CN113549578B - A strain of Bacillus siamese BsNlG13 inhibiting rice blast fungus and promoting seed germination and its application - Google Patents

Abstract

The invention discloses a strain of Siamese bacillus (BsNlG 13) for inhibiting rice blast germ and promoting seed germination and application thereof, wherein the strain is preserved in the microorganism strain preservation center of Guangdong province in 12 months of 2020, and the preservation number is GDMCC NO:61391. the research of the invention shows that the fermentation liquor of the BsNlG13 strain of the Siamese bacillus can effectively inhibit the growth of rice blast bacteria, so that hypha of the rice blast bacteria is deformed and cannot grow normally; meanwhile, the fermentation liquor of the Siamese bacillus BsNlG13 strain has remarkable promotion effect on the germination of rice seeds. The Siamese bacillus BsNlG13 strain can be used as a good biocontrol and plant production regulation resource, is used for preventing and controlling rice blast and promoting rice seed germination, and has good application prospect.

Description

A strain of Bacillus siamese BsNlG13 inhibiting rice blast fungus and promoting seed germination and its application

technical field

The invention relates to the technical field of agricultural microorganisms, in particular to a Bacillus siamese BsN1G13 strain for inhibiting rice blast fungus and promoting seed germination and application thereof.

Background technique

As a very important food crop, rice is grown all over the world. Diseases that occur during rice cultivation are currently an important limiting factor for rice yield. Rice blast is an important rice fungal disease caused by the rice blast fungus Magnaportheoryzae, and it is one of the three major diseases of rice. Rice blast occurs in all rice regions in the world, and occurs more frequently in rice leaves and nodes. When the disease occurs, it can cause a large reduction in rice yield by 10%-30% every year, resulting in serious economic losses. At present, the prevention and control methods for rice blast are mainly to study the breeding of disease-resistant rice varieties or to use chemical agents for prevention and control. However, the development cycle of disease-resistant varieties is long and often loses resistance to pathogenic bacteria due to genetic variation of pathogenic bacteria, and chemical control will also produce certain environmental problems such as drug resistance and drug residues. Therefore, the use of biological agents to treat rice blast Prevention and control can have the advantages of being environmentally friendly and not easy to produce resistance. Biological agents can be used as a good control method.

According to reports, some antagonistic bacteria have been used to prevent and control rice blast, such as Bacillus subtilis, marine Bacillus subtilis, Burkholderia and so on. Bacillus siamensis, also translated as Bacillus simum, was included as an effective species name by the publication International Journal of Systematic and Evolutionary Microbiology in 2010, belonging to the genus Bacillus in the family Bacillus. Existing studies have shown that Bacillus siamica has a certain antagonistic effect on some pathogenic bacteria, and has the prospect of being used as a biocontrol agent. Studies have also shown that Bacillus siamese promotes breeding and crop cultivation. Feeding catfish with feed containing the strain not only has no side effects, but also can promote fish growth (Meidong et al., 2017); anaerobic digestive juice is the main Raw materials, adding Bacillus siamese to develop bio-fertilizers, after application, the nitrogen use efficiency of crops is improved, and the yield is also significantly increased (Pastor-Bueis et al., 2017). Chinese patent CN105505834A discloses a strain of Bacillus siamese for preventing and treating pear ring spot and soft rot, which has a certain bacteriostatic rate on rice blast fungus. However, there are few reports on the influence of Bacillus siamese on the germination of rice seeds. reports. Due to the simultaneous evolution of rice blast fungi and the limitations of a single antagonistic strain, it is still necessary to find new strains for the control of rice blast disease.

Contents of the invention

The object of the present invention is to overcome the above-mentioned defects and deficiencies that exist in the prior art, and a kind of suppressing rice blast bacterium and promoting seed germination Bacillus siamensis (Bacillus siamensis) BsN1G13 bacterial strain are provided.

The second object of the present invention is to provide the application of said Bacillus siamensis (Bacillus siamensis) BsN1G13 bacterial strain.

Above-mentioned purpose of the present invention is given to realize by following technical scheme:

A strain of Bacillus siamensis BsNlG13, which was deposited in the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on December 25, 2020, and the strain preservation number is GDMCC NO: 61391.

Specifically, the nucleotide sequence of the 16s rDNA of the bacterial strain is shown in SEQ ID NO:1.

The present invention obtains a Bacillus siamese BsN1G13 bacterial strain from the midgut of the female adult of brown planthopper. The present invention adopts the plate confrontation method to carry out the antibacterial test through the PDA plate. Bacteria MoZC13 has a significant antagonistic effect, and has a strong inhibitory effect on the growth of Magnaporthe grisea, and BsNlG13 has a destructive effect on the hyphae of Magnaporthe grisea. Simultaneously, show by seed germination test, Bacillus siamensis (Bacillus siamensis) BsNlG13 bacterial strain has the ability of effectively promoting rice seed germination percentage, add the thalline fermentation liquid of above-mentioned BsNlG13 bacterial strain in the water soaked before sowing of rice seed, The germination rate of rice seeds can be significantly increased by 8.56%.

Therefore, the present invention also provides the application of Bacillus siamensis BsN1G13 strain or its thalline fermentation liquid in inhibiting rice blast fungus or preventing and treating rice blast.

The present invention also provides the application of Bacillus siamensis BsN1G13 strain or its thalline fermentation liquid in improving the germination rate of rice seeds.

Application of Bacillus siamensis BsN1G13 strain or its fermented liquid in the preparation of preparations for preventing and treating rice blast and/or improving the germination rate of rice seeds.

The present invention also provides a preparation for preventing and controlling rice blast and/or increasing the germination rate of rice seeds, which is characterized in that the strain according to claim 1 or its fermentation liquid is used as an active ingredient.

Preferably, the bacterial fermentation broth concentration of the preparation is 1×10 ⁷ CFU/mL.

Further preferably, the preparation method of the Bacillus siamese BsN1G13 bacterial liquid: insert the glycerol bacteria BsN1G13 stored at -80°C into fresh liquid LB medium at a ratio of 1:100, activate at 220 rpm/min at 37°C for 12 hours to obtain seed bacteria, Then take the seed bacteria at a ratio of 1:100 and inoculate them in fresh liquid LB medium, shake at 220 rpm/min at 37°C for 4 hours to obtain a highly active bacterial liquid, and measure the OD value.

The present invention also provides the application of the preparation described in claim 6 or 7 in the preparation for preventing and treating rice blast and/or improving the germination rate of rice seeds

The present invention also provides a method for improving the germination rate of rice seeds, which is characterized in that the Bacillus siamensis BsNlG13 strain is configured into an aqueous solution with a final concentration of 1×10 ⁶ CFU/mL, and the rice seeds are soaked and germinated .

Further preferably, the rice seeds are Xiuzhan 15.

Compared with the prior art, the present invention has the following beneficial effects:

The present invention provides a strain of Bacillus siamensis BsN1G13, which was deposited in Guangdong Microbial Culture Collection Center (GDMCC) on December 25, 2020, and the strain preservation number is GDMCC No: 61391. The research of the present invention shows that Bacillus siamensis (Bacillus siamensis) BsNlG13 strain has a strong inhibitory effect on the growth of rice blast fungus, and the bacteriostatic rate can reach 82.6 ± 0.5%, which provides a new strain for the biological control of rice blast resources, with good biological control potential and application prospects. Simultaneously show that Bacillus siamensis (Bacillussiamensis) BsNlG13 bacterial strain has the ability of effectively promoting the germination rate of rice seeds, adding the thalline fermentation liquid of the above-mentioned BsN1G13 bacterial strains to the water soaked before rice seed sowing can make the germination of rice seeds The seed germination rate is significantly increased by 8.56%, which provides a new method idea and application prospect for improving the germination rate of seeds.

Description of drawings

Figure 1 is a morphological diagram of the purified culture colony of Bacillus siamensis BsNlG13. A: Back view of colonies grown in LB medium for 12 hours at 37°C; B: Front view of colonies grown in LB medium for 12 hours at 37°C.

Figure 2 is a phylogenetic tree of Bacillus siamese BsN1G13 constructed based on 16s rDNA.

Fig. 3 is the test result of Bacillus siamese BsN1G13 inhibiting Magnaporthe grisea. A is the confrontation culture diagram of Bacillus siamese BsNlG13 inhibiting Magnaporthe grisea; B is the difference analysis diagram of the colony diameter of Magnaporthe grisea after the confrontation culture of Bacillus siamese BsNlG13 inhibiting Magnaporthe grisea; C is the diagram of Bacillus siamese BsNlG13 inhibiting the confrontation of Magnaporthe grisea Morphological changes of blast fungus hyphae after culture.

Fig. 4 is the test result of rice seed treated with Bacillus siamese BsN1G13. A is a graph comparing the germination effect of rice seeds treated with Bacillus siamese BsNlG13; B is a graph showing statistical differences in the germination rate of rice seeds treated with Bacillus siamese BsNlG13.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

Isolation and cultivation of embodiment 1 bacterial strain BsN1G13

Dissected and plated: BsNlG13 strain was isolated from the intestinal tract of female adults of Nilaparvata lugens, and was isolated and cultured by dilution coating method. Specifically, dissect the intestinal tract of the female adult N. lugens planthopper under a stereomicroscope, place the dissected intestinal tract in PBS, grind it into a homogenate with a hand-held electric grinder, dilute it in four concentration gradients, and spread it on three culture media (LB medium, EB medium, NA medium), each treatment was repeated three times. They were cultured in a constant temperature incubator at 37°C and observed every 24 hours.

Continuous purification and culture photography: After a single colony grows in the selective medium, first pick a single colony according to the color, size and shape of the colony, and streak and purify it on the corresponding medium for more than 5 times, and then streak the isolated strain The plate was photographed (Figure 1) and transferred to the corresponding liquid medium. When the bacteria were shaken to the exponential growth phase, they were stored in 25% aqueous glycerol solution and frozen in a -80°C refrigerator for later use.

Preparation of selective separation medium: LB medium (10g peptone, 5g yeast extract, 10g NaCl, 15g agar, dissolved in 1L sterile water, adjusted to pH 7.0, autoclaved at 115°C for 20 minutes). EB medium (3g beef extract powder, 10g tryptone, 15g yeast powder, 5g sodium chloride, 10g glucose, 15g agar, dissolved in 1L sterile water, adjusted to pH 7.2, autoclaved at 115°C for 20 minutes). NA medium (10g tryptone, 3g beef extract powder, 5g sodium chloride, 15g agar, dissolved in 1L sterile water, adjust the pH value to 7.2, autoclave at 115°C for 20 minutes).

Identification and Phylogenetic Analysis of Example 2 Bacterial Strain BsN1G13

(1) Traditional biological identification:

Observed under an optical microscope, the colony shape is round or oval, the colony is milky white and translucent, the edge is complete, the colony is moist and slightly raised. The growth rate is faster at room temperature and 37°C, and the colony is larger after 12 hours of cultivation, with a diameter of 3-5mm.

(2) Determination of physiological and biochemical characteristics:

Bacterial physiological and biochemical assays were carried out with reference to the method in the "Common Bacterial System Identification Manual", using bacterial micro-biochemical reaction tubes. Microscopic observation shows that the bacterium is a rod-shaped, Gram-positive bacterium. The physiological and biochemical characteristics of the bacteria are shown in Table 1 below.

Table 1 Biochemical characteristics of BsNlG13

Note: "+" is a positive reaction, and "-" is a negative reaction.

(3) Molecular biological identification (16S rDNA):

Bacterial genomic DNA was extracted and isolated using TIANamp Bacteria DNA Kit. The 16S rDNA universal primers 27F (27F: 5'-AGTTTGATCMTGGCTCAG-3') and 1492R (1492R: 5'-GGTTACCTTGTTACGACTT-3') were used as primers, and the extracted DNA was used as a template to amplify bacterial 16S rDNA. The PCR reaction system is shown in Table 2. Gently mix and centrifuge briefly to carry out the PCR reaction. The basic conditions of PCR reaction were set as follows: pre-denaturation at 98°C for 2min; denaturation at 98°C for 10s, annealing at 50°C for 15s, extension at 72°C for 15s, 30 cycles; 72°C for 5min; 10°C. After the PCR reaction, the PCR product was detected by 1% agarose gel, recovered and purified by cutting the gel, and then sent to Guangzhou Qingke Biotechnology Co., Ltd. for sequencing identification.

Table 2 Bacterial 16S rDNA PCR amplification system (20μL)

(4) Phylogenetic analysis of strain PxG1:

The obtained sequencing results were spliced by DNAStar, and the sequence was compared with the rRNA/ITS database in NCBI to determine the taxonomic status of the closely related bacterial strains.

After selecting and downloading the close relative sequences of the intestinal isolate BsNlG13 strain, the multiple sequence alignment analysis was performed with ClustalW software, and then the Neighbor-Joining method (Neighbor-Joining) was used to construct the phylogenetic tree with the Mega7.0 software, and the bootstrap value was adjusted and the phylogenetic tree was checked reliability. The results are shown in Figure 2. It can be seen from Figure 2 that BsNlG13 has the highest similarity with Bacillussiamensis KCTC 13613 strain PD-A10 (NR 117274.1:34-1474). Name it Bacillus siamensis BsNlG13 strain of brown planthopper Bacillus siamensis (Bacillus siamensis) BsNlG13. The preservation address is No. 100, Xianlie Middle Road, Guangzhou City, Guangdong Province.

The inhibitory activity of embodiment 3 bacillus siamese BsN1G13 to rice blast fungus

The inhibitory effect of Bacillus siamese BsN1G13 on Magnaporthe grisea was determined by plate confrontation culture method. Select solid medium as PDA medium. Inoculate the activated blast fungus MoZC13 bacterial sheet with a diameter of 5mm in the center of the PDA medium first, and inoculate the bacterial solution of the antagonistic Bacillus siamese BsN1G13 by streaking at a symmetrical place 2.5cm away from the bacterial sheet to inoculate only the blast fungus. The flat plate of MoZC13 bacterial slices was used as a control, and placed in a constant temperature incubator at 25°C for upside-down culture for 5 days under 75% humidity. The antibacterial effect of BsNlG13 was evaluated by measuring the diameter of the blast fungus colony after co-cultivation for 5 days. Perform biological replicates.

The results of plate confrontation are shown in Figure 3A and Figure 3B. The diameter of Magnaporthe grisea MoZC13 was 376±4.7mm in culture dish for 5 days. mm, the results showed that Bacillus siamese NlG13 had a significant antagonistic effect on Magnaporthe grisea MoZC13. Further, the diameter of rice blast fungus MoZC13 was 860±0.5mm after being cultured in a petri dish for 10 days, and the diameter of rice blast fungus MoZC13 and BsNlG13 cultured in a petri dish for 10 days was 150±4.1mm, showing a strong antibacterial effect , The bacteriostatic rate can reach 82.6±0.5%. Further using a microscope to observe the edge hyphae of pathogenic bacteria in confrontation culture, it was found that the growth of mycelia on the edge of pathogenic bacteria in the treatment group was deformed, and the phenomenon of mycelium rupture occurred, while the mycelia of the control group grew normally, and the thickness of mycelia was uniform, complete and smooth ( Figure 3C).

The PDA medium is 46 g of potato dextrose agar medium (without antibiotics) per 1 L of distilled water, heated until completely dissolved, and then autoclaved at 115° C. for 20 minutes.

The preparation method of the Bacillus siamese BsN1G13 bacterial liquid: insert the glycerol bacteria stored at -80°C into fresh liquid LB medium at 1:100, activate at 220rpm/min at 37°C for 12h to obtain seed bacteria, and then add 1:100 Take the seed bacteria and inoculate them in fresh liquid LB medium, shake at 220rpm/min at 37°C for 4 hours to obtain a highly active bacterial solution, measure the OD value, and prepare it to 1×10 ⁷ CFU/mL.

The influence of embodiment 4 Bacillus siamese BsN1G13 on the germination rate of rice seeds (rust accounted for 15)

Pick 100 full and plump common commercial rice seeds (Xiuzhan 15), rinse them with tap water and place them in a sterile petri dish, take 18 mL of tap water and 2 mL of Bacillus siamese BsNlG13 bacterial solution (1×10 ⁷ CFU/mL ) are mixed evenly and added to the petri dish, and the seeds are soaked and germinated. In the control group, only 100 seeds and 20 mL of tap water were added, and then the petri dish was placed in a constant temperature incubator with a temperature of 28°C and a humidity of 85% for dark cultivation. The tap water was replaced every 24 hours, and 2 mL was left in each petri dish. Only 18mL of tap water was added to the mixed liquid, and the germination of rice seeds was counted after five days of cultivation. Photographs were taken and recorded, and the experiment was repeated three times.

As shown in Figure 4A and Figure 4B, the seed germination rates of the control group and the treatment group were 89 ± 1.63% and 97.33 ± 1.25%, respectively, and compared with the control group, the seed germination rate of the treatment group was significantly increased by 8.56%. It shows that adding a certain amount of Bacillus siamese BsN1G13 bacteria solution can significantly improve the germination rate of rice seeds when soaking before germination. The results showed that the Bacillus siamese BsNlG13 bacterial solution could be used as an auxiliary bacterial agent to improve the germination rate of rice seeds, which was beneficial to improve the germination rate of rice.

In summary, a kind of Bacillus siamese BsN1G13 provided by the present invention can effectively inhibit the growth of rice blast fungus, causing its hyphae to deform and unable to grow normally, and can be used as a good biocontrol resource, with better Biocontrol prospects. In addition, the Bacillus siamese BsN1G13 provided by the present invention also has a good germination-promoting effect on rice seeds (Xiaozhan 15), can effectively increase the germination rate of rice seeds, and can be used in the pre-planting treatment of rice.

The above descriptions are only preferred embodiments of the present invention, and the above embodiments cannot limit the implementation scope of the present invention. Modifications or replacements made by those skilled in the art to the above embodiments all fall within the protection scope of the present invention.

sequence listing

<110> South China Agricultural University

<120> A Strain of Bacillus siamese BsNlG13 Inhibiting Magnaporthe grisea and Promoting Seed Germination and Its Application

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cgggaaaccg gggctaatac cggatggttg tttgaaccgc atggttcaga cataaaaggt 180

ggcttcggct accacttaca gatggacccg cggcgcatta gctagttggt gaggtaacgg 240

ctcaccaagg cgacgatgcg tagccgacct gagagggtga tcggccacac tgggactgag 300

acacggccca gactcctacg ggaggcagca gtagggaatc ttccgcaatg gacgaaagtc 360

tgacggagca acgccgcgtg agtgatgaag gttttcggat cgtaaagctc tgttgttagg 420

gaagaacaag tgccgttcaa atagggcggc accttgacgg tacctaacca gaaagccacg 480

gctaactacg tgccagcagc cgcggtaata cgtaggtggc aagcgttgtc cggaattatt 540

gggcgtaaag ggctcgcagg cggtttctta agtctgatgt gaaagccccc ggctcaaccg 600

gggagggtca ttggaaactg gggaacttga gtgcagaaga ggagagtgga attccacgtg 660

tagcggtgaa atgcgtagag atgtggagga acaccagtgg cgaaggcgac tctctggtct 720

gtaactgacg ctgaggagcg aaagcgtggg gagcgaacag gattagatac cctggtagtc 780

cacgccgtaa acgatgagtg ctaagtgtta gggggtttcc gccccttagt gctgcagcta 840

acgcattaag cactccgcct ggggagtacg gtcgcaagac tgaaactcaa aggaattgac 900

gggggcccgc acaagcggtg gagcatgtgg tttaattcga agcaacgcga agaaccttac 960

caggtcttga catcctctga caatcctaga gataggacgt ccccttcggg ggcagagtga 1020

caggtggtgc atggttgtcg tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg 1080

agcgcaaccc ttgatcttag ttgccagcat tcagttgggc actctaaggt gactgccggt 1140

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tcacaccacg agagtttgta acacccgaag tcggtgaggt aacctttatg gagccagccg 1440

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Claims (8)

1. A strain of Bacillus siamensis BsNlG13, characterized in that the strain was deposited in Guangdong Microbial Culture Collection Center (GDMCC) on December 25, 2020, and the strain preservation number is GDMCC NO: 61391. 2. The Bacillus siamensis BsN1G13 strain according to claim 1, wherein the nucleotide sequence of the 16S rDNA of the strain is shown in SEQ ID NO:1. 3. The application of the Bacillus siamensis ( Bacillus siamensis ) BsN1G13 bacterial strain or its thalline fermentation liquid described in claim 1 in improving the germination rate of rice seeds. 4. The application of the Bacillus siamensis ( Bacillus siamensis ) BsN1G13 strain or its fermented liquid according to claim 1 in the preparation of a preparation improving the germination rate of rice seeds. 5. A preparation for improving the germination rate of rice seeds, characterized in that the strain according to claim 1 or its thalline fermentation liquid is used as an active ingredient. 6. The preparation according to claim 5, characterized in that the concentration of the bacterial cell fermentation broth is 1×10 ⁷ CFU/mL. 7. A method for improving the germination rate of rice seeds, characterized in that first, Bacillus siamensis ( Bacillus siamensis ) BsNlG13 strain is fermented to prepare a bacterium liquid of 1×10 ⁷ CFU/mL, and then the bacterium liquid is configured into The rice seeds were soaked and germinated in an aqueous solution with a final concentration of 1×10 ⁶ CFU/mL. 8. The method according to claim 7, characterized in that, the rice seeds are Xiuzhan 15.

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