Application of tilletia foetida suspension in inhibition of apple black beancurd skin fungi

Abstract

The invention belongs to the technical field of biological prevention and control, and particularly relates to application of a Tilletia foetida Kuhn bacterial suspension in inhibition of apple black beancurd skin fungi. The Tilletia foetida suspension can be used for directly or indirectly inhibiting the growth of apple black beetle fungi (Valsa mali) and preventing and treating apple tree canker. Results of embodiments show that under the condition of the same medicament concentration, compared with citral and cinnamyl aldehyde, the Tilletia foetida suspension can significantly inhibit the growth of apple black beetle shells and prevent and treat apple tree canker.

Description

Technical Field

[0001] The invention belongs to the technical field of biological control, and particularly relates to application of a suspension of wheat tartaria in inhibiting apple black rot fungus. Background Art

[0002] Apple rot, also known as apple rot, is a disease caused by the infection of black rot fungus on apple trees. It mainly harms the branches and fruits of fruit-bearing trees, and can also harm young trees and seedlings. The occurrence of apple rot can cause the overall decline of the apple tree, the withering and death of the main trunk and branches, and finally the death of the entire tree, until the entire orchard is destroyed.

[0003] The pathogen of apple rot is Valsa mali, which belongs to the subdivision Ascomycota, and the asexual stage is Cytospora sp., which belongs to the subdivision Deuteromycota. At present, the prevention and control of this disease mainly relies on spraying chemical agents to inhibit the pathogen of apple rot, Valsa mali. However, due to the long-term use of fungicides, the drug resistance of pathogens is enhanced year by year, and the ecological environment is also damaged, which endangers human health. Therefore, there is an urgent need for a method for preventing and controlling apple rot, which can replace chemical pesticides, inhibit Valsa mali, and prevent and control apple rot. Summary of the invention

[0004] The invention aims to avoid the use of chemical pesticides, provide an application of a suspension of wheat smut fungi in preventing and treating apple tree rot, inhibit the growth of apple smut fungi, and prevent and treat apple tree rot.

[0005] The invention provides application of a wheat smut fungus suspension in inhibiting apple black rot fungus.

[0006] The invention also provides the use of the wheat smut fungus suspension in preventing and treating apple tree rot.

[0007] Preferably, the wheat smut fungus suspension is one or more of a wheat smut fungus suspension in the winter spore activation development period, a pre-hyphae development period, a primary basidiospore development period, a H body development period, and a secondary basidiospore development period.

[0008] Preferably, the method for preparing the bacterial suspension of the wheat tidal spores in the activated developmental period comprises: mixing the wheat tidal spores and soil extract to obtain a fungal culture solution, adding penicillin-streptomycin double antibiotics and culturing for 3 days, collecting the supernatant, and obtaining the bacterial suspension of the wheat tidal spores in the activated developmental period.

[0009] Preferably, the method for preparing the bacterial suspension of wheat tart smut fungus in the early mycelial development stage comprises: mixing the winter spores of wheat tart smut fungus and soil extract to obtain a fungal culture solution, adding penicillin-streptomycin double antibiotics and culturing for 5 days, collecting the supernatant, and obtaining the bacterial suspension of wheat tart smut fungus in the early mycelial development stage.

[0010] Preferably, the method for preparing the bacterial suspension of wheat smut at the primary basidiospore development stage comprises: mixing the winter spores of wheat smut and soil extract to obtain a fungal culture solution, adding penicillin-streptomycin double antibiotics and culturing for 6 days, collecting the supernatant, and obtaining the bacterial suspension of wheat smut at the primary basidiospore development stage.

[0011] Preferably, the method for preparing the bacterial suspension of wheat tartaria in the H body development period comprises: mixing winter spores of wheat tartaria and soil extract to obtain fungal culture solution, adding penicillin-streptomycin double antibiotics and culturing for 7 days, collecting the supernatant, and obtaining the bacterial suspension of wheat tartaria in the H body development period.

[0012] Preferably, the method for preparing the bacterial suspension of wheat smut secondary basidiospore development period comprises: mixing the winter spores of wheat smut and soil extract to obtain fungal culture solution, adding penicillin-streptomycin double antibiotics and culturing for 9 days, collecting the supernatant, and obtaining the bacterial suspension of wheat smut secondary basidiospore development period.

[0013] Preferably, the preparation method of each 1000 mL of the soil leachate comprises: filtering 75 g of nutrient soil using 900 mL of distilled water at a temperature of 100° C., and diluting the obtained filtrate to 1000 mL using distilled water;

[0014] The concentration of teliospores in the fungal culture solution is 3.5×10 6 ~5.5×10 6 pcs / L;

[0015] The culture temperature is 16°C, the pH value is 7.0, and the rotation speed is 150 rpm;

[0016] The volume ratio of the penicillin-streptomycin dual antibiotic to the fungal culture solution is 5:100;

[0017] The concentration of penicillin in the penicillin-streptomycin dual antibiotic is 100 IU / mL, and the concentration of streptomycin is 100 μg / mL.

[0018] The present invention also provides a bacterial agent for inhibiting apple black rot fungus, wherein the bacterial agent comprises wheat smut fungus and / or a wheat smut fungus suspension.

[0019] Beneficial effects:

[0020] The wheat smut fungus suspension can directly or indirectly inhibit the growth of apple black rot fungus and prevent and treat apple tree rot. The results of the embodiment show that under the same agent concentration conditions, compared with citral and cinnamaldehyde, the wheat smut fungus suspension can significantly inhibit the growth of apple black rot fungus and prevent and treat apple tree rot. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0022] Figure 1 The flow chart of the collection of lesion tissue and the separation and purification of pathogenic bacteria; wherein A is the collected lesion tissue; B is the diseased tree with the diseased skin scraped off; C is the disinfection and cultivation of the lesion tissue; D is the lesion tissue in culture; E is the strain after the first purification; F is the strain after the second purification;

[0023] Figure 2 The colony morphology of apple black rot fungus at different culture days; A is the result of 14 days of culture; B is the result of 7 days of culture;

[0024] Figure 3 The hyphae and ascospores of apple black rot fungus after different culture days; A is the result of 14 days of culture; B is the result of 7 days of culture, and the arrow in B indicates the ascospores;

[0025] Figure 4The results of the minimum inhibitory concentration test of different agents on apple black rot fungus; A1, A2, A3, and A4 are the front of the 96-well cell culture plate; B1, B2, B3, and B4 are the back of the 96-well cell culture plate; The ABC rows of A1 measure the minimum inhibitory concentration of citral, and the concentrations of citral from left to right are 4451 mg / L, 2225.5 mg / L, 1112.75 mg / L, 556.38 mg / L, 278.19 mg / L, 139.09 mg / L, 69.55 mg / L, 34.77 mg / L, 17.39 mg / L, 8.69 mg / L, 4.35 mg / L, and 0; The EFG rows of A1 measure the minimum inhibitory concentration of cinnamaldehyde, from left to right. The concentrations of right cinnamaldehyde are 2472mg / L, 1236mg / L, 618mg / L, 309mg / L, 154.50mg / L, 77.25mg / L, 38.63mg / L, 19.31mg / L, 9.66mg / L, 4.83mg / L, 2.41mg / L, and 0; the ABC rows of A2 measure the minimum inhibitory concentration of metabolites during the teliospore activation period (day 3). From left to right, the concentrations of metabolites during the teliospore activation period (day 3) are 50000.00mg / L, 25000.00mg / L, 12500.00mg / L, 6250.00mg / L, 3125.00mg / L, 1562.50mg / L, 7 81.25mg / L, 390.60mg / L, 195.31mg / L, 97.66mg / L, 48.83mg / L, 0; the EFG row of A2 measures the minimum inhibitory concentration of metabolites in the first hyphae period (5th day). From left to right, the concentrations of metabolites in the first hyphae period (5th day) are 50000.00mg / L, 25000.00mg / L, 12500.00mg / L, 6250.00mg / L, 3125.00mg / L, 1562.50mg / L, 781.25mg / L, 390.60mg / L, 195.31mg / L, 97.66mg / L, 48.83mg / L, 0; the ABC row of A3 measures The minimum inhibitory concentration of metabolites in the primary basidiospore period (day 6) is 50000.00mg / L, 25000.00mg / L, 12500.00mg / L, 6250.00mg / L, 3125.00mg / L, 1562.50mg / L, 781.25mg / L, 390.60mg / L, 195.31mg / L, 97.66mg / L, 48.83mg / L, and 0 from left to right; the EFG row of A3 measures the minimum inhibitory concentration of metabolites in the H body period (day 7), and the concentrations of metabolites in the H body period (day 7) are 50000.00mg / L, 25000.00mg / L, 12500.00mg / L, 6250.00mg / L, 3125.00mg / L, 1562.50mg / L, 781.25mg / L, 390.60mg / L, 195.31mg / L, 97.66mg / L, 48.83mg / L, and 0 from left to right.00mg / L, 12500.00mg / L, 6250.00mg / L, 3125.00mg / L, 1562.50mg / L, 781.25mg / L, 390.60mg / L, 195.31mg / L, 97.66mg / L, 48.83mg / L, 0; The ABC rows of A4 measured the minimum inhibitory concentration of metabolites during the secondary basidiospore period (9th day), from left to right, the secondary The concentrations of metabolites in the basidiospore period (day 9) were 50000.00mg / L, 25000.00mg / L, 12500.00mg / L, 6250.00mg / L, 3125.00mg / L, 1562.50mg / L, 781.25mg / L, 390.60mg / L, 195.31mg / L, 97.66mg / L, 48.83mg / L, 0.

[0026] Figure 5 The growth of black rot fungus on drug-containing plates; A1, A2, A3, A4, A5 indicate that the drug contained in the PDA plates is 0.1MIC, 0.5MIC, 1.0MIC, 1.5MIC, 2.0MIC citral; B1, B2, B3, B4, B5 indicate that the drug contained in the PDA plates is 0.1MIC, 0.5MIC, 1.0MIC, 1.5MIC, 2.0MIC cinnamaldehyde; C1, C2, C3, C4, C5 indicate that the drug contained in the PDA plates is 10g / L, 20g / L, 30g / L, 40g / L, 50g / L of winter spore activation period metabolites; D1, D2, D3, D4, D5 indicate that the drug contained in the PDA plates is 10g / L, 20g / L, 30g / L, 40g / L, 50g / L metabolites of the early hyphae period; E1, E2, E3, E4, E5 indicate that the drug contained in the PDA plate is 10g / L, 20g / L, 30g / L, 40g / L, 50g / L metabolites of the primary basidiospore period; F1, F2, F3, F4, F5 indicate that the drug contained in the PDA plate is 10g / L, 20g / L, 30g / L, 40g / L, 50g / L metabolites of the H body period; G1, G2, G3, G4, G5 indicate that the drug contained in the PDA plate is 10g / L, 20g / L, 30g / L, 40g / L, 50g / L metabolites of the secondary basidiospore period; H indicates blank control;

[0027] Figure 6 To compare the diameter of black rot fungus in apples with different concentrations of citral and cinnamaldehyde;

[0028] Figure 7 The diameter comparison results of black rot fungus of apple were obtained by applying different concentrations of culture medium of metabolites of wheat smut fungus at different periods.

[0029] Figure 8 The comparison results of the inhibition percentage of black rot fungus of apple by applying different concentrations of citral and cinnamaldehyde;

[0030] Fig. 9 To compare the inhibition percentage of black rot fungus on apple by applying different concentrations of culture medium of metabolites of wheat smut at different periods;

[0031] Figures 10-11 The regression equation curves of different agents acting on apple black rot fungus; among them, (a) is citral; (b) is cinnamaldehyde; (c) is the metabolite during the winter spore activation period; (d) is the metabolite during the pre-hyphae period; (e) is the metabolite during the primary basidiospore period; (f) is the metabolite during the H body period; and (g) is the metabolite during the secondary basidiospore period. DETAILED DESCRIPTION

[0032] The invention provides application of a wheat smut fungus suspension in inhibiting apple black rot fungus.

[0033] In the present invention, the wheat light tart smut fungus suspension is one or more of a wheat light tart smut fungus winter spore activation development period fungus suspension, a pre-hypha development period fungus suspension, a primary basidiospore development period fungus suspension, a H body development period fungus suspension, and a secondary basidiospore development period fungus suspension, and is further preferably one or more of a winter spore activation development period fungus suspension, a primary basidiospore development period fungus suspension, and a H body development period fungus suspension, and is more preferably a winter spore activation development period fungus suspension. The present invention has no strict requirements on the source of the wheat light tart smut fungus, and a conventional selection can be used.

[0034] In the present invention, the preparation method of the wheat light tartaria spore activated development period bacterial suspension preferably comprises: mixing the wheat light tartaria spores and soil extract to obtain a fungal culture solution, adding penicillin-streptomycin double antibiotics and culturing for 3 days, collecting the supernatant to obtain the wheat light tartaria spore activated development period bacterial suspension, and the collected supernatant is the product containing the wheat light tartaria spore activated development period bacterial suspension.

[0035] In the present invention, the method for preparing each 1000 mL of the soil extract preferably comprises: filtering 75 g of nutrient soil using 900 mL of distilled water at a temperature of 100° C., and the obtained filtrate is fixed to 1000 mL using distilled water. The filtration of the present invention preferably uses 8 layers of gauze. The winter spore concentration in the fungal culture solution of the present invention is preferably 3.5×10 6 ~5.5×10 6 / L, more preferably 4×10 6 ~5×10 6 / L. The culture conditions of the present invention are preferably a temperature of 16°C, a pH of 7.0, and a rotation speed of 150rpm. The volume ratio of the penicillin-streptomycin dual antibiotic and the fungal culture solution of the present invention is preferably 5:100; the concentration of penicillin in the penicillin-streptomycin dual antibiotic is preferably 100IU / mL; the concentration of streptomycin in the penicillin-streptomycin dual antibiotic is preferably 100μg / mL.

[0036] The present invention collects a fungal suspension of wheat stinking smut in the early mycelium development stage, which can inhibit the growth of apple black rot fungus. The results of the embodiment show that when the fungal suspension of wheat stinking smut in the early mycelium development stage is diluted to 10g / L-50g / L with distilled water, the growth of apple black rot fungus can be significantly inhibited, and the inhibitory effect is enhanced as the concentration increases.

[0037] In the present invention, the preparation method of the bacterial suspension of wheat smut fungus in the early mycelium development period preferably includes: mixing the winter spores of wheat smut fungus and soil extract to obtain a fungal culture solution, adding penicillin-streptomycin double antibiotics and culturing for 5 days, collecting the supernatant, and obtaining the bacterial suspension of wheat smut fungus in the early mycelium development period.

[0038] The preparation method of the soil extract of the present invention, the winter spore concentration in the fungal culture solution, the penicillin-streptomycin dual antibiotic and the volume and culture conditions of the fungal culture solution have been described above and will not be repeated here. The present invention collects the culture solution of the fungal suspension of wheat light smut in the early mycelial development period, which can inhibit the growth of apple black rot fungus. The results of the embodiment show that when the fungal suspension of wheat light smut in the primary basidiospore period is diluted to 10g / L~50g / L with distilled water, it can significantly inhibit the growth of apple black rot fungus, and the inhibitory effect increases with the increase of concentration.

[0039] In the present invention, the preparation method of the bacterial suspension of the primary basidiospore development period of wheat stinking smut preferably comprises: mixing the winter spores of wheat stinking smut and soil extract to obtain a fungal culture solution, adding penicillin-streptomycin double antibiotics and culturing for 6 days, collecting the supernatant, and obtaining the bacterial suspension of the primary basidiospore development period of wheat stinking smut.

[0040] The preparation method of the soil extract of the present invention, the winter spore concentration in the fungal culture solution, the penicillin-streptomycin dual antibiotic and the volume and culture conditions of the fungal culture solution have been described above and will not be repeated here. The present invention collects the culture solution of the primary basidiospore suspension of wheat smut fungus, which can inhibit the growth of apple black rot fungus. The results of the embodiment show that when the primary basidiospore suspension of wheat smut fungus is diluted to 10g / L to 50g / L with distilled water, it can significantly inhibit the growth of apple black rot fungus, and the inhibitory effect increases with the increase of concentration.

[0041] In the present invention, the preparation method of the bacterial suspension of wheat tartaria in the H body development period preferably includes: mixing the winter spores of wheat tartaria and soil extract to obtain a fungal culture solution, adding penicillin-streptomycin double antibiotics and culturing for 7 days, collecting the supernatant, and obtaining the bacterial suspension of wheat tartaria in the H body development period.

[0042] The preparation method of the soil extract of the present invention, the winter spore concentration in the fungal culture solution, the penicillin-streptomycin dual antibiotic and the volume and culture conditions of the fungal culture solution have been described above and will not be repeated here. The present invention collects the culture solution of the bacterial suspension of wheat smut fungus in the H body stage, which can inhibit the growth of apple black rot fungus. The results of the embodiment show that when the culture solution of the bacterial suspension of wheat smut fungus in the H body stage is diluted to 10g / L~50g / L with distilled water, it can significantly inhibit the growth of apple black rot fungus, and the inhibitory effect increases with the increase of concentration.

[0043] In the present invention, the preparation method of the bacterial suspension of wheat smut fungus in the secondary basidiospore development period preferably comprises: mixing the winter spores of wheat smut fungus and soil extract to obtain a fungal culture solution, adding penicillin-streptomycin double antibiotics and culturing for 9 days, collecting the supernatant, and obtaining the bacterial suspension of wheat smut fungus in the secondary basidiospore development period.

[0044] The preparation method of the soil extract of the present invention, the winter spore concentration in the fungal culture solution, the penicillin-streptomycin dual antibiotic and the volume and culture conditions of the fungal culture solution have been described above and will not be repeated here. The present invention collects the culture solution of the secondary basidiospore period suspension of wheat smut fungus, which can inhibit the growth of apple black rot fungus. The results of the embodiment show that when the culture solution of the secondary basidiospore period suspension of wheat smut fungus is diluted to 10g / L to 50g / L with distilled water, it can significantly inhibit the growth of apple black rot fungus, and the inhibitory effect increases with the increase of concentration.

[0045] In view of the effects of the wheat tine smut fungus suspension provided by the present invention, the use of the wheat tine smut fungus suspension in preventing and treating apple tree rot also falls within the protection scope of the present invention.

[0046] To further illustrate the present invention, the application of the wheat smut fungus suspension provided by the present invention in inhibiting apple black rot fungus is described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0047] Example 1

[0048] 1. Materials and Methods

[0049] 1.1 Test materials

[0050] wheat light tartaria, diseased branches of apple trees with obvious rot symptoms collected from the National Field Scientific Observation Station of Grassland Agricultural Ecosystem of Lanzhou University on April 10, 2023;

[0051] 1.2 Instruments and reagents

[0052] 1.2.1 Instruments

[0053] Clean bench, high-pressure steam sterilizer, 4℃ refrigerator, constant temperature incubator, electronic balance, induction cooker, small iron pot, optical microscope, 10-100μL pipette, 100-1000μL pipette, 8mm bacterial cake puncher, 96-well cell culture plate, 250mL conical flask, culture dish.

[0054] 1.2.2 Reagents

[0055] 2% sodium hypochlorite solution (weigh 2g sodium hypochlorite and dissolve it in 100mL distilled water, mix it to make it completely dissolved), 0.9% saline (weigh 0.9g sodium chloride and dissolve it in 100mL distilled water and stir it evenly), anhydrous ethanol, 75% ethanol, glucose, and agar powder.

[0056] 1.2.3 Test culture medium

[0057] (1) Preparation of PDA culture medium

[0058] Wash and peel the potatoes, cut them into pieces. Weigh 200g of the potatoes and chop them. Add 800mL of distilled water and boil for 20-30min until the potatoes are cooked. Filter with four layers of gauze, then add 20g of glucose and 20g of agar, boil until fully dissolved, then make up to 1000mL. Divide the mixture into 4 500mL conical flasks marked with the culture medium name and preparation date. Sterilize them with high-pressure steam at 121℃ for 20min. Pour them into plates after they are no longer hot.

[0059] (2) Configuration of soil leachate

[0060] Weigh 75g of plant nutrient soil produced by Hebei Devoduo Fertilizer Co., Ltd. and place it on 8 layers of gauze, pour 900mL of boiled distilled water on the nutrient soil, filter it into a beaker, and finally add distilled water to 1000mL. Divide it into 250mL conical flasks, 100mL for each conical flask, seal it with a breathable sealing film, sterilize it with high-pressure steam at 121℃ for 20min, and cool it to room temperature for use.

[0061] 2. Test methods

[0062] 2.1 Cultivation of winter spore germination of T. tritici and collection of metabolites at different stages

[0063] Referring to the prior art (He Ting. Transcriptome differential expression analysis of wheat anthers infected with wheat smut and preliminary study of the physiological response of wheat [D]. Qinghai University, 2021.), the winter spores of wheat smut were prepared. Specifically: 3 to 4 galls of wheat smut were taken, crushed, and the obtained gall powder was poured into a 2mL centrifuge tube, 2mL of sterilized water was added, and then allowed to stand overnight. Then, 150-mesh gauze was used to filter the impurities in the bacterial solution to prepare a winter spore suspension of wheat smut.

[0064] Referring to the method of the prior art (Chen Delai. Effects of wheat smut fungus on wheat anther development [D]. Gansu Agricultural University, 2021.), the above-mentioned wheat smut fungus teliospore suspension was added to the sterilized soil extract, and after mixing, the teliospore concentration was 3.5×10 6 ~5.5×10 6 spores / L of fungal culture fluid, add 0.5mL / 100mL of penicillin-streptomycin double antibiotics for cultivation; on the 3rd day of cultivation, collect the supernatant of the winter spore activation period into a centrifuge tube and store it in a 4℃ refrigerator for later use; on the 5th day of cultivation, collect the supernatant of metabolites in the pre-hyphae period into a centrifuge tube and store it in a 4℃ refrigerator for later use; on the 6th day of cultivation, collect the supernatant of metabolites in the primary basidiospore period into a centrifuge tube and store it in a 4℃ refrigerator for later use; on the 7th day of cultivation, collect the supernatant of metabolic waste in the H body period into a centrifuge tube and store it in a 4℃ refrigerator for later use; on the 9th day of cultivation, collect the supernatant of metabolites in the secondary basidiospore period into a centrifuge tube and store it in a 4℃ refrigerator for later use.

[0065] 2.2 Isolation, purification and preliminary identification of the pathogen causing apple rot

[0066] Referring to the prior art (Dou Yanan. Isolation and identification of pathogenic bacteria of apple tree rot and screening and identification of antagonistic Bacillus [D]. Northwest Normal University, 2018.), the tissue separation method was used to collect the phloem and cambium tissues of the diseased apple trees, cut the diseased tissue of about 5mm×5mm×0.2mm and put it in a sterile beaker, treat it with 2% sodium hypochlorite solution for 30s, disinfect the surface with 75% ethanol for 5s, rinse it with sterile water, place it on sterile absorbent paper to dry, transfer it to a PDA culture medium plate, and culture it at a constant temperature of 28℃ for 3-7d. The morphological characteristics of the colony were observed. After the mycelium grew mature, a sterile scalpel was used to cut a PDA thin plate with mycelium of about 5mm×5mm and placed it on a slide for microscopic observation. Referring to the external morphological characteristics of apple tree rot in the prior art (Wei Jingchao. Fungal Identification Manual. Shanghai: Shanghai Science and Technology Press, 1979:210.), a preliminary identification was made and it was identified as Valsa mali), and there is no difference with the existing black rot fungus of apples, prepare the pathogen suspension, dilute it, separate the single spores and transfer it again, after the single spores are separated, the strain grows out the colony, transfer it to the newly configured PDA medium, invert and culture it at 28℃ for 5 days, and then store it in a refrigerator at 4℃ for preparation. The flow chart of separation and purification is shown in the figure. Figure 1 As shown in Figure 2, the morphology of the colonies during the culture process is as follows: Figures 2-3 .

[0067] 2.3 Preparation of pathogen suspension

[0068] Take a PDA culture plate of apple black rot fungus cultured in a constant temperature incubator at 28°C for 5 days in step 2.2, add 5 mL of 0.9% saline, gently scrape the bacteria on the surface of the culture medium with a sterile inoculation loop, collect the bacterial suspension into a 50 mL sterile conical flask, count with a hemocytometer, and dilute to adjust the bacterial concentration to 1.0 × 10 5 cfu / mL.

[0069] 2.4 Minimum inhibitory concentration test

[0070] The minimum inhibitory concentrations of citral, cinnamaldehyde, metabolites during the activation of teliospores (day 3), metabolites during the promycelial period (day 5), metabolites during the primary basidiospore period (day 6), metabolites during the H body period (day 7), and metabolites during the secondary basidiospore period (day 9) against apple black rot fungus were determined by the MH (Mueller-Hinton medium) broth microdilution method. The results are as follows: Figure 4 shown.

[0071] according to Figure 4It can be seen that citral and cinnamaldehyde can significantly inhibit the mycelial growth of apple black rot fungus, and the inhibitory effect gradually increases with the increase of concentration. In A1 and B1, in rows 1 to 3, apple black rot fungus grows in wells 7 and thereafter, that is, the MIC value of citral to apple black rot fungus is 69.55 mg / L (100 μL 8902 mg / L citral mother solution is added to well 1, and then the drug is micro-diluted by two times); in rows 5 to 7, apple black rot fungus grows in wells 5 and thereafter, that is, the MIC value of cinnamaldehyde to apple black rot fungus is 154.50 mg / L. In A2 and B2, A3 and B3, A4 and B4, there was growth of black rot fungus, that is, the five period bacterial suspensions of winter spores of wheat light stinking powder had no obvious inhibitory effect on the pathogen of apple tree rot disease. The set concentration gradient could not measure the minimum inhibitory concentration of black rot fungus, but the inhibitory effect could be seen: metabolites of winter spore activation period (3rd day) > metabolites of pre-hyphae period (5th day) > metabolites of primary basidiospore period (6th day) > metabolites of H body period (7th day) > metabolites of secondary basidiospore period (9th day).

[0072] 2.5 Indoor toxicity test

[0073] Drug-containing PDA culture medium with different concentration gradients was prepared. In the positive control group, the concentration gradients of citral and cinnamaldehyde were 0.1MIC, 0.5MIC, 1.0MIC, 1.5MIC and 2.0MIC; in the experimental group, the concentration gradients of the bacterial suspension at the five stages of winter spore development of wheat smut fungus were: 10g / L, 20g / L, 30g / L, 40g / L, 50g / L, and PDA culture medium without drugs was used as blank control. Three parallel groups were made for each treatment. The growth rate determination method was used. An 8mm puncher was used to punch out the black rot fungus cake in the vigorous growth area at the edge of the colony. The cake was transferred to a PDA plate containing the agent for culture and inverted in a constant temperature incubator at 28°C. When the blank control group just covered the entire plate, the cross method was used to measure the colony diameter on each drug-containing plate. The average value of the three parallel groups represented the final colony diameter. The inhibition rate of mycelium growth of black rot fungus by different agents was calculated according to the following formula. The results are shown in Tables 1 and 2. Figures 5 to 9 shown.

[0074] Mycelium growth inhibition rate = (control colony diameter - treated colony diameter) / (control colony diameter - bacterial cake diameter) × 100%.

[0075] Table 1 Inhibitory effects of seven reagents on apple black rot fungus

[0076]

[0077]

[0078]

[0079] According to Table 1 and Figures 5 to 9 It can be seen that citral and cinnamaldehyde have obvious inhibitory effects on apple black rot fungus, and the inhibitory effect gradually increases with the increase of concentration. At the same concentration, citral has a better inhibitory effect on apple black rot fungus than cinnamaldehyde. When the concentration of the agent is 0.1MIC, the inhibition rates of citral and cinnamaldehyde on the mycelial growth of apple black rot fungus are 22.6% and 14.29%, respectively; when the concentration of the agent is 0.5MIC, the inhibition rates of citral and cinnamaldehyde on the mycelial growth of apple black rot fungus are 59.22% and 87.14%, respectively. When the concentration of the agent is 1.0MIC, 1.5MIC and 2.0MIC, the colony growth of citral and cinnamaldehyde on the drug-containing PDA plate is completely inhibited, and the five bacterial suspensions of the winter spore development of wheat light stinking powder fungus have a certain inhibitory effect on the mycelial growth of apple black rot fungus. When the concentration of bacterial suspension was 10 g / L, the mycelium growth inhibition rate was: winter spore activation period (44.68%) > first mycelium growth period (43.77%) > primary basidiospore period (24.68%) > H body period (1.81%) > secondary basidiospore period (0.38%); when the concentration of bacterial suspension was 20 g / L, the mycelium growth inhibition rate was: winter spore activation period (47.53%) > first mycelium growth period period (44.16%) > primary basidiospore period (27.4%) > H body period (6.49%) > secondary basidiospore period (0.91%); when the concentration of bacterial suspension was 30g / L, the mycelial growth inhibition rate was: winter spore activation period (50.52%) > first mycelial growth period (44.55%) > primary basidiospore period (30.91%) > H body period (9.87%) > secondary basidiospore period ( 1.56%); when the concentration of the bacterial suspension was 40g / L, the mycelium growth inhibition rate was: winter spore activation period (52.07%) > first mycelium growth period (44.55%) > primary basidiospore period (30.38%) > H body period (14.42%) > secondary basidiospore period (1.69%); when the concentration of the bacterial suspension was 50g / L, the mycelium growth inhibition rate was: winter spore activation period (53.64%) > first mycelium growth period (45.71%) > primary basidiospore period (36.49%) > H body period (18.83%) > secondary basidiospore period (2.47%); In summary, the mycelium growth inhibition rate of the bacterial suspension of the five periods of winter spore development of wheat smut fungus on the apple black rot shell fungus decreased with the delay of the winter spore development period, and the bacterial suspension in the same period, the inhibition effect increased with the gradual increase of concentration.

[0080] (2) The logarithm of the natural logarithm of 10 of the concentration was used as the horizontal axis, and the probability value corresponding to the inhibition rate was used as the vertical axis. The toxicity regression equation and correlation coefficient R were obtained based on the concentration of the agent and the inhibition rate of mycelial growth. According to the toxicity regression equation, the EC was calculated using the probability value corresponding to the inhibition rate of 50%. 50 The results are shown in Table 2 and Figures 10-11 shown.

[0081] Table 2 Results of 7 reagents for toxicity test on apple black rot fungus

[0082] Test drug Virulence regression equation Correlation coefficient <![CDATA[Effective medium concentration EC 50 > Citral y=3.9101x+0.5677 0.8449 83.16 (mg / L) Cinnamaldehyde y=4.0409x+0.9354 0.9386 10.60(mg / L) Metabolites during teliospore activation y=0.3241x+4.5330 0.9864 27.60(g / L) Prehyphae metabolites y=0.0572x+4.7827 0.7780 6295.06(g / L) Metabolites of primary basidiospore stage y=0.4722x+3.8154 0.9403 322.63(g / L) H body metabolites y=1.7022x+1.2203 0.9963 166.15(g / L) Secondary basidiospore metabolites y=0.9624x+1.3889 0.9828 5651.97(g / L)

[0083] According to Table 2 and Figures 10-11 It can be seen that the apple black rot fungus is more sensitive to citral and cinnamaldehyde, EC 50 The values ​​were 83.16mg / L and 27.60mg / L, respectively, and the EC 50 The smallest value is cinnamaldehyde; the sensitivity to metabolites during the activation period of teliospores is relatively high, and the EC 50 The value was 27.60 g / L; the sensitivity to metabolites in the primary basidiospore period and H body period was relatively low, EC 50 The values ​​were 322.63 g / L and 166.15 g / L respectively; the sensitivity to metabolites of the pre-hyphae period and the secondary basidiospore period was the lowest, and the EC 50 The values ​​were 6295.06 g / L and 5651.97 g / L respectively; among the metabolites of the five stages of winter spore development of wheat smut fungus, EC 50 The smallest value is in the bacterial suspension during the teliospore activation period, followed by metabolites of the H body period, metabolites of the primary basidiospore period, metabolites of the secondary basidiospore period, and metabolites of the pre-hyphae period.

[0084] Based on the above content, it can be seen that the suspension of wheat smut fungus can inhibit the growth of apple black rot fungus.

[0085] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of Tilletia laevis Kühn suspension in inhibiting Valsamali.

2. Application of Sustilbene odorifera suspension in preventing and controlling apple tree rot.

3. The use according to claim 1 or 2, characterized in that: The wheat smut fungus suspension is one or more of a wheat smut fungus suspension in the winter spore activation development period, a pre-hyphae development period, a primary basidiospore development period, a H body development period, and a secondary basidiospore development period.

4. The use according to claim 3, characterized in that: The preparation method of the bacterial suspension of wheat tidal spores in the activated developmental stage comprises: mixing the tidal spores of wheat tidal spores with soil extract to obtain fungal culture solution, adding penicillin-streptomycin double antibiotics and culturing for 3 days, collecting the supernatant, and obtaining the bacterial suspension of wheat tidal spores in the activated developmental stage.

5. The use according to claim 3, characterized in that: The preparation method of the fungal suspension of wheat tart smut fungus in the early mycelial development stage comprises: mixing winter spores of wheat tart smut fungus and soil extract to obtain fungal culture solution, adding penicillin-streptomycin double antibiotics and culturing for 5 days, collecting the supernatant, and obtaining the fungal suspension of wheat tart smut fungus in the early mycelial development stage.

6. The use according to claim 3, characterized in that: The preparation method of the bacterial suspension of wheat smut at the primary basidiospore development stage comprises: mixing winter spores of wheat smut and soil extract to obtain fungal culture solution, adding penicillin-streptomycin double antibiotics and culturing for 6 days, collecting the supernatant, and obtaining the bacterial suspension of wheat smut at the primary basidiospore development stage.

7. The use according to claim 3, characterized in that: The preparation method of the bacterial suspension of wheat tart smut fungus in the H body development period comprises: mixing winter spores of wheat tart smut fungus and soil extract to obtain fungal culture solution, adding penicillin-streptomycin double antibiotics and culturing for 7 days, collecting the supernatant, and obtaining the bacterial suspension of wheat tart smut fungus in the H body development period.

8. The use according to claim 3, characterized in that: The preparation method of the bacterial suspension of wheat smut secondary basidiospore development period comprises: mixing winter spores of wheat smut and soil extract to obtain fungal culture solution, adding penicillin-streptomycin double antibiotics and culturing for 9 days, collecting the supernatant, and obtaining the bacterial suspension of wheat smut secondary basidiospore development period.

9. The use according to any one of claims 4 to 8, characterized in that: The preparation method of each 1000 mL of the soil leachate comprises: filtering 75 g of nutrient soil using 900 mL of distilled water at a temperature of 100° C., and diluting the obtained filtrate to 1000 mL using distilled water; The concentration of teliospores in the fungal culture solution is 3.5×10 6 ~5.5×10 6 pcs / L; The culture temperature is 16°C, the pH value is 7.0, and the rotation speed is 150 rpm; The volume ratio of the penicillin-streptomycin dual antibiotic to the fungal culture solution is 5:100; The concentration of penicillin in the penicillin-streptomycin dual antibiotic is 100 IU / mL, and the concentration of streptomycin is 100 μg / mL.

10. A fungal agent for inhibiting apple black rot fungus, characterized in that: The bacterial agent includes a suspension of wheat smut fungus.

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