CN118256359A - Aureobasidium pullulans for preventing and treating pear ring rot and application thereof - Google Patents
Aureobasidium pullulans for preventing and treating pear ring rot and application thereof Download PDFInfo
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- CN118256359A CN118256359A CN202410486539.6A CN202410486539A CN118256359A CN 118256359 A CN118256359 A CN 118256359A CN 202410486539 A CN202410486539 A CN 202410486539A CN 118256359 A CN118256359 A CN 118256359A
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Classifications
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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
- C12N1/145—Fungal isolates
-
- 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
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P3/00—Fungicides
-
- 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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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- 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
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- Biotechnology (AREA)
- Mycology (AREA)
- Organic Chemistry (AREA)
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- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Virology (AREA)
- Environmental Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Plant Pathology (AREA)
- Pest Control & Pesticides (AREA)
- Biochemistry (AREA)
- Botany (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- General Chemical & Material Sciences (AREA)
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Abstract
The invention discloses a Aureobasidium pullulans strain for preventing and treating pear ring rot and application thereof. The aureobasidium pullulans YC18 is separated from the peel of the apple in the fortune city, and has good control effect on pear rot caused by ring rot. The strain YC18 does not grow at 37 ℃ and can not become human pathogenic bacteria, so that the strain YC18 has safety as a biocontrol bacterium; the strain YC18 has the advantages of low cost of large-scale production raw materials, convenient operation and stable control effect, and can effectively solve the problems of pesticide residue and environmental pollution caused by pesticide control of pear fruit diseases. Has great application potential in biological control of pear postharvest diseases and provides excellent basic strain for developing and developing microbial pesticide.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to Aureobasidium pullulans for preventing and treating pear ring rot and application thereof.
Background
Pear is one of fruit trees with long cultivation history, large area and high yield in China. The statistical that the pear is the 3 rd fruit in China, the total area of the whole country reaches 100 tens of thousands of hectares at present, the annual yield is 800 tens of thousands of tons, and the total area and the yield of the pear respectively account for 62 percent and 52 percent of the cultivation area and the yield of the pear worldwide. The pear fruit is threatened by diseases at any time during the growth, development and storage period, wherein ring rot (Botryosphaeria dothidea) is one of the most serious fruit fungus diseases and is the main cause of rotten pear fruit. Because the ring spot bacteria are latent infection in the young fruit stage, the disease is developed during the fruit ripening and storage and transportation period, and the control and prevention are very difficult.
Decay of fresh fruit after harvest is a global problem. The pear yield is reduced by about 25% each year because of the damage of pear ring disease in China, and the rotten fruit rate is up to more than 80% when serious, so that huge economic loss is caused. At present, chemical agents are often adopted for preventing and treating pear postharvest diseases, so that the ecological environment is seriously polluted, the residual quantity of toxic chemical substances in agricultural products is increased, potential harm is brought to human health, and meanwhile, the generation of drug resistance of pathogenic bacteria is easy to cause. Biological control utilizes antagonism among species to reduce occurrence of diseases, is safe to people and livestock, has good environmental compatibility and is not easy to generate drug resistance to diseases and insect pests, so that the biological control has the advantage that chemical bactericides do not have, and gradually becomes an optimal mode for replacing the chemical bactericides. Aureobasidium pullulans (Aureobasidium pullulans) are fungi resembling yeasts and are commonly found in nature, on the surface of fruits, plant leaves, in fresh water and sea water, and even on the surface of cement in urban environmental conditions. Aureobasidium pullulans can adapt to extreme environments such as saline and arctic soils, have extremely high salt and temperature stress resistance, and can also endure acidic, alkaline and oligotrophic conditions, and the characteristics provide guarantee for the Aureobasidium pullulans to be an effective biological control agent. Aureobasidium pullulans can control postharvest diseases of a variety of fruits including apples, and have been used for postharvest disease prevention and control of a variety of fruits including avocados, oranges, peaches, apples and the like (Madhupani and Adikaram,2017; klein and Kupper,2018; zhang et al, 2010), have good antagonistic activity against several common plant pathogens such as Penicillium, botrytis cinerea (Di Francesco et al.,2020;Agirman et al, 2020, iqbal et al, 2021). The microbial antagonistic bacteria can produce broad-spectrum antibacterial substances with high antagonistic activity, has extremely strong stress resistance, is nontoxic, residue-free and pathogenic, is safe to people and livestock, has good environmental compatibility, and meets the requirements of consumers on the quality and safety of agricultural products.
Disclosure of Invention
The invention aims to provide Aureobasidium pullulans for preventing and treating pear ring rot and application thereof.
Aureobasidium pullulans (Aureobusidium pullulan) YC18 has a preservation number of CGMCC No.40447. The strain is preserved in China general microbiological culture collection center (CGMCC) of China general microbiological culture Collection center (address: north Star Xiyu No.1, 3 of the Korean area North Star of Beijing city, national academy of sciences of China, and postal code 100101) at day 25 of 2022.
A microbial agent comprising the aureobasidium pullulans (Aureobusidium pullulan) YC18.
A fermentation product using a fermentation broth of aureobasidium pullulans (Aureobusidium pullulan) YC18.
The aureobasidium pullulans (Aureobusidium pullulan) YC18 and the application of the fermentation product thereof in preparing antibacterial and/or bacteriostatic products.
The antibacterial and/or bacteriostatic action is achieved by inhibiting the growth of hyphae and the formation of spores.
The bacteria are one or more of Rhizoctonia cerealis (Botryosphaeria dothidea), botrytis cinerea (Botrytis californica), sclerotinia sclerotiorum (Sclerotinia sclerotiorum) and Penicillium citrinum (Penicillium citrinum).
The application of the aureobasidium pullulans (Aureobusidium pullulan) YC18 in the prevention and treatment of ring spot disease. Preferably, the ring spot is pear ring spot.
A method for preventing and treating pear fruit ring rot comprises spraying bacterial suspension containing Aureobasidium pullulans (Aureobusidium pullulan) YC18 on pear fruit surface; the concentration of aureobasidium pullulans (Aureobusidium pullulan) YC18 in the bacterial suspension is 1-5 multiplied by 10 7 CFU/mL. Preferably, the concentration is 2X 10 7 CFU/mL.
The invention has the beneficial effects that: the Aureobasidium pullulans (Aureobusidium pullulan) are separated from the surfaces of apple fruits, and have good control effect on pear rot caused by ring rot. The thallus is used as a natural preservative, has low cost of raw materials for mass production, convenient operation and stable control effect, and can effectively solve the problems of pesticide residue and environmental pollution caused by pesticide control of pear fruit diseases. Has great application potential in biological control of pear postharvest diseases and provides excellent basic strain for developing and developing microbial pesticide.
Drawings
FIG. 1 is a morphological photograph of strain YC 18; wherein, A in FIG. 1 is a colony photograph of strain YC18 cultured for 3 days; in FIG. 1, B is a micrograph of cells of strain YC 18.
FIG. 2 is a photograph of growth for 1 week based on strain YC18 at 25℃under different nutritional conditions.
FIG. 3 is a phylogenetic tree constructed based on the ITS gene sequence of strain YC 18.
FIG. 4 is a photograph showing strain YC18 cultured for 7 days against Rhizoctonia cerealis.
FIG. 5 is a photograph showing the effect of volatile organic compounds produced by the Petri dish study strain YC18 grown for 3 days (A) and 14 days (B) on Rhizoctonia cerealis.
FIG. 6 shows that the strain YC18 volatile organic compounds inhibit the formation of the spores of Rhizoctonia cerealis.
FIG. 7 shows the effect of the strain YC18 volatile organic compounds on Botrytis cinerea.
FIG. 8 shows that the strain YC18 volatile organic compounds inhibit the formation of Botrytis cinerea spores.
FIG. 9 shows that the strain YC18 volatile organic compounds inhibit the formation of penicillium citrinum spores.
FIG. 10 shows that the strain YC18 volatile organic compounds inhibit the growth of the hyphae of Sclerotinia sclerotiorum (A) and the formation of sclerotium (B); a: co-culturing for 5 days; b: co-culturing for 11 days.
FIG. 11 shows the control of pear ring rot by strain YC 18.
FIG. 12 is a PLS-DA point cloud of Volatile Organic Compounds (VOCs) produced in a strain blank, a negative control of Aureobasidium pullulans YC18 or Rhizoctonia cerealis inoculated alone, and a YC18 and Rhizoctonia cerealis interaction headspace bottle.
FIG. 13 distribution of test statistics and p-value of PLS-DA substitution test
Fig. 14 is an S-plot of VOCs produced by different treatments.
Detailed Description
The present invention will be described more fully hereinafter in order to facilitate an understanding of the present invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
The pear variety in the following examples is "crystal pear".
Potato Dextrose Agar (PDA) medium formulation in the following examples: 200 g of potato, 20 g of glucose and 15g of agar, and distilled water is used for fixing the volume to 1 liter, and the natural pH is achieved.
Potato dextrose medium (PDB) formulation in the following examples: 200 g of potato and 20g of glucose, and distilled water is used for fixing the volume to 1 liter, and the pH is natural.
The formula of the Bengalia red agar medium in the following examples: 5g of peptone, 10 g of glucose, 1 g of potassium dihydrogen phosphate, 0.5 g of magnesium sulfate (anhydrous), 0.033 g of Bengalia, 0.1 g of chloramphenicol, 20 g of agar, and distilled water to a volume of 1 liter and a pH of 7.2-7.4.
The preparation method of YC18 suspension in the following examples comprises: YC18 was inoculated into PDB medium, and after culturing for 48hr (25 ℃ C., 150 r/min), the bacterial solution was centrifuged at 4 ℃ C., 10000r/min for 10min, the supernatant was removed, and then the concentration was adjusted to a desired concentration with sterile water.
EXAMPLE 1 isolation and identification of Brevibacterium strains
1. Isolation and purification of Strain YC18
1. Apples from different producing areas are washed and dried by distilled water, the peel is cut into small sections, 10g is added into a culture bottle containing 90mL distilled water for overnight culture. The culture solution was diluted to 10 -1、10-2 and 10 -3, respectively, 100. Mu.L of the diluted solution was spread on a Bengalia agar plate, and the culture was carried out at 25 ℃. After colonies grow out, according to the characteristic that mycelia at the edge of the colonies of the Aureobasidium pullulans are radial and the center of the colonies is yeast, different suspected Aureobasidium pullulans colonies are selected and inoculated into a PDA culture medium for streak purification culture, the purified strains are obtained after repeated times, and the purified strains are inoculated into a bevel test tube of the PDA culture medium and are stored in a refrigerator at the temperature of 4 ℃.
2. 12 Strains of Aureobasidium fungi are obtained by co-separation from apple peel of different producing places, aureobasidium fungi with biological control activity are screened by taking Rhizoctonia pyriformis as indicator bacteria, wherein 1 strain from apple in the city of fortune shows strong antagonistic activity to postharvest rot caused by Rhizoctonia pyriformis, and the strain is named as YC18.
2. Identification of Strain YC18
1. Morphological identification of strain YC18
Bacterial strain YC18 was streaked on PDA medium for 3 days, and the colony was a milky white yeast, the surface was moist and smooth, and was easy to pick up, the edge hypha of the colony was radial (A in FIG. 1), the color of the colony became dark with increasing culture time, and melanin was distributed on the surface. Strain YC18 was observed by light microscopy, and the somatic cells were oval in shape, and sporulated in a budding manner, with typical characteristics of yeast cells (B in fig. 1).
2. Physiological Properties of Strain YC18
Strain YC18 grew better at 0 ℃ and 25 ℃, where 25 ℃ was the most suitable growth temperature and failed to grow at 37 ℃. The strain YC18 can not grow in human bodies and is safe to people and livestock, and has biological safety when being used for preventing and controlling rot of fruits and vegetables after picking; the YC18 growth under different nutrition conditions is obviously changed, the diameter of a colony is about 3cm after the colony grows on a PDA culture medium for 1 week, the creamy edge is moist, and the light pink center of the colony is provided with melanin generation. YC18 also grew on 1% PDA and water agar oligotrophic medium, but colony morphology changed greatly, colony diameter 1.5cm was grown for 1 week, and colony consisted of white radial hyphae. On water agar medium, YC18 colonies were similar to those on 1% PDA, but the hyphae constituting the colonies were sparse. The YC18 can grow under oligotrophic conditions, and the characteristic ensures that the YC18 can stably grow on the surfaces of oligotrophic fruits and leaves when the YC18 is used as a biological control microbial agent, thereby playing a role in prevention and control (table 1; figure 2).
Table 1 physiological Properties of Strain YC18
+-: Poor growth +: growth-: no growth
3. Molecular characterization of Strain YC18
Extracting total DNA of the strain YC18 as a template, and carrying out PCR amplification by adopting universal primers ITS1 and ITS4 to obtain fragments of an ITS conservation region of the strain YC 18. The primer sequences were as follows:
ITS1 (upstream primer): 5'-TCCGTAGGTGAACCTGCGG-3' (SEQ ID NO: 1);
ITS4 (downstream primer): 5'-TCCTCCGCTTATTGATATGC-3' (SEQ ID NO: 2).
The ITS sequence obtained by PCR amplification is shown as a sequence table SEQ ID NO: 3. Phylogenetic tree based on ITS sequence is shown in fig. 3.
4. Preservation of strains
According to morphological characteristics, physiological and biochemical and molecular identification results and analysis, the classification of the strain YC18 is named aureobasidium pullulans (Aureobusidium pullulan), and the strain is preserved in China general microbiological culture Collection center (CGMCC, address: north Star Xiyu No. 1,3 in the Qingyang area of Beijing, and the post code 100101 of the institute of microorganisms of the national academy of sciences) at the month of 12 and 25 days 2022, and the preservation number is CGMCC No.40447.
Example 2 use of Strain YC18 in bacteriostasis or antibiosis
1. Antagonistic activity of strain YC18 on Rhizoctonia cerealis
Activating pathogenic bacteria Rhizoctonia cerealis (Botryosphaeria dothidea) on PDA culture medium, and beating the bacterial cake with 9mm puncher. Inoculating the Rhizoctonia cerealis cake on one side of a PDA plate with the diameter of 9cm by adopting a counter culture method, streaking an inoculated strain YC18 on the opposite side, and taking pathogenic strain Rhizoctonia cerealis independently inoculated on the PDA plate as a control, wherein the two inoculation points are 3cm apart. All 3 replicates were used and incubated at 25℃and when the control had grown to full dish, the treated colony diameter and zone width were measured.
As a result, as shown in FIG. 4, the plating was performed on PDA plates at 25℃for 7 days, and the plates were full of Rhizoctonia cerealis. In contrast, strain YC18 produced little zone of inhibition (FIG. 4) during the counter culture, but the Bremia lachneri could not cover the Bremia lachneri colonies and thus could not grow to a plate. Thus, the Aureobasidium pullulans YC18 inhibits the growth of the hypha of Rhizoctonia cerealis by means of space competition.
2. Antagonistic activity of volatile organic compounds produced by strain YC18 on Rhizoctonia cerealis
The influence of volatile organic compounds generated by Aureobasidium pullulans YC18 on the growth of the Rhizoctonia cerealis is determined by adopting a double-dish buckling method. Strain YC18 was streaked on PDA plates while other PDA plates were inoculated centrally with blocks of ring spot bacteria 9mm in diameter. Removing 2 culture dish covers, buckling the bottom of the dishes together, and sealing by using a sealing film. PDA plates not inoculated with strain YC18 were paired with plates inoculated with the schlieren block as controls. The 2 treated snap plates were incubated at 25℃with 3 replicates. The growth conditions of Aureobasidium pullulans YC18 and the ring spot bacteria are observed, and the diameter of the ring spot bacteria is measured in different growth periods.
As a result, as shown in FIG. 5, the growth of the myc 18-treated Rhizoctonia cerealis was significantly inhibited after 3 days of co-culture in the plate-pair inoculated with Rhizoctonia cerealis and Aureobasidium pullulans, with an inhibition rate of 28.8% (A in FIG. 5). And at the late stage of co-cultivation, the strain YC18 treated ring spot bacteria colonies were whiter, while the control ring spot bacteria colonies were filled with dark green spores (B in FIG. 5). Sampling was performed at the edges of the colonies, respectively, and microscopic examination results showed that the strain YC18 treated with the Rhizoctonia cerealis showed no significant change in mycelium morphology, but greatly inhibited sporangium formation (FIG. 6). The 2 fungi were spatially separated, indicating that strain YC18 produced volatile antifungal material during growth, strongly inhibiting the growth of the Rhizoctonia hyphae and spore production.
3. Antibacterial spectrum of strain YC18
The inhibition effect of the strain YC18 on other common plant pathogenic bacteria is studied by taking Botrytis cinerea (Botrytis californica), sclerotinia sclerotiorum (Sclerotinia sclerotiorum) and Penicillium citrinum (Penicillium citrinum) as the plant pathogenic bacteria to be tested. The method comprises the following specific steps:
Activating various pathogenic bacteria on PDA culture medium, and taking out bacterial cake with 6mm puncher to obtain bacterial cake. And (3) inoculating the bacterial cakes to one side of the separation culture dish containing the PDA by adopting the separation culture dish, and streaking an inoculated strain YC18 to the opposite side, wherein pathogenic bacteria are singly inoculated to one side of the separation culture dish for comparison. 3 replicates were used, and after 5 days of incubation at 25℃the diameter of each pathogenic colony was measured, and the inhibitory activity was expressed as = [ (control colony diameter-treated bacteria diameter)/control colony diameter ] ×100%.
As shown in FIGS. 7 and 9, the strain YC18 did not significantly affect the growth of Botrytis cinerea (Botrytis californica) and Penicillium citrinum mycelia, but showed a strong inhibitory effect on the production of spores of 2 pathogenic bacteria. The bacterial colony is earthy yellow and is full of spores when the Botrytis cinerea is cultured alone (A in FIG. 8), while the bacterial colony is white and no spores are generated under the action of the strain YC18 (B in FIG. 8). When penicillium citrinum is cultured alone, the surface of the bacterial colony is covered with green spores, and almost no green spores are generated under the action of the bacterial strain YC18, which shows that the bacterial strain YC18 strongly inhibits the generation of the spores of Botrytis cinerea and penicillium citrinum.
The capsicum sclerotinia is caused by sclerotinia sclerotiorum (Sclerotinia sclerotiorum), can cause plant withering and death, and is a destructive disease produced by the capsicum in a protected area. The strain YC18 can inhibit the growth of the white cotton flocculent hyphae of the sclerotinia sclerotiorum, and the inhibition rate of the hyphae is 65.38 percent when the strain is co-cultured for 5 days (A in figure 10); the formation of sclerotium was also inhibited, indicating that the strain YC18 had good inhibitory activity against Sclerotinia sclerotiorum (B in FIG. 10). The strain YC18 has broad-spectrum inhibition activity on the formation of spores and sclerotium of common plant pathogenic bacteria, and can be used for biological control of various diseases.
Example 3 application of Strain YC18 in prevention and treatment of pear fruit ring rot
1) Pretreatment of pear
Selecting fruits with neat appearance, no plant diseases and insect pests and no trauma, washing the fruits with tap water, spraying and sterilizing the fruits with 75% alcohol, and wiping the surfaces with filter paper for later use after 2 min.
2) YC18 suspension treatment
Two wounds of as uniform depth as possible were symmetrically made on the fruit surface with a sterile punch (diameter 9 mm), and 50 μl 2×10 7 CFU/ml YC18 bacterial suspension (YC 18 treated group, YC 18) and an equal amount of sterile water (control group, ck) were inoculated in the wounds, respectively, and each treatment of 6 fruits was repeated 3 times and left at room temperature for one day.
3) Inoculating pathogenic bacteria
And (3) inoculating the ring spot bacteria blocks with the diameter of 9mm at the wounds of the YC18 treatment group and the control group in the step (2), placing the inoculated ring spot bacteria blocks into a PE closed box for culture at 25 ℃ and RH >90%, observing and recording the results at regular time, and calculating the average incidence (%) and the average spot diameter (cm).
The result of the control effect of the strain YC18 on the pear ring spot is shown in FIG. 11 and Table 2, and compared with the control group, the disease condition of the pear ring spot is obviously weakened in the YC18 treatment group. After the third day of the grafting of the ring spot bacteria, the control group starts to attack, and the YC18 treatment can completely inhibit the generation of the ring spot of pear fruits; after the grafting of the leaf scald bacteria for 5 days, 100% of the disease occurs in the control group, and the YC18 treatment can still inhibit the generation of the leaf scald disease of pear fruits by 100%; after 7 days of pathogen inoculation, the YC18 treatment group starts to appear lesions; the rotten area of the control group is more than 50%, the average diameter is about 6.5cm, the average diameter of the pear fruit spot of the YC18 treatment group is 1.8cm, the rotten area is reduced by 72.3% compared with the control group, and the occurrence and development of pear ring rot are effectively slowed down by the YC18 treatment, so that the control effect is good.
Table 2, YC18 effects of preventing and treating pear fruit ring rot
EXAMPLE 4 Volatile Organic Compounds (VOC) against pathogenic bacteria produced by Strain YC18
1) Collection of volatile materials
Collection of volatile materials: aureobasidium pullulans YC18 and Rhizoctonia cerealis are respectively inoculated into a PDA culture medium and are placed in a 25 ℃ incubator for culture. 1.5mL PDA is added into a20 mL headspace bottle, the inclined bottle body is put into an inclined plane, after the inclined plane is solidified, the rotary bottle body is put into an inclined plane after 1.5mL PDA is added at the other side. The next day of inspection confirmed that the two bevels did not touch. And (3) streaking and inoculating aureobasidium pullulans YC18 on the inclined plane of the PDA of the headspace bottle, screwing the cap, and horizontally placing the cap in a 25 ℃ incubator for 2d of culture. 0.6cm of ring spot bacteria sheet is inoculated on the inclined surface of the PDA on the other side, 25 co-culture is carried out for 3 days, a headspace bottle without inoculating bacteria is used as a blank control, a headspace bottle with aureobasidium pullulans YC18 or ring spot bacteria is independently inoculated as a negative control, and 3 replicates are arranged for each treatment.
Adsorption and analysis of volatile substances: the SPME extraction head is arranged at the sample inlet of the gas chromatograph
Aging for 15min at 250 ℃ for standby; placing the headspace bottle after 3d culture in a constant temperature water bath kettle at 60 ℃ for incubation for 10min, then inserting an SPME needle into 1/3 of the headspace bottle, and carefully pushing out the extraction head; extracting at 60deg.C for 40min, taking out, inserting into GC-Q/TOF (Agilent 7890B-7200) sample inlet of gas chromatography-quadrupole/time-of-flight mass spectrometer, desorbing at 250deg.C for 3min, and performing sample analysis.
2) Method for analyzing volatile substance
GC-Q/TOF detection conditions: gas chromatography column HP-5MS UI (30 m×0.25mm×0.25 μm), no split sample injection, sample inlet temperature 250 ℃, helium as carrier gas, 1.5mL/min column flow, and temperature increase program: starting 50 ℃ (3 min), 3 ℃/min rise to 140 ℃ (2 min), 25 ℃/min rise to 280 ℃ (5 min). EI (Electron impact) source, ionization energy 70eV, ion source temperature 230 deg.C, quadrupole temperature 150 deg.C, and mass scan range 50-600m/z.
3) Data acquisition and analysis
The volatile components in the sample are qualitatively analyzed by adopting a combination mode of mass spectrum library retrieval (MS) and Retention Index (RI) 2 methods. Mass spectral library retrieval (MS): the components entering the mass spectrum detector are compared with a NIST 17 database, agilent MassHunter Unknows software is utilized for analysis, and substances with matching degree more than or equal to 60 are selected as identification components; retention Index (RI): and (3) taking the C7-C30 series alkane as an external standard, calculating the actual retention index of each component according to the peak time of the target object and the peak time of the series alkane under the same temperature rising condition, and comparing and analyzing with the theoretical retention index reported in the literature.
The 4 sets of data determined were subjected to peak alignment for the identified compounds using MPP 15.1 software, screening data rules: data with a frequency of occurrence of more than 50% for only one set of data is retained. The compounds of the different groups were analyzed by PLS-DA method pairs to find metabolites with significant differences between the groups.
The blank control was analyzed by SPME-GC-Q/TOF technique, and 83 volatile compounds were identified in total by inoculating the negative control of Aureobasidium pullulans YC18 or Rhizoctonia cerealis alone and the Volatile Organic Compounds (VOC) produced in the headspace bottle in which Aureobasidium pullulans YC18 and Rhizoctonia cerealis interacted. Analysis of VOCs generated by different groupings using OPLS-DA, as can be seen in fig. 12, the distribution of samples from different treatment groups over different areas, the 4 groups being able to be distinguished significantly, indicating that there may be significant differences in VOCs between the 4 groups.
The displacement test of PLS-DA selects the prediction accuracy (prediction accuracy) as the test statistic, and the observed test statistic is on the right side of the random distribution (observed values are significantly larger than random values), with p-values less than 0.05, indicating that PLS-DA can significantly distinguish between different packets, i.e. metabolites that should have significant differences between packets (fig. 13).
VIP values were calculated for each variable in VOCs (fig. 14), and in general, metabolites with VIP greater than 1 were differential metabolites from group to group, with large differences between groups. As shown in fig. 4, metabolites with metabolite names marked in the yellow region were metabolites with p values less than 0.05 and vip greater than 1 after correction, which were significantly different between the groups. By analysis of these differential metabolites, it was found that 1-octen-3-ol (1-Octen-3-ol), 2-n-pentylfuran (Furan, 2-pentyl-), 3-nonen-2-one (3-Nonen-2-one) and Styrene (Styrne) may be VOCs that exert bacteriostatic activity against Aureobasidium pullulans YC 18.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (8)
1. Aureobasidium pullulans (Aureobusidium pullulan) YC18 is characterized in that the preservation number is CGMCC No.40447.
2. A microbial agent comprising aureobasidium pullulans (Aureobusidium pullulan) YC18 of claim 1.
3. A fermentation product, wherein the fermentation product employs a fermentation bacterium that is aureobasidium pullulans (Aureobusidium pullulan) YC18.
4. Use of aureobasidium pullulans (Aureobusidium pullulan) YC18 or a fermentation product thereof as claimed in claim 1 for the preparation of an antibacterial and/or bacteriostatic product.
5. The use of aureobasidium pullulans (Aureobusidium pullulan) YC18 or a fermentation product thereof according to claim 4 for the preparation of an antibacterial and/or bacteriostatic product, wherein the fungus is one or more of the group consisting of colletotrichum sp (Botryosphaeria dothidea), botrytis cinerea sp (Botrytis californica), sclerotinia sclerotiorum sp (Sclerotinia sclerotiorum) and penicillium citrinum sp (Penicillium citrinum).
6. Use of aureobasidium pullulans (Aureobusidium pullulan) YC18 as claimed in claim 1 for controlling ring rot.
7. The use of aureobasidium pullulans (Aureobusidium pullulan) YC18 according to claim 6 for controlling ring rot, wherein the ring rot is pear fruit ring rot.
8. A method for preventing and treating pear fruit ring rot is characterized in that bacterial suspension containing aureobasidium pullulans (Aureobusidium pullulan) YC18 is sprayed on the surface of pear fruit; the concentration of aureobasidium pullulans (Aureobusidium pullulan) YC18 in the bacterial suspension is 1-5 multiplied by 10 7 CFU/mL.
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