CN108101972A - Extraction and activity determination method of a kind of money tree with maize ear rot bacterium nosotoxin - Google Patents
Extraction and activity determination method of a kind of money tree with maize ear rot bacterium nosotoxin Download PDFInfo
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- CN108101972A CN108101972A CN201711273128.5A CN201711273128A CN108101972A CN 108101972 A CN108101972 A CN 108101972A CN 201711273128 A CN201711273128 A CN 201711273128A CN 108101972 A CN108101972 A CN 108101972A
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- C—CHEMISTRY; METALLURGY
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
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- G01N33/5014—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity
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- G01—MEASURING; TESTING
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
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Abstract
The invention discloses a kind of extraction of money tree with maize ear rot bacterium nosotoxin and activity determination methods, include the following steps：1) disease sites on the money tree with root rot is taken to extract Sickle-shaped rice and pythium spp Raw toxin and are identified；2) Sickle-shaped rice and pythium spp Raw toxin extracted to step 1) carries out protein content determination；3) Sickle-shaped rice and pythium spp Raw toxin that extract step 1) are configured to the solution of various concentration respectively, smear healthy money tree seedling leaves by semar technique, carry out wilt activity measure.The present invention has effectively drawn the protein content of Sickle-shaped rice and pythium spp Raw toxin and the influence to money tree, technical support is provided with the prevention of maize ear rot bacterium for later stage money tree for extraction and activity determination method of the money tree with maize ear rot bacterium nosotoxin.
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
Money tree is called ornamental plant Malabar chestnut, is to belong to perennial Bombacaceae arbor, once by the United Nations's environmental protection
Tissue is chosen as one of ten big world indoor appreciation flowers and trees.It is extremely expanded at money tree rhizome, the bodily form is tall and big, tree-like peculiar, blade
Palmate, tree-like gracefulness is evergreen all the year round, and implied meaning is good.But regrettably, though the fast root system of money tree growth is undeveloped, every year because of root
Maize ear rot is dead or consumer abandons and the waste and old tree of generation causes the serious wasting of resources and environmental pollution.
Root rot is the general name of a major class disease as caused by a variety of pathogens and environmental activity.Cause the germ of root rot
There are many kinds, and cause root rot is pathogen of soybean root rot, and the germ of Strawberry Root Rot is caused to claim Fusarium oxysporum
With the Cochliobolus sativus that causes root rotof flax etc..Money tree root rot also known as Malabar chestnut root rot, main harm rootstock
Following xylem, susceptible initial stage, there is the scab of taupe in root cortex before this, then developed to fibrous root, lateral root, main root
With rotting for xylem, when root rot proceeds to certain stage, blade can be sagging therewith, withered until complete stool is dead.
Whatsoever plant is likely to be infected be subject to disease in growth course.The patient of aerial part can make plant
Object loses ornamental value and Greening functions, can also cause some problems, including：Form is incomplete, and part tree body is withered.But root
Disease can make plant completely dead, cause urban greening species, wooden woods and scenic forest are large stretch of dead, and caused harm is huge
With it is destructive.
So we will recognize the symptom of root rot：It can be divided into that butt portion is rotten and root cortex rots, the rotten master of base portion
Large-scale fructification is born at rotten place.Often show as that color is light and foliation is slow, leaf color is abnormal.Growth potential fails, leaf
Type becomes smaller.Completely dead after first part is withered, the process of morbidity is gradual, some big trees can postpone as long as the several years.
Sickle-like bacteria is one of most important parasitic disease fungus to harm the crops.Most powder is found that early in Link in 1809
Red sickle-like bacteria, and the classification in belonging to is established therefore.Sickle-like bacteria except can cause the root of plant, stem, leaf rot it is withered outer, and also
A variety of secondary metabolites can be generated in growth and development process --- mycotoxin.Such as：With very supervirulent volt horse bacterium
Element and AAL toxin, can directly kill the nucleus of plant or concentrate in the seed of grain, after people and domestic animal eat
Internal organ failure can be caused.Exactly because sickle-like bacteria is so big to the harmfulness of the mankind and animals and plants, make many top in the world
Scientific research expert necessity of research is all generated to it.It is also at present very more for the research of sickle-like bacteria, but in many aspects
Perhaps research deficiency.Such as：Colonial morphology and identification to sickle-like bacteria, microorganism are very indistinguishable from form, and
The spore of sickle-like bacteria contains the sexual and asexual stage, but it is very may be used that spore device wherein sexual to fungi, which carries out Morphological Identification,
A kind of capable method.Further, it is also possible to using specific antibiotic to the principle of the selection index system of specific fungi, with reference to micro- skill
Art, this is also a kind of method for identifying sickle-like bacteria.But for can definitely identify a kind of unified method in world of sickle-like bacteria also
Do not find out.Generally speaking, sickle-like bacteria species is various, and host distribution scope is wide, universal geography complex distribution, and breeds
Ability is strong.There is destructive harm to plant, there is greatly harm influence, serious harm and people on the mankind and animal
The closely bound up grain of class, generates many Food Securities.Therefore, the pathological research of sickle-like bacteria is always the heat of academia
Point.
Pythium spp is a kind of low biology, and the species of pythium spp is very various, and more than 200 kinds of corruption was reported in the whole world at present
Mould, wherein what is recognized extensively has more than 150 to plant, it is more than 20 kinds that can cause plant disease.Rotten mould root rot is pythium spp danger
A kind of more serious disease of evil, mainly endangers seedling, can also be endangered after strain.Rotten mould root rot early stage shows as a
Other supporting root and fibrous root are susceptible, and gradually expand to main root, and with the propulsion of time, main root rots to aggravate, absorb nutrient and
The deterioration of moisture, particularly yellow leaf, young leaves, makes here plant withers.Pythium spp can not only cause the diseases such as plant root rot
Evil causes crop failure and is detrimental to health, moreover it is possible to toxic substance is generated in its growth growth course --- pythium spp
Toxin, it should be noted that its toxicity is very strong.Its mechanism of action is as follows：Aflatoxin B1 can inhibit DNA, courier first
The synthesis of DNA and protein reduce the action activity of RNase, cause chromosome abnormality, such as generate core change, the unordered conjunctions of DNA
Into the fracture of, chromosome chain and Sister chromatid exohange etc..This is an example, the mechanism of action of other toxin and aspergillus flavus
Plain B1's is similar, but still the mechanism of action of some toxin is unclear, requires study.At present for pythium spp
Research is also one of hot spot of the major scientific research field in the world, and toxin, type and quantity on pythium spp etc. has very deep
The research entered, but be also there are many insufficient places, for pythium spp as the research of sickle-like bacteria in many aspects
Identification can only carry out a general identification again without a set of relatively more unified exact saying by physics and chemical method.
Generally speaking, pythium spp and the characteristics of sickle-like bacteria：Yield is few, and strong toxicity, host range is extensive, and species is various,
Fertility is strong.Crops and plant are seriously endangered, endangers the health of human and animal, and then generates many unpredictalbe ask
Topic, so research pythium spp and sickle-like bacteria have become current keep away not as good as one of problem treated.
Toxin is the metabolite of pathogen, has apparent pathogenic or murder by poisoning to make during phytopathogen occurrence and development
With there is apparent damaging action to host.The plant of selection index system or contratoxin sensitivity is whether there is to host's kind or kind according to toxin
The phytopathy original can be divided into two major classes by object scope：One kind is Non-host-specific toxin, and another kind of is host speciality poison
Element.Non- host speciality speed does not have serious host selection and specialization, has one to host plant and non-host plant
Fixed physiological activity brings it about part or all of symptom.Opposite Host-specific toxin is one kind to susceptible variety and host
Plant has more highly pathogenic.50 various plants pathogen fungies can generate Non-host-specific toxin so far, and generate
The phytopathy original fungi of Host-specific toxin has more than 20 kinds.
For the metabolite toxin of pathogen its essence is protein and lipopolysaccharide complex, wherein protein, which has, causes disease of withering
Change acts on, and the ingredient of no wilt activity is fat and polysaccharide, and germ infects host and causes Disease symptoms and protein in toxin
Its essence is the same for the mechanism of action.Therefore, how to carry out quick accurate biological activity to phytopathy original toxin is one ten
Divide important link.
The nosotoxin of money tree root rot is mainly sickle-like bacteria and pythium spp.Therefore, how effectively to extract money tree with
The nosotoxin of maize ear rot bacterium simultaneously carries out determination of activity, has great meaning with the prevention of maize ear rot bacterium nosotoxin to research money tree
Justice.
The content of the invention
It is an object of the invention to be directed to the deficiency of above-mentioned technology, a kind of money tree carrying with maize ear rot bacterium nosotoxin is provided
It takes and activity determination method.
The technical solution used in the present invention is：Extraction and determination of activity side of a kind of money tree with maize ear rot bacterium nosotoxin
Method includes the following steps：
1) disease sites on the money tree with root rot is taken to extract Sickle-shaped rice and pythium spp Raw toxin simultaneously
Identification；
2) Sickle-shaped rice and pythium spp Raw toxin extracted to step 1) carries out protein content determination；
3) Sickle-shaped rice and pythium spp Raw toxin that extract step 1) are configured to the solution of various concentration respectively,
Healthy money tree seedling leaves are smeared by semar technique, carry 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=0.0064x, R2=0.9503.
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：The present invention is directed to extraction and determination of activity side of the money tree with maize ear rot bacterium nosotoxin
Method has effectively drawn the protein content of Sickle-shaped rice and pythium spp Raw toxin and the influence to money tree, is sent out for the later stage
Wealth tree provides technical support with the prevention of maize ear rot bacterium.
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.
Extraction and activity determination method of a kind of money tree with maize ear rot bacterium nosotoxin, include the following steps：
1) disease sites on the money tree with root rot is taken to extract Sickle-shaped rice and pythium spp Raw toxin simultaneously
Identification.By to one of which germ morphological analysis in culture medium and bacterium colony microscopy：Morphological feature has for aerial hyphae
Every branch is transparent, and in flocculence or spider's thread shape, usual white, pale band rosiness, bacterium colony reverse side is red, shallow redness of the skin or complexion or is in
Micro- grey, sclerotium has or nothing, into green or black-and-blue if having；Microscopy analysis is raw microconidia on aerial hyphae,
Head, the high 3~5mm of bacterium colony, microconidia are born on single raw bottle stalk, are often polymerized to pelletizing, unit cell, ovum on bottle stalk top
Shape, and sickle-like bacteria meets features above, therefore it is accredited as sickle-like bacteria.By to another germ morphological analysis in culture medium and
Bacterium colony microscopy is understood：Morphological feature is filiform, rip-panel shape, spherical or avette sporangium raw on mycelia, sporangium basidixed
Or life, the sporangiophore without special differentiation；Microscopy analysis be trophosome be it is flourishing, colourless, nothing every mycelium, picking white cotton
Floccule microscopy, it is observed that the mycelium and zoosporangium of germ, zoosporangium is slightly thicker than mycelium, some long cylinders
Shape, some have rip-panel shape branch, it is raw mycelial top or centre, the sporangiophore substantially not broken up, but sporangium with
There is membrane to separate between mycelia, and pythium spp meets features above, therefore it is accredited as pythium spp.
2) Sickle-shaped rice and pythium spp Raw toxin extracted to step 1) carries out protein content determination, specifically
Ground, by the map data in the following table 1, standard protein is under ultraviolet specrophotometer, the OD that is surveyed595Value be respectively
0.000,0.180,0.332,0.415,0.504,0.592, the regression equation of standard curve is：Y=0.0064x, R2=
0.9503, related coefficient is more than 0.9, shows the highly reliable of standard curve, can be as the foundation of following proteins assay.
The OD of 1 protein standard curve of table595Value
3) Sickle-shaped rice and pythium spp Raw toxin that extract step 1) are configured to the solution of various concentration respectively,
Healthy money tree seedling leaves are smeared by semar technique, carry out wilt activity measure.Specifically, the processing of sickle-like bacteria is 1 to 4
Group, extension rate are respectively 10 times, 50 times, 100 times, 150 times；The processing of pythium spp is 5 to 8 groups, and extension rate is respectively 10
Again, 50 times, 100 times, 150 times.Every 3 days statistic record tikka areas, obtain such as the following table 2 data, utilizing formula：Cause a disease percentage
Than (%)=total lesion area/total blade face area, and the scope for accounting for according to lesion area the percentage of total blade face area is different, draws
It is divided into 6 grades, obtains table 3.From the data in table 3, it can be seen that Different Kinds of Pathogens object withers influence situation not to the cause of healthy money tree seedling
Together.In the case where the time is the same, Raw toxin content is identical, sickle-like bacteria withers to the cause of healthy money tree effect bigger.Same
Under conditions of kind Raw toxin, with the increase of Raw toxin concentration, the time that money tree seedling withers shortens.Processing 1 and processing 5
Money tree seedling has withered after 15 days, and with 50 times of dilution, 100 times, 150 times of sickle-like bacteria rugosity element culture solution and with diluting
50 times, the money tree seedling of 100 times of pythium spp Raw toxin culture solution processing after 15 days also very close to withered, only with dilute
The processing of pythium spp Raw toxin culture solution and control group the speed for releasing 150 times is to be sufficiently close to, in state of withering naturally.
2 lesion area of table accounts for percentage (%) record sheet of total blade face area
The toxin of 3 sickle-like bacteria of table and pythium spp is to the measure of the wilt activity of money tree seedling
Note：-：Substantially have no change (lesion area accounts for the gross area 0%)；
+：Leaves water loss, symptom unobvious (tikka area accounts for gross area 0-10%)；
++：Blade starts Wei Yan, and symptom is apparent (tikka area accounts for gross area 10-30%)；
+++：Blade Wei Yan turn to be yellow, and symptom is apparent (tikka area accounts for gross area 30-50%)；
++++：There is withered, plant and starts Wei Yan in blade (tikka area accounts for gross area 50-70%)；
+++++：Blade is withered, plant Wei Yan (tikka area accounts for gross 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 activity determination method of a kind of money tree with maize ear rot bacterium nosotoxin, which is characterized in that include the following steps：
1) disease sites on the money tree with root rot is taken to extract Sickle-shaped rice and pythium spp Raw toxin and are identified；
2) Sickle-shaped rice and pythium spp Raw toxin extracted to step 1) carries out protein content determination；
3) Sickle-shaped rice and pythium spp Raw toxin that extract step 1) are configured to the solution of various concentration respectively, pass through
Semar technique smears healthy money tree seedling leaves, carries out wilt activity measure.
2. extraction and activity determination method of a kind of money tree according to claim 1 with maize ear rot bacterium nosotoxin, special
Sign is：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 activity determination method of a kind of money tree according to claim 1 with maize ear rot bacterium nosotoxin, special
Sign is：Step 2) the protein content uses spectrophotometer measurement, and the regression equation of standard curve is y=0.0064x,
R2=0.9503.
4. extraction and activity determination method of a kind of money tree according to claim 1 with maize ear rot bacterium nosotoxin, special
Sign is：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 |
|---|---|---|---|---|
| CN101186638A (en) * | 2007-12-13 | 2008-05-28 | 华南农业大学 | Method for preparing rice basal decay pathogenic bacteriotoxin and application thereof |
| CN106244632A (en) * | 2016-07-29 | 2016-12-21 | 河南工业大学 | A kind of extracting method of sesame wilt germs toxin |
-
2017
- 2017-12-04 CN CN201711273128.5A patent/CN108101972A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101186638A (en) * | 2007-12-13 | 2008-05-28 | 华南农业大学 | Method for preparing rice basal decay pathogenic bacteriotoxin and application thereof |
| CN106244632A (en) * | 2016-07-29 | 2016-12-21 | 河南工业大学 | A kind of extracting method of sesame wilt germs toxin |
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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