CN106244632A - A kind of extracting method of sesame wilt germs toxin - Google Patents
A kind of extracting method of sesame wilt germs toxin Download PDFInfo
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Abstract
The invention belongs to plant disease prevention technique field, be specifically related to the patent application of the extracting method of a kind of sesame wilt germs toxin.The method includes the steps such as bacterial strain preculture, strain culturing, extraction sesame wilt germs toxin.The innovative point of the present invention is mainly reflected in (1) strain culturing time long enough, it is ensured that can produce sesame wilt germs toxin, consequently facilitating the preparation of subsequent process and extraction；(2) sesame wilt germs Toxic extraction process is simple and easy to do, and extraction effect is stable；The invention mainly comprises the brief steps such as centrifugal filtration, ethyl acetate extraction and vacuum evaporation, simple and easy to do, there is preferable operability；(3) it is that Semen Sesami Resistence research has established good basis；By extracting sesame wilt germs toxin, and set up a kind of evaluation model according to this, can be that Semen Sesami growth effect is laid the foundation by accurate evaluation sesame wilt germs, the most alternatively screening of Semen Sesami anti-blight study mechanism and anti-Semen Sesami droop germ plasm resource lays the foundation.
Description
Technical field
The invention belongs to plant disease prevention technique field, be specifically related to the extracting method of a kind of sesame wilt germs toxin
Patent application.
Background technology
Semen Sesami (Sesamum indicumL., 2n=26) belong to Pedaliaceae linum and belong to, it is the most ancient excellent
Matter oil crop, the characteristic oil crop that Ye Shi China is important.
Semen Sesami droop (Sesame Fusarium wilt) by fusarium oxysporum sesame (Fusarium oxysporum f.sp.sesami, FOS) infect and cause, select rot the referred to as big main fungal of Semen Sesami two with stem
Disease.China's Semen Sesami wilt disease occurs mainly in northeast, North China, northwest, the Yellow River and Huai He River and Yangze river and Huai river some areas, long-term incidence rate
About 15%, have a strong impact on Sesame Seed Yield and quality.
For verifying sesame wilt germs pathogenesis, the most successively wilt separation, qualification and pathogen are carried out
Pathogenicity authentication methods etc. are studied.Within 2014, Henan Academy of Agricultural Sciences establishes sesame wilt germs pathogenicity indoor mirror first
Determining technical system, and Semen Sesami anti-blight study mechanism pathogenic for Semen Sesami FOS provides technical support, and (enemy deposits uncut jade etc., Semen Sesami
Pathogen of Fusarium Wilt pathogenicity indoor appraising method, 2014, Plant Pathology, 44 (01): 26-35).
Previous investigation shows, Fusarium oxysporum multipotency after invading plant tissue produces toxin, and causes plant
Toxic action.Tai Lianmei in 2006 etc. utilize Toxin of Fusarium Oxysporum filtrate to have rated disease-resistant to root rot of different soybean varieties
Property, and the method is used for the primary dcreening operation work of Semen sojae atricolor disease-resistant plant.Preliminary study and inside information show in the recent period, Semen Sesami droop
Bacterium FOS is similar with other Fusarium oxysporums, and after infecting Semen Sesami, mycelium gradually blocks vascular bundle, cutting moisture and nutrient substance
Supply, thus cause plant to fall ill.Meanwhile, FOS pathogen can produce the hydrolytic enzyme such as pectase, cellulase, plants Semen Sesami
Dissemination (Henan Academy of Agricultural Sciences's scientific and technical research archive is recorded) is played in strain.But up to now, the clearest and the most definite sesame wilt germs
Whether can produce toxin, also have no, about FOS toxin, Semen Sesami poisoning symptom is disclosed report.Therefore, one currently it is badly in need of
Extract the technology of sesame wilt germs toxin, to verify sesame wilt germs pathogenesis, and for promoting Semen Sesami disease basis to grind
Offer technical support is provided.
Summary of the invention
Present invention aim at providing the extracting method of a kind of sesame wilt germs toxin, thus grind for relevant Semen Sesami disease
Study carefully and lay the foundation.
Details are as follows for the technical solution used in the present invention.
A kind of sesame wilt germs Toxic extraction method, comprises the following steps:
(1) bacterial strain preculture, particularly as follows:
By sesame wilt germs (i.e. fusarium oxysporum sesame) inoculation in PDA plate culture medium, 28 DEG C, cultivation
About 7 d, check strain activity；
Then, get a diameter of 8 mm bacterium sheets along colony edge, access in the triangular flask equipped with PD culture fluid, every bottle graft 2 ~ 3,
28 DEG C, under the conditions of 120 rpm, 4d is cultivated in concussion；
(2) strain culturing, particularly as follows:
Using 2 layers of aseptic 2 layer lens paper as filter screen, cultivation in step (1) is terminated rear bacterium solution and filters, filter mycelia,
Due to containing a large amount of sesame wilt germs spores in filtrate, in this, as connecing mother bacterial liquid；
To connect mother bacterial liquid and access Richard's culture medium, 28 DEG C, under the conditions of 120 rpm, isothermal vibration cultivates 40 ~ 50d；
(3) sesame wilt germs toxin is extracted, particularly as follows:
First, using 2 layers of sterile gauze as filter screen, culture fluid in step (2) is filtered, to filter spore, mycelia and thin
The foreign material such as born of the same parents' fragment；Filtrate 4000rpm is centrifuged 15min, takes supernatant；
Secondly, above-mentioned supernatant is slowly dripped in the solvent filter equipped with 0.45 μm water system filter membrane, vacuum filtration, collect
Filtrate, and with 2N(2mol/L) hydrochloric acid solution regulation pH value is between 3.0 ~ 3.5；
Finally, in filtrate, add the ethyl acetate equal with filtrate volume to extract as extractant；Take upper organic phase,
Dried with anhydrous sodium sulfate, filter sodium sulfate, carry out rotary evaporation in vacuo, to remove ethyl acetate, final that brown color is solid
Body product, is the thick product (abbreviation Raw toxin) containing sesame wilt germs toxin.
Preliminary Determination shows, with medium pathogenicity (20≤DI≤50), (with reference to Chou Cunpu etc., Semen Sesami Pathogen of Fusarium Wilt causes
Sick power indoor appraising method, Plant Pathology, 2012), as a example by sesame wilt germs, said method, 200mL bacterium solution are used
Finally can extract the thick product of the toxin containing sesame wilt germs about 90 ~ 120 milligrams.
Further, the Raw toxin extracted can be used for evaluating the impact that Semen Sesami is grown by sesame wilt germs, tentatively tries
Testing and show, when Raw toxin concentration is 1-10 μ g/mL, the growth of Characteristics of Sesame i.e. can substantially be suppressed.
Further, the Raw toxin extracted is for evaluating sesame wilt germs to evaluation side during Semen Sesami growth effect
Method (Raw toxin toxicity test method to Semen Sesami in other words) is to comprise the steps:
(1) seed disinfection and inoculation, particularly as follows:
Select the sesame seed of full health, first rinse the surface of the seed 3 ~ 5 times with clear water；
Then seed is soaked in 30 s in 75% ethanol, aseptic water washing 3 times；
Subsequently soak 10 min, aseptic water washing seed 3 ~ 4 times with 3% sodium hypochlorite；
Seed after sterilization is put into and equipped with shaken cultivation in the triangular flask of sterilized water (with sterilized water just submergence seed is overnight
Preferably)；
Select the sesame seed showed money or valuables one carries unintentionally, be seeded on MS solid medium, 25 DEG C, under the conditions of L//D=14 h//10 h illumination/d
Cultivate 1 week；
(2) configuration MS culture medium containing Raw toxin, particularly as follows:
Configuration MS culture medium (MS minimal medium, 1.5% agar powder), is separately added into above-mentioned prepared Raw toxin, makes Raw toxin
Concentration is that specific gradient concentration is distributed, as Raw toxin concentration is respectively 1 μ g/mL, 5 μ g/mL and 10 μ g/mL；
(3) containing Raw toxin inoculation of medium Characteristics of Sesame, particularly as follows:
Under aseptic condition, cut Characteristics of Sesame rhizome base portion above section (without root) that step (1) is cultivated, be inoculated in respectively
In the MS culture medium containing Raw toxin prepared in step (2), blank MS culture medium is set simultaneously and compares as recessiveness；Phase is set
When closing experiment group and comparison group, it may be noted that be the needs meeting statistics and accurate evaluation, it is proposed that can each process set
Putting 3 repetitions, the strain of each repeated inoculation 6 is without root；
(4) character statistics and result judge, particularly as follows:
After inoculation was without root 2 weeks in step (3), add up Characteristics of Sesame plant height, the data such as number, root length of taking root, and Taking Pictures recording,
The suppression degree that Characteristics of Sesame is grown by final overall merit Raw toxin, that is, the toxic degree that Raw toxin is to Semen Sesami.
In general, the innovative point of the present invention is mainly reflected in following aspects:
(1) strain culturing time long enough, it is ensured that can produce sesame wilt germs toxin, by using preculture in the present invention
Mode, in conjunction with incubation time long enough, it is ensured that corresponding bacterial strain can produce enough toxin, consequently facilitating the preparation of subsequent process
And extraction；
(2) sesame wilt germs Toxic extraction process is simple and easy to do, and extraction effect is stable；With existing activated carbon method (Tai Lianmei etc.,
2004) etc. extractive technique is compared, and the invention mainly comprises the brief steps such as centrifugal filtration, ethyl acetate extraction and vacuum evaporation
Suddenly, method is simple for the method, has preferable operability；For the most comprehensive, the sesame wilt germs toxin of the present invention
Extraction efficiency is high, repeatability is preferable, extraction effect is relatively stable, thus has preferably technology creation；
(3) it is that Semen Sesami Resistence research has established good basis；By extracting sesame wilt germs toxin, and set up one according to this and comment
Valency model, can be that Semen Sesami growth effect is laid the foundation by accurate evaluation sesame wilt germs, the most alternatively Semen Sesami anti-blight
The screening of study mechanism and anti-Semen Sesami droop germ plasm resource lays the foundation.
Accompanying drawing explanation
Fig. 1 is fusarium oxysporum sesame bacterial strain HSFO07021 preculture result in PDA plate culture medium；
Fig. 2 is the fusarium oxysporum sesame bacterial strain HSFO07021 bacterium sheet for cultivating；
Fig. 3 is the sesame wilt germs toxin being extracted with ethyl acetate acquisition；
Fig. 4 is that under 5 μ g/mL Raw toxin treatment conditions, No. 11 growth of seedling of Henan sesame are suppressed situation；Wherein 1,2 figures are respectively
No. 11 seedling of Henan sesame and root growth situation under clear water comparison condition of culture；3, under 4 figures are respectively 5 μ g/mL Raw toxin treatment conditions
No. 11 seedling of Henan sesame and root growth situation, it can be seen that seedling and root growth are substantially suppressed.
Detailed description of the invention
Below in conjunction with embodiment, the application is further explained explanation, before introducing specific embodiment, to following enforcement
Example relates to part biological material and culture medium situation is briefly introduced and is described as follows.
Biomaterial:
Henan sesame No. 11, is provided by Sesame germplasm preservation center, academy of agricultural sciences of Henan Province, can obtain from open channel；
Fusarium oxysporum sesame bacterial strain HSFO07021 employed in following embodiment, by academy of agricultural sciences of Henan Province Semen Sesami
Center provides, and can be obtained by open channel；In following embodiment, extracting method is only tested as a example by this bacterial strain, should not be understood
It is defined in this bacterial strain in the most specific for technical scheme specific dependency.
Culture medium:
In following embodiment, involved culture medium is culture medium commonly used in the art, mainly has:
PDA plate culture medium: Rhizoma Solani tuber osi 200g, glucose 20g, agar 15-18g, distilled water 1000mL；
PD culture fluid: compared with PDA plate culture medium, without agar；
Richard's culture medium: KNO310g, KH2PO45g, MgSO4·7H2O 2.5g, FeCl30.02g, sucrose 50g, distilled water
1000mL。
Embodiment 1
As a example by fusarium oxysporum sesame bacterial strain HSFO07021, with regard to sesame wilt germs toxin provided herein
Details are as follows for extracting method.
(1) bacterial strain preculture, particularly as follows:
With sterile toothpick, sesame wilt germs (i.e. fusarium oxysporum sesame) bacterial strain HSFO07021 is seeded in PDA to put down
In plate culture medium, 28 DEG C, cultivate 7 d.Cultivation results as it is shown in figure 1, it can be seen that bacterial strain vigor is higher, Ke Yiyong
In subsequent experimental.
Aseptic condition, gets a diameter of 8 mm bacterium sheet (as shown in Figure 2) along colony edge, accesses equipped with 200 mL PD trainings
In 500 mL triangular flasks of nutrient solution, every bottle graft 2 ~ 3,28 DEG C, under the conditions of 120 rpm, 4d is cultivated in concussion.
(2) strain culturing, particularly as follows:
Under aseptic condition, using 2 layers of aseptic 2 layer lens paper as filter screen, cultivation in step (1) is terminated rear bacterium solution and carried out
Filter, filters mycelia, due to containing a large amount of sesame wilt germs spores in filtrate, in this, as connecing mother bacterial liquid；
Under aseptic condition, take and meet mother bacterial liquid 2mL, access in the 500mL triangular flask equipped with 200mL Richard's culture medium, 28 DEG C,
Under the conditions of 120 rpm, isothermal vibration cultivates 40 ~ 50d.
(3) sesame wilt germs toxin is extracted, particularly as follows:
First, under aseptic condition, using 2 layers of sterile gauze as filter screen, culture fluid in step (2) is filtered, to filter spore
The foreign material such as son, mycelia and cell debris；Filtrate being loaded in 50mL centrifuge tube, 4000rpm is centrifuged 15min in batches, collects and closes
And supernatant；
Secondly, above-mentioned supernatant is slowly dripped in the solvent filter equipped with 0.45 μm water system filter membrane, vacuum filtration, collect
And merging filtrate, with 2N(2mol/L) hydrochloric acid solution regulation pH=3.0；
Finally, in filtrate, add the ethyl acetate equal with filtrate volume to extract as extractant；Leak in 500mL separatory
Bucket extracts 3 times, extracts 15 ~ 20min every time；Merge upper organic phase, add anhydrous sodium sulfate and be dried, after filtering sodium sulfate,
Carry out rotary evaporation in vacuo, to remove ethyl acetate, final yellow-brown solid product (as shown in Figure 3), it is containing Semen Sesami
The thick product (abbreviation Raw toxin) of wilt HSFO07021 bacterial strain toxin.
Final measurement result shows, can extract the sesame of about 90 ~ 120mg from above-mentioned 200mL sesame wilt germs filtrate
Fiber crops wilt toxin.
Embodiment 2
To the Raw toxin product prepared by embodiment 1, to the toxicity of Semen Sesami, (Raw toxin is to sesame to this Raw toxin product for inventor
The impact of Taro Aso's length) carry out concrete evaluation, evaluation methodology is as described below.
(1) seed disinfection and inoculation, particularly as follows:
Select 500, No. 11 seeds of Henan sesame of full health, first rinse the surface of the seed 3 ~ 5 times with clear water；
Then seed is soaked in 30 s in 75% ethanol, aseptic water washing 3 times；
Subsequently soak 10 min, aseptic water washing seed 3 ~ 4 times with 3% sodium hypochlorite；
Seed after sterilization is put in the triangular flask equipped with 10 mL sterilized water shaken cultivation overnight；
Select the sesame seed showed money or valuables one carries unintentionally, be seeded on MS solid medium, 50 seeds that show money or valuables one carries unintentionally of every part of inoculation of medium；25
DEG C, cultivate 1 week under the conditions of L//D=14 h//10 h illumination/d；
(2) configuration MS culture medium containing Raw toxin, particularly as follows:
Configuration MS culture medium (MS minimal medium, 1.5% agar powder), is separately added into above-mentioned prepared Raw toxin, makes Raw toxin
Concentration is respectively 1 μ g/mL, 5 μ g/mL and 10 μ g/mL；
(3) containing Raw toxin inoculation of medium Characteristics of Sesame, particularly as follows:
Under aseptic condition, cut Characteristics of Sesame rhizome base portion above section (without root) that step (1) is cultivated, be inoculated in respectively
On the solidified MS media containing Raw toxin prepared in step (2), blank MS culture medium is set simultaneously and compares as recessiveness；Often
Individual process arranges 3 repetitions, and the strain of each repeated inoculation 6 is without root；
(4) character statistics and result judge, particularly as follows:
After inoculation was without root 2 weeks in step (3), add up Characteristics of Sesame plant height, the data such as number, root length of taking root, and Taking Pictures recording,
The suppression degree (as shown in Figure 4) that Characteristics of Sesame is grown by final overall merit Raw toxin, that is, the toxicity that Raw toxin is to Semen Sesami
Degree.
Characteristics of Sesame is processed by the sesame wilt germs toxin (Raw toxin) using this technical method to be extracted.From figure
It can be seen that after 2 weeks, in clear water matched group, Characteristics of Sesame process 1 is to leaf period in 4, root of hair quantity is many at 3-7, is about 3-
4cm, growing way is normal.The Characteristics of Sesame poor growth that 5 μ g/mL wilt toxin process, is in cotyledon and launches period, have no root
Growth, shows that seedling is suppressed by more serious.This result shows, uses the sesame wilt germs poison that this technical method extracts
Element effect toxic to Semen Sesami.
Claims (4)
1. a sesame wilt germs Toxic extraction method, it is characterised in that comprise the following steps:
(1) bacterial strain preculture, particularly as follows:
By sesame wilt germs inoculation in PDA plate culture medium, cultivate, check strain activity；
Then, getting bacterium sheet along colony edge, access PD culture fluid, concussion is cultivated；
(2) strain culturing, particularly as follows:
Cultivation in step (1) being terminated rear bacterium solution filter, filtrate is as connecing mother bacterial liquid；
To connect mother bacterial liquid and access Richard's culture medium, isothermal vibration cultivates 40 ~ 50d；
(3) sesame wilt germs toxin is extracted, particularly as follows:
First, being filtered by culture fluid in step (2), filtrate is centrifuged, and takes supernatant；
Secondly, above-mentioned supernatant is dripped in the solvent filter equipped with 0.45 μm water system filter membrane, vacuum filtration, collect filter
Liquid, and regulate pH=3.0 ~ 3.5 with hydrochloric acid solution；
Finally, in filtrate, add organic extractant to extract；Take upper organic phase, carry out rotary evaporation in vacuo, final
Yellow-brown solid product, is the thick product containing sesame wilt germs toxin.
2. sesame wilt germs Toxic extraction method as claimed in claim 1, it is characterised in that in step (3), described organic extraction
Taking agent is ethyl acetate.
3. sesame wilt germs Toxic extraction method as claimed in claim 1, it is characterised in that in step (1), described Semen Sesami is withered
The pathogenic bacteria bacterial strain that withers is fusarium oxysporum sesame bacterial strain HSFO07021.
4. utilize described in claim 3 the sesame wilt germs toxin prepared by sesame wilt germs Toxic extraction method slightly to produce
Product.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108101972A (en) * | 2017-12-04 | 2018-06-01 | 佛山科学技术学院 | Extraction and activity determination method of a kind of money tree with maize ear rot bacterium nosotoxin |
| CN108118080A (en) * | 2017-11-23 | 2018-06-05 | 江苏省农业科学院 | A kind of rice brown sclerotium bacteria Raw toxin identification method |
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- 2016-07-29 CN CN201610610584.3A patent/CN106244632B/en active Active
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104388319A (en) * | 2014-10-24 | 2015-03-04 | 中国农业科学院柑桔研究所 | Extraction method for citrus brown blotch germ crude toxin and toxin production condition |
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| 董友磊: "番茄灰霉病菌的分离与毒素的提取、纯化及生物测定", 《中国优秀硕士学位论文全文数据库》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108118080A (en) * | 2017-11-23 | 2018-06-05 | 江苏省农业科学院 | A kind of rice brown sclerotium bacteria Raw toxin identification method |
| CN108101972A (en) * | 2017-12-04 | 2018-06-01 | 佛山科学技术学院 | Extraction and activity determination method of a kind of money tree with maize ear rot bacterium nosotoxin |
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