CN105861340B - Pichia anomala for controlling postharvest diseases of citrus and grapes - Google Patents
Pichia anomala for controlling postharvest diseases of citrus and grapes Download PDFInfo
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- CN105861340B CN105861340B CN201610225320.6A CN201610225320A CN105861340B CN 105861340 B CN105861340 B CN 105861340B CN 201610225320 A CN201610225320 A CN 201610225320A CN 105861340 B CN105861340 B CN 105861340B
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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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- 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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- 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
Abstract
The invention discloses a helmet shape for controlling postharvest diseases of citrus and grapesPichia pastoris, belonging to the biological prevention and control field of fruit postharvest diseases. Identified as Pichia anomala (Pichia manshurica) Is named asPichia manshuricaY4, accession number: CGMCC 2.5415. When in use, the Pichia anomala is activated, cultured and centrifuged to obtain thallus, which is diluted with sterile water to 1.0 × 108Bacterial suspension per mL. Spraying the mixture to the outer surface of the fruit by a watering can, air-drying, placing the fruit in a plastic basket, sealing by a preservative film, and storing at room temperature. The incidence of citrus green mold and grape black aspergillosis and gray mold treated by the strain is obviously reduced. Therefore, the pichia kluyveri can replace a chemical bactericide to prevent and control the postharvest diseases of the citrus and the grape, avoids the harm to people caused by the use of the chemical bactericide, and has remarkable economic and social benefits.
Description
Technical Field
The invention relates to a Pichia anomala (Pichia manshurica) Y4 strain, which can control the green mold of citrus after-harvest, the black aspergillosis and the gray mold of grape after-harvest, and belongs to the field of biological control of fruit after-harvest diseases.
Background
The serious loss of fruit due to postharvest decay has long been a global problem, according to conservative estimates, 10-20% of fresh fruit is lost in postharvest decay in developed countries, while in developing countries lacking storage and transportation refrigeration equipment, the rate of decay is as high as 40-50%, and the fruit decay in China is generally 20-30%.
China is the biggest world in fruit production and sale, the variety of fruits is various, and the yield accounts for 80% of the world fruit yield. Since the fruit is produced in a place far from urban areas and the maturation period is relatively concentrated, a great deal of postharvest loss is caused by rotting, water loss, nutrient loss, and the like during storage and transportation. According to the early data, the fruit rot in China exceeds 8000 million tons every year, so the economic loss reaches 750 million yuan, which accounts for more than 30% of the whole yield value, and the current value should be higher.
Factors causing the postharvest loss and quality deterioration of fruits are many, including weight loss and withering due to water and nutrition consumption; physiological metabolic disorders due to postharvest stress; collapse of fruit tissue structure due to postharvest ripening and senescence, decay due to pathogen infestation, and the like. Wherein decay by pathogenic infection is believed to be a major cause of postharvest fruit loss.
The species of mold that causes decay during storage of fruits is many, but the predominant pathogenic bacteria of each fruit are generally 2-3. For example, (1) diseases caused by fungi after grape harvest are mainly black aspergillosis caused by Aspergillus sp and gray mold caused by Botrytis cinerea, wherein the gray mold caused by Botrytis cinerea is the most harmful disease in grape production, and the yield is reduced by 20-40% and seriously reaches more than 60% all the year round. The botrytis cinerea can not only cause field loss, but also cause the pathogenic bacteria remaining on the grapes to be latently infected in the storage period, so as to cause the grapes to rot, thresh and brown in the storage process, shorten the storage period and reduce the quality, and is one of the important pathogenic bacteria in the grape storage. (2) After the citrus is harvested, the rot of the citrus is mainly Penicillium italicum (Penicillium italicum) caused Penicillium and Penicillium digitatum (Penicillium digitatum) caused green mold, wherein the rot caused by the Penicillium digitatum is particularly serious, and the pathogenic bacteria are widely distributed, and a large amount of generated conidia are diffused in the air in a way of saprophytic growth on organic matters, so that the conidia are diffused to fruits to cause the fruits to rot, and the loss is extremely high. The reasons for the occurrence of postharvest diseases of fruits are very complex, and in addition, diseases in the field before harvest, mechanical damage after harvest, physiological disorder caused by improper management of storage temperature and humidity conditions and the like are all factors contributing to the disease.
The harm of pathogenic fungi to harvested fruits is not only to cause the fruits to decay and lose in a large amount, but also to cause serious food safety problems due to the secretion of various secondary metabolites by many pathogenic fungi, such as ochratoxin a (ota) produced by Aspergillus sp, which is two of the main pathogens on grapes, and glucuronicamycin (borrydial) produced by botrytis cinerea. The OTA has various toxicities, particularly nephrotoxicity, and certain teratogenicity, carcinogenicity and mutagenicity, and is considered as one of the main factors of the Barr's stem endemic nephropathy, so that the OTA has great potential harm to the health of animals and human bodies.
For a long time, the main measure for controlling postharvest diseases of fruits and vegetables is to use bactericides. According to the latest data, the bactericide accounts for about 26 percent of the crop disease control market, and the amount of the bactericide sold in 2011 is up to $ 133 hundred million. The chemical bactericide has the advantages of low cost, simplicity, convenience, clear action mechanism, obvious effect and the like. The chemical bactericide for controlling postharvest diseases of fruits is wide in variety. For example, the chemical bactericides for controlling the grape diseases mainly comprise dimethomorph, carbendazim, codin, Kejia, pyrimethanil, 20 percent difenoconazole water dispersible granules and the like. The prevention and control of the green mold during the storage of the citrus mainly depends on chemical agents such as javanine, thiabendazole, azoxystrobin, texadol, sodium o-phenolate, sec-butylamine, benzimidazole bactericide and the like.
However, over the past decade, there has been an increasing concern among the public and scientific community about the problem of pesticide (especially fungicide) poisoning in food and the environment. The long-term and large-scale use of the bactericide seriously pollutes the environment and is harmful to human health. Reports from the National Academy of Sciences (NAS) (1987) indicate that fungicides constitute 60% of the tumorigenic risks of all pesticides used in foods. In addition, the high frequency of the use of a single type of bactericide causes the problem of drug resistance of pathogenic bacteria, and the control effect of the bactericide is greatly reduced. According to the earlier investigation of California in the United states, the antifungal Java strain of the penicillium digitatum appears after 5 years of continuous use on citrus as a regeneration product of benzimidazole and sec-butylamine medicaments. According to domestic investigation, in areas where carbendazim is used alone for many years, penicillium digitatum has severe resistance to the carbendazim, so that the rotting rate of citrus is increased, and meanwhile, the drug-resistant strains have cross resistance to three benzimidazole bactericides, such as thiophanate methyl, benlate, thiabendazole and the like. Thus, in many developed countries and regions, some chemical fungicides have begun to be restricted from use, and several fungicides, such as Captan (Captan) and benomyl (benomyl), have been banned by the united states environmental protection agency.
Since 2001, after world trade organization is added in China, fruit trade is becoming active in the world, competition of fruit market is also becoming more fierce, and a serious obstacle is set for export of agricultural products (including grapes and oranges) in China by using 'green barriers' in some developed countries. Therefore, the research and the utilization of the substances or organisms which are safer and more environment-friendly and can replace chemical bactericides are utilized to prevent and control the postharvest diseases of the fruits and the vegetables, and the method is a new research direction for the postharvest storage and the fresh keeping of the fruits and the vegetables. There is currently increasing international interest in the use of beneficial microorganisms for the biological control of postharvest disease in fruit, and this is considered one of the most promising alternatives to chemical fungicides. The researches have profound significance for reducing pesticide residues in fruit and vegetable products, protecting the environment and human health and increasing the economic benefits of the fruit and vegetable products.
In recent years, biological control has attracted attention from countries around the world, and the use of antagonistic microorganisms has proven to be an effective approach. The yeast has the advantages of good antagonistic effect, no toxin, capability of being used together with a chemical bactericide and the like, and becomes a research hotspot. Chalitz and the like separate a Debaryomyces hansenii strain from the surface of lemon, and the like, and has obvious control effect on citrus green mold, penicilliosis and the like. Torres et al reported that Pantoeaagglomerans had a good control effect on Penicillium digitatum. Arras reports that Candidafamata has a remarkable control effect on citrus green mold, and discusses the action mechanism of Candidafamata. At present, part of antagonistic bacteria are commercially or semi-commercially used, and mainly comprise Candida oleophilica oleophila, Candida guilliermondii, Pichia guilliermondii and Cryptococcus albidus. The products are mainly concentrated in western developed countries, such as BioSave100, 110 and Aspire which are biological control series products applied to fruit postharvest diseases and are developed in the United states for the first time in 1995; subsequently, south africa developed two biocontrol agents of avogren and Yield Plus, and at present, China failed to realize the commercial application of biological preservation. The main reason is the lack of highly effective biocontrol strains.
The pichia pastoris is considered as one of the safest yeasts, and researches prove that the pichia pastoris can obviously reduce the diseases of the picked citrus, thereby achieving the aim of storing and preserving fruits.
Disclosure of Invention
The invention aims to provide a Pichia anomala (Pichia manshurica) Y4 separated from a vineyard, which can efficiently control the postharvest green mold of citrus, the postharvest black aspergillosis of grapes and the gray mold, can be widely used for biological control of postharvest diseases of citrus and grapes, reduces the loss caused by the postharvest diseases, and has potential application value.
The technical scheme adopted by the invention
The yeast strain for preventing and treating the postharvest green mold of the citrus and the post-harvest gray mold and the black aspergillosis of the grapes, which is provided by the invention, is separated from a vineyard with full stand capacity in Zhenjiang city, Jiangsu province, is cultured on a NYDA solid culture medium plate and in a malt juice liquid culture medium at 28 ℃, and morphological observation is carried out; the 5.8S rDNA-ITS zone sequence of said strain is analyzed, and the molecular biological identification is made, and said strain is preserved in China general microbiological culture Collection center.
The Pichia manshurica (Pichia manshurica) Y4 is currently deposited in the institute of microbiology of china academy of sciences No. 3, north chen west road No. 1, north kyo, china, the common microbiological center of the committee for culture collection of microorganisms (CGMCC), and the number of deposits is: CGMCC No.2.5415, with a preservation date of 2016, 3 and 31 months, and is named as Pichia anoshimeria according to classification.
The method comprises the following steps of (1) utilizing pichia pastoris to prevent and control postharvest diseases of fruits and store and keep fresh: activating, fermenting and culturing the pichia pastoris, and centrifuging to obtain thalli; diluting the thallus with sterile water to 1.0 × 108CFU/mL of bacterial suspension; spraying the mixture to the outer surface of the fruit by using a spray can, and then air-drying; placing in plastic basket, sealing with plastic wrap, and storing at room temperature.
The invention has the advantages that:
(1) the Pichia manshurica (Pichia manshurica) Y4 used in the invention is screened by the laboratory, has strong antagonistic effect and is harmless to human body.
(2) The Pichia pastoris (Pichia manshurica) Y4 used in the invention has a wide antibacterial spectrum, and can control the disease loss caused by pathogenic bacteria after grape and orange are picked.
(3) The Pichia manshurica (Pichia manshurica) Y4 used in the present invention has better antagonistic bacteria biocontrol effect than that described in the related patents.
(4) The invention uses the Pichia pastoris (Pichia manshurica) Y4 to replace a chemical bactericide to prevent and treat the postharvest diseases of fruits, avoids the harm of the chemical bactericide to people, and has obvious economic and social benefits.
The invention will be explained in more detail by means of the following examples. The following examples are illustrative only, and the present invention is not limited by these examples.
Drawings
FIG. 1 is a diagram showing the sequence evolution relationship of 5.8S rDNA-ITS region of Pichia anomala according to the present invention.
FIG. 2 shows the inhibitory effect of Pichia kluyveri on citrus green mold. Note: CK: sterile distilled water, i.e. control group; pm: 1X 108cells/mL Pichia pastoris suspension. Different letters represent significant differences (P)<0.05)。
FIG. 3 shows the inhibitory effect of Pichia kluyveri on Botrytis cinerea and Aspergillus niger. Note: CK: sterile distilled water, i.e. control group; pm: 1X 108cells/mL Pichia pastoris suspension. Different letters represent significant differences (P)<0.05)。
Detailed Description
Example 1: the microbiological properties of the Pichia anomala (Pichia manshurica) strain Y4 were as follows:
1. morphological characteristics
(1) Culturing in NYDA culture medium (beef extract 0.8%, yeast extract 0.5%, glucose 1%, agar 2%, and wet heat sterilizing at 121 deg.C for 20min) at 28 deg.C for 48 hr to obtain circular colony, smooth edge, and relatively wet colony. The cells are in an oblong shape and are scattered singly.
(2) After culturing in NYDB liquid culture medium for 24h, no pellicle was formed, the bacterial liquid was turbid, there was a precipitate, the microscopic yeast cells were oval, and budding.
2. Molecular biological identification
5.8S rDNA-ITS region sequence of screened strain Pichia anoploides is analyzed, and searched on GenBank to determine Pichia anoplosa (Pichia manshurica). Based on the retrieved homologous strains, a tree of biological evolutionary relationships was constructed as shown in FIG. 1 using the Mege5.1 program of DNAStar software.
The yeast Pichia manshurica (Pichia manshurica) Y4 is deposited in the institute of microbiology of China academy of sciences No. 3, Xilu No. 1 Beijing north township, China general microbiological culture Collection center (CGMCC), with the preservation number: CGMCC No.2.5415, with a preservation date of 2016, 3 and 31 months, and is named as Pichia anoshimeria according to classification.
Example 2: control effect of pichia kluyveri on citrus green mold
One) test protocol
The fruits to be tested are selected carefully, the fruits are selected as uniformly as possible, and no bruise or mechanical damage and insect damage are caused on the surfaces of the fruits. Using a sterile punch, 3 holes were punched at the equator of the fruit, and the surface wound of the citrus was 5mm (diameter) x 3mm (depth). Equal amounts of 30 μ L of the following treatment solutions were added to each wound: (1) 1X 108cells/mL of a Pichia pastoris suspension; (2) sterile distilled water. After 2h, 30 μ L of 1 × 10 seed solution was inoculated to the citrus wound5A suspension of spores/mL of penicillium digitatum (p.digitatum) spores. After natural drying, the fruits were placed in a plastic basket and sealed with a plastic wrap, cultured in a constant temperature incubator (humidity 95%) and placed at room temperature (20 ℃). Each treatment was repeated 3 times, 12 fruits per repetition. The whole experiment was repeated 2 times. And recording the fruit morbidity after 7 days of culture, and evaluating the bacteriostatic effect of the pichia anomala.
The formula for the incidence of disease is as follows:
incidence (%) of disease ═ total number of fruits/fruit affected × 100%
II) test results
According to the test of the steps, the result of counting the rotting rate of the fruits is as follows:
inhibitory effect on Citrus green mold
As shown in fig. 2, the incidence of control fruit was about 99.6% and pichia pastoris treated citrus was 45.3%. Compared with a control, the incidence rate of the citrus treated by the pichia kluyveri is obviously lower than that of the control (P <0.05), so that the pichia kluyveri can effectively control the postharvest green mold of the citrus caused by the penicillium digitatum at the temperature of 20 ℃.
Example 3: control effect of pichia kluyveri on grape gray mold and black aspergillosis
One) test protocol
Inoculating the activated yeast into a triangular flask containing 50mL of NYDB (yeast extract 5g, beef extract 8g, glucose 10g and distilled water 1000mL), and performing shake culture at 28 ℃ and 180r/min for 20 h. Centrifuging the bacteria liquid at 8000r/min for 10min, discarding the supernatant, repeatedly cleaning with sterile water for 3 times, and counting with a blood counting plate to obtain a solution with a concentration of 1 × 108one/mL of bacterial suspension, and sterile water as a control group. Punching 1 hole with diameter of 3m (width) × 3mm (depth) on fruit waist with puncher, injecting yeast suspension with concentration of above 15 μ L into each hole, and injecting sterile water 15 μ L into control fruit. After 2h, 10. mu.L of 1X 10 cells were inoculated to the grape wound5spore suspension of Botrytis cinerea (B. cinerea) or 5X 10 of spores/mL4Spors/mL of Aspergillus niger spore suspension. And (4) putting the fruits into a plastic basket after airing, sealing the plastic basket by using a preservative film, keeping the humidity at 95%, storing the fruits at room temperature, and counting the incidence rate of the fruits after 5 days. Each treatment was repeated 3 times, with 36 fruits repeated each time. The whole experiment was repeated 2 times.
The formula for the incidence of disease is as follows:
incidence (%) of disease ═ total number of fruits/fruit affected × 100%
II) test results
According to the test of the steps, the result of counting the rotting rate of the fruits is as follows:
1. inhibitory Effect on grape Gray mold
As shown in fig. 3, the incidence of the control group was 94.4%, and the incidence of the grapes treated with pichia klysteroids was 5.6%, so pichia klysteroids was able to effectively control post harvest gray mold disease of grapes caused by botrytis cinerea at room temperature.
2. Inhibitory Effect on grape Black aspergillosis
As shown in fig. 3, the incidence of the control group was 100% and the incidence of the grapes treated with pichia klysteroids was 11.1%, so pichia klysteroids was able to effectively control the post harvest black aspergillosis disease of grapes caused by aspergillus niger at room temperature.
The result is obviously better than the antagonistic bacteria biocontrol effect described in the related patents, for example, the Chinese invention patent "prevention and treatment method for postharvest diseases or fruit grain shedding of grapes" and its special prevention and treatment agent (application No. 200910078858.9) discloses a technology for preventing and treating grape diseases by using the combination of Cryptococcus laurentii and sodium borate, after grapes treated by Cryptococcus laurentii and sodium borate are placed for three days at room temperature, the rotting rate caused by gray mold is only reduced by about 50% compared with the control, and the inhibition effect on postharvest black aspergillosis of grapes is not shown; the Chinese invention patent application No. CN201410231417.9 provides a technology for preventing and treating postharvest diseases of grapes by using yarrowia lipolytica and degrading ochratoxin A, and introduces that the yarrowia lipolytica has a remarkable inhibition effect on Penicillium amphenicum, but does not show that the yarrowia lipolytica has an inhibition effect on postharvest black aspergillosis and gray mold of the grapes.
Example 4: preservation of Pichia anomala
The preferred medium is NYDA medium, but potato agar medium can also be used: 200 g of potato, 20 g of glucose, 15-20 g of agar, 1000ml of tap water, 7.0-9.0 of pH, 26-28 ℃ and culturing for 36 h.
Claims (4)
1. Pichia anomala (Pichia pastoris) (II)Pichia manshurica) Y4, the preservation number is CGMCC number 2.5415.
2. The use of a pichia kluyveri strain according to claim 1, for controlling the post-harvest green mold of citrus, the post-harvest black aspergillosis of grape and the gray mold, and for storage and preservation.
3. The method for controlling postharvest diseases and storing and refreshing the pichia kluyveri as claimed in claim 1 for citrus and grapes comprises the following steps: activating Pichia anomala, culturing with NYDB culture medium, centrifuging to obtain thallus, diluting with sterile water to obtain 1 × 108cell/mL of bacterial suspension; uniformly spraying the bacterial suspension on the fruits, and naturally drying; placing in a plastic basket, sealing with preservative film, and storing at room temperature.
4. The method for preventing and treating postharvest diseases of citrus and grapes by using pichia pastoris according to claim 3, wherein the NYDB culture medium is: 5g of yeast extract, 10g of glucose, 8g of beef extract and 1000ml of distilled water, wherein the pH is natural, and the beef extract is sterilized for 30min at 121 ℃.
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| CN107937289B (en) * | 2017-12-15 | 2021-06-25 | 北京工商大学 | Application of pichia pastoris BY27 strain in prevention and treatment of postharvest diseases of fruits |
| CN113801799B (en) * | 2021-08-13 | 2023-05-02 | 西南大学 | Yeast SLL12 and application thereof in preparation of biological control agent for controlling postharvest diseases of jujube fruits |
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