A biocontrol yeast strain and its application

Abstract

The present invention provides a biocontrol yeast strain and an application thereof. The yeast strain is named Meyerozyma caribbica G31887. The Meyerozyma caribbica G31887 was deposited in the General Microbiology Center of China Culture Collection Administration on November 3, 2021, with the address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number being CGMCC No. 23714. The Meyerozyma caribbica G31887 can grow normally under the conditions of a temperature of 10°C-50°C, a pH of 3-11, and the addition of 0.1%-10% NaCl to the culture medium. The yeast fermentation liquid of the present invention 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, has certain temperature, acid, alkali and salt tolerance, and has a good control effect on grape gray mold, providing a strain screening source for biological control of grape gray mold.

Description

Technical field

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

[0002] Caused by Botrytis cinerea, grape gray mold is a devastating disease of grapes during storage, causing up to 50% post-harvest losses annually (Zhang Di et al., 2017). It primarily attacks inflorescences and fruit, sometimes also affecting leaves and new shoots. Initially, the inflorescence develops light brown, water-soaked lesions, which gradually darken, causing soft rot in the affected tissue. When exposed to moisture, a gray mold coat forms on the surface. Fruit becomes infected near maturity, initially developing light brown spots that gradually expand into sunken brown lesions. Cracks develop on the skin, causing rapid rot and the growth of a mouse-gray mold coat (Zhang Wei et al., 2019). Currently, the primary control method for grape gray mold is chemical fungicides. However, long-term use of chemical fungicides not only easily leads to resistance, but also pesticide residues that pollute the environment and threaten human health. Green control technologies, such as ecological control and biological control, aim to achieve safe production and reduce the use of chemical pesticides, thereby establishing environmentally friendly biological control measures (Ke Yang et al., 2017; Smilanick et al., 2010; Chen et al., 2016). With increasing consumer concern about pesticide residues in food and environmental safety, the demand for alternative disease control methods is also increasing (Rosslenbroich et al., 2000; Romanazzi et al., 2011). Therefore, researching and identifying effective and safe biological control technologies and methods for gray mold in O. otao is an important direction for green grape cultivation. [Summary of the invention]

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

[0004] The present invention is achieved in that:

[0005] A biocontrol yeast strain, the yeast strain is named Meyerozyma caribbica G31887, and the Meyerozyma caribbica G31887 was deposited in the General Microbiology Center of the China Culture Collection Administration on November 3, 2021, with the address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 23714.

[0006] Furthermore, the Meyerozyma caribbica G31887 can grow normally under the conditions of a temperature of 10° C.-50° C., a pH of 3-11, and the addition of 0.1%-10% NaCl to the culture medium.

[0007] Furthermore, the Meyerozyma caribbica G31887 is used to inhibit 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.

[0008] Furthermore, the Meyerozyma caribbica G31887 is used for preventing and treating grape gray mold.

[0009] The present invention has the following advantages:

[0010] The fermentation broth of the Meyerozyma caribbica G31887 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, has certain temperature, acid, alkali and salt tolerance, and has a good control effect on grape gray mold, providing a source for strain screening for biological control of grape gray mold.

Brief Description of the Drawings

[0011] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0012] Figure 1 Graph showing the colony and bacterial morphology of strain G31887 in the examples of the present invention. [Specific implementation method]

[0013] The present invention relates to a biocontrol yeast strain, which is named Meyerozyma caribbica G31887. The Meyerozyma caribbica G31887 was deposited in the General Microbiology Center of the China Culture Collection Administration on November 3, 2021, with the address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 23714.

[0014] The Meyerozyma caribbica G31887 can grow normally under the conditions of a temperature of 10° C.-50° C., a pH of 3-11, and a culture medium containing 0.1%-10% NaCl.

[0015] The present invention relates to one of the applications of the above-mentioned biocontrol yeast strain. The Meyerozyma caribbica G31887 is used to inhibit 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.

[0016] The present invention relates to the second application of the above-mentioned biocontrol yeast strain, wherein the Meyerozyma caribbica G31887 is used for preventing and controlling grape gray mold.

[0017] The technical solution of the present invention will be clearly and completely described below in conjunction with the accompanying drawings and specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0018] Example

[0019] The Meyerozyma caribbica G31887 yeast strain of the present invention is isolated and screened from Jianyang grape fruits in Nanping City, Fujian Province to obtain a strain with antagonistic effect. The fermentation liquid of the Meyerozyma caribbica G31887 yeast strain has a good 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, has a certain temperature, acid, alkali and salt tolerance, and has a good prevention and control effect on banana anthracnose.

[0020] 1. Isolation and Screening of Yeast Strain G31887

[0021] (1) The grape skins were collected from the vineyards in Jianyang District, Nanping City, Fujian Province. 9 mL of sterile water was added and the skins were gently ground. After sufficient shaking, 1 mL was drawn for gradient dilution. The dilution degree was 10 -4 , 10 -5 or 10 -6 dilution

[0022] (2) Take 100 μL and evenly spread it on a YEPD plate, incubate it at 28°C for 2-3 days, and observe the colony morphology;

[0023] (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 -1Conditions were used for 48 h, and the culture fluid was collected;

[0024] (4) Antagonistic screening of strains from the collected culture medium was performed using the test pathogen, Botrytis cinerea, using the inhibition rate method. Specifically, the cultured bacterial solution was added to the culture medium and mixed evenly, then 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 using the cross-cross method, and antagonistic strains were screened by calculating the inhibition rate. Strain G31887 had the best inhibitory effect on Botrytis cinerea.

[0025] Inhibition rate of different yeasts against grape gray mold

[0026]

[0027] The components of the YEPD medium are: 10 g yeast extract powder, 20 g peptone, 20 g glucose, 1000 mL water, pH 6.0.

[0028] 2. Identification of Yeast Strain G31887

[0029] (1) Morphological identification

[0030] The main morphological observation of yeast strain G31887 showed that the main morphology of the strain was as follows: the colonies were round, opaque, white, and the single colonies were spherical and convex, with smooth surfaces and regular edges. Scanning electron microscopy observation of the bacterial morphology of strain G31887 showed that the cells were oval and 1.5-2.0 μm × 1.7-3.0 μm in size. Figure 1 shown.

[0031] (2) Sequencing and analysis of the 26S rDNA D1 / D2 region of yeast strain G31887

[0032] The genomic DNA of the yeast strain G31887 was extracted according to the instructions of the kit provided by Shanghai Jierui Bioengineering Co., Ltd. The genomic DNA was synthesized by Shanghai Boshang Biotechnology Co., Ltd.

[0033] Forward primer: 5'-GCATATCAATAA GCGGAGGAAAAG-3' (as shown in SEQ ID NO. 1);

[0034] Reverse primer: 5'-GGTCCGTGTT TCAAGACGGG-3' (as shown in SEQ ID NO. 2).

[0035] The genomic DNA of the yeast strain G31887 was used as a template, and the above primers were used as primers to amplify the full-length sequence of the 26S rDNA D1 / D2 region:

[0036] 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;

[0037] 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.

[0038] PCR product detection and sequencing analysis: 2 μL of PCR product was spotted on a 1.5% agarose gel and separated by gel electrophoresis using a 100 bp marker as the standard molecular weight, at 100 V for 30 min, and stained with EB. The PCR product was sequenced by Shanghai Boshang Biotechnology Co., Ltd. The resulting sequence is shown in SEQ ID NO. 3. After alignment analysis of the 26S rDNA D1 / D2 region sequence of strain G31887 on the NCBI website https: / / www.ncbi.nlm.nih.gov / , strain G31887 was confirmed to belong to the yeast Meyerozyma calibbica.

[0039] 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 G31887 belonged to a strain of Meyerozyma caribbica and was named Meyerozyma caribbica G31887.

[0040] 3. Determination of the inhibitory effect of the fermentation broth of Kalibikerella meyerii strain G31887 on watermelon wilt, longan scorch rot, pear anthracnose, pear ring spot, pear black spot, loquat anthracnose and grape gray mold

[0041] (1) Strain activation: Streak the Mycobacterium californicum G31887 on YEPD medium using an inoculation loop and culture in a constant temperature incubator at 28°C for 48 h;

[0042] (2) Preparation of seed solution: A single colony of the Mycobacterium kalibikerii G31887 obtained in step (1) was inoculated into 100 ml of YEPD medium (the YEPD liquid medium comprises: 10 g yeast extract powder, 20 g peptone, 20 g glucose, 1000 mL water, pH 6.0), and cultured in a constant temperature shaking incubator at 28° C. and 180 rpm / min for 48 h.

[0043] (3) Preparing a liquid fermentation broth: transferring the seed solution obtained in step (2) into sterilized YEPD medium at a transfer rate of 10% at a temperature of 28° C. and a rotation speed of 180 rpm / min for 48 h to obtain a fermentation broth of Mycobacterium kalibikerii G31887;

[0044] (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 6mm punch was used to punch out the bacterial cake. 3 ml of the fermentation broth of Kalibikerella meyerii G31887 was taken and added to 30 mL of PDA medium (components of the PDA medium: 46 g potato dextrose agar medium, 1000 mL water, pH 7.2) was mixed and prepared into plates containing antagonistic bacteria. An equal amount of sterile water was used as a control. 6 mm cakes 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 were plated on the plates. After incubation at 25°C for 5 days, the diameter of the cakes was measured using the cross-hatch method. The fermentation broth of the Kalibiker yeast strain G31887 showed an inhibition rate of 36.58% to 87.01% against different pathogens. The inhibitory effect of the fermentation broth of the Kalibiker yeast strain G31887 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 is shown in the following table:

[0045] pathogens Antibacterial rate / % Watermelon wilt pathogen 84.16±0.68 Longan scorch rot pathogen 58.31±1.18 Pear Anthracnose 86.01±0.73 Pear ring rot pathogen 87.01±0.28 Pear black spot fungus 60.80±1.80 Loquat Anthracnose 36.58±1.12 Botrytis cinerea 83.15±0.64

[0046] 4. Determination of the temperature, acid, alkali and salt tolerance of Kalibikerella meyerii strain G31887

[0047] Effect of Temperature on Strain G31887: Overnight yeast fermentation broth was added to YEPD medium at a 1% inoculum level and incubated at 10°C, 20°C, 30°C, 40°C, and 50°C with shaking at 180 rpm for 24 hours. The medium was then removed and the absorbance was measured at 600 nm using a UV spectrophotometer. Three replicates were performed for each treatment, as shown in the table below. The yeast strain maintained stable growth at temperatures between 10°C and 50°C, with maximum growth at 30°C, reaching an OD600 of 1.82.

[0048] Temperature 10 20 30 40 50 <![CDATA[OD 600 Value]]> 0.23±0.03 1.77±0.05 1.82±0.01 1.35±0.09 0.12±0.01

[0049] Effect of pH on Strain G31887: The pH of YEPD liquid medium was adjusted to 1, 3, 5, 7, 9, and 11, respectively. Overnight yeast fermentation broth was added to the medium at different pH values at a 1% inoculum. The culture was shaken at 30°C, 180 rpm, and incubated for 24 hours. The absorbance was measured at 600 nm using a UV spectrophotometer. Three replicates were performed for each treatment, as shown in the table below. Strain G31887 exhibited strong tolerance to various pH values, maintaining its ability to grow at pH values between 3 and 11. At pH 11, the OD600 of strain G31887 was 1.27.

[0050] pH 1 3 5 7 9 11 <![CDATA[OD 600 Value]]> 0.07±0.01 1.48±0.04 1.65±0.01 1.69±0.05 1.60±0.02 1.27±0.06

[0051] Salt tolerance of strain G31887: Overnight yeast fermentation broth was added at a 1% inoculum to YEPD medium supplemented with NaCl at various concentrations, including 0.1%, 0.5%, 1.0%, 2.0%, 5.0%, 10%, and 20%. YEPD medium was used as a control. Cultures were shaken at 180 rpm and 30°C for 24 hours. The absorbance was measured at 600 nm using a UV spectrophotometer. Three replicates were used for each treatment, as shown in the table below. Strain G31887 grew in the presence of 0.1% to 10% NaCl, with the highest growth observed when 0.1% NaCl was added, resulting in an OD600 of 1.96.

[0052]

[0053] 5. Determination of the Control Effect of Fermentation Broth of Kalibikerella Meyerii Strain G31887 on Grape Botrytis Cinerea

[0054] Select grape leaves of similar size and shape, rinse them three times with sterile water and dry them in the air. Use a sterile needle to prick two wounds (3 mm × 3 mm) on both sides of the back of the leaves. Apply a 6 mm cake of grape gray mold to the punctured area. After culturing for 24 hours, spray G31887 and the fermentation liquid of G31887 (the spore count is 10 8 ) with spraying of YEPD medium as a control. After culturing at 28°C and 80%-90% relative humidity for 2 and 5 days, the leaves were examined for disease and the diameter of the lesions was measured. Three replicates were performed, with five leaves in each replicate, as shown in the table below.

[0055] Control effect (%) = (incidence of control group - incidence of treatment group) / incidence of control group × 100

[0056] When grape leaf wounds were inoculated with Botrytis cinerea 24 hours after application, spraying the fermentation broth of the yeast strain G31887 with the yeast strain Callibixima significantly inhibited the growth of Botrytis cinerea mycelium. Five days after spraying, the lesion diameter in the control group was 12.93 mm, while that in the treated groups was significantly smaller, at 7.04 mm. After two and five days of incubation, the control group experienced a disease incidence rate of 70% and 83.33%, respectively. After spraying with the fermentation broth of strain G31887, the disease incidence rates were 23.33% and 33.33%, respectively. The control efficacy reached 60.00% after five days of spraying with the fermentation broth of strain G31887.

[0057]

[0058] In summary, the present invention provides a new fermentation broth of Meyerozyma caribbica G31887. The fermentation broth of the Meyerozyma caribbica strain G31887 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, has a certain tolerance to temperature, acid, alkali and salt, and has a good control effect on grape gray mold, providing a strain screening source for biological control of grape gray mold.

[0059] Although the specific embodiments of the present invention are described above, those skilled in the art should understand that the specific embodiments described are merely 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 within the scope of protection of the claims of the present invention.

Claims

1. A biocontrol yeast strain, characterized in that: The yeast strain was named Meyer yeast ( Meyerozyma caribbica ) G31887, the Kalibiker Mayer yeast ( Meyerozyma caribbica )G31887 was deposited on November 3, 2021 at the General Microbiology Center of the China Culture Collection Administration, located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 23714.

2. A use of the biocontrol yeast strain according to claim 1, characterized in that: Meyer yeast (Meyerozyma caribbica )G31887 is used to inhibit the growth of watermelon wilt fungus, longan scorch rot fungus, pear anthracnose fungus, pear ring rot fungus, pear black spot fungus, loquat anthracnose fungus and grape gray mold fungus.

3. A use of the biocontrol yeast strain according to claim 1, characterized in that: The Kalibiker yeast ( Meyerozyma caribbica )G31887 is used for the prevention and treatment of grape gray mold.

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