Metschhia pastoris strain for delaying early leaf falling of pears and application of Metschhia pastoris strain

Abstract

The invention discloses a Mitschhia pastoris strain for delaying early leaf falling of pears and application of the Mitschhia pastoris strain, and belongs to the technical field of microorganisms. The preservation number of the strain is CGMCC (China General Microbiological Culture Collection Center) NO.31969, and the preservation date is September 13, 2024. The invention has the following beneficial effects: the provided metschhia pastoris is separated from the surface of healthy pear fruit, is harmless to human body, has high safety, has strong bacteriostatic activity, and can effectively inhibit the growth of pear early leaf falling pathogenic bacteria such as fruit-growing anthracnose, alternaria alternata and clostridium microneoshell. The plant senescence retardant overcomes the defects of an existing plant growth regulator, and the application range of the plant senescence retardant is widened.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and in particular to a Maggi yeast strain capable of delaying early leaf drop in pears and an application thereof. Background Art

[0002] Early leaf drop in pears is primarily caused by Anthracnose, Alternaria alternata, Fusarium ovale, and Botrytis cinerea. This disease causes premature leaf and fruit drop, leading to a second bloom and fruiting in the fall, severely impacting pear yield and quality the following year. Production primarily relies on pesticides for control. However, the long-term and extensive use of these pesticides increases pathogen resistance and results in significant pesticide residues, posing a significant threat to the sustainable development of the pear industry. Therefore, developing effective, non-toxic methods for controlling fruit tree diseases has become a research hotspot.

[0003] Studies have confirmed that the use of antagonistic yeasts can effectively reduce the occurrence of pests and diseases. For example, Chinese patent application publication number CN112342147A discloses a strain of Pichia guilliermondii that can antagonize multiple pathogens and its use. It was found that Pichia guilliermondii can antagonize the growth of Alternaria and Desmodium molds, reducing the occurrence of fruit and vegetable diseases. Chinese patent application publication number CN108118004A discloses a strain of Pichia cacti that can effectively prevent and treat post-harvest fruit diseases and its preparation and use methods. It was found that Pichia cacti strain BY35 can inhibit the occurrence of blue mold, gray mold, and anthracnose in pear fruit, as well as citrus blue mold. Yeasts are mainly distributed in acidic environments with high sugar content, especially in fruits with a relatively rich variety of yeast species. Although the yeasts that have been discovered can effectively inhibit the occurrence of fruit and vegetable diseases, there are no reports of yeast strains that can antagonize the pathogens of pear early leaf drop disease. Summary of the Invention

[0004] The technical problem to be solved by the present invention is how to provide a yeast strain that can antagonize pear early leaf fall pathogens and screen more efficient and broad-spectrum antagonistic yeasts to make up for the problem that the existing antagonistic yeasts have a narrow antibacterial range.

[0005] The present invention solves the above technical problems through the following technical means:

[0006] The first aspect of the present invention provides a Maggi yeast strain, whose deposit number is CGMCC NO.31969 and the deposit date is September 13, 2024.

[0007] The Maggi Meichi yeast strain screened in this study was deposited in the China General Microbiological Culture Collection Center (CGMCC) on September 13, 2024. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCCNO.31969.

[0008] The 26S rRNA nucleotide sequence of the above-mentioned Maggi yeast strain is shown in SEQ ID NO: 1.

[0009] A second aspect of the present invention provides the use of the aforementioned Maggi yeast strain or its fermentation broth in inhibiting pathogens that cause early leaf fall in pears.

[0010] Preferably, the pathogenic bacteria include one or more of Colletotrichum oleraceus, Alternaria alternata, Fusarium solani, and Botrytis cinerea.

[0011] A third aspect of the present invention provides a pear early leaf drop retarding agent, the ingredients of which include the aforementioned Maggi yeast strain or its fermentation liquid.

[0012] Preferably, the fermentation broth is obtained by the following process: picking a single colony of Maggi yeast to YPDA liquid culture medium, culturing it in a shaker at 28° C. and 200 r / min for 16-24 hours, and then collecting it by centrifugation.

[0013] Preferably, the ingredients also include melatonin (MT), 6-benzyladenine (6-BA), L-proline and linoleic acid.

[0014] Preferably, its ingredients include OD 600 =0.6 of Maggi Metschneider yeast, 25 mg / L L-proline and 50 μmol / L 6-benzyladenine.

[0015] The fourth aspect of the present invention provides a method for delaying early leaf drop in pears, comprising the following steps: spraying a pear early leaf drop retarder on pear leaves after the pear fruits are harvested, and spraying a second time after a period of time.

[0016] Preferably, the amount of each spraying is specifically to ensure that liquid drips on the leaves after spraying.

[0017] The beneficial effects of the present invention are:

[0018] 1. The Maggi yeast provided by the present invention is isolated from the surface of healthy pear fruit. It is harmless to the human body and has high safety. It has strong antibacterial activity, broadens the application range of antagonistic yeast, helps to improve the broad-spectrum disease resistance of fruits and vegetables, and can effectively inhibit the growth of pear early leaf fall pathogens such as anthracnose, alternaria, new fusobacteria and Berengue's Botrytis cinerea.

[0019] 2. The applicant of the present invention has screened out a compound combination with good control effect through a large number of experiments: OD 600=0.6 Maggi Meichi yeast cells, 25mg / L L-proline, and 50μmol / L 6-benzyladenine can enhance its resistance to fruit anthracnose, alternaria, Fusarium foetida, and Botrytis cinerea. The present invention overcomes the shortcomings of existing plant growth regulators and broadens the application range of plant aging retardants. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The results of the nucleotide evolution analysis of the 26S rRNA of the yeast Megacena in Example 1 of the present invention are as follows;

[0021] Figure 2 The morphological observation results of Maggi Metschini yeast in Example 1 of the present invention, wherein A is a morphological characteristic diagram of Maggi Metschini yeast on YPDA, and B is a morphological characteristic diagram of Maggi Metschini yeast under 10x and 40x objective lenses of an electron microscope;

[0022] Figure 3 The figures show the inhibitory effects of Maggi Meiqi yeast on pear anthracnose, black spot, leaf spot and ring rot in Example 2 of the present invention, wherein A is a graph showing the effect of Maggi Meiqi yeast on the growth of pear anthracnose, B is a graph showing the effect of Maggi Meiqi yeast on the growth of pear Alternaria, C is a graph showing the effect of Maggi Meiqi yeast on the growth of Botrytis cinerea, D is a graph showing the effect of Maggi Meiqi yeast on the growth of Fusilis minor, and E is a graph showing the lesion area of ​​the four fungi in the Maggi Meiqi yeast and control treatment groups at 2 days and 4 days;

[0023] Figure 4 The figures are as follows: Figure 3 shows the inhibitory effect of the fermentation broth of Maggi Meiqi yeast on pear anthracnose, black spot, leaf spot and ring rot pathogens in Example 3 of the present invention, wherein A is a graph showing the effect of the supernatant of Maggi Meiqi yeast on the growth of pear anthracnose, B is a graph showing the effect of the supernatant of Maggi Meiqi yeast on the growth of pear Alternaria, C is a graph showing the effect of the supernatant of Maggi Meiqi yeast on the growth of grape syringa, D is a graph showing the effect of the supernatant of Maggi Meiqi yeast on the growth of small new shell Fusora, E is a graph showing the lesion diameter of fruit anthracnose in the Maggi Meiqi yeast supernatant and control treatment groups at 3d and 6d, F is a graph showing the lesion diameter of Alternaria in the Maggi Meiqi yeast supernatant and control treatment groups at 5d and 10d, G is a graph showing the lesion diameter of grape syringa in the Maggi Meiqi yeast supernatant and control treatment groups at 2d and 4d, and H is a graph showing the lesion diameter of small new shell Fusora in the Maggi Meiqi yeast supernatant and control treatment groups at 2d and 4d;

[0024] Figure 5These are the resistance evaluation graphs for anthracnose, black spot, and leaf spot of pear leaves in the five treatment groups and one control treatment group in Example 4 of the present invention, wherein A is a resistance evaluation graph for Huangguan pear leaves in the five growth regulator treatment groups and the control group, B is a graph showing the diameter of anthracnose lesions of Huangguan pear in the five growth regulator treatment groups and the control group, C is a graph showing the diameter of black spot lesions of Huangguan pear in the five growth regulator treatment groups and the control group, and D is a graph showing the diameter of leaf spot lesions of Huangguan pear in the five growth regulator treatment groups and the control group;

[0025] Figure 6 These are the resistance evaluation graphs for different compound combination treatments to anthracnose, black spot, and leaf spot of pear leaves in Example 4 of the present invention, wherein A is a resistance evaluation graph for Huangguan pear leaves in the five growth regulator compound treatment groups and the control group, B is a graph showing the diameter of Huangguan pear anthracnose lesions in the five growth regulator compound treatment groups and the control group, C is a graph showing the diameter of Huangguan black spot lesions in the five growth regulator compound treatment groups and the control group, and D is a graph showing the diameter of Huangguan leaf spot lesions in the five growth regulator compound treatment groups and the control group;

[0026] Figure 7 The effects of different compound combination treatments on early leaf drop of pears in Example 4 of the present invention are shown in Figure A, which shows the leaf drop of Huangguan pear trees in the five compound combinations and the control treatment group, and Figure B shows the leaf drop of Cuiyu pear trees in the five compound combinations and the control treatment group;

[0027] Figure 3-6 In the table, * represents significant differences at the P < 0.05 level, ** represents extremely significant differences at the P < 0.01 level, *** represents extremely significant differences at the P < 0.001 level, and **** represents extremely significant differences at the P < 0.001 level. DETAILED DESCRIPTION

[0028] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0029] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources.

[0030] If no specific techniques or conditions are specified in the examples, they can be carried out according to the techniques or conditions described in the literature in the field or according to the product instructions.

[0031] Example 1 Isolation, Identification and Morphological Observation of Maggi Meichi Yeast

[0032] The isolated samples were healthy Zaosu pear fruits, which were collected from the pear germplasm resource garden of Anhui Agricultural University High-tech Agricultural Park. The peels of Zaosu pears were placed in a conical flask filled with YPDA liquid medium, ensuring that the YPDA liquid medium completely submerged the pear peels. The fruit was cultured in a shaker at 28°C at 200 rpm for 6 h, and then the fruit was cultured according to the 10 -1 , 10 -2 and 10 -3 Dilute the solution, then take 100 μL and plate it. Incubate the plate at 28°C for 2-3 days. After a single colony grows, pick out individual colonies of varying morphology and streak them for purification. Then, select round, creamy or milky white colonies and streak them onto WL medium. Incubate at 28°C in a shaker for 3-5 days. Identify yeast grown on WL medium using 26S rRNA gene sequence primers:

[0033] Forward NL-1: 5'-GCATATCAATAAGCGGAAAAG-3' (SEQ ID NO: 2)

[0034] Reverse NL-4: 5'-GGTCCGTGTTTCAAGACGG-3' (SEQ ID NO: 3) was amplified. The amplification system used was 25 μL, and the amplification program was as follows: 94°C for 10 min, 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s, for 30 cycles; and 72°C for 10 min. The amplified product was sent to Hefei Youkang Gene Biotechnology Co., Ltd. for sequencing.

[0035] The sequencing results were compared with the GenBank database and the phylogenetic analysis was performed. The results showed that the strain was clustered into a branch with the known yeast strains Metschnikowia pulcherrima isolate (UTAD1429) and Metschnikowia pulcherima isolate (NXU 20-245). Figure 1 The strain was named as Mp1 and deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with the accession number of CGMCCNO.31969.

[0036] The single colony of Maggi yeast was streaked on YPDA plate medium and the colony and cell morphology were observed after culturing for 2 days. The results showed that the surface of the single colony of the strain was smooth and opaque, with a pink-white creamy round shape, convex, neat colony edges, and red pigment (such as Figure 2 As shown in A). Observation under 10x and 40x microscopes revealed that the strain was oval or nearly spherical, with unilateral or multi-sided budding (as shown in Figure 2 B).

[0037] Example 2 Antibacterial test of Maggi yeast

[0038] Pick a single colony of Maggi yeast and place it in YPDA liquid medium. Incubate it in a shaker at 28℃ and 200r / min for 16-24h. 600 =1.5~2.0, collect yeast cells, dilute the collected cells with sterile water to a concentration of OD 600 =1.0 yeast suspension, 4 circular filter papers with a pore size of 5 mm were placed on the four sides of a 60 mm culture dish, 5 μL of yeast suspension was dropped on each filter paper, and 5 μL of sterile water was dropped on each control. Then, 5 mm-sized fruit anthracnose (isolated from the fruit of Autumn Pear of Henan Academy of Agricultural Sciences in 2024), Alternaria (isolated from the crystal pear leaves of Anhui Agricultural University High-tech Agricultural Park in 2016, strain name PL2, NCBI GenBank accession number: KX016032) and Fusarium spp. (isolated from the Cuiguan pear leaves of Anhui Agricultural University High-tech Agricultural Park in 2023) and Botrytis ciliata (kindly donated by Professor Wang Liping of the College of Plant Science and Technology of Huazhong Agricultural University, strain name: LW-1) cakes were placed in the center of the culture dish and cultured at 28°C for 2d and 4d and photographed.

[0039] Figure 3 The results showed that compared with the control, yeast cells could significantly inhibit the growth of mycelium of Colletotrichum, Alternaria alternata, Fusarium solani and Botrytis cinerea at 2 and 4 days of treatment, further indicating that yeast cells can secrete active antibacterial substances.

[0040] Example 3 Antibacterial test of Maggi yeast fermentation broth

[0041] In order to explore the inhibitory effect of the fermentation broth on the pathogenic bacteria of early leaf fall, a single colony of Maggi yeast was picked and placed in YPDA liquid medium, and cultured at 28 ° C in a shaker at 200 r / min for 16-24 h. 600 =1.5-2.0, the fermentation supernatant was collected by centrifugation and added to YPDA solid culture medium at volume ratios of 0%, 10%, 25% and 50%. Then, a 5mm-sized cake of Colletotrichum oleraceus, Alternaria alternata, Fusarium spp. and Botrytis cinerea was placed in the center of the culture dish. Figure 4 As shown in the figure, when the fermentation broth concentration reaches 10%, the growth of the four bacteria can be significantly inhibited, and the diameter of the lesion tends to decrease with the increase of the fermentation broth concentration.

[0042] Example 4 Effect of Maggi yeast on hyphal growth of pear early leaf fall pathogen

[0043] In order to explore the antibacterial effect of Maggi yeast and growth regulators on the pathogens of early leaf fall in pear, this study

[0044] First, the Maggi yeast cells (OD600 =1.0), 25mg / L L-proline, 50μmol / L 6-BA, 50mg / L linoleic acid, and 0.1mmol / L MT were sprayed on pear leaves after fruit harvest. The second spraying was carried out 7 days later, for a total of 2 times. The amount of each spray was determined according to the size of the tree, and the specific amount was to ensure that liquid dripped on the leaves after spraying. Anthracnose, black spot, and leaf spot resistance were evaluated 2 days after treatment. The results showed that exogenous spraying of these substances could significantly inhibit the spread of pathogens (such as Figure 5 These substances can induce plant resistance.

[0045] These substances were further compounded with Maggi yeast to design a total of 5 treatment combinations:

[0046] Maggi Meiqi yeast cells (MJMQ) and H1: 0.1 mmol / L MT and 50 μmol / L 6-BA;

[0047] MJMQ and H2: 25 mg / L L-proline and 50 μmol / L 6-BA;

[0048] MJMQ and H3: 50 mg / L linoleic acid, 25 mg / L L-proline, and 50 μmol / L 6-BA;

[0049] MJMQ and H4: 25 mg / L L-proline, 0.1 mmol / L MT, and 50 μmol / L 6-BA;

[0050] MJMQ and H5: 50 mg / L linoleic acid, 25 mg / L L-proline, 0.1 mmol / L MT, and 50 μmol / L 6-BA.

[0051] The five treatment groups and one control group (the control group was sprayed with sterile water) were sprayed exogenously on pear leaves. The second spraying was performed 7 days later, for a total of 2 times. Anthracnose, black spot, and leaf spot resistance were evaluated 2 days after treatment. The results showed that the combination of Maggi yeast with H1, 2, 3, and 4 could significantly reduce the lesion diameter of the three pathogens, and the MJMQ and H2: 25 mg / L L-proline and 50 μmol / L 6-BA treatment groups had the best antibacterial effect (such as Figure 6 shown).

[0052] By observing the leaf-fall phenotype of the treated Cuiyu and Huangguan pear trees in the field, it was found that the leaves of the control group began to yellow and fall off, while most of the leaves of the treated group remained green (e.g. Figure 7 ), indicating that the present invention has a good control effect in delaying early leaf drop of pears.

[0053] Maggi Metschneiderian yeast 26S rRNA nucleotide sequence: (SEQ ID NO: 1)

[0054] Although the above embodiments have been described in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments based on this embodiment without inventiveness, and these embodiments all fall within the scope of protection of the present invention.

[0055] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A strain of Maggi yeast, characterized in that Its deposit number is CGMCC NO.31969 and the deposit date is September 13, 2024.

2. The Maggi yeast strain according to claim 1, characterized in that The 26S rRNA nucleotide sequence of the strain is shown in SEQ ID NO:

1.

3. Use of the Maggi yeast strain or its fermentation liquid according to claim 1 in inhibiting pathogenic bacteria causing early leaf fall of pear.

4. The use according to claim 3, characterized in that: The pathogenic bacteria include one or more of Colletotrichum, Alternaria alternata, Fusarium solani, and Botrytis cinerea.

5. A pear early leaf fall delay agent, characterized in that: The ingredients include the Maggi yeast strain or its fermentation liquid as claimed in claim 1.

6. The agent for delaying early leaf fall of pear according to claim 5, characterized in that The fermentation broth is obtained by the following process: a single colony of Maggi yeast is picked up and placed in a YPDA liquid culture medium, and the culture is carried out at 28° C. and 200 r / min for 16-24 hours, and then the culture broth is collected by centrifugation.

7. The agent for delaying early leaf fall of pear according to claim 5, characterized in that: It also includes melatonin (MT), 6-benzyladenine (6-BA), L-proline, and linoleic acid.

8. The agent for delaying early leaf fall of pear according to claim 7, characterized in that: Its ingredients include OD 600 =0.6 of Maggi yeast, 25 mg / L L-proline and 50 μmol / L 6-benzyladenine.

9. A method for delaying early leaf fall of pears, characterized in that: The following steps are involved: Spray the pear early leaf fall retardant on the pear leaves after the pear fruits are harvested, and spray it a second time after a period of time.

10. The method for preventing and controlling early leaf fall of pear according to claim 9, characterized in that: The specific amount of each spraying is to ensure that liquid drips on the leaves after spraying.

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