Yeast strain and application thereof

Abstract

The invention provides a yeast strain and application thereof. The yeast strain is named as Starmerella bacillaris G17515, and the yeast (Starmerella bacillaris) G17515 is used to inhibit the growth of watermelon wilt pathogen, longan scorch rot pathogen, pear anthracnose pathogen, pear ring rot pathogen, pear black spot pathogen, loquat anthracnose pathogen and grape gray mold pathogen. The yeast fermentation liquid of the invention has an inhibitory effect on watermelon wilt pathogen, longan scorch rot pathogen, pear anthracnose pathogen, pear ring rot pathogen, pear black spot pathogen, loquat anthracnose pathogen and grape gray mold pathogen, and has a good prevention and control effect on grape gray mold.

Description

[Technical field]

[0001] The invention relates to a yeast strain and application thereof. [Background technology]

[0002] Grape gray mold is caused by infection by Botrytis cinerea. It is a devastating disease in the storage process of grapes, which can cause up to 50% post-harvest losses of grapes each year (Zhang Di et al., 2017). Grape gray mold mainly harms inflorescences and fruits, and sometimes leaves and new shoots. In the early stage of inflorescence infection, light brown water-soaked lesions appear in the infected parts, which gradually darken, and the diseased tissue rots softly. When the outside is humid, a gray mold layer appears on the surface. The fruit is infected when it is close to maturity, and light brown spots appear in the early stage, which then gradually expand, and brown sunken lesions appear. Cracks appear on the skin, and the fruit quickly rots and grows a mouse-gray mold layer (Zhang Wei et al., 2019). At present, the prevention and control of grape gray mold is still mainly based on chemical fungicides, but the long-term use of chemical fungicides is not only easy to produce drug resistance, but also pesticide residues lead to environmental pollution and threaten human health. Green control technologies such as ecological control and biological control use methods to achieve safe production and reduce the use of chemical pesticides, thereby constructing environmentally friendly biological control measures (Ke Yang et al., 2017; Smilanick et al., 2010; Chen et al., 2016). With the increasing attention of consumers to pesticide residues in food and environmental safety, the demand for developing alternative methods of disease control is also increasing (Rosslenbroich et al., 2000; Romanazzi et al., 2011). Therefore, researching and finding effective and safe biological control technologies and methods for grape gray mold is one of the important directions to solve the green cultivation of grapes. [Summary of the invention]

[0003] The technical problem to be solved by the present invention is to provide a yeast strain and application thereof.

[0004] The present invention is achieved in that:

[0005] A yeast strain, the yeast strain is named yeast (Starmerella bacillaris) G17515, the yeast (Starmerella bacillaris) G17515, on November 03, 2021, deposited in the General Microbiological Center of China Microorganism Culture Collection Administration, the address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 23713

[0006] Furthermore, the yeast (Starmerella bacillaris) G17515 is used to inhibit the growth of watermelon wilt pathogen, longan scorch rot pathogen, pear anthracnose pathogen, pear ring rot pathogen, pear black spot pathogen, loquat anthracnose pathogen and grape gray mold pathogen.

[0007] Furthermore, the yeast (Starmerella bacillaris) G17515 is used for preventing and treating grape gray mold.

[0008] The present invention has the following advantages:

[0009] The yeast (Starmerella bacillaris) G17515 fermentation liquid has an inhibitory effect on watermelon wilt pathogen, longan scorch rot pathogen, pear anthracnose pathogen, pear ring spot pathogen, pear black spot pathogen, loquat anthracnose pathogen and grape gray mold pathogen, and has a good control effect on grape gray mold, providing a strain screening source for biological control of grape gray mold.

Brief Description of the Drawings

[0010] The present invention will be further described below in conjunction with embodiments with reference to the accompanying drawings.

[0011] Figure 1 1 is a colony and bacterial morphology characteristic diagram of strain G17515 in the embodiment of the present invention. [Specific implementation method]

[0012] The present invention relates to a yeast strain, the yeast strain is named yeast (Starmerella bacillaris) G17515, and the yeast (Starmerella bacillaris) G17515 is deposited in the General Microbiological Center of China Microorganism Culture Collection Administration on November 03, 2021, with the address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 23713

[0013] The invention also relates to one of the applications of yeast strains. The yeast (Starmerella bacillaris) G17515 is used for inhibiting the growth of watermelon wilt pathogen, longan scorch rot pathogen, pear anthracnose pathogen, pear ring spot pathogen, pear black spot pathogen, loquat anthracnose pathogen and grape gray mold pathogen.

[0014] The present invention also relates to the second application of the yeast strain, wherein the yeast (Starmerella bacillaris) G17515 is used for preventing and treating grape gray mold.

[0015] The technical scheme of the present invention will be clearly and completely described below in conjunction with the accompanying drawings and specific embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0016] Example

[0017] The yeast strain (Starmerella bacillaris) G17515 of the invention is separated and screened from grape fruits in the vineyard of Yannong Ecological Agriculture Co., Ltd. in Mingxi County, Sanming City, Fujian Province, and a strain with antagonistic effect is obtained. The fermentation liquid of the yeast strain G17515 has good inhibitory effects on watermelon wilt pathogen, longan scorch rot pathogen, pear anthracnose pathogen, pear ring rot pathogen, pear black spot pathogen, loquat anthracnose pathogen and grape gray mold pathogen, and has good prevention and control effect on banana anthracnose.

[0018] 1. Isolation and screening of yeast strain G17515

[0019] (1) Fresh grapes picked from the vineyard of Yannong Ecological Agriculture Co., Ltd. in Mingxi County, Sanming City, Fujian Province were fermented at 28°C. Samples were taken at the early (7d), middle (15d) and late (25d) stages of fermentation, for a total of 15 fermentation samples.

[0020] (2) Dilute with sterile water, spread 100 μL evenly on a YEPD plate, incubate at 28°C for 2-3 days, and observe the colony morphology;

[0021] (3) Each strain obtained by culture in step (2) was inoculated into YEPD medium and cultured at a temperature of 28°C and a rotation speed of 180 r / min -1 The culture was carried out under the same conditions for 48 h, and the culture fluid was collected;

[0022] (4) The test pathogen, Botrytis cinerea, was used to screen the strains in the collected culture solution by the inhibition rate method. Specifically, the cultured bacterial solution was mixed with the culture medium, and the plate was inverted for later use. A 6 mm Botrytis cinerea cake was placed in the center of the mixed plate. After culturing in a 25°C incubator for 6 days, the diameter of the cake was measured by the cross method, and the antagonistic strains were screened by calculating the inhibition rate. Strain G17515 had the best inhibitory effect on Botrytis cinerea.

[0023] The components of YEPD medium include: 10 g yeast extract powder, 20 g peptone, 20 g glucose, 1000 mL water, pH 6.0.

[0024] Inhibition rate of different yeasts against Botrytis cinerea

[0025]

[0026] 2. Identification of Yeast Strain G17515

[0027] (1) Morphological identification

[0028] The main morphology of yeast strain G17515 was observed, and the main morphology of the strain was as follows: the colonies were round, opaque, milky white, and the single colonies were flat, with smooth surfaces and regular edges. The bacterial morphology of strain G17515 was observed under scanning electron microscopy, and the cells were rod-shaped and 1.3-2.0 μm×2.0-5.0 μm in size.

[0029] (2) Sequencing and analysis of the 26S rDNA D1 / D2 region of yeast strain G17515

[0030] The genomic DNA of the yeast strain G17515 was extracted according to the instructions in the kit of Shanghai Jierui Bioengineering Co., Ltd. Shanghai Boshang Biotechnology Co., Ltd. was commissioned to synthesize:

[0031] Forward primer: 5'-GCATATCAATAA GCGGAGGAAAAG-3' (as shown in SEQ ID NO. 1);

[0032] Reverse primer: 5'-GGTCCGTGTT TCAAGACGGG-3' (as shown in SEQ ID NO. 2).

[0033] The genomic DNA of the yeast strain G17515 obtained by extraction was used as a template, and the above primers were used as primer pairs to PCR amplify the full-length sequence of its 26S rDNA D1 / D2 region:

[0034] PCR reaction system (25 μL): 2.5 μL 10× Buffer, 0.5 μL 10 mM dNTP, 1 μL forward primer, 1 μL reverse primer, 0.3 μL (5 U / μL) Taq enzyme and 1 μL DNA template;

[0035] PCR reaction program: pre-denaturation at 95°C for 5 min; then denaturation at 94°C for 1 min, annealing at 52°C for 1 min, extension at 72°C for 1 min 20 s, for a total of 35 cycles; finally extension at 72°C for 10 min.

[0036] PCR product detection and sequencing analysis: Take 2 μL of PCR product, spot it on 1.5% agarose gel, use 100 bp Marker as standard molecular weight, 100 V voltage, electrophoresis for 30 min, EB staining, and perform gel electrophoresis separation and inspection. The PCR product is entrusted to Shanghai Boshang Biotechnology Co., Ltd. for sequencing, and the obtained sequence is shown in SEQ ID NO. 3. After comparing and analyzing the 26S rDNA D1 / D2 region sequence of strain G17515 on the NCBI website https: / / www.ncbi.nlm.nih.gov / , it is confirmed that strain G17515 belongs to yeast (Starmerella bacillaris) in taxonomy.

[0037] Based on the above 26S rDNA D1 / D2 region sequence determination and analysis combined with morphological, physiological and biochemical characteristics, it was finally determined that strain G17515 belonged to the yeast (Starmerella bacillaris) strain and was named yeast (Starmerella bacillaris) G17515.

[0038] 3. Determination of the inhibitory effect of the fermentation broth of yeast strain G17515 on watermelon wilt, longan scorch rot, pear anthracnose, pear ring spot, pear black spot, loquat anthracnose and grape gray mold

[0039] (1) Activation of strains: streak yeast strain G17515 on YEPD medium using an inoculation loop and culture in a constant temperature incubator at 28°C for 48 h;

[0040] (2) Preparation of seed solution: A single colony of yeast G17515 obtained in step (1) was inoculated into 100 ml of YEPD medium (the components of the YEPD liquid medium: 10 g yeast extract powder, 20 g peptone, 20 g glucose, 1000 mL water, pH 6.0), and placed in a constant temperature shaking incubator for 48 h at 28° C. and 180 rpm / min.

[0041] (3) Preparing liquid fermentation broth: transferring the seed liquid obtained in step (2) into sterilized YEPD medium at a transfer rate of 10%, culturing at a temperature of 28° C. and a rotation speed of 180 rpm / min for 48 h to obtain yeast G17515 fermentation broth;

[0042] (4) Watermelon wilt pathogen, longan scorch rot pathogen, pear anthracnose pathogen, pear ring spot pathogen, pear black spot pathogen, loquat anthracnose pathogen and grape gray mold pathogen were inoculated on PDA medium and cultured for 7 days. After the mycelium covered the plate, a 6 mm punch was used to punch holes to take out the bacterial cake. 3 ml of yeast G17515 fermentation liquid was added to 30 mL PDA medium (the PDA medium components: potato dextrose agar medium 46 g, water 1000 mL, pH 7.2), mixed and prepared into a plate containing antagonistic bacteria, and the plate was plated with equal amounts of 5% ethanol and 1% ethanol. 6 mm watermelon wilt pathogen, longan scorch rot pathogen, pear anthracnose pathogen, pear ring rot pathogen, pear black spot pathogen, loquat anthracnose pathogen and grape gray mold pathogen were cultured on the plate at 25°C for 5 days, and the diameter of the bacterial cake was determined by the cross method. The antibacterial rate of the fermentation liquid of the yeast strain G17515 on watermelon wilt pathogen, longan scorch rot pathogen, pear anthracnose pathogen, pear ring rot pathogen, pear black spot pathogen, loquat anthracnose pathogen and grape gray mold pathogen was 38.76%-87.44%, as shown in the following table:

[0043] Pathogens Antibacterial rate / % Fusarium wilt 38.76±0.81 Longan scorch rot pathogen 79.24±1.22 Colletotrichum sphaeroides 82.45±0.49 Pear ring spot 77.13±1.39 Alternaria obesa 73.00±0.52 Loquat Anthracnose 53.75±1.06 Botrytis cinerea 87.44±0.19

[0044] 4. Determination of the control effect of yeast strain G17515 fermentation broth on grape gray mold

[0045] Select grape leaves of similar size and shape, rinse them with sterile water three times and dry them, use a sterile needle to pierce two wounds (3 mm × 3 mm) on both sides of the back of the leaves, apply a 6 mm grape gray mold cake to the pierced position, incubate for 24 hours, and spray the fermentation liquid of G17515 and G31887 (spore count is 10 8 ), spraying YEPD medium as control, 28 ℃, relative humidity 80%-90% moisturizing culture for 2 days and 5 days, investigate the disease situation of leaves, and measure the diameter of lesions. Set 3 replicates, each replicate 5 leaves.

[0046] Control effect (%) = (incidence of control group - incidence of treatment group) / incidence of control group × 100

[0047] The wound of grape leaves was inoculated with Botrytis cinerea for 24 hours, and then the fermentation liquid of yeast strain G17515 was sprayed, which could significantly inhibit the growth of Botrytis cinerea hyphae. After 5 days of spraying, the diameter of the lesions in the control group was 12.93 mm, and the diameter of the lesions in the treatment group was significantly smaller than that in the control group, which was 6.80 mm respectively. After 2 days and 5 days of cultivation, the incidence rate of the control group was 70% and 83.33%, and after spraying the fermentation liquid of strain G17515, the incidence rate was 13.33%; the control effect after spraying the fermentation liquid of strain G17515 for 5 days was 84.03%, as shown in the following table.

[0048]

[0049] In summary, the present invention provides a new yeast strain G17515, the fermentation liquid of the yeast strain G17515 has inhibitory effects on watermelon wilt pathogen, longan scorch rot pathogen, pear anthracnose pathogen, pear ring rot pathogen, pear black spot pathogen, loquat anthracnose pathogen and grape gray mold pathogen, and has good control effect on grape gray mold, providing a strain screening source for biological control of grape gray mold.

[0050] Although the specific implementation modes of the present invention are described above, those skilled in the art should understand that the specific implementation modes described are only illustrative and are not intended to limit the scope of the present invention. Equivalent modifications and changes made by those skilled in the art in accordance with the spirit of the present invention should be included in the scope of protection of the claims of the present invention.

Claims

1. A yeast strain, characterized in that: The yeast strain was named Saccharomyces Starmerella bacillaris G17515, the yeast Starmerella bacillaris G17515 was deposited on November 3, 2021 at the General Microbiology Center of China Culture Collection Administration, located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No.23713.

2. A use of the yeast strain according to claim 1, characterized in that: The yeast Starmerella bacillaris G17515 is used for the prevention and treatment of grape gray mold.

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