CN112342173A - Bacillus belgii and application thereof - Google Patents
Bacillus belgii and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of microorganisms, and particularly discloses bacillus beleisi and application thereof. The Bacillus belgii G1S3504 has a preservation number of CGMCC No.20629, and is separated from ginseng root. Indoor plate experiments show that the strain shellfish has strong antagonistic action on 13 fungi such as ginseng root rot, ginseng rust rot, ginseng black spot, gentian root rot, citrus fruit soft rot and the like, the inhibition rate is higher than 64.37%, and the strain shellfish has a phosphorus-dissolving characteristic and has great development prospects. The field biocontrol experiment shows that the G1S3504 strain can effectively prevent and treat ginseng root rot, and has an obvious growth promoting effect on ginseng.
Description
Technical Field
The invention belongs to the field of microorganisms, and particularly relates to bacillus beleisi and application thereof.
Background
Ginseng (Panax ginseng C.A.Mey) is a perennial root herbaceous plant of Panax of Araliaceae, has high medicinal value and economic value, and is a traditional and rare Chinese medicinal material in China. The ginseng cultivation history in China is long, however, the ginseng cultivation technology is not perfect enough, so that the problem of ginseng fungal diseases is frequent, the ginseng yield is obviously reduced, the quality is reduced, the diseases are aggravated, and even the ginseng is extremely harvested.
At present, the prevention and treatment of ginseng diseases are mainly chemical prevention and treatment, although the effect is quick and good, pesticide residue, environmental pollution, ecological balance damage and germ drug resistance are easily caused, especially, the pesticide residue problem has great influence on the quality of ginseng products in China, the world first of the ginseng yield in China is caused, but the output value is far from the embarrassing situation in Korea. Therefore, it is necessary to reduce the use of chemical pesticides for ginseng, and to search and develop biopesticides capable of replacing chemical pesticides, which is an important measure for realizing zero increase in the use of chemical fertilizers of pesticides by 2020 in response to the proposal of Ministry of agriculture.
The fungal diseases of ginseng mainly include root rot, rust rot, black spot and the like. Among them, the root rot is particularly harmful to ginseng. Ginseng root rot is caused by Fusarium sp, which is common in ginseng gardens and can cause root rot, seriously affecting yield. At present, chemical agents such as mancozeb, diniconazole, triadimefon and the like are mainly adopted in China for prevention and treatment, and except for biological prevention and treatment by adopting agents used by large crops, no commercial biocontrol agent special for ginseng exists. The pesticide residue can exceed the standard after long-term application of chemical pesticides, pathogenic bacteria can easily generate drug resistance, and the prevention and treatment effect is reduced. In addition, the long-term, repeated and large-scale use of chemical pesticides can cause pollution to soil, water and atmosphere, and simultaneously kill other beneficial microorganisms, thereby destroying ecological balance. Therefore, it is very urgent to screen high-efficiency strains for fungal diseases of ginseng by biological control and simultaneously screen strains with disease prevention and growth promotion effects of biocontrol bacteria on ginseng.
Disclosure of Invention
In view of the above, the present invention aims to provide bacillus belgii and an application thereof, aiming at the problems existing in the prior art, the bacillus belgii provided by the present invention can prevent and treat ginseng root rot, ginseng rust rot and ginseng black spot, the inhibition effect on ginseng root rot is 67.97%, the inhibition effect on ginseng black spot is 68.41%, the relative prevention effect on ginseng rust rot is 64.37%, and the bacillus belgii has an obvious growth promotion effect on ginseng and a phosphorus dissolving characteristic.
The invention adopts the following technical scheme for achieving the aim of the invention.
The invention provides a strain, namely Bacillus velezensis, the preservation number of which is CGMCC No. 20629.
The Bacillus belgii is separated from the inside of the root of ginseng in the left test base of the institute of specialty of Chinese academy of agricultural sciences, Changchun, Jilin province and named as G1S3504, and the separation method comprises the following steps:
uniformly placing the treated tissue blocks of the ginseng root on a culture medium containing NA according to 7 blocks per dish, repeating 30 dishes per tissue, and placing in a constant-temperature incubator at 28 ℃ for culture. Observing newly grown bacterial colonies around the ginseng tissue on 2 nd, 5 th and 7 th days respectively, selecting a single bacterial colony for separation and purification, observing the forms of the purified bacterial strains for numbering, transferring the bacterial strains to a PDA (PDA) slant culture medium, culturing in a constant temperature incubator at 28 ℃, and screening bacterial strains which have obvious inhibiting effects on ginseng root rot, ginseng rust rot and ginseng black spot pathogenic bacteria through a plate confrontation experiment, wherein the bacterial strains are named as G1S 3504.
Morphological characteristics of the G1S3504 strain: gram staining was positive, sporulated, single or double cross-grown, and the size was 0.8- (1.2) -1.3X 2.3- (3.0) -4.2. mu.m (see FIG. 1). On the NA plate, colonies of the strain were irregularly round, yellowish, wrinkled, opaque, and often ragged (see fig. 2).
According to the analysis result of the G1S3504 combined with the 16S rDNA sequence (the 16S rDNA sequence is shown in SEQ ID NO: 1), the G1S3504 strain was identified as Bacillus belgii, and the strain was classified as Bacillus sp when it was deposited.
The invention also provides fermentation liquor which is obtained by fermenting the bacillus beilesensis G1S 3504.
The invention also provides a preparation method of the fermentation liquor, which comprises the steps of inoculating the Bacillus beiLeisi G1S3504 in a PDA slant culture medium, and culturing for 48h at 28 ℃; picking colonies on the inclined plane, and placing the colonies into a PDB culture medium to perform shake cultivation for 48 hours at 30 ℃ to obtain a seed suspension; inoculating the seed suspension into a fermentation culture medium at 25-32 ℃ and 0.5kg/cm2And fermenting for 36h under the condition of 200 r/min.
In some embodiments, the fermentation medium consists of, in mass percent: 2.5% of yeast extract powder, 1% of glucose, 1.5% of dipotassium hydrogen phosphate, 2.5% of ammonium sulfate and the balance of water.
The invention verifies that the strain is a broad-spectrum biocontrol bacterium through a confrontation culture method, has strong inhibition effect on ginseng root rot, ginseng rust rot and ginseng black spot, and also has biocontrol effect on gentian root rot, citrus fruit soft rot, cinnamomum camphora leaf spot, zinnia purple spot, peach tree branch blight, curculigo latifolia leaf spot, orange gray mold, mountain white tree leaf spot, carrot black spot and rice blast. The results of field biocontrol tests show that the biocontrol microbial inoculum prepared from the strain has strong biocontrol capability and simultaneously improves the saponin content.
The invention also provides application of the Bacillus belgii G1S3504 or fermentation liquor thereof in preventing and treating plant diseases.
In some embodiments, the plant disease is ginseng root rot, ginseng rust rot, ginseng black spot, gentian root rot, citrus fruit soft rot, cinnamomum camphora leaf spot, zinnia pertussis purpura, peach branch blight, curculigo major leaf spot, orange gray mold, mountain tree leaf spot, carrot black spot, or rice blast.
The field biocontrol experiment result shows that the Bacillus beijerinckii G1S3504 has obvious growth promoting effect on ginseng, so the invention also provides the application of the Bacillus beijerinckii or the fermentation liquor thereof in preparation of promoting plant growth.
The phosphorus-dissolving property of the microorganism means that the insoluble phosphate in the soil is converted into water-soluble phosphorus which can be absorbed and utilized by plants. The fertilizer can improve the utilization rate of the fertilizer, can prevent soil hardening caused by phosphate fertilizer, avoids soil acidification and harmful substance accumulation, and can be used as a soil conditioner. Experiments prove that the Bacillus belgii G1S3504 has good phosphorus dissolving property.
Based on the results, the invention also provides the function of the bacillus beijerinckii or the fermentation liquor thereof in dissolving phosphorus and the application of the bacillus beijerinckii or the fermentation liquor thereof in preparing soil conditioners. The soil conditioner has the regulation function of regulating the soil environment and avoiding the problems of soil hardening, harmful substance accumulation, acidification and the like caused by phosphate fertilizers.
In some embodiments, in the above application, the effective concentration of bacillus beleisi G1S3504 is 1 × 105-1×108cfu/mL, preferably 1X 105-1×106cfu/mL, the application amount is 0.5-0.8L/m2. In some embodiments, the effective concentration of Bacillus belgii G1S3504 is 1 × 105cfu/mL. In some embodiments, the effective concentration of Bacillus belgii G1S3504 is 1 × 106cfu/mL, applied at 0.6L/m2。
The invention also provides a biocontrol microbial inoculum which comprises the Bacillus belgii G1S3504 or fermentation liquor thereof and acceptable auxiliary materials.
The skilled person can select suitable auxiliary materials acceptable in the art to prepare the bacillus beijerinckii G1S3504 or the fermentation liquor thereof into aqueous agent, wettable powder or coating agent. Can also be made into other various dosage forms by selecting appropriate adjuvants according to actual needs.
The invention also provides a method for preventing and treating plant diseases, which applies the bacillus, or the fermentation liquor or the biocontrol microbial inoculum thereof, and the concentration of the applied viable bacteria is 1 multiplied by 106cfu/mL, applied at 0.7L/m2。
The Bacillus belgii has a preservation number of CGMCC No.20629, and is separated from ginseng roots. Indoor plate experiments show that the strain can prevent and treat ginseng root rot, rust rot and black spot, has the inhibition effect on the ginseng root rot of 67.18-70.49%, the inhibition effect on the ginseng black spot of 71.14-73.02% and the relative prevention effect on the ginseng rust rot of 64.37%, has obvious growth promotion effect on ginseng, has a phosphorus dissolving characteristic and has great development prospects. In addition, the biological control agent also has biological control effects on gentian root rot, citrus fruit soft rot, cinnamomum camphora leaf spot, zinnia, peach branch blight, curculigo major leaf spot, orange gray mold, leaf spot of mountain white tree, carrot black spot and rice blast.
Biological preservation Instructions
Biological material G1S3504, taxonomic nomenclature: the Bacillus sp is preserved in China general microbiological culture Collection center on 9-11 month 2020 with the addresses as follows: the collection number of the microbial research institute of Chinese academy of sciences is CGMCC No. 20629.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 shows a colony morphology of Bacillus belgii G1S 3504;
FIG. 2 shows a morphology of Bacillus belgii G1S3504 under transmission electron microscopy;
FIG. 3 is a graph showing the phosphorus solubilizing effect of Bacillus belgii G1S 3504;
FIG. 4 shows a Neighbor-Joining phylogenetic tree constructed based on the 16s rDNA gene;
FIGS. 5 to 6 show the bacteriostatic activity of the G1S3504 strain on pathogenic bacteria, wherein the pathogenic bacteria represented by each sequence number: 1: ginseng root rot; 2: black spot of ginseng; 3: rust rot of ginseng; 4: gentian root rot; 5: citrus fruit soft rot; 6: rice blast; 7: leaf spot of cinnamomum camphora; 8: leaf spot of mountain white tree; 9: leaf spot of Curculigo latifolia; 10: orange gray mold; 11: black spot of carrot; 12: (ii) zinnia purpurea; 13: peach branch blight.
Detailed Description
The invention discloses Bacillus belgii and application thereof. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and products of the present invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications of the methods described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of the present invention without departing from the spirit and scope of the invention.
In order to further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The reagents involved in the examples of the present invention are all commercially available products, and all of them can be purchased from commercial sources.
The invention is further illustrated by the following examples.
Example 1 isolation and identification of Bacillus belgii G1S3504
The Bacillus beleisi G1S3504 is separated from the inside of the root of ginseng in the left experimental base of the institute of specialty of Chinese academy of agricultural sciences, Changchun, Jilin, and the separation method comprises the following steps:
uniformly placing the treated tissue blocks of the ginseng root on a culture medium containing NA according to 7 blocks per dish, repeating 30 dishes per tissue, and placing in a constant-temperature incubator at 28 ℃ for culture. Observing newly grown bacterial colonies around the ginseng tissue on 2 nd, 5 th and 7 th days respectively, selecting a single bacterial colony for separation and purification, observing the forms of the purified bacterial strains for numbering, transferring the bacterial strains to a PDA (PDA) slant culture medium, culturing in a constant temperature incubator at 28 ℃, and screening out a bacterial strain G1S3504 which has antagonistic action on root rot, rust and black spot of ginseng through a plate confrontation experiment.
Morphological characteristics of the G1S3504 strain: gram staining reaction positive, spore rod shape, single or two cross, size of 0.8- (1.2) -1.3X 2.3- (3.0) -4.2 μm. On the NA plate, colonies of the strain were irregularly round, yellowish, wrinkled, opaque, and often ragged (see fig. 1). The morphology of the strain was observed under a lens and the results are shown in FIG. 2. A clear phosphate solubilizing loop was observed when cultured on phosphate-solubilizing medium (see FIG. 3).
Sequence 16S rRNA analysis studies were performed on strain G1S 3504. After the representative strain is cultured in the NA medium for 24h at the constant temperature of 28 ℃ in the dark by adopting a streaking culture method, a single colony is picked by a pipette gun to a 1.5mL centrifuge tube, 40 muL ddH20 is added, and the mixture is subjected to vortex oscillation to be used as a template for PCR amplification. And (3) PCR amplification: the 16S rRNA gene was amplified using the bacterial universal primer pair 27F (5'-AGAGTTTGATCCTGGCTCA-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). The reaction system is 25 μ L: mu.L of genomic DNA, 1.25. mu.L of forward and reverse primers, 12.5. mu.L of 2 XTaq PCR StarMix (Genstar, Beijing), 8. mu.L of ddH2And O. Amplification parameters: pre-denaturation at 95 ℃ for 3min, denaturation at 94 ℃ for 30s, annealing at 52 ℃ for 1min, extension at 72 ℃ for 1min, 35 cycles, and extension at 72 ℃ for 10 min. The PCR product was detected on 1% agar gel containing nucleic acid dye and then purified and sequenced by Shanghai Producer corporation.
The sequence analysis method is the same as the fungal sequence analysis method. The corrected sequences were analyzed by homology comparison in the EZTaxon (http:// EZtaxon-e. ezbiocloud. net /) database. And the strain with high homology is downloaded into Phydit software for comparative analysis. Finally, phylogenetic trees were constructed (see FIG. 4) and their species were identified.
According to the morphological characteristics of the G1S3504 strain and the results of 16S rRNA sequence analysis, the G1S3504 strain is identified to be Bacillus belgii.
Example 2 preparation of a Bacillus beilesiensis fermentation broth
(1) Strain culture
Test tube culture (in weight percent below)
Putting PDA culture medium (glucose 20G, potato extract powder 4G, agar powder 15G) into a test tube, adding distilled water to 1000mL, sterilizing, making into plate, inoculating Bacillus beijerinckii G1S3504 strain, and culturing at 28 deg.C for 48 h.
(2) Fermentation of
The specific fermentation production process is as follows:
a. preparation of seed suspension: selecting colony on the inclined plane, placing into PDB culture medium (glucose 20g, potato extract powder 4g, placing into test tube, adding distilled water to 1000mL, sterilizing), and shake culturing at 30 deg.C for 48 hr to obtain seed suspension;
b. inoculation: inoculating the seed suspension to a fermentation tank through an inoculation port by using pressure difference (the culture medium of the fermentation tank comprises yeast extract powder 2.5%, glucose 1%, dipotassium hydrogen phosphate 1.5%, and ammonium sulfate 2.5%, and distilled water is used for supplementing to 1000mL, and the pH is 8.0 before sterilization, the temperature of the fermentation tank is 25-32 deg.C, and the pressure of the fermentation tank is 0.5kg/cm2The stirring speed in the tank is 200r/min), and attention is paid to avoid pollution; the pot pressure can not exceed 1.5kg/cm during pressure increase2；
c. Liquid fermentation:
after inoculation, the fermentation tank is cultured for 36 hours until the number of bacteria is not increased any more, the fermentation tank is immediately placed, and the bacteria content is determined by plate counting and reaches 180 hundred million cfu/mL.
Example 3 preparation of biocontrol microbial inoculum-wettable powder
(1) Preparation of fermentation broth the same as in example 2 to obtain fermentation broth;
(2) adding a filling agent: pressing the fermentation liquid into a storage tank, adding diatomite (light calcium carbonate) as filler according to the following calculation formula, and stirring for 30 min;
the addition amount (kg) of the filler is (fermentation broth number (hundred million/ml) X tank volume (liter) X yield)/finished product number of bacteria-residue (kg) in the fermentation broth. Then making the above-mentioned materials undergo the processes of press-filtering by using plate frame, pulping, drying and pulverizing so as to obtain the invented wettable powder for field application.
(3) Plate-frame filtration: using 2kg/cm2The pressure of the filter cake is adjusted at any time, the bacteria content in the filtrate is not more than 0.2 hundred million/ml, and a filter cake and the filtrate are obtained;
(4) pulping: discharging the filter cake into a pulping tank, adding concentrated milk No. 100 according to 8% of the calcium carbonate, or adding SDS according to 3%, adding adjuvant (CPL) and appropriate amount of filtrate, and stirring for 30 min;
(5) and (3) drying: placing the slurry in a drying room below 60 ℃ for ventilation drying until the water content is 5% -8%, and obtaining a semi-finished product;
(6) crushing: crushing the semi-finished product, wherein the discharging temperature is less than or equal to 60 ℃ during crushing;
(7) and (3) measuring the quality index of the finished product: the determination of the bacteria content, the water content, the suspension percentage and the fineness is carried out according to the national enterprise standard (Q/KWL02-2003), the bacteria content is 18 hundred million/g, and other indexes all meet the standard.
Example 4 testing of bacteriostatic action of Bacillus beiesei in a plate confrontation test
Activating and culturing the G1S3504 strain and thirteen pathogenic fungi, respectively taking fungus cakes with the diameter of 6mm from the edges of fungus colonies by using a puncher, placing the pathogenic fungus cakes in the center of a PDA culture medium with the diameter of 90mm, respectively transferring G1S3504 single colonies with different dispersed beads at the position with the distance of 15mm, taking the rest blank as a control, and repeating the treatment for 3 times. And then placing the culture dish in a constant-temperature dark incubator at 25 ℃ for culture, growing hyphae with a control, measuring the bacteriostatic distance (the distance between the colony edge of the pathogenic bacteria and the colony edge of the endophytes), and determining the bacteriostatic activity of each endophyte.
The inhibition rate of the G1S3504 strain on the pathogenic bacteria was measured and calculated by taking the pathogenic bacteria inoculated only as a control and the average diameter of the colonies as the observation result on the 7 th day after inoculation.
The formula for calculating the bacteriostasis rate is as follows:
the bacteriostatic ratio (%) - (control pathogen colony diameter-treated pathogen colony diameter)/control pathogen colony diameter × 100%
The experimental results are shown in FIGS. 5 to 6 and Table 1. In fig. 5 to 6, the pathogenic bacteria represented by the respective numbers are: 1: ginseng root rot; 2: black spot of ginseng; 3: rust rot of ginseng; 4: gentian root rot; 5: citrus fruit soft rot; 6: rice blast; 7: leaf spot of cinnamomum camphora; 8: leaf spot of mountain white tree; 9: leaf spot of Curculigo latifolia; 10: orange gray mold; 11: black spot of carrot; 12: (ii) zinnia purpurea; 13: peach branch blight.
3 test results and analysis
TABLE 1 bacteriostatic Activity of G1S3504 Strain against pathogenic bacteria
As shown in the results of Table 1 and FIGS. 5-6, Bacillus belgii G1S3504 has strong antagonistic action on 13 fungi, has an inhibition rate higher than 64.37%, and has broad-spectrum antibacterial activity. Wherein, the inhibition effect on ginseng root rot is strongest, and the inhibition rate reaches 68.65%; the inhibition effect on the black spot of the ginseng is the next time, and the inhibition rate is 68.52%; the inhibition effect of the ginseng rust rot is weaker than the antagonism effect of other pathogenic bacteria, and the inhibition rate is 64.37%.
Example 5 potted plant test for detecting the prevention and treatment effects of the G1S3504 strain on ginseng root rot
The greenhouse conditions are controlled to be 28-30 ℃, the humidity is 80-90%, natural illumination is carried out, biennial ginseng seedlings with consistent growth vigor are selected, the nutrition bowls are filled with 1 kg/bowl, and the ginseng seedlings are planted in the nutrition bowls. The experiment was set to 7 treatments:
treatment 1: cutting root of Ginseng radix by soaking in clean water for 30min, and planting in flowerpot filled with 1kg of soil. Pouring 500mL of clear water to fix roots, and pouring the clear water every 7 days.
And (3) treatment 2: cutting root of Ginseng radix by soaking in a solution containing spore concentration of 1 × 105cfu/mL) for 30min, and planted in a flowerpot filled with 1kg of soil. Pouring 500mL of clear water to fix roots, and pouring the clear water every 7 days. Each 30 strains were treated.
And (3) treatment: cutting root of Ginseng radix by soaking in a solution containing spore concentration of 1 × 105cfu/mL) for 30min, and planted in a flowerpot filled with 1kg of soil. After diluting the initial G1S3504 microbial inoculum by 50 times with clear water, 500mL of diluent is poured to fix roots, and 500mL of fermentation liquor of example 2 diluted by 10 times with clear water is poured once every 7 days, wherein 30 strains are treated.
And (4) treatment: cutting root of Ginseng radix by soaking in G1S3504 bacterial solution (spore concentration adjusted to 1 × 10)5cfu/mL) for 30min, seeded with 1k of seedg, adding the biocontrol microbial inoculum into the soil flowerpot. The plants were post-watered with 450mL of G1S3504 initial inoculum, rooted in clear water, and watered every 7 days with 500mL of G1S3504 initial inoculum diluted 50 times in clear water. Each 30 strains were treated.
And (4) treatment 5: cutting root of Ginseng radix by soaking in a solution containing spore concentration of 1 × 105cfu/mL) for 30min, and planting the seeds in a soil flowerpot filled with 1kg of the biocontrol microbial inoculum. Plant post-watering 450mL, rooting with clear water, and watering every 7 days with 500mL of clear water. Each 30 strains were treated.
And (6) treatment: cutting root of Ginseng radix by soaking in a solution of root rot fungi (spore concentration is adjusted to 1 × 10)5cfu/mL) for 30min, and planting the seeds in a flowerpot filled with 1kg of soil added with the biocontrol microbial inoculum. The soil treatment method comprises the following steps: diluting 10mL of initial microbial inoculum by 5 times, uniformly mixing with 100g of wormcast, and then mixing into soil. The plants were post-watered with 450mL of G1S3504 initial inoculum, rooted in clear water, and watered every 7 days with 500mL of G1S3504 initial inoculum diluted 50 times in clear water. Each 30 strains were treated.
And (7) treatment: cutting root of Ginseng radix by soaking in a solution of root rot fungi (spore concentration is adjusted to 1 × 10)5cfu/mL) for 30min, and planting the seeds in a soil flowerpot filled with 1kg of the biocontrol microbial inoculum. Wherein the soil treatment method comprises the following steps: taking 50mL of clear water, uniformly mixing with 100g of wormcast, and then mixing into soil. Plant post-watering 450mL, rooting with clear water, and watering every 7 days with 500mL of clear water. Each 30 strains were treated. The pathogenic bacteria treatment method comprises the following steps:
activating and culturing root rot fungus on PDA plate for 5 days, washing with sterile water to remove pathogenic spore, making spore suspension, examining under microscope, and diluting the spore suspension to 1 × 105cfu/mL concentration. During the test period, other management measures are consistent. Sampling and recording the rotten root degree and normal human parameters of each processed ginseng plant and calculating the disease index grade at the 90 th day of ginseng transplantation.
The disease index and the disease prevention effect are calculated according to the following formula:
disease index ∑ (grade of disease x number of plants at that grade)/test plant
Grading the disease condition: the 0-grade ginseng root is completely disease-free; grade 1 indicates that the ginseng root has small disease spots or slight burning beard; grade 2, the scab accounts for 1/5 or part of the burn and beard; grade 3 is 1/3 each for the diseased spots of ginseng root and begins to rot or most of the fibrous febrile disease; grade 4 is rotten root 1/2 or only partial main lateral root; grade 5 is rotten roots greater than 1/2 or leaving only a portion of the main root.
The results are shown in Table 2.
TABLE 2
| Treatment of | Index of disease condition |
| CK | 0 |
| G1S3504 | 0 |
| Root rot of pathogenic bacteria | 4.9 |
| G1S3504+ root rot pathogen | 1.7 |
Example 6 field biocontrol test
Test site: the control effect test of the field of the experiment base of the left special institute of agricultural science institute of the left city of Jilin province. Ginseng is selected to be planted in a field with serious disease throughout the year, a test for preventing and controlling ginseng root rot diseases by biocontrol bacteria is carried out, and water and fertilizer management is unified. The cells are 2 rows of regions in one bed, the area is 20 square meters, and 3 repeats are arranged at random for each treatment.
A G1S3504 treatment fluid was prepared as in example 2 for the following treatments:
setting 3 treatments of raw G1S3504 treatment liquid, carbendazim pesticide and clear water, diluting by 100 times with the G1S3504 treatment liquid, and then carrying out root irrigation treatment on the ginseng, wherein the G1S3504 treatment liquid is applied every 20 square meters, and is applied once every 7 days for 3 times continuously.
Meanwhile, stock solution and G1S3504 treatment solution diluted by 10 times and 100 times are set for multiple treatments of the ginseng watering seedlings and clear water in contrast, and the treatment solution is applied once every 7 days and is continuously applied for 5 times.
The survey criteria are as follows:
the disease grade standard of ginseng root rot is as follows:
grading the disease condition: the 0-grade ginseng root is completely disease-free; grade 1 indicates that the ginseng root has small disease spots or slight burning beard; grade 2, the scab accounts for 1/5 or part of the burn and beard; grade 3 is 1/3 each for the diseased spots of ginseng root and begins to rot or most of the fibrous febrile disease; grade 4 is rotten root 1/2 or only partial main lateral root; grade 5 is rotten roots greater than 1/2 or leaving only a portion of the main root.
Disease index ═ Σ (number of diseased plants at each stage × relative stage value) × 100/(number of total investigated plants × highest stage value)
Relative control effect (increase rate of disease finger in control area-increase rate of disease finger in treatment area)/increase rate of disease finger in control area x 100%
The test results are shown in tables 3-4.
TABLE 3 prevention and treatment results of root rot of ginseng in the left test base of the institute of specialty of Chinese academy of agricultural sciences, Jilin province
| Numbering | Treatment method | Index of disease condition | The control effect is% |
| 1 | CK | 73.6±4.5a | -- |
| 2 | G1S3504 treating liquid | 23.5±1.2c | 54.93±2.3a |
| 3 | Carbendazim | 43.4±0.9b | 36.34±1.7b |
Note: the same column of lower case letters indicates significant difference (P < 0.05).
TABLE 4 growth promoting effect of biocontrol bacteria G1S3504 on Ginseng radix
Note: the same column of lower case letters indicates significant difference (P < 0.05).
The result shows that the G1S3504 strain can effectively prevent and treat ginseng root rot, and has a remarkable promoting effect on ginseng growth.
Example 7
The test takes 3-year-old ginseng seedlings as materials, and 6 ginseng seedlings are planted in each pot. Control (CK) 30mL of sterilized PDB medium was inoculated; treatment group, 30mL of G1S3504 strain (1X 10 concentration)5cfu/mL), wherein Bacillus megaterium BM (Bacillus megaterium) and Bacillus subtilis are inoculated and purchased from China center for Industrial culture Collection of microorganisms.Filling 1.5kg of soil in each pot, repeating for 3 times, reserving ginseng seedlings with approximately same growth vigor after the ginseng is completely spread and thinned, inoculating bacterial liquid near the roots of the ginseng plants in a drip irrigation mode, and watering every 7 days during the growth period of the ginseng plants. Sampling and measuring various biomass indexes and ginsenoside content when the ginseng plants grow to the red fruit stage (90 d). The data are analyzed by SAS9.2 statistical software and Excel2010, and the results are shown in tables 5-6.
TABLE 5 Biomass indices of different treated ginseng plants
Note: lower case letters in the same column indicate P < 0.05.
TABLE 6 Effect of different treatments on ginsenoside content (%) of Ginseng radix
Note: lower case letters in the same column indicate P < 0.05.
The result shows that compared with other bacilli, the Bacillus belgii G1S3504 provided by the invention can promote the growth of ginseng and improve the content of ginsenoside (p < 0.01).
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
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<120> Bacillus belgii and application thereof
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cgcggcgcat tagctagttg gtgaggtaac ggctcaccaa ggcaacgatg cgtagccgac 240
ctgagagggt gatcggccac actgggactg aaacacggcc caaactccta cgggaggcag 300
cagtagggaa tcttccgcaa tggacgaaag tctgacggag caacgccgcg tgagtgatga 360
aggttttcgg atcgtaaagc tctgttgtta gggaagaaca agtgccgttc aaatagggcg 420
gcaccttgac ggtacctaac cagaaagcca cggctaacta cgtgccagca gccgcggtaa 480
tacgtaggtg gcaagcgttg tccggaatta ttgggcgtaa agggctcgca ggcggtttct 540
taagtctgat gtgaaagccc ccggctcaac cggggagggt cattggaaac tggggaactt 600
gagtgcagaa gaggagagtg gaattccacg tgtagcggtg aaatgcgtag agatgtggag 660
gaacaccagt ggcgaaggcg actctctggt ctgtaactga cgctgaggag cgaaagcgtg 720
gggagcgaac aggattagat accctggtag tccacgccgt aaacgatgag tgctaagtgt 780
tagggggttt ccgcccctta gtgctgcagc taacgcatta agcactccgc ctggggagta 840
cggtcgcaag actgaaactc aaaggaattg acgggggccc gcacaagcgg tggagcatgt 900
ggtttaattc gaagcaacgc gaagaacctt accaggtctt gacatcctct gacaatccta 960
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Claims (10)
1. Bacillus velezensis (Bacillus velezensis) with the preservation number of CGMCC No. 20629.
2. A fermentation broth obtained by fermentation of the Bacillus belgii strain of claim 1.
3. The method for producing a fermentation broth according to claim 2, wherein Bacillus belgii according to claim 1 is inoculated into PDA slant culture medium and cultured at 28 ℃ for 48 hours; picking colonies on the inclined plane, and placing the colonies into a PDB culture medium to perform shake cultivation for 48 hours at 30 ℃ to obtain a seed suspension; and inoculating the seed suspension into a fermentation culture medium for fermentation for 36 hours at the temperature of 28-34 ℃ and under the condition of 160-200 r/min.
4. Use of the Bacillus belgii of claim 1 or the fermentation broth of claim 2 for controlling plant diseases.
5. The use according to claim 4, wherein the plant disease is ginseng root rot, ginseng rust rot, ginseng black spot, gentian root rot, citrus fruit soft rot, cinnamomum camphora leaf spot, zinnia pertussis purpura, peach tree branch blight, curculigo orchioides leaf spot, orange gray mold, mountain white tree leaf spot, carrot black spot or rice blast.
6. Use of a bacillus beiLeisi according to claim 1 or a fermentation broth according to claim 2 for solubilizing phosphorus.
7. Use of the Bacillus belgii of claim 1 or the fermentation broth of claim 2 for the preparation of a plant growth promoter or a soil conditioner.
8. The use according to any one of claims 4 to 7, wherein the effective concentration of Bacillus belgii is 1 x 105～1×108cfu/mL, the application amount is 0.5-1.0L/m2。
9. A biocontrol microbial inoculum comprising the Bacillus belgii of claim 1 or the fermentation broth of claim 2 and acceptable excipients.
10. A method for controlling plant diseases, characterized in that the Bacillus of claim 1, the fermentation broth of claim 2 or the biocontrol microbial agent of claim 9 is applied at a viable bacteria concentration of 1X 106cfu/mL, applied at 0.5L/m2。
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| CN115595278A (en) * | 2022-08-18 | 2023-01-13 | 吉林农业大学（Cn） | Ginseng disease-preventing growth-promoting bacterial fertilizer and application thereof |
| CN116987613A (en) * | 2023-04-03 | 2023-11-03 | 吉林农业大学 | Bacillus bailii YZ-375 and application thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115595278A (en) * | 2022-08-18 | 2023-01-13 | 吉林农业大学（Cn） | Ginseng disease-preventing growth-promoting bacterial fertilizer and application thereof |
| CN115595278B (en) * | 2022-08-18 | 2023-12-22 | 吉林农业大学 | Ginseng disease-preventing growth-promoting bacterial fertilizer and application thereof |
| CN116987613A (en) * | 2023-04-03 | 2023-11-03 | 吉林农业大学 | Bacillus bailii YZ-375 and application thereof |
| CN116987613B (en) * | 2023-04-03 | 2024-04-26 | 吉林农业大学 | Bacillus bailii YZ-375 and application thereof |
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