CN115024338B - Application of resuscitating promoting factor protein in preventing and treating crop continuous cropping diseases and promoting crop growth and application method - Google Patents
Application of resuscitating promoting factor protein in preventing and treating crop continuous cropping diseases and promoting crop growth and application method Download PDFInfo
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- CN115024338B CN115024338B CN202210766436.6A CN202210766436A CN115024338B CN 115024338 B CN115024338 B CN 115024338B CN 202210766436 A CN202210766436 A CN 202210766436A CN 115024338 B CN115024338 B CN 115024338B
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Abstract
The invention relates to application of resuscitating and promoting factor protein in preventing and treating continuous cropping diseases of crops and promoting the growth of the crops and an application method thereof. The method can be used for singly using the resuscitating and accelerating factor RPF or mixing the resuscitating and accelerating factor RPF protein with amino acid and microelements and then carrying out flushing or spraying. The invention discovers that the reviving promoting factor protein can effectively activate dormant gram-positive bacteria on soil or crops through leaf surface spraying, promote the rapid propagation of the gram-positive bacteria, increase the number of beneficial bacteria on soil and crop plants, prevent and treat diseases of underground or leaf parts, and can effectively improve the micro-ecological environment of the soil through ground spraying or flushing the reviving promoting factor, thereby having the functions of preventing and treating continuous cropping obstacle of crops and preventing and treating soil-borne diseases of root parts of crops.
Description
Technical Field
The invention relates to application and an application method of resuscitating and promoting factor protein in preventing and treating continuous cropping diseases of crops and promoting the growth of the crops, and belongs to the technical field of agricultural application.
Background
Mukamolova et al in 1998 found a factor in M.luteus that promotes resuscitative growth of bacteria in a dormant, non-multiplying state, designated resuscitating promoter (Rpf). Since it functions like a eukaryotic growth factor, it is also called a bacterial cytokine. Rpf is the first factor discovered so far that resuscitates and promotes growth of bacteria in dormant state. Rpf has a molecular weight of about 16-17 KD, in picograms (10) -12 g) Levels promote revitalization and growth of dormant bacteria in autocrine and paracrine forms.
Rpf protein refers to a generic term for a family of Rpf proteins containing Rpf-like conserved domains. Rpf is widely found in gram-positive bacteria, and there is a certain difference in the secretion of Rpf proteins by different types of bacteria, but with the Rpf conserved domain (NCBI: CDD: 284212), collectively referred to as Rpf proteins. Experiments prove that the conserved region is an important structure for Rpf resuscitation, and an individual Rpf domain has a biological function consistent with that of an Rpf protein.
Soil microorganisms and enzymes are important components of the biochemical characteristics of the soil, play an important role in the aspects of soil nutrient transformation circulation, organic matter decomposition and the like, are an important index of soil fertility, are often used for evaluating the biological characteristics of soil quality, and are now one of research hotspots in the soil science. At present, microorganisms in soil are supplemented or enriched mainly by directly adding biological bacteria into the soil, but the effect is general. Research on culturing VBNC state bacteria by utilizing Rpf protein resuscitation is mainly focused on the aspects of discovery of potential pathogenic bacteria, vaccine development, detection of food-borne pathogenic bacteria and the like in the fields of medicine and epidemiology, and application in the field of agricultural production is not reported.
The soil continuous cropping problem is more remarkable because the crop re-cropping index of China is relatively higher, the microecology of the soil is unbalanced, the number of harmful bacteria in the soil is increased, the number of beneficial bacteria is reduced, so that the soil-borne diseases are serious, at present, the problems that the harmful bacteria are killed by using some chemical pesticides or the beneficial bacteria are supplemented by using biological pesticides to improve the soil continuous cropping problem to reduce the crop diseases are solved, but excessive pesticide use easily causes soil pollution, the crop pesticide residues exceed the standard and the like are solved; no related report is currently seen on improving the soil continuous cropping problem, reducing crop diseases and the like by using pure biological Rpf protein.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides application and an application method of resuscitating and promoting factor protein in preventing and treating continuous cropping diseases of crops and promoting the growth of the crops.
The technical scheme of the invention is as follows:
the application of the resuscitating and promoting factor protein in preventing and treating crop continuous cropping diseases and promoting crop growth.
According to a preferred embodiment of the present invention, the resuscitation facilitator protein NCBI has a CAB09664.2 ID.
According to the invention, the preparation method of the resuscitation facilitating factor protein is as follows:
(1) Inoculating Streptomyces fradiae spore with strain preservation number of CGMCC No.11963 into slant culture medium, culturing at 28deg.C for 7 days, adding sterile water, and uniformly dispersing the spore in the sterile water to obtain a concentrated solution with concentration of 10 8 ～10 9 individual/mL spore suspension;
(2) Inoculating the spore suspension in the step (1) into a liquid fermentation culture medium, wherein the inoculum size is 2% of the volume of the liquid fermentation culture medium, and culturing for 24 hours at 30 ℃ and the rotating speed of 180 r/min to obtain liquid fermentation seed liquid;
(3) Inoculating the liquid fermentation seed liquid in the step (2) into a liquid culture medium, wherein the inoculation amount is 10% of the mass of the fermentation culture medium, and culturing for 4 days at 30 ℃ to obtain fermentation mother liquor containing Rpf protein;
(4) Heating the Rpf protein fermentation mother liquor in the step (3) to 80 ℃, preserving heat for 20 minutes, adding 0.1% filter aid, carrying out plate-frame filtration, and discarding the precipitate, wherein the obtained supernatant is a stock solution containing the Rpf protein;
(5) Adding ammonium sulfate into the stock solution containing RPF protein in the step (4), precipitating for 4 hours at 0 ℃, taking precipitate after plate-frame filter pressing, dissolving with a solution containing 8mol/L urea, performing gradient dialysis, removing urea in the protein solution by adopting a method of gradually reducing urea concentration, naturally renaturating the denatured protein in the process, firstly dialyzing overnight with a buffer solution containing 6mol/L urea at 4 ℃, then dialyzing for 4 hours at 4 ℃ with a buffer solution containing 4mol/L, 3mol/L, 2mol/L, 1mol/L and 0mol/L urea respectively, purifying the protein solution after dialysis is finished by adopting a weak anion exchange column DEAE, collecting different gradient eluents for SDS-PAGE analysis, and finally dialyzing for 4 hours at 4 ℃ with PBS buffer solution to obtain the resuscitation promoting factor protein.
According to the invention, in the step (1), the slant culture medium comprises (m/v) 2% of soybean meal powder, 0.5% of yeast powder, 1% of corn powder and 1.5% of agar powder; in the step (2) and the step (3), the liquid fermentation medium (m/v) comprises corn flour 3%, glucose 2%, monopotassium phosphate 0.03%, peptone 0.5%, soybean cake powder 1.5%, potassium nitrate 0.5%, soybean oil 1% and calcium chloride 0.05%.
According to a preferred embodiment of the present invention, in step (4), the filter aid is one or more of diatomaceous earth, bentonite, kaolin
According to a preferred embodiment of the invention, in step (5), the ratio of the mass of ammonium sulphate to the volume of Rpf protein-containing stock solution is 1:2, units: g/mL.
Preferably, according to the present invention, the crop continuous cropping diseases include root knot nematode disease, root rot disease, bacterial wilt disease, damping off disease, epidemic disease and the like.
Preferably according to the invention, the crop is radish, strawberry, melon, eggplant, cucumber, tomato, celery or ginger.
A method for preventing and treating the continuous cropping diseases of crops and promoting the growth of crops can be used singly or after the resuscitating and promoting factor Rpf protein is mixed with amino acid and trace elements, the resuscitating and promoting factor Rpf protein is applied by flushing or spraying.
According to the present invention, the resuscitation facilitating factor protein is preferably used at a concentration of 50 to 150mg/L.
Further preferably, the resuscitation facilitator protein is used at a concentration of 100mg/L.
According to the invention, preferably, the amino acid is one or more of lysine, arginine, glutamic acid, leucine, isoleucine, valine, alanine, glycine, aspartic acid, threonine, serine, cystine, methionine, tyrosine, phenylalanine, histidine and proline, and the concentration is 150-200 mg/L.
Further preferably, the amino acid is a combination of glutamic acid and aspartic acid, used at a concentration of 200mg/L.
According to the invention, preferably, the trace elements are a combination of ferrous citrate, zinc EDTA, copper EDTA and boric acid, and the concentration is 40mg/L.
According to the invention, the flushing or spraying is specifically: the crops are punched or sprayed for 1 to 3 times in the seedling stage, and the punching or spraying amount is 1 to 3 liters per mu.
The invention has the technical characteristics that:
about 90% of microorganisms in the soil are dormant, while in some unhealthy continuous cropping soil, the dormant microorganisms reach over 95%. Bacteria in soil that cause plant lesions are mostly gram-negative bacteria, while gram-positive bacteria are mostly of the group beneficial to crops. The Rpf protein can effectively activate gram-positive bacteria and promote rapid propagation of the gram-positive bacteria by matching with amino acid and trace elements, can increase the number of beneficial bacteria in soil, and can inhibit propagation of gram-negative bacteria, so that the soil micro-ecological environment is improved, a healthy soil micro-ecological system is reconstructed, and the problem of continuous cropping of various soils can be solved. Along with the mass propagation of soil microorganisms, healthy soil microecological balance is gradually established, and the mutual symbiotic relationship between the microorganisms and plants is reestablished, so that the continuous cropping disease of crops is prevented and controlled and the growth of the crops is promoted.
The invention has the beneficial effects that:
1. the invention discovers that the reviving promoting factor protein can effectively activate dormant gram-positive bacteria on soil or crops by means of spraying or beating, promote the rapid propagation of the gram-positive bacteria, increase the number of beneficial bacteria on soil and crop plants, prevent and treat diseases of underground or leaf parts, and can effectively improve the micro-ecological environment of the soil by means of spraying or beating the reviving promoting factor on the ground, thereby having the functions of preventing and treating continuous cropping obstacle of the crops and preventing and treating soil-borne diseases of the roots of the crops.
2. The invention discovers that the resuscitation promoting factor protein can further promote the reproduction of gram positive bacteria by being matched with amino acid and trace elements, has ecological niche competition advantage for most pathogenic bacteria, promotes the growth of crops, reduces diseases, improves and improves the quality of agricultural products, and simultaneously plays roles in preventing premature senility and rejuvenation of old trees of the crops.
3. Compared with the direct single use of the resuscitation promoting factor protein in combination with amino acid and trace elements, the invention has the advantages of generating a synergistic effect, having more obvious and durable effect, effectively improving the crop yield, increasing the yield of the radish by 32.71%, increasing the yield of the strawberry by 15.68%, increasing the yield of the tomato by 14.38% and increasing the yield of the celery by 18.81%, and being widely applied to agricultural production.
4. The invention discovers that no matter the recovery promoting factor is used alone or is matched with amino acid trace elements, the problem of continuous cropping diseases of crops can be effectively reduced, the rate of the large Jiang Qing blight is respectively reduced by 75.2 percent and 85.2 percent, the rate of stem rot is reduced by 83.2 percent and 87.8 percent, and the rate of root knot nematode disease is reduced by 79.3 percent and 85.3 percent; melon root rot is reduced by 82.3% and 85.7%, respectively, nematode disease is reduced by 76.5% and 84.3%; the root rot of the strawberry red center pillar is respectively reduced by 82.1 percent and 87.5 percent, and the anthracnose is respectively reduced by 80.3 percent and 85.2 percent; tomato bacterial wilt is reduced by 82.2 percent and 88.5 percent respectively, gray mold is reduced by 75.6 percent and 88.2 percent respectively, and epidemic disease is reduced by 77.1 percent and 82.5 percent respectively; the celery damping-off is reduced by 75.2% and 80.6%, and the sclerotinia is reduced by 76.7% and 89.2%, respectively.
Drawings
FIG. 1 is a photograph showing comparison of the number of Bacillus in soil after administration of resuscitation inducing factors in example 4.
FIG. 2 is a comparative photograph of radish after administration of resuscitation inducing factors in example 4.
FIG. 3 is a photograph showing comparison of the number of Bacillus in soil after administration of resuscitation inducing factors in example 5.
Fig. 4 is a comparative photograph of strawberry in example 5 after administration of a resuscitation inducing factor.
Detailed Description
The invention will now be described in further detail with reference to the drawings and specific examples, which should not be construed as limiting the invention. The experimental procedures and reagents not shown in the formulation of the examples were all in accordance with the conventional conditions in the art.
Example 1
A preparation method of RPF protein comprises the following specific steps:
(1) Inoculating Streptomyces fradiae spore with strain preservation number of CGMCC No.11963 into slant culture medium, culturing at 28deg.C for 7 days, adding sterile water, uniformly dispersing spores in sterile water without agglomeration, and making into concentrated solution with concentration of 10 8 Individual/ml spore suspension;
wherein the slant culture medium comprises (m/v) bean pulp powder 2%, yeast powder 0.5%, corn flour 1% and agar powder 1.5%;
(2) Inoculating the spore suspension in the step (1) into a liquid fermentation culture medium, wherein the inoculum size is 2% of the volume of the liquid fermentation culture medium, and culturing for 24 hours at 30 ℃ and the rotating speed of 180 r/min to obtain liquid fermentation seed liquid;
wherein the liquid fermentation culture medium (m/v) comprises corn flour 3%, glucose 2%, monopotassium phosphate 0.03%, peptone 0.5%, soybean cake powder 1.5%, potassium nitrate 0.5%, soybean oil 1% and calcium chloride 0.05%;
(3) Inoculating the liquid fermentation seed liquid in the step (2) into a liquid culture medium, wherein the inoculation amount is 10% of the mass of the fermentation culture medium, and culturing for 4 days at 30 ℃ to obtain fermentation mother liquor containing Rpf protein;
(4) Heating the Rpf protein fermentation mother liquor in the step (3) to 80 ℃, preserving heat for 20 minutes, adding 0.1% diatomite, carrying out plate frame filtration, and discarding the precipitate, wherein the obtained supernatant is a stock solution containing the Rpf protein;
(5) Adding the Rpf protein-containing stock solution in the step (4) according to 1:2 (g/mL), precipitating for 4 hours at 0 ℃, carrying out plate-frame filter pressing, taking precipitate, dissolving with a solution containing 8mol/L urea, carrying out gradient dialysis, removing urea in the protein solution by adopting a method of gradually reducing urea concentration, naturally renaturating the denatured protein in the process, firstly dialyzing overnight with a buffer solution containing 6mol/L urea at 4 ℃, then dialyzing for 4 hours with a buffer solution containing 4mol/L, 3mol/L, 2mol/L, 1mol/L and 0mol/L urea at 4 ℃, purifying the protein solution after the dialysis is finished by adopting a weak anion exchange column DEAE, collecting different gradient eluents for SDS-PAGE analysis, and finally dialyzing for 4 hours with a PBS buffer solution at 4 ℃ to obtain the resuscitation promoting factor protein.
In the embodiment, the strain preservation number is CGMCC No.11963 which is disclosed and preserved in the Chinese patent document CN 109749961A.
Example 2
Preparation of Rpf protein, amino acid and trace element complex:
the Rpf protein of example 1 was formulated into a Rpf protein stock solution with a concentration of 100mg/L, and then a complex amino acid stock powder consisting of lysine, arginine, glutamic acid, leucine, isoleucine, valine, alanine, glycine, aspartic acid, threonine, serine, cystine, methionine, tyrosine, phenylalanine, histidine, proline was added to the Rpf protein stock solution at a ratio of (m/v) 10%, zinc EDTA 5g/L, copper EDTA 5g/L, boric acid 8g/L, and a complex solution of Rpf protein, amino acid and trace elements was obtained.
Meanwhile, the compound amino acid raw powder, the trace element ferrous citrate, the EDTA zinc, the EDTA copper and the boric acid are added into water according to the same mass-volume ratio to prepare an amino acid and trace element compound solution without the Rpf protein, which is used in the subsequent examples.
Example 3
Disease prevention and continuous cropping resistance test of ginger. The test site is located in Anchu Linghe town of Weifang, the ginger variety is ginger, the land is continuous cropping land, the last crop is ginger, jiang Qing blight, nematodiasis, stem rot and other continuous cropping diseases are serious, and the number of dead plants is large.
Test setup: the experiment set up three treatments as follows: the blank, the Rpf protein stock solution in example 2 (treatment 1) and the complex solution of Rpf protein, amino acid and trace element in example 2 (treatment 2) were used 2 times in total for the administration of ginger, and each time was administered 2 liters/mu/time before the emergence period and the hilling, the Rpf protein concentration was 100mg/L, and the ginger yield and disease occurrence were shown in table 1 below.
TABLE 1
And respectively counting the yield and the occurrence of ginger continuous cropping diseases in the harvest period, and detecting the content of bacillus in the soil. As can be seen from Table 1, the recovery promoting factor increases the yield of ginger by 28.32%, increases the number of beneficial bacillus in soil by 685%, reduces Jiang Qing blight by 75.2%, reduces stem rot by 83.2%, reduces root knot nematode by 79.3%, obviously improves the yield of ginger, and reduces continuous cropping diseases. By using the RPF protein solution containing amino acid and trace elements, the yield of ginger is increased by 35.28%, the number of beneficial bacillus in soil is increased by 820%, and Jiang Qing blight is reduced by 85.2%; the stem rot is reduced by 87.8%, the root knot nematode disease is reduced by 85.3%, and compared with the single RPF protein, the yield of ginger is further improved, and the continuous cropping disease is reduced.
The method for detecting bacillus in soil is carried out by referring to the following method: respectively weighing soil sample of control field and test field, adding into 100ml sterilized water, shaking for 30min at 200r/min on shaking table to obtain soil suspension, respectively sucking 1ml soil mother liquor with sterile pipette, adding into 9ml sterilized water, diluting according to 1-10 gradient, and respectively making into 10, 10 2 、10 3 、10 4 The soil bacteria suspension with gradient dilution multiple is added to the prepared bacteria culture medium flat plate by taking 0.1ml of each concentration, the flat plate is uniformly coated by a sterile coating rod, and the flat plate is placed in a 37 ℃ incubator for culture overnight, and the bacterial colony number is counted.
Example 4
And (5) carrying out radish growth promotion test. Test site: and (3) in the east region of the Yi city river, test treatment: the Shi Datian radish was washed with the Rpf protein stock solution of example 2 at a concentration of 100mg/L, with a fresh water treatment as a control, at a dose of 2 liters/mu, and was washed once during the seedling stage of the radish.
At the time of harvesting, the yields were counted and the content of bacillus in the soil was examined, and the results are shown in fig. 1 to 2 and table 2.
TABLE 2
As can be seen from FIGS. 1-2 and Table 2, the number of bacillus in the soil after the RPF protein is used is increased by 865% and the yield of radish is increased by 32.71% compared with the control, and the radish has regular shape and improved product quality.
Example 5
Quality test of strawberry growth promoting and disease preventing extracts. Test site: temporary to Yi city temporary to county, test treatment: shi Datian strawberries were washed with Rpf protein stock in example 2 at a concentration of 100mg/L, with a control of 2 liters/mu of fresh water treatment, and twice in the seedling stage.
At the time of harvesting, the yields were counted and the content of bacillus in the soil was examined, and the results are shown in fig. 3 to 4 and table 3.
TABLE 3 Table 3
As can be seen from figures 3-4 and Table 3, compared with the control, the number of bacillus in the soil after using the Rpf protein is increased by 1140%, the yield of strawberries is increased by 15.68%, the sugar content is increased by 11%, the Vc content is increased by 6%, the root rot of the red middle column of strawberries is reduced by 81.7%, the anthracnose is reduced by 77.5%, which shows that the RPF protein has obvious effects of increasing yield, improving quality and preventing diseases.
Example 6
Disease prevention test of muskmelon. Test site: hebei Shijia village. Test treatment: the RPF protein stock solution (treatment 1) in example 2, the RPF protein, the complex solution of amino acid and trace element (treatment 2) in example 2, and the complex solution of amino acid and trace element (treatment 3) containing no RPF protein were used to wash Shi Datian melon, respectively, with a blank control set at a dose of 2 liters/mu, and the results of the two-time washing at the seedling stage were shown in table 4.
TABLE 4 Table 4
As can be seen from Table 4, at harvest, the actinomycetes in the soil are increased by 50% and 58% respectively, the melon yield is increased by 18.52% and 25.18% respectively, melon root rot is reduced by 82.3% and 85.7% respectively, nematode disease is reduced by 76.5% and 84.3%, melon yield can be obviously improved, root rot and nematode disease can be reduced, the influence of treatment 3 on soil microbial communities and the prevention and treatment effects of root rot and nematode disease are not obvious by using treatment 3 alone, and the combination of Rpf protein can obviously improve yield and the prevention and treatment effects of root rot and nematode disease.
Example 7
Eggplant growth promotion test. Test site: anhui province, homeland state. Test treatment: the Rpf protein stock solution in example 2 (treatment 1) and the complex solution of Rpf protein, amino acid and trace element in example 2 (treatment 2) were used to rinse Shi Datian eggplants, respectively, at a concentration of 100mg/L, while a blank control treatment was provided at a dose of 2 liters/mu, and the results of the two rinses at the seedling stage are shown in table 5.
TABLE 5
As shown in Table 5, the actinomycetes increased by 184.6% and 215.8% respectively in the soil and the eggplants increased by 21.5% and 25.6% respectively when harvesting compared with the blank.
Example 8
Disease prevention test of cucumber. Test site: river north Zunhua city, test treatment: the Shi Datian cucumber was washed with the Rpf protein stock solution of example 2 (treatment 1) and the complex solution of Rpf protein, amino acid and trace element of example 2 (treatment 2) at a concentration of 100mg/L, respectively, while a blank control treatment was provided at a dose of 2 liters/mu, and the results of the washing at the seedling stage were shown in table 6.
TABLE 6
As can be seen from Table 6, the actinomycetes in the soil increased by 500% and 650% respectively, cucumber gummy stem blight reduced by 81.5% and 88.6%, nematode disease reduced by 86.5% and 90.2%, cucumber fusarium wilt reduced by 78.2% and 86.5%, respectively, when harvesting, compared with the blank control group.
Example 9
Strawberry disease prevention test. Test site: and (3) in the presence of Yi city, juxian county, test treatment: the RPF protein stock solution (treatment 1) of example 2 and the complex solution (treatment 2) of RPF protein, amino acid and trace element of example 2 were used to wash Shi Datian strawberries, respectively, at a concentration of 100mg/L, while a blank control treatment was provided, the amounts were 2 liters/mu, and the results of the washing at the seedling stage were shown in table 7.
TABLE 7
As shown in Table 7, the yield of beneficial bacillus in the soil is increased by 48.4% and 180.6%, the yield of strawberry is increased by 25.2% and 36.5%, the sugar degree is increased by 10% and 12%, the Vc content is increased by 8% and 9%, the root rot of strawberry is reduced by 82.1% and 87.5%, and the anthracnose is reduced by 80.3% and 85.2% respectively, compared with the blank control group, and the method has obvious effect of improving continuous cropping and preventing diseases.
Example 10
Disease prevention test of tomatoes. Test site: and (3) testing and processing the Shandong Linyi city and Yinan county: the Rpf protein stock solution (treatment 1) in example 2, the Rpf protein, the complex solution of amino acid and trace element (treatment 2) in example 2, and the complex solution of amino acid and trace element (treatment 3) containing no Rpf protein were used to rinse Shi Datian tomatoes, respectively, while a blank control treatment was provided at a dose of 2 liters/mu, and the results of the two rinses at the seedling stage are shown in table 8.
TABLE 8
As can be seen from Table 8, the Bacillus amounts in treatments 1 and 2 increased by 708.3% and 825.5%, respectively, and the tomato yield increases by 14.38% and 21.65%, respectively, when harvested, as compared to the control group. The tomato bacterial wilt is reduced by 82.2% and 88.5% respectively, the gray mold is reduced by 75.6% and 88.2% respectively, the epidemic disease is reduced by 77.1% and 82.5% respectively, the treatment 3 only shows a certain growth promoting effect on the yield, no obvious effect is exerted on soil beneficial bacteria and continuous cropping diseases, the prevention and control effect of the bacterial wilt and the epidemic disease is 0, and the sum of the yield increasing effects of the treatment 1 and the treatment 3 and the quantity of soil bacillus is smaller than that of the treatment 2, which indicates that the Rpf protein and the amino acid and the trace element are not simply overlapped in the actual use process, but generate a synergistic effect, and the tomato yield and the quantity of the soil bacillus are further increased compared with the Rpf protein, the amino acid and the trace element which are used respectively.
Example 11
Disease prevention test of celery. Test site: the method for testing the river in the east of Yi city comprises the following steps: the solutions of the RPF protein stock solution (treatment 1) in example 2 and the complex solution (treatment 2) of the RPF protein, amino acid and trace element in example 2 were used to wash Shi Datian celery, respectively, at a concentration of 100mg/L, and a blank control treatment was set at the same time, the amounts of which were 2 liters/mu, and the results of the washing twice in the seedling stage were shown in Table 9.
TABLE 9
As can be seen from Table 9, celery was increased by 18.81% and 22.5% and celery damping-off was reduced by 75.2% and 80.6% and sclerotium was reduced by 76.7% and 89.2% respectively, when harvested as compared to the blank.
Claims (9)
1. The application of the resuscitating and accelerating factor protein in preventing and controlling continuous cropping diseases of crops and accelerating the growth of the crops is characterized in that the preparation method of the resuscitating and accelerating factor protein is as follows:
(1) Inoculating Streptomyces fradiae spore with strain preservation number of CGMCC No.11963 into slant culture medium, culturing at 28deg.C for 7 days, adding sterile water, and uniformly dispersing the spore in the sterile water to obtain a concentrated solution with concentration of 10 8 ~10 9 individual/mL spore suspension;
(2) Inoculating the spore suspension in the step (1) into a liquid fermentation culture medium, wherein the inoculum size is 2% of the volume of the liquid fermentation culture medium, and culturing for 24 hours at 30 ℃ and the rotating speed of 180 r/min to obtain liquid fermentation seed liquid;
(3) Inoculating the liquid fermentation seed liquid in the step (2) into a liquid fermentation culture medium, wherein the inoculation amount is 10% of the mass of the liquid fermentation culture medium, and culturing for 4 days at 30 ℃ to obtain fermentation mother liquor containing resuscitation promoting factor proteins;
(4) Heating the fermentation mother liquor containing the resuscitation facilitating factor protein in the step (3) to 80 ℃, preserving heat for 20 minutes, adding 0.1% filter aid, carrying out plate frame filtration, and discarding the precipitate, wherein the obtained supernatant is a stock solution containing the resuscitation facilitating factor protein;
(5) Adding ammonium sulfate into the stock solution containing the resuscitation promoting factor protein in the step (4), precipitating for 4 hours at 0 ℃, taking the precipitate after plate-and-frame filter pressing, dissolving the precipitate by using a solution containing 8mol/L urea, performing gradient dialysis, removing urea in the protein solution by adopting a method of gradually reducing urea concentration, naturally renaturating the denatured protein in the process, firstly dialyzing overnight at 4 ℃ by using a buffer solution containing 6mol/L urea, then respectively dialyzing for 4 hours at 4 ℃ by using a buffer solution containing 4mol/L, 3mol/L, 2mol/L, 1mol/L and 0mol/L urea, purifying the protein solution by using a weak anion exchange column DEAE after the dialysis is finished, collecting different gradient eluents for SDS-PAGE analysis, and finally, dialyzing for 4 hours at 4 ℃ by using a PBS buffer solution to obtain the resuscitation promoting factor protein.
2. The use according to claim 1, wherein in step (1), the slant medium consists of (m/v) soybean meal 2%, yeast 0.5%, corn flour 1%, agar powder 1.5%; in the step (2) and the step (3), the liquid fermentation medium (m/v) comprises corn flour 3%, glucose 2%, monopotassium phosphate 0.03%, peptone 0.5%, soybean cake powder 1.5%, potassium nitrate 0.5%, soybean oil 1% and calcium chloride 0.05%.
3. The use according to claim 1, wherein in step (4) the filter aid is one or more of diatomaceous earth, bentonite, kaolin; in the step (5), the ratio of the mass of the ammonium sulfate to the volume of the stock solution containing the resuscitation facilitating factor protein is 1:2, units: g/mL.
4. The use according to claim 1, wherein the crop continuous cropping diseases comprise root knot nematode disease, root rot, bacterial wilt, epidemic disease, wilt and damping off; the crops are radishes, strawberries, melons, eggplants, cucumbers, tomatoes, celery or ginger.
5. A method for preventing and controlling continuous cropping diseases of crops and promoting the growth of the crops can be used independently or the recovery promoting factor protein is mixed with amino acid and microelements and then applied in a flushing way or a spraying way; the using concentration of the resuscitation facilitating factor protein is 50-150 mg/L; the flushing or spraying specifically comprises the following steps: the method comprises the steps of (1) carrying out punching or spraying for 1-3 times in a seedling stage of crops, wherein the punching or spraying amount is 1-3 liters/mu;
the preparation method of the resuscitation facilitating factor protein comprises the following steps:
(1) Inoculating Streptomyces fradiae spore with strain preservation number of CGMCC No.11963 into slant culture medium, culturing at 28deg.C for 7 days, adding sterile water, and uniformly dispersing the spore in the sterile water to obtain a concentrated solution with concentration of 10 8 ~10 9 individual/mL spore suspension;
(2) Inoculating the spore suspension in the step (1) into a liquid fermentation culture medium, wherein the inoculum size is 2% of the volume of the liquid fermentation culture medium, and culturing for 24 hours at 30 ℃ and the rotating speed of 180 r/min to obtain liquid fermentation seed liquid;
(3) Inoculating the liquid fermentation seed liquid in the step (2) into a liquid fermentation culture medium, wherein the inoculation amount is 10% of the mass of the liquid fermentation culture medium, and culturing for 4 days at 30 ℃ to obtain fermentation mother liquor containing resuscitation promoting factor proteins;
(4) Heating the fermentation mother liquor containing the resuscitation facilitating factor protein in the step (3) to 80 ℃, preserving heat for 20 minutes, adding 0.1% filter aid, carrying out plate frame filtration, and discarding the precipitate, wherein the obtained supernatant is a stock solution containing the resuscitation facilitating factor protein;
(5) Adding ammonium sulfate into the stock solution containing the resuscitation promoting factor protein in the step (4), precipitating for 4 hours at 0 ℃, taking the precipitate after plate-and-frame filter pressing, dissolving the precipitate by using a solution containing 8mol/L urea, performing gradient dialysis, removing urea in the protein solution by adopting a method of gradually reducing urea concentration, naturally renaturating the denatured protein in the process, firstly dialyzing overnight at 4 ℃ by using a buffer solution containing 6mol/L urea, then respectively dialyzing for 4 hours at 4 ℃ by using a buffer solution containing 4mol/L, 3mol/L, 2mol/L, 1mol/L and 0mol/L urea, purifying the protein solution by using a weak anion exchange column DEAE after the dialysis is finished, collecting different gradient eluents for SDS-PAGE analysis, and finally, dialyzing for 4 hours at 4 ℃ by using a PBS buffer solution to obtain the resuscitation promoting factor protein.
6. The method of claim 5, wherein the resuscitation facilitator protein is used at a concentration of 100mg/L.
7. The method of claim 5, wherein the amino acid is one or more of lysine, arginine, glutamic acid, leucine, isoleucine, valine, alanine, glycine, aspartic acid, threonine, serine, cystine, methionine, tyrosine, phenylalanine, histidine, proline, and the concentration is 150-200 mg/L.
8. The method of claim 7, wherein the amino acid is a combination of glutamic acid and aspartic acid at a concentration of 200mg/L.
9. The method of claim 5, wherein the trace element is a combination of ferrous citrate, zinc EDTA, copper EDTA and boric acid at a concentration of 40mg/L.
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