CN111004726B

CN111004726B - Penicillium with bacteriostatic action and application

Info

Publication number: CN111004726B
Application number: CN201911355891.1A
Authority: CN
Inventors: 王洪凯; 李红叶; 林福呈; 王秀青
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2019-12-25
Filing date: 2019-12-25
Publication date: 2021-05-14
Anticipated expiration: 2039-12-25
Also published as: CN111004726A

Abstract

The invention discloses penicillium with an antibacterial effect and application thereof. The Penicillium is named as Penicillium sp strain number Z863, and the preservation number is CCTCC NO: m2019870. According to the invention, a penicillium Z863 with an antibacterial effect is obtained through screening and separation, and is identified through morphology and DNA biological labels, so that the penicillium Z863 is a new species of penicillium, and the penicillium Z863 has obvious inhibition effects on rice blast, citrus black spot, citrus anthracnose and citrus black spot, and can be used as a biocontrol microbial inoculum.

Description

Penicillium with bacteriostatic action and application

Technical Field

The invention relates to the technical field of prevention and control of plant pathogenic bacteria, in particular to penicillium with bacteriostatic action and application thereof.

Background

The orange is an important fruit, and a plurality of pathogenic bacteria are harmful to the orange, thereby bringing great threat to the safe production of the orange. The citrus anthracnose and citrus black spot are important pathogens on citrus, and cause citrus anthracnose and citrus black spot respectively. In the production process, the two diseases are harmful to fruits, leaves and branches, and bring serious economic loss.

For example, the invention with the publication number of CN108184875A discloses a composition containing copper abietate and captan, a preparation method and application thereof, belonging to the technical field of pesticides. The composition containing the copper abietate and the captan comprises the following raw materials in parts by weight: 1-30 parts of copper rosinate and 1-70 parts of captan. The invention adopts the copper abietate and the captan as raw materials to prepare the composition, can generate higher synergistic action, expand the broad-spectrum sterilization and overcome and delay the drug resistance of pathogenic bacteria. Especially has obvious synergistic effect on citrus red spider, citrus rust tick, citrus resinoid disease, citrus anthracnose, citrus downy mildew, citrus canker, citrus gray mold and citrus whitefly aphid, and has good control effect.

The invention with publication number CN108184879A discloses a composition containing oxine-copper and propineb, a preparation method and application thereof, and belongs to the technical field of pesticides. The composition containing oxine-copper and propineb comprises the following raw materials in parts by weight: 1-50 parts of oxine-copper and 1-50 parts of propineb. The invention adopts oxine-copper and propineb as raw materials to prepare the composition, can generate higher synergistic effect, expand sterilization broad spectrum, and overcome and delay the generation of drug resistance of pathogenic bacteria. Especially has obvious synergistic effect on citrus red spider, citrus rust tick, citrus resinoid disease, citrus anthracnose, citrus downy mildew, citrus canker, citrus gray mold and citrus whitefly aphid, and has good control effect.

The traditional pesticide control method usually pollutes the environment, and pesticide residues exist in citrus fruits, so that the citrus fruits are harmful to human health. Screening beneficial fungi for biological control is an important method for improving the safety production of the oranges.

The fungus is of various types, wherein the fungus of the penicillium is often found in the environment, and the traditional morphological classification has many disputes due to the fact that the classification features are few and the morphological features of different species are overlapped. With the development of modern molecular biology, the concept of phylogenetic species multi-site sequence type (genetic species) is proposed in the classification of penicillium fungi, species identification is carried out through sequence information of multiple gene sites, DNA biomarkers of penicillium fungi species are proposed for identification (DNAbcoding for identification), and DNA biomarkers of 370 species are accepted at present to classify and identify penicillium fungi.

Disclosure of Invention

The fungus of penicillium is separated, researches prove that the fungus has strong inhibiting effect on citrus anthracnose pathogen, citrus black spot pathogen and the like, and the fungus is identified by a DNA biological label, so that the fungus is a new species of penicillium.

A Penicillium with bacteriostasis is named as Penicillium sp, with strain number Z863 and the preservation number of CCTCC NO: m2019870. The penicillium strain Z863 is separated from the soil of the forest sesame town (Bei latitude 29.60146 degrees, east longitude 94.41736 degrees) in the forest sesame county of the forest sesame area in the Tibetan autonomous region. Through the sequence comparison of related genes, the strain Z863 has a closer relationship with Penicillium janczewski, P.nigricans, P.griseoazurum and P.echinatum, but does not belong to any one of the species, thereby indicating that the strain is a new species. The newly screened strain Z863 is named as Penicillium sp, with the strain number Z863, and is preserved in the China center for type culture Collection, located at the university of Wuhan, China, in 2019, 10 months and 30 days, with the preservation number of CCTCC NO: m2019870.

The invention also provides application of the penicillium in inhibiting the growth of pathogenic bacteria. The pathogenic bacteria are rice blast, citrus black spot, citrus anthracnose or citrus black spot.

The invention also provides application of the penicillium in preventing and treating pathogen infection of rutaceae plants. The pathogenic bacteria are citrus alternaria, citrus anthracnose or citrus alternaria. The Rutaceae plant may be a plant susceptible to infection by the above pathogenic bacteria, such as orange, grapefruit, mandarin orange. Preferably, the rutaceae plant is citrus. The control means may be spraying the control agent containing penicillium strain Z863 on the plants and/or in the soil near the roots of the plants.

The invention also provides application of the penicillium in preventing and treating rice pathogenic bacteria infection. The pathogenic bacteria is Magnaporthe grisea. The control method can be that the control drug containing penicillin strain Z863 is sprayed on the rice and/or sprayed in the soil near the root of the rice.

According to the invention, a penicillium Z863 with an antibacterial effect is separated by screening, and is identified through morphology and DNA biological labels, so that the penicillium Z863 is a new species of penicillium, and the strain Z863 has an obvious inhibiting effect on rice blast, citrus black spot, citrus anthracnose and citrus black spot, and can be used as a biocontrol microbial inoculum.

Drawings

FIG. 1 is a diagram showing the results of colony morphology detection of strain Z863 on different media, and four media of CYA, MEA, CZA and YES are detected together.

FIG. 2 is a diagram of the result of morphological feature detection of strain Z863, wherein the panel 1: the branching status of conidiophores; small figure 2: the vegetative state of conidia on phialides; small figure 3: conidiophores and phialides; small figure 4: the shape of the bottle stalk; small figure 5: the growing mode of the bottle stalk on the base stalk; small figure 6: conidiophore morphology, scale 10 μm.

FIG. 3 shows a phylogenetic tree constructed by combining ITS, CAM and BenA fragments (values indicate the bostrap values).

FIG. 4 is a graph showing the results of the detection of the inhibition rate of the strain Z863 on the radius of pathogenic bacteria.

FIG. 5 is a plot of the antagonistic culture of Z863 strain on the inhibitory effect of Aureobasidium pullulans, wherein A is a antagonistic culture group and B is a plot of Aureobasidium pullulans cultured alone.

FIG. 6 is a plot of the antagonistic culture of Z863 strain on the inhibitory effect of citrus alternaria alternata, wherein A is a antagonistic culture group and B is a plot of the isolated culture of citrus alternaria alternata.

FIG. 7 is a plot of the antagonistic culture of the Z863 strain against the inhibitory effect of citrus anthracnose pathogen, wherein A is the antagonistic culture group and B is a plot of citrus anthracnose pathogen alone.

FIG. 8 is a graph showing the antagonistic action of Z863 strain on Pyricularia oryzae, in which A is a antagonistic group and B is a graph showing the independent cultivation of Pyricularia oryzae.

FIG. 9 is a photograph showing the antagonistic action of Z863 strain on the inhibition of Gibberella zeae, in which A is the antagonistic group and B is a photograph showing the independent culture of Gibberella zeae.

FIG. 10 is a photograph showing the antagonistic action of Z863 strain on the inhibition of Verticillium piricolum, in which A is the antagonistic culture group and B is a photograph showing the independent culture of Verticillium piricolum.

FIG. 11 shows the results of the antagonistic culture of the Z863 strain against the inhibitory action of Rhizoctonia solani, wherein A represents a group of antagonistic cultures and B represents a single culture of Rhizoctonia solani.

Detailed Description

Example 1 isolation of bacterial species

Collecting soil samples from all over the country, and separating by adopting a selective culture medium dilution plate method, wherein the process comprises the following steps: weighing 10g of soil sample, and placing 250mL of three-component soil sample filled with small steel balls and 90mL of sterile waterIn a corner bottle, 120 r.min on a shaker^-1Shaking for 10min to disperse the soil sample fully to obtain 10 times of diluent; then sucking 1mL of 10 times of diluent, adding into a centrifuge tube of 9mL of sterile water, and fully and uniformly mixing; then 1mL of the solution was taken out and added into a 9mL sterile water centrifuge tube, and the solution was mixed well to prepare a 1000-fold diluted solution. Pouring the melted PDA culture medium into culture dishes, wherein each dish is 15 mL; after the culture medium is cooled to a flat plate, 0.1mL of soil sample diluent diluted by 1000 times is sucked and added to the flat plate of the PDA culture medium, and then the diluent is uniformly coated by using a sterile coater, and each concentration is repeated for 3 times. Observing the bacterial colony after culturing in an illumination incubator at 25 ℃ for 3 days, picking up hyphae of the bacterial colony on a new PDA plate, and transferring the bacterial colony to the PDA plate again when the diameter of the bacterial colony is 3 cm.

PDA culture medium: 200g potato, 20g glucose, 18g agar, 0.3g chloramphenicol (1000 mL distilled water).

PDB liquid medium: 200g potato, 20g glucose, 18g agar, 1000mL distilled water.

The strain Z863 is separated from the soil of the Linzhi town (Bei latitude 29.60146 degree, east longitude 94.41736 degree) in Linzhi county in the Linzhi district of the Tibetan autonomous region.

Example 2 species morphological identification

Culture medium:

chacker Concentrate (Czapek Concentrate) (100 mL): sodium nitrate (NaNO)₃)30g, potassium chloride (KCl)5g, magnesium sulfate heptahydrate (MgSO)₄·7H₂O)5g, iron sulfate heptahydrate (FeSO4 & 7H)₂O)0.1g, water (H)₂O) to 100 mL.

Trace elements stock solution (100 ml): cupric sulfate pentahydrate (CuSO)₄·5H₂O)0.5g, Zinc sulfate heptahydrate (ZnSO)₄·7H₂O)0.5g, water (H)₂O) to 100 mL.

Charpy sucrose medium (CZA): 10mL of Chacker's concentrated solution (Czapek concentrate), 30g of Sucrose (Sucrose), 1mL of Trace element stock solution (Trace elements stock solution), 20g of Agar powder Agar, and water added to 1000 mL.

Saccharose charles medium (CYA): chashi concentration10mL of a solution (Czapek concentrate), 30g of Sucrose (Sucrose), 1mL of a Trace element stock solution (Trace elements stock solution), 5g of a Yeast extract (Yeast extract), and dipotassium hydrogen phosphate (K)₂HPO₄)1g of Agar powder (Agar)20g of water (H)₂O) to 1000 mL.

Corn meal Medium (MEA): 50g of corn flour extract (Malt extract), 1mL of Trace elements stock solution (Trace elements stock solution), 20g of Agar powder (Agar) and water (H)₂O) to 1000 mL.

Yeast extract sucrose medium (YES): yeast extract (Yeast extract)20g, Sucrose (Sucrose)150g, magnesium sulfate heptahydrate (MgSO)₄·7H₂O)0.5g, Trace element stock solution (Trace elements stock solution)1mL, Agar powder (Agar)20g, and water (H)₂O) to 1000 mL.

Inoculating the Z863 strain to a 9cm flat plate, inoculating 3 inoculation points in each culture dish, culturing for 7 days at 25 ℃, and observing colony morphology; 60% lactic acid was used as a buoyant carrier, and the morphology of hyphae and conidia was observed under a microscope.

The results of colony morphology observation are shown in FIG. 1:

the colonies were cultured on CZA at 25 ℃ for 7 days with a diameter of 16-18mm, irregular colony edges, fluffy appearance, pale gray back, and lack of soluble pigments.

The bacterial colony is cultured on CYA at 25 deg.C for 7 days, has diameter of 30-32mm, has a large amount of radial wrinkles, and has a flocculent or felty texture, white to light yellow, thick felty texture, no radial wrinkles, and gray color. The colony surface has a large amount of yellow brown exudate and the soluble pigment is lack.

The colonies were cultured on MEA for 7 days at 25 ℃ with 20-30mm diameter, uneven colony, felt-like texture, white to light gray color, with a small amount of grayish green mycelial clusters. The middle colony has tapetum umbilical protrusion, no radial wrinkle, and gray white color. The colony surface has a large amount of yellow brown exudate and the soluble pigment is lack.

Incubate at 25 ℃ for 7 days on YES: the diameter is 42-44mm, and the bacterial colony has a large number of radial wrinkles, the texture is generally flocculent or felty, white to light yellow, the felty shape of the middle bacterial colony is thicker, and the bacterial colony has no radial wrinkles and is white. The surface of the colony has no exudate and the soluble pigment is deficient.

The morphological observations of hyphae and conidia are shown in FIG. 2:

on MEA, the meristematic peduncles occur in aerial hyphae or trailing hyphae, and the wall is smooth; the broom-shaped branches mainly grow in a single round, the double rounds exist less, the stem base grows singly, and the top ends of the stems are expanded. 2-8 or more bottle stems are arranged in each round, the bottle shape is typical, and the neck of each stem is short and obvious; the meristematic cleavers are spherical or nearly spherical and are obviously stabbing and rough. The sub-chains are loose, nearly cylindrical or irregular. Conidiophores 20-100X 2-2.5 μm, peduncles 7-16X 2.5-3.2 μm, phialides 6-8X 2.0-2.5 μm. Spherical to ellipsoidal, 2.6-4.5 μm.

The basic peduncle and conidia of Z863 are very similar in morphology to Penicillium janczewski. The difference between this strain and Penicillium janczewski: z863 is light in colony color on the CYA culture medium, is also grey white in 2 weeks and cannot become grey green; conidiophores are mainly generated in a single round, and are rarely generated in a double round, so that the stems are generated singly. And Penicillium janczewski is cultured on a CYA culture medium for 2 weeks, and bacterial colonies become grayish green to grayish black; the broom-shaped branches mainly have double-crop rotation, three-crop rotation and single-crop rotation, and 2-4 stem bases are arranged in each crop rotation.

Example 3 molecular characterization of the isolated strains

(1) DNA extraction

1) Culturing Z863 strain on PDA plate at 22 deg.C for 7 days, scraping the mycelium with toothpick, and placing into sterilized centrifuge tube containing 300 μ L extraction buffer solution 1.5 mL;

the formula of the extraction buffer solution is as follows: 1M KCl, 100mM Tris-HCl, 10mM EDTA, pH 8.0;

2) grinding the mycelium with electric grinder, adding 300 μ L extraction buffer solution, and shaking for 2 min;

3) centrifuging at 10000rpm for 10 min;

4) sucking the supernatant, transferring the supernatant into another new centrifugal tube, and discarding the precipitate;

5) adding isopropanol (analytically pure) with the same volume into the supernatant, slightly inverting and mixing uniformly for several times, centrifuging at 12000rpm for 10min, and precipitating nucleic acid;

6) pouring the supernatant slightly, and inverting the centrifuge tube containing the precipitate on absorbent paper to dry the water;

7) adding 300 μ L70% ethanol, slightly inverting and mixing for several times, and centrifuging at 12000rpm for 2 min;

8) gently pouring out the supernatant, and repeating the step 7) once;

9) inverting the centrifuge tube, draining water on absorbent paper, and standing at 37 deg.C for 15min to fully volatilize ethanol;

10) with 50. mu.L ddH₂And O, resuspending and precipitating to obtain Z863 genomic DNA, wherein the concentration reaches 30 ng/. mu.L.

(2) PCR amplification of fungal ribosome ITS rDNA gene, tubulin BenA gene, greening factor TEF gene, ribosome LSU rDNA gene, ribosome SSU rDNA gene, calmodulin CAM gene, RNA polymerase binding protein RPB2 gene and beta-tubulin gene

ITS primers: the sequence of the upstream primer ITS1 is as follows: 5'-TCCGTAGGTGAACCTGCGG-3', the sequence of the downstream primer ITS4 is: 5'-TCCTCCGCTTATTGATATGC-3', respectively;

tubulin BenA gene primer: the upstream primer Bt2 a: GGT AAC CAA ATC GGT GCT GCT TTC, and a downstream primer Bt2 b: ACC CTC AGT GTA GTG ACC CTT GGC, respectively;

CAM primer: upstream primer CMD 5: CCG AGT ACA AGG ARG CCT TC, downstream primer CMD 6: CCG ATR GAG GTC ATR ACG TGG, respectively;

RPB2 primer: an upstream primer 5F: GAY GAY MGW GAT CAY TTY GG, downstream primer 7 CR: CCC ATR GCT TGY TTR CCC AT, respectively;

the SSU primers: the upstream primer NS 1: GTA GTC ATA TGC TTG TCT C, downstream primer NS 4: CTT CCG TCA ATT CCT TTA AG, respectively;

LSU primer: the upstream primer LR 5: ATC CTG AGG GAA ACT TC, downstream primer LROR: ACC CGC TGA ACT TAA GC, respectively;

TEF primer: upstream primer CEFF 2: GGCTTCAACGTGAAGAACG, respectively; downstream primer CEFR 1: CCGTKCAARCCRGAGATGG, respectively;

beta-tubulin primer: upstream primer T12: TAACAACTGCTGGGCCAAGGGTCAC, respectively; a downstream primer T22; TCTGGATGTTGTTGGGAATCC are provided.

PCR amplification was carried out in a 50. mu.L reaction system containing: 2 μ M each of the upstream and downstream primers, 200 μ M dNTPs, MgCl₂1.5mM, 10 XPCR buffer 5. mu.L, template DNA 2. mu.L, Taq enzyme 2U.

The PCR amplification reaction was performed on a Langzy MG96G type PCR machine. Reaction conditions are as follows: pre-denaturation at 94 ℃ for 2min, followed by 35 cycles comprising: denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 40s, and extension at 72 ℃ for 1 min. Finally, extension is carried out for 10min after 72 ℃.

(3) Recovery and purification of PCR product

After the PCR reaction is finished, detecting the PCR product by 1% agarose gel electrophoresis, and then adopting a DNA gel purification kit of Aisijin biotechnology company according to the steps of the kit specification, wherein the steps are as follows:

1) adding 50 μ L of PCR product into 1% agarose gel sample application hole, and performing electrophoresis for 30min under 5V/CM electrophoresis condition;

2) after electrophoresis is finished, cutting the gel containing the target DNA fragment by using a blade under an ultraviolet lamp, placing the gel in a 2mL centrifuge tube, and weighing;

3) adding DE-A buffer solution into a 2mL centrifuge tube for collecting gel according to the standard of adding 3mL DE-A buffer solution into 1mg gel, and keeping the temperature at 75 ℃ for 10min, wherein the vibration is carried out for several times until the gel is completely melted;

4) adding DE-B buffer solution with the volume 0.5 time that of DE-A, and mixing uniformly;

5) putting the DNA preparation tube into a 2mL centrifuge tube, transferring the mixed solution into the DNA preparation tube, centrifuging at 12000rpm for 1min, and removing the supernatant;

6) the DNA preparation tube is put back into a 2mL centrifuge tube, 500. mu.L of buffer solution W1 is added, and centrifugation is carried out at 12000rpm for 30 s;

7) the DNA preparation tube is put back into a 2mL centrifuge tube, 700. mu.L of buffer solution W2 is added, and centrifugation is carried out at 12000rpm for 30 s;

8) repeating the step 7) once;

9) the DNA preparation tube was returned to the 2mL centrifuge tube and centrifuged at 12000rpm for 2 min. To remove the washing solution on the dry film;

10) the DNA preparation tube was returned to the 2mL centrifuge tube, and 50. mu.L of ddH was added₂O, centrifuging at 10000rpm for 1min, and eluting DThe NA was stored at-20 ℃.

(4) Sequencing and sequence analysis of genes

The purified and recovered target DNA fragment after electrophoresis detection is sent to an ABIPRISMA377 type automatic sequencer for sequencing. The sequencing results are strictly checked to obtain DNA fragment sequences shown as SEQ ID No.1-SEQ ID No.8 (fragments obtained by amplification of ITS, BenA, LSU, SSU, TEF, RPB2, CAM and Tubulin, respectively).

On the NCBI website, the nucleotide sequence of the ITS of the determined strain Z863 is searched and aligned with the homologous or similar nucleotide sequence in the GenBank database using BLAST. The comparison result shows that the similarity of the sequence with Penicillium janczewski with the accession number of MK179261, Penicillium arizonense with the accession number of MH492021, Penicillium canescens with the accession number of KX359603, Penicillium muticum with the accession number of NR _138358 and Penicillium janczewski with the accession number of KP016839 reaches 100 percent. This result suggests that the ITS region sequence does not determine to which species the Z863 strain belongs, and it is only clear that this strain is of the species in the Candida group in Penicillium Penicillium.

And the comparison result shows that the similarity of the sequence with Penicillium waksmanii with the accession number of MG832201, Penicillium griseoazureum with the accession number of KP016919 and Penicillium janczewski with the accession number of MG832199 exceeds 99 percent; the similarity with Penicillium canescens with the accession number KJ866964 and the accession number MG832186 is up to 98.8%.

And the LSU gene sequence of Z863 is used for searching a GenBank database, and the comparison result shows that the sequence has 100 percent of similarity with Penicillium radiatolobatum with the accession number of MH872986, Penicillium canescens with the accession number of MH867903 and Penicillium janczewski with the accession number of MH 877145;

a GenBank database was searched with the SSU sequence of Z863, and the results of comparison showed that the sequence had a similarity of more than 99% to Penicillium saccharomum with accession number AB027410, Penicillium chrysogenum with accession number KT601570, Penicillium solanum with accession number JN642222, and Penicillium verrucosum with accession number JN 938976.

A GenBank database was searched with the TEF sequence of Z863, and the comparison showed that the sequence had 98% similarity to Penicillium arizonense with accession XM _022626304 and 96% similarity to Penicillium chrysogenum with XM _002558686 and XM _ 016739759.

The GenBank database is searched by using the RPB2 sequence of Z863, and the comparison result shows that the similarity of the sequence and Penicillium dunnediense with the accession number of KP016850 reaches 98.8 percent; the similarity with Penicillium griseozureum with the accession number of KP016852 reaches 98.7 percent; the similarity with Penicillium janczewski with the accession number of JN406612 reaches 97.6 percent, and the similarity with Penicillium canescens with the accession number of JN121485 reaches 96.6 percent.

The GenBank database is searched by the CAM sequence of Z863, and the comparison result shows that the similarity of the sequence and Penicillium dunnediense with the accession number of KJ775405 reaches 98 percent; the similarity with Penicillium griseozureum with the accession number of KP016823 reaches 98.8 percent; the similarity with Penicillium janczewski with the accession number of EU644079 reaches 97.7 percent.

The GenBank database was searched with the tubulin sequence of Z863, and the comparison results showed that the sequence had a 97% similarity to Penicillium janczewski with accession No. JN112041, and had a similarity of 95% or less to other species.

Since the results of the above sequence similarity comparison of each individual DNA fragment are different and the classification status cannot be determined by one individual DNA fragment alone, we conducted comprehensive phylogenetic analysis using the ITS, CAM and BenA fragments with reference to the results of the study on the Penicillium sect.cancer group in other studies (Persononia 36, 2016: 247-.

TABLE 1 Gene sequences used in construction of phylogenetic trees

The obtained sequence was subjected to Blastn alignment with nucleotide sequences in GenBank databases, similar sequences were downloaded from GenBank databases (see table 1), the three sequences were aligned and corrected by bioidit, respectively, and then the three sequences were combined into one sequence, and a phylogenetic tree was constructed by the unweighted reduction method of PAUP 4.0b10 software. The stability of the branches of the phylogenetic tree was evaluated by 1000 bootstraps. We constructed phylogenetic trees with Aspergillus muricatus as the exogen, and the results of phylogenetic tree analysis are shown in FIG. 3, which shows that the Z863 strain is closely related to Penicillium janczewski, P. nigricans, P. griseoazureum, and P. echinatum, but does not belong to any of these species, indicating that it is a new species. The newly screened strain Z863 is tentatively named as Penicillium sp, strain number Z863, and is preserved in the China center for type culture Collection, located at the university of Wuhan, China, in 2019, 10 and 30 months, with the preservation number of CCTCC NO: m2019870.

Example 4 investigation of the inhibitory Effect of Z863 on plant pathogenic fungi by culture on the confrontation

Adopting a plate opposing culture method. 15mL of PDA culture medium is added into a culture dish with the diameter of 9cm, Z863 and phytopathogen are respectively inoculated into the culture dish after cooling, and the distance between two inocula is 4 cm. Then, the cells were cultured in an incubator at 25 ℃ for 1 week to examine the inhibition of pathogenic bacteria. The pathogenic bacteria tested were citrus black spot (Diaporthe citri), citrus anthracnose (Colletotrichum gloeosporioides), citrus black spot (Phyllosticta citrinariensis), rice blast (Pyricularia oryzae), wheat gibberellic disease (Fusarium graminearum), Pyricularia pyricularis (Botryosphaeria kusaki), Rhizoctonia solani (Rhizoctonia solani).

The results are shown in FIGS. 4 to 11: through detection, the strain Z863 has obvious inhibition effects on rice blast, citrus black spot, citrus anthracnose and citrus black spot, but has no inhibition effects on erwinia amylovora, rhizoctonia solani and wheat scab.

Sequence listing

<110> Zhejiang university

<120> penicillium with bacteriostatic action and application

<160> 24

<170> SIPOSequenceListing 1.0

<210> 1

<211> 586

<212> DNA

<213> Penicillium sp.)

<400> 1

ttcctccgct tattgatatg cttaagttca gcgggtatcc ctacctgatc cgaggtcaac 60

ctggaaaaaa gttttggttg atcggcaagc gccggccggg cctacagagc gggtgacaaa 120

gccccatacg ctcgaggacc ggacgcggtg ccgccgctgc ctttcgggcc cgtccccccg 180

ggaaggggga cgagacccaa cacacaagcc gggcttgagg gcagcaatga cgctcggaca 240

ggcatgcccc ccggaatacc agggggcgca atgtgcgttc aaagactcga tgattcactg 300

aattctgcaa ttcacattac gtatcgcatt tcgctgcgtt cttcatcgat gccggaacca 360

agagatccgt tgttgaaagt tttaaataat ttatatttag actcagactg caattttcat 420

acagagttca aggtgtcttc ggcgggcgcg ggcccggggg cagatgcccc ccggcggccg 480

tgaggcgggc ccgccgaagc aacaaggtac aataaacacg ggtgggaggt tgaattcaga 540

gaattctcgc tcggtaatga tccttccgca ggttcaccta cggaag 586

<210> 2

<211> 446

<212> DNA

<213> Penicillium sp.)

<400> 2

cagacagata tcgagacttt tttcgcgtca ttggttcaca atttactgac tggattacag 60

gcaaaccatc tccggtgagc acggtctcga tggcgatgga cagtaagttc aatgtggaat 120

ttcttgtggt gggttgggca gctgatatct tgttaggtac aacggtacct ccgacctcca 180

gctcgagcgc atgaacgtct acttcaacca tgtgagtaca atatgttgga attggctgct 240

taagcattat ctgacttcta tgttttgacc cctcaggccc acggtgacaa gtacgttccc 300

cgtgccgttc tcgtcgactt ggagcccggt accatggacg ctgtccgctc cggtcctttc 360

ggcaagcttt tccgccccga caacttcgtc ttcggtcagt ccggtgctgg taacaactgg 420

gccaagggtc actacacctg agggta 446

<210> 3

<211> 893

<212> DNA

<213> Penicillium sp.)

<400> 3

cagatggttc gattagtctt tcgcccctat acccaaattc gacgatcgat ttgcacgtca 60

gaaccgctac gagcctccac cagagtttcc tctggcttcg ccctattcag gcatagttca 120

ccatctttcg ggtcccaaca gctacgctct tactcaaatc catccgaaga catcaggatc 180

ggtcgatggt gcacccaaag ggttcccacc tccgttcgct ttcactgcgc gcacgggttt 240

gacacccgaa cactcgcgta gatgttagac tccttggtcc gtgtttcaag acgggtcgct 300

tacgaccatt atgccaacgt ccgagccgaa gcgcgttcct cggtctaggc aggtcgcatt 360

gcaccctcgg ctataagacg cccctggggg cgttaccttc cgagggcctt tgaccgaccg 420

cccaaaccga cgttggcccg cccgcgggga agtacaccgg cacgaatgcc ggctgaaccc 480

cgcgagcgag tctggtcgca agcgcttccc tttcaacaat ttcacgtgct ttttaactct 540

cttttcaaag tgcttttcat ctttcgatca ctctacttgt gcgctatcgg tctccggcca 600

atatttagct ttagatgaaa tttaccaccc atttagagct gcattcccaa acaactcgac 660

tcgtcgaagg agcttcacac gggcgcggac accccatccc atacgggatt ttcaccctct 720

atgacgtccc gttccaggga acttagatgg ggaccgctcc cgaagcatcc tctgcaaatt 780

acaatgcgga ccccgaagga gccagctttc aaatttgagc tcttgccgct tcactcgccg 840

ttactggggc aatccctgtt ggtttctttt cctccgctta ttgatatgct ata 893

<210> 4

<211> 1044

<212> DNA

<213> Penicillium sp.)

<400> 4

gcatgtctag tataagcaac ttgtactgtg aaactgcgaa tggctcatta aatcagttat 60

cgtttatttg atagtacctt actacatgga tacctgtggt aattctagag ctaatacatg 120

ctaaaaaccc cgactcacga aggggtgtat ttattagata aaaaaccaac gcccttcggg 180

gctccttggt gaatcataat aacttaacga atcgcatggc cttgcgccgg cgatggttca 240

ttcaaatttc tgccctatca actttcgatg gtaggatagt ggcctaccat ggtggcaacg 300

ggtaacgggg aattagggtt cgattccgga gagggagcct gagaaacggc taccacatcc 360

aaggaaggca gcaggcgcgc aaattaccca atcccgatac ggggaggtag tgacaataaa 420

tactgatacg gggctctttt gggtctcgta attggaatga gaacaattta aatcccttaa 480

cgaggaacaa ttggagggca agtctggtgc cagcagccgc ggtaattcca gctccaatag 540

cgtatattaa agttgttgca gttaaaaagc tcgtagttga accttgggtc tggctggccg 600

gtccgcctca ccgcgagtac tggtccggct ggacctttcc ttctggggaa cctcatggcc 660

ttcactggct gtggggggaa ccaggacttt tactgtgaaa aaattagagt gttcaaagca 720

ggcctttgct cgaatacatt agcatggaat aatagaatag gacgtgcggt tctattttgt 780

tggtttctag gaccgccgta atgattaata gggatagtcg ggggcgtcag tattcagctg 840

tcagaggtga aattcttgga tttgctgaag actaactact gcgaaagcat tcgccaagga 900

tgttttcatt aatcagggaa cgaaagttat gggatcgaag acgatcagat accgtcgtag 960

tcttaaccat aaactatgcc gactagggat cggacgggat tctataatga cccgttcggc 1020

accttacgag aaatcaaagt tttt 1044

<210> 5

<211> 1001

<212> DNA

<213> Penicillium sp.)

<400> 5

gtgattccat caagaacatg atcactggta cctcccaggc tgactgcgcc attctcatca 60

tcgcctccgg cactggtgag ttcgaggctg gtatctccaa ggatggccag acccgtgagc 120

acgctctgct tgccttcacc ctcggtgtta agcagctcat cgttgccctc aacaagatgg 180

acacctgcaa gtggtccgag gaccgttaca acgagattgt caaggagacc tccaacttca 240

tcaagaaggt cggctacaac cccaagtccg ttcccttcgt ccccatctcc ggcttcaacg 300

gcgacaacat gctcgagccc tcccccaact gcccctggta caagggttgg gagaaggaga 360

ccaagtccgg caagtccacc ggaaagaccc tcctcgaggc cattgacgcc atcgacaccc 420

ccgtccgtcc ctccaacaag cccctccgtc ttcccctcca ggatgtgtac aagatctccg 480

gtattggcac agttcccgtc ggccgtgtcg agactggtat cattaccccc ggcatggtcg 540

tcaccttcgc tcccgccaac gtgaccactg aagtcaagtc cgtcgagatg caccaccagc 600

agctcaaggc cggtaacccc ggtgacaacg tcggcttcaa cgtcaagaac gtttccgtca 660

aggaggttcg ccgtggtaac gtcgcttccg actccaagaa cgaccccgcc atgggctgtg 720

actccttcaa cgcccaggtc atcgtcctga accaccccgg tcaggtcggc gctggatacg 780

ctcccgttct ggactgccac actgcccaca ttgcttgcaa gttctccgag ctcctcgaga 840

agattgaccg ccgtaccggt aaggccaccg agacctcccc caagttcatc aagtccggtg 900

atgccgctat cgtcaagatg gttccctcca agcccatgtg tgttgaggcc ttcaccgact 960

accccctctc ggtcgtttcg ccgtccgtga catgcgtcag a 1001

<210> 6

<211> 1032

<212> DNA

<213> Penicillium sp.)

<400> 6

tggctggacc ccttctggcc accttttccg tgttcttttc acccgagtga cccgtgacct 60

tcaacgttac gtgcagcgca gtgtcgagac caaccgtgag atctacctca acattggtat 120

caaggcggcc accctcacag gaggtctgaa gtatgctctt gctaccggta actggggtga 180

gcagaagaag gccgccagcg ccaaggctgg tgtgtcccag gtgctgagtc gttacacttt 240

cgcctcttca ttgtcccatc tgcgacgtac caacacaccc attggcagag atggaaagat 300

tgccaaacca cgtcaactac acaacaccca ttggggtttg gtctgtccag ctgagacacc 360

ggaaggtcag gcttgtggtc tggtcaagaa cttggccctc atgtgctaca tcactgttgg 420

tacgccgagt gagcccatca ttgatttcat gattcagcga aatatggaag ttctcgagga 480

gttcgaacct caggtcacgc caaatgcaac caaggtgttt gtcaacggtg tctgggttgg 540

tattcacaga gacccttcgc atcttgtcgc tactatgcaa aatcttcgtc gacgtaacat 600

gatctcgcac gaggttagtt tgattcgtga catccgtgag cgtgaattca agattttcac 660

cgacaccggt cgcgtgtgcc gtcccctgtt cgtcatcgac aatgatccca agagtgagaa 720

ctcaggtgga ttgatcctca acaaggagca cattcgcaag cttgagcaag ataaggatct 780

accggccgat atggacccag aagaccgtcg cgagcagtac ttcggatggg atggtcttgt 840

tcgttctggt gcagttgaat atgtcgatgc tgaagaagag gagactatca tgattgtcat 900

gacgcccgaa gatctcgaga tttcccgaca actccaggct ggttacgctc tccccgagga 960

cgagaccaat gatcccaaca agcgcgttcg ctcgattctc agccagcgtg cccacacttg 1020

gacacattgt ga 1032

<210> 7

<211> 514

<212> DNA

<213> Penicillium sp.)

<400> 7

agctcagcgg cggagatgaa accgttgttg tcgcggtcga acaccttgaa cgcctcgcga 60

atctcctcct cggaatctgt gtccttcatc ttgcgggcca tcatggtcaa gaactctatc 120

gacgggtcaa tagccatctc gtttgggatc tagtggacac gggaaagtac cggggaagtc 180

aatagtgcca ttgttgtcag cgtcaacctc gttgatcatg tcctgcagct cagactcgga 240

ggggttctgg cccagagagc gcatgacggt gccaagctcc ttggtggtga tttgtcctgg 300

aggtggaatc ggtcttagta tatcgtttct ttcaattcgg gggcagcaga cactgctgcc 360

agttcaactg ggttgtcggg cgaggtcaca cttaccatcg ccatccttgt cctatttcgc 420

atcaaagccg tcagtatgtt gtccagcctt cataacttca atccacgggt ggaaacactt 480

acaaacaggg agaaggcctc cttgtactcg gaga 514

<210> 8

<211> 766

<212> DNA

<213> Penicillium sp.)

<400> 8

caggtgccga gctcgttgac caggtcatcg atgtcgtccg ccgtgaggcc gaggcttgtg 60

actgcctcca gggtttccag atcacccact ccctcggtgg tggtaccggt gccggtatgg 120

gtacactcct gatctccaag atccgtgagg agttccccga ccgtatgatg gccaccttct 180

ccgtcgttcc ttctcccaag gtttccgaca ccgttgttga gccttacaac gccaccctct 240

ccgtccacca gctcgttgag cactccgacg agaccttctg tattgataac gaggtatgca 300

atgcgtggga accagcaatg gcacatcact gacgaattta ggctctgtac gacatctgca 360

tgcgtaccct caagctgtcc cagccctcgt acggtgacct gaaccacctc gtctccgccg 420

tcatgtccgg tgtgaccacc tccctccgtt tccccggcca gctcaactcc gatctccgca 480

agctcgccgt caacatggtg cccttccccc gtctgcactt cttcatggtc ggcttcgctc 540

ccctgaccag ccgcagcggc taccagtacc gccaggtcag cgttcccgag ttgacccagc 600

agatgttcga ccccaagaac atgatggctg cttccgactt ccgtaacggc cgttacctca 660

cctgctccgc tctcttccgc ggtaaggtct ccatgaagga ggttgaggac cagatgcgca 720

acatccagaa caagaaccag agctacttcg ttgagtggat tcccaa 766

<210> 9

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

tccgtaggtg aacctgcgg 19

<210> 10

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

tcctccgctt attgatatgc 20

<210> 11

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

ggtaaccaaa tcggtgctgc tttc 24

<210> 12

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

accctcagtg tagtgaccct tggc 24

<210> 13

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> r

<222> (1)..(1)

<223> r is a/g

<400> 13

ccgagtacaa ggargccttc 20

<210> 14

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> r

<222> (1)..(1)

<223> r is a/g

<220>

<221> r

<222> (1)..(1)

<223> r is a/g

<400> 14

ccgatrgagg tcatracgtg g 21

<210> 15

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> y

<222> (1)..(1)

<223> y is c/t

<220>

<221> m

<222> (1)..(1)

<223> m is a/c

<220>

<221> w

<222> (1)..(1)

<223> m is a/t

<400> 15

gaygaymgwg atcayttygg 20

<210> 16

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> r

<222> (1)..(1)

<223> r is a/g

<220>

<221> y

<222> (1)..(1)

<223> y is c/t

<400> 16

cccatrgctt gyttrcccat 20

<210> 17

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

gtagtcatat gcttgtctc 19

<210> 18

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

cttccgtcaa ttcctttaag 20

<210> 19

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

atcctgaggg aaacttc 17

<210> 20

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

acccgctgaa cttaagc 17

<210> 21

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

ggcttcaacg tgaagaacg 19

<210> 22

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> k

<222> (1)..(1)

<223> k is g/t

<220>

<221> r

<222> (1)..(1)

<223> r is a/g

<400> 22

ccgtkcaarc crgagatgg 19

<210> 23

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

taacaactgc tgggccaagg gtcac 25

<210> 24

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

tctggatgtt gttgggaatc c 21

Claims

1. A Penicillium sp with bacteriostasis is characterized in that the Penicillium sp is named as Penicillium sp, strain number Z863, and the preservation number is CCTCC NO: m2019870.

2. The use of the penicillium sp according to claim 1 to inhibit the growth of pathogenic bacteria selected from the group consisting of rice blast, citrus black spot, citrus anthracnose and citrus black spot.

3. The use of a penicillium sp according to claim 1 for the control of infections by pathogens of rutaceae plants, selected from the group consisting of citrus alternaria, citrus anthracnose and citrus alternaria.

4. Use according to claim 3, wherein the rutaceae plant is citrus.

5. The use of the penicillium sp according to claim 1 for the control of infection by a pathogenic bacterium of rice, wherein the pathogenic bacterium is pyricularia oryzae.