CN107418910B - Biological agent for preventing and treating clubroot of cruciferous crops and application thereof - Google Patents
Biological agent for preventing and treating clubroot of cruciferous crops and application thereof Download PDFInfo
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Abstract
The invention discloses a biological agent for preventing and treating clubroot of cruciferous crops and application thereof, belonging to the field of applied microorganisms. The production strain of the biological agent is a bacillus amyloliquefaciens strainBacillus amyloliquefaciensKM68, which is preserved in China center for type culture Collection with the preservation number of CCTCC NO: M2017228. The method combines the soil solar heat treatment with the bacillus amyloliquefaciens strainBacillus amyloliquefaciensThe KM68 bacterial liquid root-irrigation treatment method can effectively prevent and treat clubroot of cruciferous crops, is beneficial to yield increase of the cruciferous crops, and has the advantages of simple operation, low cost and easy factory production of biological agents.
Description
Technical Field
The invention relates to a biological agent for preventing and treating clubroot of cruciferous crops and application thereof, belonging to the field of applied microorganisms.
Background
The clubroot of vegetables in cruciferae crops is caused by plasmodiophora brassicae (S. brassicae) ((S. brassicae))PLasmodiophora brassicaeWoron) a worldwide soil-borne disease caused by exhaust dyeing. Currently, most countries and regions occur. Because the pathogenic bacteria have strong infectivity, high propagation speed and difficult control, in recent years, clubroot of cruciferous crops spreads rapidly in most areas of China, and seriously threatens the production of cruciferous crops.
At present, the method for preventing and treating clubroot of cruciferous crops mainly comprises the following steps: preventing and treating diseases, breeding disease resistance, and biologically preventing and treating diseases. Because brassica plasmodiophora belongs to obligate parasitic bacteria, disease-resistant breeding is the most effective mode for preventing and treating the plasmodiophora brassicae theoretically, however, the brassica plasmodiophora has numerous physiological species and rapid variation, and is difficult to cultivate long-term effective disease-resistant varieties, the chemical agent prevention and treatment is still the main way for preventing and treating the plasmodiophora brassicae at present, but the chemical agent prevention and treatment not only wastes time and labor, but also leads to large accumulation of chemical pesticide residues in soil after long-term use of the chemical pesticide, and the chemical pesticide residues directly harm the health of people once entering agricultural products. In recent years, researchers apply biocontrol bacteria to the control of clubroot of cruciferous crops, and find some bacteria and fungi which have good control effect on the clubroot, such as bacillus amyloliquefaciens, bacillus subtilis, streptomyces, trichoderma, cladosporium endophytic fungi and the like. However, if the biocontrol bacteria are used alone to prevent clubroot, the control effect is not good, the yield of crops cannot be improved, and long-term use of the biocontrol bacteria can lead to change of microbial diversity in soil and have adverse effects on soil microbes.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides the biological agent for preventing and treating the clubroot of cruciferous crops, which can effectively, non-toxic, safe, residue-free and environment-friendly prevent and treat the clubroot of cruciferous crops.
The microbial strain adopted by the invention is bacillus amyloliquefaciensBacillus amyloliquefaciensKM68 strain, which has been deposited in the China center for type culture Collection on 5/2.2017, with the deposition address: the preservation number of the university of Wuhan and Wuhan in China is CCTCC NO: M2017228.
Bacillus amyloliquefaciens of the inventionBacillus amyloliquefaciensThe KM68 strain is obtained by separating soil from Songming county soil of Kunming city, Yunnan province by a dilution plate method and a plate marking method, and is a new strain of bacillus amyloliquefaciens determined by morphology, culture character and a DNA automatic identification systemBacillus amyloliquefaciensKM68。
Another object of the present invention is to use Bacillus amyloliquefaciensBacillus amyloliquefaciensThe KM68 strain is prepared into a biological agent and is applied to preventing and treating clubroot of cruciferous crops.
The application method of the biological agent for preventing and treating clubroot of cruciferous crops comprises the following specific steps:
(1) soil solar heat treatment: deeply ploughing soil, irrigating, sealing, and performing solar heat treatment for more than 30 days;
(2) greenhouse cultivation: collecting the soil sample subjected to solar heat treatment obtained in the step (1), placing the soil sample into a hole disc in a greenhouse, sowing plant seeds of cruciferous crops in the hole disc, covering the soil sample subjected to solar heat treatment, watering, cutting off redundant seedlings when the plant seeds of the cruciferous crops emerge to 3-4 leaves, reserving a healthy seedling in each hole, and performing root irrigation treatment on the seedlings of the cruciferous crops for 1 time by using a biological agent;
the biological agent is bacillus amyloliquefaciensBacillus amyloliquefaciensThe spore amount of the KM68 bacterial liquid is 4 × 105~ 1×107cfu/mL；
The biological preparation for preventing and treating clubroot of cruciferous crops, provided by the invention, has the following advantages:
(1) the invention adopts the soil solar heat treatment method combined with the bacillus amyloliquefaciensBacillus amyloliquefaciensThe KM68 biological agent treatment method can effectively prevent and treat clubroot of cruciferous crops and can obviously improve the yield of the crops;
(2) the application concentration of the bacterial liquid is low in the biological preparation treatment process, the application dosage of the bacterial liquid is small, and the root irrigation is only needed once; the method is simple to operate, low in cost and harmless to the environment, and the deteriorated soil environment can be repaired to a certain extent through solar heat treatment.
Drawings
FIG. 1 shows Bacillus amyloliquefaciens strain of the present inventionBacillus amyloliquefaciensA colony map of KM 68;
FIG. 2 shows Bacillus amyloliquefaciens strain of the present inventionBacillus amyloliquefaciensScanning picture of electron microscope of KM 68;
FIG. 3 shows Bacillus amyloliquefaciens strain of the present inventionBacillus amyloliquefaciensGrowth curve of KM 68.
Detailed Description
The invention is described in further detail below with reference to the figures and examples, but the scope of protection of the invention is not limited to the description.
Bacillus amyloliquefaciens strainBacillus amyloliquefaciensKM68 is obtained by separating and screening from soil in Songming county, Yunnan province by dilution plate method and plate marking method, and is preserved in China center for type culture Collection in 2017, 5, 2 and CCTCC (China center for type culture Collection) with the preservation number of CCTCC NO: M 2017228。
Example 1: bacillus amyloliquefaciens strainBacillus amyloliquefaciensScreening and isolation of KM68
The LB medium formula: 10 g/L of tryptone, 5 g/L of yeast extract, 5 g/L of sodium chloride and 15 g/L of agarose; the pH value is 7.4 plus or minus 0.1; weighing, adding distilled water, stirring for dissolving, subpackaging in triangular flasks, sterilizing at 121 ℃ under high temperature and high pressure, and pouring into a flat plate;
weighing 1 g of soil sample collected in Songming county, Yunnan province, placing the soil sample into a triangular flask with glass beads containing 50 mL of sterile water, shaking and shaking for 30min, sucking 1 mL of supernatant from the triangular flask by using a pipette, adding the supernatant into another test tube containing 9mL of sterile water, mixing, and respectively preparing 10 diluted concentration of the original solution according to the method-3、10-4、10-5And (2) coating 100 uL of diluted soil solution on an LB culture medium flat plate, repeating each dilution for 3 times, performing shake culture for 10 hours at 37 ℃ and 150 r/min, selecting a single colony to scribe on the LB culture medium, observing the growth condition of the colony at regular time, purifying strains by using a flat plate scribing method, respectively manufacturing inclined plane labels, and storing in a refrigerator at the temperature of 4 ℃. Through molecular phylogenetic analysis, named Bacillus amyloliquefaciens strainBacillus amyloliquefaciensKM68, sent to China Center for Type Culture Collection (CCTCC) for preservation.
Example 2: bacillus amyloliquefaciens strainBacillus amyloliquefaciensMorphology of KM68
Preparation of Bacillus amyloliquefaciens strain by gradient dilution methodBacillus amyloliquefaciensRespectively sucking 10uL of bacterial suspensions with different dilutions from KM68 bacterial suspensions, coating the bacterial suspensions on an LB culture medium flat plate for culture, and observing the characteristics of the grown single colony, such as size, shape, surface, edge, color, transparency and the like after 8-15 h;
respectively culturing different dilutions of Bacillus amyloliquefaciens strain on LB culture mediumBacillus amyloliquefaciensKM68, after culturing for 8-15 hours, respectively sucking 10uL bacterial suspension for flaking, and observing bacillus amyloliquefaciens strain under an optical microscopeBacillus amyloliquefaciensGrowth and morphological characteristics of KM 68;
the cultured and observed cells were characterized as follows: the colony is round or oval, has wrinkles, slightly bulges, is opaque and white, is gram-positive, and is in a short rod shape under a microscope (the colony graph is shown in figure 1, and the scanning picture of an electron microscope is shown in figure 2).
Example 3: bacillus amyloliquefaciens strainBacillus amyloliquefaciensIdentification of 16s rDNA Gene sequence of KM68
After a single colony is inoculated in an LB culture medium for 24 hours, 1 uL is taken for colony PCR verification. The primers used for PCR of the bacterial solution were as follows: forward primer 27F: 5，-AGAGTTTGATCMTGGCTCAG-3，(ii) a Reverse primer 1492R: 5，-GGTTACCTTGTTACGACTT-3，. And (3) PCR reaction system: 2.5 uL of 10xTaq Buffer, 0.5 uL of forward primer, 0.5 uL of reverse primer, 1 uL of bacterial liquid, 0.5 uL of dNTP (10 mmol/L), 0.5U of Taq DNA polymerase, water is added to 25uL, PCR amplification conditions are that pre-denaturation is carried out for 3 min at 94 ℃, 35 cycles are carried out according to the programs of 94 ℃, 30 s, 57 ℃, 120 s, 72 ℃ and 90 s, and finally, extension is carried out for 10 min at 72 ℃. The electrophoresis is performed by agarose gel electrophoresis, the nucleic acid dye is EB, the length of DNA Mark is 2000, and finally the PCR purified product is sent to Yunnan Shuoji Biotech limited for sequencing, and the obtained 16S rDNA sequence of strain KM68 is compared with the related sequence in NCBI nucleic acid database. The results showed that strain KM68 and strain KM68 were obtainedBacillus amyloliquefaciensstrain DM-54，Bacillus amyloliquefaciensstrain Lx-11，Bacillus amyloliquefaciensstrain LD5，Bacillus amyloliquefaciensThe similarity of strain EB19 reaches 99 percent (see table 1), and a new strain of strain KM68 belonging to the genus Bacillus is determined, namely a bacillus amyloliquefaciens strainBacillus amyloliquefaciensKM68，
Example 4: bacillus amyloliquefaciens strainBacillus amyloliquefaciensCulture growth of KM68
The LB culture medium formula is: 10 g of peptone, 5 g of sodium chloride, 10 g of yeast extract and 1L of distilled water, wherein the pH value is 7.4;
determination of Bacillus amyloliquefaciens Using LB liquid MediumBacillus amyloliquefaciensKM68 growth curve: weighing 1 g of tryptone, 0.5 g of yeast extract, 0.5 g of sodium chloride and 1.5 g of agarose, and adding water to dissolve and fix the volume to 100 mL; using 11mol/L sodium hydroxide solution to divide 7.0 +/-0.1, then dividing the prepared LB culture medium into 10 test tubes, sterilizing at high temperature and high pressure at 121 ℃, then cooling to room temperature, inoculating 10uL of bacillus amyloliquefaciens into each test tube under the aseptic environmentBacillus amyloliquefaciensCulturing KM68 bacterial solution at 37 deg.C and 150 r/min in shaking bed for 24 hr, measuring OD value of bacterial solution every 2 hr, and taking time (hr) as abscissa and OD value as ordinate to obtain Bacillus amyloliquefaciensBacillus amyloliquefaciensKM68 growth curve (see FIG. 3), the results show that Bacillus amyloliquefaciensBacillus amyloliquefaciensKM68 has the fastest propagation speed in 0-10 hours, and then bacterial colonies tend to be stable;
the liquid fermentation medium comprises the following components in percentage by mass: 3% of soybean meal, 3% of corn flour, 0.5% of sodium chloride, 0.5% of calcium carbonate and the balance of water;
bacillus amyloliquefaciens strainBacillus amyloliquefaciensTransplanting the spores of KM68 into an LB culture medium, culturing for 10 h at 37 ℃ to obtain a seed solution, inoculating the seed solution into a liquid fermentation culture medium according to the volume ratio of the seed solution to water of 1:100, culturing for 10 h at 37 ℃, and after the culture is finished, the concentration of the bacillus amyloliquefaciens liquid in the fermentation liquid can reach (1.6-3.2) × 108cfu/mL；
Liquid fermented preparation (biological preparation for preventing and treating clubroot of cruciferous crops) is prepared by measuring the bacteria content by plate counting, wherein the bacteria content is 1.6-3.2 and × 108cfu/mL。
Example 5: experiment for preventing and treating clubroot of cruciferous crops (pakchoi) and experiment for increasing yield of liquid fermentation preparation (biological preparation for preventing and treating clubroot of cruciferous crops)
Subject: chinese cabbage
Preparing the biological agent for the test according to the culture and growth method of the embodiment 4, measuring the bacterial content by plate counting, wherein the bacterial content of the bacillus amyloliquefaciens reaches (1.6-3.2) × 108cfu/mL；
Diluting the biological agent by 20 times to obtain a preparation A, diluting the biological agent by 100 times to obtain a preparation B, and diluting the biological agent by 500 times to obtain a preparation C;
medicine supply preparation and treatment:
(1) soil solar heat treatment: irrigating deeply ploughed soil for 30min to fully saturate water in the soil, covering a layer of plastic film for sealing, performing solar heat treatment for 30 days, and collecting a solar heat treatment soil sample;
(2) greenhouse cultivation: adopting a 72-hole culture hole tray, placing the soil sample subjected to solar heat treatment in the step (1) in the hole tray in a greenhouse, sowing 3-4 cruciferous crop (pakchoi) plant seeds in each hole of the hole tray, covering the soil sample subjected to solar heat treatment in the step (1), watering, allowing the cruciferous crop plant (pakchoi) seeds to emerge until 3-4 leaves grow, cutting off redundant seedlings, reserving a healthy seedling in each hole, performing root irrigation treatment on the cruciferous crop (pakchoi) seedlings for 1 time by using a biological preparation diluent, wherein the biological preparation diluent added at the root of each plant is 10 mL;
a: the diluent of the biological preparation is preparation A diluted by 20 times of the biological preparation;
b: the diluent of the biological preparation is a preparation B diluted by 100 times of the biological preparation;
c: the diluent of the biological preparation is preparation C with the biological preparation diluted by 500 times;
d: performing blank control, namely performing root irrigation treatment on seedlings of cruciferous crop plants (pakchoi) for 1 time by adopting clear water, wherein the amount of the clear water added into the roots of each plant is 10 mL;
the test method comprises the following steps: 4 treatments are set in the test, each treatment is repeated for 3 times, 12 zones are set in the test, the zones are not affected with each other, and the yield is measured in each zone during harvesting;
the investigation method comprises the following steps: investigating 108 seedlings in each 3-treated area, taking out the seedlings when the seedlings grow for 40 days, and measuring the fresh weight of the overground part of the seedlings;
the clubroot survey takes the size of root tumors as a basis, and the disease grade of clubroot of each plant is identified by referring to the department (2009):
level 0: the root has no tumor, and the root system develops normally;
level 1: the main root is not attacked or not obvious, and the diameter is less than or equal to 2 times of the base of the stem;
and 2, stage: the main root is swollen. The diameter is less than or equal to 2-3 times of the stem base;
and 3, level: the main root is obviously swollen, the diameter is less than or equal to 3-times of the stem base, and fibrous roots are absent;
incidence = infected plants/total number of plants counted
Disease index = ∑ (disease index of disease strain at each stage x disease index of strain)/(total investigated strain tree x highest grade value)
Relative control effect = (control disease index-treatment disease index) × 100/control disease index
The results of the experiment are shown in table 1.
Comparative example 1: soil-only solar heat treatment control method
Subject: chinese cabbage
(1) Soil solar heat treatment: deeply ploughing soil, irrigating water for 30min to fully saturate the water in the soil, covering a layer of plastic film for sealing, performing solar heat treatment for 30 days, and collecting a soil sample subjected to solar heat treatment;
(2) greenhouse cultivation: adopting a 72-hole culture hole tray, placing the solar heat treatment soil sample obtained in the step (1) in the hole tray in a greenhouse, sowing 3-4 cruciferous crop plant (pakchoi) seeds in each hole of the hole tray, covering the solar heat treatment soil sample obtained in the step (1), watering, allowing the cruciferous crop plant (pakchoi) seeds to emerge until 3-4 leaves grow, cutting off redundant seedlings, and keeping a healthy seedling in each hole;
the test method comprises the following steps: field management is similar to conventional production; the test is designed to be 1 treatment, 3 times of repetition, 3 zones in total, and each zone is separated to measure yield during harvesting;
the investigation method was the same as in example 5; the results of the experiment are shown in table 1.
Comparative example 2: control method using only liquid fermentation preparation
Subject: chinese cabbage
Cultivating a hole tray with 72 holes, adding a soil sample into the hole tray in a greenhouse, sowing 3-4 cruciferous crop plant (pakchoi) seeds in each hole of the hole tray, covering the soil sample, watering, allowing the cruciferous crop plant (pakchoi) seeds to emerge until 3-4 leaves grow, cutting off redundant seedlings, and reserving a healthy seedling in each hole; root irrigation treatment is carried out on seedlings of cruciferous crop plants (pakchoi) for 1 time by using a biological preparation diluent, and the biological preparation diluent added into the roots of each plant is 10 mL;
e: the diluent of the biological preparation is preparation A diluted by 20 times of the biological preparation;
f: the diluent of the biological preparation is a preparation B diluted by 100 times of the biological preparation;
g: the diluent of the biological preparation is preparation C with the biological preparation diluted by 500 times;
the test method comprises the following steps: except that the pesticide application is set at different levels, other field management is the same as the conventional production; 3 treatments are set in the test, 9 zones are totally set, and the yield is measured in each zone during harvesting;
the investigation method was the same as in example 5; the experimental results are shown in table 1;
the experimental results are as follows:
TABLE 1 prevention and treatment effect on clubroot of pakchoi
The results show that: the single solar heat treatment of the soil obviously improves the yield (P) of the seedlings of the pakchoi<0.05) but the control effect on clubroot is only 46.9 percent, and the liquid fermentation preparation (biological preparation for controlling clubroot of cruciferous crops) can also inhibit the clubroot and is used alone (for controlling clubroot of cruciferous crops)Biological preparation of disease) does not increase cabbage yield (P)>0.05), and the liquid fermentation preparations (biological preparations for preventing and treating clubroot of cruciferous crops) with different concentrations have obvious difference on the prevention and treatment effects on the clubroot, when the bacillus amyloliquefaciens in the liquid fermentation preparations (biological preparations for preventing and treating the clubroot of cruciferous crops)Bacillus amyloliquefaciensThe bacterial contents of KM68 are respectively 4 × 105cfu/mL、2×106cfu/mL、107cfu/mL, the control effect on clubroot is respectively 35.3%, 64.8% and 77.13%, and the yield (P) of the pakchoi is obviously increased by combining the solar heat treatment soil with biocontrol bacteria<0.05) and in a liquid fermentation formulation (biological formulation for controlling clubroot of cruciferous crops)Bacillus amyloliquefaciensWhen the bacteria content of KM68 is lower, the control effect on clubroot of pakchoi can be well achieved, and when bacillus amyloliquefaciens is usedBacillus amyloliquefaciensThe bacterium content of KM68 reaches 2 × 106When cfu/mL is adopted, the control effect on the clubroot of the pakchoi reaches 81.7 percent, the biocontrol bacterium concentration is continuously improved, and the control effect on the clubroot of the pakchoi is not obviously improved.
Example 6: experiment for preventing and treating clubroot of cruciferous crops (stem mustard) by using liquid fermentation preparation (biological preparation for preventing and treating clubroot of cruciferous crops)
Subject: caulis et folium Brassicae Junceae
Preparing the biological agent for the test according to the culture and growth method of the embodiment 4, measuring the bacterial content by plate counting, wherein the bacterial content of the bacillus amyloliquefaciens reaches (1.6-3.2) × 108cfu/mL；
Diluting the biological agent by 20 times to obtain a preparation A, diluting the biological agent by 100 times to obtain a preparation B, and diluting the biological agent by 500 times to obtain a preparation C;
medicine supply preparation and treatment:
(1) soil solar heat treatment: irrigating the soil of the deep ploughing soil for 30min to fully saturate the water in the soil, covering a layer of plastic film for sealing, then carrying out solar heat treatment for 35 days, and collecting a solar heat treatment soil sample;
(2) greenhouse cultivation: culturing an aperture disk with 72 holes, placing the solar heat treatment soil sample obtained in the step (1) in the aperture disk in a greenhouse, sowing 3-4 cruciferous crop (stem mustard) plant seeds in each aperture of the aperture disk, covering the solar heat treatment soil sample obtained in the step (1), watering, allowing the cruciferous crop plant (stem mustard) seeds to emerge until 3-4 leaves grow, cutting off redundant seedlings, reserving a healthy seedling in each aperture, and performing root irrigation treatment on the cruciferous crop plant (stem mustard) seedlings for 1 time by using a biological preparation diluent, wherein the biological preparation diluent added to the roots of each plant is 10 mL;
a: the diluent of the biological preparation is preparation A diluted by 20 times of the biological preparation;
b: the diluent of the biological preparation is a preparation B diluted by 100 times of the biological preparation;
c: the diluent of the biological preparation is preparation C with the biological preparation diluted by 500 times;
d: blank control, adopting clear water to perform root irrigation treatment on crucifer crop plants (stem mustard) seedlings for 1 time, wherein the amount of the clear water added into the root of each plant is 10 mL;
the test method comprises the following steps: except that the pesticide application is set at different levels, other field management is the same as the conventional production; the test is carried out by setting 4 treatments, repeating for 3 times, and totally 12 areas, wherein each area is separated for yield measurement during harvesting;
the investigation method comprises the following steps: investigating the overground biological yield of the disease condition in the harvest period of the stem mustard;
results of the experiment
TABLE 2 controlling effect on clubroot of stem mustard
Treating soil by solar heat treatment in combination with biocontrol bacterium root-irrigation, and treating with Bacillus amyloliquefaciens in liquid fermentation preparation (biological preparation for preventing and treating clubroot of cruciferous crops)Bacillus amyloliquefaciensThe KM68 can also achieve good prevention and treatment effect on clubroot of stem mustard when the bacteria content is lower, and when the bacillus amyloliquefaciens is usedBacillus amyloliquefaciensThe bacterium content of KM68 reachesTo 2 × 106And when cfu/mL is adopted, the control effect on the clubroot of the stem mustard reaches 76%, the biocontrol bacterium concentration is continuously improved, and the control effect on the clubroot of the stem mustard is not obviously improved.
Example 7: experiment for preventing and treating clubroot of cruciferous crops (common head cabbage) by using liquid fermentation preparation (biological preparation for preventing and treating clubroot of cruciferous crops)
Subject: common head cabbage
Preparing the biological agent for the test according to the culture and growth method of the embodiment 4, measuring the bacterial content by plate counting, wherein the bacterial content of the bacillus amyloliquefaciens reaches (1.6-3.2) × 108cfu/mL；
Diluting the biological agent by 20 times to obtain a preparation A, diluting the biological agent by 100 times to obtain a preparation B, and diluting the biological agent by 500 times to obtain a preparation C;
medicine supply preparation and treatment:
(1) soil solar heat treatment: irrigating the soil of the deep ploughing soil for 30min to fully saturate the water in the soil, covering a layer of plastic film for sealing, then carrying out solar heat treatment for 35 days, and collecting a solar heat treatment soil sample;
(2) greenhouse cultivation: culturing an aperture disk with 72 holes, placing the solar heat treatment soil sample obtained in the step (1) in the aperture disk in a greenhouse, sowing 3-4 cruciferous crop (common head cabbage) plant seeds in each aperture of the aperture disk, covering the solar heat treatment soil sample obtained in the step (1), watering, allowing the cruciferous crop plant (common head cabbage) seeds to emerge until 3-4 leaves grow, cutting off redundant seedlings, reserving a healthy seedling in each aperture, performing root irrigation treatment on the cruciferous crop plant (stem mustard) seedlings for 1 time by using a biological preparation diluent, wherein the biological preparation diluent added at the root of each plant is 10 mL;
a: the diluent of the biological preparation is preparation A diluted by 20 times of the biological preparation;
b: the diluent of the biological preparation is a preparation B diluted by 100 times of the biological preparation;
c: the diluent of the biological preparation is preparation C with the biological preparation diluted by 500 times;
d: blank control, adopting clear water to perform root irrigation treatment on seedlings of cruciferous crop plants (common head cabbages) for 1 time, wherein the amount of the clear water added into the roots of each plant is 10 mL;
the test method comprises the following steps: except that the pesticide application is set at different levels, other field management is the same as the conventional production; the test is carried out by setting 4 treatments, repeating for 3 times, and totally 12 areas, wherein each area is separated for yield measurement during harvesting;
the investigation method comprises the following steps: investigating the overground biological yield of the disease condition in the harvest period of the common head cabbages;
results of the experiment
TABLE 3 control of clubroot of Brassica oleracea
Treating soil by solar heat treatment in combination with biocontrol bacterium root-irrigation, and treating with Bacillus amyloliquefaciens in liquid fermentation preparation (biological preparation for preventing and treating clubroot of cruciferous crops)Bacillus amyloliquefaciensThe KM68 can also achieve good control effect on the clubroot of the common head cabbage when the bacteria content is lower, and when the bacillus amyloliquefaciens is usedBacillus amyloliquefaciensThe bacterium content of KM68 reaches 2 × 106And when cfu/mL is adopted, the control effect on the clubroot of the common head cabbage reaches 75.36%, the biocontrol bacterium concentration is continuously improved, and the control effect on the clubroot of the common head cabbage is not obviously improved.
In conclusion, the heat treatment of the sun and the biocontrol bacterium bacillus amyloliquefaciens have obvious control effect on the clubroot of cruciferae, the yield of plants can be increased, and when the concentration of the biocontrol bacterium reaches 2 × 106The control effect on clubroot can be well achieved when cfu/mL is adopted, so that the sun treatment is combined with the bacillus amyloliquefaciensBacillus amyloliquefaciensThe KM68 has good application prospect on the prevention and treatment and yield increase of the clubroot of cruciferae.
<110> university of Kunming science
<120> biological preparation for preventing and treating clubroot of cruciferous crops and application thereof
<130>1
<160>1
<170>PatentIn version 3.5
<210>1
<211>1427
<212>DNA
<213>Bacillus amyloliquefaciens KM68
<400>1
tctataatgc aagtcgagcg gacagatggg agcttgctcc ctgatgttag cggcggacgg 60
gtgagtaaca cgtgggtaac ctgcctgtaa gactgggata actccgggaa accggggcta 120
ataccggatg gttgtttgaa ccgcatggtt cagacataaa aggtggcttc ggctaccact 180
tacagatgga cccgcggcgc attagctagt tggtgaggta acggctcacc aaggcaacga 240
tgcgtagccg acctgagagg gtgatcggcc acactgggac tgagacacgg cccagactcc 300
tacgggaggc agcagtaggg aatcttccgc aatggacgaa agtctgacgg agcaacgccg 360
cgtgagtgat gaaggttttc ggatcgtaaa gctctgttgt tagggaagaa caagtgccgt 420
tcaaataggg cggcaccttg acggtaccta accagaaagc cacggctaac tacgtgccag 480
cagccgcggt aatacgtagg tggcaagcgt tgtccggaat tattgggcgt aaagggctcg 540
caggcggttt cttaagtctg atgtgaaagc ccccggctca accggggagg gtcattggaa 600
actggggaac ttgagtgcag aagaggagag tggaattcca cgtgtagcgg tgaaatgcgt 660
agagatgtgg aggaacacca gtggcgaagg cgactctctg gtctgtaact gacgctgagg 720
agcgaaagcg tggggagcga acaggattag ataccctggt agtccacgcc gtaaacgatg 780
agtgctaagt gttagggggt ttccgcccct tagtgctgca gctaacgcat taagcactcc 840
gcctggggag tacggtcgca agactgaaac tcaaaggaat tgacgggggc ccgcacaagc 900
ggtggagcat gtggtttaat tcgaagcaac gcgaagaacc ttaccaggtc ttgacatcct 960
ctgacaatcc tagagatagg acgtcccctt cgggggcaga gtgacaggtg gtgcatggtt 1020
gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca acccttgatc 1080
ttagttgcca gcattcagtt gggcactcta aggtgactgc cggtgacaaa ccggaggaag 1140
gtggggatga cgtcaaatca tcatgcccct tatgacctgg gctacacacg tgctacaatg 1200
gacagaacaa agggcagcga aaccgcgagg ttaagccaat cccacaaatc tgttctcagt 1260
tcggatcgca gtctgcaact cgactgcgtg aagctggaat cgctagtaat cgcggatcag 1320
catgccgcgg tgaatacgtt cccgggcctt gtacacaccg cccgtcacac cacgagagtt 1380
tgtaacaccc gaagtcggtg aggtaacctt ttaggagcca gccgcca 1427
Claims (1)
1. An application method of a biological agent for preventing and treating clubroot of cruciferous crops is characterized in that: the production strain of the preparation is Bacillus amyloliquefaciens strain Bacillus amyloliquefaciens KM68, the preservation number of the strain in China center for type culture collection is CCTCC NO: M2017228, and the preparation is the bacterial liquid of the Bacillus amyloliquefaciens strain KM 68; the biological agent is a bacterial liquid of bacillus amyloliquefaciens KM68 strain;
the method comprises the following specific steps:
(1) soil solar heat treatment: deeply ploughing soil, irrigating, sealing, and performing solar heat treatment for more than 30 days;
(2) greenhouse cultivation, namely collecting the soil sample subjected to solar heat treatment obtained in the step (1), placing the soil sample into a hole disc in a greenhouse, sowing plant seeds of cruciferous crops in the hole disc, covering the soil sample subjected to solar heat treatment, watering, cutting off redundant seedlings when the plant seeds of the cruciferous crops emerge to 3-4 leaves, reserving a healthy seedling in each hole, and performing root irrigation treatment on the seedlings of the cruciferous crops for 1 time by using a biological agent, wherein the spore amount of a bacterial liquid in the biological agent is 4 × 105～1×107cfu/mL。
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