CN111925963A - Application of Siamese bacillus B11 in prevention and/or treatment of cotton bamboo blast tip disease - Google Patents

Abstract

The invention discloses the application of Bacillus siamese B11 in preventing and/or treating Mianzhu dieback. In the present invention, Bacillus siamese B11 is used for preventing and/or treating Mianzhu dieback, and the effect is very good. Mianzhu dieback has good control effect, good stress resistance, high temperature resistance, low temperature resistance, drying resistance, suitable for commercial production, pollution-free, green and environmental protection, sustainable control of disease occurrence, and low production cost.

Description

Application of Bacillus siamese B11 in the prevention and/or treatment of Mianzhu dieback

technical field

The invention relates to the technical field of biological control, in particular to the application of Bacillus siamese B11 in preventing and/or treating Mianzhu dieback.

Background technique

Mianzhu (scientific name: Bambusa intermedia Hsueh et Yi) is a Gramineae, Bougainvillea is a tree-like bamboo plant. It is distributed in central and southern Yunnan, China. It is widely cultivated and also found in Sichuan and Guizhou. Tongci bamboo, put into production early and has high yield, is suitable for bamboo weaving processing, paper making and eating as bamboo shoots. Mianzhu dieback caused by Arthrinium paraphaeospermum and Arthrobotryum rickii first appeared tongue-shaped or fusiform lesions at the fork of the main branch or branches, which were light brown at first and then turned purple-brown. When the lesions surround the branches or dry out, the upper leaves turn yellow and roll vertically until they die and fall off. The degree of damage caused by diseases varies among the forests, and three types of bamboo withered, dead branches and whole plants can appear. When the diseased bamboo is cut open, it can be seen that the inner wall of the diseased spot turns brown and has white flocculent mycelium. In the spring of the following year, irregular small protrusions appear at the dead or dead branches, and then irregularly crack, and the bacteria are spread by water, wind and rain, or artificially. In the affected area, the disease is easy to spread in the hot and dry years from July to August.

The serious occurrence of Mianzhu dieback disease can cause the death of the entire Mianzhu tree, restrict the development of Mianzhu production, and pose a serious threat to China's Mianzhu industry. Mianzhu dieback caused by Fusarium mainly occurs on the trunk and main branches, and the disease spots appear wet at the beginning. At present, the prevention and control of Mianzhu dieback is mainly based on prevention, supplemented by chemical control. It can be prevented with 500 times of tamevin or 72% thiophanate-methyl solution. The main leaf-eating insect pest is bamboo borer, and the control method is Beauveria bassiana powder or 0.5% abamectin smoke agent 15 kg/ha; or 20% fenvalerate 3000 times liquid spraying. However, the use of chemical pesticides will pollute the environment, easily cause human and animal poisoning, and cause some pests to develop different degrees of drug resistance. Therefore, it is very meaningful to find an effective method for preventing and/or treating Mianzhu dieback.

SUMMARY OF THE INVENTION

In order to solve the above-mentioned problems existing in the prior art, the purpose of the present invention is to provide a kind of Bacillus siamensis, which is named B11, and was preserved in the China Common Microorganism Culture Collection on June 1, 2020 Management Center, deposit address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 19894. This Bacillus siamese B11 can effectively prevent and/or treat Mianzhu dieback.

When Bacillus siamese B11 is used to prevent and/or treat Mianzhu dieback, Bacillus siamese B11 can be made into biological preparations, such as liquid preparations or nano-emulsion preparations; liquid preparations are Bacillus siamese B11 It can be made into a liquid fermentation liquid and sprayed directly on the parts of Mianzhu that are prone to die-off; the nano-microemulsion preparation is made of Bacillus siamese B11 first into a liquid fermentation liquid, and then mixed with auxiliary materials such as emulsifiers to homogenize. Made of nano-emulsion.

Further, the weight ratio of fermentation broth and auxiliary materials is 100:15-25; preferably, the weight ratio of fermentation broth and auxiliary materials is 100:17.

Further, the auxiliary materials are emulsifier, stabilizer and antifreeze, and the weight ratio of emulsifier, stabilizer and antifreeze is 8-12:1-4:2-8; preferably the weight ratio of emulsifier, stabilizer and antifreeze 10:2:5. When these components are used as auxiliary materials, the specific preparation process of the nano-microemulsion is as follows: firstly preparing the fermentation broth with Bacillus siamese B11, mixing the fermentation broth with emulsifier, stabilizer and antifreeze agent, homogenizing, and preparing.

Further, the emulsifier is prepared by mixing Tween 80 and sodium lignosulfonate in a mass ratio of 1:3, the stabilizer is sorbic acid alcohol, and the antifreeze polyethylene glycol.

Further, the content of live spores of Bacillus siamese B11 in the Bacillus siamese B11 nano-emulsion is not less than 1×10 ⁹ cfu/g.

When using the above preparation to prevent and/or treat Mianzhu dieback, if the preparation is liquid, it can be directly smeared or sprayed; if the preparation is a nano-emulsion preparation, it is diluted and smeared, specifically diluting the preparation into a viable spore content of 1×10 ⁹ cfu/ml of diluent, then sprayed on the branches of Mianzhu, the usage amount is 10-50ml/mu, considering the cost and control effect, the recommended optimal usage amount is 30-40ml/mu.

The present invention has the following beneficial effects:

In the present invention, Bacillus siamese B11 is used for preventing and/or treating Mianzhu dieback, and the effect is very good. Mianzhu dieback has good control effect, good stress resistance, high temperature resistance, low temperature resistance, drying resistance, suitable for commercial production, pollution-free, green and environmental protection, sustainable control of disease occurrence, and low production cost.

Description of drawings

Figure 1 is a phylogenetic tree of strain B11 constructed based on the 16S rDNA sequence.

Detailed ways

The principles and features of the present invention will be described below with reference to the embodiments. The examples are only used to explain the present invention, but not to limit the scope of the present invention. If the specific conditions are not indicated in the examples, it is carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used without the manufacturer's indication are conventional products that can be purchased from the market.

Example 1 Isolation and identification of Bacillus siamese B11

1. Isolation of Bacillus siamese B11

The Bacillus siamensis B11 strain of the present invention was isolated from the rhizosphere soil of the bamboo cultivation area in Yibin, Sichuan by the plate dilution coating method on June 15, 2017. Specifically, the collected soil samples were diluted with sterile water. , dilute 10 ^-1 , 10 ^-2 , 10 ^-3 , 10 ^-4 , 10 ^-5 in 5 gradients, draw 100 μL of 10 ^-3 , 10 ^-4 , 10 ^-5 soil dilutions, respectively, and apply Cloth NA plate (beef extract 3g, peptone 10g, sodium chloride 5g, agar powder 15-20g, distilled water 1000mL, pH 7.0, sterilized at 121°C for 30min after mixing and packaging), each treatment was repeated 3 times, 27 After culturing at ℃ for 48h, single colonies with obvious differences in colony morphology were picked for primary screening.

According to the differences in colony size, color, protrusion, edge characteristics, smooth surface, transparency, etc., pick a single colony on the NA plate for streak purification, and after purification, transfer the NA slant to the NA slant and store it at 4°C for later use. According to the five-point confrontation method, inoculate a 5 mm diameter Mianzhu blight pathogenic bacteria cake in the center of the PDA plate, inoculate the above-mentioned strains to be screened at an equal distance around the four sides, and cultivate at a constant temperature of 25 °C, and each treatment has three replicates. The degree of antagonism was determined according to the size of the inhibition zone produced by each screened bacteria (Table 1, Table 2), and the strain with excellent effect was B11 (No. 11).

Table 1 Inhibitory effect of endophytic bacteria on Arthrinium paraphaeospermum

Numbering Inhibition zone (mm) Numbering Inhibition zone (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 Arthrobotryum rickii

Numbering Inhibition zone (mm) Numbering Inhibition zone (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 Bacillus siamese B11

The single colony of B11 strain cultured on NA plate medium for 2 days was milky white, round, rough surface, corrugated edge, opaque and non-sticky.

In addition to the above morphological observations, the endophyte B11 was identified as Bacillus siamese by combining physiological and biochemical indicators (Table 3) and molecular biology.

Table 3 Physiological and biochemical indicators of strain B11

Molecular biological identification (16S rDNA): PCR amplification and electrophoresis were performed by extracting bacterial DNA, and the recovered product was sent to a biotechnology company for sequencing; homology BLAST analysis was performed between the detected sequence and the sequence reported in the GenBank database, and Clustalx (1.83) The software performs multiple sequence comparison, and then uses the neighbor-joining method in the Mega4.0 software to construct a phylogenetic tree to determine the position of the B11 strain in the microbial phylogeny. Molecular biology identification obtained a DNA fragment with a length of 688 bp (as shown in SEQ ID NO. 1), the 16S rDNA sequence of the B11 strain was submitted to the GenBank database for BLAST analysis, and the bacterial 16S rDNA sequence with high homology was selected. , and used Clustalx software to perform multiple matching arrangement analysis, and used Mega analysis software to construct a phylogenetic tree. The constructed phylogenetic tree is shown in Figure 1. It can be seen from Figure 1 that the B11 (MT482698) strain and MT052693 are clustered into one branch with 99% similarity of 16S rRNA gene nucleotide sequence and high bootstrapping value, but they are far away from other Bacillus, indicating that B11 is closely related to Bacillus. Bacillus siamensis is most closely related.

Based on the above characteristics, the classification name of the strain is Bacillus siamensis B11, which has been deposited in the General Microbiology Center of the China Institute of Microbiology, Chinese Academy of Sciences, on June 1, 2020, with a deposit number of It is CGMCC No.19894.

Example 2 Preparation of Bacillus siamese B11 nano-emulsion

The Bacillus siamese B11 is fermented, mixed with an emulsifier, and homogenized to prepare a nano-microemulsion, which specifically includes the following steps:

(1) Fermentation: inoculate Bacillus siamese slanted seeds in liquid medium with an initial inoculum of 4%, and then shake and culture for 48h at 25°C, 185r/min, and aeration volume of 200L/h;

Wherein, the liquid medium contains the following components by weight: 0.5% nano-silica, 2% sucrose, 1% tryptone, 0.1% sodium chloride and 0.05% magnesium sulfate, and the rest are sterile water, liquid medium The pH is 7.2.

(2) prepare nano-microemulsion: add emulsifier, stabilizer, antifreeze to above-mentioned fermentation liquid and make the weight percentages of emulsifier, stabilizer and antifreeze in the autoclave to be 10%, 2%, 5%, respectively. %, mix, then homogenize to obtain Bacillus siamese B11 nano-emulsion; wherein, the emulsifier is prepared by mixing Tween 80 and sodium lignosulfonate in a mass ratio of 1:3, and the stabilizer is sorbic acid alcohol , antifreeze polyethylene glycol.

After the above-prepared nano-emulsion is bagged, the preventive and therapeutic effects are not affected by storing at room temperature for 1.5 years or at low temperature (4° C.) for 3 years.

Embodiment 3 The prevention and treatment experiment of Mianzhu dieback

Preparation of Bacillus siamese B11 nano-emulsion dilution: take 1 g of the Bacillus siamese B11 nano-emulsion prepared in Example 2, and dilute it with sterile water to a dilution with a viable spore content of 1×10 ⁹ cfu/ml .

Preparation of pathogenic bacteria solution: 14 days after the pathogenic bacteria were inoculated in the PDA medium, a spore suspension was prepared. A petri dish was rinsed with 50 ml of sterile water, sucked out with a pipette, filtered with absorbent cotton, filtered with a centrifuge, diluted with sterile water, observed on a hemocytometer to calculate the spore concentration, and diluted to 1×10 ⁶ cfu/ml. The pathogenic bacteria solution was prepared by mixing Arthrobotryum paraphaeospermum and Arthrobotryum rickii spore suspension 1:1 (weight ratio).

Potted plant control experiment: select healthy Mianzhu in the Yibin Mianzhu planting area and plant it in a greenhouse for a potted test (2-year-old, bamboo height 1m), respectively do the following treatments: 1) First use acupuncture method to inoculate the pathogenic bacteria liquid in the middle of the branches, each plant 100ml, 15 days later, inoculate different dosages of Bacillus siamese B11 nano-microemulsion dilution by spraying method, 100ml per strain. 2) Each strain was first inoculated with 100 ml of Bacillus siamese B11 nano-microemulsion dilution solution of different dosages, and 100 ml of pathogenic bacteria solution was inoculated after 15 days. 3) Simultaneously inoculate pathogenic bacteria and different concentrations of Bacillus siamese B11 nano-emulsion. 10 replicates per treatment, sterile water and sterile culture medium were used as controls.

The incidence of disease was observed 30 days after the treatment was completed, disease investigation and statistics were carried out, and the incidence rate, disease index and control effect were calculated, and the records were shown in Table 4.

Field control experiment: in the Mianzhu planting area and healthy Mianzhu planting area where the onset of dieback disease (caused by Arthrinium paraphaeospermum and Arthrobotryum rickii after investigation and identification) is divided into areas, according to 50ml/mu, 40ml/mu, 30ml/mu, 20ml/mu, 10ml/mu of diluent (1×10 ⁹ cfu/ml) was sprayed once every ten days for a total of 3 times, with 10 replicates per treatment, and sterile water and sterile culture medium were used as controls respectively. After 30 days, the incidence of disease was observed, disease investigation and statistics were carried out, and the incidence rate, disease index and control effect were calculated, and the records were shown in Table 5.

Disease grading standard: grade 0: no withered; grade 1: branch withered below 25%; grade 2: 25% to 50% (including 25% and 50%) branch withered; grade 3: 50% to 75% of branch withered (including 75%); Grade 4: more than 75% of branches withered.

Incidence rate (%)=(number of diseased plants/sum of number of plants)×100;

Disease index = [∑ (number of diseased plants × representative value) / sum of the number of plants × representative value of the most severe disease level] × 100;

Control effect (%)=[(growth rate of disease index of control - growth rate of disease index of treatment)/growth rate of disease index of control]×100.

Table 4 Potted control effect of B11 microemulsion on Mianzhu dieback

Table 5 Field control effect of B11 microemulsion on Mianzhu dieback

Because when sterile water and sterile culture medium are used as controls, there is no significant difference between the two, therefore, only the data when sterile water is used as a control is given in Table 4 and Table 5.

As can be seen from Table 4 and Table 5, in the test of Bacillus siamese B11 preventing and controlling Mianzhu blight disease, it was found that the preventive effect of Bacillus siamese B11 on Mianzhu blight disease was far higher than the therapeutic effect, which may be due to the biocontrol strain. Application in the early stage of crop growth can colonize the plant surface earlier, occupy the living space, and reduce the infection rate of pathogenic bacteria; after the second application in the middle stage, it can further improve its reproductive ability and inhibit the growth of pathogenic bacteria. However, it is only used when the disease is serious, because the plants have been infected by pathogenic bacteria, the colonization and reproduction ability of the biocontrol strains will inevitably decrease, thereby reducing the control effect. Therefore, it should be applied from the early growth stage of Mianzhu after transplanting. After the B11 nano-emulsion is diluted, the control effect decreases with the increase of dilution, showing different control effects. The control effect of the dosage of 10-50ml/mu is more than 50%, and the dosage of 50ml/mu can prevent and control Mianzhu dieback. The effect is the best, and the control effect is up to 98%. The recommended dosage is 30-40ml/mu for comprehensive cost and control effect.

The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection of the present invention. within the range.

sequence listing

<110> Sichuan Agricultural University

<120> Application of Bacillus siamese B11 in the prevention and/or treatment of Mianzhu dieback

<160> 1

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<210> 1

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<212> DNA

<213> Artificial Sequence

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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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