CN113801799B - Yeast SLL12 and application thereof in preparation of biological control agent for controlling postharvest diseases of jujube fruits - Google Patents
Yeast SLL12 and application thereof in preparation of 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 saccharomycete (Debaryomyces nepalensis) SLL12 with a 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 saccharomycete strain resource library, widens the application range of saccharomycetes in the field of postharvest fresh-keeping of the fruits, but also provides a new solution for preventing and controlling postharvest diseases of the jujube fruits.
Description
Technical Field
The invention belongs to the technical field of biological control of saccharomycetes and fruit postharvest diseases, relates to saccharomycetes, and also relates to application of the saccharomycetes in preparation of biological control agents for controlling jujube fruit postharvest diseases.
Background
The jujube fruits are rich in amino acids, proteins, dietary fibers, minerals, ascorbic acid and other nutrient substances, and are deeply favored by consumers. However, jujube fruits are extremely susceptible to latent infectious diseases of microorganisms during postharvest storage, and black spot is one of the main postharvest diseases. The method for preventing and treating the postharvest diseases of the jujube fruits is commonly used at present and comprises chemical bactericides such as mancozeb, carbendazim and the like. However, chemical bactericides have problems of safety, environmental pollution and the like. Therefore, the use of chemical bactericides is reduced, and effective biological control technology is actively discussed and developed, so that the method is a research hot spot for controlling the postharvest diseases of the jujube fruits.
Research shows that antagonistic microorganisms can effectively prevent and treat postharvest diseases of fruits, wherein antagonistic yeasts can be rapidly adapted to microenvironments such as low oxygen and high sugar on the surfaces of the fruits and can be produced in a large scale in a fermentation tank, so that the antagonistic microorganisms become an important research object for biological prevention and treatment of the fruits after harvest. The mechanism of antagonizing saccharomycetes to prevent and treat postharvest diseases includes mainly competition between nutrients and space, secretion of bacteriostasis matter, induction of host resistance, etc.
Disclosure of Invention
One of the purposes of the present invention is to provide a yeast; and the second aim is to provide the application of the saccharomycete in preparing biological control agents for controlling diseases of jujube fruits after picking, and the effect is clear and remarkable.
Through researches, the invention provides the following technical scheme:
1. the preservation number of the saccharomycete (Debaryomyces nepalensis) SLL12 is CGMCC No.21854.
2. The application of the saccharomycete SLL12 in preparing 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 the picked black spot of the jujube fruits by inducing the jujube fruits to generate disease resistance and competition of nutrition and space.
The microzyme SLL12 is separated from jujube garden leaves, and is preserved in China general microbiological culture Collection center (CGMCC) for 1 month of 2021 and 1 day of 3 months, wherein the preservation number is CGMCC No.21854 and the classification name is Debaryomyces nepalensis.
The research result of the invention shows that the saccharomycete SLL12 can not inhibit the growth of the jujube fruit melasma pathogenic bacteria, namely alternaria alternata (Alternaria alternate), in vitro, which shows that the saccharomycete does not produce corresponding antibacterial substances for the alternaria alternata or the quantity of the produced antibacterial substances is very low, but the saccharomycete SLL12 can effectively prevent the occurrence and the progress of the jujube fruit postharvest melasma on fruits, and the induction of host to produce disease resistance, nutrition and space competition is a main mechanism for preventing and treating the jujube fruit postharvest melasma.
The invention has the beneficial effects that: the invention provides the saccharomycete 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, not only enriches the saccharomycete strain resource library, widens the application range of saccharomycetes in the field of postharvest fresh-keeping of the fruits, but also provides a new solution for the prevention and control of the postharvest diseases of the jujube fruits.
Drawings
FIG. 1 shows the morphology of a screened portion of yeast on YEPD plates.
FIG. 2 shows the in vitro bacteriostatic effect of a portion of the yeasts screened on Alternaria alternata.
FIG. 3 shows the effect of the screened part of yeast on controlling melasma of date fruit after picking, wherein DI represents incidence and LD represents lesion diameter.
FIG. 4 shows the control effect of the yeast SLL12 heteroporous inoculation on postharvest melasma of jujube fruits, where DI represents morbidity and LD represents lesion diameter.
FIG. 5 shows growth dynamics of yeast SLL12 at a date fruit wound.
FIG. 6 shows growth dynamics of yeast SLL12 and Alternaria alternata at jujube fruit wound under scanning electron microscope, wherein A1 and A2 are jujube fruit wound tissue; b1 and B2 are growth states of saccharomycete SLL12 at the wound of the jujube fruit; c1 and C2 are the growth states of Alternaria alternata at the wound of the jujube fruit; d1 and D2 are growth dynamics of saccharomycetes SLL12 and Alternaria alternata at the wound of the jujube fruit; A1-D1 are amplified 1000 times; a2 to D2 are amplified 5000 times.
In fig. 3 and 4, control represents a Control; bars and vertical bars represent mean and standard deviation of biological replicates, respectively, and different letters at the same time point represent significant differences (P < 0.05).
Detailed Description
In order to make the objects, technical solutions and advantageous effects of the present invention more apparent, preferred embodiments of the present invention are described in detail below.
Experimental method
1. Screening of yeasts
Collecting the roots of different jujube gardens from the jujube gardens in Fen city, shanxi province, jin Zhongshi and Chongqing Bei area by adopting a five-point sampling methodInterplanting soil, leaves and young fruits. Rhizosphere soil samples of each jujube garden were mixed with rhizosphere soil at 5 sampling points, each weighing about 1kg. Taking 2.0g rhizosphere soil sample, placing into 20-30mL sterile water, shaking at 28deg.C under 200rmp for 1 hr to thoroughly mix, and gradient diluting 1mL (10 -1 ，10 -2 ，10 -3 ) And then coated on the culture medium. Immersing leaf sample and young fruit sample in sterile water, shaking at 28deg.C and 200rmp for 1 hr, taking 1ml, and performing gradient dilution (10 -1 ，10 -2 ) And then coated on the culture medium. Single colony with typical yeast morphological characteristics is selected for streak purification and glycerol seed preservation. The purified strain is placed in a refrigerator at the temperature of minus 80 ℃ for preservation.
2. In vitro bacteriostatic effect of saccharomycetes on alternaria alternata
In order to test the inhibition effect of saccharomycetes on alternaria in an in-vitro condition, a single saccharomycete colony is selected by an inoculating loop, streaked at the center of a PDA plate, cultured for 48 hours at 25 ℃, a 5d alternaria cake is inoculated at a position 2.5cm away from the saccharomycetes, a plate inoculated with only alternaria is used as a control, and the radial growth condition of the alternaria spores and hyphae is recorded for 7 d. Experiments were repeated 3 times.
3. Control effect of saccharomycetes on black spot disease of picked jujube fruits
In order to test the control effect of saccharomycetes on black spot of picked jujube fruits, cleaning and sterilizing the jujube fruits, drilling 2 holes (diameter 3mm, depth 3 mm) at equal distance on equatorial position of the fruits by using a sterile puncher, and adding 1×10 into wound of the fruits 8 20 mu L of cell/mL yeast suspension, inoculating with equal amount of sterile water as control, inoculating with Alternaria alternata spore suspension (1×10) 5 Spoe/mL), packaging single fruit after bacterial liquid is absorbed, storing at 25 ℃ and 4 ℃ (RH 90% -95%), and measuring the incidence rate and the diameter of the disease spots of the fruit. 10 fruits were treated per group. Experiments were repeated 3 times.
4. Molecular biological identification of yeasts
The yeast strains were identified by 16S rDNA sequence analysis. Extracting thallus DNA from the cultured saccharomycetes by using a genome DNA extraction kit, and carrying out 16s rDNA PCR amplification by using a 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. Mu.L of 2 XTaq Master mix (Dye), 1. Mu.L of each primer and 1. Mu.L of DNA template. The PCR reaction process is as follows: pre-denaturation, 94 ℃ for 2min; then 30 cycles: denaturation, 94 ℃,30s; annealing at 55 ℃ for 30s; extending at 72 ℃ for 30s; final extension at 72deg.C for 2min; finally, the mixture is preserved at the temperature of 4 ℃. The PCR amplified products were gel-electrophoresed using 1% agarose, followed by visualization of the bands under UV light. The PCR amplified products were committed to Shanghai Biotechnology Co.Ltd for sequencing and compared using BLAST database in NCBI.
5. Control effect of saccharomycete heteropore inoculation on black spot disease of picked jujube fruits
In order to determine the capability of saccharomycetes to induce fruits to generate disease resistance, a saccharomycete abnormal hole inoculation mode is adopted. Cleaning fructus Jujubae, sterilizing, making 2 holes (diameter 3mm, depth 2 mm) at the equatorial position of the fruit with aseptic puncher, and adding 1×10 at the wound of the fruit 8 20 mu L of cell/mL yeast suspension, taking the same amount of sterile water as a control, standing for 24 hours, then punching 1 new hole with the same size at a distance of about 1cm right of the hole punched before, and inoculating Alternaria alternata spore suspension (1×10) 6 Spoes/mL) 10. Mu.L, dried at room temperature, packaged in single fruit, and placed at 25℃to determine the incidence of fruit disease and the diameter of lesions. 10 fruits were treated per group. Experiments were repeated 3 times.
6. Growth dynamics of saccharomycetes at jujube fruit wound
The ability of yeasts to utilize fruit nutrition for growth and proliferation at jujube fruit wounds and their ability to conduct nutrition and space competition on fruits was assessed by cell counting. Cleaning fructus Jujubae, sterilizing, making 2 holes (diameter 3mm, depth 3 mm) at equal distance on equatorial position of fruit, and adding 1×10 at wound 8 And 20 mu L of cell/mL yeast suspension, taking the same amount of sterile water as a control, packaging single fruits after the bacterial liquid is absorbed, and storing at 25 ℃ and 4 ℃ (RH 90% -95%) respectively. The number of yeasts measured 1h after inoculation was taken as the initial value. During sampling, sterile punching is used3 parts of pulp tissue with the diameter of 10mm at the wound are taken out, placed in a sterile mortar containing 10mL of sterile water, ground to be homogenized, cultured for 48 hours at 25 ℃ by a dilution plate method, and counted by using a full-automatic colony counter, and the result is expressed as the number of yeasts at each wound and is expressed as Log10 cells/wound. The experiment was repeated 3 times with 10 fruits per group.
7. Growth state of saccharomycetes and alternaria alternata at wound of jujube fruit
And observing the growth states of saccharomycetes and alternaria alternata at the wound of the jujube fruits by adopting a scanning electron microscope. Cleaning fructus Jujubae, sterilizing, making 2 holes (diameter 3mm, depth 3 mm) at equal distance on equatorial position of fruit, and adding 1×10 at wound 8 20 mu L of cell/mL yeast suspension, inoculating with equal amount of sterile water as control, inoculating with Alternaria alternata spore suspension (1×10) 8 Spoe/mL), naturally airing the fruits at room temperature, packaging the fruits by using a PE film, storing the fruits in an environment with the relative humidity of 85% -90% at 20 ℃, covering the fruits by using the PE film for moisturizing treatment, and observing the growth states of saccharomycetes and alternaria in the wound positions of the fruits by using a scanning electron microscope after 2 d. Each group was treated with 15 fruits and the experiment was repeated 3 times.
Experimental results
1. Screening of yeasts
The suspected strain 135 strain of saccharomycete is obtained through co-separation from jujube gardens in the medium of Jinzhong and North medium of Chongqing in Shanxi province. The morphology of a portion of the yeast suspected strain pairs on YEPD plates is shown in FIG. 1.
2. In-vitro antibacterial effect of saccharomycetes on jujube black spot bacteria
And performing an in-vitro bacteriostasis experiment on the screened 135 suspected strains of saccharomycetes. The results show that the 135 strains have no inhibition effect on Alternaria alternata in vitro, and the in vitro inhibition effect of part of strains on Alternaria alternata is shown in figure 2. The above results indicate that the yeasts do not produce the corresponding bacteriostatic substances or produce very low amounts of bacteriostatic substances on Alternaria alternata.
3. Control effect of saccharomycetes on black spot disease of picked jujube fruits
8 suspected strains of saccharomycetes with higher growth speed and higher activity in vitro are randomly selected, and the control effect of the suspected strains on the black spot disease of the picked jujube fruits is examined. As shown in FIG. 3, 8 strains can reduce the incidence of black spot after picking jujube fruits and delay the growth of the black spot to a certain extent, wherein the strain SLL12 has the best effect.
4. Molecular biological identification of yeasts
The sequence of the 16S rDNA amplification product of the strain SLL12 is shown in SEQ ID No.3, and homology comparison of the measured sequence using BLAST program in NCBI shows that the homology of the strain SLL12 with yeast (Debaryomyces nepalensis) is 100%.
5. Control effect of saccharomycete heteropore inoculation on black spot disease of picked jujube fruits
As shown in figure 4, the yeast SLL12 heteropore inoculation can effectively reduce the incidence of postharvest melasma of jujube fruits and delay the growth of lesions, which indicates that the strain has the capability of inducing the disease resistance of the jujube fruits, and can prevent and treat the postharvest melasma of the jujube fruits by inducing the disease resistance of the jujube fruits.
6. Growth dynamics of saccharomycetes at jujube fruit wound
The growth dynamics of the saccharomycete SLL12 at the jujube fruit wound is shown in figure 5, and the saccharomycete SLL12 can effectively and rapidly colonise at the jujube fruit wound, which indicates that the competition of nutrition and space is one of mechanisms of the saccharomycete SLL12 for preventing and treating the jujube fruit postharvest diseases.
7. Growth state of saccharomycetes and alternaria alternata at wound of jujube fruit
The growth dynamics of saccharomycete SLL12 and Alternaria alternata at the wound of the jujube fruit under the scanning electron microscope are shown in figure 6, and the saccharomycete SLL12 can still be tightly attached to the wound surface of the jujube fruit after being washed for a plurality of times, and the formation of extracellular matrixes wrapping the saccharomycete can be observed. It can also be observed from FIGS. 6D1-D2 that a small number of Alternaria alternata hyphae are tightly surrounded by yeast SLL12 cells and do not effectively contact the wound surface of the jujube fruit. The results show that the saccharomycete SLL12 proliferates rapidly and has colonization capacity at fruit wounds, so that the saccharomycete SLL forms strong nutrition and space competition advantages for Alternaria, and further infection of Alternaria with jujube fruits is inhibited.
Finally, it is noted that the above-mentioned preferred embodiments are only intended to illustrate rather than limit the invention, and that, although the invention has been described in detail by means of the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention as defined by the appended claims.
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Claims (3)
1. Yeast @ sDebaryomyces nepalensis) SLL12 has a preservation number of CGMCC No.21854.
2. The use of the yeast SLL12 of claim 1 for the preparation of a biocontrol agent for controlling postharvest diseases of jujube fruits, said postharvest diseases of jujube fruits being black spots.
3. The use according to claim 2, wherein: the saccharomycete SLL12 can prevent and treat the postharvest black spot of the jujube fruits by inducing the jujube fruits to generate disease resistance and competing nutrition and space.
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| 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 |
| CN115160418B (en) * | 2022-06-24 | 2023-06-02 | 西南大学 | Pichia glabra Peptidase secretion protein and application thereof |
| 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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