CN106244632B - Method for extracting toxin of sesame wilt bacteria - Google Patents
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Abstract
The invention belongs to the technical field of plant disease prevention, and particularly relates to a patent application of a sesame wilt germ toxin extraction method. The method comprises the steps of strain pre-culture, strain culture, sesame wilt germ toxin extraction and the like. The innovation point of the invention is mainly that (1) the culture time of the strain is long enough to ensure that the toxin of the sesame wilt pathogen can be generated, thereby facilitating the preparation and extraction of the subsequent process; (2) the extraction process of the sesame wilt germ toxin is simple and easy, and the extraction effect is stable; the method mainly comprises the simple steps of centrifugal filtration, ethyl acetate extraction, vacuum evaporation and the like, is simple and easy to implement, and has better operability; (3) a good foundation is laid for the research of sesame resistance; by extracting the sesame wilt germ toxin and establishing an evaluation model according to the sesame wilt germ toxin, a foundation can be laid for accurately evaluating the influence of sesame wilt germ on the growth of sesame, and a foundation can be laid for the research of sesame wilt-resistant mechanism and the screening of sesame wilt-resistant germplasm resources.
Description
Technical Field
The invention belongs to the technical field of plant disease prevention, and particularly relates to a patent application of a sesame wilt germ toxin extraction method.
Background
Sesame seed (A)Sesamum indicumL, 2n = 26) is the oldest high-quality oil crop in the world and is also an important characteristic oil crop in China.
Sesame wilt disease (Sesame Fusarium wilt) is specialized form of Fusarium oxysporum Sesame (Sesame Fusarium wilt) (Setam)Fusarium oxysporum f. sp. sesamiFOS) infection, and culm blight and is known as two major fungal diseases of sesame. Sesame wilt in China mainly occurs in northeast, northChina, northwest, Huang-Huai and Jianghuai parts, the annual incidence rate is about 15%, and the sesame yield and quality are seriously affected.
Researches such as wilt pathogen separation and identification, pathogenic pathogen pathogenicity identification methods and the like are successively developed at home and abroad for exploring the pathogenic mechanism of sesame wilt pathogens. An indoor pathogenic force identification technology system for sesame wilt pathogens is established for the first time by the agricultural scientific college of Henan province in 2014, and a technical support is provided for sesame FOS pathogenic and sesame wilt resistance mechanism research (Chou Cupu and the like, an indoor pathogenic force identification method for sesame wilt pathogens, 2014, the report of plant pathology, 44 (01): 26-35).
Previous research results show that fusarium oxysporum can generate toxin after invading plant tissues and cause toxic action on plants. In 2006, the disease resistance of different soybean varieties to root rot is evaluated by using fusarium oxysporum toxin filtrate, such as Tailianmei, and the like, and the method is used for primary screening work of soybean disease-resistant plants. Recent preliminary studies and internal data show that sesame wilt bacteria FOS are similar to other fusarium oxysporum, and after sesame is infected, mycelia gradually block vascular bundles, and water and nutrient supply is cut, so that plants are attacked. Meanwhile, FOS pathogenic bacteria can generate hydrolytic enzymes such as pectinase, cellulase and the like, and have an infection effect on sesame plants (recorded in research and record of academy of agricultural sciences in Henan province). However, it has not been clarified yet whether sesamomyces verticillata can produce toxin, and there is no published report on toxicity symptom of FOS toxin to sesame. Therefore, a technology for extracting the toxin of the sesame wilt bacteria is urgently needed at present, so that the pathogenic mechanism of the sesame wilt bacteria is proved, and a technical support is provided for promoting the basic research of sesame diseases.
Disclosure of Invention
The invention aims to provide an extraction method of sesame wilt germ toxin, thereby laying a foundation for research of related sesame diseases.
The technical scheme adopted by the invention is detailed as follows.
A sesame wilt germ toxin extraction method comprises the following steps:
(1) the strain is pre-cultured, which specifically comprises the following steps:
inoculating sesame wilt bacteria (i.e. fusarium oxysporum sesame specialization type) strain on PDA plate culture medium, culturing at 28 deg.C for about 7 days, and checking strain activity;
then, punching out bacterial sheets with the diameter of 8 mm along the edges of bacterial colonies, inoculating the bacterial sheets into a triangular flask filled with PD culture solution, inoculating 2-3 bacterial sheets into each flask, and performing shake culture for 4d at the temperature of 28 ℃ and the speed of 120 rpm;
(2) the strain culture comprises the following steps:
taking 2 layers of sterile 2 layers of lens wiping paper as a filter screen, filtering the bacterial liquid after the culture in the step (1) is finished, and filtering hyphae, wherein the filtrate contains a large amount of sesame wilt germ spores and is taken as inoculation mother liquid;
inoculating the inoculation mother liquor into a Rickettel culture medium, and performing constant-temperature shaking culture for 40-50 days at 28 ℃ and 120 rpm;
(3) the method for extracting the sesame wilt germ toxin specifically comprises the following steps:
firstly, filtering the culture solution obtained in the step (2) by using 2 layers of sterile gauze as a filter screen to filter out impurities such as spores, hyphae, cell fragments and the like; centrifuging the filtrate at 4000rpm for 15min, and collecting supernatant;
secondly, slowly dripping the supernatant into a solvent filter with a 0.45-micron water-based filter membrane, carrying out vacuum filtration, collecting filtrate, and adjusting the pH value to be 3.0-3.5 by using a 2N (2 mol/L) hydrochloric acid solution;
finally, adding ethyl acetate with the volume equal to that of the filtrate into the filtrate as an extracting agent for extraction; and (3) drying the upper organic phase by using anhydrous sodium sulfate, filtering the sodium sulfate, performing vacuum rotary evaporation to remove ethyl acetate, and finally obtaining a brown yellow solid product, namely a crude product containing the sesame wilt germ toxin (crude toxin for short).
Preliminary determination shows that by taking sesame wilt germs with medium pathogenicity (DI is more than or equal to 20 and less than or equal to 50) (refer to enemy Cupu and the like, an indoor identification method for pathogenic bacteria pathogenicity of sesame wilt germs, the report of plant pathology, 2012) as an example, 200mL of bacterial liquid can finally extract about 90-120 mg of crude products containing sesame wilt germ toxins by adopting the method.
Furthermore, the extracted crude toxin can be used for evaluating the influence of sesame wilt bacteria on the growth of sesame, and preliminary experiments show that the growth of sesame seedlings can be obviously inhibited when the concentration of the crude toxin is 1-10 mug/mL.
Furthermore, the evaluation method of the extracted crude toxin for evaluating the influence of sesame wilt bacteria on the growth of sesame (or a method for measuring the toxicity of the crude toxin on the sesame) comprises the following steps:
(1) seed disinfection and inoculation, specifically:
selecting full and healthy sesame seeds, and washing the surfaces of the seeds for 3-5 times by using clear water;
then soaking the seeds in 75% alcohol for 30 s, and washing with sterile water for 3 times;
then soaking the seeds for 10 min by using 3% sodium hypochlorite, and washing the seeds for 3-4 times by using sterile water;
placing the sterilized seeds into a triangular flask filled with sterile water, and performing shaking culture overnight (preferably, the seeds are just immersed in the sterile water);
selecting white sesame seeds, inoculating the white sesame seeds on an MS solid culture medium, and culturing for 1 week under the conditions of 25 ℃ and L// D =14 h//10 h illumination/D;
(2) preparing an MS culture medium containing crude toxin, which specifically comprises the following steps:
preparing MS culture medium (MS minimal medium, 1.5% agar powder), and adding the crude toxin to make the crude toxin concentration in specific gradient concentration distribution, such as crude toxin concentration of 1 μ g/mL, 5 μ g/mL and 10 μ g/mL;
(3) inoculating sesame seedlings in a crude toxin-containing culture medium, which specifically comprises the following steps:
under the aseptic condition, cutting the part (without root seedling) above the root and stem base of the young sesame seedling cultivated in the step (1), respectively inoculating the cut part on the MS culture medium containing crude toxin prepared in the step (2), and simultaneously setting a blank MS culture medium as a recessive control; when setting the relevant experimental group and the control group, attention needs to be paid, in order to meet the requirements of statistics and accurate evaluation, 3 times of treatment is recommended, and 6 rootless seedlings are inoculated in each time of treatment;
(4) the character counting and result judging method specifically comprises the following steps:
and (4) after 2 weeks of inoculation of the rootless seedlings in the step (3), counting the plant height, the root number, the root length and other data of the sesame seedlings, photographing and recording, and finally comprehensively evaluating the inhibition degree of the crude toxin on the growth of the sesame seedlings, namely the toxicity degree of the crude toxin on the sesame.
In general, the innovation of the invention is mainly embodied in the following aspects:
(1) the culture time of the strain is long enough to ensure that the toxin of the sesame wilt pathogen can be generated, and the corresponding strain can be ensured to generate enough toxin by adopting a pre-culture mode and combining the culture time for long enough, thereby facilitating the preparation and extraction of the subsequent process;
(2) the extraction process of the sesame wilt germ toxin is simple and easy, and the extraction effect is stable; compared with the existing extraction technologies such as an active carbon method (Tailianmei, 2004) and the like, the method mainly comprises the simple steps of centrifugal filtration, ethyl acetate extraction, vacuum evaporation and the like, and the method is simple, convenient and easy to implement and has better operability; meanwhile, in a comprehensive aspect, the sesame wilt germ toxin extracting method has the advantages of high extraction efficiency, good repeatability and stable extracting effect, so that the method has good technical creativity;
(3) a good foundation is laid for the research of sesame resistance; by extracting the sesame wilt germ toxin and establishing an evaluation model according to the sesame wilt germ toxin, a foundation can be laid for accurately evaluating the influence of sesame wilt germ on the growth of sesame, and a foundation can be laid for the research of sesame wilt-resistant mechanism and the screening of sesame wilt-resistant germplasm resources.
Drawings
FIG. 1 shows the pre-culture result of Fusarium oxysporum sesame specialization strain HSFO07021 on PDA plate medium;
FIG. 2 is a strain HSFO07021 strain sheet of Fusarium oxysporum sesame specialization type for culture;
FIG. 3 shows the sesamum wilt toxin obtained by extraction with ethyl acetate;
FIG. 4 shows the growth of Yuzhi No. 11 seedlings under 5 μ g/mL crude toxin treatment; wherein, the pictures 1 and 2 are respectively the growth conditions of Yuzhi No. 11 seedlings and roots under the contrast culture condition of clear water; 3. FIG. 4 shows the growth of Yuzhi No. 11 seedlings and roots under 5. mu.g/mL crude toxin treatment, respectively, and it can be seen that the growth of seedlings and roots is significantly inhibited.
Detailed Description
The present application is further illustrated by the following examples, which are intended to briefly describe some of the biological materials and culture media in the following examples before describing the specific examples.
Biological material:
yuzhi No. 11, provided by sesame seed collection center of the national institute of agriculture in Henan province, and available from open channels;
the fusarium oxysporum sesame specialization strain HSFO07021 adopted in the following embodiment is provided by sesame center of agricultural academy in Henan province and can be obtained from a public channel; in the following examples, the extraction method is only exemplified by the strain, and it should not be understood that the technical scheme of the present invention is specifically dependent on or specifically limited to the strain.
Culture medium:
the culture media involved in the following examples are all media commonly used in the art, mainly:
PDA plate culture medium: 200g of potatoes, 20g of glucose, 15-18g of agar and 1000mL of distilled water;
PD culture solution: compared with a PDA plate culture medium, the culture medium does not contain agar;
richard medium: KNO310g，KH2PO45g，MgSO4·7H2O 2.5g，FeCl30.02g, 50g of cane sugar and 1000mL of distilled water.
Example 1
Taking fusarium oxysporum sesame specialization strain HSFO07021 as an example, the method for extracting the sesamum oxysporum toxin provided by the application is detailed as follows.
(1) The strain is pre-cultured, which specifically comprises the following steps:
inoculating sesame wilt bacteria (i.e. fusarium oxysporum sesame specialization type) HSFO07021 on PDA plate medium with sterile toothpick, and culturing at 28 deg.C for 7 days. The culture results are shown in figure 1, and it can be seen from the figure that the strain has high activity and can be used for subsequent experiments.
Under aseptic conditions, punching bacterial sheets (shown in figure 2) with the diameter of 8 mm along the edges of bacterial colonies, inoculating the bacterial sheets into a 500mL triangular flask containing 200mL PD culture solution, inoculating 2-3 bacterial sheets in each flask, and performing shake culture at 28 ℃ and 120 rpm for 4 days.
(2) The strain culture comprises the following steps:
under aseptic conditions, 2 layers of aseptic 2 layers of lens wiping paper are used as a filter screen, the bacterial liquid after the culture in the step (1) is filtered, hypha is filtered, and the filtrate contains a large amount of sesame wilt germ spores and is used as inoculation mother liquid;
under the aseptic condition, 2mL of the inoculum mother liquor is taken and inoculated into a 500mL triangular flask containing 200mL of Rickettd culture medium, and the inoculum is shake-cultured for 40-50 days at constant temperature under the conditions of 28 ℃ and 120 rpm.
(3) The method for extracting the sesame wilt germ toxin specifically comprises the following steps:
firstly, under aseptic conditions, filtering the culture solution in the step (2) by taking 2 layers of aseptic gauze as a filter screen to filter out impurities such as spores, hyphae, cell fragments and the like; putting the filtrate into 50mL centrifuge tubes in batches, centrifuging for 15min at 4000rpm, collecting and combining the supernatants;
secondly, slowly dropping the supernatant into a solvent filter with a 0.45 μm water system filter membrane, carrying out vacuum filtration, collecting and combining filtrates, and adjusting the pH with a 2N (2 mol/L) hydrochloric acid solution to be = 3.0;
finally, adding ethyl acetate with the volume equal to that of the filtrate into the filtrate as an extracting agent for extraction; extracting for 3 times in a 500mL separating funnel, wherein each extraction time is 15-20 min; and combining the upper organic phases, adding anhydrous sodium sulfate, drying, filtering to remove sodium sulfate, and performing vacuum rotary evaporation to remove ethyl acetate to obtain a brown yellow solid product (shown in figure 3), namely a crude product (crude toxin for short) containing the sesame wilt disease HSFO07021 strain toxin.
The final measurement result shows that about 90-120 mg of sesame wilt germ toxin can be extracted from the 200mL of sesame wilt germ filtrate.
Example 2
The inventors specifically evaluated the toxicity of the crude toxin product prepared in example 1 on sesame (the effect of the crude toxin on the growth of sesame), and the evaluation method was as follows.
(1) Seed disinfection and inoculation, specifically:
selecting 500 full and healthy Yuzhi No. 11 seeds, and washing the surfaces of the seeds for 3-5 times by using clear water;
then soaking the seeds in 75% alcohol for 30 s, and washing with sterile water for 3 times;
then soaking the seeds for 10 min by using 3% sodium hypochlorite, and washing the seeds for 3-4 times by using sterile water;
placing the sterilized seeds into a triangular flask filled with 10 mL of sterile water for shaking culture overnight;
selecting outcrop sesame seeds, inoculating the outcrop sesame seeds on an MS solid culture medium, and inoculating 50 outcrop seeds in each culture medium; culturing at 25 ℃ under L// D =14 h//10 h light/D for 1 week;
(2) preparing an MS culture medium containing crude toxin, which specifically comprises the following steps:
preparing an MS culture medium (an MS minimal medium, 1.5% agar powder), and respectively adding the prepared crude toxin to make the concentrations of the crude toxin respectively 1 mug/mL, 5 mug/mL and 10 mug/mL;
(3) inoculating sesame seedlings in a crude toxin-containing culture medium, which specifically comprises the following steps:
under the aseptic condition, cutting the part (without root seedling) above the root and stem base of the young sesame seedling cultivated in the step (1), respectively inoculating the cut part on the solid MS culture medium containing crude toxin prepared in the step (2), and simultaneously setting a blank MS culture medium as a recessive control; 3 repeats are set for each treatment, and 6 rootless seedlings are inoculated for each repeat;
(4) the character counting and result judging method specifically comprises the following steps:
and (3) after 2 weeks of inoculation of the rootless seedlings in the step (3), counting the plant height, the root number, the root length and other data of the sesame seedlings, photographing and recording, and finally comprehensively evaluating the inhibition degree of the crude toxin on the growth of the sesame seedlings (as shown in figure 4), namely the toxicity degree of the crude toxin on the sesame.
Sesame seedling is treated by the sesame wilt germ toxin (crude toxin) extracted by the technical method. As can be seen from FIG. 4, after 2 weeks, the number of the roots of the sesame seedlings in the clear water control group is 3-7 in more, the roots are about 3-4cm in length, and the growth vigor is normal for 1 pair of true leaf stages. The sesame seedlings treated by the fusarium oxysporum toxin of 5 mu g/mL grow slowly, and no root grows in the cotyledon expansion period, which indicates that the seedlings are inhibited more seriously. The results show that the sesame wilt germ toxin extracted by the technical method has toxic action on sesame.
Claims (6)
1. A crude sesame wilt germ toxin product is characterized by being prepared by the following steps:
(1) the strain is pre-cultured, which specifically comprises the following steps: inoculating sesame wilt germ strains on a PDA (personal digital Assistant) plate culture medium, culturing, and checking the activity of the strains; then, punching out bacterial sheets along the edges of bacterial colonies, inoculating PD culture solution, and performing shake culture;
(2) the strain culture comprises the following steps: filtering the bacterial liquid after the culture in the step (1) is finished, and taking the filtrate as inoculation mother liquor; inoculating the inoculation mother liquor into a Rickettd culture medium, and performing constant-temperature shaking culture for 40-50 days;
(3) the method for extracting the sesame wilt germ toxin specifically comprises the following steps:
firstly, filtering the culture solution in the step (2), centrifuging the filtrate, and taking the supernatant;
secondly, dropwise adding the supernatant into a solvent filter with a 0.45-micron water system filter membrane, carrying out vacuum filtration, collecting filtrate, and adjusting the pH to be 3.0-3.5 by using a hydrochloric acid solution;
finally, adding an organic extractant into the filtrate for extraction; and (3) taking the upper organic phase, and performing vacuum rotary evaporation to finally obtain a brown yellow solid product, namely a crude product containing the sesame wilt germ toxin.
2. The crude sesamum oxysporum toxin product of claim 1, wherein in step (3), the organic extractant is ethyl acetate.
3. The crude drug of sesamomyces oxysporum toxin of claim 1, wherein in step (1), the sesamomyces oxysporum strain is fusarium oxysporum sesame specialization strain HSFO 07021.
4. The method for extracting crude drug of sesamum wilt toxin according to claim 1, comprising the steps of:
(1) the strain is pre-cultured, which specifically comprises the following steps: inoculating sesame wilt germ strains on a PDA (personal digital Assistant) plate culture medium, culturing, and checking the activity of the strains; then, punching out bacterial sheets along the edges of bacterial colonies, inoculating PD culture solution, and performing shake culture;
(2) the strain culture comprises the following steps: filtering the bacterial liquid after the culture in the step (1) is finished, and taking the filtrate as inoculation mother liquor; inoculating the inoculation mother liquor into a Rickettd culture medium, and performing constant-temperature shaking culture for 40-50 days;
(3) the method for extracting the sesame wilt germ toxin specifically comprises the following steps:
firstly, filtering the culture solution in the step (2), centrifuging the filtrate, and taking the supernatant;
secondly, dropwise adding the supernatant into a solvent filter with a 0.45-micron water system filter membrane, carrying out vacuum filtration, collecting filtrate, and adjusting the pH to be 3.0-3.5 by using a hydrochloric acid solution;
finally, adding an organic extractant into the filtrate for extraction; and (3) taking the upper organic phase, and performing vacuum rotary evaporation to finally obtain a brown yellow solid product, namely a crude product containing the sesame wilt germ toxin.
5. The method for extracting crude drug of sesamoeba wilt toxin according to claim 4, wherein in step (3), said organic extractant is ethyl acetate.
6. The method for extracting crude drug of sesamomyces oxysporum toxin of claim 4, wherein in the step (1), the sesamomyces oxysporum strain is Fusarium oxysporum sesame specialization strain HSFO 07021.
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| 番茄灰霉病菌的分离与毒素的提取、纯化及生物测定;董友磊;《中国优秀硕士学位论文全文数据库》;20091215;正文第37-38页 * |
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