Application of streptomyces SC-3 volatile organic compounds in inhibition of botryosphaeria dothidea

Abstract

The invention discloses application of streptomyces SC-3 volatile organic compounds in inhibition of botryosphaeria dothidea, which comprises the following steps: preparing a streptomyces SC-3 wheat grain culture, inoculating SC-3 bacterial suspension to a sterilized wheat grain culture medium, culturing for 5-14 days at the temperature of 25-30 DEG C, and regularly shaking up during the culture period; the wheat grain culture is in gas-phase contact with B. dothidea by adopting a double-dish appointment method, an upper dish is a PDA culture medium containing B. dothidea fungus cakes, the wheat grain culture is placed in a lower dish, and the wheat grain culture is cultured for 2-5 days at the temperature of 25-30 DEG C. According to the invention, botryosphaeria dothidea (B. dothidea) is inhibited through volatile organic compounds (VOCs) of streptomyces SC-3, and a remarkable antibacterial effect is shown.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of bio-agriculture and related industries, in particular, to an application of volatile organic compounds of Streptomyces SC-3 in inhibiting Botryosphaeria dothidea. BACKGROUND

[0002] In the field of green control of plant diseases, microorganism volatile organic compounds (VOCs) have become an important research direction to replace traditional chemical fungicides due to their unique gas diffusion capacity, low residue and low resistance, etc. As an important source of natural antibiotics, Streptomyces has potential antibacterial activity on a variety of plant pathogenic fungi, but the specific antibacterial mechanism, effective active ingredients and optimized application conditions of VOCs produced by Streptomyces have not been fully elucidated, which limits the popularization and application of VOCs in practical disease control.

[0003] Botryosphaeria dothidea is a serious plant pathogenic fungus that can cause soft rot of kiwifruit and other fruits, resulting in significant economic losses. At present, the control of the pathogenic fungus still relies on chemical agents, which not only easily leads to pesticide residues and enhanced pathogenic resistance, but also does not meet the development needs of green agriculture.

[0004] In the prior art, the preparation method of Streptomyces VOCs lacks a standardized process, and the effects of key parameters such as culture time, inoculation amount and culture quality on antibacterial effect have not been systematically studied, resulting in unstable antibacterial activity of VOCs.

[0005] At the same time, the damage mechanism of VOCs to cell wall, cell membrane and other cell structures of B. dothidea, as well as the actual control effect and action mode on fruits are not further explored, which cannot provide reliable theoretical basis and technical support for the industrialization application of this kind of VOCs.

[0006] Therefore, it is urgent to clarify the antibacterial mechanism of Streptomyces to B. dothidea, screen key active ingredients, optimize culture and application conditions, and establish fruit disease control technology based on the VOCs, in order to solve the technical problems of high residue, strong resistance and unknown mechanism of Streptomyces VOCs in existing B. dothidea control, and promote the development of green control technology of plant diseases. SUMMARY

[0007] The main purpose of the present application is to provide an application of volatile organic compounds of Streptomyces SC-3 in inhibiting Botryosphaeria dothidea, in order to solve the problems of high residue, strong resistance, unknown antibacterial mechanism of Streptomyces VOCs and unclear application parameters in existing chemical control methods.

[0008] The Streptomyces SC-3 involved in the present application has been disclosed in Chinese patent CN118931757A. The strain is Streptomyces albidoflavus, which has been preserved in China Center for Type Culture Collection on May 11, 2024, with the preservation number CCTCC NO: M2024906 and the preservation unit address being Wuhan University, Wuhan, China.

[0009] Based on the first main aspect of the present application, the application of Streptomyces SC-3 volatile organic compounds in inhibiting Botryosphaeria dothidea is provided, including the following steps:

[0010] Prepare a wheat grain culture of Streptomyces SC-3 by inoculating a SC-3 bacterial suspension into a sterilized wheat grain medium, and culturing at 25-30°C for 5-14 days, with regular shaking during the culturing;

[0011] Perform gas phase contact between the wheat grain culture and B. dothidea by double-dish pair tapping method, with PDA medium containing B. dothidea fungus cake on the upper dish, and placing the wheat grain culture on the lower dish, and culturing at 25-30°C for 2-5 days.

[0012] As a further preferred solution, in the foregoing application, the preparation method of the SC-3 bacterial suspension is: streaking SC-3 on Gause No. 1 solid medium for 5-9 days, scraping the bacterial body and inoculating into Gause No. 1 liquid medium with a volume of 80-120 mL, and culturing at 25-30°C with 180-220 r / min shaking for 40-56 h.

[0013] As a further preferred solution, in the foregoing application, the sterilization treatment of the wheat grain medium is 121°C sterilization for 1-3 times, with a sterilization duration of 25-35 min each time.

[0014] As a further preferred solution, in the foregoing application, in the double-dish pair tapping method, the thickness of the PDA medium containing B. dothidea fungus cake is 3-7 mm, and the diameter of the B. dothidea fungus cake is 4-8 mm.

[0015] Based on the second main aspect of the present application, a method for preparing Streptomyces SC-3 volatile organic compounds is provided, including:

[0016] Wash and treat the wheat grains, and then divide and package them into containers to obtain a wheat grain medium after sterilization treatment;

[0017] Inoculate a SC-3 bacterial suspension into the wheat grain medium, and culture at 25-30°C for 5-14 days, with regular shaking during the culturing;

[0018] The volatile organic compounds produced by the culture are collected by solid phase microextraction method, the extraction fiber needle is aged for 15-25 minutes at 240-260℃, and the sample is adsorbed for 25-35 minutes.

[0019] As a further preferred solution, in the foregoing method, the wheat kernel washing treatment is boiled to cracking and then drained, the split amount is 45-55 g per 150 mL triangular bottle, and the sterilization treatment is sterilized twice at 121℃ for 25-35 min each time.

[0020] The inoculation amount of the SC-3 bacterial suspension is 80-1200 μL / 10 g of wheat kernel medium, and the culture is shaken once every 1-2 days during the culture.

[0021] Based on the third main aspect of the present application, a method for preventing and controlling B. dothidea infection of kiwifruit is provided, comprising:

[0022] After the non-injured kiwifruit is surface-disinfected and dried, a B. dothidea spore suspension is sprayed;

[0023] The fruit is placed on a perforated partition in a sealed container, and a Streptomyces SC-3 wheat kernel culture is placed at the bottom of the container, the culture amount is 80-170 g / L, and the culture is carried out at 25-30℃ for 8-12 days.

[0024] As a further preferred solution, in the foregoing method, the surface disinfection uses 70-75% alcohol wiping or soaking treatment, the B. dothidea spore suspension concentration is 1×10 6 -1×10 8 Spores / mL, the sealed container is naturally dried after being disinfected with 70-75% alcohol;

[0025] The Streptomyces SC-3 wheat kernel culture is a culture that is cultured at 25-30℃ for 7 days, the number of kiwifruits treated is 8-12 per time, and the test is repeated 2-4 times.

[0026] Based on the fourth main aspect of the present application, the synergistic application of multiple Streptomyces SC-3 volatile organic compounds in preparing a preparation for inhibiting Botryosphaeria dothidea is provided, wherein the multiple Streptomyces SC-3 volatile organic compounds include Carvone, 3,5-di-tert-butylphenol and 3-Butylidenephthalide; the preparation is prepared by mixing the multiple Streptomyces SC-3 volatile organic compounds in proportion, and then performing gas-phase contact with B. dothidea by using a two-compartment culture dish method, wherein the side of the culture medium containing the B. dothidea fungus cake is separated from the side of the mixed organic compounds, the total final concentration of the mixed organic compounds is 0.05-0.8 μL / mL, and the culture is performed at 25-30°C for 2-4 days.

[0027] As a further preselected scheme, in the aforementioned synergistic application, in the preparation raw materials of the preparation, the volume ratio of the three components of Carvone, 3,5-di-tert-butylphenol and 3-Butylidenephthalide is 1:(0.5-2):(0.5-2), and the purity of each of the three components is ≥98%; the volume of the two-compartment culture dish is 60-80 mL, the culture medium containing the B. dothidea fungus cake is 6-10 mL of PDA medium, the diameter of the fungus cake is 4-8 mm, and the growth state of the colony is observed every day during the culture.

[0028] Compared with the prior art, the present application exhibits a significant bacteriostatic effect by using Streptomyces SC-3 volatile organic compounds (VOCs) to inhibit B. dothidea. When the double-dish pair knock method is used and the amount of wheat grain culture is 20 g, the inhibition rate of B. dothidea mycelium growth can reach 94.90%, and the mycelium can appear shrinkage, perforation, intracellular substance loss and plasmolysis and other morphological structure damage, which indicates that the VOCs can effectively inhibit the growth of the pathogenic bacteria by destroying the cell structure of the pathogenic bacteria, and solves the problem that the traditional chemical agents are prone to causing drug resistance of the pathogenic bacteria.

[0029] In the application of preventing and controlling B. dothidea in kiwifruit fruits, the technical scheme of the present application can significantly reduce the infection rate of B. dothidea. When the amount of Streptomyces SC-3 wheat grain culture is 166.67 g / L, the disease incidence of kiwifruit fruits is reduced to 18.89% after being cultured at 28°C for 10 days, and the fruit hardness is increased and the weight loss rate is reduced, which effectively maintains the fruit quality while inhibiting the disease, provides a feasible means for green prevention and control of postharvest diseases of fruits, and overcomes the problem of pesticide residues in fruits caused by chemical prevention and control.

[0030] In addition, the application provides a synergistic application of the volatile organic compounds of the Streptomyces SC-3 in preparing a preparation for inhibiting B. dothidea. The synergistic effect of the volatile organic compounds, such as carvone, 3,5-di-tert-butylphenol and 3-Butylidenephthalide, produced by the Streptomyces SC-3, is utilized to form a multi-target inhibition on B. dothidea through gas phase diffusion: the carvone can strongly inhibit the mycelium growth, the 3,5-di-tert-butylphenol and 3-Butylidenephthalide can assist in destroying the cell wall and cell membrane structure of the pathogenic bacteria, and the three are mixed in proportion and jointly act through the gas phase contact mode of the two-division Petri dish. The effect is that under the conditions of a total final concentration of 0.05-0.8 μL / mL, 25-30℃ culture for 2-4 days, the antibacterial efficiency can be significantly improved, the pathogenic bacteria activity can be more reduced compared with a single component, the use amount of a single compound can be reduced, and the functions of inhibiting the mycelium growth and reducing the fruit infection rate can be stably exerted, which meets the needs of low residue, high antibacterial efficiency in green prevention and control.

[0031] Meanwhile, the VOCs can be effectively collected through the optimized solid phase microextraction method in the application, and the standardization of the culture conditions can ensure the stability and repeatability of the antibacterial effect, which lays a foundation for the industrial application of the technology. BRIEF DESCRIPTION OF DRAWINGS

[0032] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and for those skilled in the art, other drawings obtained according to these drawings without creative labor are still within the scope of the present application.

[0033] Figure 1 Fig. 1 shows the influence of the VOCs produced by the strain SC-3 on the mycelium morphology of B. dothidea in an embodiment of the present application; Figure 2 Fig. 2 shows the influence of the VOCs produced by the strain SC-3 on the ultrastructure of B. dothidea cells in an embodiment of the present application; Figure 3 Fig. 3 shows the CFW staining of B. dothidea treated by the sterile filtrate in an embodiment of the present application; Figure 4 Fig. 4 shows the PI staining of B. dothidea treated by the sterile filtrate in an embodiment of the present application. DETAILED DESCRIPTION

[0034] The preferred embodiments of the present application will be described in detail below with reference to the drawings. It should be understood that the embodiments described below are merely exemplary of the application and should not be considered limiting. It should also be understood that throughout this application, the term "comprising" is taken to mean the possibilities "including but not limited to" and the term "comprises" is taken to mean the possibilities "including, but not limited to".

[0035] In the following description, for purposes of explanation and not limitation, specific details are set forth such as to provide a thorough understanding of various disclosed embodiments. However, it will be apparent to one skilled in the art that embodiments can be practiced in other embodiments that depart from these specific details. In other instances, detailed descriptions of well-known methods associated with the present disclosure are not described in order to avoid obscuring embodiments of the present disclosure.

[0036] Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrase "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics can be combined in any suitable manner in one or more embodiments.

[0037] The following is one possible embodiment showing the application of volatile organic compounds of Streptomyces SC-3 in inhibiting Botryosphaeria dothidea.

[0038] In this embodiment, the test strains and culture media used are as follows:

[0039] Strains: Streptomyces albidoflavus, Botryosphaeria dothidea.

[0040] Culture media: PDA medium, Gause No. 1 liquid medium, sterile wheat grain medium.

[0041] The main reagents used in the test are shown in Table 1.

[0042] Table 1 Main reagents used in the test

[0043]

[0044] The main reagents used in the test are shown in Table 2.

[0045] Table 2 Main instruments used in the test

[0046]

[0047] In this embodiment, the preparation of wheat grain culture is first carried out, and one possible step is as follows:

[0048] Wheat kernels were washed and boiled until cracking, and the water was drained and divided into 150 mL triangular bottles, 50 g of wheat kernels per bottle, and sterilized at 121℃ for two times, 30 min each time, for later use.

[0049] The strain SC-3 was streaked on Gause No. 1 solid medium for 7 days, and the bacterial body was scraped and inoculated into 100 mL Gause No. 1 liquid medium, which was placed in a 28℃, 200 r / min shaker for 48 h of vibration culture.

[0050] The bacterial suspension was inoculated on the wheat kernel culture medium at a inoculation amount of 1 mL / 10 g (V / W), and was placed in a 28℃ environment for culture, and the wheat kernel culture medium was shaken once every other day to ensure uniform growth of the strain, to obtain the wheat kernel culture of the strain SC-3.

[0051] In this embodiment, the determination of the bacteriostatic activity of VOCs on B. dothidea was completed according to the following steps:

[0052] The plate-to-plate knocker method was used to detect the bacteriostatic effect of the VOCs produced by Streptomyces SC-3. A total of 5 treatments were set: (1) pathogenic bacteria + 15 g activated carbon + 15 g wheat seeds inoculated with SC-3 (Bd+C+SC-3), (2) pathogenic bacteria + 15 g ultraviolet sterilized activated carbon (Bd+C), (3) pathogenic bacteria + 15 g wheat seeds inoculated with strain SC-3 (Bd+SC-3), (4) pathogenic bacteria + 15 g sterile wheat seeds (Bd+Ws), and (5) pathogenic bacteria (Bd), i.e. the control group.

[0053] 10 mL PDA was poured into the upper dish, cooled, and inoculated with B. dothidea fungus cake (6 mm), and the lower dish was separated with a separation dish and added with different treatment groups of wheat or activated carbon. Each treatment was repeated 3 times. After the treatments were placed in a 28℃ environment for 3 d of culture, the pathogenic bacterial colony diameters of each group were measured, and the mycelium inhibition rate was calculated.

[0054] In this embodiment, the determination of the bacteriostatic activity of VOCs on B. dothidea was completed according to the following steps:

[0055] In a feasible implementation, the determination of the bacteriostatic activity of different pre-culture time wheat kernel cultures was completed by the following steps:

[0056] The double-dish knocker method was used to determine the bacteriostatic activity of VOCs of different culture time wheat kernel cultures. 10 mL PDA was poured into the upper dish, cooled, and inoculated with 6 mm B. dothidea fungus cake, and 15 g of wheat kernel cultures cultured for 3 d, 7 d, 14 d, 21 d, and 28 d, respectively, were added to the lower dish. 15 g of sterile wheat kernels were used as a control. After being placed in a 28℃ environment for 3 d of culture, the bacteriostatic activity of VOCs of different culture time wheat kernel cultures was determined. Each treatment was repeated 3 times.

[0057] In one possible implementation, the determination of the antibacterial activity of the culture of different quality wheat is accomplished by the following steps:

[0058] The antibacterial activity of the VOCs of the culture of different quality wheat grains was determined by the double-plate knock method. 10 mL PDA was poured into the upper plate, and after cooling, a 6 mm B. dothidea fungus cake was placed on the PDA surface. The lower plate contained 5 g, 10 g, 15 g, 20 g, and 25 g of the culture of wheat grains cultured for 7 days, respectively. 25 g of sterile wheat grains was used as a control. The antibacterial rate of the VOCs of the culture of different quality wheat grains was determined. Each treatment was repeated 3 times.

[0059] In one possible implementation, the determination of the antibacterial activity of the culture of different inoculation amount of wheat is accomplished by the following steps:

[0060] The antibacterial activity of the VOCs of the culture of different inoculation amount of wheat grains was determined by the double-plate knock method. The SC-3 fungus suspension was inoculated on sterile wheat seeds at an inoculation amount of 100, 250, 500, 750, and 1000 μL / 10 g, respectively, and cultured at 28°C for 7 days. 10 mL PDA was poured into the upper plate, and after cooling, a 6 mm B. dothidea fungus cake was placed on the PDA surface. The lower plate contained 15 g of the culture of wheat grains with different inoculation amounts, respectively. 15 g of sterile wheat grains was used as a control. The antibacterial rate of the VOCs of the culture of different inoculation amount of wheat grains was determined. Each treatment was repeated 3 times.

[0061] The following example illustrates the effect of VOCs on the biomass of B. dothidea mycelium:

[0062] The effect of the VOCs produced by SC-3 on the biomass of B. dothidea mycelium was determined by the double-plate knock method. 10 mL PDA was poured into the upper plate, and after cooling, a layer of glass paper was covered on the PDA surface, and a 6 mm B. dothidea fungus cake was placed on the glass paper. The lower plate contained 10 g, 15 g, and 20 g of the culture of wheat grains cultured for 7 days, respectively. 20 g of sterile wheat grains was used as a control. After being cultured at 28°C for 3 days, the mycelium inhibition rate was calculated, and the mycelium on the glass paper was scraped and weighed. Each treatment was repeated 3 times.

[0063] The following example illustrates the effect of VOCs on the mycelial morphology:

[0064] In one possible implementation, the sample preparation for scanning electron microscopy is accomplished by the following steps:

[0065] The effect of VOCs on the mycelium morphology of B. dothidea was determined by double-dish pair-docking method. Referring to the method of the effect of VOCs on the mycelium biomass of B. dothidea, the mycelium treated by 20 g of wheat grain culture was used as a treatment group, and the mycelium treated by 20 g of sterile wheat grain was used as a control. The mycelium was collected and placed in a 1.5 mL pointed EP tube, 4% glutaraldehyde fixing solution was added, and it was fixed at 4°C overnight. The sample preparation method of scanning electron microscopy was consistent with the determination of the antibacterial activity of VOCs on B. dothidea.

[0066] In a feasible implementation, the transmission electron microscopy sample preparation was completed by the following steps:

[0067] The sample preparation method was the same as that of scanning electron microscopy sample preparation. After the sample was fixed in 2.5% glutaraldehyde fixing solution at 4°C overnight, it was further fixed with 1% osmium tetroxide.

[0068] The following example shows the effect of VOCs on the cell wall of B. dothidea:

[0069] In a feasible implementation, the CFW staining was completed by the following steps:

[0070] The effect of VOCs on the cell wall of B. dothidea was determined by double-dish pair-docking method. Referring to the preparation of B. dothidea mycelium sample in the aforementioned effect of VOCs on the mycelium biomass of B. dothidea, a small amount of mycelium was picked up on a glass slide and stained with calcium fluorescent white (CFW) fluorescent dye for 5 min, then cleaned with cleaning solution, and then covered with a cover glass to observe the damage to the mycelium cell wall using a fluorescence microscope.

[0071] In a feasible implementation, the determination of cell wall related enzyme activity was completed by the following steps:

[0072] Referring to the preparation of B. dothidea mycelium sample in the aforementioned effect of VOCs on the mycelium biomass of B. dothidea, 0.1 g of mycelium treated by 10 g, 15 g, and 20 g of wheat culture for 3 d was used as a sample to be tested, and sterile wheat grains were used as a control. Each treatment was repeated three times. According to the instructions of the β-1,3-glucanase and chitinase activity detection kit, the samples were determined. The multifunctional enzyme marker was used to detect the absorbance values of different treatment groups, and the enzyme activity was calculated according to the standard curve.

[0073] The following example shows the effect of VOCs on the cell membrane of B. dothidea:

[0074] Reference to the foregoing B. dothidea mycelium biomass impact of VOCs, B. dothidea mycelium sample was prepared, a small amount of mycelium to be tested was picked up on a glass slide, and was dyed with 5 mg / L propidium iodide (PI) fluorescent dye at 37°C in the dark for 30 min, then washed with 0.1 M PBS (pH 7.2) for three times, the excess liquid was absorbed with a water-absorbing paper, a cover glass was covered, and the image was observed and collected under a fluorescence microscope.

[0075] The following example shows the impact of VOCs on B. dothidea infection pathogenesis:

[0076] Uniform size, maturity, no mechanical damage and no disease of kiwifruit fruit were selected, and the surface was sterilized with 75% alcohol, and the excess alcohol was washed off with sterile water. After natural drying, B. dothidea spore suspension (1×107 spores / mL) was sprayed on the surface of the kiwifruit, so that the spore suspension could fully wet each kiwifruit. Four dryers with a volume of about 5 L were prepared and sterilized with 70% alcohol, and were naturally dried for standby.

[0077] The above-mentioned B. dothidea spore-inoculated kiwifruit was placed on the perforated ceramic plate of the dryer, and 416.65 g, 625 g, 833.35 g of SC-3 wheat grain culture after 7 d of culture were placed in the bottom of the dryer, so that the content was 83.33 g / L, 125 g / L, 166.67 g / L, respectively. 833.35 g of sterile wheat without SC-3 inoculation was placed in the bottom of the dryer as a control group. 10 kiwifruits were used for each treatment, and the test was repeated 3 times. The dryer was sealed and placed at 28°C for 10 d, and the disease incidence was recorded.

[0078] In a feasible embodiment, the collection and identification of VOCs are completed by the following steps:

[0079] The SC-3 inoculated and cultured for 7 d wheat grain culture was used for gas chromatography-mass spectrometry (GC-MS) analysis. 5 g of sample was placed in a 50 mL centrifuge tube, sealed and placed at 28°C for 12 h. An equal amount of sterile wheat grain was used as a control, and the test environment was a blank control.

[0080] Solid phase microextraction (SPME) technology was used for sample pretreatment, and the specific operation was as follows: the extraction fiber needle was aged at 250°C in the GC injection port for 20 min, then the sample was adsorbed for 30 min, and then the fiber needle was withdrawn for testing. The detection data were compared with the NIST14 database. The substances existing in the control, blank control and SC-3 wheat grain culture at the same time were removed, and only the substances in the wheat grain culture were screened out.

[0081] The following example illustrates the fungistatic activity of volatile compounds against B. dothidea:

[0082] The fungistatic activity of carvyl ketone, 3,5-di-tert-butylphenol and 3-n-butenylphthalide against B. dothidea was determined using a two-compartment Petri dish with a volume of about 68 mL. In one compartment of the Petri dish, 8 mL of PDA medium was poured and, after cooling, a B. dothidea mycelium cake was inoculated. In the other compartment, carvyl ketone: 1.8, 3.6, 7.2, 14.4, 28.8 μL, 3,5-di-tert-butylphenol: 14.4, 28.8, 57.6, 115.2, 230.4 μL, 3-n-butenylphthalide: 42, 84, 168, 336, 672 μL were added, respectively, to make the final concentrations: carvyl ketone (0.03, 0.06, 0.12, 0.24, 0.48 μL / mL), 3,5-di-tert-butylphenol (0.24, 0.48, 0.96, 1.96, 3.92 μL / mL), 3-n-butenylphthalide (0.7, 1.4, 2.8, 5.6, 11.2 μL / mL). After adding the compounds, the Petri dish was quickly covered and sealed, and incubated at 28°C for 2 days. The colony diameters on each plate were measured, and the mycelial growth inhibition rate of B. dothidea by VOCs was calculated. Each treatment was repeated 3 times.

[0083] The following example illustrates the fungistatic activity of VOCs produced by Streptomyces SC-3 against B. dothidea:

[0084] The fungistatic effect of VOCs produced by strain SC-3 on B. dothidea was detected by the dual-dish knock test. The results showed that the colony diameters of the treatment groups Bd+Ws, Bd and Bd+C were 84.42, 82.58 and 83.25 mm, respectively, with no significant difference, indicating that the sterile wheat kernels and activated carbon had no inhibitory effect on the growth of B. dothidea. After B. dothidea was co-cultured with inoculated wheat kernels of strain SC-3, the colony diameter decreased to 12.92 mm, indicating that the VOCs produced by strain SC-3 inhibited the growth of B. dothidea. After adding activated carbon, the colony diameter of B. dothidea increased to 39.50 mm, indicating that activated carbon adsorbed part of the volatile substances, reducing their fungistatic activity, while the unadsorbed volatile substances still had fungistatic effect.

[0085] The above results show that strain SC-3 can inhibit mycelial growth through the production of VOCs without direct contact with the pathogen.

[0086] The following example illustrates the fungistatic activity of wheat kernel cultures with different pre-culturing times against B. dothidea:

[0087] The strain SC-3 was inoculated into sterile wheat grains and cultured for 3 d, 7 d, 14 d and 21 d, respectively, and the antibacterial activity of the wheat grain culture against B. dothidea was detected. The results showed that the antibacterial rates of the wheat grain culture of Streptomyces SC-3 were 45.49%, 73.68%, 80.77% and 83.82% at 3 d, 7 d, 14 d and 21 d, respectively.

[0088] It can be seen that with the increase of culture time, the antibacterial activity also increases, but there is no significant difference in the antibacterial activity when cultured for 7 d - 21 d, so the wheat culture of 7 d is selected for subsequent experiments.

[0089] The following example shows the antibacterial activity of different quality wheat grain cultures against B. dothidea:

[0090] The wheat grain culture inoculated with SC-3 for 7 d was co-cultured with B. dothidea at 5 g, 10 g, 15 g, 20 g and 25 g, respectively, and the antibacterial activity of the wheat grain culture against B. dothidea was detected. The results showed that when the SC-3 wheat culture increased from 5 g to 20 g, the antibacterial rate increased from 63.91% to 84.05%, and when the wheat culture increased from 20 g to 25 g, the antibacterial activity did not increase significantly.

[0091] The results show that increasing the quality of wheat grain culture within a certain range can improve the antibacterial activity of VOCs produced by strain SC-3, but continuous increase in quality cannot continue to improve the antifungal activity of VOCs.

[0092] In one possible embodiment, when the inoculation amount increases from 100 μL to 1000 μL, the inhibition rate of VOCs produced by strain SC-3 gradually increases from 47.1% to 92.94%. When the inoculation amount continues to increase, the carrying capacity of wheat grains to bacterial suspension reaches the upper limit, and if the water content is too high, the wheat grains are easy to be contaminated. Therefore, the inoculation amount should not exceed 1000 μL / 10 g.

[0093] The following example shows the effect of VOCs on the growth of B. dothidea mycelium:

[0094] The effect of VOCs produced by strain SC-3 on the biomass of B. dothidea mycelium was determined by double-dish knock method. The results showed that VOCs produced by strain SC-3 could significantly inhibit the growth of B. dothidea. After treatment of B. dothidea with 10 g, 15 g and 20 g of wheat grain culture, the mycelium became thin, the mycelial growth was significantly inhibited, and the fresh weight of mycelium was significantly reduced.

[0095] When the mycelium fresh weight of the control group was 186.17 mg, the mycelium fresh weight of the 10 g, 15 g and 20 g treatment groups was 41.00 mg, 23.33 mg and 9.50 mg, respectively, and the inhibition rates were 77.98%, 87.47% and 94.90%, respectively.

[0096] It can be seen that the VOCs produced by strain SC-3 can significantly inhibit the mycelial growth of B. dothidea after treatment.

[0097] The following example shows the effect of VOCs on the morphology of B. dothidea mycelium:

[0098] The scanning electron microscope observation results are as follows:

[0099] The morphology of B. dothidea mycelium treated with VOCs produced by strain SC-3 was observed by scanning electron microscope. The results are shown in Figure 1 The mycelium surface of the control group was smooth and full, but the mycelium of B. dothidea treated with VOCs was severely shrunk, perforated and had increased branches.

[0100] It can be seen that the treatment of VOCs produced by strain SC-3 can change the morphology of B. dothidea mycelium, destroy its normal physiological function and lead to its abnormal growth.

[0101] The transmission electron microscope observation results are as follows:

[0102] The effect of VOCs produced by strain SC-3 on the cell structure of B. dothidea mycelium was observed by transmission electron microscope. The results are shown in Figure 2 The cytoplasm of the control group was uniform, the organelle distribution was orderly, and the mitochondria morphology was normal; but the intracellular material of the mycelium treated with VOCs was lost, the mitochondria was swollen, the protoplast separation occurred, and vacuolization appeared.

[0103] The results show that the VOCs produced by strain SC-3 can destroy the cell structure of B. dothidea mycelium and affect its normal physiological function.

[0104] The following example shows the effect of VOCs on the cell wall of B. dothidea:

[0105] The CFW staining observation results are as follows:

[0106] The damage of B. dothidea mycelium cell wall treated with VOCs produced by strain SC-3 was observed by CFW staining. The results are shown in Figure 3As shown, the mycelium of the non-VOCs treated group grew smoothly, thickly, and had blue-white fluorescence, while the fluorescence of the mycelium cell wall of the wheat grain culture treatment group was weakened. With the increase in the quality of the wheat grain culture, the mycelium branches gradually increased, and the degree of fluorescence weakening intensified.

[0107] It was shown that VOCs fumigation treatment could cause B. dothidea mycelium to be deformed, and at the same time damage the mycelium cell wall.

[0108] The following example shows the effect of VOCs on B. dothidea cell wall related enzyme activity:

[0109] By determining the activities of β-1, 3 glucanase and chitinase, the effect of VOCs produced by strain SC-3 on the B. dothidea mycelium cell wall was detected. The results showed that, compared with the control, the β-1, 3-glucanase and chitinase activities of B. dothidea mycelium increased after VOCs fumigation treatment. The β-1, 3-glucanase was the highest after 15 g wheat grain culture treatment, while the chitinase activity was significantly increased after 15 g and 20 g wheat grain culture fumigation treatment.

[0110] It was shown that VOCs fumigation treatment could cause B. dothidea cell wall related enzyme activity to increase, leading to cell wall degradation.

[0111] The following example shows the effect of VOCs on B. dothidea cell membrane

[0112] PI staining was used to detect the damage to the B. dothidea mycelium cell membrane after VOCs treatment. The results are shown in Figure 4 As shown, the red fluorescence of the mycelium of the control group was weak, while the fluorescence intensity of the mycelium after VOCs treatment was significantly enhanced, and the fluorescence intensity increased with the increase in the quality of the wheat grain culture.

[0113] The results showed that VOCs produced by strain SC-3 could cause damage to the B. dothidea mycelium cell membrane, and the degree of damage was positively correlated with the concentration of VOCs.

[0114] The following example shows the effect of VOCs on the prevention and control of B. dothidea infection and pathogenesis:

[0115] The VOCs produced by strain SC-3 were used to fumigate the kiwi fruits inoculated with B. dothidea spores to determine the effect of VOCs on the infection ability of B. dothidea. The results showed that the control group of kiwi fruits had large disease spot area and serious softening and rotting; the disease incidence of kiwi fruits treated by fumigation with 88.33 g / L, 125 g / L and 166.67 g / L wheat grain culture was 68.89%, 24.44% and 18.89% respectively, indicating that the disease incidence of fruits treated by fumigation with VOCs was significantly reduced. Meanwhile, the hardness and weight loss rate of kiwi fruits were detected, and it was found that the hardness of fruits in the VOCs treatment group was increased and the weight loss rate of fruits was reduced compared with the control group.

[0116] The test results showed that the VOCs produced by strain SC-3 could effectively control the occurrence of soft rot of kiwi fruits, slow down the expansion speed of disease spot and the process of fruit softening and rotting, and significantly reduce the disease incidence of kiwi fruits.

[0117] In a feasible implementation, the identification of the VOCs component produced by Streptomyces SC-3 is completed by the following steps:

[0118] The VOCs produced by Streptomyces SC-3 were detected by GC-MS technology, and a total of 83 compounds were detected, of which 20 compounds with a relative peak area greater than 1% were mainly alcohol, ketone, phenol, ester and other compounds (Table 3).

[0119] Among the detected VOCs, the compound with the highest content was trans-1,10-dimethyl-trans-9-decalol, with a relative content of 10.04%, followed by 3,5-di-tert-butylphenol, isolongifolenone (2,4a-methanonaphthalen-7-one) and carvone, with relative contents of 8.40%, 6.94% and 4.21% respectively.

[0120] Table 3 Analysis of VOCs components produced by strain SC-3

[0121]

[0122] The following example shows the antibacterial activity of a single component volatile compound on B. dothidea:

[0123] Three compounds with antibacterial activity were screened from the compounds with high content by using the two-division Petri dish method, which were carvone, 3,5-di-tert-butylphenol and 3-n-butylidene phthalide.

[0124] The antibacterial test showed that the EC50 of the three compounds on B. dothidea was 0.03 μL / mL, 6.40 μL / mL, and 6.13 μL / mL, respectively. Among them, carvyl ketone had the best antibacterial effect on B. dothidea. When the concentration was 0.48 μL / mL, the B. dothidea mycelium could not grow basically.

[0125] It can be seen that carvyl ketone may be one of the main compounds in VOCs that play an antibacterial role.

[0126] The above examples show that the VOCs produced by strain SC-3 can significantly inhibit the accumulation of B. dothidea mycelial biomass, and the inhibition rate is positively correlated with the concentration of VOCs.

[0127] Through electron microscopy observation, it was found that after VOCs treatment, the mycelium surface appeared shrinkage, perforation and other phenomena, the intracellular material was lost, vacuolization occurred, mitochondria swelled, and plasmolysis occurred, indicating that VOCs affected the normal morphology and physiological function of B. dothidea.

[0128] Further through CFW staining, β-1, 3-glucanase and chitinase activity determination and PI staining, it was found that VOCs could damage the cell wall and cell membrane of B. dothidea mycelium, leading to damage of mycelial structure.

[0129] In addition, VOCs significantly reduced the incidence of soft rot of kiwifruit through fumigation, and slowed down the process of fruit rotting and softening. It was found that the antibacterial effect of VOCs may be due to the synergistic effect of multiple components and multiple targets.

[0130] Compared with traditional contact fungicides, VOCs only act on the surface of fruits and vegetables without entering the inside, which can effectively reduce pesticide residues and has broad application prospects in the prevention and control of plant pathogens.

[0131] Through GC-MS analysis, 83 VOCs produced by strain SC-3 were detected in the above examples, mainly including alcohol, ketone, phenol, terpene and acid derivatives and other compounds, among which geosmin had the highest content.

[0132] In vitro antibacterial test of available compounds found that carvyl ketone had strong inhibitory activity on B. dothidea, with an EC50 of 0.03 μL / mL. The VOCs produced by strain SC-3 were significantly different in composition and content compared with other Streptomyces.

[0133] For example, after 7 days of culture of S. albidoflavus strain ML27, 21 VOCs were detected, among which 4-ethyl-1, 2-dimethoxybenzene showed broad-spectrum antifungal activity S. globisporus strain JK-1 produced 41 VOCs, with geosmin being the most abundant S. salmonis strain PSRDC-09 produced 14 VOCs, with L-linalool being the main volatile component.

[0134] The above results show that the types and contents of VOCs released by different strains under specific conditions are significantly different, which may be related to the genetic background, metabolic pathways, and culture conditions of the strains. These VOCs with antibacterial activity have significant advantages in reducing the use of chemical pesticides and environmental protection.

[0135] In the above examples, the technical terms, technical principles or technical means related to the technical solutions of the present application are not described in detail in the above content, which are known to those skilled in the art or common means.

[0136] The above shows and describes the basic principles and main features of the present application and the advantages of the present application. Those skilled in the art should understand that the present application is not limited by the above examples, and the above examples and descriptions in the specification are only to illustrate the principles of the present application. Without departing from the spirit and scope of the present application, various changes and improvements can be made to the present application, and these changes and improvements all fall within the scope of the present application. The scope of protection of the present application is defined by the appended claims and their equivalents.

Claims

1. Use of Streptomyces SC-3 volatile organic compounds in inhibiting Botryosphaeria dothidea, characterized in that, The method comprises the following steps: Preparation of Streptomyces SC-3 wheat grain culture, which is prepared by inoculating SC-3 bacterial suspension into sterilized wheat grain culture medium and culturing at 25-30°C for 5-14 days, and the culture is shaken regularly during the culturing; The wheat grain culture is contacted with B. dothidea by double-plate tapping method, the upper plate is PDA medium containing B. dothidea bacterial cake, and the lower plate is placed with the wheat grain culture, and the culture is carried out at 25-30°C for 2-5 days.

2. The use of Streptomyces SC-3 volatile organic compounds for inhibiting Botryosphaeria dothidea according to claim 1, characterized in that, The preparation method of the SC-3 bacterial suspension is that SC-3 is streaked on Gao's first solid medium and cultured for 5-9 days, the bacterial body is scraped and inoculated into Gao's first liquid medium with a volume of 80-120 mL, and the culture is carried out at 25-30°C and 180-220 r / min for 40-56 h.

3. The use of Streptomyces SC-3 volatile organic compounds for inhibiting Botryosphaeria dothidea according to claim 1, characterized in that, The sterilization treatment of the wheat grain culture medium is 121°C sterilization for 1-3 times, and the sterilization time is 25-35 min each time.

4. The use of Streptomyces SC-3 volatile organic compounds for inhibiting Botryosphaeria dothidea according to claim 1, characterized in that, In the double-plate tapping method, the thickness of the PDA medium containing B. dothidea bacterial cake is 3-7 mm, and the diameter of the B. dothidea bacterial cake is 4-8 mm.

5. A method for preparing volatile organic compounds of Streptomyces SC-3, characterized by, The method comprises the following steps: The washed wheat grains are divided into containers and sterilized to obtain wheat grain culture medium; The SC-3 bacterial suspension is inoculated into the wheat grain culture medium, and the culture is carried out at 25-30°C for 5-14 days, and the culture is shaken regularly during the culturing; The volatile organic compounds generated by the culture are collected by solid-phase microextraction method, the extraction fiber needle is aged at 240-260°C for 15-25 min, and the sample is adsorbed for 25-35 min. 6.The method for preparing volatile organic compounds of Streptomyces SC-3 according to claim 5, characterized in that, The washed wheat grains are boiled to crack and then drained, the divided amount is 45-55 g per 150 mL triangular flask, and the sterilization treatment is 121°C sterilization for 2 times, 25-35 min each time; The inoculation amount of the SC-3 bacterial suspension is 80-1200 μL / 10 g of wheat grain culture medium, and the culture is shaken once every 1-2 days during the culturing.

7. A method of preventing and controlling B. dothidea infection of kiwifruit fruit, characterized by, The method comprises the following steps: The intact kiwi fruits are selected, surface-disinfected, dried, and then sprayed with B. dothidea spore suspension; The fruits are placed on the perforated partition of a sealed container, and Streptomyces SC-3 wheat grain culture is placed at the bottom of the container, the culture amount is 80-170 g / L, and the culture is carried out at 25-30°C for 8-12 days.

8. The method for preventing and controlling B. dothidea from invading kiwi fruits according to claim 7, wherein The surface disinfection was performed by wiping or soaking with 70-75% alcohol, and the concentration of B. dothidea spore suspension was 1 x 10 6 -1 x 10 8 spores / mL, and the sealed container was naturally dried after being disinfected with 70-75% alcohol; The Streptomyces SC-3 wheat grain culture is a culture prepared by culturing at 25-30°C for 7 days, the number of kiwi fruits treated is 8-12 per time, and the test is repeated 2-4 times.

9. The synergistic use of a plurality of Streptomyces SC-3 volatile organic compounds for the preparation of a preparation for inhibiting Botryosphaeria dothidea, characterized in that, The volatile organic compounds of the Streptomyces sp.SC-3 include Carvone, 3,5-di-tert-butylphenol and 3-Butylidenephthalide; the preparation is prepared by mixing the volatile organic compounds of the Streptomyces sp.SC-3 in proportion, then performing gas phase contact with B.dothidea by using a two-compartment culture dish method, and the side of the culture medium containing the B.dothidea fungus cake is separated from the side of the mixed organic compounds, the total final concentration of the mixed organic compounds is 0.05-0.8 μL / mL, and the culture is performed at 25-30 ℃ for 2-4 days.

10. The synergistic use of the plurality of Streptomyces SC-3 volatile organic compounds in the preparation of a preparation for inhibiting B. dothidea according to claim 9, characterized in that, In the preparation of the preparation raw materials, the volume ratio of the three components of Carvone, 3,5-di-tert-butylphenol and 3-Butylidenephthalide is 1:(0.5-2):(0.5-2), and the purity of the three components is all ≥98%; the volume of the two-compartment culture dish is 60-80 mL, the culture medium containing the B.dothidea fungus cake is 6-10 mL of PDA culture medium, the diameter of the fungus cake is 4-8 mm, and the growth state of the colony is observed daily during the culture.

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