CN114276938B - Paecilomyces EJKS strain, Fusarium E-9 strain and application thereof - Google Patents

Abstract

The invention discloses a paecilomyces EJKS strain which is preserved in the Guangdong province microorganism strain collection, wherein the preservation number is: GDMCC No:61996, the preservation time is: 2021, 10 and 18, the deposit unit addresses are: the institute of science and microorganisms in Guangdong province. The invention also discloses fusarium E-9 strain with the preservation number of: GDMCC No:62167, the preservation time is: 2021, 12, 28. The invention also discloses application of the paecilomyces EJKS strain and/or the fusarium E-9 strain in plant antagonism leaf spot disease or scientific research. The invention lays a research foundation for solving the problem of diseases in large-area cultivation of the Guangxi curcuma zedoary, protects wild resources, ensures sustainable utilization of the Guangxi curcuma zedoary, and has important significance for recovering and developing the Guangxi curcuma zedoary industry.

Description

Paecilomyces EJKS strain, fusarium E-9 strain and application thereof

Technical Field

The invention belongs to the technical field of plant disease resistance, and relates to paecilomyces EJKS strain, fusarium E-9 strain and application thereof.

Background

Guangxi rhizoma Curcumae (Curcuma kwangsiensis) is a plant of Curcuma (Curauna L) belonging to Zingberaceae, has long history of medicinal use, and is recorded in Chinese pharmacopoeia to have the effects of promoting qi circulation, removing blood stasis and relieving pain, and is the five most valuable medicinal plants of Curcuma, rhizoma Curcumae, radix Curcumae and Curcuma in China. Along with the development and utilization of the medicinal value of the curcuma zedoary, the Guangxi curcuma zedoary becomes a medicinal material with huge development potential and wide market prospect, and the curcuma zedoary is planted in Guangxi places. However, in recent years, due to factors such as expansion of planting area, continuous planting throughout the year, variation of surrounding environment, irregular cultivation management and the like, diseases of Curcuma xanthorrhiza have become more serious, and at present, only Jiang Ni and the like are related to pathogenic bacteria causing the leaf spot of Curcuma xanthorrhiza ^[3] The reported leaf spot disease of zedoary in Guangxi Longan county caused by the infection of soybean phomopsis (Phomopsis longicolla) appears in yellow brown spots at the leaf tip or leaf edge. At present, the disease control of the curcuma zedoary is mainly chemical control, so that the environment is polluted, the chemical pesticide can damage beneficial microorganisms in the soil, and the original biological chain in the soil can be damaged, so that the ecological system is unbalanced. Chemical pesticides have great contribution in the aspects of resisting disease control of traditional Chinese medicinal materials and guaranteeing agricultural production, but the unscientific use mode can seriously destroy the ecological environment and threaten the health of human beings. Endophytic fungi (endophytic fungi) are fungi that are an important component of the plant microbial community that do not cause significant disease symptoms to plant tissue during some or all cycles within healthy plant tissue. Endophytic fungi have abundant biodiversity, can grow asymptomatically in different healthy tissues such as stems, leaves, roots and the like of plants, naturally exist in temperate and tropical rain forests, and are distributed with about 30 ten thousand land host plants. Bioactive compounds (active compounds without host plants) produced by endophytes for enhancing endophytes and host plantsThe adaptability of the material, such as tolerance to biotic and abiotic stress, and the like, has important significance. In addition, the compounds can induce and generate a large number of known and novel bioactive secondary metabolites, have various functions, promote plant growth, improve drought resistance, salt resistance, disease resistance and insect resistance of seedlings, and have important medicinal value for the generation of medicinal effects of medicinal plants and the like. Therefore, the development of biological pesticides is a hot spot of current research by utilizing the antibacterial activity of endophytic fungi.

Disclosure of Invention

It is an object of the present invention to address at least the above problems and/or disadvantages and to provide at least the advantages described below.

It is still another object of the present invention to provide an EJKS strain of Paecilomyces.

It is still another object of the present invention to provide a method for culturing the fungus EJKS of Paecilomyces.

Another object of the invention is to provide a strain of Fusarium E-9.

It is still another object of the present invention to provide a method for culturing fusarium E-9 strain.

The invention also aims to provide the application of the paecilomyces EJKS strain and/or the fusarium E-9 strain in antagonizing leaf spot disease of plants or scientific research.

For this purpose, the technical scheme provided by the invention is as follows:

paecilomyces EJKS strain, paecilomyces Simplicillium sympodiophoum EJKS strain was deposited at the Guangdong province microorganism strain collection under the accession number: GDMCC No:61996, the preservation time is: 2021, 10 and 18, the deposit unit addresses are: building 5, guangzhou City first middle road No. 100, university, no. 59, guangdong province scientific microbiological institute.

Preferably, the partial sequence of the 28S rRNA gene of the Paecilomyces EJKS strain is shown in SEQ ID NO. 5.

The culture method of the paecilomyces EJKS strain comprises the following steps:

1) Inoculating the mycelium of the paecilomyces fungus EJKS into a potato dextrose agar medium for culture.

Preferably, in the method for culturing the paecilomyces EJKS strain, the culture temperature of the antagonistic fungus EJKS is about 28 ℃, and the culture time is 5-7 days.

Fusarium E-9 strain, fusarium E-9 strain, deposited in Guangdong province microorganism strain collection, accession number: GDMCC No:62167, the preservation time is: 2021, 12, 28, deposit unit address: building 5, guangzhou City first middle road No. 100, university, no. 59, guangdong province scientific microbiological institute.

The method for culturing fusarium E-9 strain comprises inoculating mycelium of fusarium E-9 strain into potato dextrose agar medium for culturing.

The paecilomyces EJKS strain and/or the fusarium E-9 strain are applied to plant antagonism leaf spot disease or scientific research.

Preferably, in said application, said application comprises the preparation of a biological pesticide comprising said strain of paecilomyces EJKS.

Preferably, in said application, said plant is zedoary in Guangxi province.

The invention at least comprises the following beneficial effects:

the invention lays a research foundation for solving the problem of diseases in large-area cultivation of the Guangxi zedoary, and the most direct social benefit is that the large-area cultivation of the Guangxi zedoary is ensured, thereby meeting the requirements of the medical and health industry, protecting wild resources and ensuring sustainable utilization of the Guangxi zedoary. Secondly, the invention researches the pathogenic bacteria inhibition effect of antagonistic fungi on the leaf spot disease of the Guangxi zedoary, lays a foundation for developing a biocontrol preparation for preventing and treating the disease, and has important significance for recovering and developing the Guangxi zedoary industry.

Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.

Drawings

FIG. 1 is a phylogenetic tree of pathogenic F.E-9 strains in one embodiment of the invention.

FIG. 2 is a morphological characterization of a F.E-9 colony of pathogenic bacteria in one embodiment of the invention, wherein a is the front of the F.E-9 colony, b is the back of the F.E-9 colony, and c is the F.E-9 megaspore; d is Fusarium E-9 microspore.

FIG. 3 shows the pathogenicity of pathogenic bacteria Fusarium E-9 in one embodiment of the invention, wherein a is the positive disease symptoms of the leaf and b is the negative disease symptoms of the leaf.

FIG. 4 is a graph of colony growth of the Paecilomyces EJKS strain in one embodiment of the invention, wherein A: front of EJKS bacterial colony; b: bacterial colony back of EJKS strain; c: spores.

FIG. 5 is a phylogenetic tree of antagonizing the fungus Paecilomyces EJKS strain in one embodiment of the invention.

FIG. 6 is a graph showing an experiment of pathogenic bacteria of the leaf spot disease of Curcuma xanthorrhiza infected with the strain EJKS in one embodiment of the present invention, wherein A is not added.

FIG. 7 is a graph showing hyphal growth in the test of pathogenic bacteria of EJKS infected Guangxi zedoary leaf spot disease of the strain of FIG. 6, wherein A is the normal hypha of the pathogenic bacteria E-9 of the control, and B is the abnormal hypha of the pathogenic bacteria E-9.

Detailed Description

The present invention is described in further detail below with reference to the drawings to enable those skilled in the art to practice the invention by referring to the description.

It will be understood that terms, such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.

Endophytic fungi (endophytic fungi) are fungi that are an important component of the plant microbial community that do not cause significant disease symptoms to plant tissue during some or all cycles within healthy plant tissue. Endophytic fungi have abundant biodiversity, can grow asymptomatically in different healthy tissues such as stems, leaves, roots and the like of plants, naturally exist in temperate and tropical rain forests, and are distributed with about 30 ten thousand land host plants. Bioactive compounds produced by endophytes (active compounds free of host plants) are of great importance for increasing the adaptability of endophytes and host plants, such as tolerance to biotic and abiotic stresses. In addition, the compounds can induce and generate a large number of known and novel bioactive secondary metabolites, have various functions, promote plant growth, improve drought resistance, salt resistance, disease resistance and insect resistance of seedlings, and have important medicinal value for the generation of medicinal effects of medicinal plants and the like. Therefore, the development of biological pesticides is a hot spot of current research by utilizing the antibacterial activity of endophytic fungi.

The invention provides a paecilomyces EJKS strain, wherein the paecilomyces Simplicillium sympodiophoum EJKS strain is preserved in the microorganism strain collection of Guangdong province, and the preservation number is as follows: GDMCC No:61996, the preservation time is: 2021, 10 and 18, the deposit unit addresses are: building 5, guangzhou City first middle road No. 100, university, no. 59, guangdong province scientific microbiological institute.

In some embodiments of the present invention, preferably, the partial sequence of the 28S rRNA gene of the strain of Paecilomyces EJKS is shown in SEQ ID NO. 5.

The invention also provides a method for culturing the paecilomyces EJKS strain, which comprises the following steps:

1) Inoculating the mycelium of the paecilomyces fungus EJKS into a potato dextrose agar medium for culture.

In some embodiments of the invention, the antagonistic fungus EJKS is preferably cultured at a temperature of about 28℃for a period of 5-7 days.

The invention also provides a fusarium E-9 strain, which is deposited in the Guangdong province microorganism strain collection with the deposit number: GDMCC No:62167, the preservation time is: 2021, 12, 28, deposit unit address: building 5, guangzhou City first middle road No. 100, university, no. 59, guangdong province scientific microbiological institute.

The invention also provides a method for culturing fusarium E-9 strain, which comprises the step of inoculating mycelium of the fusarium E-9 strain into a potato dextrose agar culture medium for culturing.

The invention also provides application of the paecilomyces EJKS strain and/or the fusarium E-9 strain in plant antagonism leaf spot disease or scientific research.

In some embodiments of the invention, preferably, the use comprises preparing a biological pesticide comprising the paecilomyces EJKS strain.

In some embodiments of the invention, preferably, the plant is zedoary in Guangxi province.

For a better understanding of the technical solution of the present invention, the following examples are now provided for illustration: 1 materials and methods

1.1 test materials

1.1.1 test strains

Fusarium E-9 strain of test pathogen was collected from the Gunyang county of Guangxi Zhuang nationality at 2021, 7 months, and Fusarium E-9 strain was deposited at the microorganism strain collection in Guangdong province with the accession number: GDMCC No:62167, the preservation time is: 2021, 12, 28, deposit unit address: building 5, guangzhou City first middle road No. 100, university, no. 59, guangdong province scientific microbiological institute.

The test antagonistic fungus strain EJKS (Simplicillium sympodiophoumr EJKS) was isolated from healthy Guangxi zedoary leaf blades and was identified by the national institute of medicinal plant garden phytopathology at NCBI under strain accession No. OK175703. Strain deposit No. GDMCC No:61996, the preservation time is: 2021, 10 and 18, the deposit unit addresses are: building 5, guangzhou City first middle road No. 100, university, no. 59, guangdong province scientific microbiological institute.

1.1.2 test Medium

Potato Dextrose Agar (PDA): 300g/L of potato (from which the extract powder is extracted), 20g/L of glucose, 15g/L of agar and a final pH of 5.6.+ -. 0.2.

1.1.3 Main test Agents

0.1% mercuric solution, 75% ethanol, sterile water, agarose, 1×TAE solution, and the total kit for identifying pathogenic bacterial molecules was purchased from Yu Bao day doctor materials technology Co., ltd, and the details of the reagents are shown in Table 1.

TABLE 1 molecular reagents

1.2 test methods

1.2.1 isolation, purification and identification of pathogenic bacteria

1.2.1.1 pathogen isolation and purification

Selecting diseased tissues at the disease-healthy juncture of the Guangxi zedoary leaf, cutting into small blocks with the length of 5mm multiplied by 5mm, soaking for 30s by 75% ethanol, sterilizing for 2min by 0.1% mercuric chloride, rinsing for 3 times by sterile water, culturing at 28 ℃ on a PDA culture medium, picking the edges of colonies after hyphae grow out, purifying, and separating the purified colonies by monospore to obtain a pure culture isolate.

1.2.1.2 determination of pathogenicity of pathogenic bacteria

The isolated and purified mycelium blocks (1 cm. Times.1 cm) were inoculated to the leaves of Curcuma xanthorrhiza which were healthy and close in growth, and inoculated with sterile PDA medium (1 cm. Times.1 cm) as a control, each treatment was repeated three times, placed in a petri dish with wet filter paper sheets, cultured with moisture, and observed periodically for onset. And timely separating and purifying pathogenic bacteria from the pathogenic tissues, and observing whether the pathogenic bacteria are identical with inoculated strains.

1.2.1.3 morphological identification of pathogenic bacteria

And (3) observing colony characteristics of pathogenic bacteria on a PDA culture medium, observing morphological characteristics of hyphae, conidia and the like by using an optical microscope, and carrying out morphological identification on the pathogenic bacteria.

1.2.1.4 molecular biological identification

Transplanting the purified pathogenic bacteria strain on PDA plate culture medium, culturing for 5d, collecting mycelium, and extracting with DNA reagent by SDS cracking methodThe cassette (Shanghai) extracts the fungal genomic DNA. ITS amplification primer sequences were rDNA-TUB sequences of universal primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') (SEQ ID NO: 1) and ITS4 (5'-TCCTCCGCT TATTGATATGC-3') (SEQ ID NO: 2) (White et al 1990) and beta-tubulin genes (beta-tubulin) TUB1 and TUB2 (Weir et al 2012) for the amplification of pathogenic bacteria. The amplification reaction was carried out in a 25. Mu.L reaction system with a Template (genomic DNA 20-50 ng/. Mu.L) of 0.5. Mu.L, 10 XBuffer (with Mg) ²⁺ ) 2.5. Mu.L of dNTPs (2.5 mM each) 1. Mu.L, 0.2. Mu.L of enzyme, 0.5. Mu.L of primers, and double distilled H were added ₂ O to 25. Mu.L. The thermal cycling reaction procedure was set as follows: initial denaturation at 94℃for 4min, followed by denaturation at 94℃for 45sec, primer annealing at 55℃for 45sec, extension at 72℃for 60sec, and repair extension at 72℃for 10min after 30 cycles. Water was used instead of DNA template for the amplification as a blank. The result of the PCR reaction was detected by gel electrophoresis, 4. Mu.L of the PCR product was pipetted into a 1% (W/V) agarose gel, and the gel was placed in a 1 XTBE buffer (0.9M Tris-Borate,0.01M EDTA, pH=8.3) and electrophoresed at 110V for 40min. The amplified DNA product was sent to the biological engineering (Shanghai) Co., ltd for purification sequencing. And the sequencing results were blast aligned with sequences in GenBank. And constructing a pathogenic bacteria phylogenetic tree by using a Neighbor-Joining method of MEGA4.0 software by using ITS similar sequences.

1.2.2 isolation, purification and identification of antagonistic fungi

1.2.2.1 isolation of antagonistic fungi

Selecting healthy and disease-free Guangxi zedoary leaf from different plants, adopting a conventional tissue separation method (Fangzhong, 1998), cutting the tissue into 5cm fragments, sequentially sterilizing with 75% ethanol and 0.1% mercuric chloride on the surface for 2.5min, and washing with sterile water for 3 times; the tissue blocks are cut into tissue blocks with the size of about 5mm by using sterile forceps and a scalpel, the tissue blocks are placed on a PDA (medium containing streptomycin) plate, 7 pieces of Guangxi zedoary tissues from different plants are placed on each plate by adopting a five-point sampling method, 5 plates are repeatedly treated for each experiment, and whether the surface sterilization is thorough is detected by adopting an ultra-clean workbench sterile detection method, a rinse solution sterile detection method and a tissue blotting sterile detection method so as to ensure the accurate separation of endophytic fungi in leaves of the Guangxi zedoary (Zhu Hongjian and the like, 2012). Culturing in a constant temperature incubator at 28deg.C, transferring to PDA plate for culturing after mycelia grow out at the edge of tissue block, repeatedly purifying to obtain single pure colony, transferring to slant culture medium, and preserving in a refrigerator at 4deg.C. The isolated and purified endophytic fungi are classified into different morphological types according to colony culture characteristics.

(1) Morphological identification

The purified pathogenic bacteria strain is transferred onto a new potato dextrose agar culture medium, placed in an illumination incubator at 28 ℃ and RH65% for culture, the form (mycelium form, color and the like) of the bacterial colony is observed on time, recorded and described, and photographed and recorded after the bacterial colony grows into a flat-plate culture dish of 8.5 cm. After 10 days of culture, spores are picked up, spore morphology is observed under an optical microscope, and preliminary identification is carried out on pathogenic bacteria.

(2) Molecular biological identification

After the purified strain to be tested is cultured for 5 days in an illumination incubator, a molecular kit 28S rDNA sequence is adopted to entrust a biological engineering (Shanghai) stock company to sequence an amplification product, the sequence obtained by the measurement is subjected to blast comparison in a nucleotide sequence in NCBI to analyze the sequence homology of related fungi, and then a MEGA4.0 software is utilized to construct a phylogenetic tree for antagonizing fungi. The method comprises the following specific steps:

(1) preparation of PCR products

The total volume of the PCR reaction system was 50. Mu.L (see Table 2)

TABLE 2 composition of PCR reaction System

(2) The PCR amplification step was performed at 98℃for 2min,98℃for 10s,60℃for 45s,68℃for 1min, and 68℃for 10min for 35 cycles.

(3) PCR product run electrophoresis

2.5. Mu.l of DL 2000DNA Marker and 2.5. Mu.l of 6×loading buffer were used, 2.5. Mu.l of PCR product and 2.5. Mu.l of 6×loading buffer were mixed, spotted into 1% agarose gel, placed in 1xTAE electrode buffer, run for 30min at a set voltage of 130V, and after detection by electrophoresis, the target band was observed at 302nm of a dark box type ultraviolet reflectometer, and the PCR product showing clear and bright bands was then sent to the division of biological engineering (Shanghai) for sequencing.

(4) Analysis of product sequence

And (3) carrying out blast comparison on the detected sequence by using a nucleotide sequence in NCBI (http:// blast. NCBI. Lm. Nih. Gov /), analyzing the sequence homology of related fungi, finding the sequence of a fungus isolate with higher sequence coverage rate from the result, and downloading and storing.

(5) Phylogenetic analysis

The strain sequences and the downloaded related strain sequences were stored in txt text, and in order to maximize the homology of all sequences, sequence alignment was performed using clustalx software, the irregular sequences at both ends were deleted, the file was additionally saved as txt text, and a phylogenetic tree antagonistic to the fungal strain was constructed with MEGA 4.0.

1.3 determination of the inhibition of EJKS Strain on the pathogenic bacteria of the leaf spot of Curcuma Guangxi

The antibacterial effect of the strain EJKS on the pathogenic bacteria of the Curcuma xanthorrhiza by adopting a plate counter method is measured. Plate facing method: placing pathogenic bacteria tissue blocks with the diameter of 0.5cm on one side of a PDA culture medium, inoculating bacterial strain EJKS tissue blocks at equal distance on the other side of the PDA culture medium, and taking only inoculated pathogenic bacteria as a control, wherein each treatment is repeated for 3 times; when the control had grown over the whole plate, colony diameters were measured and inhibition was calculated. Inhibition (%) = (control colony diameter-treated colony diameter)/control colony diameter x 100.

2 results and analysis

2.1 concerning pathogenic bacteria

1) Nucleotide alignment of the sequence of the pathogen gene sequences measured at NCBI relative to the sequence of the fungus showed that strain E-9 was homologous to Fusarium oxysporum (NCBI accession number: MH 85768.1) is up to 100% similar. The sequences measured in this experiment were submitted to NCBI database to obtain the accession sequence numbers of each gene, and Fusarium strain sequences containing ITS and TUB genes were searched and downloaded, and two genes (ITS and TUB) representing the strains were combined to construct a multi-gene phylogenetic tree of pathogenic bacteria, which showed that E-9 and Fusarium seminude were co-clade and the support rate was 99%, as shown in FIG. 1. According to morphological characteristics of pathogenic bacteria and polygenic phylogenetic tree, determining that the pathogenic bacteria causing leaf spot disease of Curcuma xanthorrhiza is Fusarium, and the accession numbers are OK175677 and OK326873 respectively. The Fusarium fusarium strains were deposited with the Guangdong province microorganism strain collection under the accession number: GDMCC No:62167, the preservation time is: 2021, 12, 28, deposit unit address: building 5, guangzhou City first middle road No. 100, university, no. 59, guangdong province scientific microbiological institute.

SEQ ID NO:3：

>Seq Fusariumincarnatum isolate E-9 18S ribosomal RNA gene,partial sequence；internal transcribed spacer 1,5.8S ribosomal RNA gene,and internal transcribed spacer 2,complete sequence；and 28S ribosomal RNA gene,partial sequence

TGCGGAGGGATCATTACCGAGTTTACAACTCCCAAACCCCTGTGAACATACCTATACGTTGCCTCG

GCGGATCAGCCCGCGCCCCGTAAAACGGGACGGCCCGCCCGAGGACCCCTAAACTCTGTTTTTAG

TGGAACTTCTGAGTAAAACAAACAAATAAATCAAAACTTTCAACAACGGATCTCTTGGTTCTGGC

ATCGATGAAGAACGCAGCAAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAAT

CTTTGAACGCACATTGCGCCCGCCAGTATTCTGGCGGGCATGCCTGTTCGAGCGTCATTTCAACCC

TCAAGCTCAGCTTGGTGTTGGGACTCGCGGTAACCCGCGTTCCCCAAATCGATTGGCGGTCACGT

CGAGCTTCCATAGCGTAGTAATCATACACCTCGTTACTGGTAATCGTCGCGGCCACGCCGTAAAAC

CCCAACTTCTGAATGTTGACCTCGGATCAGGTAGGAATACCCGCTGAACTTAAGCATATCAATA

SEQ ID NO:4

>Seq Fusariumincarnatum strain E_9T beta-tubulin(tub)gene,partial cds

CCACCCTGTTCACCTTCAGACCGGTCAGTGCGTTTTCAGGTCCACGTCAGGTCGGCCAATGTGTA

AGGCTTACCCACCAAAAAACTGTTTTGAGGGAAATGTGTTGAGTTGATGATTTTTGCAGGGCAAC

CAAGTCGGTTCCAGCTTCTGGTAGGTGTTTTACAGCTTGAATTAACTTTTCAACAGTCAATTGAGC

TTTTGCTAACGTGTTTTCAGGAACACCGTCGGTAACCAAATTGGTGCTGCTTTCTGGCAAACCATC

TCTGGCGAGCACGGTCTCGACAGCAATGGTGTTTACAACGGTACCTCCGAGCTCCAGCTCGAGCG

TATGAGCGTCTACTTCAACGAGAGCGCATCAACGTCTACTTCGCTGAGGTAAGCTAACCGCAAAC

TCTTATGAGTTTGTTTACTAACGTATCCAGCGTTCCGGTAACAAGTATGTTCCCCGTGCCGTCCTCG

TCGATCTCGAGCCCGGTACCATGGACGCCGTCCGTGCCGGTCCTTTCGGACAGCTTTTCCGTCCCG

ACAACTTCGTTTTCGGTCAATCCGGTGCCGGAAACAACTGGGCCAAGGGGCCCTACACTGAGGGT

ACTTACAACCCCCCCCCCTTTTTAA

2) E-9 bacterial colony is regular circular growth, hypha cashmere-like, the primary color is white, the back is light yellow, the late hypha becomes light pink, the back is orange yellow in the middle, and the outer ring is pink. The size of the conidium has no obvious limit on aerial hyphae, most of which are mesospores, sickle-shaped, 3-5-partition, and small conidium oblong, 1-3-partition. Based on the morphological characteristics described above, pathogenic bacteria E-9 are primarily considered to be Fusarium fungi, as shown in FIG. 2.

3) Pathogenicity determination pathogenic bacteria E-9 the leaf is inoculated 2-3 days after, and the tender leaf and the old leaf are both infected, and the tender leaf is generally first infected. Early symptoms of disease occurrence are manifested by water-stain-like yellowish lesions on the leaves, and obvious disease-health interphase, the lesions are continuously enlarged with continued infection of pathogenic bacteria, irregular lesions with yellow edges and dark centers are formed later, furthermore, the lesions are wound around yellow halos, and the lesions are connected into pieces when serious, so that the whole leaves turn yellow, as shown in fig. 3. According to the Koch's law, it was thus determined that the strain E-9 is a pathogenic bacterium causing leaf spot of zedoary.

2.2 antagonizing fungi

2.2.1 morphological features and molecular characterization of Strain EJKS

Bacterial strain EJKS colony is white to light yellow, the edge is neat, the texture is villiated, the back of the bacterial colony is yellow, and the bacterial colony grows slowly; the bacterial strain EJKS spores are characterized in that conidium is in a sphere shape and the size is 1-2 mu m. As shown in fig. 4.

Molecular biological characteristics the EJKS strain was PCR amplified and sequenced with the universal primer 28S rDNA sequence to obtain a 583bp nucleotide sequence. BLAST comparison is carried out on the sequence and the sequence of GenBank in NCBI, and the result shows that the EJKS strain has high homology with Simplicillium sympodiophoumr, and the similarity reaches 100.0%. The phylogenetic tree is constructed by adopting MEGA4.0 to 28S rDNA sequence of EJKS strain, as shown in figure 5, and the result shows that the EJKS strain and S.sympodiophoumNG 068548 strain sequence are in the same branch on the phylogenetic tree, belonging to S.sympodiophoumr with sequence accession number OK175703.

SEQ ID NO:5

>Seq Simplicillium sympodiophorum EJKS28S rRNA gene,partial sequence

CAATTGGCATTGCCCCAGTACGGCGAGTGAGCGGCAACAGCTCAAATTTGAAATCTGGCTCCTAGAGTCCGAATTGTAATTTGCAGAGGATGCTTTTGATGCGGTGCCTTCCGAGTTCCCTGGAACGGGACGCCATAGAGGGTGAGAGCCCCGTCTGGTCGGATGCCAAATCTCTGTAAAGCTCCTTCGACGAGTCGAGTAGTTTGGGAATGCTGCTCTAAATGGGAGGTATATGTCTTCTAAAGCTAAATATTGGCCAGAGACCGATAGCGCACAAGTAGAGTGATCGAAAGATGAAAAGCACTTTGAAAAGAGGGTTAAAAAGTACGTGAAATTGTTGAAAGGGAAGCGCCTATGACCAGACTTGGGCCCGGTGAATCATCCAGCGTTCTCGCTGGTGCACTTTGCCGGGCACAGGCCAGCATCAGTTTGGCTCGGGGGAAAAAGGCTTTGGGAATGTAGCTCCCCTCGGGGAGTGTTATAGACCATTGCACAATACCCTGGGCCGGACTGAGGTTCGCGCATCTGCAAGGATGCTGGCGTAATGGTCATCAGCGATCCGTCTTGAAACCACGGACCACTG

2.3 antagonism of EJKS strain on the pathogenic bacteria of the leaf spot of Curcuma xanthorrhiza

From fig. 6 and 7, the strain EJKS has obvious inhibition effect on the curculigo guangxi leaf spot pathogenic bacteria E-9, and the inhibition rate reaches 56.47%. Under an optical microscope, the growth deformity of the mycelia of the pathogenic bacteria E-9 influenced by the strain EJKS is observed, the mycelia are enlarged and thickened to form a beaded shape, and the normal mycelia are smooth and fine and straight, so that the inhibition effect of the strain EJKS on the mycelia of the pathogenic bacteria is obvious.

The number of modules and the scale of processing described herein are intended to simplify the description of the present invention. Modifications and variations of the present invention will be apparent to those skilled in the art.

Although embodiments of the present invention have been disclosed above, it is not limited to the details and embodiments shown and described, it is well suited to various fields of use for which the invention would be readily apparent to those skilled in the art, and accordingly, the invention is not limited to the specific details and illustrations shown and described herein, without departing from the general concepts defined in the claims and their equivalents.

With references

Weir BS,Johnston PR,Damm U(2012)The Colletotrichum gloeosporioides species complex.Stud Mycol 73:115–180.

White TJ,Bruns T,Lee S,Taylor J(1990)Amplification and directsequencing of fungal ribosomal RNA genes for phylogenetics.In:Innis MA,Gelfand DH,Sninsky JJ,WhiteTJ(eds)PCR protocols,a guide to methods and applications.Academic Press,San Diego,pp 315–322.

The national formulary committee 2020. First pharmacopoeia of the people's republic of China [ M ]. Beijing: chinese medical science and technology Press Ma Rui pathogen identification, biological Properties and indoor agent screening of two diseases of Curcuma wenyujin Hainan province [ D ] university of Hainan, 2018. Jiang Ni, liu Lihui, hu Fengyun, liu Wei, liu Fan. Guangxi Curcuma zedoaria leaf spot disease pathogen identification and biological Properties research [ J ] Chinese modern traditional Chinese medicine, 2016,18 (05): 616-619.

Huo Huanran the antibacterial activity of herba Artemisiae Annuae extract on four plant pathogens Fusarium [ D ]. University of northwest, 2021. Liu Cai. Pollution problem of agricultural chemical used in crop pest control and control countermeasure [ J ]. Seed science, 2021,39 (02): 75-76.

SEQUENCE LISTING

<110> Guangxi Zhuang nationality medicinal plant garden

<120> Paecilomyces EJKS strain, fusarium E-9 strain and uses thereof

<130> 2020

<160> 5

<170> PatentIn version 3.5

<210> 1

<211> 19

<212> DNA

<213> Synthesis

<400> 1

tccgtaggtg aacctgcgg 19

<210> 2

<211> 20

<212> DNA

<213> Synthesis

<400> 2

tcctccgctt attgatatgc 20

<210> 3

<211> 523

<212> DNA

<213> Fusarium incarnatum E-9

<400> 3

tgcggaggga tcattaccga gtttacaact cccaaacccc tgtgaacata cctatacgtt 60

gcctcggcgg atcagcccgc gccccgtaaa acgggacggc ccgcccgagg acccctaaac 120

tctgttttta gtggaacttc tgagtaaaac aaacaaataa atcaaaactt tcaacaacgg 180

atctcttggt tctggcatcg atgaagaacg cagcaaaatg cgataagtaa tgtgaattgc 240

agaattcagt gaatcatcga atctttgaac gcacattgcg cccgccagta ttctggcggg 300

catgcctgtt cgagcgtcat ttcaaccctc aagctcagct tggtgttggg actcgcggta 360

acccgcgttc cccaaatcga ttggcggtca cgtcgagctt ccatagcgta gtaatcatac 420

acctcgttac tggtaatcgt cgcggccacg ccgtaaaacc ccaacttctg aatgttgacc 480

tcggatcagg taggaatacc cgctgaactt aagcatatca ata 523

<210> 4

<211> 615

<212> DNA

<213> fusarium incarnatum E-9

<400> 4

ccaccctgtt caccttcaga ccggtcagtg cgttttcagg tccacgtcag gtcggccaat 60

gtgtaaggct tacccaccaa aaaactgttt tgagggaaat gtgttgagtt gatgattttt 120

gcagggcaac caagtcggtt ccagcttctg gtaggtgttt tacagcttga attaactttt 180

caacagtcaa ttgagctttt gctaacgtgt tttcaggaac accgtcggta accaaattgg 240

tgctgctttc tggcaaacca tctctggcga gcacggtctc gacagcaatg gtgtttacaa 300

cggtacctcc gagctccagc tcgagcgtat gagcgtctac ttcaacgaga gcgcatcaac 360

gtctacttcg ctgaggtaag ctaaccgcaa actcttatga gtttgtttac taacgtatcc 420

agcgttccgg taacaagtat gttccccgtg ccgtcctcgt cgatctcgag cccggtacca 480

tggacgccgt ccgtgccggt cctttcggac agcttttccg tcccgacaac ttcgttttcg 540

gtcaatccgg tgccggaaac aactgggcca aggggcccta cactgagggt acttacaacc 600

cccccccctt tttaa 615

<210> 5

<211> 583

<212> DNA

<213> Simplicillium sympodiophorum EJKS

<400> 5

caattggcat tgccccagta cggcgagtga gcggcaacag ctcaaatttg aaatctggct 60

cctagagtcc gaattgtaat ttgcagagga tgcttttgat gcggtgcctt ccgagttccc 120

tggaacggga cgccatagag ggtgagagcc ccgtctggtc ggatgccaaa tctctgtaaa 180

gctccttcga cgagtcgagt agtttgggaa tgctgctcta aatgggaggt atatgtcttc 240

taaagctaaa tattggccag agaccgatag cgcacaagta gagtgatcga aagatgaaaa 300

gcactttgaa aagagggtta aaaagtacgt gaaattgttg aaagggaagc gcctatgacc 360

agacttgggc ccggtgaatc atccagcgtt ctcgctggtg cactttgccg ggcacaggcc 420

agcatcagtt tggctcgggg gaaaaaggct ttgggaatgt agctcccctc ggggagtgtt 480

atagaccatt gcacaatacc ctgggccgga ctgaggttcg cgcatctgca aggatgctgg 540

cgtaatggtc atcagcgatc cgtcttgaaa ccacggacca ctg 583

Claims (5)

1. Paecilomyces EJKS strain, paecilomyces Simplicillium sympodiophoum EJKS strain was deposited at the Guangdong province microorganism strain collection under the accession number: GDMCC No:61996, the preservation time is: 2021, 10 and 18, the deposit unit addresses are: building 5, guangzhou City first middle road No. 100, university, no. 59, guangdong province scientific microbiological institute.

2. The paecilomyces EJKS strain according to claim 1, wherein the partial sequence of the 28S rRNA gene of the paecilomyces EJKS strain is shown in SEQ ID No. 1.

3. A method for culturing a strain of Paecilomyces EJKS, comprising the steps of:

1) The method for culturing paecilomyces EJKS mycelium according to claim 1, wherein the mycelium is inoculated into potato dextrose agar medium.

4. A method of culturing an EJKS strain of paecilomyces as claimed in claim 3, wherein the culturing temperature is 28 ℃ and the culturing time is 5-7 days.

5. Use of the strain EJKS of paecilomyces for antagonizing fusarium incarnatum E-9, which is a causative agent of the leaf spot disease of zedoary in Guangdong province, wherein the strain fusarium incarnatum E-9 is deposited at the microorganism strain deposit center of guangdong province under the accession number: GDMCC No:62167, the preservation time is: 2021, 12, 28, deposit unit address: building 5, guangzhou City first middle road No. 100, university, no. 59, guangdong province scientific microbiological institute.