CN111961622A - Siamese bacillus B11 and application thereof in prevention and/or treatment of hybrid bamboo stem-based rot disease - Google Patents
Siamese bacillus B11 and application thereof in prevention and/or treatment of hybrid bamboo stem-based rot disease Download PDFInfo
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Abstract
The invention discloses Siamese bacillus B11 and application thereof in preventing and/or treating hybrid bamboo stem rot. The Siamese bacillus B11 is used for preventing and/or treating the hybrid bamboo dry base rot disease, the effect is very good, after the Siamese bacillus B11 is prepared into nano water dispersible granules, the storage time is long, the survival rate of strains is high, the Siamese bacillus B11 has good prevention effect on the hybrid bamboo dry base rot disease, the stress resistance is good, the Siamese bacillus B11 can resist high temperature, low temperature and drying, the Siamese bacillus B11 is suitable for commercial production, is pollution-free, green and environment-friendly, can sustainably control the disease generation, and has low production cost.
Description
Technical Field
The invention relates to the technical field of biological control, in particular to Siamese bacillus B11 and application thereof in preventing and/or treating hybrid bamboo stem rot.
Background
Hybrid bamboo (bamboo pervariabilis. times. Dendrocalamopsis grandis) refers to a general name of 2 or more than 2 bamboo species, seeds are produced through artificial flower pollination, excellent plants are selected through seeding and seedling raising, and a plurality of excellent clone new bamboo species are cultured through vegetative propagation. The bamboo crossbreeding work in China began in the last 60 th century, and enjoyable results were obtained in the 80 th century. The excellent clone hybrid bamboo seeds popularized and applied in production at present have the numbers of shou Lv No. 3, Ma version No. 6, shou Ma Qing No. 15, etc.
The hybrid bamboo-based rot disease is a new disease on the young bamboo in the current year. The distribution of southern hybrid bamboo was found to varying degrees. The rot disease of the hybrid bamboo stem base caused by Diaporter acaciarum and Exomassilinia calospora is mainly characterized in that the rot disease of the hybrid bamboo stem base is firstly appeared on the bamboo walls of the third to fifth sections of the diseased bamboo stem base, and is initially in a yellow brown to purple brown punctate small spot shape, star-shaped or strip-shaped distribution. At this time, the bamboo shoot sheaths wrap the base parts of the bamboo stalks, so that the bamboo shoots are not easy to find. These small lesions spread rapidly upwards and combine with each other into large patches in the form of strips or blocks. The infected bamboo flesh tissue turns light brown and spreads upward much faster than the lesions on the outer wall. When the scab reaches the bamboo joint, the scab rapidly develops transversely, so that the whole circle or most of the bamboo joint is changed into brown, and the bamboo joint is taken as a base point, greatly expands upwards along the conduction tissue in the bamboo culm and gradually spreads from the interior of the bamboo culm to the outer wall. The bamboo stalk can be seen with block or strip-shaped moire spots developed from bamboo joints, which are black brown at first and then light brown. After the tender bamboo is infected with diseases, the disease spots rapidly spread and expand, and the base of the whole tender bamboo can be rotten and die immediately if the disease spots are serious; when the mild tender bamboo is diseased and becomes bamboo, one or more block-shaped or banded rotten spots are left at the base of the bamboo rod and are easy to turn over when meeting wind, thereby obviously weakening the growth potential and stress resistance of the hybrid bamboo and reducing the utilization value of the bamboo forest. Therefore, it is very useful to find a method for effectively preventing and/or treating the rot disease of the hybrid bamboo stem base.
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, the North China province has a preservation number of CGMCC No.19894, and the Siamese Bacillus (Bacillus simensis) B11 (which can be abbreviated as Siamese Bacillus B11) can effectively prevent and/or treat the hybrid bamboo stem rot.
When the Siamese bacillus B11 is used for preventing and/or treating the rotting disease of the hybridized bamboo stem base, the Siamese bacillus B11 can be made into a biological preparation, such as a liquid preparation or a solid preparation; the liquid preparation is prepared by preparing Siamese bacillus B11 into liquid fermentation liquor, and directly spraying the liquid fermentation liquor on the part of the hybridized bamboo which is easy to form dry-base rot; the solid preparation is prepared by firstly preparing Siamese bacillus B11 into liquid fermentation liquor, then mixing the liquid fermentation liquor with auxiliary materials such as a binder and the like to prepare a solid object, and specifically preparing the solid object into nano water dispersible granules, wherein the specific preparation process comprises the following steps: preparing fermentation liquor from Siamese bacillus B11, mixing with auxiliary materials, and performing extrusion granulation to obtain the Siamese bacillus B11.
Further, the weight ratio of the fermentation liquor to the auxiliary materials is 100: 45-55; preferably, the weight ratio of the fermentation liquid to the auxiliary materials is 100: 52.
Further, the auxiliary materials comprise a dispersing agent, a wetting agent, a disintegrating agent, a binder and a carrier, wherein the weight ratio of the dispersing agent to the wetting agent to the disintegrating agent to the binder to the carrier is 3-5:8-12:2-3:0.3-0.6: 32-38; preferably, the weight ratio of the dispersing agent, wetting agent, disintegrant, binder and carrier is 4:8:2:0.5: 35. When the components are used as auxiliary materials, the nano water dispersible particles are prepared by the following specific steps: preparing fermentation liquor from Siamese bacillus B11, mixing the fermentation liquor with a dispersing agent, a wetting agent, a disintegrating agent and a binding agent to form suspension, mixing the suspension with a carrier after spray drying, extruding, granulating and drying to obtain the Siamese bacillus B11.
Further, the carrier is kaolin, the dispersing agent is polyethylene glycol, the wetting agent is tween, the disintegrating agent is corn starch, and the binder is polyvinylpyrrolidone.
Further, the content of the living spores of the Siamese bacillus B11 in the Siamese bacillus B11 nano water dispersible particles is not less than 1 multiplied by 109cfu/g。
When the preparation is used for preventing and/or treating rot of hybrid bamboo stems, the preparation can be directly applied if the preparation is liquid, or applied after being dissolved and diluted if the preparation is solid, the non-diseased hybrid bamboo is applied with the preparation diluent to the stems once in spring, and the diseased hybrid bamboo is applied with the preparation diluent to the scabs once in spring and autumn.
The invention has the following beneficial effects:
the Siamese bacillus B11 is used for preventing and/or treating the hybrid bamboo dry base rot disease, the effect is very good, after the Siamese bacillus B11 is prepared into nano water dispersible granules, the storage time is long, the survival rate of strains is high, the Siamese bacillus B11 has good prevention effect on the hybrid bamboo dry base rot disease, the stress resistance is good, the Siamese bacillus B11 can resist high temperature, low temperature and drying, the Siamese bacillus B11 is suitable for commercial production, is pollution-free, green and environment-friendly, can sustainably control the disease generation, 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, peptone 10g, sodium chloride 5g, agar powder 15-20g, distilled water 1000mL, pH 7.0, autoclaved at 121 ℃ for 30min after mixing and split charging), each treatment was repeated 3 times, cultured at 27 ℃ for 48h, and single colonies with significant differences in colony morphology were picked and placed in 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 a cross-bred bamboo trunk rot disease pathogenic bacteria cake with the diameter of 5mm at the center of a PDA (potato dextrose agar) flat plate according to a five-point opposing method, inoculating the strains to be screened at equal intervals around the center, culturing at constant temperature of 25 ℃, and repeating 3 treatments each. The antagonistic degree (table 1, table 2) was determined according to the size of the zone of inhibition produced by each selected bacterium, and the strain with excellent effect was B11.
TABLE 1 inhibitory Effect of endophytic bacteria on hybrid bamboo Stem rot pathogen Diaporthe acaciarum
TABLE 1 inhibitory Effect of endophytic bacteria on hybrid bamboo Stem rot pathogen Exomassarinula calospora
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 Water-dispersible nanoparticles of Bacillus siamensis B11
The preparation method comprises the following steps of fermenting, drying, crushing and granulating Siamese bacillus B11, drying again, and granulating to prepare nano water dispersible granules, and specifically comprises the following steps:
(1) fermentation: inoculating Siamese bacillus slant seeds into a liquid culture medium, wherein the initial inoculation amount is 6%, and then performing shake culture for 40h under the conditions of 27 ℃, 185r/min and 100L/h of ventilation;
wherein, the liquid culture medium contains the following components in percentage by weight: 0.1% of nano silicon dioxide, 2% of glucose, 1% of peptone, 0.1% of potassium chloride and 0.05% of magnesium sulfate, and the balance of sterile water, wherein the pH value of the liquid culture medium is 7.0.
(2) Preparing nano water dispersible particles: adding polyethylene glycol, tween, corn starch and polyvinylpyrrolidone into the fermentation liquor to enable the weight percentages of the polyethylene glycol, tween, corn starch and polyvinylpyrrolidone to be 4%, 8%, 2% and 0.5% respectively, uniformly mixing to form a suspension, spraying and drying the suspension, mixing the suspension with kaolin, wherein the weight percentage of the kaolin is 35%, fully grinding the suspension into powder, adding water, stirring, granulating, drying and sieving to obtain the product.
After the nano water dispersible particles are bottled, the prevention and treatment effects are not influenced after the nano water dispersible particles are stored for 1 year at normal temperature or stored for 2 years at low temperature (4 ℃).
Example 3 prevention and treatment experiment of rot disease of hybrid bamboo Stem
Preparing a Siamese bacillus B11 nano water dispersible granule diluent: 1g of the Siamese bacillus B11 nano water dispersible granules prepared in the embodiment 2 are respectively diluted by sterile water by 50 times, 100 times, 500 times and 1000 times to prepare diluent, and the spore content of the diluent is respectively 2 multiplied by 107cfu/ml、1× 107cfu/ml、2×106cfu/ml、1×106cfu/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 50 ml 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 bacterial solutions were prepared from Diaporthe acaciarum and Exomassilinia calospora spore suspensions 1: 1 (weight ratio).
Prevention and control experiment: selecting healthy 2-year-old seedlings of hybrid bamboo with the plant height of about 60-70cm in a Yibin hybrid bamboo introduction area, and respectively treating the seedlings as follows: 1) in spring and autumn (3-5 months and 9-10 months), inoculating pathogen bacteria solution at the base part by adopting a needle punching method, wherein each strain is 100ml, spraying water dispersible granule diluent of Siamese bacillus B11 nano-particles with different concentrations for 1 time respectively in the original place by adopting a spraying method after 15 days, wherein the two seasons are 2 times, and each strain is 100 ml. The spraying method is that bamboo shoot sheaths at the base parts of the bamboo shoots are firstly peeled off, and then the bamboo shoot sheaths are sprayed aiming at the base parts of the bamboo poles, and the same is carried out. 2) Spraying the basal part of the dried bamboo 1 time by using water dispersible granule diluent of Siamese bacillus B11 nano water with different concentrations in spring every year (3-5 months), wherein each strain is 100ml, and inoculating pathogen suspension with 100ml after 15 days. 10 replicates of each treatment were run with sterile water and sterile culture medium 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.
Grading the disease condition standard: healthy or nearly healthy is grade I (0); the scab is obvious, but the scab does not go deep into the base of the bamboo trunk and is I-grade (1); the disease spot penetrates into the base of the bamboo trunk, and the width of the disease spot does not exceed 1/6 of the bamboo periphery and is II-level (2); the width of the scab deep into the base of the bamboo trunk is IV grade (3) between 1/6-1/2 of the bamboo periphery; the width of the scab deep into the base of the bamboo trunk is above 1/2 of the bamboo periphery and is V-grade (4), and the value in the bracket is a representative value.
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 4B 11 field control of rot disease of hybrid bamboo bases by water-dispersible granules
Since there was no significant difference between sterile water and the sterile culture medium as controls, only the data for the sterile water as a control are shown in Table 4.
As can be seen from table 4, in the test for preventing and treating the dry rot of the hybrid bamboo by using the siamesed bacillus B11, the prevention effect of the siamesed bacillus B11 on the dry rot of the hybrid bamboo is far higher than the treatment effect, which is probably because the biocontrol strain can be earlier colonized on the plant surface when being applied in the early stage of crop growth, occupies living space and reduces 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 stage of growth after the transplantation of the hybrid bamboo. After the B11 nano water dispersible particles are diluted, the control effect is reduced along with the increase of the dilution, different control effects are shown, the control effects of 50, 100, 500 and 1000 times of dilution are all over 50 percent, the control effect of 50 times of dilution on the rot disease of the hybrid bamboo stem base is the best, and the control effect can reach as high as 100 percent. The preparation is applied to prevent the hybrid bamboo dry base rot disease from occurring, the prevention effect can be achieved by applying 200 times of liquid once in spring, and the treatment effect can be achieved by applying 100 times of liquid once in spring and autumn.
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
Siamese bacillus B11 and application thereof in prevention and/or treatment of hybrid bamboo stem-based rot disease
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ccgacctgag agggtgatcg gccacactgg gactgagaca cggcccagac tcctacggga 300
ggcagcagta gggaatcttc cgcaatggac gaaagtctga cggagcaacg ccgcgtgagt 360
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gggcggcacc ttgacggtac ctaaccagaa agccacggct aactacgtgc cagcagccgc 480
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tttcttaagt ctgatgtgaa agcccccggc tcaaccgggg agggtcattg gaaactgggg 600
aacttgagtg cagaagagga gagtggaatt ccacgtgtag cggtgaaatg cgtacagatg 660
tggaggaaca ccagtggcga aggcgagt 688
Claims (10)
1. A Siamese Bacillus (Bacillus siamensis) B11 is preserved in China general microbiological culture collection center in 6.1.2020 with the preservation number of CGMCC No. 19894.
2. Use of Bacillus siamensis (Bacillus siamensis) B11 according to claim 1 for preventing and/or treating rot disease of hybrid bamboo stem base.
3. The use according to claim 2, wherein Bacillus siamensis B11 is formulated as a liquid or solid formulation for preventing and/or treating dry rot of hybrid bamboo.
4. A formulation for preventing and/or treating dry rot of hybrid bamboo, comprising Bacillus siamensis (Bacillus siamensis) B11 according to claim 1.
5. The formulation for preventing and/or treating dry rot of hybrid bamboo according to claim 4, wherein the formulation is Siamese Bacillus (Bacillus siemensis) B11 liquid fermentation broth.
6. The formulation for preventing and/or treating dry rot of hybrid bamboo as claimed in claim 4, wherein the formulation is water dispersible granules of Siamese Bacillus (Bacillus siemensis) B11 nanoparticles.
7. The formulation for preventing and/or treating dry rot of hybrid bamboo as claimed in claim 6, wherein the Siamese Bacillus (Bacillus simensis) B11 nano water dispersible granule has Siamese Bacillus (Bacillus simensis) B11 live spore content not less than 1 x 109cfu/g。
8. The method for preparing an agent for preventing and/or treating rot of hybrid bamboo culm as set forth in claim 6 or 7, comprising the steps of:
preparing Siamese Bacillus (Bacillus siamensis) B11 into liquid fermentation liquor, mixing with auxiliary materials, extruding and granulating to obtain the Siamese Bacillus fermentation liquor.
9. The method of claim 8, wherein the excipients include a dispersing agent, a wetting agent, a disintegrant, a binder, and a carrier.
10. The method for preventing and/or treating rot of hybrid bamboo bases by using the preparation according to any one of claims 4 to 7, characterized in that the branches of non-diseased hybrid bamboo are coated with the preparation diluent once in spring, and the diseased hybrid bamboo is coated with the preparation diluent once each time in spring and autumn.
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| CN111925963A (en) * | 2020-08-21 | 2020-11-13 | 四川农业大学 | Application of Siamese bacillus B11 in prevention and/or treatment of cotton bamboo blast tip disease |
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| TOUSEEF HUSSAIN 等: "Determining the antifungal activity and characterization of Bacillus siamensis AMU03 against Macrophomina phaseolina (Tassi) Goid", 《INDIAN PHYTOPATHOLOGY》 * |
| 林志楷 等: "暹罗芽孢杆菌研究进展", 《亚热带植物科学》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111925963A (en) * | 2020-08-21 | 2020-11-13 | 四川农业大学 | Application of Siamese bacillus B11 in prevention and/or treatment of cotton bamboo blast tip disease |
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