CN107881121B - Saccharomyces cerevisiae BY23 for controlling postharvest diseases of fruits and preparation and use methods thereof - Google Patents
Saccharomyces cerevisiae BY23 for controlling postharvest diseases of fruits and preparation and use methods thereof Download PDFInfo
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Abstract
The invention discloses a Saccharomyces cerevisiae BY23 with wide antibacterial spectrum and stable effect for controlling postharvest disease control of fruits, and a use method and application thereof. The number of the strain preserved in the China general microbiological culture Collection center is CGMCC No. 14905. The application method of the saccharomyces cerevisiae comprises the following steps: activating the strain, fermenting and culturing with YPD, centrifuging, and preparing the thallus into 1 × 10 with sterile water8cells/mL of bacterial suspension; putting fruits such as apples, pears, grapes or oranges into the bacterial suspension, soaking for 30 seconds, taking out, and air-drying; putting into a fresh-keeping box, and storing at normal temperature. The saccharomyces cerevisiae strain can simultaneously control and control penicilliosis, botrytis cinerea and black spot of apples, botrytis cinerea, grape black spot, fusarium, anthracnose and stalk point mildew of pears, and penicilliosis of citrus, reduces loss caused by postharvest diseases, and has good application prospect.
Description
Technical Field
The invention relates to the field of biological control of fruit postharvest diseases, in particular to Saccharomyces cerevisiae (Saccharomyces cerevisiae) for biological control of fruit postharvest diseases, and the Saccharomyces cerevisiae has obvious control effects on main postharvest diseases of apples, pears, grapes and oranges.
Background
While the deterioration of fresh fruit quality is influenced by a number of factors, disease is the most prominent cause. Among them, rotting and deterioration caused by fungal diseases are the most serious factors in postharvest loss of fruits. Although fruit postharvest diseases can be controlled by many ways such as agricultural control, physical control, chemical control and biological control, the main current measure is chemical control (Eckert)&Ogawa, 1985, 1988). However, long-term use of chemical pesticides not only results in resistance to pathogenic bacteria and reduced bactericidal effect (Prusky et al, 1985;
as et al.，1991；Holmes&eckert, 1999), and frequent use of high concentrations of chemicals also increases the amount of pesticide left on the fruit, seriously threatens human health, and causes environmental pollution (gulion @)&Kuijipers, 1994). Therefore, the development of a new safe, efficient, non-toxic, and low-resistant technology for controlling postharvest disease of fruits has become the focus of research in various countries of the world (Falik et al, 1995; Tian et al, 2001; Kulakiotiet al, 2004), in which the control by using bio-antagonistic bacteria is a new method that has been proven safe and effective at present (Wilson)&Wisniewski，1989；Janisiewicz&Koersten，2002)。
So far, many bacteria, yeasts and small filamentous fungi with obvious bacteriostatic effects on postharvest pathogenic fungi of fruits have been screened at home and abroad, wherein the prevention and control of postharvest diseases of fruits by using antagonistic yeasts is a new safe and efficient technology which is proved at present, mainly because most pathogenic bacteria invade fruits through wounds, the yeasts mainly prevent and control diseases by carrying out nutrition and space competition with the pathogenic bacteria, and the antagonistic yeasts can adapt to postharvest storage conditions of fruits such as low temperature, low oxygen, high carbon dioxide and the like (Wang Youguo, 2012).
However, although there are nearly a hundred kinds of antagonistic yeasts reported at home and abroad, the biocontrol effect of most of the antagonistic yeasts is only verified on a few fruits. Since the biocontrol effect of different strains of the same yeast is very different (Filonowet al, 1996), most antagonistic yeasts lack strains with wide bacteriostatic spectrum and stable effect.
Disclosure of Invention
Aiming at the technical problems, the invention aims to provide a Saccharomyces cerevisiae strain BY23 with wide antibacterial spectrum and stable effect for biological control of postharvest diseases of fruits, and a preparation method and application thereof. The saccharomyces cerevisiae BY23 for biological prevention and treatment of fruit postharvest diseases has the preservation strain number of CGMCC No.14905 in China general microbiological culture Collection center.
The method for preventing and treating the postharvest diseases of the fruits and storing and refreshing the saccharomyces cerevisiae strain comprises the following steps: activating Saccharomyces cerevisiae, fermenting and culturing with YPD liquid culture medium, centrifuging to obtain thallus, and preparing the thallus with sterile water to concentration of 1 × 108cells/mL of bacterial suspension; putting fruits such as apples, pears, grapes and oranges into the bacterial suspension, soaking for 30 seconds, taking out, and air-drying; putting into a fresh-keeping box, sealing, and storing at room temperature. The Saccharomyces cerevisiae BY23 is prepared BY taking out strain from refrigerator at-80 deg.C, activating with YPDA culture medium, picking single colony to YPD liquid culture medium, culturing at 26 deg.C and 200r/min for 24 hr, centrifuging at 4000rpm for 5min, collecting thallus, and washing with sterile water for 3 times. The YPDA culture medium is: 10g of yeast extract powder, 20g of peptone, 20g of glucose, 18g of agar and 1000ml of deionized water, and sterilizing at 121 ℃ for 30min under natural pH. The saccharomyces cerevisiae strain can be simultaneously used for controlling and simultaneously controlling penicilliosis, botrytis cinerea and black spot of apples, botrytis cinerea of pear fruits, grape black spot, fusarium wilt, anthracnose and stalk point mildew, and penicilliosis of citrus.
The strain provided BY the invention is the saccharomyces cerevisiae BY23 which has obvious prevention and treatment effects on the postharvest diseases of apples, pears, grapes and oranges and is screened and separated from the naturally fermented wine-making millstone persimmon mash, can be widely used for preventing and treating the postharvest diseases of fruits, reduces the loss caused BY the postharvest diseases, and has good application prospects.
The invention has the advantages that: (1) the saccharomyces cerevisiae BY23 provided BY the invention is obtained BY screening from fermented liquor of the persimmons millettii in the laboratory, is harmless to human bodies and has high safety. (2) The saccharomyces cerevisiae BY23 used in the invention has a wide antibacterial spectrum, and can simultaneously control penicilliosis, botrytis cinerea and black spot of apples, botrytis cinerea, grape black spot, fusarium, anthracnose and stalk point mildew of pears, and penicilliosis of citrus. (3) The saccharomyces cerevisiae BY23 provided BY the invention has good growth in YPD medium, easy culture and stable properties, and the bacterial suspension with a certain concentration can effectively prevent and treat postharvest diseases of various fruits BY being used alone, so that the use cost is low and the market prospect is wide. (4) The saccharomyces cerevisiae BY23 provided BY the invention can replace a chemical bactericide to prevent and treat postharvest diseases of fruits, avoids harm to people caused BY the use of the chemical bactericide, does not pollute the environment, and has remarkable social and ecological benefits.
The present invention is illustrated in more detail by the following examples. The following embodiments are merely illustrative, and the present invention is not limited to these embodiments.
Drawings
FIG. 1 is a diagram showing the nucleotide sequence of the 26S rDNA D1/D2 region of Saccharomyces cerevisiae BY23 according to the present invention.
FIG. 2 shows the inhibitory effect of Saccharomyces cerevisiae BY23 on Penicillium, Botrytis and black spot of apple. Note: control: sterile water, i.e. control group; s.c: 1X 108cells/mL of Saccharomyces cerevisiae suspension. Different letters represent significant differences (P < 0.05).
FIG. 3 shows the effect of Saccharomyces cerevisiae BY23 on the inhibition of gray mold of pear fruit BY Saccharomyces cerevisiae. Note: control: sterile water, i.e. control group; s.c: 1X 108cells/mL of Saccharomyces cerevisiae BY23 bacterial suspension. Different letters represent significant differences (P < 0.05).
FIG. 4 shows the inhibitory effect of Saccharomyces cerevisiae BY23 on grape black spot, Fusarium mold, anthracnose, and stalk mold. Note: control: sterile water, i.e. control group；S.c：1×108cells/mL of Saccharomyces cerevisiae BY23 bacterial suspension. Different letters represent significant differences (P < 0.05).
FIG. 5 shows the effect of Saccharomyces cerevisiae BY23 on the inhibition of penicilliosis in citrus. Note: control: sterile water, i.e. control group; s.c: 1X 108cells/mL of Saccharomyces cerevisiae BY23 bacterial suspension. Different letters represent significant differences (P < 0.05).
Detailed Description
Example 1: biological Properties of Saccharomyces cerevisiae BY23
1. Morphological characteristics
(1) Culturing on YPDA medium (yeast extract powder 1%, peptone 2%, glucose 2%, agar 1.8%, sterilizing at 121 deg.C for 20min) at 26 deg.C for 48 hr to obtain circular and white colony with smooth and round edge. The cell morphology is ellipsoidal.
(2) After culturing in YPDA liquid medium for 24h, no pellicle is formed, the bacterial liquid is turbid, precipitates exist, the microscopic yeast cells are oval, and buds grow.
2. Molecular biological identification
The nucleotide sequence of the region of Saccharomyces cerevisiae BY 2326S rDNA D1/D2 was amplified BY PCR using the universal forward primer NL-1 (5'-GCATATCAATAAGCGGAGGAAAAG-3') and reverse primer NL-4 (5'-GGTCCGTGTTTCAAGACGG-3'), the sequencing result of the PCR product was inputted into the website www.NCBI.nlm.nih.gov for BLAST, the homologous sequence was downloaded from the GenBank database, the evolutionary tree was constructed BY MEGA6 software as shown in FIG. 1, and the strain selected was determined to be Saccharomyces cerevisiae (Saccharomyces cerevisiae).
The Saccharomyces cerevisiae BY23 is preserved in the common microorganism center of the China Committee for culture Collection of microorganisms of China academy of sciences, institute of microbiology, No.1, Xilu, No. 3, North Cheng, Chaoyang, China, the preservation time is 11 and 15 days in 2017, the preservation number is CGMCC No.14905, and the suggested classification is Saccharomyces cerevisiae.
EXAMPLE 2 inhibitory Effect of Saccharomyces cerevisiae BY23 on Penicillium and Botrytis cinerea
1. Experimental protocol
Taking out Saccharomyces cerevisiae BY23 from-80 deg.C refrigerator, and soaking in YPDA culture medium (yeast extract)10g of powder, 20g of peptone, 20g of glucose, 18g of agar, 1000ml of deionized water, natural pH, sterilization at 121 ℃ for 30min), selecting single colonies into a YPD liquid culture medium, culturing at 26 ℃ and 200r/min for 24h, centrifuging at 4000rpm for 5min, discarding supernatant, repeatedly cleaning collected thalli with sterile water for 3 times, and counting by a blood counting plate to prepare the mixture with the concentration of 1 × 108cells/mL of Saccharomyces cerevisiae BY23 bacterial suspension.
Activating Penicillium expansum, Botrytis cinerea or Alternaria alternata on PDA culture medium plate, culturing at 26 deg.C for 7-14 days, scraping appropriate amount of spores, and preparing with sterile water to obtain the final product with concentration of 5 × 104cells/mL of Penicillium, Botrytis, or Alternaria spore suspension.
Sterilizing healthy and undamaged apple fruits for 5min by using 2% sodium hypochlorite, washing with deionized water, drying, and punching 5 holes in the equator of the fruits by using an aseptic puncher, wherein the surface wound is 2mm (diameter) multiplied by 2mm (depth). Equal amounts of 20 μ L of the following treatment solutions were added to each wound: (1) 1X 108cells/mL of saccharomyces cerevisiae BY23 bacterial suspension; (2) sterile distilled water. After 4h, 20. mu.L of a suspension of the spores of Penicillium, Botrytis or Alternaria alternata was inoculated. After air drying, the fruits are placed in a plastic box, the relative humidity is kept at 95%, and the incidence rate of the fruits is recorded after the fruits are placed at room temperature (25 ℃) for 4 days, so that the bacteriostatic effect of the saccharomyces cerevisiae BY23 is evaluated. The formula for calculating the incidence of disease is: incidence (%) is the total number of fruits/fruit affected × 100%.
2. Test results
According to the test of the steps, the result of counting the incidence rate of the apples is as follows:
(1) inhibitory effect of Saccharomyces cerevisiae BY23 on penicilliosis of apple
As shown in figure 2, the control group apple penicilliosis incidence rate is 100%, and the apple penicilliosis incidence rate treated BY saccharomyces cerevisiae BY23 is 33%, so saccharomyces cerevisiae BY23 can effectively control apple penicilliosis at room temperature.
(2) Inhibition effect of saccharomyces cerevisiae BY23 on apple gray mold
As shown in figure 2, the incidence rate of the gray mold of the apple in the control group is 100%, and the incidence rate of the gray mold of the apple treated BY the saccharomyces cerevisiae BY23 is 20%, so that the saccharomyces cerevisiae BY23 can effectively control the gray mold of the apple at room temperature.
(3) Inhibitory effect of Saccharomyces cerevisiae BY23 on apple black spot
As shown in figure 2, the incidence rate of the apple black spot disease of the control group is 100%, and the incidence rate of the apple black spot disease treated BY the saccharomyces cerevisiae BY23 is 33%, so that the saccharomyces cerevisiae BY23 can effectively control the apple black spot disease at room temperature.
EXAMPLE 3 inhibitory Effect of Saccharomyces cerevisiae BY23 on Botrytis cinerea
1. Experimental protocol
Taking out Saccharomyces cerevisiae BY23 from-80 deg.C refrigerator, activating with YPDA culture medium (yeast extract powder 10g, peptone 20g, glucose 20g, agar 18g, deionized water 1000ml, natural pH, sterilizing at 121 deg.C for 30min), selecting single colony to YPD liquid culture medium, culturing at 26 deg.C and 200r/min for 24h, centrifuging at 4000rpm for 5min, discarding supernatant, cleaning collected thallus with sterile water repeatedly for 3 times, counting with counting blood cell plate to obtain the final product with concentration of 1 × 108cells/mL of Saccharomyces cerevisiae BY23 bacterial suspension.
Activating Botrytis cinerea on PDA culture medium plate, culturing at 26 deg.C for 7-14 days, scraping appropriate amount of spore, and preparing with sterile water to obtain the product with concentration of 5 × 104cells/mL of Botrytis cinerea spore suspension.
Sterilizing healthy and undamaged pomes with 2% sodium hypochlorite for 5min, washing with deionized water, air drying, and punching 5 holes at the equator of the pomes with an aseptic puncher, wherein the surface wound is 2mm (diameter) × 2mm (depth). Equal amounts of 20 μ L of the following treatment solutions were added to each wound: (1) 1X 108cells/mL of saccharomyces cerevisiae BY23 bacterial suspension; (2) sterile distilled water. After 4h, 20. mu.L of Botrytis cinerea spore suspension was inoculated. After air drying, the fruits are placed in a plastic box, the relative humidity is kept at 95%, and the incidence rate of the fruits is recorded after the fruits are placed at room temperature (25 ℃) for 4 days, so that the bacteriostatic effect of the saccharomyces cerevisiae BY23 is evaluated. The formula for calculating the incidence of disease is: incidence (%) is the total number of fruits/fruit affected × 100%.
2. Test results
According to the test of the steps, the result of counting the incidence rate of the pear fruits is shown in fig. 3, the incidence rate of the gray mold of the pear fruits in the control group is 100%, and the incidence rate of the gray mold of the pear fruits treated BY the saccharomyces cerevisiae BY23 is 73%, so that the saccharomyces cerevisiae BY23 can effectively control the gray mold of the pear fruits at room temperature.
EXAMPLE 4 Effect of Saccharomyces cerevisiae BY23 on post-harvest disease inhibition of grape
1. Experimental protocol
Taking out Saccharomyces cerevisiae BY23 from-80 deg.C refrigerator, activating with YPDA culture medium (yeast extract powder 10g, peptone 20g, glucose 20g, agar 18g, deionized water 1000ml, natural pH, sterilizing at 121 deg.C for 30min), selecting single colony to YPD liquid culture medium, culturing at 26 deg.C and 200r/min for 24h, centrifuging at 4000rpm for 5min, discarding supernatant, cleaning collected thallus with sterile water repeatedly for 3 times, counting with counting blood cell plate to obtain the final product with concentration of 1 × 108cells/mL of Saccharomyces cerevisiae BY23 bacterial suspension.
Activating Alternaria alternata (Alternaria eichhorniae), Fusarium fragrans (Fusarium redolen), Colletotrichum fructicola (Colletotrichum fructicola) or Phoma sp on a PDA culture medium plate, culturing at 26 ℃ for 7-14 days, scraping a proper amount of spores, preparing into a culture medium with sterile water to obtain a culture medium with a concentration of 5 × 104cells/mL of Alternaria alternata, Fusarium fragrans, Colletotrichum fructicola or Phoma stipulatum spore suspensions.
Sterilizing healthy and undamaged pomes with 2% sodium hypochlorite for 5min, washing with deionized water, air drying, and punching 1 hole at the equator of the pome with an aseptic puncher, wherein the surface wound is 2mm (diameter) × 2mm (depth). Equal amounts of 20 μ L of the following treatment solutions were added to each wound: (1) 1X 108cells/mL of Saccharomyces cerevisiae strain BY23 bacterial suspension; (2) sterile distilled water. After 4h, 20. mu.L of Alternaria alternata, Fusarium fragrans, Colletotrichum fructicola or Phoma stipulatum spore suspension was inoculated. After air drying, the fruits are placed in a plastic box, the relative humidity is kept at 95%, and the incidence rate of the fruits is recorded after the fruits are placed at room temperature (25 ℃) for 4 days, so that the bacteriostatic effect of the saccharomyces cerevisiae BY23 is evaluated. The formula for calculating the incidence of disease is: incidence (%) is the total number of fruits/fruit affected × 100%.
2. Test results
According to the test of the steps, the result of counting the morbidity of the grape fruits is as follows:
(1) inhibitory effect of Saccharomyces cerevisiae BY23 on grape fruit melasma
As shown in FIG. 4, the incidence of grape fruit black spot in the control group was 100%, and the incidence of grape fruit black spot BY Saccharomyces cerevisiae BY23 was 67%, so Saccharomyces cerevisiae BY23 was able to control grape fruit black spot disease at room temperature.
(2) Inhibition effect of Saccharomyces cerevisiae BY23 on fusarium head blight of grape fruit
As shown in FIG. 4, the incidence rate of the fusarium mildew of the grape fruits in the control group is 100%, and the incidence rate of the fusarium mildew of the grape fruits treated BY the saccharomyces cerevisiae BY23 is 53%, so that the saccharomyces cerevisiae BY23 can effectively control the fusarium mildew of the grape fruits at room temperature.
(3) Inhibitory effect of Saccharomyces cerevisiae BY23 on grape fruit anthracnose
As shown in FIG. 4, the control group has 100% of grape fruit anthracnose, and the control group has 67% of grape fruit anthracnose treated BY Saccharomyces cerevisiae BY23, so that Saccharomyces cerevisiae BY23 can effectively control grape fruit anthracnose at room temperature.
(4) Inhibitory effect of Saccharomyces cerevisiae BY23 on grape fruit stalk point mildew
As shown in FIG. 4, the incidence rate of the grape fruit stem point mildew in the control group is 100%, and the incidence rate of the grape fruit stem point mildew treated BY the Saccharomyces cerevisiae strain BY23 is 53%, so that the Saccharomyces cerevisiae BY23 can effectively control the grape fruit stem point mildew at room temperature.
EXAMPLE 5 inhibitory Effect of Saccharomyces cerevisiae BY23 on Penicillium citrinum
1. Experimental protocol
Taking out Saccharomyces cerevisiae BY23 from a refrigerator at-80 deg.C, activating with YPDA culture medium (yeast extract powder 10g, peptone 20g, glucose 20g, agar 18g, deionized water 1000ml, natural pH, sterilizing at 121 deg.C for 30min), picking single colony to YPD liquid culture medium, culturing at 26 deg.C and 200r/min for 24h, centrifuging at 4000rpm for 5min,discarding supernatant, repeatedly cleaning collected thallus with sterile water for 3 times, and counting with blood counting plate to obtain the final product with concentration of 1 × 108cells/mL of Saccharomyces cerevisiae BY23 bacterial suspension.
Activating Penicillium (Penicillium italicum) on PDA culture medium plate, culturing at 26 deg.C for 7-14 days, scraping appropriate amount of spore, and preparing with sterile water to obtain the final product with concentration of 5 × 104cells/mL of Penicillium spore suspension.
Sterilizing healthy and undamaged strawberry fruits by using 2% sodium hypochlorite for 5min, washing with deionized water, drying, and punching 1 hole at the equator of the fruits by using an aseptic puncher, wherein the surface wound is 2mm (diameter) multiplied by 2mm (depth). Equal amounts of 20 μ L of the following treatment solutions were added to each wound: (1) 1X 108cells/mL of saccharomyces cerevisiae BY23 bacterial suspension; (2) sterile distilled water. After 4h, 20. mu.L of the Penicillium spore suspension was inoculated. After air drying, the fruits are placed in a plastic box, the relative humidity is kept at 95%, and the incidence rate of the fruits is recorded after the fruits are placed at room temperature (25 ℃) for 4 days, so that the bacteriostatic effect of the saccharomyces cerevisiae BY23 is evaluated. The formula for calculating the incidence of disease is: incidence (%) is the total number of fruits/fruit affected × 100%.
2. Test results
According to the test of the steps, the result of counting the incidence rate of the citrus fruits is shown in fig. 5, the incidence rate of the penicilliosis of the control group citrus fruits is 73%, and the incidence rate of the penicilliosis of the citrus fruits treated BY the saccharomyces cerevisiae BY23 is 27%, so the saccharomyces cerevisiae BY23 can effectively control the penicilliosis of the citrus fruits at room temperature.
Claims (4)
1. Saccharomyces cerevisiae (A) for preventing and treating fruit postharvest diseasesSaccharomyces cerevisiae) BY23 strain, characterized BY: the BY23 strain is submitted to preservation in China general microbiological culture Collection center, and the preservation number of the strain is CGMCC No. 14905.
2. The use of the BY23 strain of claim 1 for the control of postharvest disease in fruit: the method comprises the following steps: activating the BY23 strain, fermenting and culturing in YPD liquid culture medium, and centrifuging to obtainThe obtained thallus is prepared with sterile water to concentration of 1 × 108cells/mL of bacterial suspension; putting fruits into the bacterial suspension, soaking for 30 seconds, taking out, and air-drying; putting into a fresh-keeping box, sealing, and storing at normal temperature;
the BY23 strain is used for controlling penicillium expansum (A) of applePenicillium expansum) Botrytis cinerea (A), (B), (C), (B), (C), (B), (C)Botrytis cinerea) Or Alternaria tenuissima (A), (B), (C)Alternaria tenuissima) Botrytis cinerea (Botrytis cinerea) of pear fruitBotrytis cinerea) Alternaria cucurbitae of Vitis vinifera (Alternaria cucurbitae, Inc.)Alternaria eichhorniae) Fusarium fragrans: (A) and (B)Fusarium redolens) Fruit anthrax bacteria (A), (B)Colletotrichum fructicola) Or Phoma mold (a)Phoma sp.) Penicillium italicum of citrus (Penicillium italicum)。
3. The use as claimed in claim 2, wherein the BY23 strain is removed from a-80 ℃ refrigerator, activated BY YPDA medium, single colony is picked up to YPD liquid medium, cultured at 26 ℃ and 200r/min for 24h, centrifuged at 4000rpm for 5min to collect the thallus, and washed 3 times with sterile water.
4. Use according to claim 3, characterized in that the YPDA medium is: 10g of yeast extract powder, 20g of peptone, 20g of glucose, 18g of agar and 1000ml of deionized water, and sterilizing at 121 ℃ for 30min under natural pH.
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| CN112143659B (en) * | 2020-06-10 | 2022-11-04 | 南京万拓生物科技有限公司 | Pichia kluyveri for green production of fruit wine in whole process and application of pichia kluyveri |
| CN112111416B (en) * | 2020-06-10 | 2022-11-04 | 南京万拓生物科技有限公司 | Issatchenkia orientalis strain for whole-process green production of fruit wine and application thereof |
| CN111887293B (en) * | 2020-07-14 | 2022-10-28 | 江苏大学 | Application of saccharomyces cerevisiae in disease control, storage and fresh keeping of picked baby cabbage |
| CN113943664B (en) * | 2021-08-31 | 2023-03-03 | 福州大学 | Yeast P4 for controlling postharvest diseases of bananas and application method thereof |
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