CN105112314A - Separation and identification of Lysobacter gummosus OH17 with wide spectrum and high efficiency antagonistic action on plant pathogenic fungi and oomycete - Google Patents

Abstract

The invention relates to a biocontrol bacterial strain OH17 with strong antagonistic activity to plant pathogenic fungi and oomycetes. The strain is characterized in that: the strain is Lysobacter? gummosus, belonging to Xanthomonadaceae, Lysobacter, CGMCC? No. 8649. Colony morphology: different forms on different media. On NA medium, the colony is light yellow, the surface of the bacterium is round and shiny, and the surface is prominent; on LB, the growth is slow, and the colony is always yellow; on 10% TSA, the colony White, flat surface; does not grow in TSA. Physiological characteristics: Gram-negative, rod-shaped, without flagella; optimum growth temperature: 28°C. The strain has broad-spectrum and high-efficiency antagonistic effects on the tested plant pathogenic fungi and oomycetes.

Description

Isolation and identification of a broad-spectrum and high-efficiency antagonistic phytopathogenic fungus and oomycete Lysobacter colloidus OH17

(1) Technical field

The invention belongs to the field of microorganisms, and in particular relates to the isolation and identification of a bacterial strain with antagonistic activity to plant pathogenic fungi and oomycetes.

(2) Background technology

Diseases caused by pathogenic fungi account for about 70-80% of plant diseases and are one of the main obstacles affecting agricultural output, quality and benefit in our country. Chemical pesticides are generally used to prevent and control fungal diseases in production. However, food quality and safety issues and ecological security issues caused by chemical pesticide residues have become the focus of attention from all walks of life.

The development of ecologically safe and environmentally friendly biopesticides is one of the effective ways to solve the above problems. The biopesticides currently used are mainly microbial pesticides and agricultural antibiotics. The former mainly use living microorganisms as active ingredients, while the latter use microbial secondary metabolites as active ingredients. The development of these two types of biopesticides depends on the identification and mining of microbial resources with biocontrol potential.

Lysobacter (Lysobacterspp.) belongs to Xanthomonadaceae, Lysobacter genus. As of July 2014, there are 32 species of Lysobacterium bacteria identified internationally, including Lysobacterenzymogenes, Lysobacterantibioticus, Lysobactergummosus and Lysobacterium browning ( Four species of Lysobacterbrunescens) have high antibacterial activity and plant disease biocontrol potential, and are a new type of plant disease biocontrol bacteria. These bacteria have significant antagonistic effects on plant pathogenic fungi, oomycetes, Gram-negative bacteria (G ^- ), positive bacteria (G ⁺ ) and nematodes. The mechanism of action is: (1) produce small molecule antibacterial secondary Metabolism substances; (2) secrete and produce a large number of extracellular enzymes, including chitinase, β-1,3-glucan, protease and cellulase; (3) induce plant disease resistance; (4) better colonization ability.

In summary, the isolation and identification of new strains of lysobacteria with important biocontrol potential is of great significance for the construction of biocontrol lysobacteria resource bank and the development of new, efficient and broad-spectrum biopesticides (agricultural antibiotics).

(3) Contents of the invention

In the present invention, a bacterial strain OH17 with antagonistic activity against fungi and oomycetes was screened from the rhizosphere soil of crops with Saccharomyces cerevisiae as a target bacterium by means of dilution coating, and was identified as colloidal lysate by morphology, 16SrDNA and other methods. Bacillus (Lysobacter gummosus). The strain has strong antagonistic activity against various plant pathogenic fungi and oomycetes tested.

The bacterial strain OH17 provided by the present invention has been preserved on December 27, 2013 at No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, the General Microbiology Center of the Chinese Microbiological Culture Preservation Management Committee (abbreviated as CGMCC), the preservation number is CGMCCNo.8649, and the classification name is Lysobactergummosus colloidus.

(4) Description of drawings

Figure 1 The colony morphology of OH17 on different media

Figure 2 SEM observation picture of OH17

Figure 3 The antagonistic effect of OH17 on pathogenic fungi and oomycetes

Figure 4 16SrDNA PCR electrophoresis of OH17

Phylogenetic tree of Figure 5OH17

(5) Specific implementation methods

The present invention will be further elaborated below through specific implementation, but the present invention is not limited.

1 Materials and methods

1.1 Test strains, medium

Test strains: Saccharomyces cerevisiae, Magnaporthegrisea, Fusarium graminearum, Sclerotias clerotiorum, Verticillium dahliae, Rhizoctorziasolani, Alternaria alternata, Colletotrichumgloeosporioides, Botryosphaeria berengeriana, Alternariasolani, Fusarium solani, Fusarium moniliforme, Pepper blight Phytophthoracapsici, Pythiumaphanidermatum.

Medium:

NA medium: 3g of beef extract, 5g of peptone, 1g of Yeastextract, 10g of sucrose, 1L of distilled water, pH7.2, add 1.5g/100mL of agar after subpackaging the solid medium.

10% TSA: 3 g of Tryptic-Soytone-Broth-Medium, 1 L of distilled water, and 1.88 g/100 mL of agar after aliquoting.

TSA: 30 g of Tryptic-Soytone-Broth-Medium, 1 L of distilled water, and 1.88 g/100 mL of agar after aliquoting.

LB: Tryptone 10g, NaCl 10g, YeastExtract 5g/L, add water to dissolve, finally adjust the volume to 1L, adjust pH=7.0-7.2, after subpackaging, add 1.5g/100mL agar powder to LB solid medium.

1.2 Isolation of rhizosphere soil bacteria

Take 10 parts of crop rhizosphere soil samples, sieve the collected soil samples through a 2mm soil sieve, weigh 10g into a conical flask filled with 90mL sterile water, add an appropriate amount of antibiotics, shake the shaker for 30min, and then dilute 10 times in a gradient of 5 Second-rate. Pipette 100 μl of the diluted solution and spread it on LB plates, and spread each dilution on 3 plates repeatedly, and incubate at 28°C for 2 days. The appropriate dilution is 100-300 colonies on each plate. Pick different types of colonies on LB plates for purification, and store them at -70°C after purification for 3 times.

1.3 Determination of antagonistic activity of antagonistic bacteria against pathogenic fungi and oomycetes

A single colony of OH17 was picked and cultured in LB liquid medium for 24-48 hours to obtain a bacterial liquid with OD ₆₀₀ =1.0. Using the plate confrontation method, place the fungus or oomycelium block for testing in the center of the PDA plate. Take 3 μl of antagonistic bacteria and drop them around the plate, repeat three times, incubate upside down at 28°C until the antagonistic effect appears, observe and take pictures.

1.4 Identification of antagonistic bacterial strains

1.4.1 Colony Morphological Observation and Electron Microscopic Observation

Colony morphology: Take 3 μl of OH17 bacteria liquid and spot them in the center of 10% TSA, TSA, NA, and LB medium respectively, culture at 28°C for 2-4 days, and observe the morphology. On NA medium, the colony is light yellow, the surface of the bacterium is round and shiny, and the surface is prominent; on LB, the growth is slow, and the colony is always yellow; on 10% TSA, the colony is white and the surface is flat; it does not grow on TSA.

Electron microscope observation:

(1) Streak OH17 on the LB medium (the age of the bacteria should not be too large), wash the colonies with a small amount of sterilized water in the place where the colonies grow fresh, and gently shake the culture dish to make the sterilized water repeatedly around the fresh colonies Rinse and prepare a bacterial suspension.

(2) Add 1-2 drops of fresh PTA negative stain solution on the paraffin plate.

(4) Take a new copper mesh to absorb the bacterial suspension, and put it on a clean filter paper to dry.

(5) When the bacterial suspension on the copper mesh is 70% to 80% dry, touch the side of the copper mesh that absorbs the bacterial suspension to the negative dye solution, and float and dye for 15 seconds (if the time is too long, the color will be darker).

(6) Take out the copper mesh sheet from the negative dye solution, blot the excess negative dye solution with filter paper, and bake it under the fluorescent lamp for 45 seconds to make it dry.

(7) Place the prepared sample in a transmission electron microscope Hitachi H-650, and observe the cell morphology at 80 kV.

OH17 is rod-shaped and has no flagella.

1.4.216SrDNA identification

The OH17 DNA was extracted using the TIANGEN Bacterial Genomic DNA Extraction Kit as a template for 16S rDNA amplification. Primers were designed using Primer 5.0 software, upstream primer 16S-F: AGAGTTTGATCATGGCTCAG, downstream primer 16S-R: ACGGTTACCTTGTTACGACTT, the primer sequence was synthesized by Yingwei Jieji (Shanghai) Trading Co., Ltd., and the amplified band was about 1500bp. PCR reaction system (25μl): 10×buffer2.5μL; Mg ²⁺ (25mM) 2μL; dNTP (1.25mM) 2μL; forward primer (20pmol/μL) 0.5μL; reverse primer (20pmol/μL) 0.5μL; Trans-Taq polymerase 0.3 μL; gDNA (10 ng/μL) 0.5 μL; ddH ₂ O 16.7 μL. PCR reaction conditions: pre-denaturation at 95° for 5 min, denaturation at 94° for 30 s, annealing at 55° for 30 s, extension at 72° for 1 min and 30 s, 30 cycles, and extension at 72° for 8 min. The PCR reaction product was detected by 1% agarose coagulation electrophoresis. Compared with DL2000marker, the fragment size was correct, which was the target fragment, as shown in Figure 4.

The PCR product containing the target fragment was purified and recovered using the AXYGENPCRcleanup kit, and the purified product was sent to Yingwei Jieji (Shanghai) Trading Co., Ltd. for sequencing. The length of the obtained sequence was 1428bp.

The obtained sequence was compared with the NCBI nucleic acid database for homology comparison analysis, and the results showed that the sequence similarity between strain OH17 and Lysobactergummosus KCTC12132 was as high as 99%. Some of the strains with high similarity and some other strains of Lysobacterium and Xanthomonas were selected for phylogenetic analysis, and a phylogenetic tree was constructed by using the Neighbor-Joining method in the MEGA software, as shown in FIG. 5 . It can be seen from the figure that the genetic distance between OH17 and Lysobactergummosus is the shortest, and the genetic distance with other species of Lysobactergummosus is far. According to the results of homology comparison and phylogenetic analysis, OH17 is Lysobacter colloidis.

OH1716SrDNA sequence, the length is 1428bp:

caccgtggcagcgccctcccgaaggttaagctacctgcttctggtgcaacaaactcccat

ggtgtgacgggcggtgtgtacaaggcccgggaacgtattcaccgcagcaatgctgatctg

cgattactagcgattccgacttcacggagtcgagttgcagactccgatccggactgagat

agggtttctgggattggcttgccctcgcgggtttgcagccctctgtccctacattgtag

tacgtgtgtagccctggccgtaagggccatgatgacttgacgtcatccccaccttcctcc

ggtttgtcaccggcggtctccttagagttcccaccattacgtgctggcaactaaggacaa

gggttgcgctcgttgcgggacttaacccaacatctcacgacacgagctgacgacagccat

gcagcacctgtctcacggttcccgaaggcaccaatccatctctggaaagttccgtggatg

tcaaggccaggtaaggttcttcgcgttgcatcgaattaaaccacatactccaccgcttgt

gcgggcccccgtcaattcctttgagtttcagtcttgcgaccgtacttcccaggcggcgaa

cttaacgcgttagcttcgatactgagagccaagttgctcccaacatccagttcgcatcgt

ttagggcgtggactaccagggtatctaatcctgtttgctccccacgctttcgtgcctcag

tgtcagtgctggtccaggtagtcgccttcgccacagatgttcctcccgatatctacgcat

ttcactgctacaccgggaattccactacccctctaccgcactctagtcagccagtttccaa

tgccattcccaggttgagcccagggctttcacatcagacttaacaaaccacctacgcacg

ctttacgcccagtaattccgagtaacgcttgcacccttcgtattaccgcggctgctggca

cgaagttagccggtgcttattcttccggtaccgtcatgacatcggggtattaacccaatg

cttttctttccggacaaaagtgctttacaacccgaaggccttcttcacacacgcggcatg

gctggatcaggcttgcgcccattgtccaatattccccactgctgcctcccgtagtagtct

ggaccgtgtctcagttccagtgtggctgatcatcctctcagaccagctacggatcgtcgc

cttggtgggcctttaccccgccaactagctaatccgacgtcggctcatctatctgcgcga

agcccgaaggtcctccgctttcacccgtaggtcgtatgcggttattagcgtaagtttccct

acgttatcccccacaaataggcagattccgacgtattcctcacccgtccgccactcgcca

cccaaggagcaagctcctctgtgctgccgttcgacttgcatgtgttag

Claims (2)

1. A biocontrol bacterial strain with strong antagonistic activity against phytopathogenic fungi and oomycetes, characterized in that the bacterial strain is Lysobacter colloidus, the preservation registration number is: CGMCC No.8649, named LysobactergummosusOH17. 2. the application of the bacterium described in claim 1 is characterized in that to rice blast fungus (Magnaporthegrisea), wheat scab fungus (Fusarium graminearum), rape sclerotiorum (Sclerotias clerotiorum), cotton Verticillium dahliae (Verticillium dahliae), rice grain Rhizoctorziasolani, Alternariaalternata, Colletotrichumgloeosporioides, Botryosphaeriaberengeriana, Alternariasolani, Fusarium solani, Bakanae rice Fungi such as Fusarium moniliforme and plant pathogenic oomycetes such as Phytophthora capsici and Pythiumaphanidermatum have strong antagonistic activity.