Application of Freshwater Fungus Neocucurbitaria quercina in Controlling Pear Brown Rot Disease

Abstract

The application belongs to the technical field of microbial inoculants, and particularly relates to application of a freshwater fungus Neocucurbitaria quercina in prevention and treatment of pear ring rot disease. The freshwater fungus N. quercina is derived from a natural freshwater environment, and metabolites produced by the fungus are safe to the environment and have good inhibitory activity on the pathogenic fungus of the pear ring rot disease, Botryosphaeria dothidea. The metabolites can effectively inhibit the growth of mycelium of the fungus and effectively inhibit the infection of the fungus on pear fruits, thereby having a good prevention and treatment effect on the pear ring rot disease, and can reduce the use of traditional chemical synthetic pesticides, and have important significance for protecting the environment and producing green and safe agricultural products.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microbial agents, and particularly relates to application of freshwater fungus Neocucurbitaria quercina in prevention and treatment of pear ring rot disease. BACKGROUND

[0002] Pear ring rot disease is one of the main diseases in the growth period and postharvest storage period of pear production areas all over the world, which harms branches, fruits and leaves of pear trees, and causes early decline of tree vigor and fruit rot. When fruits are infected, water-stained brown spots are generated around the skin pores, the disease spots gradually change from light brown to red-brown, and concentric rings appear, and the fruits will soon rot with tea-brown juice flowing out. Some diseased fruits can become black-brown after water loss. The pathogen of pear ring rot disease is mainly Botryosphaeria dothidea, which belongs to Ascomycetes. In addition to parasitizing pear, it also harms apple, apricot and kiwi fruit trees. Its harm range includes more than 20 provinces in the north and south of China. The pear trees in the south have a higher incidence, which leads to a reduction in fruit yield and serious economic losses, and poses a great threat to the development of the fruit industry.

[0003] At present, the main microbial agents for preventing and treating pear ring rot disease are chemical synthetic pesticides such as carbendazim, aluminum ethylphosphonate and tebuconazole. The use method is mainly to spray the pesticides 10 days after flowering or after flowering. Specifically, 80% carbendazim wettable powder 800-1000 times liquid can be used to smear the disease scars. From the beginning of May to the end of August, that is, during the fruit expansion period, the spraying time can be determined according to the specific disease condition, and 80% carbendazim wettable powder 800 times liquid + 90% aluminum ethylphosphonate soluble powder 600 times liquid can be sprayed. Although such chemical synthetic pesticides have obvious effects in preventing and treating diseases and are widely used in production. However, due to the unscientific use or long-term use of chemical synthetic pesticides, problems such as pesticide resistance, pesticide residues, deterioration of ecological environment and reduction of biological diversity level are emerging, and the inhibitory activity of the pathogen of pear ring rot disease is greatly reduced. SUMMARY

[0004] The purpose of the present application is to provide the application of freshwater fungus Neocucurbitaria quercina in prevention and treatment of pear ring rot disease. The microbial agent containing the freshwater fungus Neocurbitaria quercina and / or its metabolites has good inhibitory activity on the pathogen Botryosphaeria dothidea of pear ring rot disease, good effect in preventing and treating pear ring rot disease, and is green, safe and pollution-free.

[0005] The present application provides the application of a microbial agent containing freshwater fungus Neocurbitaria quercina and / or metabolites of freshwater fungus Neocurbitaria quercina in prevention and treatment of pear ring rot disease.

[0006] Preferably, the microbial agent comprises an organic extract of a fermentation product of the freshwater fungus Neocucurbitaria quercina.

[0007] Preferably, the organic extract of the fermentation product comprises an organic extract of a rice fermentation product.

[0008] The present application also provides an organic extract of a rice fermentation product of the freshwater fungus Neocucurbitaria quercina, wherein the organic extract of the rice fermentation product comprises a methanol extract of a rice fermentation product or an ethyl acetate extract of the methanol extract of the rice fermentation product.

[0009] The present application also provides a preparation method of the organic extract of the rice fermentation product according to the above technical solution, comprising the following steps:

[0010] Inoculating an activated strain of the freshwater fungus Neocurbitaria quercina into a PDB liquid culture medium to perform seed culture, to obtain a seed culture solution;

[0011] Inoculating the seed culture solution into a rice culture medium to perform fermentation culture, to obtain a rice fermentation product;

[0012] Mixing the rice fermentation product with a methanol aqueous solution to perform cold soaking extraction, and reducing pressure concentration of the obtained cold soaking extraction solution, to obtain a methanol extract of the rice fermentation product;

[0013] Mixing the methanol extract of the rice fermentation product with water to obtain a methanol extract suspension;

[0014] Mixing the methanol extract suspension with ethyl acetate to perform extraction, reducing pressure concentration of the obtained organic phase, to obtain an ethyl acetate extract of the methanol extract of the rice fermentation product.

[0015] Preferably, the methanol aqueous solution comprises a methanol aqueous solution with a volume fraction of 95-99%;

[0016] The mass of the rice fermentation product to the volume of the methanol aqueous solution is 1 kg:(3-6) L;

[0017] The number of times of the cold soaking extraction is 4-6, and the extraction time of each time is 1-3 days.

[0018] Preferably, the concentration of the methanol extract suspension is 20-100 g / L; the volume ratio of the methanol extract suspension to ethyl acetate is 1:(0.5-1.5); and the number of times of the extraction is 3-5.

[0019] The application further provides a fungicide for preventing and treating pear ring rot, which comprises the rice fermentation organic extract or the rice fermentation extract prepared by the preparation method.

[0020] Preferably, the solvent comprises one or more of water, ethanol and Tween.

[0021] Preferably, the concentration of the rice fermentation extract in the fungicide is 312.5 μg / mL to 250 mg / mL.

[0022] Beneficial effects:

[0023] The application provides application of a fungicide containing freshwater fungus Neocucurbitaria quercina and / or metabolic products of the freshwater fungus N. quercina in prevention and treatment of pear ring rot. The freshwater fungus N. quercina in the application is derived from a natural freshwater environment, and the metabolic products produced by the freshwater fungus are safe to the environment and have good inhibitory activity on the pathogenic fungus Botryosphaeria dothidea. The metabolic products can effectively inhibit the growth of mycelium of the fungus Botryosphaeria dothidea and effectively inhibit the infection of the fungus on pear fruits, thereby having a good prevention and treatment effect on pear ring rot, and can reduce the use of traditional chemical synthetic pesticides, which has important significance for protecting the environment and producing green and safe agricultural products. BRIEF DESCRIPTION OF DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the drawings needed in the embodiments will be briefly introduced.

[0025] Figure 1 Figure for mycelium growth inhibition of the rice fermentation extract of the freshwater fungus N. quercina on the pear ring rot fungus in Examples 1-2 and Comparative Example 1;

[0026] Figure 2 Figure for mycelium growth inhibition of the ethyl acetate extract of the rice fermentation methanol extract of the freshwater fungus N. quercina on the pear ring rot fungus in Examples 3-6 and Comparative Example 2;

[0027] Figure 3 Figure for mycelium morphology inhibition of the rice fermentation methanol extract of the freshwater fungus N. quercina on the pear ring rot fungus in Example 3 and Comparative Example 2;

[0028] Figure 4 Figure for the prevention and treatment effect of the ethyl acetate extract of the rice fermentation methanol extract of the freshwater fungus N. quercina on the pear ring rot in Example 7. DETAILED DESCRIPTION

[0029] The application provides application of a microbial agent containing freshwater fungus Neocucurbitaria quercina and / or fermentation product of the freshwater fungus N. quercina in prevention and treatment of pear ring rot disease. The prevention and treatment of the pear ring rot disease preferably comprises inhibition of growth and / or infection of pear fruit by the pathogen of the pear ring rot disease. The freshwater fungus N. quercina is referred to as freshwater fungus N. quercina.

[0030] The pathogen of the pear ring rot disease is Botryosphaeria dothidea (B. dothidea). The strain type and source of the B. dothidea are not particularly limited. The microbial agent containing the freshwater fungus N. quercina and / or fermentation product of the freshwater fungus N. quercina has an inhibitory effect on the B. dothidea in the art.

[0031] The freshwater fungus N. quercina is collected by the laboratory, and is identified as N. quercina by means of morphology, ITS specific sequence and phylogenetic tree comparison, and has been disclosed in Jaklitsch, WM, Checa, et al. A preliminary account of the Cucurbitariaceae [J]. STU D MYCOL, 90: 71-118 (2018).

[0032] The microbial agent preferably comprises fermentation product organic extract of the freshwater fungus N. quercina, further preferably comprises rice fermentation product organic extract, and more preferably comprises rice fermentation product methanol extract or ethyl acetate extract of the rice fermentation product methanol extract.

[0033] The freshwater fungus N. quercina is derived from a natural freshwater environment, has great potential to produce novel secondary fermentation products, can be used as a decomposer for waste and wastewater remediation, promotes nutrient and carbon cycling, maintains the balance of the freshwater ecosystem, and is safe to the environment. The fermentation product extract obtained by extracting the fermentation product of the freshwater fungus N. quercina has high inhibitory activity on the pathogen B. dothidea of the pear ring rot disease, and can effectively prevent and treat the pear ring rot disease.

[0034] The application further provides a rice fermentation product organic extract of the freshwater fungus N. quercina, wherein the rice fermentation product organic extract comprises rice fermentation product methanol extract or ethyl acetate extract of the rice fermentation product methanol extract, and is more preferably ethyl acetate extract of the rice fermentation product methanol extract.

[0035] The application further provides a preparation method of the rice ferment organic extract, comprising the following steps:

[0036] The activated fresh water fungus N.quercina strain is inoculated into a PDB liquid culture medium for seed culture to obtain a seed culture solution;

[0037] The seed culture solution is inoculated into a rice culture medium for fermentation culture to obtain a rice ferment;

[0038] The rice ferment is mixed with a methanol aqueous solution for cold soaking extraction, and the obtained cold soaking extraction solution is reduced pressure concentrated to obtain a rice ferment methanol extract;

[0039] The rice ferment methanol extract is mixed with water to obtain a methanol extract suspension;

[0040] The methanol extract suspension is mixed with ethyl acetate for extraction, and the obtained organic phase is reduced pressure concentrated to obtain an ethyl acetate extract of the rice ferment methanol extract.

[0041] Preferably, the fresh water fungus N.quercina is inoculated into a PDA solid culture medium for activation culture in the application. The temperature of the activation culture is preferably 20-30 DEG C, and more preferably 25 DEG C. The time of the activation culture is preferably 0.5-3 weeks, and more preferably 2 weeks. The activation culture is preferably dark culture. The activation culture is preferably carried out in a constant temperature incubator.

[0042] After the activation culture, the strain after the activation culture is preferably subcultured to obtain the activated fresh water fungus N.quercina strain. The subculture conditions are preferably the same as the activation culture conditions to achieve the purpose of expanding culture and rejuvenating the strain.

[0043] After obtaining the activated fresh water fungus N.quercina strain, the activated fresh water fungus N.quercina strain is inoculated into a PDB liquid culture medium for seed culture to obtain a seed culture solution. The seed culture is preferably carried out in dark conditions and is preferably shock culture. The temperature of the seed culture is preferably 20-30 DEG C, and more preferably 25 DEG C. The rotation speed is preferably 120-180 rpm, and more preferably 150 rpm. The time is preferably 10-20 d, and more preferably 14 d.

[0044] After obtaining the seed culture solution, the seed culture solution is inoculated into the rice culture medium for fermentation culture to obtain a rice fermentation product. The rice fermentation product of the present application is preferably long rice culture medium of N. quercina mycelium. The inoculation amount of the seed culture solution of the present application is preferably 1-10% of the volume of the rice culture medium, and more preferably 5%. The fermentation culture of the present application is preferably carried out in the dark, and is preferably static culture. The temperature of the fermentation culture of the present application is preferably 20-30°C, and more preferably 25°C; the time is preferably 40-80 days, and more preferably 60 days.

[0045] The preparation method of the rice culture medium of the present application preferably comprises: mixing rice and water, and then high-pressure steam sterilization to obtain the rice culture medium. The mass-volume ratio of the rice and water of the present application is preferably 100g: 110mL; and the water is preferably deionized water. The temperature of the high-pressure steam sterilization of the present application is preferably 121°C, and the time is preferably 15-25min, and more preferably 20min. The pH value of the rice culture medium of the present application is preferably the natural value.

[0046] After obtaining the rice fermentation product, the rice fermentation product is preferably cut into rice fermentation product debris. The particle size of the rice fermentation product debris of the present application is not particularly limited, and the conventional size in the art can be used for convenient cold extraction.

[0047] After obtaining the rice fermentation product debris, the rice fermentation product debris is preferably mixed with a methanol aqueous solution for cold extraction. The methanol aqueous solution of the present application preferably comprises a methanol aqueous solution with a volume fraction of 95-99%. The rice fermentation product debris is preferably soaked in the methanol aqueous solution for cold extraction. The temperature of the cold extraction of the present application is preferably room temperature; the cold extraction is preferably 4-6 times, and more preferably 5 times; and the time of each cold extraction is preferably 1-3 days, and more preferably 2 days. The 4-6 times of cold extraction of the present application preferably comprises: after each cold extraction, the cold extraction liquid is taken out, and the methanol aqueous solution is continuously added to the remaining cold extraction material for the next cold extraction, and the cold extraction liquids obtained by the 4-6 times of cold extraction are combined. The mass of the rice fermentation product debris to the volume of the methanol aqueous solution is preferably 1kg: (3-6)L, and more preferably 1kg: 5L.

[0048] After the cold extraction, the obtained cold extraction liquid is reduced pressure concentrated to obtain a rice fermentation product methanol extract. The specific conditions of the reduced pressure concentration of the present application are not particularly limited, and the cold extraction liquid can be reduced pressure concentrated to dryness.

[0049] After obtaining the rice fermentation methanol extract, the present application mixes the rice fermentation methanol extract with water to obtain a methanol extract suspension. The concentration of the methanol extract suspension of the present application is preferably 20-100 g / L, more preferably 50 g / L.

[0050] After obtaining the methanol extract suspension, the present application extracts the methanol extract suspension by mixing with ethyl acetate. The volume ratio of the methanol extract suspension to ethyl acetate of the present application is preferably 1:(0.5-1.5), more preferably 1:1. The volume fraction of the ethyl acetate of the present application is preferably ≥99.5%. The number of extractions of the present application is preferably 3-5 times, more preferably 4 times.

[0051] After the extraction, the present application mixes the obtained organic phase and concentrates under reduced pressure to obtain a rice fermentation methanol extract ethyl acetate extract. The present application does not have special limitations on the specific conditions of the reduced pressure concentration, and the reduced pressure concentration can be performed until dryness.

[0052] The rice fermentation extract of the freshwater fungus N. quercina of the present application is an extract obtained from the rice fermentation product of the freshwater fungus N. quercina, has a good effect of inhibiting the growth of pathogenic bacteria, and the rice fermentation methanol extract and the rice fermentation methanol extract ethyl acetate extract of the freshwater fungus N. quercina are natural microbial source components without residue problems. They can be used as pollution-free, safe, and green biopesticides, reducing the use of traditional chemical synthetic pesticides, and having important significance for protecting the environment and producing green and safe agricultural products.

[0053] Based on the above advantages, the present application also provides a fungicide for preventing and treating pear annular canker, which comprises the rice fermentation organic extract of the above technical solution or the rice fermentation extract prepared by the preparation method of the above technical solution and a solvent. The solvent of the present application preferably comprises one or more of water, ethanol, and Tween, more preferably ethanol or ethanol and water. The ethanol of the present application is preferably an ethanol solution, more preferably an ethanol aqueous solution with a volume fraction of 50%. The concentration of the rice fermentation extract in the fungicide of the present application is preferably 312.5 μg / mL-250 mg / mL, more preferably 2.5-250 mg / mL, and specifically can be preferably 0.3125, 0.625, 1.25, 2.5, 31.25, 50, 62.5, 125, or 250 mg / mL.

[0054] The present application also provides a preparation method of the above fungicide for preventing and treating pear annular canker, which comprises mixing the rice fermentation extract of the above technical solution or the rice fermentation extract prepared by the preparation method of the above technical solution with a solvent. The mixing method of the present application is not particularly limited and can be a conventional mixing method in the art.

[0055] The application does not have special limitations on the use mode of the bacterial agent, and the application mode known to those skilled in the art can be used.

[0056] In order to further illustrate the present application, the technical solutions provided by the present application are described in detail below in combination with the drawings and examples, but they should not be understood as limiting the scope of protection of the present application.

[0057] Example 1

[0058] Mycelial growth inhibition activity of methanol extract of rice fermentation of N. quercina on V. pirina

[0059] 1. Preparation of methanol extract of rice fermentation of N. quercina

[0060] The strain N. quercina preserved in a 4℃ cryotube on PDA slant medium (the composition of PDA medium: 200g of potato, 20g of glucose, 20g of agar and 1000mL of deionized water, natural pH) was inoculated into a PDA culture dish and activated and cultured in a 25℃ dark constant temperature incubator for 2 weeks. Under the same culture conditions, the strain was subcultured for 2 weeks and then inoculated into PDB liquid medium, and shaken at 25℃, 150rpm, in the dark for 14d as a seed culture. Then the seed culture containing mycelium and bacterial liquid was uniformly poured into a tissue culture bottle containing sterilized rice medium (the composition of rice medium: 100g of rice and 110mL of deionized water, natural pH value, prepared by high-pressure steam sterilization at 121℃ for 15-20min) in advance, and cultured at 25℃ in the dark for 60d to obtain a rice culture, i.e. rice medium with long mycelium.

[0061] The rice medium with long mycelium was cut and extracted by cold soaking with a 95-99% methanol aqueous solution at room temperature, 2d each time, for a total of 5 times. The methanol extract obtained was concentrated to dryness under reduced pressure to obtain methanol extract of rice fermentation of N. quercina.

[0062] Preparation of methanol extract of rice fermentation of N. quercina mother liquor: 5g of methanol extract of rice fermentation of N. quercina was dissolved in an aqueous solution containing 50% ethanol by volume to obtain a methanol extract of rice fermentation of N. quercina mother liquor with a concentration of 50mg / mL.

[0063] 2、Preparation of the test V. pirina mycelium pieces: The V. pirina (B. dothidea) preserved in a 4°C refrigerator was taken out, inoculated into fresh PDA medium, and activated and cultured in a 25°C constant-temperature incubator. After 3 days of culture, 3 mycelium pieces were punched out using a 7-mm puncher.

[0064] The PDA medium was potato dextrose agar medium, and the composition was: potato 200 g, glucose 20 g, agar 20 g, and deionized water 1000 mL, natural pH.

[0065] 3、0.3 mL of the N. quercina rice fermentation methanol extract mother liquor prepared in step 1 was added to 30 mL of PDA medium (the temperature of the medium was about 50°C) and mixed uniformly to prepare a drug-containing medium with a concentration of 500 μg / mL of the N. quercina rice fermentation methanol extract, which was poured into three culture dishes to set up three parallel experiments.

[0066] The mycelium pieces prepared in step 2 were inoculated in the center of the drug-containing medium culture dishes, with the mycelium side down, and one mycelium piece was placed in each culture dish, which was placed in a 25°C constant-temperature incubator for 3 days of culture. Three parallel experiments were set up. The diameter (mm) of the colony was measured by the cross method, and the inhibition rate was calculated.

[0067] Example 2

[0068] Inhibition activity of the ethyl acetate extract of the N. quercina rice fermentation methanol extract on the mycelium growth of V. pirina

[0069] 1、Preparation of the ethyl acetate extract of the N. quercina rice fermentation methanol extract:

[0070] The preparation process of the N. quercina rice fermentation methanol extract was the same as in Example 1.

[0071] The obtained methanol extract was suspended in water to a concentration of 50 g / L, and extracted 4 times with ethyl acetate at a volume ratio of 1:1. The organic phase was combined and concentrated to dryness under reduced pressure to obtain the ethyl acetate extract of the N. quercina rice fermentation methanol extract.

[0072] Preparation of the mother liquor of the ethyl acetate extract of the methanol extract of the rice fermentation product of N. quercina: 100 mL of an ethanol aqueous solution having a volume fraction of 50% was measured, and 5 g of the ethyl acetate extract of the rice fermentation product of N. quercina was dissolved in the ethanol aqueous solution to obtain a mother liquor of the ethyl acetate extract of the rice fermentation product of N. quercina at a concentration of 50 mg / mL.

[0073] 2. Preparation of the test V. pirina cake: the test pathogenic fungus V. pirina stored in a refrigerator at 4°C was taken out, inoculated into fresh PDA medium, and activated and cultured in a constant-temperature incubator at 25°C. After 3 days of culture, 3 cakes were punched out using a 7-mm puncher.

[0074] 3. 0.3 mL of the mother liquor of the ethyl acetate extract of the methanol extract of the rice fermentation product of N. quercina was added to 30 mL of PDA medium (the temperature of the medium was about 50°C) to prepare a drug-containing medium with the ethyl acetate extract of the rice fermentation product of N. quercina at a concentration of 500 μg / mL, and then the medium was mixed uniformly and poured into three culture dishes to set up three parallel experiments.

[0075] The cakes prepared in step 2 were inoculated in the center of the culture dishes with the drug-containing medium, with the mycelium side facing down, and one cake was placed in each culture dish. The culture dishes were placed in a constant-temperature incubator at 25°C and cultured for 3 days. Three parallel experiments were set up. The diameters of the colonies (mm) were measured by the cross method, and the inhibition rate was calculated.

[0076] Comparative Example 1

[0077] 0.3 mL of an ethanol aqueous solution having a volume fraction of 50% was added to 30 mL of PDA medium (the temperature of the medium was about 50°C) to prepare a control PDA medium containing ethanol aqueous solution having a volume fraction of 0.5%, and then the medium was mixed uniformly and poured into three culture dishes. The remaining conditions were the same as in Example 1.

[0078] The inhibitory effects of the components in Example 1, Example 2, and Comparative Example 1 on the mycelial growth of V. pirina were analyzed, and the inhibition rate was calculated according to the following formula:

[0079] Inhibition rate = (diameter of the control colony - diameter of the treated colony) / (diameter of the control colony) x 100.

[0080] The diameter of the colony = the diameter of the colony - the diameter of the cake. In the formula for calculating the inhibition rate, the diameter of the control colony was the diameter of the colony of Comparative Example 1 - the diameter of the cake, and the diameter of the treated colony was the diameter of the colony in Example 1 or Example 2 - the diameter of the cake.

[0081] The results of the mycelial growth inhibition of the fresh water fungus N. quercina rice fermentation extract of Example 1, Example 2 and Comparative Example 1 on V. pirina are shown in Table 1. Figure 1 The results of the determination are shown in Table 1.

[0082] Table 1 Inhibition of mycelial growth of V. pirina by the fresh water fungus N. quercina rice fermentation extract

[0083]

[0084] From Figure 1 It can be concluded that the fresh water fungus N. quercina rice fermentation extract in Example 1 and Example 2 has a higher inhibitory effect on the mycelial growth of V. pirina than that of Comparative Example 1, and the inhibitory effect of the ethyl acetate extract of the methanol extract of the rice fermentation of Example 2 is more obvious. In combination with Table 1, it can be concluded that the microbial source component of the fresh water fungus N. quercina rice fermentation extract has a more obvious inhibitory effect on the mycelial growth of V. pirina, and the antibacterial activity of the ethyl acetate extract is better than that of the methanol extract. When the concentration of the ethyl acetate extract of the methanol extract of the fresh water fungus N. quercina rice fermentation is 500 μg / mL, the inhibition rate of V. pirina is as high as 66.67%, which is 25% higher than that of the methanol extract. Therefore, the antibacterial activity of the ethyl acetate extract of the methanol extract of the fresh water fungus N. quercina rice fermentation is better.

[0085] Example 3

[0086] Inhibitory activity of gradient concentrations of the ethyl acetate extract of the methanol extract of the fresh water fungus N. quercina rice fermentation on the mycelial growth and morphology of V. pirina

[0087] Preparation of the ethyl acetate extract of the methanol extract of the fresh water fungus N. quercina rice fermentation: the preparation of the ethyl acetate extract of the methanol extract of the fresh water fungus N. quercina rice fermentation in Example 2.

[0088] Preparation of the ethyl acetate extract mother liquor of the fresh water fungus N. quercina rice fermentation: 25 g of the ethyl acetate extract of the methanol extract of the fresh water fungus N. quercina rice fermentation was dissolved in 100 mL of an ethanol aqueous solution with a volume fraction of 50% to obtain an ethyl acetate extract mother liquor with a concentration of 250 mg / mL, and the ethyl acetate extract mother liquor was diluted with the ethanol aqueous solution with a volume fraction of 50% to obtain a series of solutions of the ethyl acetate extract of the methanol extract of the fresh water fungus N. quercina rice fermentation with concentrations of 125, 62.5 and 31.25 mg / mL.

[0089] In 30 mL of PDA medium (temperature of the medium is about 50°C), 0.3 mL of the ethyl acetate extract solution of the methanol extract of the rice fermentation product of the aquatic fungus N. quercina having a concentration of 62.5 mg / mL was added to prepare a drug-containing medium having a concentration of 0.625 mg / mL, and then the mixture was uniformly poured into three Petri dishes, and the rest of the conditions were the same as in Example 3.

[0090] The test pathogenic fungus V. pyri-reliani, which was stored in a refrigerator at 4°C, was taken out, inoculated into fresh PDA medium, and activated and cultured in a constant-temperature incubator at 25°C. After 3 days of culture, a 7-mm puncher was used to take a fungus cake to inoculate the center of the drug-containing medium in a Petri dish with the mycelium side downward, and one fungus cake was placed in each Petri dish, and then the Petri dishes were placed in a constant-temperature incubator at 25°C and cultured for 3 days. The diameters (mm) of the colonies were measured by the cross method, and the inhibition rate was calculated.

[0091] Example 4

[0092] In 30 mL of PDA medium (temperature of the medium is about 50°C), 0.3 mL of the ethyl acetate extract solution of the methanol extract of the rice fermentation product of the aquatic fungus N. quercina having a concentration of 62.5 mg / mL was added to prepare a drug-containing medium having a concentration of 0.625 mg / mL, and then the mixture was uniformly poured into three Petri dishes, and the rest of the conditions were the same as in Example 3.

[0093] Example 5

[0094] In 30 mL of PDA medium (temperature of the medium is about 50°C), 0.3 mL of the ethyl acetate extract solution of the methanol extract of the rice fermentation product of the aquatic fungus N. quercina having a concentration of 62.5 mg / mL was added to prepare a drug-containing medium having a concentration of 0.625 mg / mL, and then the mixture was uniformly poured into three Petri dishes, and the rest of the conditions were the same as in Example 3.

[0095] Example 6

[0096] In 30 mL of PDA medium (temperature of the medium is about 50°C), 0.3 mL of the ethyl acetate extract solution of the methanol extract of the rice fermentation product of the aquatic fungus N. quercina having a concentration of 62.5 mg / mL was added to prepare a drug-containing medium having a concentration of 0.625 mg / mL, and then the mixture was uniformly poured into three Petri dishes, and the rest of the conditions were the same as in Example 3.

[0097] Comparative Example 2

[0098] The same as in Comparative Example 1.

[0099] The inhibition of the mycelial growth of V. pirina by the ethyl acetate extract of the methanol extract of the rice ferment of N. quercina of Examples 3 to 6 and Comparative Example 2 was analyzed, and the inhibition rate was calculated according to the following formula:

[0100] Inhibition rate = (diameter of control colony - diameter of treated colony) / (diameter of control colony) x 100.

[0101] Diameter of colony = diameter of colony - diameter of colony, and the diameter of control colony in the inhibition rate formula is the diameter of colony - diameter of colony of Comparative Example 2 in the present application.

[0102] The results of the inhibition of the mycelial growth of V. pirina by the ethyl acetate extract of the methanol extract of the rice ferment of N. quercina of Examples 3 to 6 and Comparative Example 2 are shown in Table 2. Figure 2 wherein the results of the inhibition of the mycelial growth of V. pirina by the ethyl acetate extract of the methanol extract of the rice ferment of Comparative Example 2, Example 6, Example 5, Example 4 and Example 3 are shown from left to right, respectively, and the results are shown in Table 2.

[0103] Table 2 Inhibition of the mycelial growth of V. pirina by the ethyl acetate extract of the methanol extract of the rice ferment of N. quercina

[0104]

[0105]

[0106] The inhibition rate in Table 2 was converted into the inhibition probability value according to the biological statistical probability value conversion table, and is shown in Table 2. The concentration logarithm set in the experiment was taken as the abscissa, and the inhibition probability value was taken as the ordinate, and the toxicity regression straight line equation of the ethyl acetate extract of the methanol extract of the rice ferment of N. quercina for V. pirina was fitted as y = -0.86 - 1.2x, and the EC 50 value was calculated when the inhibition rate was 50%, and the median concentration EC 50 value was 188 μg / mL, and the correlation coefficient r was 0.995.

[0107] As can be seen from Table 2, Figure 2 compared with Comparative Example 2, the ethyl acetate extract of the methanol extract of the rice ferment of N. quercina in Examples 3 to 6 of the present application had a certain inhibitory effect on the mycelial growth of V. pirina, and the inhibitory effect of the ethyl acetate extract of the methanol extract of the rice ferment of Example 3 on the mycelial growth of V. pirina was more obvious.

[0108] Mycelia from the edges of colonies in Example 3 and Comparative Example 2 were observed under an optical microscope. The results of the inhibition of mycelial morphology against *Pyrus pyriformis* in Example 3 and Comparative Example 2 are shown in the figure. Figure 3 As shown.

[0109] Depend on Figure 3 It can be concluded that: the mycelial image of Comparative Example 2 shows that the mycelium grows fully and spread out, with uniform contents; the mycelial image of Example 3 shows that the mycelium is obviously swollen and deformed, with increased and shorter branches (indicated by red arrows), and there are also cavities inside the mycelium and uneven color of the contents (indicated by blue arrows). This indicates that the ethyl acetate extract of the methanol extract of the freshwater fungus N. quercina rice fermentation significantly affects the mycelial morphology and contents of N. quercina, inhibits the mycelial growth of N. quercina, and has a significant inhibitory effect on N. quercina.

[0110] Combination Figure 2 Table 2 and Figure 3 It can be concluded that the ethyl acetate extract of the methanol extract of the freshwater fungus N. quercina rice fermentation product, a microbial component of this invention, has good inhibitory activity on the mycelial growth and morphology of *N. quercina*, the causal agent of pear ring rot. The inhibition efficiency is concentration-dependent; the higher the concentration, the better the inhibitory activity. At a concentration of 2.5 mg / mL, the ethyl acetate extract of the methanol extract of the freshwater fungus N. quercina rice fermentation product exhibits an inhibition rate of 91.38% on the mycelial growth of *N. quercina*, indicating significant application potential.

[0111] Example 7

[0112] The effect of ethyl acetate extract of methanol extract from rice fermentation product of freshwater fungus N. quercina on the control of pear ring rot disease.

[0113] Gong pears of similar size and condition were selected from the local fruit market. After cleaning, punctures were made near the equator using a punch and inoculated with pear ring rot fungus (5 mm in diameter). The fungus preparation method was the same as in Example 1. After air-drying for 2 hours, 20 μL of an ethyl acetate extract of the methanol extract of rice fermentation by the freshwater fungus N. quercina (10 mg / mL) was inoculated at the inoculation site. The mixture was then incubated at 25°C with 95% humidity, and this group was designated as the experimental group.

[0114] Meanwhile, several Gong pears were inoculated with 20 μL of aqueous solution at the sites where pear ring rot pathogens were inoculated, serving as a control group.

[0115] The disease incidence of Guo pears treated with the aqueous solution of rice fermentation ethyl acetate extract of N. quercina after inoculation was counted on 1, 2, 3, 4, 5 and 6 days after inoculation, and the lesion diameter (mm) was recorded by cross method. The inhibition rate was calculated according to the following formula: inhibition rate = (diameter of control lesion - diameter of treated lesion) / (diameter of control lesion - 5) x 100. The results are shown in Table 3 and Figure 4 , wherein Figure 4 is the prevention effect of rice fermentation ethyl acetate extract of N. quercina on pear fruit pear ring rot disease, from left to right representing 1, 2, 3, 4, 5 and 6 days after inoculation, wherein Figure 4 is the rice fermentation ethyl acetate extract of N. quercina in methanol extract of rice fermentation of N. quercina in the present application.

[0116] Table 3 Prevention effect of rice fermentation ethyl acetate extract of N. quercina on pear ring rot disease

[0117]

[0118] According to Table 3, the microbial source component rice fermentation ethyl acetate extract of N. quercina in the present application can also effectively inhibit the infection of pear ring rot fungus in pear living body, and has good prevention effect, with a lesion inhibition rate of 40.88% on 6 days after inoculation, and the inhibition rate shows an upward trend with the increase of days.

[0119] From the above examples, it can be concluded that the microbial agent containing N. quercina and / or metabolites of N. quercina has high inhibitory activity on pear ring rot fungus, and can effectively prevent and reduce the occurrence of pear ring rot disease. At the same time, the microbial agent is derived from natural microorganisms, has its degradation pathway in nature, has no residue problem, and is in harmony with the environment.

[0120] Although the above examples have made a detailed description of the present application, it is only a part of the examples of the present application, not all the examples, and other examples can be obtained according to the present examples without creativity, which all belong to the protection scope of the present application.

Claims

1. Contains freshwater fungi Neocucurbitaria quercina Application of microbial agents containing metabolites in the control of pear ring rot disease; said microbial agents include freshwater fungi. Neocucurbitaria quercina The organic extract of rice fermentation product, comprising a methanol extract of rice fermentation product or an ethyl acetate extract of a methanol extract of rice fermentation product; the freshwater fungus Neocucurbitaria quercina The strain number is CBS 115095.

2. The application according to claim 1, characterized in that, The preparation method of the rice ferment organic extract includes the following steps: activating freshwater fungi... Neocucurbitaria quercina The strain was inoculated into PDB liquid medium for seed culture to obtain seed culture solution; The seed culture solution was inoculated into a rice culture medium for fermentation to obtain rice fermentation product; The rice fermentation product was mixed with a methanol aqueous solution and then subjected to cold soaking extraction. The resulting cold soaking extract was concentrated under reduced pressure to obtain a methanol extract of the rice fermentation product. The rice fermentation product methanol extract was mixed with water to obtain a methanol extract suspension; The methanol extract suspension was mixed with ethyl acetate and extracted. The resulting organic phase was concentrated under reduced pressure to obtain the ethyl acetate extract of the rice fermentation methanol extract.

3. The application according to claim 2, characterized in that, The methanol-water solution comprises a methanol-water solution with a volume fraction of 95-99%; the mass ratio of the rice fermentation product to the volume of the methanol-water solution is 1 kg: (3-6) L; the cold soaking extraction is performed 4-6 times, and each extraction takes 1-3 days.

4. The application according to claim 3, characterized in that, The concentration of the methanol extract suspension is 20~100g / L; the volume ratio of the methanol extract suspension to ethyl acetate is 1:(0.5~1.5); and the number of extractions is 3~5.

5. A fungicide for controlling pear ring rot disease, characterized in that, Including freshwater fungi Neocucurbitaria quercina Organic extracts and solvents from rice fermentation products; The preparation method of the rice ferment organic extract includes the following steps: activating freshwater fungi... Neocucurbitaria quercina The strain was inoculated into PDB liquid medium for seed culture to obtain seed culture solution; The seed culture solution was inoculated into a rice culture medium for fermentation to obtain rice fermentation product; The rice fermentation product was mixed with a methanol aqueous solution and then subjected to cold soaking extraction. The resulting cold soaking extract was concentrated under reduced pressure to obtain a methanol extract of the rice fermentation product. The rice fermentation product methanol extract was mixed with water to obtain a methanol extract suspension; The methanol extract suspension was mixed with ethyl acetate and extracted. The resulting organic phase was concentrated under reduced pressure to obtain the ethyl acetate extract of the rice fermentation methanol extract. The freshwater fungi Neocucurbitaria quercina The strain number is CBS 115095.

6. The microbial agent according to claim 5, characterized in that, The solvent includes one or more of water, ethanol, and Tween.

7. The microbial agent according to claim 5 or 6, characterized in that, The concentration of the rice ferment extract in the inoculum is 312.5 μg / mL to 250 mg / mL.

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