CN115024338A

CN115024338A - Application of resuscitation promotion factor protein in preventing and treating crop continuous cropping diseases and promoting crop growth and application method

Info

Publication number: CN115024338A
Application number: CN202210766436.6A
Authority: CN
Inventors: 孟祥坤; 于新; 徐文凤; 徐文华; 邢芳芳; 于文华; 朱孔杰; 林先贵; 宋涛; 邹朋
Original assignee: Guangdong Qingyuan Jinzhengda Agricultural Research Institute Co ltd; HEZE KINGENTA ECOLOGICAL ENGINEERING CO LTD; Kingenta Ecological Engineering Group Co Ltd
Current assignee: Guangdong Qingyuan Jinzhengda Agricultural Research Institute Co ltd; HEZE KINGENTA ECOLOGICAL ENGINEERING CO LTD; Kingenta Ecological Engineering Group Co Ltd
Priority date: 2022-06-30
Filing date: 2022-06-30
Publication date: 2022-09-09
Anticipated expiration: 2042-06-30
Also published as: CN115024338B

Abstract

The invention relates to application of a resuscitation promoting factor protein in preventing and treating continuous cropping diseases of crops and promoting crop growth and an application method thereof. The method can be used for singly using the resuscitation promoting factor RPF or mixing the resuscitation promoting factor RPF protein, amino acid and trace elements and then flushing or spraying. The invention discovers that the leaf surface spraying of the resuscitation promoting factor protein can effectively activate dormant gram-positive bacteria on soil or crops, promote the rapid propagation of the gram-positive bacteria, increase the number of beneficial bacteria on the soil and crop plants, and prevent and treat underground or leaf diseases, the ground spraying or flushing application of the resuscitation promoting factor can effectively improve the soil micro-ecological environment, and the invention has the effects of preventing and treating continuous cropping obstacles of the crops and preventing and treating soil-borne diseases of the roots of the crops.

Description

Application of resuscitation promotion factor protein in preventing and treating crop continuous cropping diseases and promoting crop growth and application method

Technical Field

The invention relates to application of a resuscitation promotion factor protein in preventing and treating crop continuous cropping diseases and promoting crop growth and an application method, belonging to the technical field of agricultural application.

Background

Mukamolova et al discovered a factor in m.luteus in 1998 that promotes the growth of resuscitation of bacteria in a dormant, non-multiplying state, named resuscitation-promoting factor (Rpf). It is also known as a bacterial cytokine because it functions similarly to a eukaryotic growth factor. Rpf is the first factor discovered to date that can resuscitate and promote the growth of dormant bacteria. The molecular weight of Rpf is about 16-17 KD, and the molecular weight is 10 picogram ^-12 g) Levels promote the reactivation and growth of dormant bacteria in autocrine and paracrine forms.

The Rpf protein refers to the generic term for the Rpf protein family containing the Rpf-like conserved domain. Rpf is widely found in gram-positive bacteria, and Rpf proteins secreted by different types of bacteria have certain differences, but have an Rpf conserved domain (NCBI: CDD:284212), collectively referred to as Rpf proteins. Experiments prove that the conserved region is an important structure for promoting resuscitation by Rpf, and an individual Rpf structural domain has a biological function consistent with Rpf protein.

Soil microorganisms and enzymes are important components of soil biochemical characteristics, play an important role in aspects such as soil nutrient conversion cycle, 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 one of the research hotspots of the soil academia. At present, the microorganisms in the soil are supplemented or enriched mainly by directly adding the biological agent into the soil, but the effect is general. The research of utilizing Rpf protein to resuscitate and culture VBNC state bacteria mainly focuses on the aspects of discovery of potential pathogenic bacteria, vaccine development, detection of food-borne pathogenic bacteria and the like in the medical and epidemiological fields, and the application in the agricultural production field is not reported.

Because the crop replanting index is relatively high in China, the problem of soil continuous cropping is relatively outstanding, soil micro-ecology is unbalanced, the number of harmful bacteria in soil is increased, and the number of beneficial bacteria is reduced, so that the soil-borne diseases are serious, at present, the problems that some chemical pesticides are used for killing the harmful bacteria or biological bactericides are used for supplementing the beneficial bacteria to improve the soil continuous cropping problem and reduce the crop diseases are mainly used, but excessive pesticides are used to easily cause soil pollution, crop pesticide residue exceeds the standard and the like; relevant reports on improving the soil continuous cropping problem and reducing crop diseases and the like by using the pure biological Rpf protein are not seen at present.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides the application of the resuscitation 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 technical scheme of the invention is as follows:

the application of the resuscitation promoting factor protein in preventing and treating continuous cropping diseases of crops and promoting the growth of the crops.

Preferably, according to the invention, the resuscitation-promoting factor protein NCBI has an ID CAB 09664.2.

According to the invention, the preparation method of the resuscitation promoting factor protein is as follows:

(1) inoculating Streptomyces fradiae spore with strain preservation number of CGMCC No.11963 to slant culture medium, culturing at 28 deg.C for 7 days, adding sterile water, and uniformly dispersing the spore in sterile water to obtain product with concentration of 10 ⁸ ～10 ⁹ Spores per mL of spore suspension;

(2) inoculating the spore suspension obtained in the step (1) into a liquid fermentation culture medium, wherein the inoculation amount is 2% of the volume of the liquid fermentation culture medium, and culturing for 24 hours at the temperature of 30 ℃ and the rotating speed of 180 r/min to obtain a liquid fermentation seed solution;

(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 liquid containing Rpf protein;

(4) heating the Rpf protein fermentation mother liquor in the step (3) to 80 ℃, preserving the heat for 20 minutes, then adding 0.1% of filter aid, performing plate-frame filtration, discarding the precipitate, and obtaining supernatant as stock solution containing the Rpf protein;

(5) adding ammonium sulfate into the stock solution containing the RPF protein in the step (4), precipitating for 4 hours at the temperature of 0 ℃, taking the precipitate after plate-frame filter pressing, dissolving the precipitate with a solution containing 8mol/L urea, performing gradient dialysis, removing the urea in the protein solution by adopting a method for gradually reducing the concentration of the urea to naturally renature the denatured protein in the process, dialyzing the denatured protein at the temperature of 4 ℃ overnight with a buffer solution containing 6mol/L urea, dialyzing the denatured protein for 4 hours at the temperature of 4 ℃ respectively with buffer solutions containing 4mol/L, 3mol/L, 2mol/L, 1mol/L and 0mol/L urea, purifying the protein solution by adopting a weak anion exchange column DEAE after the dialysis is finished, collecting different gradient eluents, performing SDS-PAGE analysis, and finally dialyzing for 4 hours at the temperature of 4 ℃ with a PBS buffer solution to obtain the resuscitation promotion factor protein.

Preferably, in step (1), the slant culture medium consists of (m/v) soybean meal powder 2%, yeast powder 0.5%, corn flour 1%, agar powder 1.5%; in the step (2) and the step (3), the liquid fermentation medium (m/v) comprises 3% of corn flour, 2% of glucose, 0.03% of monopotassium phosphate, 0.5% of peptone, 1.5% of soybean cake powder, 0.5% of potassium nitrate, 1% of soybean oil and 0.05% of calcium chloride.

Preferably, in step (4), the filter aid is one or more of diatomite, bentonite and kaolin

Preferably, in step (5), the ratio of the mass of ammonium sulfate to the volume of the Rpf protein-containing stock solution is 1: 2, unit: g/mL.

Preferably, the crop continuous cropping diseases comprise root knot nematode diseases, root rot diseases, bacterial wilt diseases, fusarium wilt diseases, rhizoctonia rot diseases, epidemic diseases 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 continuous cropping diseases of crops and promoting the growth of crops can be realized by using a recovery promoting factor Rpf alone or by mixing a recovery promoting factor Rpf protein, amino acid and trace elements and then flushing or spraying.

According to the invention, the use concentration of the resuscitation promoting factor protein is preferably 50-150 mg/L.

Further preferably, the concentration of the resuscitation promoting factor protein is 100 mg/L.

According to the invention, 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 using concentration is 150-200 mg/L.

Further preferably, the amino acid is a combination of glutamic acid and aspartic acid, and the concentration is 200 mg/L.

According to the invention, the trace element is a composition of ferrous citrate, zinc EDTA, copper EDTA and boric acid, and the concentration is 40 mg/L.

According to the invention, the rinsing or spraying is preferably as follows: the fertilizer is applied or sprayed for 1-3 times in the seedling stage of crops, and the application amount is 1-3 liters per mu.

The invention has the technical characteristics that:

about 90% of microorganisms in the soil are in a dormant state, and in some unhealthy continuous cropping soil, the microorganisms in the dormant state reach more than 95%. The bacteria causing plant diseases in the soil are mostly gram-negative bacteria, and the gram-positive bacteria are mostly beneficial to crops. The Rpf protein can effectively activate gram-positive bacteria and promote the 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 the propagation of gram-negative bacteria, thereby improving the soil micro-ecological environment, reconstructing a healthy soil micro-ecosystem and further solving the problem of continuous cropping of various soils. Along with the mass propagation of soil microorganisms, healthy soil micro-ecological balance is gradually established, and a mutual symbiotic relationship is reestablished between the microorganisms and plants, so that the aims of preventing and treating continuous cropping diseases of crops and promoting the growth of the crops are achieved.

The invention has the beneficial effects that:

1. the invention discovers that the dormant gram-positive bacteria on the soil or crops can be effectively activated by spraying or spraying the resuscitation promoting factor protein, the rapid propagation of the gram-positive bacteria is promoted, the number of beneficial bacteria on the soil and crops can be increased, the underground or leaf diseases can be prevented and controlled, the micro-ecological environment of the soil can be effectively improved by spraying or spraying the resuscitation promoting factor on the ground, and the effects of preventing and controlling continuous cropping obstacles of the crops and preventing and controlling soil-borne diseases of roots of the crops can be realized.

2. The invention finds that the resuscitation promoting factor protein can be matched with amino acid and trace elements to be used to further promote the propagation of gram-positive bacteria, form ecological niche competitive advantage for most pathogenic bacteria, promote the growth of crops, reduce diseases, improve and enhance the quality of agricultural products, and simultaneously play a role in preventing the premature senility of crops and rejuvenating old trees.

3. Compared with the use of the recovery promoting factor protein matched with amino acid and trace elements, the recovery promoting factor protein is directly and independently used, the synergistic effect is generated, the effect is more obvious and lasting, the crop yield is effectively improved, the radish yield is increased by 32.71%, the strawberry yield is increased by 15.68%, the tomato yield is increased by 14.38%, and the celery yield is increased by 18.81%, so that the recovery promoting factor protein can be widely applied to agricultural production.

4. The invention finds that the problem of continuous cropping diseases of crops can be effectively reduced no matter the recovery promoting factors are used alone or the recovery promoting factors are matched with amino acid trace elements, ginger bacterial wilt is respectively reduced by 75.2 percent and 85.2 percent, stem root rot is reduced by 83.2 percent and 87.8 percent, and root knot nematode is reduced by 79.3 percent and 85.3 percent; the root rot of the melon is respectively reduced by 82.3 percent and 85.7 percent, and the nematode disease is reduced by 76.5 percent and 84.3 percent; the strawberry red stele root rot 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; the tomato bacterial wilt is respectively reduced by 82.2 percent and 88.5 percent, the gray mold is respectively reduced by 75.6 percent and 88.2 percent, and the blight is respectively reduced by 77.1 percent and 82.5 percent; the celery rhizoctonia rot is reduced by 75.2 percent and 80.6 percent, and the sclerotinia rot is reduced by 76.7 percent and 89.2 percent respectively.

Drawings

FIG. 1 is a photograph showing a comparison of the number of Bacillus species in soil after the resuscitation inducing agent of example 4 was applied thereto.

FIG. 2 is a photograph showing a comparison of radish after administration of resuscitation-promoting factors in example 4.

FIG. 3 is a photograph showing a comparison of the number of Bacillus bacteria in soil after the resuscitation inducing factors of example 5 were applied.

FIG. 4 is a photograph comparing strawberries after administration of resuscitation-promoting factors in example 5.

Detailed Description

The invention is described in further detail below with reference to the figures and the examples, but the invention should not be construed as being limited thereto. The experimental methods and reagents of the formulations not specified in the examples are in accordance with the conventional conditions in the art.

Example 1

A preparation method of RPF protein comprises the following steps:

(1) inoculating Streptomyces fradiae spore with strain preservation number of CGMCC No.11963 to slant culture medium, culturing at 28 deg.C for 7 days, adding sterile water, uniformly dispersing the spore in sterile water, and making into product with concentration of 10 ⁸ Spore suspension of each ml;

wherein the slant culture medium comprises (m/v) 2% of soybean meal, 0.5% of yeast powder, 1% of corn flour and 1.5% of agar powder;

wherein the liquid fermentation medium (m/v) comprises 3% of corn flour, 2% of glucose, 0.03% of potassium dihydrogen phosphate, 0.5% of peptone, 1.5% of soybean cake powder, 0.5% of potassium nitrate, 1% of soybean oil and 0.05% of calcium chloride;

(4) heating the Rpf protein fermentation mother liquor in the step (3) to 80 ℃, preserving the heat for 20 minutes, then adding 0.1% of diatomite, filtering a plate frame, removing the precipitate, and obtaining supernatant as stock solution containing the RPF protein;

(5) adding the Rpf protein-containing stock solution in the step (4) according to the ratio of 1: 2, adding ammonium sulfate in a mass-to-volume ratio (g/mL) of the solution, precipitating for 4 hours at 0 ℃, carrying out plate-frame filter pressing, taking the precipitate, dissolving the precipitate by using a solution containing 8mol/L urea, carrying out gradient dialysis, removing the urea in a protein solution by adopting a method for gradually reducing the concentration of the urea, naturally renaturing the denatured protein in the process, dialyzing the denatured protein by using a buffer solution containing 6mol/L urea at 4 ℃ overnight, then respectively dialyzing the denatured protein by using buffer solutions containing 4mol/L, 3mol/L, 2mol/L, 1mol/L and 0mol/L urea at 4 ℃ for 4 hours, purifying the protein solution by using a weak anion exchange column DEAE after the dialysis is finished, collecting different gradient eluents, carrying out SDS-PAGE analysis, and finally dialyzing the protein by using PBS buffer solution at 4 ℃ for 4 hours to obtain the resuscitation promotion factor protein.

The strain preservation number of the embodiment is CGMCC No.11963 which is disclosed and preserved in Chinese patent document CN 109749961A.

Example 2

Preparing an Rpf protein, amino acid and trace element compound:

the Rpf protein of example 1 is prepared into Rpf protein stock solution with the concentration of 100mg/L, then composite amino acid raw powder is added into the Rpf protein stock solution according to the proportion (m/v) of 10%, the composite amino acid raw powder is composed of lysine, arginine, glutamic acid, leucine, isoleucine, valine, alanine, glycine, aspartic acid, threonine, serine, cystine, methionine, tyrosine, phenylalanine, histidine and proline in a composite mode, 10g/L of trace element ferrous citrate, 5g/L of EDTA zinc, 5g/L of EDTA copper and 8g/L of boric acid are added according to the proportion (m/v), and a composite solution of RPF protein, amino acid and trace element is obtained.

And simultaneously adding the compound amino acid raw powder, the trace elements ferrous citrate, EDTA zinc, EDTA copper and boric acid into water according to the same mass-volume ratio to prepare the amino acid and trace element compound solution without Rpf protein for the subsequent embodiment.

Example 3

Disease prevention and continuous cropping resistance test of ginger. The test site is located in the Anqiling river town of Weifang city, the ginger variety is ginger, the field is a continuous cropping land, the upper crop is ginger, and continuous cropping diseases such as ginger bacterial wilt, nematode disease, stem base rot and the like are serious and more dead plants are generated.

And (3) experimental setting: the experiment was set up with three treatments: blank control, Rpf protein stock solution (treatment 1) in example 2 and Rpf protein, amino acid and trace element compound solution (treatment 2) in example 2 are applied to the ginger by flushing for 2 times, the dosage is 2 liters/mu/time, the RPF protein application concentration is 100mg/L, and the ginger yield and the disease occurrence condition are detailed in the following table 1.

TABLE 1

And (4) respectively counting the yield and the occurrence condition of the ginger continuous cropping diseases in the harvesting period, and detecting the content of bacillus in the soil. As can be seen from Table 1, after the resuscitation promoting factors are used, the ginger yield is increased by 28.32%, the number of beneficial bacilli in soil is increased by 685%, ginger bacterial wilt is reduced by 75.2%, stem root rot is reduced by 83.2%, root knot nematode disease is reduced by 79.3%, the ginger yield is obviously improved, and continuous cropping diseases are reduced. By using the RPF protein solution containing amino acid and trace elements, the ginger yield is increased by 35.28%, the number of beneficial bacilli in soil is increased by 820%, and ginger bacterial wilt is reduced by 85.2%; the stem rot is reduced by 87.8 percent, the root knot nematode disease is reduced by 85.3 percent, and compared with the single use of RPF protein, the yield of the ginger is further improved, and the continuous cropping diseases are reduced.

The detection method of bacillus in soil is carried out according to the following method: respectively weighing 10g of soil sample from the soil of the control field and the soil of the test field, adding the soil sample into 100ml of sterilized water, shaking the soil sample on a shaking table at a speed of 200r/min for 30min to prepare soil suspension, then respectively sucking 1ml of soil mother liquor by using a sterile pipette, adding the soil mother liquor into 9ml of sterilized water, diluting the soil mother liquor according to a gradient of 1-10 to respectively prepare 10 and 10 ² 、10 ³ 、10 ⁴ And (3) taking 0.1ml of soil bacteria suspension with gradient dilution times, adding each concentration to a prepared bacterial culture medium plate, uniformly coating the plate by using an aseptic coating rod, placing the plate in an incubator at 37 ℃ for overnight culture, and counting the number of bacillus colonies.

Example 4

Radish growth promotion test. Test site: in the eastern region of linyi city, test treatment: the raw liquid of the Rpf protein in the embodiment 2 with the concentration of 100mg/L is used for rushing field radishes, the contrast is clear water treatment, the using amount is 2 liters/mu, and the rushing application is carried out once in the seedling stage of the radishes.

During harvesting, the yields were counted and the bacillus content in the soil was examined, and the results are shown in fig. 1-2 and table 2.

TABLE 2

As can be seen from FIGS. 1-2 and Table 2, compared with the control, the number of bacillus in the soil after the RPF protein is used is increased by 865%, the yield of the radish is increased by 32.71%, the radish shape is regular, and the product quality is improved.

Example 5

Strawberry growth promoting, disease preventing and quality improving tests. Test site: the test treatment comprises that: the Rpf protein stock solution in example 2 with the concentration of 100mg/L is used for rushing field strawberries, and the contrast is clear water treatment, 2 liters/mu, and rushing twice in the seedling stage.

During harvesting, the yields were counted and the bacillus content in the soil was examined, with the results shown in fig. 3-4 and table 3.

TABLE 3

As can be seen from FIGS. 3-4 and Table 3, compared with the control, the number of bacilli in the soil after the application of the Rpf protein is increased by 1140%, the yield of strawberry is increased by 15.68%, the sugar degree is increased by 11%, the Vc content is increased by 6%, the strawberry red center pillar root rot is reduced by 81.7%, the anthracnose is reduced by 77.5%, and the effect of improving the RPF protein on obvious yield and disease prevention is demonstrated.

Example 6

Melon disease prevention test. Test site: hebei Shijiazhuang city. And (3) test treatment: the cantaloupe was subjected to two-time flushing at the seedling stage using a stock solution of RPF protein in example 2 (treatment 1), a solution of RPF protein, amino acid and trace element complex in example 2 (treatment 2), and a solution of amino acid and trace element complex without RPF protein (treatment 3) at a concentration of 100mg/L, respectively, while setting blank controls, and the amounts were 2 liters/acre, as shown in table 4.

TABLE 4

As can be seen from table 4, in harvest, compared with the blank control, the number of the actinomycetes in the soil in the treatment 1 and the treatment 2 is respectively increased by 50% and 58%, the yield of the melons is respectively increased by 18.52% and 25.18%, the root rot of the melons is respectively reduced by 82.3% and 85.7%, the nematode disease is reduced by 76.5% and 84.3%, the yield of the melons can be obviously increased, the root rot and the nematode disease can be reduced, the influence of the treatment 3 on the soil microbial community and the control effects of the root rot and the nematode disease are not obvious, and the yield can be obviously increased and the control effects of the root rot and the nematode disease can be obviously improved by combining the Rpf protein.

Example 7

Eggplant growth promotion test. Test site: dormitory city of Anhui province. And (3) test treatment: eggplant plants were fertilized with 2 liters/mu of the Rpf protein stock solution (treatment 1) in example 2 and the Rpf protein-amino acid-trace element complex solution (treatment 2) in example 2 at a concentration of 100mg/L, respectively, while a blank control treatment was set, and the results are shown in table 5.

TABLE 5

As can be seen from Table 5, the number of the actinomycetes in the soil is increased by 184.6% and 215.8% and the yield of the eggplant is increased by 21.5% and 25.6% respectively when the eggplant is harvested compared with a blank control.

Example 8

Disease prevention test of cucumber. Test site: hebei Zunhua city, test treatment: the field cucumbers were applied by flushing with the Rpf protein stock solution (treatment 1) in example 2 and the Rpf protein, amino acid and trace element complex solution (treatment 2) in example 2 at a concentration of 100mg/L, respectively, while a blank control treatment was set, with an amount of 2 liters/acre, and the results are shown in table 6.

TABLE 6

As can be seen from Table 6, at the time of harvest, the number of released nematode germs in the soil was increased by 500% and 650%, respectively, the cucumber gummy stem blight was decreased by 81.5% and 88.6%, respectively, nematode disease was decreased by 86.5% and 90.2%, and cucumber fusarium wilt was decreased by 78.2% and 86.5%, respectively, as compared to the blank control group.

Example 9

And (5) strawberry disease prevention test. Test site: the test treatment comprises the following steps of: the field strawberries were flushed with 100mg/L RPF protein stock solution (treatment 1) in example 2 and RPF protein, amino acid and trace element complex solution (treatment 2) in example 2, respectively, while a blank control treatment was set, the amount of each of the two solutions was 2L/mu, and the results are shown in table 7.

TABLE 7

As can be seen from Table 7, compared with a blank control group, the number of beneficial bacilli in soil is respectively increased by 48.4% and 180.6%, the yield of strawberry is increased by 25.2% and 36.5%, the sugar degree is respectively increased by 10% and 12%, the Vc content is respectively increased by 8% and 9%, the red pillar root rot of strawberry is respectively reduced by 82.1% and 87.5%, the anthracnose is respectively reduced by 80.3% and 85.2%, and the effect of improving continuous cropping and disease prevention is obvious.

Example 10

Tomato disease prevention test. Test site: test treatment in Yinan county, Shandong Linyi city: the field tomatoes were washed with 100mg/L Rpf protein stock solution (treatment 1) in example 2, Rpf protein, amino acid-trace element complex solution (treatment 2) in example 2, and amino acid-trace element complex solution (treatment 3) without Rpf protein, while blank control treatment was set, and the amount was 2 liters/acre, and washed twice in the seedling stage, and the results are shown in table 8.

TABLE 8

As can be seen from Table 8, the number of Bacillus bacteria in treatment 1 and treatment 2 was increased 708.3% and 825.5%, respectively, and the yield of tomato was increased 14.38% and 21.65%, respectively, when compared with the blank control group at harvest. Tomato bacterial wilt is reduced by 82.2% and 88.5% respectively, gray mold is reduced by 75.6% and 88.2% respectively, epidemic diseases are reduced by 77.1% and 82.5% respectively, the treatment 3 only shows a certain growth promotion effect on yield, no obvious effect is exerted on soil beneficial bacteria and continuous cropping diseases, the control effect of bacterial wilt and epidemic diseases is 0, and the sum of the yield increase effects of the treatment 1 and the treatment 3 and the sum of soil spore quantity are both smaller than the treatment 2, which shows that Rpf protein, amino acid and trace elements are not simply superposed in the actual use process, but synergistic effect is generated, and the tomato yield and the soil spore quantity are further increased compared with the respective use of Rpf protein, amino acid and trace elements.

Example 11

Celery disease prevention test. Test site: in the eastern district of Linyi city, test method: the field celery was applied by flushing with the RPF protein stock solution (treatment 1) in example 2 and the compound solution (treatment 2) of RPF protein, amino acid and trace elements in example 2 at a concentration of 100mg/L, respectively, while setting blank control treatment, the amount was 2 liters/mu, and the results are shown in table 9.

TABLE 9

As can be seen from Table 9, when harvested, compared with the blank control, the celery yield is increased by 18.81% and 22.5%, the celery rhizoctonia rot is reduced by 75.2% and 80.6%, and the sclerotinia rot is reduced by 76.7% and 89.2%, respectively.

Claims

1. The application of the protein of the resuscitation promoting factor in preventing and treating continuous cropping diseases of crops and promoting the growth of the crops, wherein the ID of the protein of the resuscitation promoting factor NCBI is CAB 09664.2.

2. The use of claim 1, wherein the resuscitation-promoting factor protein is prepared by the following method:

(1) inoculating Streptomyces fradiae spore with strain preservation number of CGMCC No.11963 to slant culture medium, culturing at 28 deg.C for 7 days, adding sterile water, and uniformly dispersing the spore in sterile water to obtain product with concentration of 10 ⁸ ～10 ⁹ Spore suspension per mL;

(4) heating the RPF protein fermentation mother liquor in the step (3) to 80 ℃, preserving heat for 20 minutes, then adding 0.1% of filter aid, performing plate-frame filtration, removing precipitate, and obtaining supernatant as stock solution containing RPF protein;

(5) adding ammonium sulfate into the stock solution containing the RPF protein in the step (4), precipitating for 4 hours at the temperature of 0 ℃, taking the precipitate after plate-frame filter pressing, dissolving the precipitate with a solution containing 8mol/L urea, performing gradient dialysis, removing the urea in the protein solution by adopting a method for gradually reducing the concentration of the urea to naturally renature the denatured protein in the process, dialyzing the denatured protein overnight at the temperature of 4 ℃ with a buffer solution containing 6mol/L urea, dialyzing for 4 hours at the temperature of 4 ℃ with buffer solutions containing 4mol/L, 3mol/L, 2mol/L, 1mol/L and 0mol/L urea respectively, purifying the protein solution by using a weak anion exchange column DEAE after dialysis is finished, collecting different gradient eluents to perform SDS-PAGE analysis, and dialyzing for 4 hours at the temperature of 4 ℃ with a PBS buffer solution to obtain the resuscitation promotion factor protein.

3. The method according to claim 2, wherein in step (1), the slant culture medium comprises (m/v) 2% of soybean meal, 0.5% of yeast powder, 1% of corn meal, and 1.5% of agar powder; in the step (2) and the step (3), the liquid fermentation medium (m/v) comprises 3% of corn flour, 2% of glucose, 0.03% of monopotassium phosphate, 0.5% of peptone, 1.5% of soybean cake powder, 0.5% of potassium nitrate, 1% of soybean oil and 0.05% of calcium chloride.

4. The preparation method according to claim 2, wherein in the step (4), the filter aid is one or more of diatomite, bentonite and kaolin; in the step (5), the ratio of the mass of the ammonium sulfate to the volume of the stock solution containing the RPF protein is 1: 2, unit: g/mL.

5. The use of claim 1, wherein the crop continuous cropping diseases comprise root knot nematode disease, root rot disease, bacterial wilt disease, epidemic disease, wilt disease and damping off disease; the crops are radishes, strawberries, melons, eggplants, cucumbers, tomatoes, celery or gingers.

6. A method for preventing and treating continuous cropping diseases of crops and promoting the growth of crops can be realized by using a recovery promoting factor Rpf alone or by mixing a recovery promoting factor RPF protein, amino acid and trace elements and then flushing or spraying.

7. The method of claim 6, wherein the resuscitation-promoting factor protein is administered at a concentration of 50-150 mg/L;

8. The method of claim 6, 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, used at a concentration of 150-200 mg/L;

9. The method of claim 6, wherein the trace element is a combination of ferrous citrate, zinc EDTA, copper EDTA and boric acid used at a concentration of 40 mg/L.

10. The method according to claim 6, wherein the flushing or spraying is in particular: the fertilizer is applied or sprayed for 1-3 times in the seedling stage of crops, and the application amount is 1-3 liters per mu.