CN111925963A - Application of Siamese bacillus B11 in prevention and/or treatment of cotton bamboo blast tip disease - Google Patents
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Abstract
The invention discloses application of Siamese bacillus B11 in prevention and/or treatment of cotton bamboo blast tip disease. The Siamese bacillus B11 is used for preventing and/or treating cotton bamboo blast tip disease, the effect is very good, after the Siamese bacillus B11 is prepared into nano microemulsion, the Siamese bacillus B11 has long storage time, high strain survival rate, good prevention effect on the cotton bamboo blast tip disease, good stress resistance, high temperature resistance, low temperature resistance and drying resistance, is suitable for commercial production, is pollution-free, green and environment-friendly, can sustainably control the generation of diseases, and has low production cost.
Description
Technical Field
The invention relates to the technical field of biological control, in particular to application of Siamese bacillus B11 in prevention and/or treatment of cotton bamboo blast tip disease.
Background
Mianzhu (scientific name: Bambusa intermedia Hsueh et Yi) is a gramineae Acanthopanax bamboo plant, is distributed in the middle and south of Yunnan China, is widely cultivated, is also found in local areas of Sichuan and Guizhou, has basically the same fiber degree and toughness as Sasa albo-marginata, is early in production and high in yield, and is suitable for bamboo weaving and processing, papermaking and eating as bamboo shoots. The blight of Mikania sinensis caused by Arthronium parahaeospermum and Arthrobotrys rickii firstly appears tongue-shaped or spindle-shaped disease spots at the joint of main branches or branches, and the head is light brown and then turns into purple brown. When the lesion spot surrounds the branch or trunk for a circle, the upper leaf turns yellow, and the lesion spot is longitudinally curled until withered and falls off. In forests, the damage degree of diseases is different, and the bamboos can be withered and dead in shoots, branches and plants. When the diseased bamboo is cut open, the inner wall of the diseased spot becomes brown and white flocculent mycelium grows on the diseased bamboo. In spring of the next year, small irregular bulges appear at dead tips or dead branch nodes, then the cracks are irregular, and germs are spread by water, wind and rain or artificially spread. In the affected area, the disease is easy to spread in the dry years with high temperature in 7-8 months.
The serious disease of the cotton bamboo blight can cause the death of the whole cotton bamboo tree, restrict the development of cotton bamboo production and seriously threaten the Chinese cotton bamboo industry. The cotton bamboo blight caused by fusarium mainly occurs on the trunk and main branches, the disease spots are wet at first, and the cotton bamboo blight is mainly prevented and assisted by chemical prevention and chemical prevention at present. The use of 500 times of carbendazim or 72% thiophanate methyl aqueous solution can prevent the disease. The main leaf eating pest is bamboo borer, and the prevention and control method is beauveria bassiana powder or 0.5 percent abamectin smoke agent 15 kg/hectare; or spraying 3000 times of 20% of cyanovalerian ester. However, the use of chemical pesticides can pollute the environment, easily cause poisoning of people and livestock, and cause certain pests to have drug resistance to different degrees. Therefore, it would be of great interest to find an effective way to prevent and/or treat the blight of Mianzhu.
Disclosure of Invention
In order to solve the above problems in the prior art, the present invention aims to provide a siamenobacillus siamenosis, wherein the siamenobacillus is named as B11, and is preserved in the china general microbiological culture collection management center at 6.1.2020 with the preservation address: the microorganism research institute of China academy of sciences No. 3 of Xilu No.1 of Beijing, Chaoyang, with the preservation number of CGMCC No.19894, the Siamese bacillus B11 can effectively prevent and/or treat the cotton bamboo blast tip disease.
When the Siamese bacillus B11 is used for preventing and/or treating the cotton bamboo blast tip disease, the Siamese bacillus B11 can be prepared into a biological preparation, such as a liquid preparation or a nano micro-emulsion preparation; the liquid preparation is prepared by preparing Siamese bacillus B11 into liquid fermentation liquor, and directly spraying the liquid fermentation liquor on a part of Mianzhu which is easy to form a withered tip disease; the nano microemulsion preparation is prepared by preparing Siamese bacillus B11 into liquid fermentation liquor, mixing with auxiliary materials such as emulsifier, homogenizing, and making into nano microemulsion.
Further, the weight ratio of the fermentation liquor to the auxiliary materials is 100: 15-25; preferably, the weight ratio of the fermentation liquid to the auxiliary materials is 100: 17.
Further, the auxiliary materials are an emulsifier, a stabilizer and an antifreeze, and the weight ratio of the emulsifier to the stabilizer to the antifreeze is 8-12:1-4: 2-8; preferably the weight ratio of emulsifier, stabilizer and antifreeze is 10:2: 5. When the components are used as auxiliary materials, the nano-microemulsion is prepared by the following specific steps: preparing fermentation liquor from Siamese bacillus B11, uniformly mixing the fermentation liquor with an emulsifier, a stabilizer and an antifreezing agent, and homogenizing to obtain the Siamese bacillus B11.
Further, the emulsifier is prepared by mixing Tween 80 and sodium lignosulfonate in a mass ratio of 1:3, and the stabilizer is sorbitol and an antifreeze polyethylene glycol.
Further, the content of the viable spore of the Siamese bacillus B11 in the Siamese bacillus B11 nano microemulsion is not less than 1 multiplied by 109cfu/g。
When the above-mentioned preparation is used for prevention and/or treatmentWhen the Mianzhu withered shoot is detected, the preparation can be directly applied or sprayed if the preparation is liquid, or the preparation is a nano microemulsion preparation and is applied after being diluted, specifically, the preparation is diluted to have the content of viable spores of 1 × 109The diluted solution of cfu/ml is sprayed on the cotton bamboo branches, the using amount is 10-50 ml/mu, the comprehensive cost and the prevention effect are achieved, and the optimal using amount is 30-40 ml/mu.
The invention has the following beneficial effects:
the Siamese bacillus B11 is used for preventing and/or treating cotton bamboo blast tip disease, the effect is very good, after the Siamese bacillus B11 is prepared into nano microemulsion, the Siamese bacillus B11 has long storage time, high strain survival rate, good prevention effect on the cotton bamboo blast tip disease, good stress resistance, high temperature resistance, low temperature resistance and drying resistance, is suitable for commercial production, is pollution-free, green and environment-friendly, can sustainably control the generation of diseases, and has low production cost.
Drawings
FIG. 1 is a phylogenetic tree of strain B11 constructed based on the 16S rDNA sequence.
Detailed Description
The principles and features of this invention are described below in conjunction with embodiments, which are included to explain the invention and not to limit the scope of the invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
Example 1 isolation and identification of Bacillus siamensis B11
1. Isolation of Bacillus siamensis B11
The invention relates to a Siamese Bacillus (Bacillus siamensis) B11 strain which is separated from rhizosphere soil of Sichuan Yibin bamboo cultivation area by adopting a flat plate dilution coating method in 2017, 6 and 15 days, in particular to a method for diluting a collected soil sample by using sterile water and diluting by 10-1，10-2，10-3，10-4，10-5Total 5 gradients, 100. mu.L 10 aspirated-3，10-4，10-5The 3 gradient soil dilutions were coated with NA plates (beef extract 3g, protein) respectively10g of peptone, 5g of sodium chloride, 15-20g of agar powder, 1000mL of distilled water and pH of 7.0, uniformly mixing, subpackaging, sterilizing at 121 ℃ for 30min under high pressure, repeating each treatment for 3 times, culturing at 27 ℃ for 48h, and selecting single colonies with obvious colony morphology difference to enter a primary screen.
According to the difference of colony size, color, protrusion, edge characteristic, smooth surface, transparency, etc., selecting single colony, streaking and purifying on NA plate, and transferring the NA slant to 4 deg.c for storage. Inoculating the pathogenic bacteria cake of Mianzhu blight with diameter of 5mm at the center of PDA plate, inoculating the strains to be screened at equal intervals, culturing at constant temperature of 25 deg.C, and repeating each treatment for 3 times. The degree of antagonism was determined from the size of the zone of inhibition by each selected bacterium (Table 1, Table 2), and the strain having excellent effects was B11 (No. 11).
TABLE 1 inhibitory Effect of endophytic bacteria on Arthronium parahaeospermum
| Numbering | Antibacterial circle (mm) | Numbering | Antibacterial circle (mm) |
| 1 | 0 | 9 | 0 |
| 2 | 0 | 10 | 4.0 |
| 3 | 5.5 | 11 | 21.2 |
| 4 | 0 | 12 | 0 |
| 5 | 11.0 | 13 | 0 |
| 6 | 10.5 | 14 | 0 |
| 7 | 0 | 15 | 8.8 |
| 8 | 0 | 16 | 6.9 |
TABLE 1 inhibitory Effect of endophytic bacteria on Arthrobotrys rickii
| Numbering | Antibacterial circle (mm) | Numbering | Antibacterial circle (mm) | |
| 1 | 0 | 9 | 0 | |
| 2 | 15.2 | 10 | 0 | |
| 3 | 13.3 | 11 | 19.8 | |
| 4 | 0 | 12 | 0 | |
| 5 | 0 | 13 | 0 | |
| 6 | 4.5 | 14 | 0 | |
| 7 | 7.5 | 15 | 0 | |
| 8 | 0 | 16 | 6.0 |
2. Identification of Siam bacillus B11
A single colony of the B11 strain cultured on the NA plate medium for 2 days is milky white, round, rough in surface, wavy in edge, opaque and non-adhesive.
In addition to the above morphological observations, the endophyte B11 was identified as Bacillus siamensis by combining physiological and biochemical criteria (Table 3) and molecular biology.
TABLE 3 physiological and biochemical indices of Strain B11
Molecular biological identification (16S rDNA): extracting bacterial DNA to perform PCR amplification and electrophoresis, and recycling a product to a biotechnology company for sequencing; the homology BLAST analysis of the determined sequence with the sequences already reported in the GenBank database was carried out, and multiple sequence comparisons were carried out with Clustalx (1.83) software, and phylogenetic trees were constructed by the adjacency method in Mega4.0 software to determine the phylogenetic status of the B11 strain in microorganisms. Molecular biology identification to obtain a DNA fragment (shown as SEQ ID NO. 1) with the length of 688bp, submitting the 16S rDNA sequence of the B11 strain to a GenBank database for BLAST analysis, selecting a bacterial 16S rDNA sequence with higher homology, performing multiple matching arrangement analysis by using Clustalx software, constructing a phylogenetic tree by using Mega analysis software, and obtaining the constructed phylogenetic tree shown in figure 1. As can be seen from FIG. 1, the strain B11(MT482698) and MT052693 supported a nucleotide sequence similarity of 99% of the 16SrRNA gene and a higher self-expansion value for a total of 1 branch, but were more distant from other Bacillus, indicating that the relationship between B11 and Bacillus siamensis is closest.
Based on the characteristics, the strain is classified and named as Siamese Bacillus (Bacillus siamensis) B11, and has been preserved in China general microbiological culture Collection center of the institute of microbiology, China academy of sciences, China Committee for culture Collection of microorganisms, 6.1.2020, with the preservation number of CGMCC No. 19894.
EXAMPLE 2 preparation of Bacillus siameses B11 Nano microemulsion
The preparation method comprises the following steps of fermenting Siamese bacillus B11, mixing with an emulsifier, homogenizing, and preparing into the nano microemulsion, wherein the preparation method specifically comprises the following steps:
(1) fermentation: inoculating Siamese bacillus slant seeds into a liquid culture medium, wherein the initial inoculation amount is 4%, and then performing shake culture for 48h under the conditions of 25 ℃, 185r/min and 200L/h of ventilation;
wherein, the liquid culture medium contains the following components in percentage by weight: 0.5% of nano silicon dioxide, 2% of cane sugar, 1% of tryptone, 0.1% of sodium chloride and 0.05% of magnesium sulfate, the balance being sterile water, and the pH value of the liquid culture medium being 7.2.
(2) Preparing the nano microemulsion: adding an emulsifier, a stabilizer and an antifreeze into the fermentation liquor in a high-pressure mixing kettle to ensure that the weight percentages of the emulsifier, the stabilizer and the antifreeze are respectively 10%, 2% and 5%, uniformly mixing, and homogenizing to obtain a Siamese bacillus B11 nano microemulsion; wherein the emulsifier is prepared by mixing Tween 80 and sodium lignosulfonate in a mass ratio of 1:3, and the stabilizer is sorbitol and an antifreeze polyethylene glycol.
The obtained nanometer microemulsion can be stored at room temperature for 1.5 years or at low temperature (4 deg.C) for 3 years without affecting the prevention and treatment effects.
Example 3 prevention and treatment experiment of Mianzhu blight
Preparing a Siamese bacillus B11 nano microemulsion diluent: 1g of the Siamese bacillus B11 nano microemulsion prepared in the embodiment 2 is diluted into the Siamese bacillus B11 nano microemulsion with sterile water, and the content of the viable spores is 1 multiplied by 109Dilutions of cfu/ml.
Preparing a pathogenic bacterium solution: after the inoculation of the pathogens on PDA medium for 14 days, a spore suspension was prepared. Washing a culture dish with 50ml sterile water, sucking with pipette, filtering with absorbent cotton, filtering with centrifuge, diluting with sterile water, observing with blood counting plate to calculate spore concentration, and diluting to 1 × 106cfu/ml. Pathogenic bacteria were obtained from Arthronium parahaeospermum and Arthrobotrys rickii spore suspension 1: 1 (weight ratio).
Pot control experiment: selecting healthy Mianzhu from Mianzhu planting area to plant in greenhouse for pot experiment (2 years, bamboo height 1m), and respectively treating as follows: 1) inoculating pathogenic bacteria liquid in the middle of a branch by adopting a needle punching method, wherein each strain is 100ml, and respectively inoculating different dosages of Siamese bacillus B11 nano microemulsion diluent in the original position by adopting a spraying method after 15 days, wherein each strain is 100 ml. 2) Each strain is inoculated with 100ml of Siamese bacillus B11 nanometer microemulsion diluent with different dosage, and inoculated with 100ml of pathogen solution 15 days later. 3) Simultaneously inoculating pathogenic bacteria and Siamese bacillus B11 nanometer micro-emulsion with different concentrations. Each treatment was repeated 10 times, and sterile water and sterile culture were used as controls.
Disease incidence is observed 30 days after treatment, disease investigation statistics is carried out, and disease incidence, disease index and prevention and treatment effect are calculated and recorded as shown in Table 4.
Field control experiment: spraying diluent (1 × 10) at 50 ml/mu, 40 ml/mu, 30 ml/mu, 20 ml/mu and 10 ml/mu on the cotton bamboo planting area and the healthy cotton bamboo planting area with blight (caused by Arthronium parahaeospermum and Arthrobotryum rickii) according to the ratio of 50 ml/mu, 40 ml/mu, 30 ml/mu, 20 ml/mu and 10 ml/mu9cfu/ml) were administered once every ten days for 3 times, 10 replicates per treatment, with sterile water and sterile culture fluid as controls, respectively. Disease incidence is observed after 30 days, disease investigation statistics is carried out, and disease incidence, disease index and prevention and treatment effect are calculated and recorded as shown in Table 5.
Grading the disease condition standard: level 0: no withering; level 1: less than 25% of withered branches; and 2, stage: 25-50% (including 25% and 50%) of paraquat; and 3, level: 50-75% of paraquat (containing 75%); 4, level: over 75 percent of the plant leaves are withered.
Incidence (%) × 100 (number of diseased plants/total number of plants);
the disease index [ Σ (disease-grade number of plants × representative value)/total number of plants and × representative value of the most serious grade of disease ] × 100;
the preventing and treating effect (%) is [ (control disease index increasing rate-treatment disease index increasing rate)/control disease index increasing rate ] × 100.
TABLE 4 potted plant control of Michelia lanchoe blight with microemulsion of B11
TABLE 5B 11 field control effect of microemulsion on Mianzhu blight
Since there was no significant difference between sterile water and the sterile culture medium as controls, only the data of the sterile water as a control are shown in tables 4 and 5.
As can be seen from tables 4 and 5, in the experiments on controlling cotton bamboo blast tip disease by siamesed bacillus B11, it is found that the prevention effect of siamesed bacillus B11 on cotton bamboo blast tip disease is far higher than the treatment effect, which may be because the biocontrol strain applied in the early stage of crop growth can be colonized on the plant surface earlier, occupy the living space and reduce the infection rate of pathogenic bacteria; after the second application in the middle period, the reproductive capacity of the feed is further improved, and the growth of pathogenic bacteria is inhibited. And only when the disease is serious, because the plants are infected by pathogenic bacteria, the colonization and reproduction capability of the biocontrol strain is inevitably reduced, so that the control effect is reduced. Therefore, the application should be started from the early growth stage after the transplantation of Mirabilis bambusoides. After the B11 nano microemulsion is diluted, the control effect is reduced with the increase of the dilution, different control effects are shown, the control effect of the dosage of 10-50 ml/mu exceeds 50%, the control effect of the dosage of 50 ml/mu is the best to the control effect of the cotton bamboo blight, the control effect reaches as high as 98%, the comprehensive cost and the control effect are realized, and the recommended dosage is 30-40 ml/mu.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Sequence listing
<110> Sichuan university of agriculture
Application of Siamese bacillus B11 in prevention and/or treatment of cotton bamboo blast tip disease
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 688
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
tgcaagtcga gcggacagat gggagcttgc tccctgatgt tagcggcgga cgggtgagta 60
acacgtgggt aacctgcctg taagactggg ataactccgg gaaaccgggg ctaataccgg 120
atggttgttt gaaccgcatg gttcagacat aaaaggtggc ttcggctacc acttacagat 180
ggacccgcgg cgcattagct agttggtgag gtaacggctc accaaggcga cgatgcgtag 240
ccgacctgag agggtgatcg gccacactgg gactgagaca cggcccagac tcctacggga 300
ggcagcagta gggaatcttc cgcaatggac gaaagtctga cggagcaacg ccgcgtgagt 360
gatgaaggtt ttcggatcgt aaagctctgt tgttagggaa gaacaagtgc cgttcaaata 420
gggcggcacc ttgacggtac ctaaccagaa agccacggct aactacgtgc cagcagccgc 480
ggtaatacgt aggtggcaag cgttgtccgg aattattggg cgtaaagggc tcgcaggcgg 540
tttcttaagt ctgatgtgaa agcccccggc tcaaccgggg agggtcattg gaaactgggg 600
aacttgagtg cagaagagga gagtggaatt ccacgtgtag cggtgaaatg cgtacagatg 660
tggaggaaca ccagtggcga aggcgagt 688
Claims (9)
1. The application of Siamese Bacillus (Bacillus siamensis) B11 in preventing and/or treating long-stalked bamboo blight is characterized in that the Siamese Bacillus (Bacillus siamensis) B11 is preserved in China general microbiological culture collection center in No. 6 and 1 of 2020, and the preservation number is CGMCC No. 19894.
2. The use according to claim 1, wherein Bacillus siamensis B11 is formulated as a liquid or milk formulation for the prevention and/or treatment of cotton bamboo culm blight.
3. A formulation for preventing and/or treating cotton bamboo blight, comprising Bacillus siamensis (Bacillus siamensis) B11 according to claim 1.
4. The formulation for preventing and/or treating long bamboo blight according to claim 4, wherein the formulation is Siamese Bacillus (Bacillus siamensis) B11 liquid fermentation broth.
5. The formulation for preventing and/or treating cotton stalk blight according to claim 3, wherein said formulation is Siamese Bacillus B11 nano microemulsion.
6. The method for preparing the preparation for preventing and/or treating cotton shoot blight according to claim 5, comprising the steps of:
preparing Siamese Bacillus (Bacillus siamensis) B11 into liquid fermentation liquor, mixing with auxiliary materials, and homogenizing to obtain the Siamese Bacillus Siamensis fermentation liquor.
7. The method of claim 6, wherein the auxiliary materials include an emulsifier, a stabilizer, and an antifreeze.
8. The process according to claim 6 or 7, wherein the amount of viable spores of Bacillus siameses B11 in the Bacillus siameses B11 nanomicroemulsion is not less than 1X 109cfu/g。
9. Method for preventing and/or treating cotton shoot blight using the preparation according to any one of claims 3 to 5, wherein the preparation is diluted to a viable spore content of 1 x 109And (3) spraying the cfu/ml diluent on the cotton bamboo branches, wherein 10-50ml of the diluent is sprayed per mu.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111849843A (en) * | 2020-08-21 | 2020-10-30 | 四川农业大学 | Application of Siamese bacillus B11 in prevention and/or treatment of bamboo shoot dry blight |
| CN111909877A (en) * | 2020-08-21 | 2020-11-10 | 四川农业大学 | Application of Siamese bacillus B11 in prevention and/or treatment of chestnut branch blight |
| CN111961622A (en) * | 2020-08-21 | 2020-11-20 | 四川农业大学 | Siamese bacillus B11 and application thereof in prevention and/or treatment of hybrid bamboo stem-based rot disease |
| CN112646745A (en) * | 2020-12-28 | 2021-04-13 | 中国林业科学研究院森林生态环境与保护研究所 | Siamese bacillus, microbial inoculum and application thereof |
| CN115418337A (en) * | 2022-09-30 | 2022-12-02 | 中国科学院东北地理与农业生态研究所 | Lignin degrading bacterium and application thereof in rice straw micro-storage |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111849843A (en) * | 2020-08-21 | 2020-10-30 | 四川农业大学 | Application of Siamese bacillus B11 in prevention and/or treatment of bamboo shoot dry blight |
| CN111909877A (en) * | 2020-08-21 | 2020-11-10 | 四川农业大学 | Application of Siamese bacillus B11 in prevention and/or treatment of chestnut branch blight |
| CN111961622A (en) * | 2020-08-21 | 2020-11-20 | 四川农业大学 | Siamese bacillus B11 and application thereof in prevention and/or treatment of hybrid bamboo stem-based rot disease |
| CN112646745A (en) * | 2020-12-28 | 2021-04-13 | 中国林业科学研究院森林生态环境与保护研究所 | Siamese bacillus, microbial inoculum and application thereof |
| CN112646745B (en) * | 2020-12-28 | 2022-04-12 | 中国林业科学研究院森林生态环境与保护研究所 | Siamese bacillus, microbial inoculum and application thereof |
| CN115418337A (en) * | 2022-09-30 | 2022-12-02 | 中国科学院东北地理与农业生态研究所 | Lignin degrading bacterium and application thereof in rice straw micro-storage |
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