CN107988088B - Application of pichia deserticola in prevention and treatment of postharvest diseases of fruits - Google Patents
Application of pichia deserticola in prevention and treatment of postharvest diseases of fruits Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
- C12N1/165—Yeast isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/84—Pichia
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
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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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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
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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
Abstract
The invention discloses a Pichia pastoris strain BY25 with wide antibacterial spectrum and stable effect for preventing and treating postharvest diseases of fruits and vegetables, and a use method and application thereof. The number of the strain preserved in China general microbiological culture Collection center is CGMCC No. 14906. The application method of the pichia pastoris 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 and vegetables such as apples, pears, grapes, strawberries, oranges or cherry tomatoes 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 pichia pastoris strain can simultaneously control the penicilliosis, the gray mold and the black spot of apples, the penicilliosis and the gray mold of pears, the gray mold, the aspergillosis, the black spot, the pink mildew and the stalk spot mold of grapes, the gray mold and the aspergillosis of strawberries, the penicilliosis of oranges and tangerines, and the gray mold and the aspergillosis of cherry tomatoes, so that the loss caused by the postharvest diseases is reduced, and the pichia pastoris strain has a good application prospect.
Description
Technical Field
The invention relates to the field of biological control of fruit postharvest diseases, in particular to application of Pichia deserticola (Pichia pastoris) in control of fruit postharvest diseases.
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 disease control can be achieved by many means including agricultural control, physical control, chemical control and biological control, the main current practice is chemical control (Eckert & Ogawa, 1985, 1988). However, long-term use of chemical pesticides not only causes resistance to pathogenic bacteria and reduces the bactericidal effect (Prusky et al, 1985; Vi ň as et al, 1991; Holmes & Eckert, 1999), but frequent use of high concentrations of chemical agents also increases the residual amount of pesticides on fruits, seriously threatens human health, and causes environmental pollution (Gullion & Kuijipers, 1994). Therefore, the development of a new technology for safe, efficient, non-toxic, low-resistant fruit postharvest disease control is currently the focus of research in countries of the world (Falik et al, 1995; Tian et al, 2001; Kulakiotiet al, 2004), where the use of bio-antagonistic bacteria for control is a new method that has been proven safe and effective at present (Wilson & Wisnewski, 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 the application of a Pichia stipitis (Pichia desublicola) BY25 strain in the disease control of fruit postharvest, wherein the BY25 strain is obtained BY screening and separating a naturally fermented wine grape mash and submitted to preservation in the China general microbiological culture Collection center, and the number of the preserved strain is CGMCC No. 14906; the method comprises the following steps: activating BY25 strain, fermenting and culturing in YPD liquid culture medium, centrifuging to obtain thallus, and preparing into 1 × 10 concentration with sterile water8Bacterial suspension per mL; 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 room temperature.
Preferably, the fruits are apples, pears, grapes, strawberries, citrus and cherry tomatoes.
Preferably, the bacterial suspension is prepared by: taking out the strain from a 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.
Preferably, 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.
Preferably, the BY25 strain is used to control apple penicilliosis, gray mold and black spot disease, pear penicilliosis and gray mold, grape gray mold, aspergillosis, black spot disease, pink powder disease and stalk point mold, strawberry gray mold and aspergillosis, citrus penicilliosis, and cherry tomato gray mold and aspergillosis simultaneously.
The strain provided BY the invention is the pichia pastoris BY25 which has remarkable prevention and treatment effects on the postharvest diseases of apples, pears, grapes, strawberries, oranges and cherry tomatoes and is screened and separated from the naturally fermented wine grape 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 prospect.
The invention has the advantages that: (1) the pichia pastoris BY25 provided BY the invention is obtained BY screening from wine fermentation mash in a laboratory, is harmless to human bodies and has high safety. (2) The Pichia pastoris BY25 used in the invention has a wide antibacterial spectrum, and can simultaneously control the penicilliosis, gray mold and black spot of apples, the penicilliosis and gray mold of pears, the gray mold of grapes, the aspergillosis, the black spot, the pink disease and the stalk point mold, the gray mold and aspergillosis of strawberries, the penicilliosis of citrus, and the gray mold and aspergillosis of cherry tomatoes. (3) The pichia pastoris BY25 provided BY the invention grows well in a YPD culture medium, is easy to culture and stable in property, and can effectively prevent and treat postharvest diseases of various fruits BY singly using the bacterial suspension with a certain concentration, so that the use cost is low, and the market prospect is wide. (4) The pichia pastoris BY25 provided BY the invention can replace a chemical bactericide to prevent and control fruit postharvest diseases, avoids the harm of the chemical bactericide to people, 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 evolution of the 26S rDNA D1/D2 region of Pichia stipitis BY 25.
FIG. 2 shows the inhibitory effect of Pichia pastoris BY25 on penicilliosis, gray mold and black spot of apple. Note: control: sterile water, i.e. control group; p.d: 1X 108one/mL of pichia pastoris BY25 bacterial suspension. Different letters represent significant differences (P)<0.05)。
FIG. 3 shows the effect of Pichia pastoris BY25 on the inhibition of pear penicilliosis and gray mold BY Pichia pastoris. Note: control: sterile water, i.e. control group; p.d: 1X 108one/mL of pichia pastoris BY25 bacterial suspension. Different letters represent significant differences (P)<0.05)。
FIG. 4 shows the inhibitory effect of Pichia pastoris BY25 on grape gray mold, aspergillosis, black spot, pink disease and stalk mold. Note: control: sterile water, i.e. control group; p.d: 1X 108one/mL of pichia pastoris BY25 bacterial suspension. Different letters represent significant differences (P)<0.05)。
FIG. 5 shows the inhibitory effect of Pichia pastoris BY25 on gray mold and aspergillosis of strawberry. Note: control: sterile water, i.e. control group; p.d: 1X 108individual/mL pichia yeast suspension. Different letters represent significant differences (P)<0.05)。
FIG. 6 shows the effect of Pichia pastoris BY25 on the inhibition of penicilliosis in citrus. Note: control: sterile water, i.e. control group; p.d: 1X 108individual/mL pichia yeast suspension. Different letters represent significant differences (P)<0.05). FIG. 7 shows the inhibitory effect of Pichia pastoris BY25 on gray and aspergillosis of cherry tomato. Note: control: sterile water, i.e. control group; p.d: 1X 108individual/mL pichia yeast suspension. Different letters represent significant differences (P)<0.05)。
Detailed Description
Example 1: biological Properties of Pichia deserticola Pichia pastoris Strain BY25
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
PCR was performed using the universal forward primer NL-1 (5'-GCATATCAATAAGCGGAGGAAAAG-3') and reverse primer NL-4 (5'-GGTCCGTGT TTCAAGACGG-3') to amplify the nucleic acid sequence of region 26S rDNA D1/D2, the sequencing results of the PCR products were entered into the website www.NCBI.nlm.nih.gov for BLAST, the homologous sequences were downloaded from the GenBank database, the evolutionary tree was constructed by MEGA6 software as shown in FIG. 1, and the screened strain was identified as Pichia deserticola (Pichia deserticola).
The Pichia pastoris BY25 is preserved in the common microorganism center of the China Committee for culture Collection of microorganisms of the institute of microbiology of China academy of sciences, No. 3 of West Lu 1, North Cheng, No. 3, in Beijing, China, the preservation time is 11 months and 15 days in 2017, the preservation number is CGMCC No.14906, and the proposed classification is named as the desert Pichia pastoris.
Example 2 inhibitory Effect of Pichia pastoris BY25 on Penicillium, Botrytis and Black Spot disease in apple
Experimental protocol
Taking out Pichia pastoris BY25 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 final product with concentration of 1 × 108one/mL of pichia pastoris BY25 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 × 104Per mL of a suspension of spores of Penicillium, Botrytis or Alternaria.
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 108The strain per mL of pichia pastoris BY25 bacterial suspension; (2) sterile distilled water. After 4h, 20. mu.L of Penicillium, Botrytis or Alternaria 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 pichia pastoris BY25 is evaluated. The formula for calculating the incidence of disease is: incidence (%) is the total number of fruits/fruit affected × 100%.
Test results
According to the test of the steps, the result of counting the incidence rate of the apples is as follows:
(1) inhibition effect of pichia pastoris BY25 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 pichia pastoris BY25 is 53%, so pichia pastoris BY25 can effectively control apple penicilliosis.
(2) Inhibition effect of pichia pastoris BY25 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 pichia pastoris BY25 is 6%, so that the pichia pastoris BY25 can effectively control the gray mold disease of the apple.
(3) Inhibition effect of pichia pastoris BY25 on apple black spot
As shown in fig. 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 BY25 is 61%, so BY25 can effectively control the apple black spot disease.
Example 3 inhibitory Effect of Pichia pastoris BY25 on Penicillium and Blackspot disease in pear
Experimental protocol
Taking out Pichia pastoris BY25 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 final product with concentration of 1 × 108one/mL of pichia pastoris BY25 bacterial suspension.
Activating Penicillium expansum or 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 5 × 104Per mL of a suspension of spores of Penicillium or Botrytis.
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 108The strain per mL of pichia pastoris BY25 bacterial suspension; (2) sterile distilled water. Inoculating 20 μ L of Penicillium bacteria or ash after 4 hrAnd (3) a mildew spore suspension. 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 pichia pastoris BY25 is evaluated. The formula for calculating the incidence of disease is: incidence (%) is the total number of fruits/fruit affected × 100%.
Test results
According to the test of the steps, the result of counting the incidence rate of the pear fruits is as follows:
(1) inhibition effect of pichia pastoris BY25 on pear penicilliosis
As shown in figure 3, the incidence rate of the fruit penicilliosis of the pear in the control group is 100%, and the incidence rate of the fruit penicilliosis treated BY the pichia pastoris BY25 is 73%, so that the pichia pastoris BY25 can effectively control the fruit penicilliosis of the pear.
(2) Inhibition effect of pichia pastoris BY25 on gray mold of pear fruit
As shown in figure 3, the incidence rate of the gray mold of the pear in the control group is 100%, and the incidence rate of the gray mold of the pear treated BY the pichia pastoris BY25 is 0, so that the pichia pastoris BY25 can effectively control the gray mold of the pear.
Example 4 inhibitory Effect of Pichia pastoris BY25 on post-harvest disease of grape
Experimental protocol
Taking out Pichia pastoris BY25 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 final product with concentration of 1 × 108one/mL of pichia pastoris BY25 bacterial suspension.
Activating Botrytis cinerea pathogenic bacteria such as Botrytis cinerea (Botrytis porri), Aspergillus aculeatus (Aspergillus aculeatus), Alternaria alternata (Alternaria ehhorniae), Trichothecium roseum (Trichothecium roseum) or Phoma sp on a PDA culture medium plate, culturing at 26 ℃ for 7-14 days, scraping a proper amount of spores, and preparing into a culture medium with a concentration of 5 × 10 by using sterile water4Per mL of a suspension of spores of Botrytis cinerea, Aspergillus aculeatus, Alternaria alternata, Tricholoma roseum or Phoma longissima.
Sterilizing healthy and undamaged grape fruits by using 2% sodium hypochlorite for 5min, washing with deionized water, drying, and punching 1 hole at the equator of the grape fruits by using a sterile 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 108The strain per mL of pichia pastoris BY25 bacterial suspension; (2) sterile distilled water. After 4h, 20. mu.L of a spore suspension of Botrytis cinerea, Aspergillus aculeatus, Alternaria alternata, Tricholoma roseum or Phoma longituba spores 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 pichia pastoris BY25 is evaluated. The formula for calculating the incidence of disease is: incidence (%) is the total number of fruits/fruit affected × 100%.
Test results
According to the test of the steps, the result of counting the morbidity of the grape fruits is as follows:
(1) inhibition effect of pichia pastoris BY25 on grape fruit gray mold
As shown in FIG. 4, the incidence rate of the gray mold of the grape fruits in the control group is 100%, and the incidence rate of the gray mold of the grape fruits treated BY the Pichia pastoris BY25 is 0, so that the Pichia pastoris BY25 can effectively control the gray mold of the grape fruits.
(2) Inhibition effect of pichia pastoris BY25 on grape fruit aspergillosis
As shown in FIG. 4, the incidence rate of the grape fruit aspergillosis of the control group is 100%, and the incidence rate of the grape fruit aspergillosis treated BY the Pichia pastoris BY25 is 35%, so that the Pichia pastoris BY25 can effectively control the caused grape fruit aspergillosis disease.
(3) Inhibition effect of pichia pastoris BY25 on grape fruit black spot
As shown in fig. 4, the incidence rate of black spot disease of grape fruits in the control group is 100%, and the incidence rate of black spot disease of grape fruits passing through pichia pastoris BY25 is 67%, so pichia pastoris BY25 can control the black spot disease of grape fruits.
(4) Inhibition effect of pichia pastoris BY25 on grapevine pink disease
As shown in FIG. 4, the incidence rate of the fusarium botrytis of the control group is 100%, and the incidence rate of the fusarium botrytis treated BY the Pichia pastoris BY25 is 80%, so that the Pichia pastoris BY25 can effectively control the fusarium botrytis disease.
(5) Inhibition effect of pichia pastoris BY25 on grape fruit stalk point mildew
As shown in FIG. 4, the incidence rate of the grape stem point mildew in the control group is 100%, and the incidence rate of the grape stem point mildew treated BY the Pichia pastoris BY25 is 67%, so that the Pichia pastoris BY25 can effectively control the grape stem point mildew disease.
Example 5 inhibitory Effect of Pichia pastoris BY25 on Gray mold and aspergillosis of strawberry fruit
Experimental protocol
Taking out Pichia pastoris BY25 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 final product with concentration of 1 × 108one/mL of pichia pastoris BY25 bacterial suspension.
Activating Botrytis porari (Botrytis porri) or Aspergillus aculeatus (Aspergillus aculeatus) of gray mold pathogenic bacteria on a PDA culture medium plate, culturing at 26 ℃ for 7-14 days, scraping a proper amount of spores, and preparing into 5 × 10 sterile water4Per mL of a suspension of Botrytis cinerea or Aspergillus aculeatus spores.
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 108The strain per mL of pichia pastoris BY25 bacterial suspension; (2) sterile distilled water. After 4h, 20. mu.L of Botrytis cinerea or Aspergillus aculeatus spore suspension was inoculated. After air drying, the fruits were placed in a plastic box with a relative humidity of 95% and left at room temperature (25 ℃) for 4 daysAnd then recording the fruit morbidity, and evaluating the bacteriostatic effect of the pichia pastoris BY 25. The formula for calculating the incidence of disease is: incidence (%) is the total number of fruits/fruit affected × 100%.
Test results
According to the test of the steps, the result of counting the incidence rate of the strawberry fruits is as follows:
(1) inhibition effect of pichia pastoris BY25 on gray mold of strawberry fruits
As shown in FIG. 5, the incidence rate of gray mold of strawberry fruits in the control group is 69%, and the incidence rate of gray mold of strawberry fruits treated BY Pichia pastoris BY25 is 20%, so that the Pichia pastoris can effectively control gray mold of strawberry fruits.
(2) Inhibition effect of pichia pastoris BY25 on strawberry fruit aspergillosis
As shown in fig. 5, the incidence rate of strawberry fruit aspergillosis in the control group is 60%, and the incidence rate of strawberry fruit aspergillosis treated BY pichia pastoris BY25 is 0, so pichia pastoris can effectively control strawberry fruit aspergillosis diseases.
Example 6 inhibitory Effect of Pichia pastoris BY25 on Penicillium citrinum
Experimental protocol
Taking out Pichia pastoris BY25 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 final product with concentration of 1 × 108one/mL of pichia pastoris BY25 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 × 104Sterilizing healthy and undamaged citrus fruits with 2% sodium hypochlorite for 5min, washing with deionized water, air drying, perforating 1 hole at equator of the fruit with sterile perforator, and equally adding 2mm (diameter) x 2mm (depth) on surface woundAdd 20. mu.L of the following treatment: (1) 1X 108The strain per mL of pichia pastoris BY25 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 pichia pastoris BY25 is evaluated. The formula for calculating the incidence of disease is: incidence (%) is the total number of fruits/fruit affected × 100%.
Test results
According to the test of the steps, the result of counting the incidence rate of the citrus fruits is shown in fig. 6, 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 pichia pastoris BY25 is 27%, so that the pichia pastoris BY25 can effectively control the penicilliosis of the citrus fruits.
Example 7 inhibitory Effect of Pichia pastoris BY25 on Gray mold and aspergillosis of cherry tomato
Experimental protocol
Taking out Pichia pastoris BY25 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 final product with concentration of 1 × 108one/mL of pichia pastoris BY25 bacterial suspension.
Activating Botrytis porari (Botrytis porri) or Aspergillus aculeatus (Aspergillus aculeatus) of gray mold pathogenic bacteria on a PDA culture medium plate, culturing at 26 ℃ for 7-14 days, scraping a proper amount of spores, and preparing into 5 × 10 sterile water4Per mL of a suspension of Botrytis cinerea or Aspergillus aculeatus spores.
Sterilizing health cherry tomato with 2% sodium hypochlorite for 5min, washing with deionized water, air drying, and perforating 1 hole with sterile perforator at the equator of the cherry tomato with surface wound of 2mm (diameter) × 2mm (depth). Equal amounts of 20 μ L of the following treatment solutions were added to each wound: (1) 1X 108The strain per mL of pichia pastoris BY25 bacterial suspension; (2) sterile distilled water. Inoculation after 4h20 μ L of Botrytis cinerea (Botrytis porri) or Aspergillus aculeatus (Aspergillus aculeatus) spore suspension. 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 pichia pastoris strain BY25 is evaluated. The formula for calculating the incidence of disease is: incidence (%) is the total number of fruits/fruit affected × 100%.
Test results
According to the test of the steps, the result of counting the morbidity of the cherry tomatoes is as follows:
(1) inhibition effect of pichia pastoris BY25 on gray mold of cherry tomato
As shown in fig. 7, the incidence rate of gray mold of cherry tomatoes in the control group was 97%, and the incidence rate of gray mold of cherry tomatoes treated BY pichia pastoris BY25 was 53%, so pichia pastoris BY25 was able to effectively control gray mold disease of cherry tomatoes caused BY gray mold.
(2) Inhibition effect of pichia pastoris BY25 on cherry tomato aspergillus
As shown in fig. 7, the incidence rate of the cherry tomato aspergillus of the control group was 100%, and the incidence rate of the cherry tomato aspergillus treated BY pichia pastoris BY25 was 26%, so pichia pastoris BY25 was able to effectively control the cherry tomato aspergillus disease.
Claims (4)
1. Pichia stipitis (A. stipitis) for preventing and treating postharvest diseases of fruitsPichia deserticola) BY25 strain, characterized BY: the BY25 strain is obtained BY screening and separating naturally fermented wine grape mash and submitted to preservation in China general microbiological culture Collection center with the preservation number of CGMCC No. 14906.
2. The use of the BY25 strain according to claim 1 for the control of postharvest disease in fruit, characterized in that: the method comprises the following steps: activating BY25 strain, fermenting and culturing in YPD liquid culture medium, centrifuging to obtain thallus, and preparing into 1 × 10 concentration with sterile water8Bacterial suspension per mL; putting fruits into the bacterial suspension, soaking for 30 seconds, taking out, and air-drying; putPutting the mixture into a fresh-keeping box, sealing the fresh-keeping box, and then putting the fresh-keeping box into a room temperature for storage; said fruit is selected from the group consisting of apple, pear, grape, strawberry, citrus and cherry tomato;
the BY25 strain is used for controlling penicillium expansum (A) of applePenicillium expansum) Botrytis cinerea (A), (B), (CBotrytis cinerea) Or Alternaria tenuissima (A), (B), (C)Alternaria tenuissima) Penicillium expansum of pear fruit (Penicillium expansum) Or Botrytis cinerea (Botrytis cinerea) Garlic blind species of grape (Botrytis cinerea)Botrytis porri) Aspergillus aculeatus (A.aculeatus)Aspergillus aculeatus) Alternaria cucurbitae: (Alternaria eichhorniae) Trichotheca pinicola (A) and (B)Trichothecium roseum) Or Phoma mold (a)Phoma sp.) Garlic Blind species of strawberry, Botrytis cinerea (Botrytis porri) Or Aspergillus aculeatus (Aspergillus aculeatus) Penicillium italicum of citrus (Penicillium italicum) And Vitaceae, a blind species of garlic of cherry tomato: (Botrytis porri) Or Aspergillus aculeatus (Aspergillus aculeatus)。
3. The use according to claim 2, wherein the bacterial suspension is prepared by: taking out the strain from a 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.
4. Use according to claim 3, wherein 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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