CN112159767B - Method for improving biological control efficiency of antagonistic yeast of acid pathogenic fungi after fruit and vegetable harvest and application - Google Patents
Method for improving biological control efficiency of antagonistic yeast of acid pathogenic fungi after fruit and vegetable harvest and application Download PDFInfo
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Abstract
The invention relates to a method for improving the biological control efficiency of antagonistic yeast of acid pathogenic fungi after fruit and vegetable harvest, which comprises the following steps: activating a biological yeast; performing liquid culture on the activated biocontrol saccharomycetes; the biocontrol yeast is marine yeast Rhodosporidium paludigenum Fell & Tallman, the strain is IMI 394084, and organic acid is added into a culture medium to improve the capability of antagonizing acid pathogenic fungi of the yeast after fruit and vegetable harvest, wherein the acid pathogenic fungi comprise: penicillium digitatum, penicillium italicum, penicillium expansum, botrytis cinerea and sclerotinia sclerotiorum can effectively improve the survival rate of antagonistic yeast in different organic acid stresses, improve the capability of antagonistic yeast in preventing postharvest diseases, are safe, efficient, green and pollution-free, accord with natural and environment-friendly concepts, and are easy to realize in large scale due to high growth and propagation speed of microorganisms.
Description
Technical Field
The invention belongs to the field of postharvest disease control of fruits and vegetables, relates to antagonistic yeast biological control technology, and in particular relates to a method for improving the antagonistic yeast biological control efficacy of acidic pathogenic fungi of fruits and vegetables after harvest and application thereof.
Background
Diseases of fruits and vegetables after picking are mainly caused by pathogenic fungi, so that the loss of fruit and vegetable products in China reaches hundreds of billions of primordial coins each year, and the resource and economic losses are serious. Not only does the pathogenic fungi cause a large loss in fruit quantity, but also the large quantities of secondary metabolites secreted by many pathogenic fungi may cause serious food safety problems such as patulin produced by penicillium expansum, etc.
The pathogenic fungi causing postharvest diseases of fruits and vegetables mainly include penicillium, rhizopus, alternaria, botrytis, and sclerotinia, and are classified into 2 types, i.e., acid pathogenic fungi and alkaline pathogenic fungi. Among them, the acid pathogenic fungi occupy the main body of postharvest fungal diseases, including penicillium digitatum, penicillium italicum, penicillium expansum, etc., botrytis cinerea, etc., and sclerotinia sclerotiorum capable of infecting various hosts, etc., causing the most serious loss. The acid pathogenic fungi can secrete oxalic acid, citric acid, glucuronic acid and other organic acids in the growth and infection processes, so that the environment and host tissues are acidified, toxic factors of the acid pathogenic fungi are activated, generation of hydrogen peroxide and other resistance signal molecules in peel tissues is inhibited, plant defense reaction is hijacked, and pathogenicity of the acid pathogenic fungi is further enhanced.
Among the many methods studied to replace chemical bactericides, biological control techniques for inhibiting postharvest fruit diseases using antagonistic microorganisms, particularly antagonistic yeasts, are currently one of the most recently focused novel postharvest disease control methods at home and abroad. Yeast shows better tolerance to environmental conditions such as low temperature, high temperature, drying, low oxygen level, pH value fluctuation, ultraviolet radiation and the like. However, low concentrations of organic acids have been shown to still stress yeast growth and the like, inhibiting their growth, resulting in an extended lag phase.
At present, little research is done on how to enhance the stable biological control effect of antagonistic yeast at the wound of acidic fruits, and the development of technology for directionally improving the activity of fruit biological yeast is severely limited. At present, the method for improving the acid resistance and the biological activity of the yeast is mainly focused on the aspect of genetic engineering fungus transformation, and the research on effectively enhancing the biological control efficacy of antagonistic yeast by a fermentation process optimization method in combination with acidic pathogenic fungus virulence factors (oxalic acid, citric acid, glucuronic acid and the like) has not been reported yet.
Through a search of the prior published patent documents, the following several related published patent documents are found:
1. a method for suppressing postharvest diseases of fruits by inducing resistance and a preparation (publication No. CN 106085890B) used are disclosed, wherein the preparation is composed of cell walls of rhodosporidium marine and water, and each 1L of preparation contains 0.1-10 g of cell walls of rhodosporidium marine. The invention also provides a method for inhibiting postharvest diseases of fruits by inducing resistance by using the preparation, and the pretreatment of any one of the following modes is carried out before boxing and storage of the fruits: firstly, soaking fruits in the preparation, draining, and then placing the soaked fruits in the container to keep the fruits in a sealed state for 23-25 hours; spraying the preparation on the surface of fruits before picking the fruits in the second pretreatment mode, and then placing the fruits in a container to keep the fruits in a sealed state for 23-25 hours; the pretreated fruit is taken out of the container and then is filled in a box. The invention can effectively control the postharvest diseases of fruits without using chemical bactericides.
2. A method for regulating and controlling the acid stress resistance of Torulopsis glabrata by using a transcription factor Crz1p (publication number: CN 105400770B) discloses a method for regulating and controlling the acid stress resistance of Torulopsis glabrata by using the transcription factor Crz1p, and belongs to the field of bioengineering. The invention correspondingly reduces or improves the acid stress resistance of the strain by deleting or over-expressing the Cgcrz1 gene of the torulopsis glabrata. The invention also compares the fatty acid, sterol component, proportion and permeability of cell membrane of deletion mutant strain Cgcrz1 delta under acid stress, and finds that Crz1p is an essential transcription factor of Torulopsis glabrata for resisting acid stress, and the over-expression of Crz1p can improve the acid stress resistance of Torulopsis glabrata.
3. A co-culture method for improving acid resistance of saccharomycetes (publication number: CN 109370933A) discloses a co-culture method for improving acid resistance of saccharomycetes, and belongs to the technical field of bioengineering. The method comprises the steps of culturing the cultured lactobacillus in a culture medium for 4 hours, inoculating saccharomycetes, co-culturing for 4 hours, suspending the obtained lactobacillus in physiological saline (pH=1.9 and 30 ℃) containing lactic acid for stress for 90 minutes, centrifugally washing the acid-stressed thallus, dibbling the thallus on a saccharose peptone glucose agar culture medium (containing 0.01% chloramphenicol), standing and culturing for 48 hours at 30 ℃, wherein the biomass of the saccharomycetes is respectively 7.16 times higher than that of cells which are not co-cultured. The method is simple and convenient to operate, and can be used for improving the stress resistance of the saccharomycetes.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, and provides a method for improving the biological control efficacy of acid pathogenic fungi antagonistic yeasts after fruit and vegetable picking based on microecological principles, including competition, parasitism and other comprehensive actions, which is safe and efficient to use, green and pollution-free, accords with natural and environment-friendly concepts, and is easy to implement in large scale due to the rapid growth and propagation speed of microorganisms.
The invention solves the technical problems by adopting the following technical scheme:
a method for improving the biological control efficiency of antagonistic yeast of acid pathogenic fungi after fruit and vegetable harvest comprises the following steps:
activating a biological yeast;
performing liquid culture on the activated biocontrol saccharomycetes;
the biocontrol yeast is marine yeast Rhodosporidium paludigenum Fell & Tallman, and the strain preservation number is IMI 394084;
1-500mmol L is added into the culture medium -1 An organic acid.
Moreover, the organic acid includes: oxalic acid, fumaric acid, citric acid, glucuronic acid, malic acid or ascorbic acid.
The concentration of each organic acid added was as follows: oxalic acid 1-10mmol L -1 1-100mmol L of fumaric acid -1 1-10mmol L of citric acid -1 Glucuronic acid 1-500mmol L -1 1-100mmol L malic acid -1 Or ascorbic acid 1-100mmol L -1
Moreover, in the step (A), an activation culture medium is as follows:
200 g of potato, and the weight of the potato,
glucose or fructose 0-20 g,
glucuronic acid 1-500mmol L -1 ，
20 g of agarose, which has the advantages of high purity,
1 liter of tap water and sterilizing to obtain an activated culture medium.
In the step, the liquid culture medium is as follows:
200 g of potato, and the weight of the potato,
glucose or fructose 0-20 g,
glucuronic acid 1-500mmol L -1 ，
And (3) fixing the volume to 1L by tap water, and sterilizing to obtain liquid culture.
Moreover, in the preferred activation medium and liquid medium: the addition amount of glucose or fructose is 1-20 g, and the addition concentration of glucuronic acid is 10-200mmol L -1 。
Furthermore, the optimal incubation time in liquid medium was 48h.
An application for improving the biological control efficiency of antagonistic yeast of acid pathogenic fungi after fruit and vegetable harvest is characterized in that: the application of the yeast of the biological yeast ocean Rhodosporidium paludigenum Fell and Tallman IMI 394084 in improving the capability of antagonizing the acid pathogenic fungi of the picked fruits and vegetables.
Moreover, the acid pathogenic fungi include: penicillium digitatum, penicillium italicum, penicillium expansum, botrytis cinerea of Botrytis genus.
Moreover, the method is applied to biological control of acid pathogenic fungi antagonistic yeasts after fruit and vegetable harvest in any of the following modes:
mode a: concentration is set to 10 7 Spraying antagonistic yeast of cells/mL on the surface of the fruits before picking to moisten the surfaces of the fruits;
mode B: soaking picked fruits at a concentration of 10 7 Soaking in the cell/mL antagonistic yeast suspension for 1-30 min, and standing and airing at 25 ℃ and relative humidity of 90%.
The invention has the advantages and positive effects that:
(1) The fruit disease control method based on antagonistic yeast is one of the novel postharvest disease control methods which are the most focused at home and abroad at present, is based on the principle of microecology, comprises competition, parasitism, other comprehensive actions and the like, is safe, green and pollution-free, accords with the natural and environment-friendly concepts, and is easy to realize large-scale equipment due to the rapid growth and propagation speed of microorganisms.
(2) The D-glucuronic acid is a chemical agent, is a six-carbon sugar widely existing in organisms, is easy to dissolve in water, can be widely applied to fermentation culture processes of antagonistic yeasts of different types, can reduce the damage of organic acid in the environment to the antagonistic yeasts, enhances the capability of the antagonistic yeasts for biologically preventing and treating postharvest diseases, and has the advantages of low cost, easy production, transportation and convenient use.
(3) The method for improving the efficacy of antagonistic yeast in preventing postharvest acid pathogenic fungi can effectively improve the survival rate of antagonistic yeast in different organic acid stresses and improve the capability of antagonistic yeast in preventing postharvest diseases.
Drawings
FIG. 1 shows the results of the effect of different organic acids and sugar sources on the growth of marine yeasts in the experiments of the present invention, wherein (a) is an organic acid and (b) is a different sugar source;
FIG. 2 shows the results of the effect of different optimal carbon source concentrations and application concentrations on the incidence of citrus fruit downy mildew in the experiments of the present invention, wherein (a) is the incidence of fruit wounds after treatment with different optimal carbon source concentrations, and (b) is the incidence of fruit wounds after treatment with different concentrations of yeast;
FIG. 3 shows the results of the effect of marine yeasts cultured from different nitrogen sources on the incidence of citrus fruit downy mildew in the experiments of the present invention, wherein (a) is the incidence of wound treatment and (b) is the diameter of the lesions of wound treatment;
FIG. 4 shows the effect of marine yeasts cultured from different nitrogen sources on the incidence of gray mold of tomato fruits in the experiments of the present invention, wherein (a) is the incidence of wound treatment and (b) is the diameter of the lesions of wound treatment;
FIG. 5 shows the number of surviving yeasts in the plates obtained after glucuronic acid-induced culture in the experiments of the present invention;
FIG. 6 shows the result of staining yeast PI in the solution obtained after glucuronic acid-induced culture in the experiment of the present invention.
Detailed Description
The invention is further illustrated by the following examples, which are intended to be illustrative only and not limiting in any way.
Example 1
A method for improving the biological control efficiency of antagonistic yeast of acid pathogenic fungi after fruit and vegetable harvest comprises the following steps:
activating a biological yeast;
performing liquid culture on the activated biocontrol saccharomycetes;
the biocontrol yeast is marine yeast Rhodosporidium paludigenum Fell & Tallman, and the strain preservation number is IMI 394084.
Moreover, in the step (A), an activation culture medium is as follows:
200 g of potato, and the weight of the potato,
glucose or fructose in an amount of 0g,
fumaric acid 1-100mmol L -1 ，
20 g of agarose, which has the advantages of high purity,
1 liter of tap water and sterilizing to obtain an activated culture medium.
In the step, the liquid culture medium is as follows:
200 g of potato, and the weight of the potato,
glucose or fructose in an amount of 0g,
fumaric acid 1-100mmol L -1 ，
And (3) fixing the volume to 1L by tap water, and sterilizing to obtain liquid culture.
Further, the liquid culture time was 24 hours.
The following method is applied to biological control of acid pathogenic fungi antagonistic yeasts after fruit and vegetable harvest: concentration is set to 10 7 Antagonistic yeasts of cells/mL are sprayed on the surface of fruits before picking to moisten the surface of the fruits.
Example 2
A method for improving the biological control efficiency of antagonistic yeast of acid pathogenic fungi after fruit and vegetable harvest comprises the following steps:
activating a biological yeast;
performing liquid culture on the activated biocontrol saccharomycetes;
the biocontrol yeast is marine yeast Rhodosporidium paludigenum Fell & Tallman, and the strain preservation number is IMI 394084.
Moreover, in the step (A), an activation culture medium is as follows:
200 g of potato, and the weight of the potato,
20 g of glucose or fructose, and the weight of the mixture is as follows,
malic acid 1-100mmol L -1 ，
20 g of agarose, which has the advantages of high purity,
1 liter of tap water and sterilizing to obtain an activated culture medium.
In the step, the liquid culture medium is as follows:
200 g of potato, and the weight of the potato,
20 g of glucose or fructose, and the weight of the mixture is as follows,
malic acid 1-100mmol L -1 ，
And (3) fixing the volume to 1L by tap water, and sterilizing to obtain liquid culture.
Further, the liquid culture time was 24 hours.
The following is applied to biological control of antagonistic yeast of postharvest acid pathogenic fungi of fruits and vegetables: concentration is set to 10 7 Antagonistic yeasts of cells/mL are sprayed on the surface of fruits before picking to moisten the surface of the fruits.
Example 3
A method for improving the biological control efficiency of antagonistic yeast of acid pathogenic fungi after fruit and vegetable harvest comprises the following steps:
activating a biological yeast;
performing liquid culture on the activated biocontrol saccharomycetes;
the biocontrol yeast is marine yeast Rhodosporidium paludigenum Fell & Tallman, and the strain preservation number is IMI 394084.
Moreover, in the step (A), an activation culture medium is as follows:
200 g of potato, and the weight of the potato,
15 g of glucose or fructose,
glucuronic acid 200mmol L -1 ，
20 g of agarose, which has the advantages of high purity,
1 liter of tap water and sterilizing to obtain an activated culture medium.
In the step, the liquid culture medium is as follows:
200 g of potato, and the weight of the potato,
15 g of glucose or fructose,
glucuronic acid 200mmol L -1 ，
And (3) fixing the volume to 1L by tap water, and sterilizing to obtain liquid culture.
Further, the liquid medium was cultured for 48 hours.
The method is applied to the biological control of the antagonistic yeast of the acid pathogenic fungi of the picked fruits and vegetables: soaking picked fruits at a concentration of 10 7 The cell/mL antagonistic yeast suspension is soaked for 15 minutes, and then is left to stand and air-dried under the condition of 25 ℃ and 90% relative humidity.
Experiment 1 Effect of different organic acids and carbon sources on Marine Yeast growth
1. Experimental materials:
antagonizing yeast: rhodosporidium rhodosporum Rhodosporidium paludigenum Fell & Tallman (IMI 394084)
Organic acid: d-glucuronic acid, malic acid, citric acid, oxalic acid, and ascorbic acid
2. Test method
Under aerobic conditions, L-malic acid, oxalic acid, fumaric acid, citric acid, glucuronic acid, ascorbic acid and glucose, yeast suspension and carbon-deficient nutrient yeast glucose medium (NYCB: 8g nutrient solution and 5g yeast extract in 1 liter tap water) were added to a 100-well honeycomb plate to give final concentrations of acid and glucose of 500, 200, 100 or 10mmol L -1 . Growth was monitored with bioscreen C (Oy growth Curves Ab Ltd). The optical density (OD 600 nm) was measured automatically every 30 minutes with a 600nm filter, and the data from Biosceen C was measured over 72 hours.
3. Test results
At a removal of 10mmolL -1 At all but the concentrations tested, the addition of different carbon sources (organic acids) and their concentrations significantly affected yeast growth. The yeast grows best in the medium containing glucose, and secondly, glucuronic acid and malic acid. Fumaric acid has the strongest inhibition of yeast growth or the worst availability of yeast. Wherein the glucuronic acid is at 100mmmolL -1 At a concentration of (2), the growth effect was best, similar to the amount of yeast in the group with glucose as the sole carbon source, and the optimal cultivation time was 48h (FIG. 1).
1. Experimental materials:
the fruit is mandarin orange, and the variety is ponkan orange. Antagonizing yeast: rhodosporidium maritimum R.paludigenum Fell & Tallman (IMI 394084). Culture medium: potato glucose deficient medium (200 g cooked potato and 20 g agar to volume in 1 liter tap water, 121 ℃ sterilization 20 min). Pathogenic bacteria: penicillium digitatum (Penicillium digitatum) was activated for 7 days.
2. And (3) treatment:
(1) Fruits used in the test are slightly greenish in appearance, have no mechanical wound, and are uniform in size, soaked in 0.1% sodium hypochlorite solution for sterilization for 1-2min, rinsed with tap water, and dried at room temperature for later use.
(2) Wounds of uniform size (5 mm) and as deep as possible (2 mm) were made on top of each fruit with a sterile punch. Equal amounts (50 μl) of normal antagonistic yeast and acid-adapted yeast cell suspensions were added at each wound site at different concentrations, and equal amounts of sterile water were added as controls. Then 1X 10 is cut into the original wound 4 cells/ml viridae pathogen p.digitatum30 ul. The film was sealed with PE plastic film for moisture preservation and stored in a constant temperature and humidity storage room (25 ℃ C., relative humidity higher than 95%). Disease occurrence and lesion diameter were recorded at regular intervals each day, and results were expressed as morbidity (%) and average lesion diameter (mm), and direct bacteriostatic efficacy was compared for different yeasts. 12 fruits were selected as a group/repeat, 3 replicates.
The effect of glucuronic acid induction culture on marine yeast control of citrus green mold disease is shown in figure 2 (detection time is day 2).
3. Experimental results
In potato dextrose deficiency medium, the potato is subjected to glucuronic acid (500 mmol L) -1 ) After the adaptive culture, antagonistic yeast has the best biological control effect on the incidence rate of the green mold, and the biological control capability is respectively improved by 43.7 percent and 31.3 percent. The optimal use concentration of yeast is 10 7 cells/mL (FIG. 2).
Experiment 3 Effect of different optimal carbon sources on Marine Yeast control of citrus Green mildew disease
1. Experimental materials:
the fruit is mandarin orange, and the variety is ponkan orange. Antagonizing yeast: rhodosporidium maritimum R.paludigenum Fell & Tallman (IMI 394084). Culture medium: nutrient yeast glucose liquid medium (beef extract 8g, yeast powder 5g, constant volume to 1L tap water, sterilizing at 121deg.C for 20 min). Medium carbon source: glucose, glucuronic acid. Pathogenic bacteria: penicillium digitatum (Penicillium digitatum) was activated for 7 days at 25℃for further use.
2. And (3) treatment:
(1) Fruits used in the test are slightly greenish in appearance, have no mechanical wound, and are uniform in size, soaked in 0.1% sodium hypochlorite solution for sterilization for 1-2min, rinsed with tap water, and dried at room temperature for later use.
(2) Wounds of uniform size (5 mm) and as deep as possible (2 mm) were made on top of each fruit with a sterile punch. Equal amount (50. Mu.l) of 1X 10 is added to each wound 7 Normal antagonistic yeast and acid-adapted yeast cell suspensions at cells/ml, and equal amounts of sterile water were added as controls. Then 1X 10 is cut into the original wound 4 cells/ml viridae pathogen p.digitatum30 ul. The film was sealed with PE plastic film for moisture preservation and stored in a constant temperature and humidity storage room (25 ℃ C., relative humidity higher than 95%). Disease occurrence and lesion diameter were recorded at regular intervals each day, and results were expressed as morbidity (%) and average lesion diameter (mm), and direct bacteriostatic efficacy was compared for different yeasts. Select 12The fruits were one set/repeat, 3 replicates.
The effect of glucuronic acid induction culture on marine yeast control of citrus green mold disease is shown in figure 3 (detection time is day 2).
3. Experimental results
Glucuronic acid can significantly reduce the severity of citrus fruit green mold in nutrient yeast glucose broth. Glucuronic acid (500 mmolL L) -1 ) The treatment group had 22.1% increased (incidence) of the ability of antagonistic yeasts to control citrus green mold pest over the NYDB medium treatment group, and the lesion diameter was also significantly larger than in the NYDB medium treatment group (fig. 3).
Experiment 4 Effect of different optimal carbon sources on control of Botrytis cinerea disease in tomato fruit by Marine Yeast
1. Experimental materials:
the fruit is tomato. Antagonizing yeast: rhodosporidium maritimum R.paludigenum Fell & Tallman (IMI 394084). Culture medium: potato glucose deficient medium (200 g cooked potato and 20 g agar to volume in 1 liter tap water, 121 ℃ sterilization 20 min). Medium carbon source: glucose, glucuronic acid. Pathogenic bacteria: botrytis cinerea (Botrytis cinerea) is activated for 7 days at 25 ℃ for later use.
2. And (3) treatment:
(1) The fruits used in the test are fruits in the red ripe stage, have no mechanical wound and have uniform sizes, and are soaked in 0.1% sodium hypochlorite solution for sterilization for 1-2min, washed by tap water and dried at room temperature for later use.
(2) Wounds of uniform size (5 mm) and as deep as possible (2 mm) were made on top of each fruit with a sterile punch. Equal amount (50. Mu.l) of 1X 10 is added to each wound 7 Normal antagonistic yeast and acid-adapted yeast cell suspensions at cells/ml, and equal amounts of sterile water were added as controls. Then 1X 10 is cut into the original wound 4 cell/ml Proteus griseofulvis B.cinerea30 ul. The film was sealed with PE plastic film for moisture preservation and stored in a constant temperature and humidity storage room (25 ℃ C., relative humidity higher than 95%). The occurrence of diseases and the diameter of the lesions are recorded at regular intervals every day, and the results are obtainedThe disease rate (%) and average lesion diameter (mm) represent the direct bacteriostatic efficacy of different yeasts to be compared. 12 fruits were selected as a group/repeat, 3 replicates.
The effect of glucuronic acid induction culture on marine yeast control of citrus green mold disease is shown in figure 4 (detection time is day 2).
3. Experimental results
As shown in FIG. 4, after the composite culture of glucuronic acid and glucose in the potato glucose deficiency culture medium, antagonistic yeast has better biological control effect on the incidence of gray mold, and the biological control capacity is respectively improved by 29.2 percent.
1. Experimental materials:
antagonizing yeast: rhodosporidium maritimum R.paludigenum Fell & Tallman (IMI 394084)
Organic acid: d-glucuronic acid, malic acid, citric acid, oxalic acid, and ascorbic acid
2. Test method
(1) Taking 1mL of acid-adaptive antagonistic yeast and normal antagonistic yeast as seed solution, respectively, inoculating into 500mM different organic acid PDB culture medium with an inoculum size of 1×10 6 Shaking culture at 28 deg.C and 200rpm for 48 hr, sampling and gradient diluting to 10 4 After plating on PDA plates, counting was performed after incubation for 2d at 28 ℃.
(2) Acid-adaptive antagonistic yeast and normal antagonistic yeast were inoculated into PDB medium to which 500mM organic acid was added and to which no organic acid was added, respectively, and cultured at 200rpm and 28℃for 48 hours to achieve the logarithmic phase. The mid-log yeast cells were collected under centrifugation at 12000rpm for 5 min. The yeast cells collected and cultured without acid served as a control. After washing twice with PBS, the yeast cells were resuspended in the presence of 10. Mu.g/mL Propidium Iodide (PI) and allowed to stand in the dark for 20min. Then washed twice with PBS, resuspended in an equal volume of PBS and visualized under a fluorescence microscope. PI stained cells were stimulated at 488 nm. Yeast cells that bind positively to PI (red) are dead cells with plasma membrane damage.
3. Test results
As shown in fig. 5 and 6, after 500mM glucuronic acid adaptability induction treatment, the survival rate of rhodosporidium maritimum r.paludigenum in solid plates containing different organic acids (glucuronic acid, malic acid, citric acid, ascorbic acid and oxalic acid) is obviously improved by 21.2%, 33.6%, 19.4%, 33.3% and 39.4% respectively compared with control treatment; the rate of decay in PDB liquid medium containing high concentrations (500 mM) of glucuronic acid, malic acid, citric acid, ascorbic acid and oxalic acid was significantly reduced by 4.57%, 5.41%, 4.53%, 7.66% and 13.01%, respectively, effectively alleviating the cytoplasmic membrane damage of yeast.
Although embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the disclosure of the embodiments.
Claims (5)
1. A method for improving the biological control efficiency of antagonistic yeast of acid pathogenic fungi after fruit and vegetable harvest is characterized in that: the method comprises the following steps:
activating a biological yeast;
performing liquid culture on the activated biocontrol saccharomycetes;
the biocontrol yeast is marine yeastRhodosporidium paludigenum Fell &Tallman, strain deposit number is IMI 394084;
1-500mmol L is added into the culture medium -1 An organic acid is used for preparing the organic acid,
the organic acid is as follows: oxalic acid, fumaric acid, citric acid, glucuronic acid, malic acid or ascorbic acid,
the addition concentrations of the organic acids were as follows: oxalic acid 1-10mmol L -1 1-100mmol L of fumaric acid -1 1-10mmol L of citric acid -1 Glucuronic acid 1-500mmol L -1 1-100mmol L malic acid -1 Or ascorbic acid 1-100mmol L -1 ，
The activation culture medium in the step (A) is as follows:
200 g of potato, and the weight of the potato,
glucose or fructose 0-20 g,
glucuronic acid 1-500mmol L -1 ，
20 g of agarose, which has the advantages of high purity,
1 liter of tap water, sterilizing to obtain an activated culture medium,
the liquid culture medium in the step (a) is as follows:
200 g of potato, and the weight of the potato,
glucose or fructose 0-20 g,
glucuronic acid 1-500mmol L -1 ，
And (5) fixing the volume to 1L by tap water, and sterilizing to obtain the liquid culture.
2. The method for improving the biological control efficacy of antagonistic yeasts against acid pathogenic fungi after fruit and vegetable harvest according to claim 1, which is characterized by comprising the following steps: in the activation medium and the liquid medium: the addition amount of glucose or fructose is 1-20 g, and the addition concentration of glucuronic acid is 10-200mmol L -1 。
3. The method for improving the biological control efficacy of antagonistic yeasts against acid pathogenic fungi after fruit and vegetable harvest according to claim 1, which is characterized by comprising the following steps: the optimal incubation time in liquid medium was 48h.
4. An application for improving the biological control efficiency of antagonistic yeast of acid pathogenic fungi after fruit and vegetable harvest is characterized in that: fermenting yeastRhodosporidium paludigenum Fell &Tallman IMI 394084 saccharomycetes are applied to improving the capability of antagonizing acid pathogenic fungi of fruits and vegetables after picking, wherein the acid pathogenic fungi are penicillium digitatum.
5. The use of claim 4 for improving the biological control efficacy of acid pathogenic fungi antagonistic yeasts after fruit and vegetable harvest, characterized by: the method is applied to biological control of acid pathogenic fungi antagonistic yeasts after fruit and vegetable harvest in any mode as follows:
mode a: concentration is set to 10 7 Spraying antagonistic yeast of cells/mL on the surface of the fruits before picking to moisten the surfaces of the fruits;
mode B: soaking picked fruits at a concentration of 10 7 Soaking in the cell/mL antagonistic yeast suspension for 1-30 min, and standing and airing at 25 ℃ and relative humidity of 90%.
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