Bacillus amyloliquefaciens, mildew preventer containing the bacillus amyloliquefaciens and spore preservative, and application thereof

Abstract

The present invention relates to a bacillus amyloliquefaciens KTA2108 which is deposited in CGMCC with a deposit number of CGMCC No. 24720, a mildew preventer containing the bacillus and a spore preservative and an application thereof. The spores of the bacillus amyloliquefaciens KTA2108 have a high survival rate in the preservative, and the spores preserved therein can also germinate normally under suitable conditions. The mildew preventer contains 1.4 g / L of sodium lauryl polyoxyethylene ether sulfate and the bacillus amyloliquefaciens KTA2108, has a synergistic effect, and can better inhibit the reproduction of molds.

Description

Technical Field

[0001] The present application relates to the field of microorganisms, and in particular to a Bacillus amyloliquefaciens, a mildew preventer containing the Bacillus amyloliquefaciens and a spore preservative, and applications thereof. Background Art

[0002] The natural color of wood and its products is destroyed, mainly by wood mold and discoloration fungi. Although the strength of wooden products is not greatly affected, the aesthetic value of wood discoloration is reduced, which still causes serious economic losses to the wood industry in the world today. Wooden products are easily colored by fungi, mainly because the pigments in the fungal hyphae cell cavity combine with the ray parenchyma of the wood to form plaque. At present, in the wood industry, chemical agents are mainly used to control the growth of wood fungi, but these chemical agents pose a serious threat to the environment, human health and the sustainable development of the ecosystem. Therefore, the development of natural resources and biochemical compounds for the prevention and control of wood mold has great economic significance in the wood industry.

[0003] Biological control agents have been widely used in agriculture, medicine, forestry and other fields due to their advantages of environmental protection, health and safety. Compared with other biological control microorganisms used in the environment, Bacillus, such as Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, etc., can produce a variety of active secondary metabolites that antagonize bacteria and fungi, such as bacteriocins, surfactins, iturins, fengycins, bacilysins, and because of their special endospore structure, they have superb resistance to various adverse environments such as high temperature, strong acid and alkali, and certain chemicals. With its high adaptability to the environment, it has significant advantages in production and transportation, and is currently one of the most widely used types in the field of biological control.

[0004] At present, most of the various functional Bacillus agents on the market are used in the form of powders. In order to expand the application scope of biological agents such as Bacillus and improve portability and comfort during use, it is extremely important to find a preservative that can inhibit spore germination and maintain spore activity for a long time.

[0005] At the same time, during normal use of wooden products, such as constant temperature wooden wine cabinets, due to the suitable temperature and humidity in the wine cabinets, mold in the environment is more likely to colonize and grow in the wine cabinets, forming hyphae or plaque, which not only affects the appearance of the wine cabinets, but also easily has a great impact on the quality of the red wine stored in the wine cabinets. Therefore, the development of healthy, safe and environmentally friendly mildew inhibitors for use with constant temperature wine cabinets to prevent the wooden compartments of the wine cabinets from mold has great potential in production and application. Summary of the invention

[0006] In view of the above problems, the present application provides a Bacillus amyloliquefaciens KTA2108 with anti-mildew effect, which is deposited in CGMCC with the deposit number of CGMCC No. 24720.

[0007] Different from the existing technology, the Bacillus amyloliquefaciens KTA2108 has been deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration on April 19, 2022, with the deposit number CGMCC NO 24720, the classification name is Bacillus amyloliquefaciens, the Latin classification name is Bacillus amyloliquefaciens, and the address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0008] Preferably, the Bacillus amyloliquefaciens KTA2108 has an antagonistic effect on wood mold and wood discoloration fungi.

[0009] Preferably, the wood molds include Aspergillus niger, Penicillium citrinum and Trichoderma viride.

[0010] Preferably, the wood discoloration fungi include Diplodia theobromin, Fusarium moniliforme and Alternaria alternata.

[0011] Preferably, the survival rate of the Bacillus amyloliquefaciens stored in a spore liquid preservative at room temperature (18-28° C.) for 360 days exceeds 85%, and the spore liquid preservative contains sodium lauryl polyoxyethylene ether sulfate; tetradecyl dimethyl ammonium oxide 3-sulfopropyl tetradecyl dimethyl ammonium; fatty alcohol polyoxyethylene ether-7 or a combination thereof.

[0012] Under normal temperature liquid environment, Bacillus amyloliquefaciens KTA2108 spores have a high survival rate in this preservative, and the spores preserved therein can also germinate normally under suitable conditions.

[0013] Preferably, the spore liquid preservative contains 0.14-14 g / L sodium lauryl polyoxyethylene ether sulfate or 0.1-10 g / L tetradecyl dimethyl ammonium oxide, or 0.1-10 g / L 3-sulfopropyl tetradecyl dimethyl ammonium, or 0.5-50 g / L fatty alcohol polyoxyethylene ether-7.

[0014] The present application also provides a mildew preventer, wherein the mildew preventer contains 1.4 g / L sodium lauryl alcohol polyoxyethylene ether sulfate and Bacillus amyloliquefaciens KTA2108, wherein the spore count of the Bacillus amyloliquefaciens KTA2108 is (5.0±1.0)×10 7 CFU / ml.

[0015] Different from the prior art, the 1.4 g / L sodium lauryl polyoxyethylene ether sulfate and Bacillus amyloliquefaciens KTA2108 in the mildew inhibitor have a synergistic effect and can better inhibit the reproduction of mold.

[0016] Preferably, the mildew inhibitor is used for mildew prevention of radiata pine.

[0017] Preferably, the mildew preventer is used for preventing mildew of beech wood.

[0018] Preferably, the mildew inhibitor is sprayed on the wood surface in a spraying manner, with a spraying frequency of 7 days / time.

[0019] The above-mentioned records related to the invention content are only an overview of the technical solution of the present application. In order to enable ordinary technicians in the field to more clearly understand the technical solution of the present application, and then implement it according to the text of the specification and the contents recorded in the drawings, and to make the above-mentioned purpose and other purposes, features and advantages of the present application easier to understand, the following is an explanation in combination with the specific implementation mode and drawings of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The accompanying drawings are only used to illustrate the principles, implementation methods, applications, features and effects of the specific implementation methods of the present application and other related contents, and cannot be considered as limiting the present application. In the accompanying drawings of the specification:

[0021] Figure 1 The results of the confrontation between Bacillus amyloliquefaciens KTA2108 and wood mold, color-changing fungi, and mixed molds in Example 1 (A is Aspergillus niger, B is Penicillium citrinum, C is Trichoderma viride, D is Diplodia cholerae, E is Fusarium moniliforme, F is Alternaria alternata, and G is a mixed mold);

[0022] Figure 2 The colonies and bacterial morphology of Bacillus amyloliquefaciens KTA2108 cultured on NA medium for 24 h (A and B) and 48 h (B and C) in Example 2 (the upper left corner of Figure D shows the spore morphology of KTA2108 in another field of view);

[0023] Figure 3 is a 16S rRNA phylogenetic tree diagram of Bacillus amyloliquefaciens KTA2108 in Example 2;

[0024] Figure 4 The anti-mildew effect diagram of the Bacillus mildew inhibitor in Example 5 applied to radiata pine for 0 days (A), 3 days (B), 5 days (C), and 7 days (D) (the upper end is the test end, and the lower end is the control end);

[0025] Figure 5 This is a diagram showing the anti-mildew effect of the Bacillus mildew inhibitor in Example 6 applied to beech wood at a frequency of 3 days / time for 28 days (A0 is the control group, A1 is the test group);

[0026] Figure 6 This is a diagram showing the anti-mildew effect of the Bacillus mildew inhibitor in Example 6 applied to beech wood at a frequency of 5 days / time for 28 days (B0 is the control group, and B1 is the test group);

[0027] Figure 7 This is a diagram showing the anti-mildew effect of the Bacillus mildew inhibitor in Example 6 applied to beech wood at a frequency of 7 days / time for 28 days (C0 is the control group, C1 is the test group);

[0028] Figure 8 This is the application of the Bacillus mildew inhibitor on the wooden wine cabinet in Example 7 (A is the control group, B is the test group, A1, A2, A3, B1, B2, B3 correspond to the partial pictures of the rectangular frame, and the circles are mildew spots). DETAILED DESCRIPTION

[0029] In order to explain in detail the possible application scenarios, technical principles, specific schemes that can be implemented, and the purposes and effects that can be achieved, the following is a detailed description of the specific embodiments listed in conjunction with the accompanying drawings. The embodiments described herein are only used to more clearly illustrate the technical solutions of the present application, and are therefore only used as examples, and cannot be used to limit the scope of protection of the present application.

[0030] Reference to "embodiment" herein means that the specific features, structures or characteristics described in conjunction with the embodiment may be included in at least one embodiment of the present application. The term "embodiment" appearing in various places in the specification does not necessarily refer to the same embodiment, nor does it particularly limit its independence or association with other embodiments. In principle, in the present application, as long as there is no technical contradiction or conflict, the various technical features mentioned in the embodiments can be combined in any way to form a corresponding implementable technical solution.

[0031] Unless otherwise defined, the technical terms used in this document have the same meanings as those generally understood by those skilled in the art to which this application belongs; the use of relevant terms in this document is only for describing specific embodiments and is not intended to limit this application.

[0032] In the description of this application, the term "and / or" is an expression used to describe the logical relationship between objects, indicating that three relationships may exist, for example, A and / or B, which means: A exists, B exists, and A and B exist at the same time. In addition, the character " / " in this article generally indicates that the objects before and after are in an "or" logical relationship.

[0033] In the present application, terms such as “first” and “second” are merely used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship of quantity, priority or sequence between these entities or operations.

[0034] Without further limitations, in this application, the words "include", "comprises", "has" or other similar expressions used in sentences are intended to cover non-exclusive inclusion. These expressions do not exclude the presence of additional elements in the process, method or product including the elements, so that the process, method or product including a series of elements may include not only those limited elements, but also other elements not explicitly listed, or also include elements inherent to such process, method or product.

[0035] Similar to the understanding in the Examination Guidelines, in this application, expressions such as "greater than", "less than", "exceed" and the like are understood to exclude the number itself; expressions such as "above", "below", "within" and the like are understood to include the number itself. In addition, in the description of the embodiments of this application, "multiple" means more than two (including two), and similar expressions related to "multiple" are also understood in this way, such as "multiple groups", "multiple times", etc., unless otherwise clearly and specifically limited.

[0036] Example 1 Isolation of Bacillus spores antagonistic to wood mold and discoloration fungi

[0037] Isolation and purification of Bacillus: Randomly pick 20 moldy black spots on a moldy wooden wine cabinet placed at a constant temperature of 12℃, and carefully scrape the surface and 0.80cm below the wood with a utility knife. After crushing the obtained wood sample, weigh 0.5g and put it into a sterilized 500ml triangular flask (the bottom is covered with glass beads with a diameter of about 0.3-0.5cm), pour 100ml of sterile water into the triangular flask, place it in an ultrasonic cleaner with a power of 100W for 10min, then put it in an air bath shaker, shake and mix at 30℃ and 200r / min for 30min, absorb 10ml of the supernatant, place it in a water bath at 80℃ for 10min, and then dilute the supernatant with sterile water to a suitable gradient. 200μl of each gradient dilution is evenly spread on the nutrient agar (NA) medium, and placed in a constant temperature incubator at 30℃ for 48h. Single colonies of different morphologies were picked from the culture medium and streaked onto NA medium for purification. They were cultured at 30°C for 96 h. Gram staining, malachite green spore staining and catalase test were performed on the single colonies obtained by streaking. For Gram-positive bacilli, colonies with green spores by malachite green staining and positive catalase test, they were streaked for purification 2 to 3 times again and cultured at 30°C for 24 h.

[0038] Expansion culture of Bacillus: Pick a single colony of Bacillus after purification and put it into 100 ml of nutrient broth, and culture it in an air bath shaker at a speed of 180 r / min and 30°C for 24 hours to obtain a Bacillus suspension.

[0039] Activation of wood mold and wood discoloration bacteria: Wood mold and wood discoloration bacteria purchased from Shanghai Bioresource Collection Center (SHBCC) were inoculated on potato dextrose agar (PDA) containing chloramphenicol for activation and cultured at 28°C for 3 to 5 days until the colonies grew to the edge of the culture dish. The wood molds included Aspergillus niger SHBCC D23943, Penicillium citrinum SHBCC D11599 and Trichoderma viride SHBCC D10197. The color-changing fungi included Lasiodiplodia theobromae SHBCC D64351, Fusarium moniliforme SHBCC D24889 and Alternaria alternata AS3.4255.

[0040] Preparation of mixed spore suspension of wood mold and discoloration fungi: Under sterile conditions, use an inoculation needle to pick up the mycelium and spores of three kinds of molds and three kinds of discoloration fungi, put them into a sterilized tissue grinder, add an appropriate amount of sterile water, grind them appropriately, pour them into a sterilized triangular flask with glass beads, add a small amount of sterile water, put them on a shaker and shake at a speed of 120r / min for 15 minutes to obtain a spore suspension of a single mold and discoloration fungi. After appropriately diluting the spore suspension of each mold and discoloration fungus, the number of spores in the final suspension is (1-2)×10 6 / ml, then mix the spore suspensions of the three molds and the three color-changing bacteria in equal volumes to obtain a mixed spore suspension for later use.

[0041] Preparation of mixed fungal moss of wood mold and discoloration fungi: Take 200 μl of spore suspension of wood mold and discoloration fungi, evenly spread it on a PDA plate containing chloramphenicol, and invert it in an incubator at 28°C for 3 to 5 days until the entire plate is covered with mixed fungal moss.

[0042] Confrontation test between Bacillus and wood mold, discoloration fungi, and mixed fungi: The confrontation method was adopted, and three wood molds, three wood discoloration fungi, and mixed fungi were used as indicator fungi to explore the antifungal activity of isolated Bacillus against wood fungi. Wood mold, discoloration fungi, and mixed fungi were dug out with an Oxford cup with an inner diameter of 6 mm, and the mycelium surface of the fungus disc was inoculated downward in the center of the PDA culture medium without chloramphenicol. Two Oxford cups were gently placed at a distance of 3 cm from the horizontal direction, left and right sides of the fungus disc, and 200 μl of Bacillus suspension was inoculated into them. The suspension of each strain of Bacillus was repeated in 3 plates. After the bacterial liquid was fully immersed in the culture medium, the culture plate was moved to a 28°C incubator and cultured upright for 3 to 5 days. At the same time, a plate inoculated with an equal amount of sterile water was used as a blank control. When the fungi in the blank control grew to the edge of the plate, the diameter of the inhibition zone of Bacillus against different indicator fungi was measured with a vernier caliper. The average value was taken by the cross method. The value of "inhibition zone diameter / colony diameter" was used to represent the antifungal activity of each strain of Bacillus on PDA medium. When the ratio was >1.0, it indicated that the strain had antifungal activity. The larger the ratio, the stronger the biological antagonism against the indicator fungi.

[0043] A total of 13 strains of Bacillus were isolated from the black spots on the wooden shelves of the moldy wine cabinet, which were numbered KTA2101 to KTA2113 in the order of isolation. After confrontation tests, it was found that only strains KTA2108 and KTA2111 had inhibitory effects on the six indicator fungi, and their antifungal activities are shown in Table 1. According to Table 1, Bacillus KTA2108 has a stronger inhibitory effect on wood mold and discoloration fungi than strain KTA2111, among which the confrontation results of KTA2108 on wood mold, discoloration fungi and mixed fungi are shown in Table 1. Figure 1 .

[0044] Therefore, in the present invention, Bacillus KTA2108 is used as the object to prepare a Bacillus mildew inhibitor.

[0045] Table 1 Antifungal activity of two strains of Bacillus against wood mold and discoloration fungi (inhibition zone diameter / colony diameter)

[0046]

[0047]

[0048] Example 2 Identification and preservation of mildew-resistant Bacillus KTA2108

[0049] Colony and cell characteristics of Bacillus KTA2108: When Bacillus KTA2108 was cultured at 30°C for 24 hours on NA medium, the colony morphology was as follows: Figure 2-A, the colony is translucent and convex, round, with expanded edges, milky white in color, dull, moist in texture, slightly dry and wrinkled on the surface, with a colony diameter of about 0.20-0.40 cm; Gram staining is positive, the bacteria are short rod-shaped, about 0.6-0.8μm×2-3μm (see Figure 2 -B). When strain KTA2108 was cultured for 48 h, Figure 2 -C, the colony is opaque and convex, round, with expanded edges, grayish white in color, dry, rough, and wrinkled on the surface. The colony is about 0.30-0.60 cm; Gram staining is positive, with obvious spores, about 0.6-0.8 μm × 1.0-1.6 μm, oval, mesophyll, and the bacteria do not expand after spore formation (see Figure 2 -D).

[0050] Biochemical characteristics of Bacillus KTA2108: The microbial microfermentation method was used and biochemical identification strips ( The biochemical results were input into Microgen Identification System Software (MID-60) for analysis. Meanwhile, referring to Bergey's Manual of Bacterial Identification, combined with the colony and somatic characteristics of the strain, it was preliminarily determined that Bacillus KTA2108 belonged to the Bacillus subtilis group.

[0051] Table 2 Physiological and biochemical characteristics of mildew-resistant Bacillus KTA2108

[0052]

[0053]

[0054] Note: "+" indicates that the trait is positive; "-" indicates that the trait is negative

[0055] Molecular biological identification of Bacillus KTA2108: Take an appropriate amount of fresh Bacillus KTA2108 suspension and use a bacterial genomic DNA rapid extraction kit ( The genomic DNA of KTA2108 was extracted by Sangon Biotech. The extracted DNA was used as a template and PCR amplification was performed using the universal primers 27F [5'-TACGGCTACCTTGTTACGACTT-3'] (SEQ ID NO.1) and 1492R [5'-GAATAAGTGCGACATCATCATC-3'] (SEQ ID NO.2) of the bacterial 16S rRNA gene. The product was about 1500 bp. The amplified product was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing, and it was found that the 16S rRNA gene sequence of Bacillus KTA2108 was 1409 bp. The specific gene sequence is shown in the sequence table SEQ ID NO.3. The sequencing results were submitted to the Gen Bank nucleotide sequence database of the National Center for Biotechnology Information (NCBI) of the United States. The basic local alignment search tool (BLAST) was used for homology comparison analysis. In the results of the online comparison, 15 of the top 20 sequences hit Bacillus amyloliquefaciens, and the coverage and homology with the 16S rRNA gene sequences of these strains reached 100% and 99.86%, respectively. Four model strains with high homology were selected, and the 16S rRNA gene sequences of the other 9 strains of Bacillus were downloaded from the Gen Bank nucleotide sequence database. After all DNA sequences were aligned using the Clustal-W algorithm in the MEGA6.06 biological software, the phylogenetic tree was constructed using the neighbor-joining (NJ) method. The bootstrap value analysis was set to repeat 500 times. The phylogenetic tree is shown in Figure 3 It can be clearly seen that Bacillus KTA2108 and Bacillus amyloliquefaciens W36 are clustered together, with the closest relationship and a genetic distance of 0.003. Therefore, Bacillus KTA2108 was identified from the perspective of molecular biology, and the identification result was Bacillus amyloliquefaciens, which was consistent with the physiological and biochemical identification results.

[0056] Preservation of Bacillus KTA2108: According to the physiological and biochemical identification results and molecular biological identification results of Bacillus KTA2108, it can be finally determined that KTA2108 is Bacillus amyloliquefaciens KTA2108. The fresh bacterial suspension of Bacillus amyloliquefaciens KTA2108 was inoculated into the slant culture medium with an inoculation loop, and placed in a 30°C constant temperature incubator for 24 hours to obtain the slant strain, which was then preserved in the China General Microbiological Culture Collection Center (CGMCC), and the strain preservation number was CGMCC No. 24720.

[0057] Example 3 Screening of spore liquid preservatives

[0058] A large number of studies have shown that surfactants with amphiphilic structures can bind to polar groups on spore coats, thereby inhibiting the germination of spores or directly killing spores, making it impossible for spores to grow in nutrient agar medium and form colonies. At present, based on this principle, related surfactants are mainly used in the fields of food preservatives and disinfectants, such as polylysine salts, thiamine dilauryl sulfate, and dodecyl dimethyl benzyl ammonium chloride.

[0059] Therefore, if the Bacillus preparation is to be used in liquid form, it is necessary to ensure that the Bacillus amyloliquefaciens KTA2108 spores have a high survival rate in the preservative under a room temperature liquid environment, and the spores preserved therein can also germinate normally under suitable conditions.

[0060] Preparation of sporulation medium (Difco sporulation medium, DSM): Weigh 3.2g of tryptone, 1.6g of yeast extract, and 3.2g of NaCl respectively, add 10.0ml of 1.2% (w / v) MgSO4·7H2O, 10.0ml of 10% (w / v) KCl and 0.5ml of 1mol / LNaOH, make the volume to 1000ml, and sterilize at 121℃ for 30min. After sterilization, add 1ml of 0.01mol / L MnCl2, 1mol / L Ca(NO3)2 and 1mmol / L FeSO4 filtered through a microporous membrane per liter of culture medium, and mix well.

[0061] Preparation of Bacillus amyloliquefaciens KTA2108 spores: inoculate fresh KTA2108 bacterial liquid into DSM culture medium at a 10% inoculation rate, culture in a constant temperature shaker at 30°C and 180 r / min for 72 hours, dye with malachite green to ensure that the proportion of sporulation of bacteria in the culture medium exceeds 85%, then centrifuge at 5000 r / min for 10 minutes at room temperature, discard the supernatant, wash the spore precipitate with sterile water 3 times, and set aside.

[0062] Preparation of liquid inoculum of Bacillus amyloliquefaciens KTA2108 using different preservatives: purified water or a single surfactant was selected as a liquid preservative for spores, such as a cationic surfactant dodecyldimethylbenzyl ammonium chloride (1227) [0.02-2 g / L], an anionic surfactant sodium lauryl polyoxyethylene ether sulfate (AES) [0.14-14 g / L], a zwitterionic surfactant tetradecyldimethylammonium oxide (OA-14) [0.1-10 g / L], 3-sulfopropyltetradecyldimethylammonium (STDAB) [0.1-10 g / L] or a nonionic surfactant fatty alcohol polyoxyethylene ether-7 (AEO-7) [0.5-50 g / L], a certain amount of Bacillus amyloliquefaciens KTA2108 spores was added to each preservative to obtain Bacillus amyloliquefaciens KTA2108 spores preserved by different preservatives.

[0063] Screening of components and concentrations of spore liquid preservatives: The number of spores in each preservative solution was measured using the ten-fold gradient dilution method and the pouring method, and the preservation of Bacillus amyloliquefaciens KTA2108 spores in each preservative was monitored within 360 days, as shown in Table 3.

[0064] Table 3 Effect of different concentrations of spore preservative on the spore count of Bacillus amyloliquefaciens KTA2108 (CFU / ml)

[0065]

[0066] Note: 1 1227, dodecyl dimethyl benzyl ammonium chloride; AES, sodium lauryl polyoxyethylene ether sulfate; OA-14, tetradecyl dimethyl ammonium oxide; STDAB, 3-sulfopropyl tetradecyl dimethyl ammonium; AEO-7, fatty alcohol polyoxyethylene ether-7

[0067] As shown in Table 3, after KTA2108 spores were stored in purified water for 360 days, the number of spores decreased from the original 5.5 billion to 785,000, a decrease of nearly 4 orders of magnitude, and the survival rate of spores in water was less than 1%; KTA2108 spores decayed faster in a higher concentration of cationic surfactants than in purified water. After being stored at 0.2 g / L of 1227 for 90 days, the survival rate of spores decreased by 3 orders of magnitude, and the survival rate of spores was less than 0.11%, which was lower than the survival rate of 0.27% in pure water during the same period. It is speculated that the reason may be that 1227 combines with the protein on the spore coat, the spore shell is destroyed, and after the water molecules penetrate into the core layer of the spores, the related enzymes are inactivated, making the spores unable to germinate. However, after KTA2108 spores were stored in AES, OA-14, STDAB and AEO-7 solutions at certain concentrations for one year, the decrease in spores in each preservative did not exceed one order of magnitude. When 0.14-14 g / L AES solution was used as the preservative, the survival rate of KTA2108 spores exceeded 95%; in 1 g / L and 10 g / L OA-14, the spore survival rates were 90.30% and 87.06% respectively; in 10 g / L STDAB solution, the spore survival rate was 92.53%; KTA2108 spores were stored in 0.5 g / L and 5 g / L AEO-7 for 360 days, and the spore survival rates were 97.61% and 95.58% respectively, both exceeding 95%.

[0068] In summary, according to the monitoring results of Bacillus amyloliquefaciens KTA2108 spores in different preservatives, KTA2108 spores can be stored for one year in 0.14g / L, 1.4g / L, 14g / L AES, 1g / L, 10g / L OA-14, 10g / L STDAB, or 0.5g / L, 5g / L AEO-7, with a survival rate of more than 85%, which is an extremely good alternative component and working concentration as a spore liquid preservative.

[0069] Example 4 Synergistic antifungal effect of spore liquid preservative and Bacillus amyloliquefaciens KTA2108

[0070] Confrontation test of Bacillus amyloliquefaciens KTA2108 with preservatives and wood mold, discoloration fungi, and mixed fungi: The confrontation method was used to explore the joint effects of various spore preservatives and Bacillus amyloliquefaciens KTA2108, and to observe whether the combination of the two has a synergistic and synergistic effect on wood mold and discoloration fungi, so as to screen out the best preservative for liquid preservation of Bacillus amyloliquefaciens KTA2108 at room temperature. The antifungal activity of Bacillus amyloliquefaciens liquid preparations prepared with different spore liquid preservatives against various wood molds, discoloration fungi and mixed fungi is shown in Table 4.

[0071] Table 4 Antifungal activity of Bacillus amyloliquefaciens KTA2108 containing spore preservative (inhibition zone diameter / colony diameter)

[0072]

[0073]

[0074] Note: 1 AES, sodium lauryl polyoxyethylene ether sulfate; OA-14, tetradecyl dimethyl ammonium oxide; STDAB, 3-sulfopropyl tetradecyl dimethyl ammonium; AEO-7, fatty alcohol polyoxyethylene ether-7

[0075] 2 Data in the same column with different lowercase letters on the right shoulder indicate significant differences (p<0.05), and data with the same letters or no mark indicate no significant differences (p>0.05)

[0076] As can be seen from Table 4, the spores of Bacillus amyloliquefaciens KTA2108 were used together with purified water or different concentrations of spore liquid preservatives in the test against wood mold, discoloration fungi, and mixed fungi. The effect was better when 1.4 g / L and 14 g / L AES were used in combination with strain KTA2108. Considering the long-term preservation effect of two concentrations of AES on KTA2108 spores and production costs, in the present invention, the Bacillus mildew inhibitor was obtained by adding 1.4 g / L AES to Bacillus amyloliquefaciens KTA2108 spores, where the number of KTA2108 spores was (5.0±1.0)×10 7 CFU / ml.

[0077] Example 5: Application of Bacillus mildew inhibitor on radiata pine wood blocks

[0078] The radiata pine test material was made into a 50 mm (grain length) × 20 mm × 5 mm specimen, and was divided into a control group and a test group horizontally from the middle of the specimen using a marker pen. The upper right corner of the test group was marked with a black dot, such as Figure 4 -A, the specimen was sterilized at 121℃ for 30min. The control end of the sterilized radiata pine specimen was completely immersed in sterile water for 1min, and then removed and placed horizontally for 5min. The test end was then completely immersed in the Bacillus antifungal agent for 1min, removed and placed horizontally for 5min. The radiata pine treated with the reagent was suspended in a sterile plate with two sterilized glass rods (3mm in diameter), and 100ul of a mixed spore suspension of wood mold and discoloration fungi was dripped on both ends of the specimen. The spore count was (7-8)×10 6 / ml, and finally carefully put the plate into a constant temperature and humidity incubator, relative humidity 70%, 28 ℃ culture for 3 days, 5 days and 7 days, observe the anti-mildew effect of Bacillus mildew inhibitor on radiata pine test material for one week. The test results are shown in Figure 4 .

[0079] from Figure 4 It can be seen that, at the control end treated with sterile water, a small amount of mold hyphae appeared on the 3rd day of the test, obvious mold hyphae and plaques appeared on the 5th day, and the distribution area of ​​mold plaques at the control end on the 7th day accounted for more than 3 / 4 of the total area; at the same time, at the test end treated with the Bacillus mildew inhibitor, no hyphae or plaques appeared throughout the one-week test period, proving that the Bacillus mildew inhibitor developed in the present invention has a significant mildew-proof effect on radiata pine.

[0080] Example 6 Application of Bacillus mildew inhibitor on beech wood blocks

[0081] The beech wood specimens were made into 50mm×50mm×50mm specimens. 12 clean and mold-free specimens were completely immersed in a mixed spore suspension of wood mold and discoloration fungi, with a spore count of (7-8)×106 / ml. After being treated in the spore suspension for 1 minute, they were fished out, drained, and randomly divided into 6 groups, with 2 specimens in each group. The grouping was as follows: A0, B0, and C0 groups were applied with purified water, and A1, B1, and C1 were applied with Bacillus mildew inhibitors. The application frequency of A0 and A1 was 3 days / time, B0 and B1 was 5 days / time, and C0 and C1 was 7 days / time. According to the grouping, 5ml of purified water or Bacillus mildew inhibitor was applied to the surface of each group of specimens using a spray bottle. The treated beech wood specimens were suspended in a 500ml beaker with 100ml purified water at the bottom using the wet chamber hanging method. After the beaker was sealed with a breathable sealing film, it was placed in a constant temperature and humidity incubator with a relative humidity of 70% and 28°C. Subsequently, each group of specimens was sprayed with water or mildew inhibitors on time according to the frequency of use designed in the experiment. After 28 days, the mildew prevention effect of Bacillus mildew inhibitors with different usage frequencies on beech wood was observed.

[0082] With reference to the “Test Method for the Effectiveness of Antifungal Agents on Wood Mold and Discoloration Fungi” (GB / T 18261-2013) involving the grading of the surface infection values ​​of the specimens by mold and discoloration fungi, an evaluation table for the mildew infection of the specimens was developed, as shown in Table 5.

[0083] Table 5 Evaluation table of surface infection value of samples by mold and discoloration bacteria

[0084]

[0085] Note: The lower the infection value, the better the anti-mildew effect; "-" means the anti-mildew effect is good; "+" means there is mold on the wood, the more plus signs, the worse the anti-mildew effect

[0086] The moldy beech wood specimens were treated with Bacillus mildew inhibitor at a frequency of 3 days / time, 5 days / time and 7 days / time. After 28 days, the moldy conditions of the beech wood specimens in each group were as follows: Figure 5 to Figure 7According to the mold conditions of each sample and the "Evaluation Table of Surface Infection Values ​​of Samples Infected by Mold and Discoloration Bacteria", the mold prevention effects of Bacillus mildew inhibitors with different usage frequencies on beech wood and the infection values ​​of each group of beech wood specimens are shown in Table 6.

[0087] Table 6 The anti-mildew effect of Bacillus mildew inhibitors with different usage frequencies on moldy beech wood

[0088]

[0089] Depend on Figure 5 to Figure 7 As can be seen from Table 6, for beech wood specimens inoculated with a mixed spore suspension of wood mold and discoloration fungi, Bacillus mildew inhibitor was applied at a frequency of 3 days / time, 5 days / time or 7 days / time. During the 28-day test period, the corresponding beech wood specimens had no obvious mold hyphae or mildew spots, and the infection value of mold and discoloration fungi was 0. When purified water was applied at different frequencies to the beech wood specimens inoculated with wood fungi, a large number of mildew spots appeared in the corresponding specimens, and the infection values ​​were all above 3. At the same time, the infection value increased with the increase of the frequency of purified water application. The reason may be that the frequency of purified water application is too high, which further increases the humidity of the wood, causing the growth rate of mold to accelerate.

[0090] In summary, the anti-mildew results of Bacillus mildew inhibitor on radiata pine or beech are consistent, that is, the anti-mildew effect of Bacillus mildew inhibitor on radiata pine can last for more than 7 days, and the anti-mildew effect on beech wood is similar to that of 3 days / time or 5 days / time when applied 7 days / time. Therefore, in order to save the cost of agents and labor costs, in the actual application process, the frequency of use of Bacillus mildew inhibitor can be set to 7 days / time.

[0091] Example 7 Application of Bacillus mildew inhibitor on wooden wine cabinet

[0092] Two constant temperature wine cabinets of the same model (Haier, WS053A, 497mm×585mm×820mm) were used as the objects, which were set as the control group and the test group respectively. Because the wine cabinet interlayer is made of beech wood, according to the experimental results of Example 6, the wine cabinet was filled with unopened red wine (except the bottom layer), and kept at a constant temperature of 12°C. A small atomizer was placed on the bottom layer, and purified water (control group) or Bacillus mildew inhibitor (test group) was sprayed in the wine cabinet at a frequency of 7 days / time, 5ml / time. In order to accelerate the process of mold infection of wooden interlayers in the environment, before the first 4 times of spraying mildew inhibitors, 5ml of mixed spore suspension with a spore content of about 20 / ml was uniformly inoculated into the wine cabinet, and then purified water or mildew inhibitors were sprayed into the wine cabinet at a normal frequency. After 90 days, the moldy condition of the wooden interlayers in the two groups of wine cabinets was observed.

[0093] In the artificial mold environment, the constant temperature wine cabinet with purified water as the control group has a beech wood partition. Figure 8-A shows that many black mold spots have grown, the infected area is large, and the infection value is judged to be 1; the application effect of Bacillus mildew inhibitor on wooden partitions is shown in Figure 8 -B, it can be seen from the figure that the mildew inhibitor of the present invention can make the wooden partition have an excellent mildew-proof effect. During the 90-day test period, no hyphae or plaque growth was observed throughout the entire process, and the infection value of the wooden partition was 0.

[0094] In summary, the Bacillus mildew inhibitor of the present invention can be well applied to wooden wine cabinets or other wooden furniture, and is a new choice for wood mildew prevention by a healthy, safe and environmentally friendly biological control method.

[0095] Finally, it should be noted that although the above embodiments have been described in the specification and drawings of this application, this does not limit the scope of patent protection of this application. All technical solutions generated by replacing or modifying equivalent structures or equivalent processes based on the essential concept of this application using the contents recorded in the specification and drawings of this application, as well as directly or indirectly implementing the technical solutions of the above embodiments in other related technical fields, are included in the scope of patent protection of this application.

Claims

1. A strain of Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ), which was named KTA2108 and its accession number was CGMCC No.24720.

2. A liquid preservative containing the spores of Bacillus amyloliquefaciens as claimed in claim 1, characterized in that: The spore liquid preservative also contains 0.14-14 g / L of sodium lauryl polyoxyethylene ether sulfate or 0.1-10 g / L of tetradecyl dimethyl ammonium oxide, or 10 g / L of 3-sulfopropyl tetradecyl dimethyl ammonium, or 0.5-5 g / L of fatty alcohol polyoxyethylene ether-7.

3. A mildew-proofing agent, characterized in that: The mildew inhibitor contains 1.4 g / L sodium lauryl alcohol polyoxyethylene ether sulfate and Bacillus amyloliquefaciens KTA2108 with a preservation number of CGMCC No. 24720, and the spore count of Bacillus amyloliquefaciens KTA2108 is 5.0±1.0×10 7 CFU / ml.

4. The use of the mildew inhibitor according to claim 3 on radiata pine and beech wood blocks to prevent mildew, characterized in that: The mold is a wood mold, a wood discoloration fungus, or a combination of wood mold and wood discoloration fungus; the wood mold is Aspergillus niger, Penicillium citrinum, and Trichoderma viride; the wood discoloration fungus is Diplodia theobromin, Fusarium moniliforme, and Alternaria alternata.

5. The anti-mildew application as claimed in claim 4, characterized in that: The anti-mildew agent is sprayed on the wood surface in a spraying manner, and the spraying frequency is once every 7 days.

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