CN109706082B - Biocontrol giant-cavity phoma strain P2 and application thereof - Google Patents

Abstract

The invention belongs to the technical field of agricultural microbiology, and particularly discloses a giant-cavity phoma strain and application thereofPhoma macrostoma) The strain has a preservation number of CCTCC NO: m2018726. The phoma macrostoma or the filtrate of the fermentation product of the phoma macrostoma can effectively inhibit the hypha growth of important pathogenic bacteria (sclerotinia sclerotiorum, botrytis cinerea, rhizoctonia solani and rape black shank) on rape, and the germination of conidia and sclerotium; also has broad-spectrum antifungal effect, and can inhibit the growth of hypha of most other plant pathogenic fungi. In addition, the strain P2 ferment filtrate has no toxic action on the rape seeds, and the rape seeds can normally germinate after being soaked in the ferment filtrate, so that the strain P2 can be used for treating the rape seeds to prevent and treat soil-borne diseases. Meanwhile, the bacterium has strong capabilities of high temperature resistance, acid and alkali resistance and ultraviolet radiation resistance. Therefore, the P2 strain has potential for development and utilization.

Description

Biocontrol giant-cavity phoma strain P2 and application thereof

Technical Field

The invention belongs to the technical field of agricultural microbiology, and particularly relates to a Phoma locularis (Phomamacostoma) and application thereof.

Background

Phoma (Phoma) fungi cause important plant diseases, and can cause diseases such as leaf spot, stem rot and branch withering of plants by damaging the plants of Leguminosae, Solanaceae, Brassicaceae and Magnoliaceae. For example, Phoma destructor is a pathogen causing tomato leaf and stem blight (Boerema et al,2004), while Phoma lingam is an important pathogen causing canker of the stem base of cruciferous crops (two types of leprosphereria maculons/leprosphereria abigbosa in a sexual state), and can cause rapeseed to be reduced in yield by 20% to 60%. P.andigena and p.tracheiphila of the genus phoma are listed as local quarantine pathogenic bacteria in regions such as europe, p.fovata in the southern united states and p.macconaldii in australia, respectively, and have important quarantine properties. In our country, p.exigua, p.pinodella and p.tracheiphila are listed in the "entry plant quarantine pest list of the people's republic of china" published in 2007.

The phoma fungi widely exist in the environment and play an important role in the process of degrading organic matters; in addition, some Phoma fungi can also be used as biocontrol agents to control biological plant diseases. For example, by Arie, 1999, reported Phoma globeratea, which was able to produce epoxon to inhibit the development of clubroot in cruciferous crops; in addition, White reported in 2000 that the bacterium was also capable of parasitizing powdery mildew, which produced a large number of conidiospores on the closed shell of powdery mildew, thereby inhibiting the growth of powdery mildew. Giant-cavity phoma (phomamacostomoma) capable of generating novel cyclic tetraacid (novel cyclic tetramic acid) is reported by Graupener in 2003 to be used as a herbicide to control weeds on lawns, such as broad-leaved weeds including thistle, dandelion, clematis and the like, but the biological control of phytopathogens by using giant-cavity phoma at home and abroad is not reported yet.

Disclosure of Invention

The invention aims to provide a Phoma macrostoma (Phoma macrostoma) with a preservation number of CCTCC NO: m2018726, classification name: phoma macrotoma P2.

Another purpose of the invention is to provide application of Phoma macrostoma P2, and the strain can be used for preparing biocontrol microbial inoculum or preparing a plant seed coating agent.

In order to achieve the purpose, the invention adopts the following technical measures:

the Phoma macrospora is separated from a rape black shank sample collected from Yun xi county in Hubei province, and the morphological characteristics of the Phoma macrospora are very similar to the sterile Phoma linggam of the rape black shank, so that the strain is delivered to the China center for type culture collection in Wuhan university in Wuhan city in Hubei province for collection in 2018, 10 and 30 days, and the applicant names the strain P2. The preservation number of the giant-cavity phoma is CCTCC NO: m2018726, classification name: phoma macrotoma P2.

Colony characteristics:

after the culture is carried out for 7d on an OA culture medium, the edges of the bacterial colonies are neat, the bacterial colonies are grayish green, the back surfaces of the bacterial colonies are light green, conidiospores are densely generated in the centers of the bacterial colonies, and the diameters of the bacterial colonies are 49.0 mm; after the culture is carried out for 7 days on the MA culture medium, the edges of the colonies are neat, the colonies are white villiform, the back faces are orange, no spore is generated, and the diameter of the colonies is 61.3 mm; after 7d of culture on the PDA culture medium, the edges of the colonies are neat, the colonies are gray green, the back surface of the colonies is green, the conidiospore devices are densely scattered, and the diameter of the colonies is 64.3 mm; p2 on PDA medium 14d showed a large number of conidiophores on the surface of the medium and the conidiophores appeared as orange pink spore mucus. The conidiophore is spherical and brown, the outer wall of the conidiophore is not smooth and is leather-like, and the size of the conidiophore is 320-333 mu m; the conidiophores are bottle-shaped, and the size is 6.6-11.2 mu m multiplied by 6.6-8.6 mu m; the conidiophores have an oval shape, are smooth and transparent and have no septum, the conidiophores contain 2-5 oil drops, and the size of the conidiophores is 7.3-9.3 mu m multiplied by 3.3-4.0 mu m.

The application of Phoma Macrocystis (Phoma macrostoma) P2 strain comprises preparing the strain into a control agent of plant pathogenic bacteria or a coating agent of plant seeds; the phytopathogens include, but are not limited to: pythium aphanidermatum (Pythium apanidum), Rhizopus stolonifer (Rhizopus stolonifer), Mucor hiemalis (Mucor hiemalis), Aspergillus flavus (Aspergillus flavus), Aspergillus niger (Aspergillus niger), Botrytis cinerea (Botrytis cinerea), Curvularia oryzae (Curvularia lunata), Leptosphaeria brasiliensis (Leptosphaeria bigloeosa), Sclerotia sativa (Sclerotia minor), Sclerotium sclerotiorum (Sclerotia sclerotiorum), and Rhizoctonia solani (Rhizoctonia solani).

Compared with the prior art, the invention has the following advantages:

the use of the phoma macrostoma P2 fermented product filtrate provided by the invention can effectively inhibit the growth of hyphae such as sclerotinia sclerotiorum, botrytis cinerea and the like which are important pathogenic bacteria on rape, and the germination of conidia and sclerotium; the P2 fermented product filtrate also has broad-spectrum antibacterial effect, and can effectively inhibit the growth of pathogenic fungi such as Aspergillus flavus which are harmful to human body, besides the growth of most of plant pathogenic fungi. In addition, the P2 ferment filtrate has no toxic effect on the rape seeds, and the rape seeds can normally germinate after being soaked in the ferment filtrate, so that the root irrigation of the P2 ferment filtrate can be considered when preventing and treating soil diseases, and the potential adverse effect of chemical prevention and treatment on the environment and crop safety is avoided. Finally, the bacterium has the characteristics of strong high temperature resistance, acid and alkali resistance and ultraviolet resistance, and can be used as a metabolite for formulation processing and development.

Drawings

FIG. 1 is a morphological diagram of a colony of Phoma macrostoma P2 cultured on a medium;

wherein A is the colony morphology of the OA culture medium; b is the colony morphology of the MEA culture medium; c is colony morphology of PDA culture medium; d is the culture form of PDA 14D; e is a conidiophore formed on the PDA culture medium; f is a conidiophore; g is conidiophore; h is conidium.

FIG. 2 is a diagram showing the pathogenicity of Phoma macrostoma P2 and other strains of Phoma (P1, P3 and P4) of the present invention to canola.

FIG. 3 is a graph showing the results of experiments on the confrontation of Phoma macrostoma P2 and other strains of Phoma (P1, P3 and P4) with the pathogen Leptosphaeria biglobosa.

FIG. 4 is a graphical representation of the results of experiments on the inhibition of Leptosphaeria biglobosa infestation on canola by the fermentation filtrate of Phoma macrostoma P2 and other strains of Phoma (P1, P3 and P4) of the present invention.

FIG. 5 is a schematic time diagram of the production of antifungal substances from the fermentation filtrate of Phoma macrostoma P2 according to the present invention.

FIG. 6 is a schematic diagram showing the screening of the strain for measuring the biological activity of an antifungal substance in the filtrate of Phoma macrostoma P2 fermentation product of the present invention.

FIG. 7 is a schematic diagram showing the inhibition zone of a fermentation filtrate (containing an antifungal substance) of Phoma megalocularis P2 on Leptosphaeriabaiglobosa according to the present invention;

wherein: a, a bacteriostatic circle overall diagram; b, a transparent ring boundary diagram under the body type mirror; c, drawing the hypha tip of the transparent circle under the optical microscope; d, a malformed conidiophore diagram inside the transparent ring.

FIG. 8 is a graph showing the effect of different temperature treatments on the activity of Phoma macrostoma P2 ferment filtrate (containing antifungal substances).

FIG. 9 is a schematic diagram showing the effect of acid and alkali treatment on the activity of the filtrate (containing antifungal substances) of the fermentation product of Phoma macrostoma P2.

FIG. 10 is a graph showing the effect of UV treatment on the activity of Phoma macrostoma P2 ferment filtrate (containing antifungal substances).

FIG. 11 is a graph showing the effect of ambient pH on Phoma macrostoma P2 ferment filtrate (containing antifungal substances) activity.

FIG. 12 is a schematic diagram showing the effect of different concentrations of the filtrate of the fermentation product of Phoma macrospora P2 (containing antifungal substances) on the hyphae of 4 phytopathogenic fungi of plants according to the present invention.

FIG. 13 is a diagram showing the teratogenicity of 0.6% P2 fermented product filtrate (containing antifungal substance) against hyphae of 4 pathogenic bacteria of Phoma macrostoma according to the present invention;

0.6% P2 ferment filtrate (containing antifungal substance) refers to 90mL PDA medium + 600. mu. L P2 ferment filtrate.

FIG. 14 is a schematic representation of the effect of Phoma macrostoma 10% P2 ferment filtrate (containing an antifungal substance) of the present invention on the germination of sclerotinia sclerotiorum and Botrytis cinerea sclerotium;

the 10% P2 ferment filtrate (containing antifungal substance) refers to 90mL PDA medium +10mL P2 ferment filtrate.

FIG. 15 is a schematic diagram showing the effect of Phoma macrostoma P2 ferment filtrate (containing antifungal substance) of the present invention on sclerotium germination after soaking sclerotium and Botrytis cinerea sclerotium.

FIG. 16 is a schematic representation of the effect of Phoma macrospora P2 ferment filtrate (containing an antifungal substance) of the present invention on the germination of conidia Botrytis.

FIG. 17 is a schematic diagram showing the effect of Phoma macrostoma P2 ferment filtrate (containing antifungal substances) on the germination of the conidia of Leptosphaeria brasiliensis.

FIG. 18 is a schematic diagram showing the effect of Phoma macrostoma P2 fermented product filtrate (containing antifungal substances) on sclerotinia sclerotiorum disease prevention on live rape leaves.

FIG. 19 is a schematic diagram showing the effect of the fermentation filtrate of Phoma macrospora P2 (containing antifungal substances) on the disease prevention effect of Botrytis cinerea on live rape leaves.

FIG. 20 is a schematic diagram showing the measurement of the bacteriostasis spectrum of the fermentation filtrate (containing antifungal substances) of Phoma macrospora P2 of the present invention.

FIG. 21 is a schematic diagram showing the toxicity of the fermentation filtrate of Phoma macrostoma P2 (containing antifungal substances) to rape seeds.

Detailed Description

In order to better explain the invention, the following further illustrate the main content of the invention in connection with specific examples, but the content of the invention is not limited to the following examples. Technical schemes related to the embodiments of the present invention are all conventional schemes in the art if not specifically stated; the reagents or materials, if not specifically mentioned, are commercially available.

Example 1:

separation and identification of Phoma macrospora P2 strain and biological characteristic analysis

First, separation of strain and analysis of biological characteristics thereof

The applicant selects the sick and disabled with typical symptoms from rape black shank samples collected in Yun xi county in Hubei province, and cuts tissue blocks at the diseased and healthy junction, wherein the size of the tissue blocks is about 0.5cm multiplied by 0.5 cm; treating with 75% ethanol and 5% sodium hypochlorite for 30s and 5min, respectively, washing with sterile water for 3 times, transferring to the center of PDA culture medium plate containing 25% lactic acid, culturing at constant temperature of 22 deg.C for 4d, and purifying fungi growing from the edge of diseased tissue. In addition, 4 fungi (Phoma sp.) similar to the pathogenic bacteria of the rape black shank are respectively separated from a rape black shank sample in the laboratory and named as P1, P2, P3 and P4, and the 4 Phoma strains can not cause the rape diseases through pathogenicity analysis (figure 2), so that the P1, P2, P3 and P4 strains are subjected to primary biocontrol determination by adopting a plate opponent culture method; finally, the PDB shake culture solution of the 4 Phoma strains is used for determining the influence of the PDB shake culture solution on the rape infected by the Leptosphaeria biglobosa Lb20, so as to judge whether the 4 Phoma strains have the biocontrol strain.

The test results show that 4 Phoma strains have certain inhibition on hypha growth of Lb20 and obviously reduced conidiophores compared with CK (inoculated with Lb20 only) in the culture process of confronting Leptosphaeria biglobosa 20 (FIG. 3). Test results of Lb20 infection of 4 Phoma strain fermentation filtrates on rape show that pathogenic bacteria Lb20 on P2 fermentation filtrate can not be infected on rape, Lb20 can be infected on rape after treatment of the fermentation filtrates of the other 3 Phoma strains (figure 4), CK (only inoculated with Lb20 and inoculated with drop clear water) can normally attack, and the diameter of a lesion spot after 7d inoculation reaches 8.4mm, so P2 is preliminarily judged to generate a certain antifungal substance, and the growth of Lb20 hypha can be well inhibited, the strain is delivered to the China center for type culture collection in Wuhan university in Wuhan, Hubei province in 2018 and 30 months, and the collection number is CCTCC NO: m2018726, classification name: phomamacostoma P2.

Secondly, morphological characteristics of the strain

(1) Characteristics of bacterial colony

After the culture is carried out for 7d on an OA culture medium, the edges of the bacterial colonies are neat, the bacterial colonies are grayish green, the back surfaces of the bacterial colonies are light green, conidiospores are densely generated in the centers of the bacterial colonies, and the diameters of the bacterial colonies are 49.0 mm; after the culture is carried out for 7 days on the MA culture medium, the edges of the colonies are neat, the colonies are white villiform, the back faces are orange, no spore is generated, and the diameter of the colonies is 61.3 mm; after 7d of culture on the PDA culture medium, the edges of the colonies are neat, the colonies are gray green, the back surface of the colonies is green, the conidiospore devices are densely scattered, and the diameter of the colonies is 64.3 mm; after the P2 strain is cultured on the PDA culture medium for 14 days, a large number of conidiophores are seen on the surface of the culture medium, and orange pink spore mucus is secreted on the conidiophores (figure 1).

(2) Microscopic observation results

The conidiophore is spherical and brown, the outer wall of the conidiophore is not smooth and is leather-like, and the size of the conidiophore is 320-333 mu m; the conidiophores are bottle-shaped, and the size is 6.6-11.2 mu m multiplied by 6.6-8.6 mu m; the tail end of the conidiophores is provided with an oval shape, is smooth and transparent and has no septum, the conidiophores contain 2-5 oil drops, and the size of the conidiophores is 7.3-9.3 Mum multiplied by 3.3-4.0 Mum (figure 1).

Thirdly, identifying the classification attribute of the strain P2

The P2 mycelia cultured for 4d were scraped from PDA medium spread with cellophane, put in a mortar, added with an appropriate amount of liquid nitrogen, ground sufficiently, and extracted by CTAB method for total DNA of P2 strain, which was identified based on internal transcribed spacer l (ITS1), 5.85rDNA, and internal transcribed spacer 2(ITS 2). PCR amplification is carried out on the total DNA of the strain through primers ITS1 and ITS4, the obtained sequence is subjected to Blast comparison in NCBI website, and the identification result is Phoma macrostoma, namely Phoma locusta.

Example 2:

purification and fermentation culture of Phoma macrosperma P2

(1) Strain activation: a P2 strain slant stored in a refrigerator at 4 ℃ is picked up by a sterile inoculating needle, inoculated on a PDA culture medium and cultured in an incubator at 22 ℃ for 3 d.

(2) Strain culture: transferring the P2 strain cultured for 3d in the step (1) to a fresh PDA culture medium, and culturing for 7d in a 22 ℃ incubator.

(3) Fermentation: the colony edge of the P2 strain in step (2) was punched with several bacterial cakes using a sterile punch, 5 hypha blocks were inoculated to each 100mLPDB culture medium, the culture was shake-cultured at 150rpm and 22 ℃ for 3d, 6d, 9d and 15d, and the fermentation filtrates were collected at different days after centrifugation at 6000rpm and used in the following examples.

(4) Phoma macrostoma P2 ferment filtrate: the above-mentioned Phoma macrostoma P2 fermentation filtrate was centrifuged at 6000rpm for 10min, and the supernatant was collected and filtered again through a 0.22 μm bacterial filter to obtain a fermentation filtrate, which was used in the following examples.

(5) PDB medium composition: 200g of potatoes, 20g of glucose and distilled water are added to the mixture until the volume is 1000 mL.

Example 3:

preparation of Phoma macrospora P2 fermentation filtrate and determination of antifungal substance activity thereof

Time dynamics of Phoma Macrocava P2 fermentation filtrates to produce antifungal substances

The fermentation filtrates were collected at 3d, 6d, 9d, 12d and 15d of the P2 strain by shaking, the pH was measured, and the dry weight of the mycelia was measured after drying the mycelia (Table 1). After the fermentation filtrate was filtered through a bacterial filter having a pore size of 0.22 μm, 10% (v/v) (in the present invention, this percentage means that the P2 fermentation filtrate: the culture medium was 1:9 by volume) was added to the PDA culture medium and mixed well, and the plate was inverted to obtain a plate with toxicity, and 10% (v/v) PDB solution was added as a negative control. The following strains of Sclerotinia sclerotiorum (sclerotiorum), Botrytis cinerea (Botrytis cinerea), Rhizoctonia solani (Rhizoctonia solani) and Leptosphaeria brassicae (Leptosphaeria biglobosa) having a diameter of 5mm were isolated in the laboratory as not specifically described, wherein Botrytis cinerea, Rhizoctonia solani and Leptosphaeria brassicae were used, and Botrytis cinerea (B05.10) was a model strain of Botrytis cinerea, and Sclerotinia sclerotiorum (Ss), Rhizoctonia solani (Rs) and Leptosphaeria brassicae (Lb20) identified to be consistent with the properties of the disclosed strains were inoculated onto a virus-carrying plate and a negative control plate, incubated at an isothermal temperature of 22 ℃, after the negative control plate was filled, the diameters of the colonies of the treatments were measured, and 3 replicates were set for each treatment. Calculating the bacteriostasis rate according to a formula:

as shown in FIG. 5, the amount of biomass of Phoma macrostoma P2 was the greatest when the time of shake culture was extended to 15d, the bacteriostatic activity was the greatest, the pH was increased, and the pH was raised to 8.3 when the time of shake culture was extended to 15 d. According to the results of this test, the biological activity was determined in the subsequent tests using the filtrate of the fermentation from the shake culture of P2 strain to 15 days.

TABLE 1 time dynamics of the production of antifungal substances by the fermentation filtrate of Strain P2

Note: the effect of different shake culture P2 fermentation filtrates on the hyphal growth of Sclerotinia sclerotiorum (Ss), Botrytis cinerea (B05.10), Rhizoctonia solani (Rs) and Leptosphaeria maculans (Lb20) was determined in PDB medium. After 3d, 6d, 9d, 12d and 15d of shake culture at 22 ℃ and 150rpm, respectively, the dry weight of hyphae in the fermentation product is weighed, the pH value of the fermentation product filtrate is measured on different shake culture days P2, the difference significance between treatments is analyzed by using the least significant difference method (LSD), and the letters in the same column indicate that the difference is not significant (P > 0.05).

Secondly, screening of the biological activity determination bacteria of the fermentation filtrate (antifungal substance) of Phoma macrostoma P2

Respectively culturing plant pathogenic fungi Aspergillus niger Y-1(Aspergillus niger Y-1) and botrytis cinerea RoseBc-3 (the test selects a strain with high conidium yield to detect the sensitivity of the strain to P2 antifungal substances, the strain with high conidium yield shows darker color on a culture medium, the inhibition zone is more obvious, the botrytis cinerea mode strain B05.10 has low conidium yield and is not suitable for the test) and the rape black shank bacterium Leptosphaeriiglosa (Lb20) until the conidia are produced, then washing the conidia into a sterile 50mL centrifuge tube by sterile water, filtering by four layers of mirror wiping paper to obtain conidia suspension, and adjusting the concentration to 1 × 10⁸spores/mL. Then 20mL of conidium suspension is added into every 200mL of PDA, the mixture is fully mixed and poured into a flat plate, the sensitivity measurement is carried out by adopting a pipe-disk method, then 200 mu LP2 fermentation product filtrate (15d) is added into an oxford cup (the same applies below) with the same specification and the aperture of 6mm, 200 mu LPDB solution is added into the oxford cup as a negative control, and the mixture is cultured for 3d at the constant temperature of 22 ℃. The results show that Lb20 is most sensitive to antifungal substances produced by P2, the mean diameter of the zone of inhibition is 2.9cm, the zone of inhibition is very transparent, aspergillus niger is the most sensitive, the mean diameter of the zone of inhibition is 2.1cm, the zone of inhibition is translucent, RoseBc-3 is the least sensitive, no zone of inhibition is produced, and no zone of inhibition is produced on the bacteria-carrying plate using PDB as the control (fig. 6). Finally, observing the most transparent bacteriostatic circle by an optical microscope, wherein normal hyphae (C in figure 7) and abnormal hyphae (C in figure 7) can be seen at the junction of the transparent circle, and the hyphae which are visible inside the transparent circle are in a chain shape or are already degraded (D in figure 7). Thus, the following tests were carried outThe activity of the compounds is determined by selecting Leptosphaeria biglobosa (Lb20) as test strains.

Stability determination of Phoma Macrocava P2 fermentation filtrate (antifungal substance)

(1) Thermal stability: the P2 ferment filtrate after shaking for 15 days was treated in water baths at 40 deg.C, 60 deg.C, 80 deg.C and 100 deg.C for 10min, 30min and 60min, respectively, with no heat treatment as control. The treated solution was rapidly cooled to room temperature on ice, and the bioactivity assay was performed by the tube-dish method, and the assay bacteria were Leptosphaeria biglobosa (Lb20), and the results of each treatment are shown in fig. 8. The results show that the activity of the antifungal substance produced by P2 remained almost unchanged compared to the control (without heat treatment) after treatment in a water bath at 100 ℃ for 60min, indicating that the substance is resistant to high temperature and does not lose its biological activity under high temperature conditions.

(2) Acid-base stability: adjusting pH of the P2 fermented product filtrate which is shaken for 15d to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and 13 respectively by using HCl of 4mol/L and NaOH of 4mol/L, placing the fermented product filtrate in a refrigerator of 4 ℃, adjusting pH of each treated fermented product filtrate to a normal level (pH 6) respectively after 24h, and finally performing biological activity determination by using a tube-disc method, wherein the biological activity determination bacterium is Leptosphaeria biglobosa (Lb20), and biological activity results of each treatment are shown in figure 9. The result shows that the biological activity of the P2 ferment filtrate is still unchanged after the filtrate is subjected to acid-base treatment, which indicates that the antifungal substance generated by P2 is acid-base resistant.

(3) And (3) ultraviolet resistance stability: placing the P2 fermented product filtrate after shaking culture for 15d under ultraviolet lamp (UV-C,30W) at 20cm, respectively irradiating for 0min, 1min, 5min, 15min, 20min, 30min, 40min, 60min, 80min and 100min, sampling, and performing bioactivity determination by using tube-disc method, wherein the bioactivity determination bacteria is Leptosphaeria biglobosa (Lb20), and each processing bioactivity result is shown in figure 10. The results show that the biological activity of the P2 ferment filtrate after being irradiated by ultraviolet rays for 100min is basically consistent with that of a control group (not irradiated by ultraviolet rays) without ultraviolet rays, which indicates that the antifungal substance generated by P2 is resistant to ultraviolet rays.

Example 4:

effect of ambient pH on Phoma Macrocava P2 ferment filtrate (containing antifungal substances) Activity

PDA medium is first adjusted to pH 3.5, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 with 4mol/L HCl and 4mol/L NaOH, respectively, wherein the PDA medium initially has a pH of 6, and then 20mL of 1 × 10⁸Mixing conidium solution of spores/mL rape black shank bacteria (Lb20) with 200mL PDA culture medium to prepare a bacteria-carrying plate, performing bioactivity determination by a tube-disc method, adding 200 mu L P2 of fermentation filtrate (15d) into an Oxford cup, culturing at constant temperature of 22 ℃, and observing the result after 3 d. The results show (figure 11) that the antifungal substance produced by the P2 fermented product filtrate exerts the best activity under the acidic condition, generates the largest inhibition zone, but the bioactivity is reduced along with the increase of the pH value of the culture medium, but the antifungal substance still has the activity, which indicates that the antifungal substance produced by P2 does not exert the good activity under the alkaline condition.

TABLE 2 Effect of ambient pH on the Activity of P2 ferment filtrate (containing antifungal substances)

Note: treatment-to-treatment significance of differences was analyzed using the least significant difference method (LSD), with letters in the same column indicating no significant difference (P > 0.05).

Example 5:

teratogenicity of filtrate (containing antifungal substance) of fermentation product of Phoma giganteum P2 on 4 kinds of plant pathogenic fungi hypha

The P2 ferment filtrate (15d) was mixed with the melted PDA medium at a ratio (v/v) of 0.1%, 0.2%, 0.3%, 0.6%, 1.3%, 2.5%, 5.0% and 10.0%, respectively, by the solid plate method to make a toxic plate, and PDA without the ferment filtrate added was used as a negative control. Inoculating a pathogenic bacteria (botrytis cinerea, sclerotinia sclerotiorum, rhizoctonia solani and phytophthora parasitica) hypha block with the diameter of 5mm in the center of each plate, repeating the treatment for 3 times, culturing at constant temperature of 22 ℃, and measuring the colony diameter among the treatments after negative control grows out of the plate, thereby calculating the percentage of inhibiting the growth of the pathogenic bacteria hypha.

The results are shown in the figure (figure 12), the P2 fermented product filtrate has teratogenic action on botrytis cinerea, sclerotinia sclerotiorum, rhizoctonia solani and phytophthora parasitica var brassicae, the growth of hyphae of 4 pathogenic bacteria is obviously inhibited along with the increase of the concentration of the P2 fermented product filtrate, hyphae among bacterial colonies are accumulated, aerial hyphae are developed, and the hyphae of the sclerotinia sclerotiorum and the phytophthora parasitica brassicae are obviously inhibited when the concentration of the fermented product filtrate is only 0.6 percent.

TABLE 3 influence of the concentration of the fermentation filtrate (containing antifungal substances) of Strain P2 on the growth of hyphae of 4 pathogenic bacteria

Picking a hypha tip with the final concentration of 0.6% of P2 fermentation liquor and a hypha tip of a negative control (not containing P2 fermentation liquor), and observing by using a microscope after dyeing cotton blue, wherein pathogenic bacteria hypha branches are obviously increased after the P2 fermentation liquor is treated, and the hypha is tangled into bundles and has the phenomenon of hypha overflow; however, the negative control (without P2 fermentation filtrate) had sparse tips, normal branches, and full hyphae (FIG. 13).

Example 6

Effect of Phoma macrostoma P2 fermentation filtrate (containing antifungal substance) on Nuclear germination of Sclerotinia sclerotiorum and Botrytis cinerea

Two methods were used in this test to determine the effect of antifungal substances produced by P2 ferment filtrate on sclerotinia sclerotiorum and botrytis cinerea sclerotinia germination.

The method comprises the following steps: selecting sclerotium with substantially consistent size, treating the sclerotium to be tested with 75% ethanol solution (v/v) and 5% sodium hypochlorite solution (v/v) for 1min and 5min respectively, washing with sterile water for 3 times, placing the sclerotium on sterilized filter paper to absorb water on the surface, and naturally air drying in sterile environment. Finally, the sclerotium was immersed in the P2 ferment filtrate (15d), and left to stand at room temperature for 24h with the PDB culture solution immersed in the sclerotium as a control. After 24h, each treated sclerotium was placed on a PDA plate with 4 sclerotium per plate, each treatment was repeated 4 times, the plates were incubated at 22 ℃ and the sclerotium germination was observed after 5 days.

The second method comprises the following steps: and (4) selecting sclerotium and performing surface disinfection according to the first method. Mixing the P2 ferment filtrate (15d) with molten PDA at a ratio of 10% (v/v) to make into toxic flat plate; PDA plates without P2 fermentation filtrate added were used as negative controls, and finally 4 sclerotia per plate, and each treatment was repeated 4 times, the plates were incubated at 22 ℃ and sclerotia germination was observed after 5 days.

The first test result is shown in the figure (figure 14), after sclerotinia sclerotiorum and botrytis cinerea are soaked in the P2 ferment filtrate for 24h and inoculated on a PDA plate to be cultured for 5d, from the observation result, the sclerotium of two pathogenic bacteria can germinate, but the growth of the germinated hyphae is slow or inhibited, while the sclerotium of two control groups soaked by PDB can normally germinate on the PDA plate, and the hyphae can normally grow.

The test result of the second method is shown in the figure (figure 15), after sclerotinia sclerotiorum and botrytis cinerea sclerotia are cultured on a toxic plate containing 10% (v/v) P2 fermentation filtrate for 5 days, although the sclerotium of the two pathogenic bacteria can germinate to produce hypha, the germinated hypha can not grow normally, and is obviously inhibited. On the PDA plate without the P2 fermented product filtrate, the sclerotia of the two fungi can normally germinate, and the germinated hyphae can normally grow.

Example 7:

effect of Phoma macrostoma P2 ferment filtrate (containing antifungal substance) on the germination of Botrytis cinerea and Leptosphaeria brasiliensis conidia

Washing conidia of Botrytis cinerea and Leptosphaera rapae with sterilized water to obtain conidia suspension, measuring conidia concentration with blood counting plate, and regulating conidia concentration with sterilized water to 1 × 10⁶Taking 200 mu L of conidium to drop in the center of a flat plate, uniformly coating the conidium solution by using an aseptic coater, and finally observing the germination condition of the conidium and the standard of whether the conidium germinates: length of bud tubeA spore is considered to have germinated when it grows 1/2 longer than the length of the conidium.

The P2 fermented product filtrate (15d) WAs mixed with the melted WA (containing 2% glucose) medium at a ratio (v/v) of 0.1%, 0.2%, 0.3%, 0.6%, 1.3%, 2.5%, 5.0% and 10.0%, respectively, by a solid plate method to prepare a toxic plate. Negative control plates without addition of P2 ferment filtrate; subsequently, 200. mu.L of conidia of Botrytis cinerea (B05.10) and Leptosphaeria campestris (Lb20) were dropped into the center of the WA plate, and the conidia were uniformly spread on the plate using a sterile spreader. Each treatment was coated on 3 plates as 3 replicates. All the plates are placed at 22 ℃ for culture, the germination condition of conidia on each plate is observed under an optical microscope after the conidia of the botrytis cinerea are cultured for 9h, and each plate randomly counts 100 conidia so as to count the germination rate of the conidia. And (3) observing the germination condition of the conidia of the rape phytophthora parasitica (Lb20) after the conidia are cultured for 20h, and counting the germination rate of the conidia. The results show that the germination of the germ tube of the botrytis cinerea is obviously inhibited along with the increase of the concentration of the P2 fermentation filtrate, the length of the germ tube of the botrytis cinerea is obviously shorter than that of the negative control when the concentration of the P2 fermentation filtrate is 2.5%, and the germination of the germ tube of the botrytis cinerea is obviously inhibited when the concentration of the fermentation filtrate is 10% (figure 16). While the conidia of the phytophthora parasitica var nicotianae are sensitive to the P2 ferment filtrate, the germination of the germ tube is obviously inhibited when the concentration of the ferment filtrate is 0.5% (fig. 17).

TABLE 4 Effect of the fermentation filtrate of Strain P2 (containing antifungal substances) on the Germination of the meristematic Aconitum Botrytis

TABLE 5 Effect of the fermentation filtrate of Strain P2 (containing antifungal substances) on the germination of conidia of Leptosphaeria maculans

Example 8:

disease prevention effect of Phoma macrostoma P2 fermented product filtrate (containing antifungal substance) on 2 pathogenic bacteria on living rape leaves

In indoor planting of No. 9 rape, 5 rape seeds with accelerated germination are sown in each pot (10cm multiplied by 10cm), and after the rape seedlings grow to 30 days, the seedlings are respectively used for inoculating two pathogenic bacteria of sclerotinia sclerotiorum and botrytis cinerea. Each leaf was coated with the fermented filtrate of P2 strain 15d (approximately 500. mu. L P2 of fermented filtrate was added dropwise to each leaf). The PDB solution treated leaves were used as controls, 4 replicates per treatment (4 replicates in this case means 4 pots of rape, each of which was inoculated with 4 true leaves). Then, hypha blocks with a diameter of 5mm were punched from the edges of 3 d-grown fresh sclerotinia sclerotiorum and botrytis cinerea colonies, respectively. Inoculating hypha blocks to two ends of each true leaf, placing the inoculated rape seedlings in a plastic box, sealing the top of the plastic box with a preservative film, performing moisture-preserving culture, placing the plastic box in a 22 ℃ culture room, performing light/dark (12h/12h) culture for 3d, and observing the infection condition of pathogenic bacteria.

The results show that after 3 days of inoculation, sclerotinia were inoculated under the PDB solution treatment conditions, which were able to normally infect rape leaves with lesion diameters of 14.7mm, whereas re-inoculation of rape leaves treated with the P2 ferment filtrate with sclerotinia, which was significantly limited in infestation on rape leaves with lesion diameters of only 0.9mm (fig. 18). Under the PDB solution treatment condition, botrytis cinerea is inoculated, the botrytis cinerea can normally infect rape leaves, and the diameter of the disease spot reaches 8.1mm, while the botrytis cinerea is inoculated on the rape leaves treated by the P2 fermentation product filtrate, the botrytis cinerea obviously hardly causes infection on the rape, and the diameter of the disease spot is 0mm (figure 19).

Example 9:

determination of bacteriostasis spectrum of fermentation filtrate (containing antifungal substances) of Phoma macrospora P2

And (3) detecting the bacteriostatic action of the antifungal substance generated by the P2 fermented product filtrate (15d) on the plant pathogenic fungi by adopting a hypha growth rate method. Firstly, adding the P2 ferment filtrate into PDA culture medium according to the proportion of 10% (v/v) to prepare a toxic plate. Fresh test pathogenic bacteria mycelia (d ═ 5mm in diameter) were separately inoculated into the center of the plate, and a PDA plate to which no P2 fermented product filtrate was added was used as a control. Each treatment was repeated 3 times and each pathogen was incubated at the appropriate growth temperature (22 or 28 ℃). When the control bacterial colony grows to be close to the edge of the culture dish, the diameter of the bacterial colony is measured, the bacteriostasis rate is calculated, the table 6 shows the antagonistic effect of the antifungal substance generated by the P2 fermentation product filtrate on the pathogenic bacteria of the test plant and the culture conditions and time of each bacterial strain, and fig. 20 is a bacterial colony graph of the inhibited effect of the test bacterial strain.

TABLE 6 antagonistic action of the fermentation filtrate of Strain P2 (containing antifungal substance) on pathogenic bacteria of the test plants

Example 10:

virulence determination of Brassica napus seeds by Phoma macrostoma P2 ferment filtrate (containing antifungal substances)

In order to determine whether the antifungal substances generated by the strain P2 have influence on the germination and growth of rape seeds, the test firstly treats the rape seeds of the variety Zhongshuang No. 9 with 75% ethanol solution (v/v) and 5% sodium hypochlorite solution (v/v) for 1min and 5min respectively, then washes the treated seeds with sterile water for 3 times, finally soaks the seeds in P2 fermentation filtrate (15d) and sterile water respectively for 24h, puts the seeds on a culture dish (at the moment, a sterilized filter paper sheet is paved on the culture dish), preserves moisture for culture, and observes and counts the germination and growth of the seeds in the 3 rd and the 4 th days respectively. The results are shown in the figure (fig. 21), with no significant difference in germination, seedling stem length and shoot length (P >0.05) between P2 ferment filtrate treated oilseed rape seeds and sterile water treated oilseed rape seeds, and with both treatments the seeds were able to germinate and grow normally. These results indicate that the P2 ferment filtrate has no deleterious effect on oilseed rape seed germination. The phoma macrostoma P2 and the fermentation filtrate thereof can be used as a seed coating agent for preventing the rape seeds and seedlings from being infected by phytopathogens.

Claims (5)

1. The Phoma macrospora (Phoma macrostoma) has a preservation number of CCTCC NO: and M2018726.

2. A fermentation filtrate of Phoma macrostoma (Phoma macrocystoma) as claimed in claim 1.

3. Use of a Phoma macrostoma according to claim 1 or of a ferment filtrate according to claim 2 for the preparation of a phytopathogen inhibitor.

4. Use of Phoma macrostoma according to claim 1 or of the ferment filtrate according to claim 2 for the preparation of a coating agent for plant seeds.

5. The use according to claim 3, said phytopathogen comprising: pythium aphanidermatum (Pythium aphanidermatum), Rhizopus stolonifer (Rhizopus stolonifer), Mucor hiemalis (Mucor hiemalis), Aspergillus flavus (Aspergillus flavus), Aspergillus niger (Aspergillus niger), Botrytis cinerea (Botrytis cinerea), Curvularia oryzae (Curvularia lunata), Leptosphaeria brasiliensis (Leptosphaeria biglobosa), Sclerotiella lactuca (Sclerotianum), Sclerotium sclerotiorum (Sclerotilia sclerotiorum), and Rhizoctonia solani (Rhizoctonia solani).

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