CN113801799A - Yeast SLL12 and application thereof in preparing biological control agent for controlling postharvest diseases of jujube fruits - Google Patents
Yeast SLL12 and application thereof in preparing biological control agent for controlling postharvest diseases of jujube fruits Download PDFInfo
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
- A23B7/00—Preservation or chemical ripening of fruit or vegetables
- A23B7/14—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10
- A23B7/153—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10 in the form of liquids or solids
- A23B7/154—Organic compounds; Microorganisms; Enzymes
- A23B7/155—Microorganisms; Enzymes; Antibiotics
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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Abstract
The invention discloses a yeast (Debaryomyces nepalensis) SLL12 with the preservation number of CGMCC No.21854 and application thereof in preparing a biological control agent for controlling postharvest diseases of jujube fruits, which not only enriches a yeast strain resource library, widens the application range of the yeast in the field of postharvest preservation of the fruits, but also provides a new solution for the prevention and control of the postharvest diseases of the jujube fruits.
Description
Technical Field
The invention belongs to the technical field of yeast and biological prevention and control of postharvest diseases of fruits, relates to yeast, and further relates to application of the yeast in preparation of a biological prevention and control agent for controlling postharvest diseases of jujube fruits.
Background
The jujube fruit is rich in amino acid, protein, dietary fiber, mineral substance, ascorbic acid and other nutrient substances, and is deeply loved by the consumers. However, the jujube fruits are very easy to have the latent microbial infectious diseases during the postharvest storage period, and the black spot is one of the main postharvest diseases. The current commonly used method for preventing and treating the postharvest diseases of the jujube fruits is that chemical bactericides comprise mancozeb, carbendazim and the like. But the chemical bactericide has the problems of safety, environmental pollution and the like. Therefore, the application of chemical bactericides is reduced, effective biological control technologies are actively researched and developed, and the method is a research hotspot for controlling postharvest diseases of the jujube fruits.
Research shows that antagonistic microbe can effectively prevent and treat postharvest diseases of fruits, wherein antagonistic yeast can be a key research object for biological prevention and treatment of postharvest fruits because of being capable of rapidly adapting to microenvironment such as low oxygen and high sugar on the surfaces of fruits and being capable of being produced in a fermentation tank in large quantities. The mechanism of the antagonistic yeast for preventing and treating the postharvest diseases of the fruits mainly comprises the competition of nutrition and space, the secretion of antibacterial substances, the induction of host resistance and the like.
Disclosure of Invention
One of the purposes of the present invention is to provide a yeast; the second purpose is to provide the application of the microzyme in preparing the biological control agent for controlling the postharvest diseases of the jujube fruits, and the effect is clear and remarkable.
Through research, the invention provides the following technical scheme:
1. yeast (Debaryomyces nepalensis) SLL12 with preservation number of CGMCC No. 21854.
2. The yeast SLL12 is applied to the preparation of biological control agents for controlling postharvest diseases of jujube fruits.
Further, the disease of the picked jujube fruits is black spot.
Further, the yeast SLL12 can prevent and treat postharvest black spot of the jujube fruits by inducing the jujube fruits to generate disease resistance and competition between nutrition and space.
The yeast SLL12 is separated from leaves of jujube garden, and is preserved in China general microbiological culture Collection center (CGMCC, address: No.3 of Xilu 1 of Beijing's republic of Chaoyang district), with the preservation number of CGMCC No.21854, and is classified and named as Debaryomyces nepalensis, 3.1.2021.3.
The research result of the invention shows that the yeast SLL12 can not inhibit the growth of Alternaria alternate (Alternaria alternate) which is a pathogenic bacterium of black spot of jujube fruit in vitro, which indicates that the yeast does not produce corresponding antibacterial substances to the Alternaria alternate or the amount of the generated antibacterial substances is extremely low, but the yeast SLL12 can effectively prevent and control the occurrence and the development of black spot of picked jujube fruit on the fruit, and induces the competition of disease resistance, nutrition and space of a host, which is the main mechanism for preventing and controlling the black spot of picked jujube fruit.
The invention has the beneficial effects that: the invention provides the yeast SLL12 with the preservation number of CGMCC No.21854 and the application thereof in preparing the biological control agent for controlling the postharvest diseases of the jujube fruits, thereby enriching the yeast strain resource library, widening the application range of the yeast in the field of postharvest preservation of the fruits and providing a new solution for the prevention and control of the postharvest diseases of the jujube fruits.
Drawings
FIG. 1 shows the form of the selected yeast pairs on YEPD plates.
FIG. 2 shows the in vitro bacteriostatic effect of the screened partial saccharomycetes on Alternaria alternata.
FIG. 3 is a graph showing the control effect of some selected yeasts on black spot disease after picking jujube fruits, wherein DI represents the incidence rate and LD represents the lesion diameter.
FIG. 4 shows the control effect of yeast SLL12 cross-hole inoculation on postharvest melasma of jujube fruit, wherein DI represents the incidence rate and LD represents the lesion diameter.
FIG. 5 shows the growth dynamics of yeast SLL12 on the wound of jujube fruit.
FIG. 6 shows the growth dynamics of yeast SLL12 and Alternaria alternata on the wound of a jujube fruit under a scanning electron microscope, wherein A1 and A2 are tissues of the wound of the jujube fruit; b1 and B2 indicate the growth state of yeast SLL12 on the jujube fruit wound; c1 and C2 represent the growth state of alternaria alternate on the jujube fruit wound; d1 and D2 indicate the growth dynamics of yeast SLL12 and alternaria alternate on the jujube fruit wound; A1-D1 is amplified by 1000 times; A2-D2 is 5000 times magnification.
In fig. 3 and 4, Control represents a Control; bars and vertical bars represent mean and standard deviation, respectively, of biological replicates, with different letters indicating significant differences (P <0.05) at the same time point.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, preferred embodiments of the present invention are described in detail below.
Experimental method
1. Screening of Yeast
From date gardens in the Yanfen city, the jin Zhongshi city and the Beibei Bei region in Shanxi province, five-point sampling method is adopted to collect rhizosphere soil, leaves and young fruits of different date gardens. The rhizosphere soil sample of each jujube orchard is rhizosphere soil mixture of 5 sampling points, and each rhizosphere soil sample weighs about 1 kg. Placing 2.0g rhizosphere soil sample in 20-30mL sterile water, shaking at 28 deg.C and 200rmp for 1h to mix well, and taking 1mL to perform gradient dilution (10)-1，10-2，10-3) And then spread on a culture medium. Completely immersing leaf sample and young fruit sample in sterile water, shaking at 28 deg.C and 200rmp for 1h, and taking 1ml for gradient dilution (10)-1，10-2) And then spread on a culture medium. Single colonies with typical yeast morphological characteristics were picked for streaking purification and glycerol conservation. The purified strain is stored in a refrigerator at-80 ℃.
2. In-vitro bacteriostatic effect of saccharomycetes on alternaria alternata
In order to test the inhibiting effect of the yeast on the alternaria alternate under the in vitro condition, a single yeast colony is picked by an inoculating loop and streaked on the center of a PDA (personal digital assistant) plate, after the yeast colony is cultured for 48 hours at 25 ℃, an alternaria alternate cake cultured for 5d is picked and inoculated at a position 2.5cm away from the yeast, the plate inoculated with only the alternaria alternate is used as a control, and 7d, the radial growth conditions of spores and hyphae of the alternaria alternate are recorded. The experiment was repeated 3 times.
3. Control effect of saccharomycetes on black spot of picked jujube fruits
In order to test the control effect of the yeasts on the black spot of the picked jujube fruits, the jujube fruits are cleaned and disinfected and then are subjected to black spot controlPunching 2 holes (diameter 3mm, depth 3mm) at equal intervals on fruit equator part with bacteria puncher, and adding 1 × 10 holes on fruit wound8cells/mL yeast suspension 20 u L, with equal amount of sterile water as control, 4h after inoculation of Alternaria alternata spore suspension (1X 10)5spoore/mL), after the bacterial liquid is absorbed, packaging the fruits individually, storing the fruits respectively at 25 ℃ and 4 ℃ (RH 90% -95%), and determining the disease incidence and lesion diameter of the fruits. Each group treated 10 fruits. The experiment was repeated 3 times.
4. Molecular biological identification of yeast
The yeast strains were identified by 16S rDNA sequence analysis. Extracting thallus DNA from the cultured saccharomycete with genome DNA extracting kit, and performing 16s rDNA PCR amplification with universal primer. The forward primer was 5'-TCCGTAGGTGAACCTGCGG-3' (SEQ ID No.1) and the reverse primer was 5 '-TCCTCCGCTTATTGAT-ATGC-3' (SEQ ID No. 2). The reaction system comprises: 12.5 μ L of 2 XTaq MasterMix (Dye),1 μ L of each primer and 1 μ L of DNA template. The PCR reaction process is as follows: pre-denaturation at 94 ℃ for 2 min; then 30 cycles: denaturation, at 94 ℃ for 30 s; annealing at 55 ℃ for 30 s; extension at 72 ℃ for 30 s; final extension at 72 deg.C for 2 min; finally, the mixture is stored at 4 ℃. The PCR amplification products were subjected to gel electrophoresis using 1% agarose, followed by observing the bands under a UV lamp. The PCR amplification products were subjected to sequencing by Shanghai Biotech Co., Ltd, and compared with each other using BLAST database in NCBI.
5. Control effect of yeast different-hole inoculation on black spot of picked jujube fruits
In order to determine the ability of the saccharomycetes to induce the disease resistance of fruits, a heteropore saccharomycetes inoculation mode is adopted. Cleaning and sterilizing fructus Jujubae, punching 2 holes (diameter 3mm and depth 2mm) at equal intervals on equator of fructus Jujubae with sterile puncher, and adding 1 × 10 holes on the wound820 mu L of cell/mL yeast suspension, using equivalent sterile water as a control, placing for 24h, drilling 1 new hole with the same size at the position of about 1cm away from the right side of the former punched hole, and inoculating Alternaria alternata spore suspension (1X 10) into the new hole6spores/mL)10 μ L, dried at room temperature, packaged individually, placed at 25 deg.C, and assayed for fruit incidence and lesion diameter. Each set of treatments 10And (4) fruit. The experiment was repeated 3 times.
6. Dynamic growth of yeast in jujube fruit wound
The ability of the yeast to utilize fruit nutrition for growth and proliferation at the wound of the jujube fruit and the ability of the yeast to perform nutrition and space competition on the fruit are evaluated through cell counting. Cleaning and sterilizing fructus Jujubae, punching 2 holes (diameter 3mm, depth 3mm) at equal intervals on equator of fructus Jujubae with sterile puncher, and placing 1 × 10 holes on the wound8cell/mL yeast suspension 20 μ L, with the same amount of sterile water as control, after the bacterial liquid absorption, single fruit packaging, respectively at 25 ℃ and 4 ℃ (RH 90% -95%). The number of yeasts measured 1h after inoculation was used as the starting value. In sampling, 3 parts of pulp tissue having a wound with a diameter of 10mm were taken by a sterile punch, ground in a sterile mortar containing 10mL of sterile water until homogenized, incubated at 25 ℃ for 48 hours by a dilution plate method, and counted by a full-automatic colony counter, and the result was expressed in units of Log10 cells/wind in terms of the number of yeasts per wound. Each group treated 10 fruits and the experiment was repeated 3 times.
7. Growth state of yeast and alternaria alternate in jujube fruit wound
And observing the growth states of the saccharomycetes and the alternaria alternate at the wound of the jujube fruit by adopting a scanning electron microscope. Cleaning and sterilizing fructus Jujubae, punching 2 holes (diameter 3mm, depth 3mm) at equal intervals on equator of fructus Jujubae with sterile puncher, and placing 1 × 10 holes on the wound8cells/mL yeast suspension 20 u L, with equal amount of sterile water as control, 4h after inoculation of Alternaria alternata spore suspension (1X 10)8spoore/mL) 10 mu L, naturally airing the fruits at room temperature, packaging the fruits with a PE film, storing the fruits at 20 ℃ in an environment with the relative humidity of 85-90%, covering the fruits with the PE film for moisture preservation, and observing the growth states of the yeasts and the alternaria alternata on the wounds of the fruits by using a scanning electron microscope after 2 days. Each group treated 15 fruits and the experiment was repeated 3 times.
Second, experimental results
1. Screening of Yeast
The suspected yeast strain 135 is obtained by co-separation from date gardens in the Yanfen city, the jin Zhongshi city and the Beibei Bei region of Chongqing city in Shanxi province. The morphology of a portion of the yeast suspected strains on YEPD plates is shown in FIG. 1.
2. In-vitro bacteriostatic effect of saccharomycetes on jujube black spot germs
The 135 strains of suspected yeast strains are screened out and subjected to an in vitro bacteriostasis experiment. The results show that 135 strains all have no in vitro inhibition effect on alternaria alternata, and the in vitro inhibition effect of part of the strains on alternaria alternata is shown in figure 2. The results show that the yeast does not produce corresponding bacteriostatic substances to Alternaria alternata or produces extremely low amount of bacteriostatic substances.
3. Control effect of saccharomycetes on black spot of picked jujube fruits
Randomly selecting 8 suspected yeast strains with high growth speed and high activity in vitro, and investigating the control effect of the suspected yeast strains on the black spot of the picked jujube fruits. As shown in FIG. 3, the 8 strains can reduce the incidence rate of black spot of picked jujube fruits and delay the growth of the spot to some extent, wherein the strain SLL12 has the best effect.
4. Molecular biological identification of yeast
The sequence of the 16S rDNA amplification product of the strain SLL12 was shown in SEQ ID No.3, and homology analysis using the BLAST program in NCBI showed that the strain SLL12 had 100% homology to yeast (Debaryomyces nepalensis).
5. Control effect of yeast different-hole inoculation on black spot of picked jujube fruits
As shown in figure 4, the yeast SLL12 heteropore inoculation can effectively reduce the incidence rate of the postharvest melasma of the jujube fruits and delay the growth of disease spots, which shows that the yeast SLL12 has the capability of inducing the disease resistance of the jujube fruits and can prevent and control the postharvest melasma of the jujube fruits by inducing the disease resistance of the jujube fruits.
6. Dynamic growth of yeast in jujube fruit wound
The growth dynamics of the yeast SLL12 on the jujube fruit wound is shown in figure 5, and it can be seen that the yeast SLL12 can effectively and rapidly colonize the jujube fruit wound, which indicates that the competition of nutrition and space is one of the mechanisms of the yeast SLL12 for preventing and treating the postharvest diseases of the jujube fruit.
7. Growth state of yeast and alternaria alternate in jujube fruit wound
The growth dynamics of yeast SLL12 and Alternaria alternata at the wound of the jujube fruit under a scanning electron microscope are shown in figure 6, so that the yeast SLL12 can still be tightly attached to the wound surface of the jujube fruit after being washed for multiple times, and the formation of extracellular matrix wrapping the yeast can be observed. From FIGS. 6D1-D2, it was also observed that a small number of the mycelia of Alternaria alternata were tightly surrounded by the cells of the yeast SLL12 and did not effectively contact the wound surface of the jujube fruit. The results show that the yeast SLL12 has rapid proliferation and colonization ability on the wounds of the fruits, thereby forming strong nutrition and space competitive advantages on the alternaria alternate and inhibiting the alternaria alternate from further infecting the jujube fruits.
Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.
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Claims (4)
1. Yeast (Debaryomyces nepalensis) SLL12 with preservation number of CGMCC No. 21854.
2. The use of the yeast SLL12 in claim 1 in preparing biological control agent for controlling jujube fruit postharvest diseases.
3. Use according to claim 2, characterized in that: the disease of the picked jujube fruits is black spot.
4. Use according to claim 3, characterized in that: the yeast SLL12 can prevent and treat postharvest black spot of jujube fruits by inducing the jujube fruits to generate disease resistance and competition between nutrition and space.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114521585A (en) * | 2022-01-25 | 2022-05-24 | 西南大学 | Method for preventing and treating postharvest diseases of jujube fruits by using antagonistic yeast to remold epiphytic microbial communities |
| CN115160418A (en) * | 2022-06-24 | 2022-10-11 | 西南大学 | Pichia capsulata Peptidase secretory protein and application thereof |
| CN115161252A (en) * | 2022-06-24 | 2022-10-11 | 西南大学 | Application of phenylalanine in preparation of reagent for improving antagonistic yeast biocontrol effect and preventing and treating postharvest diseases of jujube fruits |
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| CN109169881A (en) * | 2018-10-23 | 2019-01-11 | 江苏大学 | The method that ascorbic acid induces Cali's Bick Pichia pastoris prevention and treatment fruit postharvest diseases |
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| CN105861340A (en) * | 2016-04-12 | 2016-08-17 | 江苏大学 | Pichia manshurica for controlling postharvest diseases of oranges and grapes |
| CN109169881A (en) * | 2018-10-23 | 2019-01-11 | 江苏大学 | The method that ascorbic acid induces Cali's Bick Pichia pastoris prevention and treatment fruit postharvest diseases |
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| XINGMENG LEI 等: ""Debaryomyces nepalensis reduces fungal decay by affecting the postharvest microbiome during jujube storage"" * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114521585A (en) * | 2022-01-25 | 2022-05-24 | 西南大学 | Method for preventing and treating postharvest diseases of jujube fruits by using antagonistic yeast to remold epiphytic microbial communities |
| CN115160418A (en) * | 2022-06-24 | 2022-10-11 | 西南大学 | Pichia capsulata Peptidase secretory protein and application thereof |
| CN115161252A (en) * | 2022-06-24 | 2022-10-11 | 西南大学 | Application of phenylalanine in preparation of reagent for improving antagonistic yeast biocontrol effect and preventing and treating postharvest diseases of jujube fruits |
| CN115161252B (en) * | 2022-06-24 | 2024-03-15 | 西南大学 | Application of phenylalanine in preparation of reagent for improving antagonistic yeast biocontrol efficacy and preventing and treating jujube fruit postharvest diseases |
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| CN113801799B (en) | 2023-05-02 |
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