, peppers grew more robustly after inoculation with Microsporum vulga GZ1F2, with significantly higher plant height, aboveground / underground dry weight, and fresh weight than the control group. This indicates that Microsporum vulga GZ1F2 can significantly promote pepper growth and increase pepper biomass.

[0046] Table 3. Growth-promoting effects of strain GZ1F2 on chili peppers.

[0047] Note: Different lowercase letters in the table indicate significant differences in data. p <0.01).

[0048] In summary, the microsporidis var. GZ1F2 significantly inhibits the mycelial growth, zoosporangium production, zoospore motility, and rest spore germination of Phytophthora capsici. Furthermore, it can form a symbiotic relationship with pepper, thereby effectively controlling pepper blight and promoting pepper growth.

[0049] It should be noted that the present invention is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments that have the same structure and perform the same effects as the technical concept within the scope of the present invention are included within the scope of the present invention. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of the present invention, are also included within the scope of the present invention.

Claims

1. Application of Microsporum vulga GZ1F2 in the control of pepper blight and promotion of pepper growth.

2. The application according to claim 1, characterized in that, Peppers were treated with root irrigation with Microsporum villi var. GZ1F2.

3. The application according to claim 2, characterized in that, The root irrigation treatment method is as follows: prepare a spore suspension of Microsporum rhizogenes var. GZ1F2, and use the spore suspension to irrigate the roots of peppers.

4. The application according to claim 2, characterized in that, Root irrigation should be carried out when the chili pepper has 4 leaves and 1 heart leaf.

5. A microbial preparation for preventing and / or promoting the growth of peppers, characterized in that, Contains microsporum rhizopus var. GZ1F2 and / or its spore suspension and / or its fermentation broth.

6. The microbial preparation according to claim 5, characterized in that, The microbial preparation is any one of a dry powder, a wettable powder, or a liquid preparation.

7. A method for inhibiting the growth and development of Phytophthora capsici, characterized in that, Inhibition was achieved using the microsporidis var. GZ1F2.

8. The method according to claim 7, characterized in that, A fermentation broth for Microsporum var. GZ1F2 was prepared, and the fermentation broth was used to inhibit the growth and development of Phytophthora capsici.

9. The method according to claim 8, characterized in that, The fermentation broth is prepared by inoculating a block of microsporum rhizopus var. GZ1F2 into a culture medium, removing the mycelium, and then filtering the culture medium through a microporous membrane.

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