CN108101971A - A kind of extraction of tropical American hymenocallis leaf pinta nosotoxin and activity determination method - Google Patents
A kind of extraction of tropical American hymenocallis leaf pinta nosotoxin and activity determination method Download PDFInfo
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- CN108101971A CN108101971A CN201711260550.7A CN201711260550A CN108101971A CN 108101971 A CN108101971 A CN 108101971A CN 201711260550 A CN201711260550 A CN 201711260550A CN 108101971 A CN108101971 A CN 108101971A
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Abstract
Extraction and activity determination method the invention discloses a kind of tropical American hymenocallis leaf pinta nosotoxin, include the following steps：1) disease sites on the water ghost any of several broadleaf plants with leaf spot is taken to extract botrytis cinerea Raw toxin and verticillium sp Raw toxin and are identified；2) the botrytis cinerea Raw toxin and verticillium sp Raw toxin extracted to step 1) carries out protein content determination；3) botrytis cinerea Raw toxin and verticillium sp Raw toxin that step 1) extracts are configured to the solution of various concentration respectively, healthy water ghost any of several broadleaf plants seedling leaves are smeared by semar technique, carry out wilt activity measure.Extraction and activity determination method of the present invention for tropical American hymenocallis leaf pinta toxin, have effectively drawn the protein content of botrytis cinerea Raw toxin and verticillium sp Raw toxin and the influence to tropical American hymenocallis leaf pinta, the prevention of tropical American hymenocallis leaf pinta provides technical support for the later stage.
Description
Technical field
The present invention relates to domestication of plants field, the extraction of more particularly to a kind of Causative virus and activity determination method.
Background technology
Water ghost any of several broadleaf plants also known as America Hymenocallis americana, Amaryllidaceae Hymenocallis plant are a kind of perennial flowering bulbs, main to produce
Ground is concentrated in South America with Southeast Asia matto area, and original producton location is american torrid zone area.China Guangdong, Guangxi, Fujian,
The large area distribution of the south such as Yunnan, Chongqing provinces and cities.Its leaf color is verdant, leaf beauty；Pattern is pure white, and flower-shape is spoilt U.S.；With higher
Ornamental value.The Hua Yuwei " natural beauty " of water ghost any of several broadleaf plants has fine implied meaning, and appearance is elegant free from vulgarity, therefore in ornamental flower
Grass occupies certain share in the market, has certain economic value.It is a variety of containing lycorine etc. in the bulb of water ghost any of several broadleaf plants simultaneously
Alkaloid has good pharmacologic action for relaxing tendons and activating collaterals, there is higher medical value, is usually used in the symptoms such as bruise, swelling and pain.
On Greening functions, it is usually used in roadside greening, park irrigation, the shady greening of bridge, courtyard greening etc., is the main greening crop in China；
It is also the second class protection plant in China simultaneously.
Hymenocallis americana is a kind of good aromatic raw material, since it possesses light faint scent free from vulgarity, and other fragrant flowers,
Such as：Rose, frangipanis etc. can refine the fragrance such as essential oil and the fragrant medicinal extract of extraction.Its fragrant cosmetic product and health articles for use are made
Product are subject to the warm welcome and support of consumers in general, and sales volume is considerable, is sold abroad, and form an emerging industrial chain, create
Larger economic value.Meanwhile water ghost any of several broadleaf plants, as Fresh Cutting flower, because its shape is free from vulgarity, pattern is simple and elegant, similary on consumption market
The ardent of consumer is subject to pursue, with the traditional flowers such as carnation, lily, the substantial amounts of market share of the unity of possession.Thus may be used
Know, water ghost any of several broadleaf plants not only possesses ornamental effect, pharmacologic action, Greening functions, also possesses economic function.Therefore, water ghost any of several broadleaf plants is important
Ornamental crops, medicinal crop, greening crop, industrial crops, the leaf spot that it is often sent out is studied, reduce incidence be very
It is necessary to.
Water ghost any of several broadleaf plants often has leaf spot, and morbidity is generally caused by the following.(1) bacterium that seed belt leaf spot fungi is led to
Property leaf spot；(2) field germ primary infection；(3) overwintering leaf spot fungi remains in caused bacterial leaf spot on invalid body
Secondary Infection；Wherein, most important cause of disease source is thirdly, can to avoid the infringement of pathogen by cleaning rural area, drop
Low incidence.
The morbidity of leaf spot is generally related with the factors such as fertilising, sowing time, meteorology, wherein：(1) apply fertilizer：Nitrogenous fertilizer is generally right
Leaf spot plays the role of promoting morbidity, and potash fertilizer then plays the role of inhibiting morbidity；Within the specific limits, increase with the amount of application of nitrogenous fertilizer
More, the progression of the disease of leaf spot is heavier；Increase with the amount of application of potash fertilizer, the state of an illness of leaf spot gets over unobvious.It can be seen that potash fertilizer
Plant can be made to obtain stronger disease resistance, keep out disease invasion, and nitrogenous fertilizer is then opposite；(2) sowing time：The generation and sowing of disease
Phase is related, and with sowing time ahead of time with postponement, the generation of disease is also correspondingly done sth. in advance with development and postponement；But sowing time gets over
In evening, that a situation arises is more serious for disease, therefore mitigates disease, should avoid late cultivated evening kind；(3) meteorologic factor：The generation of disease
Related with meteorologic factor, temperature is higher, and humidity is bigger, is more conducive to the growth of leaf spot germ, falls ill so as to cause leaf spot
Situation is more serious；Rainfall is early, and period of disease is more early；Weather Continuous Drought has inhibitory action to the growth of leaf spot germ, thus
Disease is inhibited.The morbidity of tropical American hymenocallis leaf pinta is then mainly related with meteorologic factor, the high temperature and rainy in Pearl River Delta area
In weather, morbidity is particularly acute, this is because hot and humid weather is grown on development beneficial to tropical American hymenocallis leaf pinta pathogen,
So as to which the leaf spot as caused by botrytis cinerea with verticillium sp is serious with regard to incidence.It is acted on to the lesion for weakening leaf spot, just
The measure of various drainings, dehumidifier, temperature adjustment should be needed to pay attention to, makes to be led to that the botrytis cinerea of leaf spot, verticillium sp should not be grown, this
Sample is with regard to that can weaken the generation and development of leaf spot.
Due to Pearl River Delta area, humidity is larger throughout the year, and green plant environment ventilation is bad, water ghost any of several broadleaf plants is easily made leaf spot occur, sternly
Ornamental value, greening value and the economic value of ghost image Xiangshui County ghost any of several broadleaf plants.Existing pertinent literature is consulted, is found for tropical American hymenocallis leaf
The research of pinta quite lacks.So it is studied because the influence of disease causes ornamental value, greening value, the drop of economic value
Low, the cost of greening maintenance increases particularly important.If the occurring degree of the leaf spot of water ghost any of several broadleaf plants can be reduced, just can fundamentally drop
The cost of low various greening maintenances, such as：Using the expense of fungicide；Trim the labour cost of sick body；The greening more renewed is made
The cost of the costliness such as expense of object；Maintain ornamental, economic dispatch value again simultaneously.
By pathogen generate low molecule secondary metabolite be known as toxin, even if be one kind in the case where concentration is very low
It remains to encroach on plant, plant is caused disease occur, be both not belonging to enzyme, be also not belonging to hormone, be with planting in pathogen
The pilot process product be mutually distinguishable between object, to interact；It is to be subject to germ parasitic mostly when plant is infected be subject to pathogen
The metabolite generated in host the influence of inhibitions such as causes to grow, develop, host is just made to be unfavorable for growing to host
Development, final disease resistance are reduced and caused a disease；Therefore, we have to see clearly the relevant feature of toxin, could further study
Tropical American hymenocallis leaf pinta, and make the scheme of prevention；Toxin has following characteristics：(1) toxin is not belonging to enzyme material, while does not also belong to
It is the secondary metabolite of pathogen in hormone substance；(2) toxin has significantly damages effect to tissue, organ of plant etc.；
(3) toxin has plant apparent pathogenic effects；(4) toxin is generated by spore rudiment；(5) toxin has disease induction to make
With.
Toxin is divided into：Two kinds of Host-specific toxin and Non-host-specific toxin, Host-specific toxin is to host's
Kind or the selective effect of kind only show dissemination to partial response host, it are made symptom occur；Non- host speciality poison
For element to the kind or kind of host then without selectively acting, specialization is not strong, makes most of plant that all or part of symptom occur；
Germ generates the toxic substance with the lesion effect that causes to wither as protein, and fat and polysaccharide material are then without causing the lesion work that withers
With it is consistent, pathogenic toxin that the mechanism of action of toxin protein infects the Disease symptoms that host is led to germ in essence
Substance is only to host with wilt activity just with practical significance.
The nosotoxin of tropical American hymenocallis leaf pinta is mainly botrytis cinerea and verticillium sp.Therefore, water ghost any of several broadleaf plants how is effectively extracted
Leaf spot nosotoxin simultaneously carries out determination of activity, is of great significance to the prevention for studying tropical American hymenocallis leaf pinta nosotoxin.
The content of the invention
It is an object of the invention in view of the above shortcomings of the prior art, provide a kind of tropical American hymenocallis leaf pinta nosotoxin
Extraction and activity determination method.
The technical solution used in the present invention is：A kind of extraction of tropical American hymenocallis leaf pinta nosotoxin and determination of activity side
Method includes the following steps：
1) disease sites on the water ghost any of several broadleaf plants with leaf spot is taken to extract botrytis cinerea Raw toxin and verticillium sp Raw toxin
And it identifies；
2) the botrytis cinerea Raw toxin and verticillium sp Raw toxin extracted to step 1) carries out protein content determination；
3) botrytis cinerea Raw toxin and verticillium sp Raw toxin that step 1) extracts are configured to the molten of various concentration respectively
Liquid smears healthy water ghost any of several broadleaf plants seedling leaves by semar technique, carries out wilt activity measure.
As being further improved for said program, described be accredited as after medium culture of step 1) is swept by range estimation with Electronic Speculum
Retouch method identification.
As being further improved for said program, the step 2) protein content uses spectrophotometer measurement, standard
The regression equation of curve is y=0.006x, R2=0.950.
As being further improved for said program, the cycle that the step 2) wilt activity measures is 15 days.
The beneficial effects of the invention are as follows：Extraction and activity determination method of the present invention for tropical American hymenocallis leaf pinta toxin, have
Effect has drawn the protein content of botrytis cinerea Raw toxin and verticillium sp Raw toxin and the influence to tropical American hymenocallis leaf pinta, is the later stage
The prevention of tropical American hymenocallis leaf pinta provides technical support.
Specific embodiment
The present invention is specifically described with reference to embodiment, in order to technical field personnel to the present invention
Understand.It is necessary to it is emphasized that embodiment is only intended to, the present invention will be further described herein, it is impossible to be interpreted as to this
The limitation of invention protection domain, fields person skilled in the art, the non-intrinsically safe made according to foregoing invention content to the present invention
The modifications and adaptations of property, should still fall within protection scope of the present invention.Mentioned raw materials following simultaneously are unspecified, are
Commercial product；The processing step or preparation method not referred in detail be processing step known to a person skilled in the art or
Preparation method.
A kind of extraction of tropical American hymenocallis leaf pinta nosotoxin and activity determination method, include the following steps：
1) disease sites on the water ghost any of several broadleaf plants with leaf spot is taken to extract botrytis cinerea Raw toxin and verticillium sp Raw toxin
And identify, wherein being accredited as bacterium colony initial stage for the thick virus of botrytis cinerea as white, radioactivity mycelia continues to train with botrytis cinerea
Health is long, and bacterium colony begins to change into grey black, and mycelium color is gradually deepened, and the later stage can obtain conidium as ellipse or spherical single
Spore, conidiophore is several to grow thickly, and has membrane, raw a large amount of conidiums, and appearance is in grape spike；It is thick for verticillium sp
Toxin is accredited as bacterium colony initial stage as dark brown, more sticky, can obtain that conidiophore is several verticillate, and branches end generates the later stage
Multiple ampuliforms produce spore, and conidium is often monospore with being gathered into fugitive spore ball at the top of bottle.
2) the botrytis cinerea Raw toxin and verticillium sp Raw toxin extracted to step 1) carries out protein content determination, specifically
Ground, by the map data in the following table 1, standard protein content is under spectrophotometer, the OD that is surveyed595Value is respectively
0.000,0.180,0.332,0.415,0.504,0.592, the regression equation of standard curve is y=0.006x, R2=0.950,
Related coefficient is more than 0.9, illustrates that the standard curve is highly reliable, the foundation available for following proteins assay.
The OD of 1 protein standard curve of table595Value
3) botrytis cinerea Raw toxin and verticillium sp Raw toxin that step 1) extracts are configured to the molten of various concentration respectively
Liquid smears healthy water ghost any of several broadleaf plants seedling leaves by semar technique, carries out wilt activity measure.Specifically, it is dense by the difference of preparation
Botrytis cinerea Raw toxin solution treatment A1~A4 groups of degree, verticillium sp Raw toxin solution treatment B1~B4 groups of various concentration, often
1 water ghost any of several broadleaf plants spire wilting situation was recorded every 3 days, calculates the percentage that tikka area accounts for total blade face area, lasting record and meter
It calculates 15 days, obtains such as the following table 2 data.The percentage of total blade face area is accounted for by tikka area, divides six grades, carries out data
Statistics, obtains table 3.It is obtained by 3 data of table, Different Kinds of Pathogens object influence situation of withering on the cause of water ghost any of several broadleaf plants seedling leaves is different.
In the case where the time is the same, Raw toxin content is identical, botrytis cinerea is larger to the toxic action of water ghost any of several broadleaf plants seedling leaves, takes turns branch spore
Bacterium is then relatively weak.In the case where other conditions are the same, with the increase of pathogen Raw toxin content in solution, water ghost any of several broadleaf plants seedling
The percentage that the tikka area of blade accounts for total blade face area is bigger, until situation of withering is more apparent.After the 15th day, water ghost any of several broadleaf plants seedling leaf
Piece is substantially withered, wilts, necrosis.Formula：Cause a disease percentage (%)=total lesion area/total blade face area.
2 water ghost any of several broadleaf plants seedling lesion area of table accounts for percentage (%) record sheet of total blade face area
The toxin of 3 botrytis cinerea of table and verticillium sp is to the measure statistical form of the wilt activity of water ghost any of several broadleaf plants seedling
Note：-：Substantially what changes (lesion area accounts for total leaf area 0%) without；
+：There are slight scab, symptom unobvious in leaves water loss (lesion area accounts for total leaf area 0~10%)
++：There is apparent tikka, symptom is more apparent (lesion area accounts for total leaf area 10~30%)；
+++：Tikka is apparent, and tikka center starts to be recessed (lesion area accounts for total leaf area 30~50%)；
++++：Tikka center starts to dry up, and becomes brown, and symptom is apparent (lesion area accounts for total leaf area 50~70%)；
+++++：Blade starts withered, wilting, tikka partial necrosis (lesion area accounts for total leaf area 70~90%).
Above-described embodiment is the preferred embodiment of the present invention, all with similar technique of the invention and the equivalence changes made,
The protection category of the present invention should all be belonged to.
Claims (4)
1. extraction and the activity determination method of a kind of tropical American hymenocallis leaf pinta nosotoxin, which is characterized in that include the following steps：
1) disease sites on the water ghost any of several broadleaf plants with leaf spot is taken to extract botrytis cinerea Raw toxin and verticillium sp Raw toxin and are reflected
It is fixed；
2) the botrytis cinerea Raw toxin and verticillium sp Raw toxin extracted to step 1) carries out protein content determination；
3) botrytis cinerea Raw toxin and verticillium sp Raw toxin that step 1) extracts are configured to the solution of various concentration respectively, led to
It crosses semar technique and smears healthy water ghost any of several broadleaf plants seedling leaves, carry out wilt activity measure.
2. extraction and the activity determination method of a kind of tropical American hymenocallis leaf pinta toxin according to claim 1, it is characterised in that：
Step 1) is described to be accredited as after medium culture through range estimation and the identification of electron-microscope scanning method.
3. extraction and the activity determination method of a kind of tropical American hymenocallis leaf pinta toxin according to claim 1, it is characterised in that：
Step 2) the protein content uses spectrophotometer measurement, and the regression equation of standard curve is y=0.006x, R2=
0.950。
4. extraction and the activity determination method of a kind of tropical American hymenocallis leaf pinta toxin according to claim 1, it is characterised in that：
The cycle that the step 2) wilt activity measures is 15 days.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101822272A (en) * | 2010-04-21 | 2010-09-08 | 中国农业科学院草原研究所 | Streptomyces griseoflavus and application thereof in biological prevention and control of plant diseases |
| CN103954714A (en) * | 2014-01-03 | 2014-07-30 | 南通市产品质量监督检验所 | Method for determination of aflatoxin |
-
2017
- 2017-12-04 CN CN201711260550.7A patent/CN108101971A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101822272A (en) * | 2010-04-21 | 2010-09-08 | 中国农业科学院草原研究所 | Streptomyces griseoflavus and application thereof in biological prevention and control of plant diseases |
| CN103954714A (en) * | 2014-01-03 | 2014-07-30 | 南通市产品质量监督检验所 | Method for determination of aflatoxin |
Non-Patent Citations (1)
| Title |
|---|
| 侯丽娟等: "棉花黄萎病菌液体培养特性及其毒素的生物测定", 《陕西农业科学》 * |
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