Use of imazapyr in the control of fungal competing weeds in edible fungi

Abstract

The present application relates to the application of imazalil in preventing and treating fungal competitive weeds in edible fungi. The present application finds that imazalil unexpectedly has a significant inhibitory effect on fungal competitive weeds in edible fungi, and has little effect on the growth of edible fungi such as shiitake mushrooms, oyster mushrooms and pleurotus eryngii at a reasonable concentration, based on which it can be used to prevent and treat fungal competitive weeds in edible fungi.

Description

Technical Field

[0001] This invention relates to the field of microbiology, and in particular to the application of imazalil in the control of fungal competitive contaminants in edible fungi. Background Technology

[0002] With increasing attention to healthy eating, edible fungi, due to their unique flavor and rich nutritional value, have become an indispensable part of the global food structure. Among the many varieties of edible fungi, shiitake mushrooms (…) Lentinula edodes ), oyster mushroom ( Pleurotus ostreatus ) and king oyster mushroom ( Pleurotus eryngii As a typical wood-rotting edible fungus, mushrooms, using wood, sawdust, and other lignocellulosic materials as a substrate, possess characteristics such as not competing with grain crops for land, low investment, short cycle, and quick returns, occupying an important position in the edible fungus industry. With the increasingly mature cultivation techniques of shiitake, oyster, and king oyster mushrooms, and the promotion of large-scale, factory-style production models, the market supply capacity of these varieties has been greatly improved. They are not only delicious dishes but also highly valued for their rich content of protein, dietary fiber, vitamins, minerals, polysaccharides, triterpenoids, and other bioactive substances. Studies have shown that their abundant bioactive substances have significant immunomodulatory, anti-tumor, antioxidant, lipid-lowering, and antiviral effects, possessing important medicinal and health-promoting value.

[0003] However, in the cultivation of wood-rotting edible fungi such as shiitake, oyster, and king oyster mushrooms, especially in intensive and large-scale production environments, the substrate is rich in nutrients and has high humidity, making it extremely easy for various competing fungi to grow, among which Trichoderma (… Trichoderma sp.), Neurospora ( Neurospora sp.) and Penicillium ( Penicillium (Sp.) are the most threatening competitive fungi to edible fungi. Infection by these competitive fungi directly leads to slow or stagnant mycelial growth, delayed fruiting, deformed, small, and few fruiting bodies, or even complete fruiting. Statistics show that fungal contamination is one of the main causes of reduced yield, decreased quality, and even crop failure in edible fungi, resulting in hundreds of millions of yuan in economic losses to the industry annually. Currently, the control of fungal contamination in edible fungi mainly relies on strict sterilization processes such as high-temperature and high-pressure steam sterilization, strict control of the production environment, and the selection of highly resistant strains. While biological control technology has made some progress, its effectiveness is greatly affected by environmental factors, and its onset speed is slow, making it difficult to meet the needs of large-scale production for efficient and rapid control. Since edible fungi and the aforementioned fungi both belong to the fungal family, generally speaking, chemical pesticides that inhibit the growth of fungi are also toxic to edible fungi, affecting their normal growth and fruiting. Summary of the Invention

[0004] This invention provides the application of imazalil in the prevention and control of fungal competitive contaminants in edible fungi.

[0005] In one specific embodiment, the edible fungus is at least one fungus in the order Agaricales.

[0006] In one specific embodiment, the edible fungus is at least one fungus from the Omphalotaceae and / or Pleurotaceae families.

[0007] In one specific embodiment, the edible fungus is a member of the genus Lentinus (Lentinula edodes). Lentinula sp.) and / or Pleurotus (sp.) and / or Pleurotus genus ( Pleurotus At least one fungus of the species (sp.).

[0008] In one specific embodiment, the fungal competing contaminant is at least one fungus belonging to the phylum Ascomycota.

[0009] In one specific embodiment, the fungal competing contaminants are at least one fungus belonging to the classes Sordariomycetes and / or Eurotiomycetes.

[0010] In one specific embodiment, the fungal competing contaminant is at least one fungus from the families Hypocreaceae, Sordariaceae, and Aspergillaceae.

[0011] In one specific embodiment, the fungal competing contaminant is Trichoderma ( Trichoderma sp.), Neurospora ( Neurospora sp.) and Penicillium ( Penicillium At least one fungus of the species (sp.).

[0012] In one specific embodiment, the fungal competing contaminant is *Trichoderma viride* (…). Trichoderma atroviride Neurospora tetrasporium ( Neurospora tetrasperma ) and Penicillium citrinum ( Penicillium citrinum At least one fungus in the group consisting of .

[0013] In one specific embodiment, the concentration of the imidacloprid used against the airborne fungal competing bacteria is 0.5 to 25 micrograms per milliliter, at which point it can effectively inhibit the growth of Trichoderma viride and has almost no effect on the growth of edible fungi such as shiitake mushrooms.

[0014] In one specific embodiment, the concentration of the imidacloprid used against the airborne fungal competing bacteria is 1 to 25 micrograms per milliliter, at which point it can effectively inhibit the growth of Trichoderma viride and / or Penicillium citrinum, and has almost no effect on the growth of edible fungi such as shiitake mushrooms.

[0015] In one specific embodiment, the concentration of the imidacloprid used against the airborne fungal competing bacteria is 5 to 25 micrograms per milliliter, at which point it can effectively inhibit the growth of Trichoderma viride and / or Penicillium citrinum, and has almost no effect on the growth of edible fungi such as shiitake mushrooms.

[0016] In one specific embodiment, the concentration of the imidacloprid used against the airborne fungal competing bacteria is 10 to 25 micrograms per milliliter, at which point it can effectively inhibit the growth of at least one of Trichoderma viride, Neurospora tetraspora, and Penicillium citrinum, and has almost no effect on the growth of edible fungi (e.g., shiitake mushrooms).

[0017] In one specific embodiment, the concentration of the imidacloprid used against the airborne fungal competing bacteria is 0.5 to 10 micrograms per milliliter, which can effectively inhibit the growth of Trichoderma viride and has no effect on the growth of edible fungi (such as shiitake mushrooms).

[0018] In one specific embodiment, the concentration of the imidacloprid used against the airborne fungal competing bacteria is 1 to 10 micrograms per milliliter, which can effectively inhibit the growth of Trichoderma viride and / or Penicillium citrinum, and has no effect on the growth of edible fungi (e.g., shiitake mushrooms).

[0019] In one specific embodiment, the concentration of the imidacloprid used against the airborne fungal competing bacteria is 5 to 10 micrograms per milliliter, which can effectively inhibit the growth of Trichoderma viride and / or Penicillium citrinum, and has no effect on the growth of edible fungi (such as shiitake mushrooms).

[0020] In one specific embodiment, the concentration of the imidacloprid used against the airborne fungal competing bacteria is 10 micrograms / ml, which can effectively inhibit the growth of at least one of Trichoderma viride, Neurospora tetraspora, and Penicillium citrinum, and has no effect on the growth of edible fungi (e.g., shiitake mushrooms).

[0021] In one specific embodiment, the concentration of the imidacloprid used to inhibit the growth of at least one of Trichoderma viride, Neurospora tetraspora, and Penicillium citrinum in the solid component of the culture medium used to cultivate the edible fungi is 40 micrograms / mL. At this concentration, it can effectively inhibit the growth of Trichoderma viride, Neurospora tetraspora, and Penicillium citrinum, and has almost no effect on the growth of the edible fungi.

[0022] The beneficial effects of this invention are as follows: This invention has discovered that imazalil unexpectedly has a significant inhibitory effect on fungal competitive bacteria in edible fungi, while at reasonable concentrations it has almost no effect on the growth of edible fungi such as shiitake mushrooms, oyster mushrooms and king oyster mushrooms. Based on this, it can be used to prevent and control fungal competitive bacteria in edible fungi. Attached Figure Description

[0023] Figure 1 The results show the inhibitory effect of imazalil on the mycelial growth of Penicillium citrinum in an indoor toxicity assay. In the assay, 1 represents the blank control, 2 represents the concentration of imazalil (0.15 μg / mL), 3 represents the concentration of imazalil (0.20 μg / mL), 4 represents the concentration of imazalil (0.25 μg / mL), 5 represents the concentration of imazalil (0.30 μg / mL), 6 represents the concentration of imazalil (0.35 μg / mL), and 7 represents the concentration of imazalil (0.40 μg / mL).

[0024] Figure 2 The results show the inhibitory effect of imazalil on the mycelial growth of Trichoderma viride in an indoor toxicity assay; where 1: blank control, 2: imazalil concentration 0.8 μg / mL, 3: imazalil concentration 1.6 μg / mL, 4: imazalil concentration 3.2 μg / mL, 5: imazalil concentration 6.4 μg / mL, and 6: imazalil concentration 12.8 μg / mL.

[0025] Figure 3 The results showed the inhibitory effect of imazalil on the mycelial growth of Neurospora tetraspora in an indoor toxicity assay; where 1: blank control, 2: imazalil concentration 1.6 μg / mL, 3: imazalil concentration 3.2 μg / mL, 4: imazalil concentration 6.4 μg / mL, 5: imazalil concentration 12.8 μg / mL, and 6: imazalil concentration 25.6 μg / mL.

[0026] Figure 4 The study showed the effect of imazalil on the mycelial growth of shiitake mushrooms in an indoor toxicity assay. Among them, 1: blank control, 2: imazalil concentration of 1.6 μg / mL, 3: imazalil concentration of 3.2 μg / mL, 4: imazalil concentration of 6.4 μg / mL, 5: imazalil concentration of 12.8 μg / mL, and 6: imazalil concentration of 25.6 μg / mL.

[0027] Figure 5The study demonstrated the inhibitory effect of imidacloprid on the mycelial growth of Penicillium citrinum using the double-plate inverted method. In the table, 1: blank control; 2: imidacloprid concentration 0.05 μg / mL; 3: imidacloprid concentration 0.1 μg / mL; 4: imidacloprid concentration 0.5 μg / mL; 5: imidacloprid concentration 1.0 μg / mL; 6: imidacloprid concentration 5.0 μg / mL; 7: imidacloprid concentration 10.0 μg / mL; 8: imidacloprid concentration 25.0 μg / mL; and 9: imidacloprid concentration 50.0 μg / mL.

[0028] Figure 6 The study demonstrated the inhibitory effect of imidacloprid on the mycelial growth of Trichoderma viride using the double-plate inverted method. In the figures, 1: blank control; 2: imidacloprid concentration 0.05 μg / mL; 3: imidacloprid concentration 0.1 μg / mL; 4: imidacloprid concentration 0.5 μg / mL; 5: imidacloprid concentration 1.0 μg / mL; 6: imidacloprid concentration 5.0 μg / mL; 7: imidacloprid concentration 10.0 μg / mL; 8: imidacloprid concentration 25.0 μg / mL; and 9: imidacloprid concentration 50.0 μg / mL.

[0029] Figure 7 The study demonstrated the inhibitory effect of imidacloprid on the mycelial growth of Neurospora tetraspora using the double-plate inverted method; where 1: blank control, 2: imidacloprid concentration 0.5 μg / mL, 3: imidacloprid concentration 1.0 μg / mL, 4: imidacloprid concentration 5.0 μg / mL, 5: imidacloprid concentration 10.0 μg / mL, 6: imidacloprid concentration 25.0 μg / mL, 7: imidacloprid concentration 50.0 μg / mL, 8: imidacloprid concentration 75.0 μg / mL, and 9: imidacloprid concentration 100.0 μg / mL.

[0030] Figure 8 The effect of imidacloprid on the mycelial growth of shiitake mushroom was shown by the double-plate inverted method; where 1: blank control, 2: imidacloprid concentration 0.5 μg / mL, 3: imidacloprid concentration 1.0 μg / mL, 4: imidacloprid concentration 5.0 μg / mL, 5: imidacloprid concentration 10.0 μg / mL, 6: imidacloprid concentration 25.0 μg / mL, 7: imidacloprid concentration 50.0 μg / mL, 8: imidacloprid concentration 75.0 μg / mL, and 9: imidacloprid concentration 100.0 μg / mL.

[0031] Figure 9The experiment showed the inhibitory effect of 20% imazalil emulsion on the spread and growth of Trichoderma viride mycelium in solid matrix bottles in a solid matrix simulation test. Among them, 1: blank control, 2: 20,000 times dilution, 3: 10,000 times dilution, and 4: 5,000 times dilution.

[0032] Figure 10 The experiment showed the inhibitory effect of 20% imazalil emulsion on the spread and growth of Penicillium citrinum hyphae in solid matrix bottles in a solid matrix simulation test. Among them, 1: blank control, 2: 20,000 times dilution, 3: 10,000 times dilution, and 4: 5,000 times dilution.

[0033] Figure 11 The experiment showed the inhibitory effect of 20% imazalil emulsion on the spread and growth of Nematospora tetraspora hyphae in solid matrix bottles in a solid matrix simulation test. Among them, 1: blank control, 2: 20,000 times dilution, 3: 10,000 times dilution, and 4: 5,000 times dilution.

[0034] Figure 12 The experiment showed the effect of 20% imazalil emulsion on the spread and growth of shiitake mushroom mycelium in a solid substrate bottle in a solid substrate simulation experiment. Among them, 1: blank control, 2: 20,000 times dilution, 3: 10,000 times dilution, and 4: 5,000 times dilution.

[0035] Figure 13 The experiment showed the effect of 20% imazalil emulsion on the spread and growth of oyster mushroom mycelium in a solid substrate bottle in a solid substrate simulation experiment. Among them, 1: blank control, 2: 20,000 times dilution, 3: 10,000 times dilution, and 4: 5,000 times dilution.

[0036] Figure 14 The experiment showed the effect of 20% imazalil emulsion on the spread and growth of Pleurotus eryngii mycelium in a solid substrate bottle in a solid substrate simulation experiment. Among them, 1: blank control, 2: 20,000 times dilution, 3: 10,000 times dilution, and 4: 5,000 times dilution. Detailed Implementation

[0037] The present invention will be further described in detail below through preferred embodiments, but these embodiments do not constitute a limitation thereof.

[0038] Unless otherwise specified, the strains and reagents used in the embodiments of this invention can be purchased commercially.

[0039] Solid substrate culture medium for shiitake mushrooms, Penicillium citrinum, Trichoderma viride and Neurospora tetraspora: 79 g oak wood chips, 20 g wheat bran, 1 g CaSO4·2H2O, mixed evenly, 65 mL distilled water, mixed well, dispensed into each culture bottle, pressed to flatten the surface, and sterilized at 121 degrees Celsius and 0.12 MPa for 30 min.

[0040] Solid substrate culture medium for king oyster mushroom: 70 g oak wood chips, 25 g wheat bran, 3 g corn flour, 1 CaSO4·2H2O molecule, 1 g CaO. Mix well, add 65 mL distilled water, mix well, dispense into each culture bottle, press to make the surface flat, and sterilize by steaming at 121 degrees Celsius and 0.12 MPa for 30 min.

[0041] Solid substrate culture medium for oyster mushrooms: 92 g cottonseed hulls, 6 g wheat bran, 2 g CaO, mix well, add 65 mL distilled water, mix well, dispense into each culture bottle, press to flatten the surface, and sterilize at 121 degrees Celsius and 0.12 MPa for 30 min.

[0042] Shiitake mushrooms, king oyster mushrooms, and oyster mushrooms were purchased from the market, and the experimental strains were isolated and purified in the laboratory.

[0043] Trichoderma viride ( Trichoderma atroviride Neurospora tetrasporium ( Neurospora tetrasperma ) and Penicillium citrinum ( Penicillium citrinum All samples were isolated from contaminated shiitake mushroom logs in edible mushroom cultivation areas in Shunping County and Pingquan County, Baoding City, Hebei Province, and identified by morphological and ITS sequence determination.

[0044] Potato culture medium (PDA): Cut 200 g of potatoes into pieces, add distilled water and boil for 15 min, filter, add 20 g of glucose and 15 g of agar powder to the filtrate, and make up to 1000 mL with water, then steam sterilize at 121 degrees Celsius for 20 min.

[0045] 98% imazalil, 99.9% fludioxonil, 98% prochloraz, 98% oxadixyl, 99.8% cyazofamid, 99.76% chlorothalonil, and 99.6% prothioconazole were purchased from Shanghai Bide Pharmaceutical Technology Co., Ltd.

[0046] 96% tebuconazole, 97% tebuconazole, and 97% thiram were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0047] 98% azoxystrobin was purchased from Nantong Taihe Chemical Co., Ltd.

[0048] 95% difenoconazole was purchased from TCI (Shanghai) Chemical Industry Development Co., Ltd. Example 1: Effect of imazalil on Penicillium citrinum

[0049] Indoor toxicity was determined using the colony growth rate method.

[0050] Under aseptic conditions, 98% imazalil technical grade was prepared into imazalil solution using anhydrous ethanol.

[0051] Add imidazolium solution to PDA culture medium to make the final concentration of the active ingredient imidazolium in the culture medium 0.15 μg / mL, 0.20 μg / mL, 0.25 μg / mL, 0.30 μg / mL, 0.35 μg / mL, and 0.40 μg / mL to prepare drug-containing PDA plates.

[0052] Penicillium citrinum was activated on PDA medium at 25°C. Then, 6 mm diameter agar plates were punched out. The mycelial side of the agar plates was placed on the center of PDA plates containing different concentrations of the drug. A PDA plate without the drug served as a blank control. Each treatment was repeated three times, and then incubated at 25°C. After 14 days of incubation, photographs were taken. (Image follows) Figure 1 As shown. Then, the diameter of each treatment colony was measured using the cross-sectional method, and the inhibition rate of imazalil on mycelial growth was calculated. Inhibition rate (%) = (control colony diameter - treatment colony diameter) × 100 / (control colony diameter - mycelial plate diameter).

[0053] Using the mass concentration (µg / mL) and inhibition rate of imazalil as variables, IBM SPSS Statistics 27 software was used to analyze the data, obtaining the toxicity regression equation and correlation coefficient (R²). 2 ) and inhibition of medium concentration (EC) 50 Among them, EC 50 The results (µg / mL) are shown in Table 1. Example 2: Effects of imazalil on Trichoderma viride, Neurospora tetrasporium, or Lentinus edodes

[0054] Penicillium citrinum was replaced with Trichoderma viride, Neurospora tetraspora, or Lentinus shiitake, and different concentration gradients were set, with culture times of 3 days, 2 days, and 15 days, respectively. All other procedures were the same as in Example 1. The inhibition rate was calculated, and the virulence regression equation and correlation coefficient (R²) were obtained using SPSS software analysis. 2 ) and inhibition of medium concentration (EC) 50 (Photos as shown) Figures 2 to 4 As shown, EC 50 The results (µg / mL) are shown in Table 1. Example 3: Effects of other chemical agents on shiitake mushrooms and competing fungal contaminants

[0055] Imazalil was replaced with 5 to 6 different concentrations of fludioxonil, prochloraz, tebuconazole, azoxystrobin, tebuconazole, thiram, oxadixyl, cyazofamid, difenoconazole, chlorothalonil, and prothioconazole to test their inhibition rates on the mycelial growth of *Lentinula edodes*, *Trichoderma viride*, *Neurospora tetraspora*, or *Penicillium citrinum*. Further analysis yielded the regression equations and correlation coefficients (R²) for their toxicity against these fungi. 2 ) and inhibition of medium concentration (EC) 50 EC 50 The results (µg / mL) are shown in Table 1.

[0056] Table 1

[0057] Table 1 shows that the toxicity of the 12 antifungal agents against shiitake mushrooms and three contaminating fungi—Trichoderma viride, Neurospora tetraspora, and Penicillium citrinum—exhibited significant selectivity differences. Specifically, the EC50 values ​​of tebuconazole, azoxystrobin, tebuconazole, thiram, oxadixyl, cyazofamid, chlorothalonil, and prothioconazole against shiitake mushrooms were all lower than their EC50 values ​​against competing fungi. 50 The values ​​or equivalent indicate that these agents are more toxic to shiitake mushrooms than to competing fungi; while the three antifungal agents, prochloraz, fludioxonil, and difenoconazole, have an EC value of [missing value] on shiitake mushrooms. 50 Although the value was higher than the EC value for three competing bacteria. 50 The value, but the selectivity index of prochloraz, fludioxonil, and difenoconazole to shiitake mushrooms (EC50 of shiitake mushrooms) is relatively high. 50 / Competitive bacteria EC 50 The values ​​were 17.6 to 3378, 1.5 to 20.4, and 2.1 to 11.0, respectively. This means that while these three antibacterial agents can effectively inhibit competing bacteria, they do not significantly affect the EC50 of shiitake mushrooms. 50 The value was also lower, and compared with the EC of competing bacteria. 50 The low differential concentration makes it difficult to select a concentration that effectively inhibits these competing bacteria without affecting the growth of shiitake mushrooms, and it easily poses a safety risk to shiitake mushrooms. Surprisingly, imazalil showed strong toxicity against the three competing bacteria: *Trichoderma viride*, *Neurospora tetrasporium*, and *Penicillium citrinum*, with EC50 values. 50 The values ​​were 1.343 μg / mL, 0.541 μg / mL, and 0.256 μg / mL, respectively, but the toxicity to shiitake mushrooms was low. (EC) 50 The value was 205.235 μg / mL. The selectivity index of this antibacterial agent against shiitake mushrooms and three competing fungi ranged from 152.8 to 801.7. The toxicity of this agent against shiitake mushrooms and the three competing fungi varied greatly. It is suitable for use in the cultivation of shiitake mushrooms to control these three fungal competing fungi. Example 4: Simulation study of the inhibitory effect of imazalil on airborne competitive bacteria using the double-plate inverted method.

[0058] The inhibitory effect of imazalil on airborne fungal competitive bacteria was simulated using the double-plate inverted method.

[0059] According to Penicillium citrinum EC 50 A concentration gradient of the active ingredient, imazalil, was established. PDA plates containing imazalil at final concentrations of 0 μg / mL (blank control), 0.05 μg / mL, 0.1 μg / mL, 0.5 μg / mL, 1.0 μg / mL, 5.0 μg / mL, 10.0 μg / mL, 25.0 μg / mL, and 50.0 μg / mL were used as bottom plates (placed at the bottom) and PDA plates without imazalil as top plates (placed at the top). Activated *Penicillium citrinum* spawn discs with a diameter of 6 mm were punched. The mycelial side of the discs was placed in the center of the top plate. The two plates were then interlocked and sealed with sealing film. They were incubated at 25°C, with each treatment repeated three times. After 14 days of incubation, the growth of *Penicillium citrinum* colonies in the PDA plates containing imazalil (bottom plate) was observed and recorded. The results are shown in the attached images. Figure 5 .

[0060] According to Trichoderma EC 50 A concentration gradient of the active ingredient, imazalil, was established. PDA plates containing imazalil at final concentrations of 0 μg / mL (blank control), 0.05 μg / mL, 0.1 μg / mL, 0.5 μg / mL, 1.0 μg / mL, 5.0 μg / mL, 10.0 μg / mL, 25.0 μg / mL, and 50.0 μg / mL were used as bottom plates (placed at the bottom), and PDA plates without imazalil were used as top plates (placed at the top). Activated Trichoderma viride mycelium discs with a diameter of 6 mm were punched out. The mycelial side of the mycelium disc was placed in the center of the top plate. The top and bottom plates were then interlocked and sealed with sealing film. The plates were incubated at 25°C, with each treatment repeated three times. After 10 days of incubation, the growth of Trichoderma viride colonies in the PDA plates containing imazalil (bottom plate) was observed and recorded. The results are shown in the attached photographs. Figure 6 .

[0061] According to Neurospora tetraspora EC 50A concentration gradient of the active ingredient, imazalil, was established. PDA plates containing imazalil at final concentrations of 0 μg / mL (blank control), 0.5 μg / mL, 1.0 μg / mL, 5.0 μg / mL, 10.0 μg / mL, 25.0 μg / mL, 50.0 μg / mL, 75.0 μg / mL, and 100.0 μg / mL were used as bottom plates (placed at the bottom), and PDA plates without imazalil were used as top plates (placed at the top). Activated *Neurospora tetraspora* fungal discs with a diameter of 6 mm were punched. The mycelial side of the discs was placed in the center of the top plate. The top and bottom plates were then interlocked and sealed with sealing film. The plates were incubated at 25°C, with each treatment repeated three times. After 3 days of incubation, the growth of *Neurospora tetraspora* colonies in the PDA plates (bottom plates) containing imazalil was observed and recorded. The results are shown in the attached images. Figure 7 .

[0062] PDA plates containing imazalil were prepared at the same concentration as those used to treat Neurospora tetraspora. The mycelial side of 6 mm diameter shiitake mushroom discs was placed onto the center of each drug-containing medium, and the plates were incubated at 25°C. Each treatment was repeated three times. Shiitake mushroom colony growth was observed after 15 days of incubation. The results are shown in the attached photographs. Figure 8 .

[0063] *Penicillium citrinum* and *Trichoderma viride* were graded according to the proportion of colony area in the petri dish to the total area of ​​the petri dish: Grade 0: no colonies; Grade 1: colony area ≤ 10%; Grade 3: colony area 11% to 25%; Grade 5: colony area 26% to 50%; Grade 7: colony area 51% to 75%; Grade 9: colony area ≥ 76%. *Neurospora tetrasporium* was graded into five levels based on the density of colony growth in the petri dish, using "-", "+", "++", "+++", and "++++". The diameter of *Vibrio edodes* colonies was measured using the cross-hatching method. The results are shown in Table 2.

[0064] The test results are shown in Figure 5 According to Table 2, when the concentration of the agent is ≥1 μg / mL, the growth of Penicillium citrinum in the lower dish is completely inhibited; Figure 6 The experimental results shown in Table 2 indicate that when the drug concentration is ≥5 μg / mL, Trichoderma viride in the lower dish is completely inhibited; Figure 7 The experimental results shown in Table 2 indicate that when the concentration of the agent is 10 μg / mL or higher, the growth of *Neurospora tetraspora* hyphae in the lower dish begins to show a significant inhibitory effect. Figure 8 The experimental results shown in Table 2 indicate that treatment concentrations of 10 μg / mL and below have no effect on the growth of shiitake mushroom colonies, while concentrations of 25 μg / mL and above have a certain inhibitory effect on the mycelial growth of shiitake mushrooms.

[0065] In summary, imazalil at concentrations of 1 μg / mL and above showed significant antibacterial activity against *Penicillium citrinum* in the lower dish; at 5 μg / mL and above, it showed significant antibacterial activity against *Trichoderma viride* and *Penicillium citrinum* in the lower dish; and at 10 μg / mL and above, it showed significant antibacterial activity against *Trichoderma viride*, *Neurospora tetraspora*, and *Penicillium citrinum* in the lower dish. It began to inhibit the mycelial growth of *Lentinula edodes* at 25 μg / mL, but had no inhibitory effect at 10 μg / mL. This indicates that imazalil has good antibacterial properties and can effectively control competing bacteria at 10 μg / mL without affecting the normal growth of the target edible fungi.

[0066] Table 2 Example 5: Solid substrate cultivation experiment simulation study on the control effect of imazalil on fungal competitive bacteria.

[0067] A solid substrate cultivation experiment was conducted to simulate actual production and to determine the control effect of imazalil on competing bacteria.

[0068] Raw materials were weighed according to the formula and mass ratio of solid matrix culture medium containing shiitake mushrooms, Trichoderma viride, Neurospora tetraspora, and Penicillium citrinum. Three concentration gradients of 20% imazalil emulsion were prepared according to the recommended dosage in the instructions and the experimental results of Example 4: 20,000 times (active ingredient content 10 μg / mL), 10,000 times (active ingredient content 20 μg / mL), and 5,000 times (active ingredient content 40 μg / mL). Diluted solutions of each concentration were obtained. The 65% (v / w) water added during the preparation of the culture medium (calculated based on the total mass (g) of the solid components as 100% of the water volume (mL)) was replaced with the diluted solutions of each concentration. A treatment using 65% distilled water to prepare the culture medium served as a blank control. Each treatment was replicated three times. After thorough mixing, the mixture was dispensed into tissue culture bottles and then sterilized by steam at 121°C and 0.12 MPa for 30 min. After sterilization, using a perforator, 6mm diameter mycelium discs were taken from the activated PDA plates containing various pathogens and shiitake mushrooms. One type of fungal mycelium disc (one Trichoderma viride disc, one Neurospora tetraspora disc, one Penicillium citrinum disc, or one shiitake mushroom disc) was inoculated into each bottle. These discs were then placed in a 25°C incubator and cultured for 4, 2, 15, and 17 days, respectively, until the mycelium in the blank control group completely covered the substrate. The results are shown in the photographs. Figures 9 to 12 .

[0069] Replace the solid substrate medium for shiitake mushrooms with solid substrate medium for king oyster mushrooms, inoculating one king oyster mushroom spawn tray into each bottle; replace it with solid substrate medium for oyster mushrooms, inoculating one oyster mushroom spawn tray into each bottle. All other operations were the same. Incubate at 25°C for 13 days. See the photos for the results. Figure 13 and 14.

[0070] The substrate was graded based on the proportion of the area where mycelium spread downwards to the total substrate area: Grade 0: no mycelium growth; Grade 1: mycelial growth area ≤ 10%; Grade 3: mycelial growth area 11% to 25%; Grade 5: mycelial growth area 26% to 50%; Grade 7: mycelial growth area 51% to 75%; Grade 9: mycelial growth area ≥ 76%. The results are shown in Table 3.

[0071] like Figure 9 , Figure 10 , Figure 11 As shown in Table 3, a 5000-fold dilution of 20% imazalil emulsion significantly inhibited the mycelial growth of *Trichoderma viride*, *Neurospora tetraspora*, and *Penicillium citrinum*. Figure 12 , Figure 13 , Figure 14 As shown in Table 3, dilutions of the pesticide at 20,000, 10,000, and 5,000 times had no effect on the growth of shiitake, oyster, and king oyster mushrooms. Therefore, a 5,000-fold dilution of 20% imazalil emulsion can be used for the control of competing fungi in edible fungi such as shiitake, oyster, and king oyster mushrooms.

[0072] Table 3

Claims

1. Application of imazalil in the control of fungal competitive contaminants in edible fungi.

2. The application according to claim 1, characterized in that, The edible fungus is at least one fungus in the order Agaricales.

3. The application according to claim 1, characterized in that, The edible fungus is at least one fungus from the Omphalotaceae and / or Pleurotaceae families.

4. The application according to claim 1, characterized in that, The edible fungus is a member of the genus Lentinus (Shiitake). Lentinula sp.) and / or Pleurotus (sp.) and / or Pleurotus genus ( Pleurotus At least one fungus of the species (sp.).

5. The application according to claim 1, characterized in that, The fungal competing contaminants are at least one fungus belonging to the phylum Ascomycota.

6. The application according to claim 1, characterized in that, The fungal competing contaminants are at least one fungus from the class Sordariomycetes and / or Eurotiomycetes.

7. The application according to claim 1, characterized in that, The fungal competing contaminants are at least one fungus from the families Hypocreaceae, Sordariaceae, and Aspergillaceae.

8. The application according to claim 1, characterized in that, The fungal competing bacteria is Trichoderma ( Trichoderma sp.), Neurospora ( Neurospora sp.) and Penicillium ( Penicillium At least one fungus of the species (sp.).

9. The application according to claim 1, characterized in that, The fungal competing bacteria is Trichoderma viride ( Trichoderma atroviride Neurospora tetrasporium ( Neurospora tetrasperma ) and Penicillium citrinum ( Penicillium citrinum At least one fungus in the group consisting of .

10. The application according to any one of claims 1 to 9, characterized in that, The concentration of the imidacloprid used against the airborne fungal competitive bacteria is 0.5 to 25 micrograms per milliliter; and / or the concentration of the imidacloprid used against the fungal competitive bacteria in the solid components of the culture medium used to cultivate the edible fungi is 40 micrograms per milliliter; Preferably, the concentration of the imidacloprid used against the airborne fungal competing bacteria is 1 to 25 micrograms per milliliter; Preferably, the concentration of the imidacloprid used against the airborne fungal competing bacteria is 5 to 25 micrograms per milliliter; Preferably, the concentration of the imidacloprid used against the airborne fungal competing bacteria is 0.5 to 10 micrograms per milliliter; Preferably, the concentration of the imidacloprid used against the airborne fungal competing bacteria is 1 to 10 micrograms per milliliter; Preferably, the concentration of the imidacloprid used against the airborne fungal competing bacteria is 5 to 10 micrograms per milliliter; Preferably, the concentration of the imidacloprid used against the airborne fungal competing bacteria is 10 micrograms per milliliter.

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