CN118374358B - A strain of Penicillium bilai for effectively preventing and controlling peanut root rot and its application - Google Patents
A strain of Penicillium bilai for effectively preventing and controlling peanut root rot and its application Download PDFInfo
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- A01C—PLANTING; SOWING; FERTILISING
- A01C1/00—Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
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- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G13/00—Protection of plants
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- A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
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Abstract
本发明公开了一株高效防控花生根腐病的比莱青霉菌及其应用，涉及微生物技术领域，其技术方案要点是：比莱青霉菌(Penicillium bilaiae)BL‑1的保藏编号为CGMCC NO.41008，保藏时间为2023年12月，保藏单位为中国微生物菌种保藏管理委员会普通微生物中心，保藏地址：北京市朝阳区北辰西路1号院3号；所述比莱青霉菌的ITS序列见SEQ ID NO：1，菌株BL‑1对引起花生青枯病的茄科雷尔氏菌、花生根腐病的尖孢镰刀菌、花生纹枯病的立枯丝核菌、花生果腐病的群结腐霉菌、花生茎腐病的可可毛色二孢菌等植物病原细菌和真菌具有明显的平板拮抗活性，对花生根腐病的温室防效达到90.36％。进一步发现，P.bilaiae BL‑1还具备促进花生幼苗生长的能力。因此，P.bilaiae BL‑1是一株具有花生促生和抑制多种花生病害潜力的高效生物防治菌。
The invention discloses a Penicillium bilaiae strain for efficiently preventing and controlling peanut root rot and its application, and relates to the field of microbial technology. The main points of the technical scheme are: the deposit number of Penicillium bilaiae BL‑1 is CGMCC NO.41008, the deposit time is December 2023, the deposit unit is the General Microbiological Center of China Microbiological Culture Collection Administration, and the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; the ITS sequence of the Penicillium bilaiae is shown in SEQ ID NO: 1, and the strain BL‑1 has obvious plate antagonistic activity against plant pathogenic bacteria and fungi such as Ralstonia solanacearum that causes peanut bacterial wilt, Fusarium oxysporum that causes peanut root rot, Rhizoctonia solani that causes peanut sheath blight, Pythium sphaeroides that causes peanut fruit rot, and Lactochromis cocoi that causes peanut stem rot, and the greenhouse prevention effect on peanut root rot reaches 90.36%. It is further found that P.bilaiae BL‑1 also has the ability to promote the growth of peanut seedlings. Therefore, P. bilaiae BL‑1 is a highly effective biocontrol bacterium with the potential to promote peanut growth and inhibit a variety of peanut diseases.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to a strain of penicillium bipolarii for effectively preventing and controlling peanut root rot and application thereof.
Background
The peanuts are annual herbaceous crops belonging to the genus peanut of the family Leguminosae, are good in high-temperature drying environment, and are suitable for growing on sandy soil with warm climate and sufficient illumination. Peanuts are native to south america brazil and are now widely planted around the world because of their extremely high economic value and market potential. China is the most important peanut planting and production base in the world, and also has the largest peanut planting area worldwide and the first peanut yield in the world. Peanut can be planted in Huang-Huai-Hai region, north China plain, yangtze river basin, southeast coast and northeast peanut region of China, wherein more peanut can be planted in regions such as Henan, shandong, hebei, north of Suwanhua and the like. Peanuts are also the third largest oil crop in China, the planting area is inferior to soybeans and rapeseeds, and according to data, 2022-year-old peanuts are planted for up to 4683.80 kilohectares, accounting for 20.03% of the total planting area of the oil crops in China, so that the important role of the peanuts in guaranteeing the edible oil safety in China and the economic and stable development in agricultural rural areas is highlighted. In addition, peanuts have high nutritional value and economic value. The peanut fruits contain rich nutrient components such as fat, protein and the like, are leisure health-care food which is rich in nutrition and is deeply favored by people, and according to the record of the schema and the sequelae, the peanut has the effects of strengthening the spleen, nourishing the stomach, moistening the lung and reducing phlegm. The protein content in the residual peanut meal after the peanut is squeezed is up to more than 50%, so that the peanut meal is a good feed and fertilizer component.
In recent years, the method is influenced by unfavorable planting modes such as continuous cropping of peanuts, environmental factors such as climate change and the like, and the peanut planting is threatened by various soil-borne diseases and leaf diseases such as bacterial wilt, leaf spot, stem rot, root rot and the like, so that the green high yield of the peanuts is seriously influenced. Disease occurrence is one of main factors limiting high and stable peanut yield, and seriously affects sustainable peanut production and trade. At present, broad-spectrum chemical bactericides such as carbendazim, kresoxim-methyl, chlorothalonil and mancozeb are still mainly relied on in production to prevent and treat peanut diseases, however, as the planting year of peanuts increases, the occurrence frequency of diseases is improved, the use frequency and the use amount of the bactericides are also continuously improved, the planting and growing environment of the peanuts are influenced to a certain extent, and pesticide residues are easily caused to exceed standards, so that the quality and the safety of the peanuts are seriously threatened.
Biological control is one of the important ways to achieve green production of peanuts, with penicillium bipolarii (Penicillium bilaiae) as a beneficial fungus exhibiting great potential in crop disease green control and crop promotion. The penicillium bipolarii can synthesize more than 1300 antibacterial substances including antibiotics, antibacterial peptides, lysozyme and the like, and the occurrence of plant diseases is reduced by directly destroying the cell structure of pathogenic microorganisms. Especially has good inhibiting effect on pathogenic bacteria such as fusarium, pythium, rhizoctonia solani, bacterial wilt and the like. Researches show that the bipecilomyces can quickly colonize in soil environment and crop root systems to form dominant bacterial groups, and the decomposition of nutrient components in soil is accelerated by secreting various acidic substances and hydrolytic enzymes, so that the absorption and extension of the crop root systems are promoted, and the growth of crops is further accelerated. In addition, the strain can secrete various plant hormones to influence the growth and development of crops. The penicillium bipolaris gradually becoming a key strain resource for preventing and controlling pathogenic microorganisms in agricultural production, and increasing the development and utilization of the penicillium bipolaris has important significance for enriching microorganism products and promoting the increase of agricultural yield and income.
However, at present, the microbial preparation effective in preventing and controlling peanut diseases is not available, and green and efficient production of peanuts cannot be met.
Disclosure of Invention
The invention aims to solve the problems, and provides a penicillium bipolaris strain capable of effectively preventing and controlling peanut root rot and application thereof, wherein the strain BL-1 has obvious flat antagonistic activity on plant pathogenic bacteria and fungi such as Solanaceae Ralstonia (Rastonia solanacearum) which causes peanut bacterial wilt, fusarium oxysporum (Fusarium oxysporum) which causes peanut root rot, rhizoctonia solani (Rhzoctonia solani) which causes peanut sheath blight, group rot fungi (Pythium myriotylum) which cause peanut root rot, cocoa trichlrabi (Lasiodiplodia theobromae) which causes peanut stem rot and the like, has a greenhouse prevention effect of 90.36% on peanut root rot, and the P.biaiae BL-1 also has the capability of promoting peanut seedling growth.
The technical aim of the invention is realized by the following technical scheme that the method comprises the steps of effectively preventing and controlling the peanut root rot from being used as a strain of penicillium bifidum, wherein the preservation number of the penicillium bifidum (Penicillium bilaiae) BL-1 is CGMCC No.41008, the preservation time is 2023, 12 months and 19 days, the preservation unit is China general microbiological culture Collection center, the preservation address is North Star Xway No. 1,3 in the Beijing Kogyo area, the penicillium bifidum aerobic fungus can normally grow on a PDA culture medium, white hypha is arranged at the edge of a colony and dark green is arranged in the middle of the colony, and the ITS sequence of the penicillium bifidum is SEQ ID NO. 1;
SEQ ID NO:1:
GTGTCTCTTGTACCATGTTGCTTCGGCGAGCCCGCCTCACGGCCGCCGGGGGGCATCTGCCCCCGGGCCCGCGCCCGCCGAAGCCCCCTCTGAACGCTGTCTGAAGATTGCAGTCTGAGCGATAAGCAAAAATTATTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAACTAATGTGAATTGCAGAATTCAGTGAATCATCGAGTCTTTGAACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTCCGAGCGTCATTGCTGCCCTCAAGCACGGCTTGTGTGTTGGGCCTCCGTCCTCCCCCCGGGGGGACGGGCCCGAAAGGCAGCGGCGGCACCGTGTCCGGTCCTCGAGCGTATGGGGCTTTGTCACCCGCTCTGTAGGCCCGGCCGGCGCTGGCCGACCCTCCAACCCCATTTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATATCAAA.
The invention further discloses application of the penicillium bipinnatum for effectively preventing and controlling peanut root rot in any one of the following:
application of U1 and P.biliae BL-1 in preventing and/or treating peanut root rot;
U2, P.Bilaiae BL-1 in inhibiting Ralstonia solanaceae (bacterial wilt), U3, P.Bilaiae BL-1 in inhibiting fusarium oxysporum (root rot);
U4, P.biliae BL-1 in inhibiting Rhizoctonia solani (sheath blight);
u5 and P.biliae BL-1 are applied to inhibiting the pythium aphanidermatum (fruit rot);
U6 and P.Bilaiae BL-1 are used for inhibiting the cocoa hair color dichotoma (stem rot) and U7 and P.Bilaiae BL-1 are used for improving the biomass of peanut plants.
The invention further discloses application of the metabolite of the penicillium bifidum for effectively preventing and controlling peanut root rot in any one of the following:
use of metabolites of V1, p.biae BL-1 for the prevention and/or treatment of peanut root rot;
Use of metabolites of V2, p.biaae BL-1 for inhibiting ralstonia solanacearum (bacterial wilt);
The application of the metabolites of V3 and P.biae BL-1 in inhibiting fusarium oxysporum (root rot);
the application of the metabolites of V4 and P.biliae BL-1 in the inhibition of rhizoctonia solani (banded sclerotial blight);
The application of the metabolites of V5 and P.biliae BL-1 in inhibiting Pythium insidiosum (fruit rot);
Use of metabolites of V6, p.biaiae BL-1 for inhibiting conidiophore (stem rot) of cocoa;
Use of metabolites of V7, p.biliae BL-1 for increasing biomass of peanut plants.
The invention further discloses a culture of the penicillium bimanum for effectively preventing and controlling peanut root rot, wherein the culture is a substance obtained by culturing the penicillium bimanum in various microorganism culture mediums, and comprises mycelium, conidium and secretion of P.biae BL-1.
The invention further discloses an application of the culture of the penicillium bimatose for effectively preventing and controlling peanut root rot in any one of the following:
Use of a culture of B1, p.biae BL-1 for the prevention and/or treatment of peanut root rot;
use of a culture of B2, p.biliae BL-1 for inhibiting ralstonia solanacearum (bacterial wilt);
Use of a culture of B3, p.biae BL-1 for inhibiting fusarium oxysporum (root rot);
Use of a culture of B4, p.biaiae BL-1 for inhibiting rhizoctonia solani (banded sclerotial blight);
b5, P.biliae BL-1 in inhibiting Pythium insidiosum (fruit rot);
use of a culture of B6, p.biaiae BL-1 for inhibiting cola bipolaris (stem rot);
Use of B7, p.biliae BL-1 cultures for increasing biomass in peanut plants.
The invention further discloses a fermentation liquor of the penicillium bimanum for effectively preventing and controlling peanut root rot, and the preparation method of the fermentation liquor of the P.bimae BL-1 comprises the following steps:
s1, punching 3-5 bacterial cakes on a PDA plate of a freshly cultured bacterial strain P.biaiae BL-1 by using a 7mm diameter puncher, and inoculating the bacterial cakes into a PDB liquid culture medium;
s2, adding 10-15 sterile glass beads with the diameter of 5mm into the PDB liquid culture medium inoculated in the step S1;
S3, shaking culture is carried out for 5 days at 25 ℃ and 180rpm, and fermentation liquor is obtained.
The invention further discloses application of the fermentation liquor of the penicillium bimatose for effectively preventing and controlling peanut root rot in any one of the following:
The application of fermentation liquor of C1 and P.Cilaiae CL-1 in preventing and/or treating peanut root rot;
application of C2, P.Cilaiae CL-1 fermentation liquor in inhibiting Ralstonia solanacearum (bacterial wilt);
application of fermentation liquor of C3 and P.Cilaiae CL-1 in inhibiting fusarium oxysporum (root rot);
application of fermentation liquor of C4 and P.Cilaiae CL-1 in inhibiting rhizoctonia solani (banded sclerotial blight);
Application of fermentation liquor of C5 and P.Cilaiae CL-1 in inhibiting pythium aphanidermatum (fruit rot);
Application of fermentation liquor of C6 and P.Cilaiae CL-1 in inhibiting cola hairline (stem rot);
use of C7, P.Cilaiae CL-1 fermentation broth for increasing biomass of peanut plants.
In summary, the invention has the following beneficial effects:
1. The strain P.biae BL-1 is a stable, efficient and broad-spectrum plant disease biocontrol fungus, and can be effectively used for biological control of peanut root rot;
2. The biocontrol strain has obvious flat plate antagonistic activity on plant pathogenic bacteria and fungi such as Ralstonia solanaceae (Rastonia solanacearum) causing peanut bacterial wilt, fusarium oxysporum (Fusarium oxysporum) causing peanut root rot, rhizoctonia solani (Rhzoctonia solani) causing peanut sheath blight, mucor (Pythium myriotylum) causing peanut fruit rot, and cocoa trichlegmator (Lasiodiplodia theobromae) causing peanut stem rot, and has a greenhouse control effect on peanut root rot reaching 90.36 percent. It was further found that P.biae BL-1 also has the ability to promote peanut seedling growth;
3. the strain P.biliae BL-1 is a beneficial penicillium bipinnatum with peanut disease control and peanut growth promotion effects.
Drawings
FIG. 1 is a phylogenetic tree of P.biliae BL-1 and a proximal penicillium strain constructed based on ITS sequences by adopting an adjacent method;
FIG. 2 shows the plate antagonism effect of the Mucor bifidus BL-1 of the present invention on various peanut pathogens;
FIG. 3 is an effect of the Mucor bifidus BL-1 of the present invention on peanut seed germination.
Detailed Description
In order that those skilled in the art will better understand the present invention, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings, wherein it is to be understood that the illustrated embodiments are merely exemplary of some, but not all, of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, shall fall within the scope of the present invention.
It should be noted that, without conflict, the embodiments of the present invention and features of the embodiments may be combined with each other. The present invention will be described in detail with reference to examples.
The experimental methods in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The pathogenic bacteria used in the following examples may be collected from the field or may be obtained by contacting the company of Wanlong fertilizer, inc. in Baofeng county, in order to repeat the experiments related to the present invention.
The strain is characterized in that the strain is a strain of penicillium belgium with high efficiency for preventing and controlling peanut root rot, the strain is penicillium belgium (Penicillium bilaiae) BL-1, hereinafter referred to as P.biliae BL-1, the preservation number is CGMCC No.41008, the preservation time is 2023, 12 months and 19 days, the preservation unit is China general microbiological culture Collection center, the preservation address is North Star Xili No.1, 3 in the Beijing area, the strain of penicillium belgium aerobic fungus can normally grow on a PDA culture medium, the edge of a colony is white hypha, the middle is dark green, and the ITS sequence of the strain of penicillium belgium is shown as SEQ ID No. 1.
The preparation method of the culture medium used in the following examples is as follows:
PDA culture medium, potato 200g, glucose 20g, agar 18g, distilled water to 1000mL.
PDB culture medium, 200g of potato, 20g of glucose and distilled water to 1000mL.
Nutrient agar medium (NA medium) is beef extract 3g, peptone 5g, glucose 2.5g, agar 12g, pH 7.0, distilled water to 1000mL.
Nutrient broth (NB medium) was beef extract 3g, peptone 5g, glucose 2.5g, pH 7.0, distilled water to 1000mL.
Example 1 isolation and characterization of P.biliae BL-1 (CGMCC No. 41008).
1. Isolation of strains
The P.biliaiae BL-1 is separated from the root canal soil of peanut field in Baofeng county, flat mountain city, henan province. And (3) picking up the collected soil sample to remove sundries such as plant root systems, residual branches and the like, further grinding and sieving, weighing 10g of the sample, adding the sample into 90mL of sterilized normal saline, and vibrating for 60min at 25 ℃ and 180rpm to enable the soil sample to be uniformly suspended in the normal saline, so that microorganisms in the soil are promoted to be dispersed in the solution to the greatest extent. Standing for 2-3 min, taking 5mL of supernatant, and serially diluting to 10 -5 by a 10-time gradient. 200. Mu.L of each dilution was spread evenly on PDA plates, with streptomycin added to PDA medium at a final concentration of 50. Mu.g/ml, and 3 replicates were set, dried under aseptic conditions and placed in a 25℃fungal incubator for approximately 48h. After single bacterial colonies grow out, selecting bacterial colonies with single color and form, picking single hyphae, transferring the bacterial colonies to a new PDA plate containing streptomycin, and purifying and culturing to obtain the bacterial strain BL-1.
2. Identification of Strain BL-1
The isolated strain BL-1 was inoculated onto a PDA plate, placed in a thermostatic incubator at 25℃for 5d, and then observed for morphological characteristics. The strain grows fast on the PDA plate, the whole plate (90 mm) can be fully grown for 5 days, hyphae are white just at first, edge hyphae are white along with the growth of bacterial colonies, the middle part is green, hyphae are attached to the surface of a culture medium for growth, aerial hyphae are short, and are in a velvet shape, and no obvious soluble pigment is produced. The mycelium is colorless, smooth in surface, and has an endophytic diaphragm, the top end of the mycelium is a conidiophore, a plurality of branches exist, and the mycelium is broom-shaped, and colorless, transparent and nearly spherical conidiophores are grown on the mycelium, which is typical morphological characteristics of penicillium fungi.
Bacterial strain BL-1 genomic DNA was obtained using Omega fungal DNA miniprep kit, and PCR amplification was performed based on fungal ITS sequence amplification universal primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'). The PCR system contained 2 XPCR Mastermix 10. Mu.L, 1. Mu.L each of the upstream and downstream primers (10. Mu.M concentration), 50ng of DNA template, and ddH 2 O make up 20. Mu.L. The PCR amplification procedure was 95℃pre-denaturation for 5min, 95℃denaturation for 30s,55℃annealing for 30s,72℃extension for 45s,35 cycles, 72℃renaturation for 10min. The PCR product was sent to Takara Bio-engineering (Dalian) Inc. to complete the sequencing work to obtain the 16S rDNA-ITS sequence of strain BL-1 as shown in SEQ ID NO. 1. Further, by means of NCBI website online BLAST alignment, penicillium proximal to the source was searched and phylogenetic tree was constructed using MEGAX (FIG. 1). The strain BL-1 was found to be branched from Mucor bifidus (Penicillium bilaiae) and to have a recent homology, and was identified as Penicillium bilaiae BL-1. The strain is preserved in China general microbiological culture collection center (CGMCC) of China general microbiological culture Collection center (CGMCC) for 12 months and 19 days in 2023, and the registration number is CGMCCNO.41008. Hereinafter referred to as P.biliae BL-1.
Example 2 antagonistic Activity of P.biliae BL-1 against different pathogenic bacteria
The peanut pathogenic bacteria include peanut bacterial wilt pathogenic bacteria Solanaceae Ralstonia (Rastonia solanacearum), peanut root rot pathogenic bacteria Fusarium oxysporum (Fusarium oxysporum), peanut sheath blight pathogenic bacteria Rhizoctonia solani (Rhzoctonia solani), peanut fruit rot pathogenic bacteria Pythium gracile (Pythium myriotylum), peanut stem rot pathogenic bacteria cocoa hair color two spore bacteria (Lasiodiplodia theobromae).
1. Antibacterial effect of P.biliae BL-1
Bacterial inhibition activity of the strain BL-1 against the above pathogenic bacteria was analyzed by plate confrontation. The plant pathogenic fungi counter experiment method comprises the steps of taking the edge part of a bacterial colony on a fresh cultivated pathogenic fungi PDA plate by using a puncher with the diameter of 7mm, inoculating one surface of a bacterial ribbon mycelium to a blank PDA plate by using a sterilization inoculating needle, inoculating bacterial strain BL-1 bacterial cakes with the diameter of 7mm on two sides of the pathogenic fungi cakes equidistantly, then placing the bacterial cakes in a25 ℃ constant temperature fungi incubator for cultivation, and setting for 3 times of repetition. And observing the growth condition every day, respectively culturing for 3-5 days according to the growth speed of different pathogenic bacteria, and then photographing, recording and measuring the size of the antibacterial zone. The R.solanacearum plates are subjected to the opposite operation, namely 1) fresh R.solanacearum single colonies are inoculated into NB culture solution, the NB culture solution is cultured overnight at 28 ℃ and 180rpm to obtain pathogenic bacteria culture solution, 2) when the thawed NA culture medium is cooled to 40-45 ℃, the pathogenic bacteria culture solution is added into the cooled NA culture medium to enable the final concentration to be 1 multiplied by 10 7 cfu/mL, the cooled NA culture medium is uniformly shaken and poured into a culture dish to obtain a bacteria-carrying plate, 3) after the plate is completely solidified, 7mm bacterial strain BL-1 bacterial cakes are inoculated at the central position, the bacterial strain BL-1 bacterial cakes are placed in a 28 ℃ constant temperature incubator, the diameter of a bacteria inhibition zone is observed and measured after the bacterial inhibition zone is inversely cultured for 48 hours, and 3 repeats are set.
The results demonstrate that P.biliae BL-1 has obvious plate antagonistic activity on 5 plant pathogenic bacteria and fungi such as Solanaceae Ralstonia, fusarium oxysporum, rhizoctonia solani, pythium gracile, thermomyces lanuginosus and the like (figure 2), wherein the antagonistic activity of Fusarium oxysporum and Rhizoctonia solani is the highest.
2. Antibacterial effect of P.biliae BL-1 sterile fermentation broth of P.biliae
Preparing sterile fermentation liquor, namely, taking 3-5 bacterial cakes on a PDA flat plate of a freshly cultured strain P.biliae BL-1 by using a 7mm diameter puncher, inoculating the bacterial cakes into a PDB liquid culture medium, adding 10-15 sterile glass beads with the diameter of 5mm into the bacterial cakes, and shaking and culturing the bacterial cakes for 5 days at the temperature of 25 ℃ and at the speed of 180rpm to obtain a fermentation product. Further centrifuging the fermentation product at 7000 Xg at room temperature for 10min, collecting supernatant, and filtering with 0.45 μm filter membrane to remove mycelium and conidium to obtain sterile fermentation liquid. After thawing, the PDA culture medium cooled to about 45 ℃ is added with sterile fermentation liquid with the final concentration of 10%, the mixture is poured into a culture dish after uniform mixing, after solidification, fresh fungus cakes of different pathogenic fungi are inoculated at the middle position of the culture medium, and then the culture medium is poured into a fungus incubator with constant temperature of 25 ℃ for culture, and the culture is repeated for 3 times. And observing the growth condition every day, respectively culturing for 3-5 days according to the growth speed of different pathogenic bacteria, photographing and recording, measuring the colony diameter, and calculating the bacteriostasis rate. The plate bacteriostasis test of the sterile fermentation liquid on the Ralstonia solanaceae R.solanacearum is carried out by preparing a bacteria-carrying plate by the method, then inoculating 10 mu L of the sterile fermentation liquid to the middle position of the bacteria-carrying plate, airing, placing in a 28 ℃ constant temperature incubator, inversely culturing for 48 hours, observing and measuring the diameter of a bacteria inhibition zone, and setting 3 repeats.
As can be seen from the results in table 1, the p.biaie BL-1 sterile fermentation broth has a certain inhibitory effect on all 5 peanut pathogens selected in the present invention. The bacterial strain sterile fermentation liquor has better inhibition effect on pathogenic fungi, wherein the bacteriostasis rate on fusarium oxysporum causing peanut root rot and rhizoctonia solani causing sheath blight is highest, the bacteriostasis rate reaches 62.03% and 64.65% respectively, the bacteriostasis effect on pathogenic bacteria Solanaceae Ralstonia is weaker, and the plate bacteriostasis rate is only 26.43%.
TABLE 1 antibacterial Rate of P.biliae BL-1 against various peanut pathogens
Example 3 P.biliae BL-1 promotes peanut seed germination
Preparing a P.biliae BL-1 spore suspension, namely taking 3-5 bacterial cakes on a PDA flat plate of a freshly cultured bacterial strain P.biliae BL-1 by using a 7mm diameter puncher, inoculating the bacterial cakes into a PDB liquid culture medium, adding 10-15 sterile glass beads with the diameter of 5mm into the bacterial cakes, and shaking and culturing for 5 days at 25 ℃ and 180rpm to obtain fermentation liquor. The fermentation broth was filtered through 3 layers of sterilized gauze to remove mycelia, centrifuged at 7000 Xg for 10min, the supernatant was discarded, the conidia were collected, resuspended in PDB medium to obtain a conidia suspension, and the concentration of the conidia suspension was calculated by means of a hemocytometer and finally adjusted to 1X 10 8/mL.
Selecting peanut seeds with full grains, good skin luster and high uniformity, firstly soaking the peanut seeds in 75% ethanol for 30s for surface disinfection, then washing the peanut seeds with sterile water for 2-3 times, then soaking the peanut seeds in 2% sodium hypochlorite solution for 5min for secondary disinfection, and washing the peanut seeds with sterile water for 3-5 times to ensure that no sodium hypochlorite residues exist on the surfaces of the peanut seeds. And respectively soaking the peanut seeds with the surfaces disinfected into a bacterial strain BL-1 conidium suspension and a sterile fermentation liquid for 10min, taking the peanut seeds soaked in a PDB culture medium for 10min as a blank control, repeating each treatment for 3 times, repeating 10 peanut seeds each, taking out, airing the epidermis, and carrying out moisturizing and germination acceleration. The treated peanut seeds were placed in sterile petri dishes (150 mm) and incubated in a climatic chamber (light dark time period 12/10h, relative humidity 60%), light temperature 28 ℃, dark temperature 25 ℃. Adding a proper amount of sterile water for moisturizing every day, observing germination conditions, recording the germination number in 10d, measuring the indexes such as bud length, stem thickness and the like, and calculating germination rate.
All treated peanut seeds can germinate normally, and the germination rate is 100%. However, there was a significant difference in the length and size of peanut sprout formation from the different treatments after germination, and the peanut seeds treated by seed soaking with strain BL-1 spore suspension and sterile fermentation broth, and the sprouts formed after germination were significantly larger than those of the blank treatment (FIG. 3). Further measurements of germination length and diameter of the thickest part of germination revealed that the germination length of BL-1 spore suspension and sterile broth treated peanuts exceeded 60% of the blank control in the same time, the germination lengths reached 40.17mm and 36.33mm, respectively, and the diameter of the thickest part of germination exceeded 1mm of the control group (Table 2).
TABLE 2 influence of P.biliae BL-1 on peanut seed germination
Example 4 control of peanut root rot by P.biliae BL-1
Selecting full peanut seeds with uniform sizes, soaking the peanut seeds in clear water at room temperature for 12 hours to enable the seeds to fully absorb water, then keeping the peanut seeds wet by using gauze, accelerating germination at room temperature, enabling the peanut seeds to appear white after about 3-5 days, planting the peanut seeds with uniform germination in a seedling pot, and growing 2 true She Bianke seedlings until seedlings emerge.
Billenia P.Bilaiae BL-1 and Fusarium oxysporum F.spores preparation of suspensions reference is made to the preparation of spore suspensions in example 3. Mixing the prepared spore suspension with nutrient soil according to a certain proportion, and designing 4 treatments. The biological control is inoculated with P.biliae BL-1 alone, F.oxysporum which is a pathogen, and 4 treatments are respectively carried out according to the equal proportion of 1:1 on the biological control and the pathogen. The concentration of inoculated conidia in the soil inoculated with biocontrol bacteria or pathogenic bacteria is ensured to be 1X 10 6/g, and a blank control is mixed into the equal amount of PDB culture medium. Each treatment is divided into 15 pots with the diameter of 9cm, 1 peanut seedling is planted in each pot, every 5 pots are in one group, and each treatment is in 3 groups. And (3) placing the planted peanut seedlings in a manual climate chamber for normal watering management (the light and dark time is 12/10h, the relative humidity is 60%), the illumination temperature is 28 ℃, the dark temperature is 25 ℃, and the disease condition is observed and recorded every day. And when the plant grows to 7d, counting the number of plants and severity of disease, and calculating the disease rate, disease index and prevention and treatment effect.
The grading standard of the peanut root rot comprises 0 grade without obvious symptoms, 1 grade with slight discoloration, wherein the discoloration part accounts for less than 10% of the whole root system, plants do not wilt, 3 grade with obvious browning, the discoloration root system accounts for 10-30% of the whole root system, the plants show water loss symptoms, 5 grade with most browning, the discoloration root system accounts for 30-50% of the whole root system, the plants obviously wilt, 7 grade with more than 50% of the whole root system, the plants wilt but not withered, and 9 grade with dry death.
Incidence (%) = number of diseased plants per group/total number of plants per group x 100%;
Disease index = [ Σ (number of disease stages x number of disease stages) ]/(total number of investigation x highest disease stage) ×100;
control effect (%) = (control disease index-treatment disease index)/control disease index x 100%.
The results prove that the root rot of the peanut can be effectively prevented and treated by root irrigation of the strain BL-1 fermentation liquor, the disease incidence and severity are obviously reduced, and the control effect of the strain BL-1 fermentation liquor on the root rot of the peanut is proved to be more than 90% by indoor potting (Table 3).
TABLE 3 control Effect of P.biliae BL-1 on peanut root rot
The present embodiment is only for explanation of the present invention and is not to be construed as limiting the present invention, and modifications to the present embodiment, which may not creatively contribute to the present invention as required by those skilled in the art after reading the present specification, are all protected by patent laws within the scope of claims of the present invention.
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