Use of fluopyram for the control of mycosphaerella diseases

Abstract

The present application relates to the application of fluopyram in the prevention and treatment of Morel white mold disease. The present application finds that the chemical agent fluopyram has selective specificity for Pseudomonas plecoglossi relative to Morel, and therefore can be used to prevent and treat Morel white mold disease.

Description

Technical Field

[0001] This invention relates to the field of microbiology, and in particular to the application of fluopyram in the prevention and control of morel white mold disease. Background Technology

[0002] Morel mushrooms belong to the phylum Ascomycota, class Discomycetes, order Pezizales, family Morchellaceae, and genus Morl. Morchella The type species of this genus is Morchella esculenta (L.) Pers. Morels belong to the genus *Morchella* (…). Morchella Morel mushrooms (spp.) are a general term for fungi. They are named for their resemblance to sheep stomachs. With their exceptionally delicious flavor, they are known as the "King of Fungi." Rich in various nutrients, they possess edible, medicinal, and economic value, making them a precious dual-purpose fungus highly sought after by consumers. Since commercial cultivation began in 2012, morel cultivation has rapidly expanded due to its advantages of low labor intensity, short growth cycle, and high economic benefits. Cultivation areas now cover more than 20 provinces and cities across China, and cultivation methods are becoming increasingly diversified. From 2020 to 2021, the national cultivation area exceeded 80,000 hectares, but the industry still has significant room for growth.

[0003] With the continuous expansion of morel mushroom cultivation, white mold disease has become widespread in morel mushroom growing areas, becoming one of the most important diseases in morel mushroom cultivation. Once it occurs, white mold rapidly covers the morel mycelium, competing with the morel mushrooms for nutrients and space. In severe cases, it can lead to the shrinkage, deformity, and even death of young mushrooms, causing large-scale yield reduction or crop failure, and has become a key bottleneck restricting the stable development of the industry. It has been reported that there are multiple pathogens causing diseases of morel fruiting bodies, but the main pathogen causing morel white mold is *Pseudomonas longissima* (…). Pseudodiploöspora longispora *Pseudodiplosporeus* belongs to the phylum Ascomycota, class Sordariomycetes, order Hypocreales, family Pseudodiplosporeaceae, and genus *Pseudodiplosporeus*. Pseudodiploöspora Morel mushrooms and *Pseudomonas longissima* belong to the same phylum Ascomycota. Generally speaking, chemical pesticides that inhibit the growth of *Pseudomonas longissima* are also toxic to morels. Therefore, the current control of morel white mold disease still focuses on prevention, environmental control, and physical isolation. However, due to the significant advantages of chemical agents in disease control, the selective use of appropriate concentrations of chemical agents remains an important means to mitigate economic losses. Summary of the Invention

[0004] This invention provides fluopyram for the control of morel mushrooms (Morchella spp.) Morchella Application of at least one of the fungi in white mold disease.

[0005] In one specific embodiment, the morel fungus is *Morchella esculenta* (also known as *Morchella esculenta*). Morchella sextelata ).

[0006] In one specific embodiment, the white mold disease is caused by Pseudomonas longissima (… Pseudodiploöspora longispora Caused by pathogens.

[0007] In one specific embodiment, the concentration of fluopyram used is from 0.09 to 4.1 micrograms per milliliter.

[0008] In one specific embodiment, the concentration of fluopyram used is 0.1 to 4.1 micrograms per milliliter.

[0009] In one specific embodiment, the concentration of fluopyram used is 0.2 to 3.2 micrograms per milliliter.

[0010] In one specific embodiment, the concentration of fluopyram used is 0.8 to 3.2 micrograms per milliliter.

[0011] Beneficial effects of the present invention: The present invention discovers that the chemical agent fluopyram has selective specificity for Pseudomonas longissima compared to Morel, and therefore can be used to prevent and control morel white mold disease. Attached Figure Description

[0012] Figure 1 The study showed the effect of fluopyram on the mycelial growth of *Morchella esculenta* in a toxicity assay. 1: blank control; 2: fluopyram concentration 0.8 μg / mL; 3: fluopyram concentration 3.2 μg / mL; 4: fluopyram concentration 6.4 μg / mL; 5: fluopyram concentration 12.8 μg / mL; 6: fluopyram concentration 25.6 μg / mL.

[0013] Figure 2 The study showed the effect of fluopyram on the mycelial growth of *Pseudomonas longissima* in a toxicity assay. 1: blank control; 2: fluopyram concentration 0.01 μg / mL; 3: fluopyram concentration 0.05 μg / mL; 4: fluopyram concentration 0.2 μg / mL; 5: fluopyram concentration 0.8 μg / mL; 6: fluopyram concentration 3.2 μg / mL. Detailed Implementation

[0014] The present invention will be further described in detail below through preferred embodiments, but these embodiments do not constitute a limitation thereof.

[0015] Unless otherwise specified, the strains and reagents used in the embodiments of this invention can be purchased commercially.

[0016] Potato glucose agar (PDA) medium: 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, and steam sterilize at 121 degrees Celsius for 20 min.

[0017] Sixth Sister Morel Mushrooms ( Morchella sextelata Healthy morel fruiting bodies were collected from the Langfang morel cultivation base, and the experimental strains were isolated and purified in the laboratory.

[0018] Pseudomonas longissima ( Pseudodiploöspora longispora It was isolated and purified from disease samples naturally infected with white mold at the Langfang morel cultivation base, and identified by morphology and combined sequences of rDNA-ITS, rDNA-LSU, and rDNA-SSU genes.

[0019] 98% imazalil, 99.9% fludioxonil, 98% oxadixyl, 99.76% chlorothalonil, and 99.8% cyazofamid were purchased from Shanghai Bide Pharmaceutical Technology Co., Ltd.

[0020] 98% thiabendazole, 96% tebuconazole, 98% fluopyram, 97% tebuconazole, and 97% thiram were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0021] 98% metalaxyl and 95% difenoconazole were purchased from TIXIA (Shanghai) Chemical Industry Development Co., Ltd.

[0022] 98% carbendazim was purchased from Beijing Inokai Technology Co., Ltd.

[0023] 98% azoxystrobin was purchased from Nantong Taihe Chemical Co., Ltd.

[0024] 98% dimethomorph was purchased from Jiangsu Henglong Crop Protection Co., Ltd. Example 1: Effects of chemical agents on morel mushrooms

[0025] Indoor toxicity was determined using the colony growth rate method.

[0026] Under aseptic conditions, 98% imazalil, 99.9% fludioxonil, 98% hymexazol, 99.76% chlorothalonil, 99.8% boscalid, 98% thiabendazole, 96% tebuconazole, 98% fluopyram, 97% tebuconazole, 97% thiram, 98% metalaxyl, 95% difenoconazole, 98% carbendazim, 98% azoxystrobin, and 98% dimethomorph technical grade were each prepared with anhydrous ethanol to a concentration of 1×10⁻⁶. 4 The test solution of each chemical reagent at a concentration of micrograms per milliliter.

[0027] Each test solution was added to the PDA medium to prepare PDA plates with different drug concentrations. Taking fluopyram as an example, the final concentrations of the active ingredient fluopyram in the drug-containing PDA plates were 0.8 μg / mL, 3.2 μg / mL, 6.4 μg / mL, 12.8 μg / mL, and 25.6 μg / mL.

[0028] Morel mushrooms were activated at 25°C using PDA medium. Then, 6 mm diameter morel mushroom discs were punched out. The mycelial side of the discs was placed on the center of plates containing different concentrations of PDA containing the drug. A plate without the drug served as a blank control. Each plate was a replicate, and each treatment was repeated three times. The plates were then incubated at 25°C. After 4 days of incubation, photographs were taken. The photograph of the PDA plate containing fluopyram is shown below. Figure 1 As shown. Then, the diameter of each treatment colony was measured using the cross-sectional method, and the inhibition rate of fluopyram on mycelial growth was calculated. Inhibition rate (%) = (control colony diameter - treatment colony diameter) × 100 / (control colony diameter - mycelial plate diameter).

[0029] Table 1 shows the colony diameter and inhibition rate of *Morchella esculenta* in the blank control and those treated with fluopyram at various concentrations.

[0030] Table 1

[0031] According to the results in Table 1, fluopyram at a concentration of 6.4 μg / mL showed an inhibition rate of over 50% against *Morchella esculenta*, indicating a significant inhibitory effect. At a concentration of 3.2 μg / mL, the inhibition rate was 33%, which was within an acceptable range.

[0032] Using the mass concentration (µg / mL) and inhibition rate of the active ingredient in each chemical agent as variables, IBM SPSS Statistics 27 software was used to analyze the data, obtaining the toxicity regression equation and correlation coefficient (R²). 2 ), inhibiting medium concentration (EC) 50 The results are shown in Table 2.

[0033] Table 2

[0034] Table 2 shows that the toxicity of the 15 antifungal agents against *Morchella esculenta* exhibited significant selectivity differences. Based on orders of magnitude, they were divided into five groups. The group with the strongest toxicity to *Morchella esculenta* consisted of four antifungal agents: tebuconazole, fludioxonil, tebuconazole, and thiamethoxam. These four antifungal agents showed the highest EC50 concentrations in *Morchella esculenta*. 50 The values ​​were relatively low, ranging from 0.001 to 0.072 μg / mL; the group with the second highest toxicity to *Morchella esculenta* included: difenoconazole, azoxystrobin, carbendazim, and imazalil. These four fungicides showed low EC50 values ​​for *Morchella esculenta*. 50 Values ​​ranged from 0.350 to 0.727 μg / mL; the group with intermediate toxicity to *Morchella esculenta* included fluopyram, with an EC50 value of [missing value] for *Morchella esculenta*. 50 The value was 4.124 μg / mL; the group with weaker toxicity to *Morchella esculenta* included: oxadixyl, cyazofamid, and thiram. These three antibacterial agents showed relatively low EC50 values ​​against *Morchella esculenta*. 50 The values ​​ranged from 14.522 to 31.200 μg / mL; the group with the weakest toxicity to *Morchella esculenta* consisted of chlorothalonil, metalaxyl, and dimethomorph, these three antibacterial agents having the lowest EC50 values ​​against *Morchella esculenta*. 50 The range of values ​​between 90.561 and 125.882 μg / mL is the safest for *Morchella esculenta*. Example 2: Effect of chemical agents on *Pseudomonas longissima*

[0035] Morel mushrooms were replaced with Pseudomonas longissima, and 5 to 6 different concentration gradients were set up. The culture time was 14 days. 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. 2 ), inhibiting medium concentration (EC) 50 (and 95% confidence interval).

[0036] Photographs of PDA plates containing fluopyram Figure 2 As shown in Table 3; the colony diameter and inhibition rate of *Pseudomonas longissima* in the blank control and at various concentrations of fluopyram are shown in Table 3; the toxicity regression equations, correlation coefficients, and EC50 values ​​of each chemical agent are also shown in Table 3. 50 The 95% confidence intervals are shown in Table 4.

[0037] Table 3

[0038] Table 4

[0039] Table 3 shows that fluopyram at a concentration of 3.20 μg / mL exhibited an inhibition rate of 89.38% against *Pseudomonas longissima*, indicating that the disease caused by *Pseudomonas longissima* was essentially under control at this concentration. Table 4 shows that fluopyram's EC50 against *Pseudomonas longissima*... 50 The value was 0.093 micrograms / mL, which is a relatively effective level of inhibition against Pseudomonas longissima.

[0040] Table 4 shows that the toxicity of the 15 antifungal agents against *Pseudomonas longissima* also exhibited significant selectivity differences. Based on orders of magnitude, they were divided into five groups. The group with the strongest toxicity to *Pseudomonas longissima* consisted of fluopyram and cyprodinil, with these two antifungal agents showing the highest EC500 against *Pseudomonas longissima*. 50 The values ​​were relatively low, ranging from 0.093 to 0.015 μg / mL; the group with the second strongest toxicity to *Pseudomonas longissima* included: imazalil, tebuconazole, tebuconazole, and thiamethoxam, these four fungicides showing EC50 values ​​against *Pseudomonas longissima*. 50 The values ​​ranged from 0.167 to 0.822 μg / mL; the group with intermediate toxicity to *Pseudomonas longissima* included: difenoconazole, azoxystrobin, carbendazim, and boscalid. These four fungicides showed EC50 values ​​against *Pseudomonas longissima*. 50 Values ​​ranged from 1.667 to 8.427 μg / mL; the group with weaker toxicity to *Pseudomonas longissima* included oxadixyl, with EC50 values ​​against *Pseudomonas longissima*. 50 The value was 17.190 μg / mL; the group with the weakest toxicity to *Pseudomonas longissima* included: thiram, chlorothalonil, dimethomorph, and metalaxyl. These three antifungal agents showed the lowest EC50 concentrations against *Pseudomonas longissima*. 50 The values ​​ranged from 137.991 to 423.343 μg / mL. Example 3: Selection of suitable chemical agents for the prevention and control of morel white mold caused by *Pseudomonas longissima*

[0041] Based on the effects of various chemical agents on *Morchella esculenta* EC 50 and Pseudomonas longissimus EC 50 The selectivity index (SI) of each chemical agent was calculated. A higher SI value indicates that the chemical agent effectively inhibits *Pseudomonas longissima* while having less impact on *Morchella esculenta* mycelial growth, thus indicating higher safety for *Morchella esculenta*. The formula is as follows: Selectivity Index SI = *Morchella esculenta* EC 50 / Pseudomonas longissima EC 50 The results are shown in Table 5.

[0042] Table 5

[0043] As shown in Table 5, among the 15 tested chemical agents, fluopyram exhibited a significantly higher selectivity index (SI) of 44.344, indicating that it has selective specificity against *Pseudomonas longissima* compared to *Morchella esculenta*. In other words, while six of the 15 chemical agents are safer than fluopyram in terms of overall safety against *Morchella esculenta*, these six agents also have weaker toxicity against *Pseudomonas longissima*, even weaker than against *Morchella esculenta*. Although one chemical agent has comparable toxicity to fluopyram and four are slightly less toxic than fluopyram in terms of toxicity against *Pseudomonas longissima*, except for imazalil, the other four agents are all more toxic than *Pseudomonas longissima*. This is reflected in the selectivity index (SI). Therefore, these chemical agents are not suitable for controlling white mold disease in morel mushrooms. As for imazalil, its selectivity index is second only to fluopyram, but its selectivity index is only 4.353, indicating that its toxicity to *Morchella esculenta* and *Pseudomonas longissima* is not significantly different. In actual production, it is difficult to select a dosage that has almost no effect on *Morchella esculenta* but can significantly control white mold. Moreover, the selectivity index of fluopyram is 10 times that of imazalil. Therefore, from both a safety perspective and the perspective of controlling white mold, the use of fluopyram can achieve a balance.

Claims

1. Fluopyram is used to control morel mushrooms (Morchella spp.) Morchella Application of at least one of the fungi in white mold disease.

2. The application according to claim 1, characterized in that, The morel fungus mentioned is *Morchella spp.* (also known as *Morchella spp.* Morchella sextelata ).

3. The application according to claim 1, characterized in that, The white mold disease is caused by Pseudomonas longissimum (… Pseudodiploöspora longispora Caused by pathogens.

4. The application according to any one of claims 1 to 3, characterized in that, The concentration of fluopyram used is from 0.09 to 4.1 micrograms per milliliter.

5. The application according to any one of claims 1 to 3, characterized in that, The concentration of fluopyram used is 0.1 to 4.1 micrograms per milliliter.

6. The application according to any one of claims 1 to 3, characterized in that, The concentration of fluopyram used is 0.2 to 3.2 micrograms per milliliter.

7. The application according to any one of claims 1 to 3, characterized in that, The concentration of fluopyram used is 0.8 to 3.2 micrograms per milliliter.

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