CN107099478A - One plant of Lycoris aurea endogenetic bacteria bulkholderia cepasea HDXY 02 and its application - Google Patents

Abstract

The present invention discloses an endophytic bacterium that antagonizes various phytopathogenic fungi, and specifically relates to Burkholderia sp. Microbiology Center, its deposit number is CGMCC No 14054, and the deposit date is April 20, 2017. The invention also discloses the cultivation method and application of the above-mentioned Burkholderia sp. HDXY-02. The Burkholderia sp. HDXY‑02 provided by the invention has a strong antagonistic effect on rice sheath blight bacteria, has good inhibitory activity on various pathogenic fungi, has a wide antibacterial spectrum, and can effectively inhibit bacteria silk grows. The present invention also provides a biocontrol bacterial agent for preventing and treating fungal diseases of crops, that is, the bacterial agent obtained by inoculating HDXY‑02 strains in culture medium and fermenting and cultivating them. The bacterial strain can be applied to microbial bacterial agents for inhibiting plant diseases, and has good development and application prospects.

Description

An Endophytic Bacteria Burkholderia HDXY-02 and Its Application

technical field

The invention belongs to the field of microorganisms, in particular to an endophytic Burkholderia sp. HDXY-02, and also relates to a Burkholderia sp. HDXY-02 for preventing and treating fungal diseases of plants cultivation methods and uses.

Background technique

Rice sheath blight, also known as moiré, is infected by Rhizoctonia solani, and mostly occurs under high temperature and high humidity conditions. Sheath blight is particularly serious in southern rice areas, and is one of the main diseases in rice production. The disease can cause rice to fail to ear, or there will be more grains for earing, and the grain weight will decrease. Due to the lack of rice varieties resistant to rice sheath blight, chemical fungicides are still effective means to control rice sheath blight, among which Jinggangmycin and Amy are the main pesticides used, and the large use of Amy will pollute the environment , and the occurrence rate of Aimi-resistant sheath blight bacteria has also increased greatly; in addition, in recent years, with the continuous use of Jinggangmycin, strains resistant to Jinggangmycin have also been found in the field. Therefore, finding new measures to prevent and treat rice sheath blight has become the top priority of plant disease control work.

As a natural member of the plant micro-ecosystem, some endophytic bacteria can inhibit pathogenic bacteria by producing antibacterial active substances such as antibiotics and hydrolytic enzymes, so as to achieve the purpose of preventing and controlling plant diseases. The use of microorganisms to control plant fungal diseases has the advantages of no pollution to the environment, no harm to humans and animals, and slowing down the emergence of pathogenic bacteria resistance. It has become a hot spot in the prevention and control of plant diseases. A number of endophytic bacteria with good control effects on plant diseases have been isolated from cotton, rice, pepper, tomato and other crops, some of which have entered the application stage and have produced certain social and environmental benefits.

In the early stage, a strain of Burkholderia HDXY-02 with antifungal activity was isolated from the root of Hudixiao. This strain has good control effect on various plant pathogenic fungi, especially the inhibitory effect on rice sheath blight most. Burkholderia, as a biocontrol bacterium, can control plant diseases. It can be made into a biofungicide, which can partially replace chemical pesticides. Several strains of Burkholderia genus biocontrol bacteria have been submitted Go to the US Environmental Protection Agency (EPA) and register as a biopesticide. Therefore, the strains of this genus can play an important role in agricultural production and environmental protection, and have good application prospects.

Invention content:

The object of the present invention is to provide a strain of Burkholderia sp. HDXY-02 with good antagonistic activity to pathogenic fungi, said Burkholderia sp. HDXY-02 bacterial strain It has been preserved in the General Microbiology Center of China Microbiological Culture Collection Management Committee, the preservation number is CGMCC No.14054, the preservation date is April 20, 2017, and the preservation address is No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Chinese Academy of Sciences Institute of Microbiology.

The biological characteristics of the Burkholderia HDXY-02 are: negative Gram staining (G ^─ ), short rod-shaped cells, no spores, aerobic, positive oxidase test, positive gelatin liquefaction reaction, The contact enzyme reaction is positive; the colony on the solid medium of beef extract peptone medium is pale yellow, round, with neat edges, smooth surface, and raised.

The Burkholderia HDXY-02 is derived from healthy roots of Hudixiao, and has a good biocontrol effect on rice sheath blight. The 16S rDNA sequence of the strain HDXY-02 was amplified, compared with NCBI online, and combined with the results of physiological and biochemical tests, it was determined that the strain HDXY-02 was Burkholderia.

Another object of the present invention is to provide a method for cultivating Burkholderia HDXY-02 for preventing and controlling plant fungal diseases. A step of culturing Burkholderia HDXY-02 in a fermentation medium is included.

The application of the Burkholderia HDXY-02 or the bacterium agent containing Burkholderia HDXY-02 as an active ingredient in production also belongs to the protection scope of the present invention.

Another object of the present invention is to provide a method for preparing a biocontrol agent, the active ingredient of which is the Burkholderia HDXY-02. By preparing the fermentation liquid containing Burkholderia HDXY-02 or the sterile fermentation supernatant obtained after filtering the fermentation liquid containing Burkholderia HDXY-02, the above aseptic fermentation The supernatant obtained by centrifuging the fermentation broth of Burkholderia HDXY-02 was used as the supernatant, and then filtered through a bacterial filter. The preparation method is simple and easy, no organic solvent is used, and the environment is safe and pollution-free.

The present invention also provides the application of the above-mentioned Burkholderia HDXY-02 in inhibiting rice sheath blight. The fermentation product of the Burkholderia HDXY-02 can effectively inhibit the growth of rice sheath blight mycelia.

The application of the above-mentioned endophytic bacterium HDXY-02 in inhibiting pathogenic fungi, that is, the inhibitory effect of Burkholderia HDXY-02 on pathogenic fungi also includes: rice blast fungus, wheat scab, and cotton wilt fungus and Sclerotinia sclerotiorum, therefore, the endophytic bacterium HDXY-02 can be used to control rice sheath blight, rice blast, wheat head blight, cotton wilt and rape sclerotinia.

The Burkholderia HDXY-02 provided by the present invention is an endophytic bacterium strain of spp., which has a strong antibacterial effect on rice sheath blight. Strain HDXY-02 has antibacterial effect on a variety of pathogenic fungi, and has a very broad antibacterial spectrum. The above-mentioned bacterial strain HDXY-02 has simple culture conditions, is easy to store and produce, and has a fast growth rate. It plays a very important role in the biological control of plant diseases, especially for the control of rice sheath blight. It can be used as a A broad-spectrum fungicide with good development and application prospects.

Description of drawings:

Figure 1 is the colony morphology of Burkholderia HDXY-02.

Figure 2 is the electrophoretic pattern of the 16S rDNA sequence of Burkholderia HDXY-02.

Fig. 3 is a graph showing the antagonistic effect of Burkholderia HDXY-02 on rice sheath blight.

Figure 4 is the effect of Burkholderia HDXY-02 aseptic fermentation supernatant on the growth of rice sheath blight mycelia.

detailed description:

In the following, the specific implementation manner of the present invention will be further described in detail in conjunction with the examples and accompanying drawings.

The medium formula used in the following examples is:

PDA solid medium formula: potato 200g, sucrose 20g, agar 20g, distilled water to 1L, pH natural;

NA medium formula: beef extract 3g, peptone 10g, sodium chloride 5g, agar 20g, distilled water to 1L, pH7.3;

NB medium formula: beef extract 3g, peptone 10g, sodium chloride 5g, distilled water to 1L, pH 7.3.

Example 1: Isolation of endophytes

Take an appropriate amount of Hudixiao root, soak it in 70% ethanol for 1 minute on a clean bench, then soak it in 0.1% mercury chloride for 6 minutes, optionally use 70% ethanol for 1 minute, and rinse it with sterile water for 3-5 times. Weigh 1g of surface-sterilized root, soak it in 10mL water and grind it in a sterile mortar. After standing still, take the supernatant and make gradient dilution with sterile water. Take an appropriate dilution and spread it evenly on the NA plate. After drying, culture it upside down at 28°C. After 2-3 days, pick colonies of different sizes, colors and shapes to a new NA plate for streaking and purification, number the isolated colonies to be tested, and store them in glycerol The purified colonies were stored in an ultra-low temperature refrigerator for later use.

Embodiment 2: the antagonism of bacterial strain HDXY-02 to various pathogenic fungi

NA medium activates the isolated endophytic bacteria, conducts antagonism test, adopts PDA plate medium, adopts plate confrontation method, and uses a sterile puncher to punch out the test fungus cake with a diameter of 6 mm, and put it on a PDA plate with a diameter of 90 mm. In the center of the plate, after 2-3 days of culture, inoculate the activated endophytes on both sides 3cm away from the bacteria block, and culture at a constant temperature of 28°C for 3-5 days. Take only the pathogenic bacteria and no isolated strains as the control, observe regularly, and measure the width of the inhibition zone and colony radius between the endophytic bacteria and the tested pathogenic fungi, as an index to judge their antagonistic ability. An endophyte HDXY-02 with antagonistic effect on various plant pathogenic bacteria was obtained. Fig. 3 is a graph showing the antagonistic effect of strain HDXY-02 on rice sheath blight.

The test results show that Burkholderia HDXY-02 of the present invention is effective against rice sheath blight (Rhizoctorziasolani), wheat scab (Fusariumt graminearumt), rice blast fungus (Magnaportheoryzae), rape sclerotinia (Sclerotinia libertiana) ) and Fusarium oxysporumf.sp.vasinfectum, the five pathogenic fungi, all had antibacterial effects. The results are shown in Table 1, especially the inhibitory effect on rice sheath blight is the best, and the inhibition rate reaches more than 72%. Therefore, strain HDXY-02 is a strain with strong inhibitory effect on pathogenic fungi.

Table 1 Results of confrontation test between bacterial strain HDXY-02 and various plant pathogenic fungi

Embodiment 3: Cloning analysis of the 16S rDNA sequence of bacterial strain HDXY-02 of the present invention

Genomic DNA of strain HDXY-02 was extracted and used as a template to amplify its 16SrDNA fragment by PCR with bacterial universal primers. After the PCR products were tested by 1% agarose gel electrophoresis, the PCR products were recovered and purified, and sent to Shanghai Sangon for sequencing. The primers used in PCR were: 16S-27: 5'-AGAGTTTGATCCTGGCTCAG-3'; 16S-1492: 5'-GGTTACCTTGTTACGACTT-3'. PCR reaction system (25 μL): 1 μL of diluted DNA template; 2 μL of dNTP; 1 μL of primer 16S-27; 2 μL of primer 16S-1492; 2.5 μL of 10×PCR Buffer; 0.2 μL of Taq DNA polymerase; The PCR reaction conditions were 94°C for 5min; 28 cycles of 94°C for 30s, 52°C for 30s, 72°C for 1.5min; 72°C for 10min.

The result of BLAST analysis of the 16S rDNA sequence (shown in SEQ ID No.1) shows that the strain of the present invention has a homology of 99% with Burkholderia sp. Combining the colony morphology, physiological and biochemical test results and 16SrDNA sequence analysis of the strain, the strain HDXY-02 was identified as Burkholderia sp.

Embodiment 4: the culture method of bacterial strain HDXY-02

The specific steps are as follows: A. Pick the strain HDXY-02 and activate it in 5mL NB culture solution for 22-26h at a temperature of 28-30°C and a shaker speed of 180-220r/min;

B. Draw 1mL (inoculum size 2%) of the activated bacterial strain HDXY-02 culture solution in step (A) and inoculate it into 50mL fermentation medium (fermentation medium formula: peptone 10-20g, glycerol 5-15mL, dihydrogen phosphate Potassium 1-3g, magnesium sulfate 0.5-2g, dilute to 1L, pH 6.5-7.5) in a 250mL Erlenmeyer flask, temperature 28-30°C, shaker speed 180-220r/min, shake culture for 48h-72h;

C. Centrifuge the fermented liquid obtained in step (B) at 5000-8000 rpm for 10-20 min at 4° C., and then filter through a bacterial filter (0.22 μm) to obtain a sterile fermentation supernatant;

D. Wash the thallus 3-5 times with sterile water to make its bacterium suspension. Store the aseptic fermentation supernatant and bacterial suspension at 4°C for later use.

Embodiment 5: the inhibitory effect of the aseptic fermented liquid of bacterial strain HDXY-02 on rice sheath blight mycelia growth

A. Inoculate the strain HDXY-02 into 5mL NB culture medium and activate for 24h, inoculate 1mL of activated HDXY-02 into a 250m1 Erlenmeyer flask containing 50mL fermentation medium, and culture it with shaking at 28°C and 200rpm for 48h. The fermentation broth was placed at 4° C., centrifuged at 8000 rpm for 10 min, and the supernatant was filtered with a bacterial filter (0.22 μm) to obtain a sterile fermentation broth.

B. Add the fermentation supernatant to the PDA solid medium in proportions of 1%, 5%, 10%, 15%, 20%, 25% and 30%, and inoculate a 5mm mycelium block with a diameter of 5mm on each plate , each treatment was repeated 3 times, and the results of 3 parallel experiments were obtained, cultured at 28°C, and the colony diameter of rice sheath blight was measured after 5 days and related data were recorded.

The test results are shown in Figure 4. Different concentrations of the sterile fermentation broth of the strain HDXY-02 can inhibit the growth of rice sheath blight mycelia, and the inhibition rate gradually strengthens with the increase of the concentration. Observing the inhibitory effect of different concentrations of sterile fermentation filtrates of strain HDXY-02 on the growth of rice sheath blight fungus mycelium on the PDA plate shows that the growth of mycelium is inhibited and the growth is slow.

SEQUENCE LISTING

<110> Institute of Botany, Chinese Academy of Sciences, Jiangsu Province

<120> An Endophytic Bacteria Burkholderia HDXY-02 and Its Application

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 1437

<212>DNA

<213> Burkholderia sp. HDXY-02

<400> 1

attggcggct gcctttccat gcaagtcgaa cggcagcacg ggtgcttgca cctggtggcg 60

agtggcgaac gggtgagtaa tacatcggaa catgtcctgt agtgggggat agcccggcga 120

aagccggatt aataccgcat acgatctacg gatgaaagcg ggggaccttc gggcctcgcg 180

ctatagggtt ggccgatggc tgattagcta gttggtgggg taaaggccca ccaaggcgac 240

gatcagtagc tggtctgaga ggacgaccag ccacactggg actgagacac ggcccagact 300

cctacggggag gcagcagtgg ggaattttgg acaatgggcg aaagcctgat ccagcaatgc 360

cgcgtgtgtg aagaaggcct tcgggttgta aagcactttt gtccggaaag aaatcctgag 420

ggctaatatc cttcggggat gacggtaccg gaagaataag caccggctaa ctacgtgcca 480

gcagccgcgg taatacgtag ggtgcgagcg ttaatcggaa ttactgggcg taaagcgtgc 540

gcaggcggtt tgttaagacc gatgtgaaat ccccgggctc aacctgggaa ctgcattggt 600

gactggcaag ctagagtatg gcagagggggg gtagaattcc acgtgtagca gtgaaatgcg 660

tagagatgtg gaggaatacc gatggcgaag gcagccccct gggccaatac tgacgctcat 720

gcacgaaagc gtggggagca aacaggatta gataccctgg tagtccacgc cctaaacgat 780

gtcaactagt tgttggggat tcatttccctt agtaacgtag ctaacgcgtg aagttgaccg 840

cctggggagt acggtcgcaa gattaaaact caaaggaatt gacggggacc cgcacaagcg 900

gtggatgatg tggattaatt cgatgcaacg cgaaaaacct tacctaccct tgacatggtc 960

ggaaccttgg agagatccga gggtgctcga aagagaaccg atacacagt gctgcatggc 1020

tgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg caacgagcgc aacccttgtc 1080

cttagttgct acgcaagagc actctaggga gactgccggt gacaaaccgg aggaaggtgg 1140

ggatgacgtc aagtcctcat ggcccttatg ggtagggctt cacacgtcat acaatggtcg 1200

gaacagagggg tcgccaaccc gcgaggggga gctaatccca gaaaaccgat cgtagtccgg 1260

attgcactct gcaactcgag tgcatgaagc tggaatcgct agtaatcgcg gatcagcatg 1320

ccgcggtgaa tacgttcccg ggtcttgtac acaccgcccg tcacaccatg ggagtgggtt 1380

ttaccagaag tggctagtct aaccgcaagg aggacggtca cccacggtag gatctac 1437

Claims (6)

1. Burkholderia sp. (Burkholderia sp.) HDXY-02, this strain was preserved in the General Microbiology Center of China Committee for the Collection of Microbial Cultures on April 20, 2017, and its preservation number is: CGMCC No.14054 . 2. the bacterial strain culture that Burkholderia sp. (Burkholderia sp.) HDXY-02 as claimed in claim 1 obtains, fermentation broth or fermentation broth aseptic supernatant. 3. Burkholderia sp. (Burkholderia sp.) HDXY-02 described in claim 1 is the biological bacterium agent of active ingredient, it is characterized in that, this biocontrol bacterium agent adopts the following method to prepare: the described in claim 1 The seed liquid of Burkholderia sp. (Burkholderia sp.) HDXY-02 is inoculated in the culture medium, the temperature is 28-30°C, the rotation speed of the shaking table is 180-220r/min, and the shaking culture is 48h-72h; The formula is: peptone 10-20g, glycerin 5-15mL, dipotassium hydrogen phosphate 1-3g, magnesium sulfate 0.5-2g, dilute to 1L, pH 6.5-7.5. 4. The application of Burkholderia sp. HDXY-02 described in claim 1 in antagonizing pathogenic fungi. 5. The culture of Burkholderia sp. (Burkholderia sp.) HDXY-02 described in claim 2, the application of the fermentation broth or the aseptic supernatant of the fermentation broth in antagonizing pathogenic fungi. 6. The application according to claim 4 or 5, characterized in that the pathogenic fungi are rice sheath blight, wheat head blight, rice blast fungus, cotton fusarium wilt and Sclerotinia sclerotiorum.