CN106818754B - Application of the plant polyphenol after preparation prevention and treatment citrus is adopted in green mold medicine - Google Patents
Application of the plant polyphenol after preparation prevention and treatment citrus is adopted in green mold medicine Download PDFInfo
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- CN106818754B CN106818754B CN201710106316.2A CN201710106316A CN106818754B CN 106818754 B CN106818754 B CN 106818754B CN 201710106316 A CN201710106316 A CN 201710106316A CN 106818754 B CN106818754 B CN 106818754B
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/02—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms
- A01N43/04—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom
- A01N43/14—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings
- A01N43/16—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings with oxygen as the ring hetero atom
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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
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Abstract
The invention discloses application of the plant polyphenol after preparation prevention and treatment citrus is adopted in green mold medicine.Tannin is preparing the application in anti-penicillium digitatum Penicillium digitatum drug.Tannin of the present invention is a kind of natural polyphenol class compound, it all has good inhibiting effect to the spore germination of citrus common green mold bacterium penicillium digitatum Penicillium digitatum and mycelia growth, the generation that citrusfruit adopts rear green mould can be significantly reduced, effectively extend the storage period of citrusfruit, and citrusfruit quality is not influenced, is had a good application prospect in citrusfruit anti-corrosive fresh-keeping field.
Description
Technical field
The invention belongs to fruit prevention and control of plant diseases, pest control fields, and in particular to plant polyphenol green mould after preparation prevention and treatment citrus is adopted
Application in drug.
Background technique:
Citrus common green mold is the most important disease of citrus post-harvest fruits, caused by loss account for about the 90% of total losses with
On.Production at present is upper mainly to prevent and treat citrus common green mold using chemical bactericides such as probenazole, imazalils, however these are used for a long time
Medicament can often cause the resistance problems of germ, and control efficiency is caused to be gradually reduced, or even failure.In addition, long-term a large amount of uses
Chemical bactericide is easy to cause damages to environment and human health.
Tannin is the natural polyphenol substance that a kind of higher plant generates.Existing research shows that tannin is thin to a variety of cause of diseases
Bacterium, fungi have preferable bacteriostatic activity.The invention reduces the means of prevention of abundant citrus common green mold to chemical sterilization
The dependence of agent improves the edible safety of citrusfruit, increases the economic value added of tannin.
Summary of the invention:
The first purpose of the invention is to provide a kind of tannin to prepare anti-penicillium digitatum Penicillium
Application in digitatum drug.
The present invention is found through experiments that tannin is to citrus common green mold bacterium penicillium digitatum Penicillium digitatum
With good inhibiting effect, minimal inhibitory concentration MIC is 1mg/ml.And tannin is able to suppress penicillium digitatum
The sprouting of Penicillium digitatum has good control efficiency to citrus common green mold.
Therefore, the present invention provides tannin in preparing anti-penicillium digitatum Penicillium digitatum drug
Using.
Second purpose of the invention is to provide application of the tannin in preparation prevention and treatment citrus common green mold drug.
Third purpose of the present invention is to provide a kind of prevention and treatment citrus method for adopting rear green mould, which is characterized in that including with
Lower step:
The citrusfruit without wound and disease-free pityriasis simplex trace is selected, be soaked in 1 in the tannin aqueous solution of 200~800 μ g/ml~
3min is subsequently placed in naturally dry at shady and cool ventilation, the packaging storage of single fruit bagging.
The tannin aqueous solution of 200~800 μ g/ml be tannin is soluble in water, be configured to respective concentration.
It is preferred that being 2min in the tannin aqueous solution for be soaked in citrusfruit 500 μ g/ml.500 μ g/ml tannin are to citrus
The inhibiting rate of green mould is 70.3%, and the citrus common green mold disease incidence of processing is only 2.3%.And the citrus of clear water control treatment
Green mould disease incidence is 7.6%.
Tannin of the present invention is a kind of natural polyphenol class compound, to citrus common green mold bacterium penicillium digitatum
The spore germination of Penicillium digitatum and mycelia growth all have good inhibiting effect, can significantly reduce mandarin orange
Tangerine fruit adopts the generation of rear green mould, effectively extends the storage period of citrusfruit, and does not influence citrusfruit quality, in mandarin orange
Tangerine fruit anti-corrosive fresh-keeping field has a good application prospect.
Preservation process method of the invention is simple, and operation is easy.
Detailed description of the invention:
Fig. 1 is the inhibitory effect that various concentration tannin grows citrus common green mold bacterium mycelia；
Fig. 2 is influence of the various concentration tannin to citrus common green mold bacterium spore germination；
Fig. 3 be inoculation after tannin processing to citrus common green mold bacterium control efficiency；
Fig. 4 is influence of the tannin processing to citrusfruit scab citrus common green mold bacterium DNA content；
Fig. 5 is influence of the tannin to citrus common green mold bacterium cell wall integrity.
Specific embodiment:
The following examples are further illustrations of the invention, rather than limiting the invention.
Embodiment 1:
One, experimental material and method
Experimental material:
Citrus common green mold bacteria strain Penicillium digitatum is inoculated in potato dextrose agar
On the inclined-plane (PDA, potato 200g, glucose 20g, agar 20g, water 1L), 25 DEG C of culture 7d, 4 DEG C are saved backup.
Tannin is purchased from Tianjin good fortune morning chemical reagent factory.
Experimental method:
1, bacterium colony growth inhibition test
By spore suspension (the 5 μ l 1 × 10 of citrus common green mold bacterium Penicillium digitatum6spores/ml)
It being inoculated on the PDA plate containing 200,400,600,800 and 1000 μ g/ml tannin, addition equivalent sterile water, which is used as, to be compareed, and 25 DEG C
Cultivate 7d.Crossing method measures colony diameter.3 repetitions of each experimental setup.Inhibiting rate (%)=(it is straight that water compares bacterium colony
Diameter-tannin processing colony diameter)/water control colony diameter × 100%.
By citrus common green mold bacterium Penicillium digitatum spore suspension (5 μ l 1 × 106Spores/ml it) connects
In equivalent sterile water on the PDA plate containing 200,400,600,800 and 1000 μ g/ml tannin, is added as control, 25 DEG C are trained kind
Support culture 3d.The growing state for observing citrus common green mold bacterium Penicillium digitatum, leads to citrus common green mold bacterium
The minimum tannin concentration that Penicillium digitatum mycelia cannot grow, as tannin grow citrus common green mold bacterium
Three repetitions are arranged in minimum inhibitory concentration (minimum inhibitory concentration, MIC), each concentration.
2, spore germination inhibits test
By spore suspension (the 10 μ l 1 × 10 of citrus common green mold bacterium Penicillium digitatum4spores/ml)
It is inoculated on the PDA culture medium plate containing 200,400 and 600 μ g/ml tannin respectively, equivalent sterile water is added as control.25
DEG C culture 12h, micro- sem observation count spore germination situation.It is considered as spore when germ tube length is greater than twice of spore length to sprout
Hair.Spore germination rate (%)=sprouting spore count/(sprout spore count+spore count is not sprouted) × 100%.Each processing is at least seen
Examine 100 spores.
3, inoculation test
Select the Ponkan fruit without wound and disease-free pityriasis simplex trace.Ponkan fruit 5min, clear water punching are impregnated with 2% liquor natrii hypochloritis
It washes three times, dries.A wound is pricked on Ponkan fruit equatorial plane position with transfer needle.By citrus common green mold bacterium
Penicillium digitatum spore suspension (10 μ l 1 × 105Spores/ml it) is inoculated in Ponkan wound, is inoculated with fruit
It is real to place 2h at room temperature, then impregnate 2min in the aqueous solution containing 500 μ g/ml tannin, isometric clear water leaching fruit is cooked pair
According to.Naturally dry is fitted into plastic casing, moisturizing of spraying water, room temperature storage.It stores 7d and observes disease incidence.It is each to handle in triplicate,
10 fruits of each reprocessing.
Wound scab is scraped with scalpel, measures scab weight.Total DNA at scab is extracted using CTAB method.It takes appropriate
Scab sample, be added 1ml CTAB, 65 DEG C of warm bath 1h, 10000g, be centrifuged 10min.Supernatant is taken, isometric chloroform/different is added
Amylalcohol (24:1), concussion mix.10000g is centrifuged 1min.Supernatant is taken, isometric isoamyl alcohol precipitating is added.10000g, centrifugation
10min.The rinsing of 75% ethyl alcohol of 1ml is added.10000g is centrifuged 10min.Supernatant is abandoned, is dried.It is added 50 μ l water dissolving DNAs, -20
DEG C save.
By citrus common green mold bacterium Penicillium digitatum spore suspension (500 μ l 1 × 106spores/ml)
It being inoculated in 100ml PDB culture medium, 25 DEG C, 180rpm shaken cultivation 3d, sterile gauze is collected by filtration mycelia, liquid nitrogen grinding,
Citrus common green mold bacterium Penicillium digitatum mycelia genomic DNA is extracted using CTAB method, Nanodrop 2000 surpasses
Micro-spectrophotometer measures DNA concentration.Citrus common green mold bacterium Penicillium digitatum mycelia genomic DNA is used
ddH2O is diluted to the standard items of various concentration.It usesPremix Ex TaqTMII fluorescence quantitative kit, with standard
Product are as template, with Actin-F (CGTTCGTGACATCAAGGA) and Actin-R (ATACCGCCAGACTCAAGA) for primer,
InQuantitative fluorescent PCR is carried out on 480II fluorescence quantitative PCR instrument.10 μ l quantitative fluorescent PCR systems: 1 μ l of template,
Quantitative fluorescent PCR upstream and downstream primer each 0.4 μ l, Premix Ex Taq II 5 μ l, ddH2O 3.2μl.PCR response procedures are as follows: 95
DEG C initial denaturation 30s, 95 DEG C of 5s, 60 DEG C of 30s are recycled 40 times.60-95 DEG C of temperature programming detects melt curve analysis.According to fluorescent value
Variation, establishes citrus common green mold bacterium Penicillium digitatum Actin gene absolute fluorescence quantitative PCR standard curve,
Detection is repeated 3 times.
Quantitative fluorescent PCR is carried out according to DNA of the above method to citrus common green mold bacterium at scab, each DNA sample does three
A repetition.According to starting copy number, the content of citrus common green mold bacterium DNA at scab is calculated by standard curve.
4, mechanism study
By citrus common green mold bacterium Penicillium digitatum spore suspension (5 μ l 1 × 106Spores/ml it) connects
Kind on the PDA plate containing 250 μ g/ml tannin and 0.15 ‰ SDS, tannin and SDS is not added as compareing, 25 DEG C of culture 7d.
Crossing method measurement colony diameter simultaneously calculates bacterium colony growth inhibition ratio.3 repetitions of each experimental setup.
Two, experimental result:
1, tannin obviously inhibits citrus common green mold bacterium mycelia to grow
Fig. 1 is that various concentration tannin imitates the inhibition that citrus common green mold bacterium Penicillium digitatum mycelia grows
Fruit.Compared with sterile water control, 400 μ g/ml tannin grow citrus common green mold bacterium Penicillium digitatum mycelia
Inhibiting rate is 40.8 ± 2.6%.600 μ g/ml tannin grow citrus common green mold bacterium Penicillium digitatum mycelia
Inhibiting rate is 67.2 ± 4.0%.When the final concentration of tannin in PDA culture medium reaches 800 μ g/ml, inhibiting rate is up to 83.9 ±
2.0%.
MIC experiment shows the minimal inhibitory concentration that tannin grows citrus common green mold bacterium Penicillium digitatum
MIC is 1mg/ml.
2, tannin inhibits the sprouting of citrus common green mold bacterium spore
Germination rate of the citrus common green mold bacterium Penicillium digitatum spore in the PDA culture medium without tannin
It is 92%.And citrus common green mold bacterium Penicillium digitatum spore is in the PDA culture medium containing 400 μ g/ml tannin
Germination rate is 45%, and corresponding inhibition of germination is 51%.When the final concentration of tannin in PDA culture medium reaches 600 μ g/ml
When, the spore of citrus common green mold bacterium is all without sprouting (Fig. 2).Also, spore expands deformation.
3, tannin is substantially reduced citrus common green mold bacterium infecting in citrusfruit wound
Citrus common green mold bacterium Penicillium digitatum spore suspension is inoculated on citrusfruit respectively, is connect
Kind 2h, with the leaching fruit processing of 500 μ g/ml tannin aqueous solutions.Room temperature moisturizing culture observes incidence.It is inoculated with 2d, inoculation citrus is green
There is apparent water stain shape in soak after the mildew bacterium citrusfruit wound of processing, and is inoculated with citrus common green mold bacterium
There is no above-mentioned symptoms for the citrusfruit wound of tannin immersion treatment after Penicillium digitatum.It is inoculated with 7d, tannin
The citrusfruit scab of processing is smaller, and lesion diameter is only 60% or so (Fig. 3) for compareing (immersion is handled after inoculation).
Meanwhile this experiment utilizes the content of citrus common green mold bacterium DNA at fluorescent quantitative PCR technique detection scab.From experiment
As a result the regression equation for obtaining standard curve is Y=-3.0716X+22.954, regression coefficient R2=0.995.Solubility curve table
It is now single peak.According to starting copy number, the content of citrus common green mold bacterium DNA at scab is calculated by standard curve.Inoculation
7d is inoculated with after citrus common green mold bacterium the citrusfruit wound citrus common green mold bacterium Genome DNA content of processing of soaking and is up to
205.83 ± 7.44 μ g, and 500 μ g/ml tannin are 90.5% to the inhibiting rate of artificial infection citrus common green mold bacterium, it is corresponding
Citrus common green mold bacterium Genome DNA content is 19.58 ± 1.72 μ g (Fig. 4).
4, research of the tannin to citrus common green mold bacterium antifungal mechanism
It is coerced using SDS, studies tannin to citrus common green mold bacterium Penicillium digitatum cell wall integrity
It influences (Fig. 5).When not adding tannin, the inhibiting rate that 0.15 ‰ SDS grow citrus common green mold bacterium bacterium colony is only 29.3%.When
When adding 250 μ g/ml tannin, the inhibiting rate that 0.15 ‰ SDS grow citrus common green mold bacterium bacterium colony is up to 58.9%.As a result table
Bright, tannin destroys the integrality of citrus common green mold bacterium cell wall, causes citrus common green mold bacterium higher to the sensibility of SDS.Table
Bright tannin inhibits the growth of citrus common green mold bacterium by the integrality of destruction cell wall.
In conclusion tannin causes nutriment intracellular to exosmose, in fact by destroying citrus common green mold bacterium cell wall integrity
Now to the inhibiting effect of bacterium colony growth and spore germination.Vitro Inoculation Technique experiment and storage experiment show 500 μ g/ml tannin to citrus
Green mould has good control efficiency.
Embodiment 2
Select the granulated sugar tangerine fruit without wound and disease-free pityriasis simplex trace.Adopt non-spraying pesticide of the previous moon.It is soaked in 500 μ g/
2min in the tannin aqueous solution of ml, is placed in naturally dry at shady and cool ventilation, is fitted into plastic casing, room temperature storage.It unites after storage 7d
Count disease incidence.It is each to handle in triplicate, 100 fruits of each reprocessing.The tannin aqueous solution of the 500 μ g/ml is
The tannin of 500 micrograms is placed in 1ml water, obtains the tannin aqueous solution of 500 μ g/ml after mixing.Storage experiment result table
Bright, the citrus common green mold disease incidence of clear water control treatment is 7.6%.Inhibition of the 500 μ g/ml tannin aqueous solutions to citrus common green mold
Rate is 70.3%, and the citrus common green mold disease incidence of processing is only 2.3%.
Embodiment 3
Selection maturity reaches the citrusfruit of commercial requirements, is soaked in the tannin aqueous solution of 200 μ g/ml
3min is placed in naturally dry at shady and cool ventilation, single fruit bagging packaging, storage.Disease incidence is counted after storage 7d.Each processing repeats
Three times, 100 fruits of each reprocessing.The tannin aqueous solution of the 200 μ g/ml is that the tannin of 200 micrograms is placed in 1ml
In water, the tannin aqueous solution of 200 μ g/ml is obtained after mixing.Storage experiment the result shows that.200 μ g/ml tannin are green to citrus
The inhibiting rate of mildew is preferable, and the citrus common green mold disease incidence of processing is lower.
Embodiment 4
Selection maturity reaches the citrusfruit of commercial requirements, is soaked in the tannin aqueous solution of 800 μ g/ml
1min is placed in naturally dry at shady and cool ventilation, single fruit bagging packaging, storage.Disease incidence is counted after storage 7d.Each processing repeats
Three times, 100 fruits of each reprocessing.The tannin aqueous solution of the 800 μ g/ml is that the tannin of 800 micrograms is placed in 1ml
In water, the tannin aqueous solution of 800 μ g/ml is obtained after mixing.800 μ g/ml tannin are preferable to the inhibiting rate of citrus common green mold,
The citrus common green mold disease incidence that it is handled is lower.
Claims (4)
1. tannin is preparing answering in anti-penicillium digitatum Penicillium digitatum drug as sole active agent
With.
2. application of the tannin as sole active agent in preparation prevention and treatment citrus common green mold drug.
3. a kind of method that prevention and treatment citrus adopts rear green mould, which comprises the following steps:
Select the citrusfruit without wound and disease-free pityriasis simplex trace, be soaked in the tannin of 200~800 μ g/ml as sole active at
1~3min in the aqueous solution divided, is subsequently placed in naturally dry at shady and cool ventilation, is fitted into box and stores.
4. according to the method described in claim 3, it is characterized in that, be using citrusfruit be soaked in the tannin of 500 μ g/ml as
2min in the aqueous solution of sole active agent.
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| CN113455515A (en) * | 2021-06-24 | 2021-10-01 | 广东省农业科学院果树研究所 | Application of cantharidin in preparation of pesticide bactericide for preventing and treating postharvest chloromycete of citrus |
| CN115119872A (en) * | 2022-06-29 | 2022-09-30 | 中国热带农业科学院海口实验站 | Application of polyphenol protein nanoparticles |
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Non-Patent Citations (4)
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| Biological activities of the extracts from wild growing Origanum vulgare L;Braho Z. Licina等;《Food Control》;20131231;第33卷;第498-504页 * |
| Braho Z. Licina等.Biological activities of the extracts from wild growing Origanum vulgare L.《Food Control》.2013,第33卷第498-504页. * |
| Phytochemical Analysis and Antimicrobial Activity of Mangrove Leaves;Foukia E. Mouafi等;《World Applied Sciences Journal》;20141231;第29卷(第4期);第547-554页 * |
| Phytochemical analysis, antifungal;Hussan Ara Begum等;《Pure Appl. Biol.》;20160630;第5卷(第2期);第183-192页 * |
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