Candida tropicalis and application of preparation thereof in prevention and treatment of tobacco mildew

Abstract

The invention provides application of candida tropicalis and a preparation thereof in prevention and treatment of tobacco mildew, and belongs to the technical field of microorganisms. A strain of candida tropicalis is named as candida tropicalis 2-17, the preservation number of the candida tropicalis 2-17 is CCTCC NO: M 20242184, a microbial preparation prepared from the candida tropicalis can inhibit growth of main mould in tobacco leaves, and the candida tropicalis has good application potential for biological prevention and treatment of tobacco leaf mould.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to application of a tropical Candida and a preparation thereof in preventing and controlling tobacco mildew. Background Art

[0002] In the process of tobacco preparation and processing, especially the preparation of cigars, the production links are complex, there are many processes, and there are many high-humidity environments, which are very conducive to the growth of mold. With the increasing scale of cigar production, the problem of cigar mildew has gradually become prominent, restricting the high-quality development of cigars. The prevention and control of cigar mildew has become a common problem that needs to be solved urgently in the entire industry.

[0003] The main molds that cause cigar mildew include Aspergillus and Penicillium, but there are differences in different regions. The main molds in Hubei production area include Aspergillus, Penicillium, and Blue Fungus, etc.; the main molds in Hainan production area include Blue Fungus, Scopulariopsis, and Aspergillus, etc.; Jinan Cigar Factory reported that the main mold strains are Aspergillus fumigatus and Aspergillus monteri. The moldy cigar samples from various regions were separated and identified, and the main molds identified included: Aspergillus monteri, Aspergillus pseudogrey green, Trichoderma longipes, Aspergillus versicolor, Aspergillus chevalieri, Aspergillus polydophorus, Blue Fungus, Penicillium chrysogenum, etc. Finding antagonistic strains that are effective against these main molds is of great significance for the biological control of cigar mildew.

[0004] The Chinese patent application with the application date of April 28, 2024 and the application publication number CN118497012A discloses a strain of Pichia terricola N-4 and its use in inhibiting the mildew of cigar tobacco leaves. The strain can inhibit the growth of various molds, and spraying the strain on tobacco leaves can also inhibit the mildew of cigar tobacco leaves. However, the types of molds involved are not representative, mainly involving Penicillium citrinum, Aspergillus chevalieri, Aspergillus flocculus, Cladosporium, Aspergillus ochratus, Aspergillus flavus, Aspergillus polydophorus, Rope-shaped blue fungus, Aspergillus fumigatus, Aspergillus niger, Penicillium chrysogenum or Penicillium, and the inhibition rate is between 28.36% and 82.15%, and the inhibition effect on mold is limited. Summary of the invention

[0005] To solve the above problems, the first object of the present invention is to provide a tropical Candida, which is an antagonistic strain effective against the main molds on tobacco leaves and can inhibit the growth of the main molds in cigars.

[0006] The second object of the present invention is to provide a microbial preparation.

[0007] The third object of the present invention is to provide the use of tropical Candida and its preparation in preventing and controlling tobacco mildew.

[0008] To achieve the above object, the present invention provides a strain of Candida tropicalis using the following technical solution:

[0009] A tropical Candida, named as Candida tropicalis 2-17, with a preservation number of CCTCC NO:M 20242184.

[0010] The beneficial effect of the above technical scheme is that a yeast strain was obtained from cigar tobacco leaves by a targeted screening method, and the strain was identified as tropical Candida tropicalis 2-17 through morphological identification and molecular biological identification, and in subsequent analysis experiments, it was found that the Candida tropicalis 2-17 strain can inhibit the growth of major molds in tobacco leaves, thereby inhibiting the mildew of tobacco leaves, thereby achieving effective prevention and control of mildew of tobacco leaves, especially cigar tobacco leaves.

[0011] To achieve the above object, the present invention provides a technical solution adopted by a microbial preparation:

[0012] A microbial preparation comprising Candida tropicalis 2-17.

[0013] The beneficial effect of the above technical scheme is that the microbial preparation prepared by using tropical Candida suspension has antagonistic ability to the main molds of cigar tobacco, and can inhibit the growth of Penicillium chrysogenum, Aspergillus monteritus, Blue Fungus, Aspergillus polydorum, Aspergillus pseudogray green, Aspergillus versicolor, Trichoderma longipes, and Aspergillus shevastatin. Except for Aspergillus versicolor and Penicillium chrysogenum, the growth inhibition rate of other molds is more than 90%.

[0014] To achieve the above object, the present invention provides the use of tropical Candida and its preparation in preventing and controlling tobacco mildew.

[0015] The present invention is a pioneering invention. The beneficial effect of the above technical solution is that tropical Candida and its preparation can effectively inhibit the mildew of tobacco leaves. The surface of the tobacco leaves treated with tropical Candida is bright, without mildew, and has a sweet fragrance when smelled, indicating that the tropical Candida and its preparation can be effectively used for the biological control of cigar tobacco mildew.

[0016] Preferably, the molds include Aspergillus monteritus, Aspergillus pseudoglaucus, Trichoderma longipes, Aspergillus versicolor, Aspergillus shevarium, Aspergillus polydophorus, Blue-shaped funiculus, and Penicillium chrysogenum.

[0017] Preferably, the tobacco leaves are fumigated with volatile substances produced by Candida tropicalis 2-17. The fumigation treatment can achieve contactless prevention and control of mildew, and is more flexible in application. It can be used to fumigate and kill mildew during the drying process of cigar tobacco leaves or in the drying room.

[0018] Preferably, the tobacco leaves comprise cigar tobacco leaves. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a colony morphology diagram of Candida tropicalis in Example 1 of the present invention;

[0020] Figure 2 The phylogenetic tree constructed based on the ITS gene sequence in Example 1 of the present invention;

[0021] Figure 3 This is a graph showing the antibacterial results of Candida tropicalis in Example 3 of the present invention;

[0022] Figure 4 This is a diagram showing the effect of using tropical Candida in Example 4 of the present invention in inhibiting cigar mildew. DETAILED DESCRIPTION

[0023] The invention mainly aims at the technical problem that the prior art cannot effectively inhibit the growth of main molds in tobacco leaves, provides a tropical Candida and its preparation for use in preventing and controlling tobacco mildew, and achieves the inhibition of Aspergillus monteritus, Aspergillus pseudogrey green, Trichoderma longipes, Aspergillus versicolor, Aspergillus shevarium, Aspergillus polydophorus, blue fungus and Penicillium chrysogenum, and the inhibition effect reaches 71.34%-100%.

[0024] The technical solution of the present invention is further described below in conjunction with specific embodiments.

[0025] Unless otherwise specified, the terms used in the present invention are commonly used terms in the relevant field, the technical means used in the embodiments are conventional means well known to those skilled in the art, and the reagents and products used are also commercially available.

[0026] In the following examples, primer sequence synthesis and gene sequencing were provided by Beijing Liuhe BGI Biotechnology Co., Ltd.

[0027] Preparation of YPD solid culture medium: prepare according to the ratio of 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, and 15 g / L agar, and sterilize at 115°C for 30 min.

[0028] Preparation of YPD test tube culture medium: prepare in the following proportions: 10 g / L, 20 g / L peptone, and 20 g / L glucose, and sterilize at 115°C for 30 min.

[0029] Preparation of PDA medium: Select the potato dextrose agar (PDA) medium (Cat. No. 021050) from Guangdong Huankai Microbiological Technology Co., Ltd., configure it at 40.1 g / L, and sterilize it at 115°C for 30 min.

[0030] Biomaterials:

[0031] Cigar tobacco leaves: the main cigar varieties from Hubei cigar producing areas.

[0032] Strains: Aspergillus montenegro, Aspergillus pseudoglaucus, Trichoderma longipes, Aspergillus versicolor, Aspergillus shevarium, Aspergillus polydophora, Blue-spored fungus, and Penicillium chrysogenum were isolated from the surface of cigar leaves from Sichuan China Tobacco, Hubei China Tobacco, Yunnan production areas, and Hainan production areas, and the number accounted for a relatively large proportion.

[0033] Example 1

[0034] This example is the screening and identification of Candida tropicalis, and the specific implementation operations are as follows:

[0035] Take 5.0g of cigar tobacco leaves and add 45mL of sterile PBS buffer, shake at 28℃, 180rpm for 30min, and let stand at room temperature for 1min to obtain the bacterial suspension stock solution. The bacterial suspension stock solution was diluted in 10-fold gradients to obtain a dilution of 10 -1 , 10 -2 The yeast suspension was spread on the YPD solid culture medium plate, and 100 μL of the yeast suspension of different dilutions was taken and spread on the plate. The plates were cultured at 28°C. After purification for 2-3 generations to obtain the strain to be identified, the single strain to be identified was inoculated into the YPD test tube culture medium again, and cultured at 28°C and 180 rpm for 24 h to obtain the seed solution. The seed solution of the strain to be screened was spread on the YPD culture medium to obtain the yeast strain 2-17.

[0036] 1) Morphological identification

[0037] The yeast strain 2-17 obtained by the above screening was inoculated onto a YPD solid culture medium plate and cultured at 28°C for 2 days before observing the colony morphology. Figure 1 As shown, the colonies of yeast strain 2-17 are relatively round, white in color, with neat edges and a relatively moist surface.

[0038] 2) Molecular biological identification

[0039] The yeast strain 2-17 was subjected to ITS sequencing, and the ITS nucleotide sequence of the yeast strain 2-17 was measured as shown in SEQ. No 1. The sequencing results were submitted to the NCBI database for comparison and strain identification. The identification results showed that the ITS sequence of the yeast strain 2-17 was 100% similar to the ITS sequence of Candida tropicalis (MK 409681.1), and a phylogenetic tree was constructed using MEGA 11.0 software. The phylogenetic tree is shown in Figure 2 As shown. Figure 2It can be seen that yeast strain 2-17 belongs to the tropical Candida clade. Combining morphological characteristics and ITS sequence analysis, the yeast strain 2-17 was identified as tropical Candida (Candida tropicalis), named Candida tropicalis 2-17, and was deposited in the China Center for Type Culture Collection (CCTCC) on October 14, 2024, with the deposit number CCTCC NO: M 20242184, and the deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0040] Example 2

[0041] This example is the preparation of Candida tropicalis 2-17 microbial preparation

[0042] A single colony of tropical Candida 2-17 was inoculated into a YPD test tube medium and cultured in a horizontal shaker at 28°C and 180 rpm for 24 h to obtain a seed solution. Subsequently, the inoculation volume was transferred to a YPD liquid medium and cultured for 12 h, and the tropical Candida 2-17 suspension was obtained by diluting with sterile water. The concentration of the suspension was adjusted to 1×10 8 CFU / mL, and obtained the Candida tropicalis 2-17 microbial preparation.

[0043] Example 3

[0044] This example is the application of Candida tropicalis 2-17 in inhibiting the growth of major molds. The specific implementation operations are as follows:

[0045] The plate-pairing method was used to verify the antagonistic ability of tropical Candida 2-17 strain against major molds and to determine the inhibitory effect of tropical Candida 2-17 on molds.

[0046] Take 100 μL of the Candida tropicalis 2-17 microbial preparation prepared in Example 2 and spread it on a YPD solid medium plate. Take another 1 μL of 1×10 6 CFU / mL mold seed liquid was inoculated into the PDA solid culture medium plate, and the inoculated YPD solid culture medium plate and the PDA fixed culture medium plate were buckled and sealed, and cultured at 28°C for 4 days. The diameter d after the mold antagonism was measured and compared with the control diameter D to obtain the inhibition rate, and the inhibition rate (%) = (Dd) / D. Among them, the blank YPD solid culture medium plate was used as the control. The inhibition effect of mold is as follows Figure 3 As shown in the figure, "after antagonism of 2-17" refers to "after fumigation of tropical Candida 2-17 strain", and the inhibition results of different molds are shown in Table 1.

[0047] Table 1 The results of inhibition of tropical Candida against different molds in cigars

[0048]

[0049]

[0050] Note: D represents the growth diameter of the mold in the control group; d represents the growth diameter of the mold in the treatment group.

[0051] From the data in Table 1, it can be seen that tropical Candida 2-17 has a significant inhibitory effect on the growth of major molds such as Penicillium chrysogenum, Aspergillus monteritus, Blue Fungus, Aspergillus polydophorus, Aspergillus pseudogray green, Aspergillus versicolor, Trichoderma longibrachiatum, and Aspergillus shevastatin in cigars. Among them, except for Aspergillus versicolor and Penicillium chrysogenum, the growth inhibition rate of other molds is more than 90%. It can be seen that the tropical Candida 2-17 provided by the present invention has a significant inhibitory effect on the main molds in cigars, and has great application potential for the biological control of cigar mildew.

[0052] Example 4

[0053] This example is the application of Candida tropicalis 2-17 in preventing and controlling cigar mildew. The specific implementation operation is as follows:

[0054] A single colony of tropical Candida 2-17 was inoculated into a YPD test tube medium and cultured in a horizontal shaker at 28°C and 180 rpm for 24 h to obtain a seed solution. The solution was diluted with sterile water to obtain a concentration of 1×10 8 CFU / mL bacterial suspension. 100 μL tropical Candida 2-17 bacterial suspension was spread on YPD solid medium, and the control group used sterile YPD solid medium. Cigar tobacco leaves were sterilized at 121°C for 15 minutes and then placed in a blank sterile culture dish with an infection concentration of 1×10 6 CFU / mL of mold spore suspension, and the control group used sterile YPD solid culture medium and the culture dish with tobacco leaves. The treatment group and the control group were placed at 28℃ for 5 days, and the mildew of cigar tobacco leaves in the treatment group and the control group were compared. The results are as follows: Figure 4 shown.

[0055] Comparing the control group with the experimental group, it can be seen that the cigar tobacco leaves in the control group are obviously moldy and have a musty smell; the surface of the cigar tobacco leaves in the treatment group is bright, without mold, and has a sweet fragrance. This shows that the fumigation treatment of tropical Candida 2-17 can significantly inhibit the mildew of cigar tobacco leaves. Therefore, the tropical Candida 2-17 provided by the embodiment of the present invention can effectively inhibit the mildew of cigar tobacco leaves.

[0056] Therefore, the embodiment of the present invention obtains a tropical Candida tropicalis 2-17 through a targeted screening method. The tropical Candida tropicalis 2-17 strain can inhibit the growth of major molds such as Penicillium chrysogenum, Aspergillus monteritus, Blue Fungus, Aspergillus polydophorus, Aspergillus pseudogray green, Aspergillus versicolor, Trichoderma longipes, and Aspergillus shevarium in cigars, and has a more obvious inhibitory effect on the main molds in cigars; and can inhibit the mildew of cigars.

[0057] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, ordinary technicians in the relevant field should understand that the specific implementation methods of the present invention can still be modified or some technical features can be replaced by equivalents without departing from the spirit of the technical solution of the present invention, which should be included in the scope of the technical solution for protection of the present invention.

Claims

1. A strain of Candida tropicalis, characterized in that Candida tropicalis Candida tropicalis 2-17, the accession number is CCTCC NO: M 20242184.

2. A microbial preparation, characterized in that: Including the tropical Candida Candida tropicalis 2-17.

3. Use of the tropical Candida albicans according to claim 1 and the microbial preparation according to claim 2 in preventing and controlling tobacco mildew.

4. The use according to claim 3, characterized in that: Molds include Aspergillus monteritus, Aspergillus pseudoglaucus, Trichoderma longiflorum, Aspergillus versicolor, Aspergillus shevarium, Aspergillus polydophorus, Blue-shaped fungus, and Penicillium chrysogenum.

5. The use according to claim 3, characterized in that: Using the tropical yeast Candida tropicalis The volatile substances produced by 2-17 are used to fumigate tobacco leaves.

6. The use according to claim 5, characterized in that: The tobacco leaves include cigar tobacco leaves.

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