Penicillium oxalicum and application thereof

Abstract

The application provides a penicillium oxalicum strain and application thereof, and relates to the technical field of microorganisms. Penicillium oxalicum HN-7 is obtained through screening in the application, and it is proved that the penicillium oxalicum strain has inhibiting effects on multiple plant disease pathogens such as tobacco phytophthora, pythium ultimum, botrytis cinerea and pseudomonas syringae, has broad-spectrum antibacterial performance, can stably colonize in plant roots and stems, and can efficiently prevent and control multiple diseases such as bacterial wilt, thereby providing a new biocontrol strain for biological control of pesticides.

Description

Technical Field

[0001] This application relates to the field of microbial technology, and particularly to a strain of Penicillium oxalicum and its applications. Background Art

[0002] Globally, crop yield losses caused by pathogens and pests are a serious problem, posing a major threat to food security and economic development. It is estimated that this loss ranges from 17% to 30%. Plant pathogens include viruses, bacteria, oomycetes, and fungi, etc., which infect plants in different ways and affect crop growth and yield. Among agricultural disease control measures such as breeding disease-resistant varieties, biological control, and chemical control, chemical control is still the main means to control plant diseases at present, but it also causes problems such as drug resistance, pesticide residues, and environmental pollution. Compared with traditional chemical control, biocontrol microorganisms have the advantages of environmental friendliness, no chemical residues, and high biosafety, and have become the core strategy for the green control of plant fungal diseases. At present, it is urgent to strengthen the research and development of biocontrol methods and products.

[0003] Using beneficial microorganisms for crop disease control is one of the important development directions of green control. Trichoderma ([[]] Trichoderma spp. ), Penicillium ([[]] Penicillium spp. ), Bacillus ([[]] Bacillus spp. ), etc. have good effects in inhibiting pathogenic fungi and bacteria. The action mechanisms of biocontrol strains mainly include antibiotic action, competition for colonization sites, nutrients or minerals, parasitism, and bacteriophage action. Among them, the most common strategy is to exert antibiotic action on pathogenic fungi by secreting antifungal metabolites (AFMs). Currently, the antifungal metabolites studied more are mainly antibiotics, enzymes, and volatiles.

[0004] Using natural microbial antibacterial extracts is one of the safe means to reduce or replace fungicides. Microbial volatile compounds (VOCs) are small signaling molecules (<C15) produced by organisms such as bacteria and fungi, with characteristics such as low molecular mass (<300 Da), high vapor pressure, and low boiling point. Volatile compounds are usually lipophilic compounds. These molecules are volatile at 25°C and 1 atm pressure and can easily diffuse through the water- and gas-filled pores in the soil and rhizosphere environment, and have the effects of promoting plant growth, inhibiting nematode activity, inducing plants to produce disease resistance, and inhibiting the growth and spore germination of pathogenic bacteria. Due to the characteristics of high-efficiency antibacterial, rich components, easy volatilization, and no residue on the plant surface of volatile substances, they play an important role in agriculture, environment, and healthcare. Summary of the Invention

[0005] In view of the above problems, the present application aims to provide a new biocontrol fungus Penicillium oxalicum (HN-7) Penicillium oxalicum )HN-7, and it is clear that its main antibacterial volatile substance is 1-octene-3-ol and linalool, which has broad-spectrum antibacterial activity against a variety of pathogenic bacteria, and the prepared Penicillium oxalicum fungicide has a significant control effect on Ralstonia solanacearum.

[0006] In one aspect, the present application provides a Penicillium oxalicum (HN-7) Penicillium oxalicum )HN-7, which is preserved in the China General Microbiological Culture Collection Center (CGMCC) with a preservation number of CGMCC No.42167.

[0007] Preferably, the Penicillium oxalicum (HN-7) Penicillium oxalicum )HN-7 has a bacteriostatic effect and can stably colonize in plant roots and stems.

[0008] In a preferred embodiment, the plant is tomato. In the present application, tomato is taken as an example for experiment, and those skilled in the art can conceive its application in other plants.

[0009] In another aspect, the present application also provides a microbial preparation containing the Penicillium oxalicum.

[0010] Preferably, the Penicillium oxalicum includes Penicillium oxalicum and its related products, such as:

[0011] A1) Penicillium oxalicum;

[0012] A2) Penicillium oxalicum fungicide;

[0013] A3) Penicillium oxalicum dead fungus suspension;

[0014] A4) Penicillium oxalicum mycelium;

[0015] A5) Penicillium oxalicum spores.

[0016] In a preferred embodiment, the number of live Penicillium oxalicum HN-7 in the fungicide is ≥50 billion / g.

[0017] In another aspect, the present application also provides the use of the Penicillium oxalicum or the microbial preparation in the preparation of an antibacterial agent.

[0018] Preferably, the antibacterial agent includes volatile metabolites produced by Penicillium oxalicum.

[0019] More preferably, the volatile metabolites include one or more of anisole, 1-octene-3-ol, linalool, and m-dimethyl ether.

[0020] Further, the antibacterial agent comprises one or more of anisole, 1-octen-3-ol, linalool, m-dimethoxybenzene.

[0021] Preferably, the antibacterial agent comprises 1-octen-3-ol and / or linalool.

[0022] It is first discovered in the present application that Penicillium oxalicum (P. Penicillium oxalicum ) HN-7 can produce anisole, 1-octen-3-ol, linalool, m-dimethoxybenzene, and it is also confirmed that anisole, 1-octen-3-ol, linalool, m-dimethoxybenzene are the main active ingredients for the antibacterial effect of Penicillium oxalicum (P. Ralstonia solanacearum ) HN-7.

[0023] Preferably, the antibacterial agent can be used to inhibit the growth activity of pathogenic bacteria, pathogenic oomycetes and / or pathogenic fungi.

[0024] The pathogenic bacteria comprise one or more of Pseudomonas solanacearum (P. Phytophthora nicotianae );

[0025] The pathogenic oomycetes comprise one or more of Phytophthora nicotianae (P. Pythium ), Pythium ultimum (P. ultimum Pythium aphanidermatum ), Pythium aphanidermatum (P. (Botrytis cinerea) );

[0026] The pathogenic fungi comprise one or more of Botrytis cinerea (B. Fusarium ), Fusarium graminearum (F. graminearum Colletotrichum orbiculare ), Sphaerotheca fuliginea (S. Rhizoctonia ), Septoria tritici (S. cerealis Magnaporthe oryzae) ), Magnaporthe grisea (M. Alternaria alternata , Alternaria alternata (A. Phytophthora nicotianae ).

[0027] In another aspect, the present application also provides a preparation method of the antibacterial agent, the method comprising: culturing the Penicillium oxalicum or the microbial preparation.

[0028] Preferably, the skilled person in the art can culture the Penicillium oxalicum in a conventional manner.

[0029] In a preferred embodiment, the method comprises: inoculating the Penicillium oxalicum into a culture medium, and culturing at 25-30°C for 2-3 days.

[0030] Preferably, the preparation method further comprises collecting the volatile metabolites of Penicillium oxalicum.

[0031] Preferably, the preparation method further comprises a purification step.

[0032] The obtained one or more than one antibacterial active substance can be anisole, 1-octen-3-ol, linalool, m-dimethoxybenzene.

[0033] In a preferred embodiment, the culture medium can be PDA medium.

[0034] In another aspect, the present application also provides an antibacterial agent prepared by the method.

[0035] Further, the antibacterial agent comprises one or more than one of anisole, 1-octen-3-ol, linalool, m-dimethoxybenzene.

[0036] In another aspect, the present application also provides the use of the Penicillium oxalicum or the microbial preparation or the antibacterial agent in preventing and treating plant diseases and / or inhibiting the growth of pathogenic bacteria.

[0037] Further, the pathogenic bacteria are pathogenic bacteria, pathogenic oomycetes and / or pathogenic fungi.

[0038] Further, the pathogenic bacteria comprise one or more than one of Phytophthora nicotianae Pythium ultimum , Pythium ultimum Botrytis cinerea , Botrytis cinerea Ralstonia solanacearum , Pseudomonas solanacearum Pythium aphanidermatum , Pythium aphanidermatum Fusarium graminearum , Fusarium graminearum Colletotrichum orbiculare , Sphaerotheca fuliginea Rhizoctonia cerealis , Gaeumannomyces graminis Magnaporthe oryzae , Magnaporthe grisea Alternaria alternata , Alternaria alternata Phytophthora nicotianae .

[0039] Further, the plant disease is a disease caused by pathogenic bacteria, and the plant disease comprises one or more than one of black shank, sudden death, wilt, gray mold, bacterial wilt, scab, root rot, anthracnose, leaf blight, rice blast, and leaf spot.

[0040] Preferably, the plant disease is a disease caused by pathogenic bacteria, and the plant disease comprises one or more than one of black shank of tobacco, sudden death and wilt of crops, gray mold of grape, cucumber, tomato, and strawberry, bacterial wilt of Solanaceae crops, scab of wheat, root rot of tobacco, anthracnose of cucumber, leaf blight of wheat, rice blast, and leaf spot of tobacco.

[0041] Preferably, the plant disease is a disease caused by pathogenic bacteria, and the plant disease comprises one or more than one of black shank of tobacco, sudden death and wilt of crops, gray mold of grape, cucumber, tomato, and strawberry, bacterial wilt of Solanaceae crops, scab of wheat, root rot of tobacco, anthracnose of cucumber, leaf blight of wheat, rice blast, and leaf spot of tobacco. Pythium ultimum Botrytis cinerea Ralstonia solanacearum ​​) It can infect hundreds of plants such as grape, strawberry, tomato, cucumber, flowers, etc., causing rot, soft rot and gray mold on the surface, which is the pathogen of gray mold; Ralstonia solanacearum ( Pythium aphanidermatum ) It can cause the whole tomato, pepper, tobacco, potato, peanut and other crops to wither and die rapidly, and the white bacterial pus can be seen in the stem base, which is the pathogen of bacterial wilt; Pythium aphanidermatum ( Fusarium graminearum ) It causes sudden collapse and fruit rot (mushy rot) of melon (such as cucumber, watermelon), solanaceae and other crops; Fusarium graminearum ( Colletotrichum orbiculare ) It mainly causes scab disease of wheat, barley, corn and other cereal crops, and root rot of tobacco. Colletotrichum lagenarium ( Rhizoctonia cerealis ) It is the pathogen of cucumber anthracnose, which also harms watermelon, melon, pumpkin and other cucurbitaceae crops, and forms round or oval brown lesions on leaves, stems and fruits; Gaeumannomyces graminis ( Magnaporthe ) It mainly harms the leaf sheath and stem of wheat, barley and other crops, causing rotten buds, seedling wilt, rotten stems and white dead ears, and is the pathogen of Gaeumannomyces graminis; Magnaporthe grisea ( oryzae Alternaria alternata ) It mainly harms rice, causing fusiform spots on rice leaves, white ears due to neck blast, and shriveled grains, and can cause the whole field to die and reduce yield, and is the pathogen of rice blast. Alternaria alternata Phytophthora nicotianae ) It mainly harms tobacco leaves, causing spots, necrosis and early decline, leading to a decrease in effective tobacco leaves and a decrease in quality, and is the pathogen of tobacco brown spot.

[0042] Preferably, the inhibition rate of the pathogen can be more than 80%; alternatively, the inhibition rate of the pathogen can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% and above.

[0043] On the other hand, the present application also provides a pesticide, which comprises the Penicillium oxalicum or the microbial preparation or the antibacterial agent.

[0044] The skilled person in the art can select the content of microorganisms and the content of antibacterial agents in the pesticide product according to the actual situation.

[0045] In a preferred embodiment, the number of viable Penicillium oxalicum HN-7 in the pesticide is ≥50 CFU billion per gram, the addition amount is greater than or equal to 0.1 g, and the control effect on Ralstonia solanacearum is greater than or equal to 68%.

[0046] Preferably, the addition amount can be 0.1 g-10 g, the addition amount can be any of 0.1 g, 0.2 g, 0.5 g, 0.6 g, 0.7 g, 0.8 g, 0.9 g, 1 g, 2 g, 3 g, 4 g, 5 g, 6 g, 7 g, 8 g, 9 g, 10 g.

[0047] Preferably, the control effect is greater than or equal to any of 68%, 70%, 77%, 79%, and the like.

[0048] In a preferred embodiment, the concentration of the antibacterial agent in the pesticide is greater than or equal to 0.5 times the EC 50 .

[0049] Preferably, the concentration of the antibacterial agent in the pesticide for controlling Botrytis cinerea can be greater than or equal to 0.5 times, 1 times, 1.5 times, 2 times, 2.5 times, 3 times the EC 50 .

[0050] In another aspect, the application also provides the use of anisole, 1-octen-3-ol, linalool and / or m-dimethoxybenzene in controlling plant diseases and / or inhibiting the growth of pathogenic bacteria.

[0051] Further, the pathogenic bacteria are pathogenic bacteria, pathogenic oomycetes and / or pathogenic fungi.

[0052] Further, the pathogenic bacteria include one or more of Phytophthora nicotianae ( Pythium ultimum ), Pythium ultimum ( Botrytis cinerea ), Botrytis cinerea ( Ralstonia solanacearum ), Pseudomonas syringae ( Pythium aphanidermatum ), Pythium aphanidermatum ( Fusarium graminearum ), Fusarium graminearum ( Colletotrichum orbiculare ), Sphaerotheca fuliginea ( Rhizoctonia cerealis ), Septoria tritici ( Magnaporthe oryzae ), Magnaporthe grisea ( Alternaria alternata ), Alternaria alternata ( Penicillium oxalicum ).

[0053] Further, the plant disease is a disease caused by pathogenic bacteria, and the plant disease includes one or more of black foot disease, sudden collapse disease, wilt disease, gray mold disease, bacterial wilt disease, sclerotinia disease, root rot disease, anthracnose disease, sheath blight disease, rice blast disease, and leaf spot disease.

[0054] The application has the following beneficial effects:

[0055] 1. In the present application, a new biocontrol fungus Penicillium oxalicum is obtained by screening. Figure 1HN-7, and it is proved that it has inhibitory effect on many plant disease pathogens such as Phytophthora nicotianae, Pythium ultimum, Botrytis cinerea and Pseudomonas solanacearum, has broad-spectrum antibacterial performance, and can stably colonize in plant roots and stems, efficiently prevent and control many diseases such as bacterial wilt, thereby providing a new biocontrol strain for biological control of pesticides;

[0056] 2, The antibacterial performance of the biocontrol fungus is further studied in the application, and it is confirmed that the antibacterial effective components are anisole, 1-octene-3-ol, linalool and m-dimethyl ether, wherein 1-octene-3-ol and linalool have inhibitory effect on many common plant pathogens such as Phytophthora nicotianae, Botrytis cinerea, Pseudomonas solanacearum, Pythium aphanidermatum, Fusarium graminearum, Sphaeropsis fuliginea, Septoria tritici, Pyricularia oryzae and Alternaria alternata, and can be widely applied in plant disease prevention and control process, safe and efficient, thereby providing a new effective component for the field of pesticides. BRIEF DESCRIPTION OF DRAWINGS

[0057] The drawings described herein are used to provide further understanding of the application, and constitute a part of the application. The illustrative embodiments of the application and their descriptions are used to explain the application, and do not constitute an improper limitation on the application. In the drawings:

[0058] Figure 2 is the colony morphology characteristic diagram of fungus HN-7 on PDA plate;

[0059] Figure 3 is the phylogenetic tree result diagram of fungus HN-7;

[0060] Figure 4 is the plate opposite test result diagram of Penicillium oxalicum HN-7 and Phytophthora nicotianae, Pythium ultimum, Botrytis cinerea and Pseudomonas solanacearum;

[0061] Figure 5 is the inhibition rate statistical diagram of Penicillium oxalicum HN-7 on Phytophthora nicotianae, Pythium ultimum and Botrytis cinerea;

[0062] Figure 6 is the antibacterial effect diagram of four volatile substances on Botrytis cinerea;

[0063] Figure 7 is the inhibition rate statistical diagram of four volatile substances on Botrytis cinerea;

[0064] Figure 8 is the antibacterial effect diagram of linalool and 1-octene-3-ol on common pathogenic fungi;

[0065] Figure 9Regression equations for the toxicity of linalool and 1-octen-3-ol against *Pythium spp.* and *Botrytis cinerea*, where (A) is the regression equation for the toxicity of linalool against *Pythium spp.*; (B) is the regression equation for the toxicity of linalool against *Botrytis cinerea*; (C) is the regression equation for the toxicity of 1-octen-3-ol against *Pythium spp.*; and (D) is the regression equation for the toxicity of 1-octen-3-ol against *Botrytis cinerea*.

[0066] Figure 10 The diagram shows the control effects of linalool and 1-octen-3-ol on Botrytis cinerea infection in grapes.

[0067] Figure 11 The image shows the effect of Penicillium oxalate HN-7 inoculant on controlling bacterial wilt under potted conditions on day 11. In this image, (A) is a top view of each group of potted plants, and (B) is a front view of each group of potted plants.

[0068] ​ The graph shows the colonization effect of Penicillium oxalate HN-7 in tomato rhizomes on day 11 under potted conditions. (A) is the relative biomass of Penicillium oxalate in the roots, and (B) is the relative biomass of Penicillium oxalate in the stems.

[0069] Preservation of biological materials:

[0070] A strain of Penicillium oxalicum ( Penicillium oxalicum HN-7 was deposited on August 15, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 42167. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, China, 100101, China. Detailed Implementation

[0071] To more clearly illustrate the overall concept of this application, a detailed description is provided below with reference to the accompanying drawings and embodiments. Numerous specific details are set forth in the following description to provide a more thorough understanding of the invention. However, it will be apparent to those skilled in the art that the invention can be practiced without one or more of these details. In other instances, certain technical features well-known in the art have not been described to avoid confusion with the invention.

[0072] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention. Test methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the respective manufacturers.

[0073] It is to be understood that the terms used herein are for the purpose of describing particular embodiments and are not intended to limit exemplary embodiments according to the present application. As used herein, the singular forms are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and / or "comprising," when used in this specification, specify the presence of stated features, steps, operations, devices, components and / or combinations thereof, but do not preclude the presence or addition of one or more other features, steps, operations, devices, components and / or combinations thereof.

[0074] When embodiments give numerical ranges, it is to be understood that every numerical range encompassing the minimum and maximum values, and any numerical value between the minimum and maximum values, is contemplated unless otherwise indicated herein. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Except in the Examples, or where otherwise explicitly indicated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Any method, device, material, or the like that is similar in principle to those described for the examples and functionality similar thereto can be employed as such technical persons will be able to grasp, in light of the present description and the state of the art.

[0075] In the following examples, reagents or instruments not specifically mentioned are commercially available and are conventional products, unless otherwise specified.

[0076] Unless otherwise specified, the experimental methods, detection methods, and preparation methods disclosed in the present application employ conventional microbiological, biochemical, analytical chemistry, cell culture, and related arts techniques.

[0077] In addition, the "water" described in the present application includes deionized water, distilled water, ion exchange water, double distilled water, high purity water, purified water, and any feasible water that can be used in the art.

[0078] In the following examples, wt% means weight percent, unless otherwise specified.

[0079] The culture media used in the following examples are as follows:

[0080] PDA liquid medium: 6 g / L of potato infusion powder, 20 g / L of glucose, 1 L of distilled water;

[0081] PDA flat plate medium: 6 g / L of potato infusion powder, 20 g / L of glucose, 20 g / L of agar, 1 L of distilled water;

[0082] NA flat plate medium: 10 g / L of peptone, 5 g / L of sodium chloride, 3 g / L of beef extract powder, 20 g / L of agar, 1 L of distilled water;

[0083] NB liquid medium: 10 g / L proteose peptone, 5 g / L sodium chloride, 3 g / L beef extract powder, 1 L distilled water;

[0084] Preparation method of OA medium: 30 g oat, after boiling with distilled water, cook for 15-20 min, filter with gauze, then add distilled water to 1 L, add 16-18 g agar and stir evenly;

[0085] Phytophthora nicotianae (Coker) Schroter, Phytophthora nicotianae Pythium ultimum Trow, Pythium ultimum Botrytis cinerea Persoon, Botrytis cinerea Pseudomonas syringae van Hall, Ralstonia solanacearum Pythium aphanidermatum Edson, Pythium Fusarium graminearum Schwabe, aphanidermatum Colletotrichum lagenarium (Pass.) Ellis et Halsted, Fusarium graminearum Magnaporthe grisea Shearer, Colletotrichum Gaeumannomyces graminis (Sacc.) Schrot, orbiculare Magnaporthe grisea Shearer, Rhizoctonia cerealis Pyricularia grisea (Cooke) Sacc. Magnaporthe oryzae Alternaria alternata (Fr.) Keissler Alternaria alternata were purchased from the National Agricultural Environmental Microbial Germplasm Repository (Shandong).

[0086] Example 1 Isolation and identification of biocontrol strain HN-7

[0087] 1. Strain isolation

[0088] Strain HN-7 was isolated from the saline-alkali soil of Hainan mangrove. The specific isolation method is as follows: 0.1 g of collected soil sample was placed in a sterilized 2 mL centrifuge tube, about 1.5 mL of PDA liquid medium was taken with a pipette gun, mixed as a stock solution, then diluted with sterile water, and the dilution multiples were 10 -2 and 10 -3 , respectively. 100 μL of the stock solution and the diluted sample were evenly coated on PDA plates, respectively, and different gradients were repeated 3 times, and placed at 28℃ for 2-3 d to grow single colonies. Different colonies in shape, size and color were picked, numbered and purified.

[0089] 2. Morphological observation

[0090] The strain numbered HN-7 was inoculated into PDA solid medium and cultured at 28℃ for 3 d, as shown in Figure 1 , the colony center of HN-7 on PDA medium was green to dark green, the edge was white to grayish white, the edge gradually blurred, forming a radial growth pattern, the colony surface was villous and relatively loose, especially the central part of the mycelium was relatively dense.

[0091] 3. Molecular identification

[0092] The strain HN-7 is subjected to CTAB method for genome total DNA extraction, and is subjected to ITS1 / ITS4 gene amplification and sequencing. The PCR amplification system is 50 μL: template DNA (36 ng / μL) 2 μL, primer ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') (10 μmol / L) and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') (10 μmol / L) each 2 μL, 2xPhantaFlash Mix (Dye Plus) 25 μL, and double distilled water 19 μL. The PCR amplification procedure is 95℃ for 5 min, 95℃ for 15 s, 56℃ for 15 s, 72℃ for 10 s, 34 cycles; and 72℃ for 10 min. The PCR product is sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The ITS1 / ITS4 sequence part fragment length of HN-7 is 562 bp, and is specifically shown in SEQ ID No. 1.

[0093] 14 standard strain sequences are obtained from the NCBI (GenBank) database, the ITS1 / ITS4 sequences of the isolated strains and reference strains are analyzed by using MEGA 11, and a Neighbor-joining phylogenetic tree is constructed, as shown in Figure 2 It can be seen that the HN-7 provided by the present application has the closest genetic relationship with Penicillium oxalicum In combination with the colony characteristics of the strain on the plate, the isolated strain HN-7 is identified as Penicillium oxalicum (P. oxalicum), named as Penicillium oxalicum HN-7, and is preserved in the China General Microbiological Culture Collection Center (CGMCC) on August 15, 2025, with a preservation number of CGMCC No. 42167. Penicillium oxalicum

[0094] Example 2: Inhibition activity determination of Penicillium oxalicum HN-7

[0095] 1. Plate inverted test of Penicillium oxalicum HN-7 on tobacco Phytophthora infestans, Pythium ultimum, Botrytis cinerea and Ralstonia solanacearum

[0096] ​First, select the activated state of the oxalic acid penicillium HN-7 in example 1, use puncher to take 5mm mycelium cake at the edge of the mycelium, inoculate in the center of PDA medium, cultivate for 3d at 28℃. Take 5mm pathogenic fungus cake to inoculate in the center of PDA plate, then put the inoculated tobacco blight, ultimate rot fungus or gray grape rot plate and the inoculated oxalic acid penicillium HN-7 plate upside down, seal with parafilm sealing film, put the inoculated oxalic acid penicillium HN-7 PDA plate down, put the inoculated pathogenic fungus PDA plate up, replace the inoculated oxalic acid penicillium HN-7 plate with blank PDA plate as blank control, repeat 3 times for each treatment. Cultivate for 2d in 25℃ constant temperature incubator in dark, observe the results, the results are shown in Figure 3 The calculation method is as follows, the results are shown in Figure 4

[0097] Colony diameter (cm) = measured colony diameter-0.5;

[0098] Inhibition rate (%) = (blank control colony diameter-treatment colony diameter) / blank control colony diameter x 100.

[0099] In this example, the Ralstonia solanacearum is also used as pathogenic fungus, inoculate on NA plate for 12h at 28℃, pick single colony in NB liquid medium, cultivate for 12h at 28℃, 180rpm. Centrifuge for 3min at 6000rpm, remove the medium, dilute the bacterial liquid to OD 600 value of 0.3 with sterile water. Use sterile small spray bottle to spray Ralstonia solanacearum liquid on NA plate, then put 5mm oxalic acid penicillium HN-7 cake in the center of PDA plate, put the two plates upside down and seal, put the inoculated oxalic acid penicillium HN-7 plate down, cultivate for 2d at 28℃, observe the results, the results are shown in Figure 3

[0100] Figure 3 It can be seen from the results that oxalic acid penicillium HN-7 has inhibitory effect on tobacco blight, ultimate rot fungus, gray grape rot and Ralstonia solanacearum. Figure 4 It can be seen from the results that the inhibition rate of oxalic acid penicillium HN-7 on tobacco blight, ultimate rot fungus and gray grape rot is 87.32%, 81.87% and 99.45% respectively. The above results prove that the volatiles produced by oxalic acid penicillium HN-7 can effectively inhibit the growth of tobacco blight, rot fungus, gray grape rot and Ralstonia solanacearum.

[0101] 2. Determination of oxalic acid penicillium HN-7 volatile components

[0102] ​​Accurately pipette 10 mL of the PDA medium which has not been cooled, pour into a 20 mL sterilized brown headspace bottle, and tilt the bottle body at an angle of 15 degrees. After the medium cools and solidifies, inoculate 5 mm mycelium cake of Penicillium oxalicum HN-7 on the medium slope, quickly cover, and place the headspace bottle in a 28°C incubator in the dark for 5 days. In order to exclude the interference of volatile substances released by the medium and the loss of the coating of the extraction head on the identification results of bacterial volatile substances, set the treatment without coating of bacterial suspension as a blank control. Each sample has 3 replicates. After the headspace bottle is equilibrated at room temperature for 20 min, insert the aged extraction head (50 / 70 μm DVB / CAR / PDMS, Supelco, USA) into the upper 1 / 3 of the headspace bottle, and sample at room temperature for 30 min. After sampling, immediately insert the extraction head into the sample inlet of the gas chromatograph, desorb at 250°C for 5 min, and perform GC / MS detection.

[0103] GC / MS analysis conditions: chromatographic column DB-5MS elastic capillary chromatographic column with a size of 30 m x 0.25 mm x 0.25 μm; injection port 250°C: non-split mode; carrier gas is 99.999% helium with a flow rate of 1.2 mL / min; column oven: initial temperature 45°C, hold for 1 min, increase to 180°C at a rate of 5°C / min, then increase to 280°C at a rate of 20°C / min, hold for 5 min, total running time is 38 min; EI ion source, electron energy 70 ev; ion source temperature 230°C, quadrupole temperature 150°C, transfer line temperature 280°C; full scan mode, scan range 30-400 m / z.

[0104] The volatile components of Penicillium oxalicum HN-7 were identified by GC-MS analysis, and 4 main compounds, anisole, 1-octene-3-ol, linalool, and m-dimethyl ether, were screened out (as shown in Table 1).

[0105] Table 1 Volatile components

[0106]

[0107] 3. Inhibition test of volatile components of Penicillium oxalicum HN-7 on Botrytis cinerea

[0108] The volatile organic compounds identified by GC-MS analysis (anisole, 1-octene-3-ol, linalool, and m-dimethyl ether) were purchased, and the inhibition activity of the volatile organic compounds was detected by taking Botrytis cinerea as an example.

[0109] The fumigation method was used to evaluate the antibacterial activity of the volatile compounds against Botrytis cinerea. The B. cinerea strain was inoculated on a PDA plate (5 mm mycelium cake), and then a 10 mm × 10 mm sterilized filter paper was pasted on the inner center of the Petri dish cover, 6.5 μL of the volatile compound (final concentration 100 μL / L) was added on the filter paper, the Petri dish cover was quickly covered, and the Petri dish was sealed with Parafilm. The filter paper without the volatile compound was used as a control. Each treatment was set up in triplicate. The Petri dish was placed in a 25℃ constant temperature incubator in the dark for 3 d, and the diameter (mm) of the strain colony was accurately measured. The inhibition rate was calculated by calculation. The inhibition effect was as shown in Table 2 and Figure 5 Figure 6 .

[0110] Colony diameter (cm) = measured colony diameter - 0.5;

[0111] Inhibition rate (%) = (control colony diameter - treatment colony diameter) / control colony diameter × 100.

[0112] Table 2

[0113]

[0114] Note: In the table, “a”, “b”, and “c” represent significant differences (P < 0.05) by Duncan's new multiple range test. P <0.05).

[0115] As shown in Table 2 and Figure 5 the results show that anisole, 1-octen-3-ol, linalool, and m-dimethyl ether have inhibitory effects on B. cinerea.

[0116] As shown in Table 2 and Figure 6 the results show that 1-octen-3-ol and linalool have the highest inhibitory effect on B. cinerea at 0.107 μL / cm 3 , with an inhibition rate of 100%, which is significantly better than m-dimethyl ether and anisole.

[0117] 4. Effects of volatile compounds 1-octen-3-ol and linalool of Penicillium oxalicum HN-7 on the growth of common pathogenic fungi

[0118] In this example, the inhibitory effects of 1-octen-3-ol and linalool on common pathogenic fungi were further tested, including Phytophthora nicotianae (the pathogen of tobacco black shank), Botrytis cinerea (the pathogen of grape, cucumber, tomato, and strawberry gray mold), Pseudomonas solanacearum (the pathogen of solanaceous crops and cucumber crops), Pythium aphanidermatum (the pathogen of solanaceous and cucumber crops), Fusarium graminearum (the pathogen of wheat scab and tobacco root rot), Sphaeropsis fuliginea, Septoria tritici, Magnaporthe grisea, and Alternaria alternata (the pathogen of tobacco brown spot). The test method is as follows: ​

[0119] Each 6 μL of volatile (l-octen-3-ol or linalool) was dissolved in 9 mL of PDA, OA medium, poured into a plate, and 9 mL of PDA, OA medium was poured into the other side of the two-division plate. After cooling and solidification, 5 mm pathogenic fungus cakes were inoculated. Botrytis cinerea, Pythium aphanidermatum, Fusarium graminearum, Sphaerotheca fuliginea, Guignardia humuli, Magnaporthe grisea, Rhizoctonia solani, and Alternaria alternata were cultured on PDA medium, and Phytophthora nicotianae and Aspergillus niger were cultured on OA medium. Plates without the addition of volatile were used as blank controls. Each treatment was repeated three times. The plates were cultured in a constant temperature incubator at 28°C in the dark for 2-3 days, the antibacterial effect was counted, and the results are shown in Table 3. The antibacterial rate was determined, and the calculation method was the same as above. The results are shown in Table 3. Figure 7

[0120] In this example, the antibacterial effect of volatile linalool and l-octen-3-ol of Penicillium oxalicum HN-7 on eight pathogenic fungi, including Phytophthora nicotianae, Botrytis cinerea, Pythium aphanidermatum, Fusarium graminearum, Sphaerotheca fuliginea, Guignardia humuli, Magnaporthe grisea, Rhizoctonia solani, and Alternaria alternata, was determined. As shown in Table 3, Figure 7 linalool (0.093 μg / cm 3 ) and l-octen-3-ol (0.090 μg / cm 3 ) had inhibitory effects on a variety of pathogenic fungi, with the best inhibitory effect on Botrytis cinerea and Pythium aphanidermatum.

[0121] Table 3. Inhibition rate of linalool and l-octen-3-ol on common pathogenic fungi

[0122]

[0123] Determination of virulence regression equation: 4 μL, 3 μL, 2 μL, 1.5 μL, and 0.75 μL of volatile linalool and l-octen-3-ol were respectively dissolved in 9 mL of PDA medium, poured into a plate, and 9 mL of PDA medium was poured into the other side of the two-division plate. After cooling and solidification, 5 mm Pythium aphanidermatum or Botrytis cinerea cakes were inoculated. Plates without the addition of volatile were used as blank controls. Each treatment was repeated three times. The plates were cultured in a constant temperature incubator at 28°C in the dark for 2 days, the antibacterial effect was counted, and the inhibition rate was calculated according to the above method.

[0124] In this example, the inhibitory concentration of linalool and l-octen-3-ol on Pythium aphanidermatum and Botrytis cinerea was determined, as shown in Table 3. Figure 8 The virulence regression equation of linalool on Pythium aphanidermatum was y = 4.5966x + 3.7111 (R 2 ​= 0.9894), where "x" is the lg value of linalool concentration and "y" is the bioprobability statistic corresponding to the inhibition rate of linalool against Pythium spp. Substituting the bioprobability statistic corresponding to 50% into the equation, the median inhibitory concentration EC of linalool against Pythium spp. is calculated. 50 =0.030 μg / CM 3 And it increases in a dose-dependent manner. For example... Figure 8 As shown in Figure B, the regression equation for the toxicity of linalool to Botrytis cinerea is y = 1.6877x + 4.6757 (R²). 2 = 0.988), EC 50 =0.024 μg / CM 3 ;like Figure 8 As shown in Figure C, the regression equation for the toxicity of 1-octen-3-ol to *Pythium spp.* is y = 1.6035x + 4.1535 (R²). 2 = 0.9839), EC 50 =0.051 μg / CM 3 ;like Figure 8 As shown in Figure D, the regression equation for the toxicity of 1-octen-3-ol to Botrytis cinerea is y = 1.0291x + 5.2255 (R0). 2 = 0.9948), EC 50 =0.0092 μg / CM 3 .

[0125] 5. Fruit disease prevention test of Penicillium oxalate HN-7 volatiles against Botrytis cinerea.

[0126] Healthy grapes of equal weight and uniform shape were selected, disinfected with 75% ethanol, washed thoroughly with sterile water, and air-dried. A hole (Ø=2 mm, d=2 mm) was pierced at the maximum circumference of each grape using a sterile toothpick, and then inoculated with *Botrytis cinerea* mycelium at a diameter of Ø=2 mm. All treated samples were then placed in sterile tissue culture flasks, and volatiles (linalool or 1-octen-3-ol) were added to the flasks at amounts corresponding to their EC50 and EC50 values. 50 The fungicides were diluted 0.5, 1.5, and 3 times with the active ingredient and cultured at 25℃ for 3 days, with 5 fruits per group, repeated 3 times. The control group consisted of water. The diameter of the lesions was measured, and the inhibition rate was calculated based on the lesion diameters of the experimental and control groups using the following formula:

[0127] Lesion diameter (mm) = Measured lesion diameter - 2;

[0128] Lesion growth inhibition rate (%) = (diameter of lesions in control group - diameter of lesions in treatment group) / (diameter of lesions in control group).

[0129] The results are shown in Table 4 and Figure 9As shown in the fruit disease prevention test, linalool can effectively inhibit the fruit damage caused by Botrytis cinerea, and the damage inhibition rate has a dose-dependent relationship with the concentration. At 1.5×EC 50 concentration, it can inhibit 84.8%, and at 3×EC 50 concentration, it can completely inhibit. 1-octen-3-ol can effectively inhibit the fruit damage caused by Botrytis cinerea, and the damage inhibition rate has a dose-dependent relationship with the concentration. At 3×EC 50 , it can inhibit 90.06%.

[0130] Table 4 Prevention and treatment effect of linalool and 1-octen-3-ol on grape infected by Botrytis cinerea

[0131]

[0132] Note: a / b / c indicates that the difference is significant (P<0.05) by Duncan's new range test. P <0.05).

[0133] Example 3 Potting prevention and treatment effect of Penicillium oxalicum HN-7 microbial agent

[0134] 1. Biocontrol potting test of Penicillium oxalicum HN-7 microbial agent

[0135] Tomato seeds were sown in a seedling tray and cultured for about 15 days. Three treatment groups were set up, 80 g of substrate soil was weighed in each pot, and 0.1 g, 0.2 g and 0.5 g of Penicillium oxalicum microbial agent (≥50 billion / gram) was added respectively, and then stirred uniformly and placed in a 9×9×8 cm plastic flowerpot. Tomato seedlings with consistent growth were taken, the root soil was shaken off carefully, about 5 cm long roots were reserved, the root substrate was cleaned, and then transplanted into the flowerpot. There were 10 tomato seedlings in each treatment group, and the substrate soil without the addition of Penicillium oxalicum was used as a control. After 3 days of transplanting, 10 mL (OD 600 =0.1) of Pseudomonas solanacearum was inoculated, and the incidence rate and disease index were counted 11 days after the inoculation of Pseudomonas solanacearum, and the disease prevention effect was calculated.

[0136] Pseudomonas solanacearum disease grading standard: 0 level: no disease on the whole plant; 1 level: occasional yellowing spots on the stem, or 1 / 2 or less leaf wilting on the diseased side; 3 level: black striping on the stem, but not more than 1 / 2 of the stem height, or 1 / 2-2 / 3 leaf wilting on the diseased side; 5 level: black striping on the stem more than 1 / 2 of the stem height, but not reaching the top of the stem, or more than 2 / 3 leaf wilting on the diseased side; 7 level: black striping on the stem reaching the top of the stem, or all leaves wilting on the diseased plant; 9 level: the diseased plant is basically dead.

[0137] Disease index=[ (disease level of each plant×disease level value) / (total number of plants surveyed×highest level value)]×100;

[0138] The disease prevention effect = [(disease index of the control group - disease index of the treatment group) / disease index of the control group] * 100.

[0139] As shown in Table 5 and Figure 10 The pot experiment confirmed that the bacterial agent of Penicillium oxalicum HN-7 had obvious prevention and treatment effect on bacterial wilt. As shown in Table 5, Figure 10 The prevention and treatment effect of adding 0.2 g of the bacterial agent of Penicillium oxalicum per pot was significantly better than that of adding 0.1 g and 0.5 g of the bacterial agent of Penicillium oxalicum per pot on the 11th day after treatment, and the disease index was 10.33, and the prevention and treatment effect was 79.19%.

[0140] Table 5 Disease index and prevention and treatment effect of the pot experiment on the 11th day

[0141]

[0142] Note: a / b / c / d represents significant difference (P<0.05) by Duncan's new multiple range test. P <0.05).

[0143] 2. Penicillium oxalicum HN-7 in tomato plants

[0144] For the tomato plants of step 1, the roots and stems of the tomato plants were taken 11 days after inoculation of bacterial wilt, and total DNA was extracted using a FastDNA™ Spin Kit for Soil (MP) kit. The primers of β-actin of Penicillium oxalicum HN-7 were β-actin-F (5'-GCCACTCAACAGCCTGATCT-3') and β-actin-R (5'-GCTTGGTGTCTACGGAGCTT-3'), and the internal reference genes were tomato actin ACT genes SLACT-F (5'-CGGTGACCACTTTCCGATCT-3') and SLACT-R (5'-TCCTCACCGTCAGCCATTTT-3'). The relative biomass of the Penicillium oxalicum gene in each tissue was detected using 2xSYBR Green PCR Mix reagent with the DNA of each tissue of the roots and stems as a template. The 10 μL qRT-PCR reaction system was 2xSYBR Green PCR Mix 5 μL, upstream primer 0.4 μL, downstream primer 0.4 μL, cDNA template 1.0 μL (200 ng / μL), and ddH2O 3.2 μL; the reaction condition was: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 5 s, 56℃ annealing for 45 s, and 40 cycles. The relative expression of the Penicillium oxalicum gene in different tissues of the tomato was analyzed by 2 -△△Ct As shown in Table 5 and Figure 11 As shown in Table 5 and Figure 11

[0145] On the 11th day of the pot experiment, Penicillium oxalicum HN-7 could colonize in the roots and stems of tomato. As shown in Fig. 2A, the relative biomass of Penicillium oxalicum HN-7 0.1, 0.2, 0.5 g / plant in the roots was 2.47, 1.93, 0.30. As shown in Fig. 2B, the relative biomass of Penicillium oxalicum HN-7 in the stems was 6.63, 7.51, 7.85. It indicated that Penicillium oxalicum HN-7 could colonize in the rhizosphere of tomato and gradually transfer to the stems, thereby effectively preventing and treating bacterial wilt. Figure 11 Figure 11

[0146] The above only describes the embodiments of the present application and is not intended to limit the present application. The present application can have various modifications and changes for those skilled in the art. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application shall be included in the scope of claims of the present application.​​

Claims

1. A strain of Penicillium oxalicum ( Penicillium oxalicum HN-7 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 42167.

2. A microbial preparation containing Penicillium oxalate as described in claim 1.

3. The use of Penicillium oxalate as described in claim 1 or the microbial preparation as described in claim 2 in the preparation of antibacterial agents.

4. The application according to claim 3, characterized in that, The antibacterial agent includes volatile metabolites produced by Penicillium oxalate, and the volatile metabolites include one or more of anisole, 1-octen-3-ol, linalool, and m-phenylenediamine.

5. A method for preparing an antibacterial agent, characterized in that, The method includes: culturing Penicillium oxalate as described in claim 1 or a microbial preparation as described in claim 2.

6. An antibacterial agent, characterized in that, The antibacterial agent comprises Penicillium oxalate as described in claim 1.

7. The application of the *Penicillium oxalicum* as described in claim 1, the microbial preparation as described in claim 2, or the antibacterial agent as described in claim 6 in the prevention and control of plant diseases and / or inhibition of pathogen growth, characterized in that... The pathogens include Phytophthora indicum ( Phytophthora nicotianae ), ultimate phytosis ( Pythium ultimum Botrytis cinerea ( ), Botrytis cinerea ( Botrytis cinerea Ralstonia solanacearum ( Ralstonia solanacearum ), fruit mold ( Pythium aphanidermatum Fusarium graminearum ( ), Fusarium graminearum ), cucumber anthracnose bacteria ( Colletotrichum orbiculare ), wheat sheath blight fungus ( Rhizoctonia cerealis ), rice blast fungus ( Magnaporthe oryzae Alternaria ( Alternaria alternata One or more of the following: the plant disease is a disease caused by a pathogen, and the plant disease includes one or more of the following: black shank, damping-off, wilt, gray mold, bacterial wilt, scab, root rot, anthracnose, sheath blight, rice blast, and red spot disease.

8. A pesticide, characterized in that, The pesticide includes Penicillium oxalicum as described in claim 1, or a microbial preparation as described in claim 2, or an antibacterial agent as described in claim 6.

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