CN106916854B - Tobacco black shank bacterium fermentation broth pathogenic active ingredient and preparation method thereof - Google Patents
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Abstract
The invention relates to a tobacco black shank bacterium fermentation broth pathogenic active component and a preparation method thereof. The method comprises the following steps: (1) shaking culturing the bacterial hyphae of the phytophthora parasitica in a liquid culture medium by a shaking table; collecting the sterile fermentation liquor, centrifuging, and filtering and sterilizing the supernatant for later use; (2) performing membrane filtration separation on the sterilized black shank bacteria sterile fermentation liquor obtained in the step (1) through a microfiltration hollow cellulose membrane and an ultrafiltration hollow cellulose membrane to obtain clear and transparent ultrafiltrate; loading the ultrafiltrate into a column, enriching with macroporous adsorbent resin, and eluting the components adsorbed on the resin with ethanol to obtain ethanol eluate, i.e. the pathogenic active component of the fermentation broth of black shank bacteria. The method has the advantages of simple operation, reliable result, strong pathogenic capability of the pathogenic active component of the black shank bacterium fermentation liquor and good stability, and can be efficiently used for screening the tobacco black shank bacterium resistant varieties.
Description
Technical Field
The invention belongs to the field of pathology, and particularly relates to a tobacco black shank bacterium fermentation broth pathogenic active ingredient and a preparation method thereof.
Background
Tobacco black shank was first discovered in java, indonesia. The disease is reported to occur in Huang-Huai-Yan district in China for the first time in 1950, and is harmful for a long time and the disease condition is more and more serious. At present, except for sporadic outbreaks in the northeast tobacco zone, the tobacco black shank commonly occurs in other tobacco zones in China, and the economic loss caused each year is serious and is only second to viral diseases.
At present, the commonly used chemical agents for preventing and treating the black shank of the tobacco mainly comprise wettable powder of daphniphora, Bordeaux mixture, thiophanate-methyl, wettable powder of fenaminosulf, wettable powder of alum M-8 and the like, and a plurality of agents can be jointly applied in the period, are sprayed in different periods and phases, and have ideal effects of preventing diseases and increasing yield. In addition, a new control method is found to overcome the problems of pesticide residue in tobacco leaves and drug resistance of germs, the diversity of the biological control method determines the diversity of the control method, and the diversity of biological control ways of tobacco black shank is also ensured. Foreign research shows that: in the control work of tobacco black shank, the trichoderma has outstanding antagonistic effect, and is mainly caused by competition with black shank bacteria after the trichoderma occupies infection points in soil, so that the infection probability of the tobacco black shank bacteria is reduced.
With the continuous development of the tobacco industry to the green and pollution-free direction, the prevention and control of the tobacco black shank only stays in a comprehensive prevention and control measure taking a rational wheel as a main part and taking ridging cultivation and chemical prevention as an auxiliary part, and the development requirement cannot be met. Although the strain for antagonizing the tobacco black shank is searched in biological control, the tobacco black shank can be effectively controlled to a certain extent, but only the disease resistance of tobacco varieties is the basis for controlling the diseases, so that the research on resistant varieties is a hot topic in the tobacco field at present.
Disclosure of Invention
The invention aims to provide a tobacco phytophthora parasitica fermentation broth pathogenic active component and a preparation method thereof. The preparation method disclosed by the invention is simple to operate, reliable in result, strong in pathogenic capability and good in stability, and can be efficiently used for screening tobacco black shank bacterium resistant varieties.
A preparation method of a tobacco phytophthora parasitica fermentation broth pathogenic active component comprises the following steps:
(1) shaking culturing the bacterial hyphae of the phytophthora parasitica in a liquid culture medium by a shaking table; collecting the sterile fermentation liquor, centrifuging, and filtering and sterilizing the supernatant for later use;
(2) performing membrane filtration separation on the sterilized black shank bacteria sterile fermentation liquor obtained in the step (1) through a microfiltration hollow cellulose membrane and an ultrafiltration hollow cellulose membrane to obtain clear and transparent ultrafiltrate; loading the ultrafiltrate into column, performing macroporous adsorption resin enrichment, eluting the components adsorbed on the resin with 80-95% ethanol to obtain 80-95% ethanol eluate, i.e. the pathogenic active components of the fermentation broth of Heiguobacter nigra.
As a further improvement, the liquid culture medium in the step (1) is a potato liquid culture medium.
As a further improvement, the shake culture conditions in step (1): at 26-30 ℃ and 180 ℃ at 250rpm, preferably at 28 ℃ and 220 rpm.
As a further improvement, in the step (1), a sterile fermentation liquid is collected from 5d to 7d after the culture, preferably 7 d.
As a further improvement, the centrifugation is carried out in the step (1) at 4 ℃ and 6000-10000rpm for 10-20min, preferably at 8000rpm for 15 min; filtering the supernatant with 0.22um bacteria filter, sterilizing, and storing in 4 deg.C refrigerator for use.
As a further improvement, the 5KDa micro-filtration hollow cellulose membrane and the 3KDa ultra-filtration hollow cellulose membrane are adopted in the step (2) for membrane filtration separation.
As a further improvement, D101 type macroporous adsorption resin is adopted in the step (2).
As a further improvement, the ethanol concentration used in step (2) was 95%.
A tobacco phytophthora parasitica fermentation broth pathogenic active component is prepared by the method.
The pathogenicity of the active components of the black shank bacterium fermentation liquor is measured as follows: firstly, adopting an in-vitro leaf injection method, pricking 3 holes at asymmetric positions on two sides of a main vein of a tobacco leaf by using a 1ml sterile injector needle, then pressing the punctured hole part by using an injection hole of a sterile injector filled with an active component of black shank bacterium fermentation liquor, and slowly injecting the fermentation liquor into the space between epidermis, wherein the injection amount of each hole is controlled to be about 0.2 ml; and then wrapping the tobacco leaf stalks with sterile filter paper, putting the tobacco leaf stalks into a culture dish, putting the tobacco leaf stalks into a culture room for culture, spraying water on the filter paper at the room temperature of about 25 ℃, illuminating for 12 hours, observing and recording the change of the injection area of the tobacco leaves, and judging the pathogenicity of the induced black shank bacteria according to the morbidity condition of the injection area of the leaves, namely HR reaction (existence, size and morning and evening of the disease spots).
The preparation method disclosed by the invention is simple to operate, reliable in result, strong in pathogenic capability and good in stability, and can be efficiently used for screening tobacco black shank bacterium resistant varieties.
Drawings
FIG. 1 shows the tobacco leaf HR reaction of the phytophthora parasitica fermentation broth in V8 culture medium;
from left to right, the HR reaction of the sterile fermentation broth after the black shank is subjected to shaking culture for 0d, 5d, 6d and 7d respectively;
FIG. 2 shows the tobacco leaf HR reaction of the phytophthora parasitica fermentation broth in a carrot culture medium;
from left to right, the HR reaction of the sterile fermentation broth after the black shank is subjected to shaking culture for 0d, 5d, 6d and 7d respectively;
FIG. 3 is a tobacco leaf HR reaction of a phytophthora parasitica fermentation broth in an oat culture medium;
from left to right, the HR reaction of the sterile fermentation broth after the black shank is subjected to shaking culture for 0d, 5d, 6d and 7d respectively;
FIG. 4 is a tobacco leaf HR reaction of a phytophthora parasitica fermentation broth in a tobacco leaf juice culture medium;
from left to right, the HR reaction of the sterile fermentation broth after the black shank is subjected to shaking culture for 0d, 5d, 6d and 7d respectively;
FIG. 5 shows the tobacco leaf HR reaction of the phytophthora parasitica fermentation broth in the potato medium;
from left to right, the HR reaction of the sterile fermentation broth after the black shank is subjected to shaking culture for 0d, 5d, 6d and 7d respectively;
FIG. 6 shows HR reactions of tobacco leaves treated differently with the black shank bacterium fermentation broth;
FIG. 7 is a screening comparison of the optimal selection pressures of the anther anti-phytophthora parasitica stock solution and the fermentation broth active ingredients of the tobacco high potassium line HKDN-2;
FIG. 8 is a screening comparison of the optimal selection pressures of the anther anti-phytophthora parasitica stock solution and the fermentation broth active ingredients of the tobacco high-potassium line HKDN-5;
FIG. 9 shows the resistance identification of black shank of the active ingredient anther culture seedling of the blackleg bacterium fermentation liquor;
(Note: H.R represents high antibody; M.R represents medium antibody; A represents potting identification; B represents field; C represents field individual)
FIG. 10 shows the resistance identification of phytophthora parasitica stock solution;
(Note: H.S indicates a high-grade strain; A indicates identification of a pot transfer; B indicates a field; and C indicates a field individual).
Detailed Description
The following examples are intended to further illustrate the invention, but not to limit it.
Preparing a black shank bacterium fermentation liquor and separating active components of the black shank bacterium fermentation liquor: firstly, selecting a proper amount of mycelium blocks from an inoculating loop, putting the mycelium blocks into a potato liquid culture medium, culturing the mycelium blocks in a shaking table at 28 ℃ and 220rpm, collecting sterile fermentation liquor from the 7 th day into a 10ml sterile centrifuge tube, centrifuging the fermentation liquor for 15min at 4 ℃ and 8000rpm, and filtering the supernatant through a 0.22um bacterial filter to obtain black shank bacteria fermentation liquor; then processing the filtrate by a 5KDa microfiltration hollow cellulose membrane and a 3KDa ultrafiltration hollow cellulose membrane to obtain clear and transparent ultrafiltrate; and finally, passing the filtrate through 6kg of D101 type macroporous adsorption resin, and then flushing the components adsorbed on the resin down by using 95% ethanol to obtain 95% ethanol eluent.
Example 1: pathogenic effect of black shank bacterium sterile fermentation liquor
(1) Pathogenic effect of black shank bacteria sterile fermentation liquor cultured by V8 culture medium
As can be seen from fig. 1: the fermentation broth obtained after 5 days of shake culture of the phytophthora parasitica had caused the leaf injection zone to appear slightly concave with white circles compared to Control (CK) (CK is 0 days of culture), and after a period of shake culture, the leaf injection zone appeared concave and circles due to the taken fermentation broth after 7 days. However, no obvious HR necrotic spots appear in the tobacco leaves in the whole.
(2) Pathogenic effect of black shank bacterium sterile fermentation liquid cultured by carrot culture medium
As can be seen from fig. 2: the injection result of the black shank bacterium sterile fermentation liquor obtained after the black shank bacterium is cultured for 5 days on the leaves has no HR reaction as the Control (CK), the obtained fermentation liquor causes slight white spots to appear around the injection hole of the leaves after the black shank bacterium is cultured for 6 days, and the white spots are increased and the area is enlarged compared with the culture for 6 days after the black shank bacterium is cultured for 7 days, and the tobacco leaves in the injection area are sunken in a large range. And (4) no obvious HR necrotic spot phenomenon is generated by integrating the whole culture period.
(3) Pathogenic effect of black shank bacterium sterile fermentation liquid cultured by oat culture medium
As can be seen from fig. 3: the black shank bacterium sterile fermentation broth cultured for 0 day (CK) has no HR reaction on tobacco leaves. The fermentation liquor of the black shank bacterium cultured for 5 days has slight influence on the tobacco leaves, and is accompanied with the appearance of white spots, compared with the fermentation liquor of the black shank bacterium cultured for 6 days, the white spots generated in the tobacco leaf injection area are larger, and after the fermentation liquor of the black shank bacterium cultured for 7 days has a trend of generating HR reaction, but is not obvious even if the yellow spots generated in the tobacco leaf injection area.
(4) Pathogenic effect of black shank bacteria sterile fermentation liquor cultured by tobacco juice culture medium
As can be seen from fig. 4, the black shank bacterium sterile fermentation broth cultured for 0 day (CK) does not produce an obvious injection effect on tobacco leaves, after the black shank bacterium is cultured for 5 days, the obtained black shank bacterium sterile fermentation broth is white depression in the tobacco leaf injection area, accompanied by white patches and yellowing phenomenon of partial patches, and the injection effects of the black shank bacterium sterile fermentation broths obtained after 6 days and 7 days on leaves are unchanged compared with those obtained after 5 days of culture, and no obvious HR necrotic spots are produced.
(5) Pathogenic effect of black shank bacterium sterile fermentation broth cultured by potato culture medium
As can be seen from fig. 5: the black shank bacterium sterile fermentation broth (CK) cultured for 0 day has no obvious damage phenomenon to tobacco leaves; the sterile fermentation liquor obtained after the phytophthora parasitica is cultured for 5 days has slight necrotic spot phenomenon on the injection effect of tobacco leaves and is accompanied with small-range yellow spots; compared with the sterile fermentation liquid obtained after the culture of the phytophthora parasitica for 7 days, the sterile fermentation liquid obtained after the culture of the phytophthora parasitica for 6 days has the advantages that spots at the injection position of tobacco leaves gradually become bigger and yellow, obvious anti-sensitive necrotic spots appear, and HR reaction is generated.
(6) Comparison of pathogenic effects of the sterile fermentation broth of the phytophthora parasitica cultured in 5 culture media
In summary (Table 1): the injection effect of the phytophthora parasitica sterile fermentation broth obtained by culturing in a potato culture medium (PDA) on tobacco leaves is most obvious, the secondary effect obtained by culturing in a tobacco juice culture medium is the worst effect obtained by culturing in a V8 culture medium; respectively culturing the phytophthora parasitica for 5, 6 and 7 days, taking sterile fermentation liquor, and performing in-vitro injection on tobacco leaves, wherein the injection effect of the phytophthora parasitica sterile fermentation liquor obtained after culturing for 7 days is the best; 5 kinds of black shank bacteria sterile fermentation liquor (culture medium stock solution) cultured for 0 day are injected in vitro to the leaf blade respectively, no necrotic spot phenomenon exists, and the pathogenicity of the culture medium is eliminated.
Pathogenic effect of black shank bacterium sterile fermentation broth of table 15 culture media
| | |
5d | 6d | 7d | ||
| V8 | - | - | - | - | ||
| Carrot (Carex sativus L.) | - | - | - | + | ||
| Oat | - | - | + | + | ||
| Tobacco leaf juice | - | - | + | + | ||
| PDA | - | ++ | ++ | +++ |
Note: -represents no HR response of tobacco leaves; + indicates that the tobacco leaves have slight HR reaction; + represents HR reaction of tobacco leaves; and +++ represents significant HR response in tobacco leaves.
Example 2: determination of pathogenicity of active ingredients of black shank bacterium fermentation liquor
As can be seen from table 2 and fig. 6: after ultrafiltrate obtained by treating the sterile fermentation broth of the phytophthora parasitica with 5KDa microfiltration hollow cellulose membrane and 3KDa ultrafiltration hollow cellulose membrane is injected by leaves, the tobacco leaves can generate obvious HR necrotic spots, and the average value of the areas of the diseased spots reaches 0.87cm2The average value of the area of the lesion spots is increased by 0.32cm compared with the area of the lesion spots after the injection of the stock solution into the leaves2The difference between the two reaches the significance level of 5 percent; after the ultrafiltrate is processed by the open chromatographic column, the obtained 70%, 80% and 95% ethanol eluents can enable the tobacco leaves to generate HR reaction, compared with the HR reaction of the stock solution and the ultrafiltrate, the ethanol eluents cause more obvious necrotic spots, darker color and yellowish appearance, and the average value of the area of the scab reaches 0.88-0.93cm2(ii) a Wherein the difference between the average necrotic spot area of 80% (C4) and 95% (C5) ethanol eluents and the ultrafiltrate also reached a significance level of 5%; although the difference between the average necrotic spot area of the 70% (C3) ethanol eluate and the ultrafiltrate was not significant, the coefficient of variation (9.075) was less than that of the ultrafiltrate (11.49).
TABLE 2 area difference of HR necrotic spot of tobacco leaves treated differently with Heiguobacterium nigrum fermentation broth
EXAMPLE 3 acquisition of anther anti-blackleg mutant
(1) Screening comparison of optimal selection pressure of anther anti-phytophthora parasitica stock solution and active ingredients of fermentation liquor of the invention
(ii) tobacco high potassium line HKDN-2
First, as can be seen from fig. 7 and table 3: along with the increase of the concentration of the active ingredients in the black shank bacterium fermentation liquor, the germination rate of the anther of HKDN-2 is gradually reduced; compared with a control group (CK, 0%), the germination rate of the anther in the culture medium containing 1% of the active ingredient concentration of the phytophthora parasitica fermentation liquor of the invention is 12.0%, and the two have no difference significance; when the concentration of the active ingredients in the black shank bacterium fermentation liquor is 2%, the anther germination rate is obviously lower than that of a control group, but the anther germination rate does not reach the extremely significant level; when the concentration of the active ingredients in the black shank bacterium fermentation liquor reaches 3%, the anther germination rate is remarkably different from the low concentration (1% and 2%) and is extremely remarkably different from the high concentration (4% and 5%) treatment, so that the concentration of 3% is selected as the optimal selection pressure for screening the HKDN-2 black shank resistant mutant.
Second, differences of 5% level were achieved between the 1% and 2% and 4% and 5% stock solution concentration treatments and the 3% treatment, thus determining 3% as the optimal selection pressure for the blackleg bacterium stock solution.
Thirdly, the average number of seedlings which are treated by stock solutions with different concentrations is 41.4, and the germination rate is 55.2%; the average number of seedlings germinated by the active ingredients of the black shank bacterium fermentation liquor with different concentrations is 5.2, and the germination rate is 6.93%.
TABLE 3 germination rates of HKDN-2 anther black shank resistant mutants at different selective pressure concentrations
② tobacco high potassium line HKDN-5
First, as can be seen from fig. 8 and table 4: when the active ingredient concentration of the phytophthora parasitica fermentation liquor of the invention in the anther culture medium is 1%, the germination rate is very different from that of a control group (0%); when the concentration of the active ingredient of the black shank bacterium fermentation liquor is 2%, the germination rate is 12.0%, no significant difference is generated compared with the germination rate of low-concentration treatment (1%), and the germination rate of high-concentration treatment (3%, 4% and 5%) is extremely significant, which indicates that 2% is the concentration of the ultimate pathogenic factor which can be tolerated by anther culture of flue-cured tobacco HKDN-5, so the optimum selection pressure for screening the black shank resistant mutant of HKDN-5 is preliminarily considered to be 2%.
Secondly, differences of 5% level were achieved between the 1%, 2% and 3% and 5% stock solution concentration treatments and the 4% treatment, thus determining 4% as the optimal selection pressure for the blackleg bacterium stock solution.
Thirdly, the average number of seedlings which are treated by stock solutions with different concentrations is 40.0, and the germination rate is 53.3%; the average number of seedlings germinated by the active ingredients of the black shank bacterium fermentation liquor with different concentrations is 8.6, and the germination rate is 11.46%.
TABLE 4 germination rates of HKDN-5 anther black shank resistant mutants at different selective pressure concentrations
(2) Verification results of potted plants and fields
After 2-3% of active ingredients of the black shank bacterium fermentation liquor and 4% of the survival anther cultured by the black shank bacterium stock solution are differentiated, rooted, doubled and trained respectively through the determined optimal selection pressure, the black shank resistance is identified through stem inoculation, and the result shows that: the former treated, tobacco plants showed varying degrees of resistance with an incidence of only 3.6% (fig. 9); the latter treatment, the incidence of tobacco plants was higher, with an incidence rate of up to 45.3% (fig. 10).
Claims (8)
1. A preparation method of a tobacco phytophthora parasitica fermentation broth pathogenic active component is characterized by comprising the following steps:
(1) shaking culturing the bacterial filaments of the phytophthora parasitica in a potato liquid culture medium by using a shaking table; collecting the sterile fermentation liquor at 5d-7d, centrifuging, and filtering and sterilizing the supernatant for later use;
(2) performing membrane filtration separation on the sterilized phytophthora parasitica sterile fermentation broth obtained in the step (1) through a 5KDa microfiltration hollow cellulose membrane and a 3KDa ultrafiltration hollow cellulose membrane to obtain clear and transparent ultrafiltrate; loading the ultrafiltrate into column, performing macroporous adsorption resin enrichment, eluting the components adsorbed on the resin with 80-95% ethanol to obtain 80-95% ethanol eluate, i.e. the pathogenic active components of the fermentation broth of Heiguobacter nigra.
2. The method for preparing a pathogenic active ingredient of a tobacco phytophthora parasitica fermentation broth according to claim 1, wherein the fermentation broth is a mixture of tobacco phytophthora parasitica fermentation broth and tobacco phytophthora parasitica fermentation broth,
the shake culture conditions in the step (1) are as follows: 26-30 ℃ and 180 rpm.
3. The method for preparing a pathogenic active ingredient of a tobacco phytophthora parasitica fermentation broth according to claim 1, wherein the fermentation broth is a mixture of tobacco phytophthora parasitica fermentation broth and tobacco phytophthora parasitica fermentation broth,
the shake culture conditions in the step (1) are as follows: 28 ℃ and 220 rpm.
4. The method for preparing a pathogenic active ingredient of a tobacco phytophthora parasitica fermentation broth according to claim 1, wherein the fermentation broth is a mixture of tobacco phytophthora parasitica fermentation broth and tobacco phytophthora parasitica fermentation broth,
centrifuging at 4 deg.C and 6000-.
5. The method for preparing the pathogenic active ingredient of the tobacco phytophthora parasitica fermentation broth according to claim 4, wherein the fermentation broth is prepared by mixing the components,
centrifuging at 8000rpm for 15min in step (1).
6. The method for preparing a pathogenic active ingredient of a tobacco phytophthora parasitica fermentation broth according to claim 1, wherein the fermentation broth is a mixture of tobacco phytophthora parasitica fermentation broth and tobacco phytophthora parasitica fermentation broth,
d101 type macroporous absorption resin is adopted in the step (2).
7. The method for preparing a pathogenic active ingredient of a tobacco phytophthora parasitica fermentation broth according to claim 1, wherein the fermentation broth is a mixture of tobacco phytophthora parasitica fermentation broth and tobacco phytophthora parasitica fermentation broth,
the ethanol concentration used in step (2) was 95%.
8. A tobacco phytophthora parasitica fermentation broth pathogenic active ingredient, characterized in that it is prepared by the method of any one of claims 1 to 7.
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| CN103081678A (en) * | 2013-01-14 | 2013-05-08 | 中国农业科学院烟草研究所 | Rapid identification method of tobacco black shank resistance |
| CN106318999A (en) * | 2016-08-23 | 2017-01-11 | 义乌章舸生物工程有限公司 | Method for preparing pumpkin seed meal molecular peptide through solid state fermentation on pumpkin seed cake meal by virtue of mixed bacteria |
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|---|---|---|---|---|
| CN103081678A (en) * | 2013-01-14 | 2013-05-08 | 中国农业科学院烟草研究所 | Rapid identification method of tobacco black shank resistance |
| CN106318999A (en) * | 2016-08-23 | 2017-01-11 | 义乌章舸生物工程有限公司 | Method for preparing pumpkin seed meal molecular peptide through solid state fermentation on pumpkin seed cake meal by virtue of mixed bacteria |
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