Penicillium bilaense W2 and application thereof in coupling antagonism and growth promotion of peanut aspergillus flavus
By screening and identifying Penicillium bilaiae W2, the strain and its fermented metabolites not only have significant antagonistic effects of Aspergillus aflatoxin, but also promote peanut seed germination and seedling growth, solving the problem that the existing technology cannot take into account pest control and crop yield improvement, and achieving effective application in agricultural production.
Patent Information
- Application Number
- CN202411887652.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-20
- Publication Date
- 2025-04-11
AI Technical Summary
The existing Aspergillus antagonist microorganisms cannot effectively promote peanut seed germination and seedling growth while inhibiting Aspergillus aflatoxin, and cannot meet the needs of modern agriculture for both pest control and crop yield improvement.
A Penicillium bilaiae W2 strain and its fermented metabolites were screened and identified, and this strain and its fermented metabolites had significant antagonistic effects and could promote peanut seed germination and seedling growth.
Penicillium Bilai W2 fermentation broth almost completely inhibits the germination of Aspergillus aflatoxin spores and the growth of mycelium at 50% concentration, significantly antagonizes Aspergillus aflatoxin contamination, and significantly promotes peanut seed germination and seedling growth under 100-fold dilution.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of harmful microbial antagonists, and in particular relates to Penicillium billais W2 and application thereof in coupled antagonism and growth promotion of Aspergillus flavus in peanuts. Background Art
[0002] Aflatoxins are bifuranoid toxins produced by fungi such as Aspergillus flavus and Aspergillus parasiticus. To date, nearly 20 types of aflatoxins have been identified. Among them, aflatoxin B1 is particularly toxic, about 10 times more toxic than potassium cyanide and 68 times more toxic than arsenic, and has been classified as a Class I carcinogen by the International Agency for Research on Cancer.
[0003] Peanuts are one of the agricultural products and foods that are extremely susceptible to aflatoxin contamination. During the planting, harvesting, and subsequent storage and transportation of peanuts, they may be attacked by aflatoxin, resulting in the production of a large amount of aflatoxin. Regarding prevention and control measures, the current short-term effective methods for aflatoxin control mainly include physical, chemical and biological methods. Biological control uses mutual inhibition between organisms or the production of specific active substances to achieve prevention and control effects. Due to its advantages such as environmental friendliness, low toxicity, strong selectivity, low residue and low resistance to drugs, it has become the research focus of aflatoxin control. Against the backdrop of the current severe food and oil security situation, the demand for sustainable agriculture is growing. How to increase crop yields while preventing and controlling pests and diseases has become a new challenge for the development of modern agriculture.
[0004] At present, through purification and separation technology, a variety of microorganisms have been obtained, such as Bacillus subtilis, lactic acid bacteria, yeast, mold, actinomycetes and non-toxigenic Aspergillus flavus, which are used for biological control against toxigenic Aspergillus flavus. However, the existing antagonistic microorganisms cannot fully meet the new needs of the development of modern agricultural industry to promote plant growth while inhibiting Aspergillus flavus. Summary of the invention
[0005] The invention provides a Penicillium bilaiae W2, which is an antagonistic bacterial strain of Aspergillus flavus. The strain and its fermentation metabolites can promote peanut seed germination and seedling growth, and significantly antagonize Aspergillus flavus contamination, and has good application prospects in agricultural production.
[0006] In order to achieve the above-mentioned purpose, the present invention provides a Penicillium bilaiae W2, which was deposited in the General Microbiological Center of China Culture Collection Administration on July 12, 2024, with a deposit number of CGMCCNO: 41463.
[0007] Preferably, the nucleotide sequence is shown in SEQ ID NO:1.
[0008] The present invention also provides a use of Penicillium bilaiaeW2 according to the above technical solution in inhibiting the spore germination and / or hyphae growth of Aspergillus flavus.
[0009] Preferably, when the concentration of Penicillium bilaiae W2 fermentation broth is 50%, the spores and / or hyphae of Aspergillus flavus almost stop germinating and / or growing.
[0010] The present invention also provides a use of Penicillium bilaiae W2 according to the above technical solution in preventing Aspergillus flavus from infecting stored peanuts / extending the storage period of peanuts.
[0011] Preferably, the concentration of Penicillium bilaiae W2 fermentation broth is 50%.
[0012] The present invention also provides a use of Penicillium bilaiae W2 according to the above technical solution in promoting peanut seed germination and / or seedling growth.
[0013] The present invention also provides a use of Penicillium bilaiae W2 according to the above technical scheme in promoting peanut seed germination and / or seedling growth while antagonizing peanut aflatoxin.
[0014] Preferably, the concentration of Penicillium bilaiae W2 fermentation broth is 15%.
[0015] The present invention also provides a peanut biocontrol fungicide, characterized in that it contains the Penicillium bilaiae W2 described in the above technical scheme as a main raw material.
[0016] Compared with the prior art, the advantages and positive effects of the present invention are:
[0017] The present invention screened a Penicillium bilaiae W2, which is an antagonistic bacteria strain of Aspergillus flavus. The strain and its fermentation metabolites have both the functions of antagonizing Aspergillus flavus and promoting peanut growth. Not only can it almost completely inhibit the germination of Aspergillus flavus spores and the growth of hyphae when the fermentation liquid concentration is 50%, and significantly antagonizes the contamination of Aspergillus flavus, but the W2 fermentation liquid diluted 100 times can also significantly promote the germination of peanut seeds and the growth of seedlings, and has good application prospects in agricultural production. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1The antagonistic effect of strain W2 provided in the embodiment of the present invention on Aspergillus flavus;
[0019] Figure 2 Phylogenetic tree analysis of strain W2 provided in the embodiment of the present invention;
[0020] Figure 3 The effect of 50% W2 fermentation liquid provided in the embodiment of the present invention on the germination of Aspergillus flavus spores;
[0021] Figure 4 The addition of 80% of the W2 fermentation liquid provided in the embodiment of the present invention has an inhibitory effect on Aspergillus flavus in peanuts;
[0022] Figure 5 This is a diagram showing the effect of the 100-fold diluted W2 fermentation liquid provided in an embodiment of the present invention on the germination of peanut seeds. DETAILED DESCRIPTION
[0023] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0024] Example 1 Isolation of Penicillium bilaiae W2
[0025] The fungi were isolated and screened from the healthy soil of the peanut experimental field in Laixi, Qingdao, Shandong Province. The specific steps are as follows:
[0026] a. Under sterile operation, weigh 10g of soil and add it to 90mL of PDB liquid medium. After activation culture at 200r / min for 3h, separate single colonies on PDA solid medium by dilution plate method. The dilution gradient is set to 10 -1 , 10 -2 , 10 -3 .
[0027] b. After obtaining a single colony, purify and isolate it on a separation plate 2-3 times to obtain a purified strain.
[0028] c. The punch method was used for quantification. The candidate antagonistic bacteria cake and Aspergillus flavus were selected and inoculated into two PDA culture media respectively. The two PDA plates were placed side by side for culture. The candidate antagonistic bacteria were inoculated as a blank control. The plates were cultured in the dark at 28°C for 3 to 5 days. The growth of Aspergillus flavus colonies was observed, the growth diameter of Aspergillus flavus colonies was measured, and the inhibition rate was calculated. The results are shown in Table 1.
[0029] d. Use the punching method to quantify, pick the fungus cake and inoculate it on the PDA medium, 2 cm away from the edge of the culture dish, and inoculate the Aspergillus flavus cake on the other side of the culture dish 2 cm away from the edge of the culture dish for confrontation and culture. Use the blank control without inoculating the candidate antagonistic strain, and culture it in the dark at 28℃ for 3-5 days, observe the growth of the Aspergillus flavus colony, determine the growth radius of the Aspergillus flavus colony, and calculate its antibacterial rate. The above tests were repeated 3 times. The results are shown in Table 1
[0030] The calculation formula is as follows:
[0031] Antibacterial rate (%) = (Rp-Rt) / Rp×100%
[0032] Among them, Rp represents the growth radius of Aspergillus flavus in the control group; Rt represents the growth radius of Aspergillus flavus in the treatment group.
[0033] e. The strain screened was found to have the most advantageous antagonistic effect against Aspergillus flavus under the conditions of confrontation and confrontation, and was named W2.
[0034] Table 1 Inhibitory effect of Penicillium bilainum W2 on the growth of Aspergillus flavus
[0035]
[0036] like Figure 1 As shown in the figure, W2 and Aspergillus flavus were cultured in confrontation for 4 days. At the confrontation interface, W2 grew vigorously, while the growth of Aspergillus flavus was restricted, and an obvious inhibition zone was produced in the middle, indicating that the antagonistic strain W2 had obvious antagonistic properties against the growth of Aspergillus flavus under confrontation culture conditions. W2 could secrete extracellular active ingredients, which could inhibit the growth of Aspergillus flavus. Combined with the data in Table 1, it can be seen that the inhibition rate of W2 on Aspergillus flavus reached 57.54%, which confirmed that W2 had a good inhibitory effect on Aspergillus flavus.
[0037] Example 2 ITS Sequencing of Antagonistic Bacteria to Aspergillus flavus
[0038] The genomic DNA of the candidate aflatoxin antagonistic strain W2 was extracted and amplified and sequenced using the laboratory ITS universal primers ITS1 / ITS4, where ITS1: 5'-TCCGTAGGTGAACCTGCGG-3', ITS4: 5'-TCCTCCGCTTATTGATATGC-3'. The amplified sequence was subjected to Blast analysis and homology comparison with the sequence in the gene library, and the phylogenetic tree was constructed using Neihbor-Joinhing in MEGA5.1 software, as shown in Figure 2. Figure 2As shown. Strain W2 has the highest homology with Penicillium bilaiae, reaching 99%. After growing in a 28°C incubator for 4 days, the colony diameter of W2 was 1.67±0.21cm, gray-green and opaque, with a dry and wrinkled surface and wavy edges, which is consistent with the characteristics of Penicillium. Combining the results of identification such as bacterial morphology and growth conditions, it was identified as Penicillium bilaiae and named W2. It was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on July 12, 2024, with the deposit number CGMCC NO: 41463.
[0039] Sequencing revealed that the ITS sequence of the Penicillium billais W2 strain was as shown in SEQ ID NO:1.
[0040] Example 3 Biocontrol effect experiment of Penicillium bilai W2
[0041] The inhibitory effect of Penicillium bilainum W2 fermentation broth on Aspergillus flavus was investigated, and the specific steps were as follows:
[0042] a. Preparation of fermentation broth of biocontrol strain W2: Use a pick to pick up the frozen biocontrol strain W2, transfer and streak it onto the surface of the solid culture medium containing PDA, culture for 4 days to activate the strain, cut 2mm×2mm bacterial blocks and add them to 100mL of PDB culture medium, culture at 28℃ with shaking for 96h, centrifuge at 1000r / min for 10min, and filter the supernatant with a filter membrane to obtain sterile fermentation broth with a concentration of 50%.
[0043] b. Preparation of Aspergillus flavus spore suspension: Use sterile distilled water containing 0.1% Tween-80 to wash the Aspergillus flavus spores cultured on the PDA slant and filter through absorbent cotton to prepare an Aspergillus flavus spore suspension. The obtained spores are counted by hemocytometer and diluted to the required concentration with sterile distilled water containing 0.1% Tween-80.
[0044] c. Determination of the effect of W2 fermentation broth on the germination of Aspergillus flavus spores: Dilute the fermentation broth with PDB medium to prepare a series of culture solutions with final concentrations of 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, and 0%, and add 2 mL of each culture solution to a 12-well plate. Add 20 μL of 1×10 7 The suspension of Aspergillus flavus spores was prepared in a 28°C constant temperature incubator at 130 rpm for 12 h. The germination of each spore was observed under a microscope, and the germination was considered to be completed when the length of the germ tube was greater than or equal to the short diameter of the spore. Three parallels were set for each group of experiments, and the experiment was repeated three times. The results are shown in Table 2.
[0045] Spore germination inhibition rate = (spore germination rate of control group - spore germination rate of treatment group) / spore germination rate of control group × 100%
[0046] Table 2 Effect of Penicillium bilainum W2 fermentation broth on the germination of Aspergillus flavus spores
[0047]
[0048]
[0049] From the data in Table 2, it can be seen that after 12 hours of cultivation, the spores in the control group containing 0% fermentation liquid basically germinated, while after adding the fermentation liquid, the spore germination rate decreased. As the concentration of the fermentation liquid increased, the antibacterial effect became more obvious. Figure 3 The results also showed that when the fermentation broth content reached 50%, almost no spore germination was observed.
[0050] d. Effect of W2 fermentation broth on the growth of Aspergillus flavus hyphae: 20 μL of PDB medium with a concentration of 1×10 7 spore suspension of Aspergillus flavus at 1000 g / mL, incubated in a constant temperature incubator at 28°C and 130 rpm for 24 h. After centrifugation to remove the supernatant, the Aspergillus flavus hyphae were transferred to a series of PDB culture media with fermentation liquid contents of 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, and 0%, and continued to be cultured for 24 h at 28°C and 130 rpm. Centrifuged at 4000 r / min for 10 min, the supernatant was removed, and the collected hyphae were weighed. The experiment was repeated 3 times. The results are shown in Table 3.
[0051] Mycelium growth inhibition rate = (mycelium weight of control group - mycelium weight of treatment group) / mycelium weight of control group × 100%
[0052] Table 3 Effect of Penicillium bilainum W2 fermentation broth on the mycelial growth of Aspergillus flavus
[0053] W2 fermentation broth concentration (%) Inhibition rate (%) 0 -- 10 85.46±7.77 20 58.79±9.46 30 32.45±2.45 40 10.34±1.23 50 0.23±0.01 60 0.10±0.01 70 0.02±0.06 80 0.03±0.21 90 0 100 0
[0054] From the data in Table 3, it can be seen that the addition of fermentation liquid affects the growth of mycelium. The higher the concentration of fermentation liquid, the more inhibited the growth of mycelium. When the content of fermentation liquid reaches 50%, the mycelium of Aspergillus flavus almost stops growing.
[0055] e. Antagonism of the fermentation liquid of biocontrol strain W2 against Aspergillus flavus in peanuts: Take 20 peanuts and put them into two sterilized culture dishes, numbered A and B. Take 3 mL of sterilized PDB medium and add 20 μL of 1×10 7 spores of Aspergillus flavus, mixed thoroughly and added to A as the control group; 3 mL of the fermentation broth after filtration and sterilization was taken, and 20 μL of 1×10 7 spores of Aspergillus flavus were added to medium B after being thoroughly mixed. The mixture was cultured at 28°C for 6 days. Three parallel experiments were set up for each group of experiments, and the experiment was repeated 3 times.
[0056] The results are as follows Figure 4 As shown, the surface of the peanuts in A is covered with Aspergillus flavus, while the surface of the peanuts in B is not covered with Aspergillus flavus, indicating that the fermentation broth of Penicillium birei W2 can significantly inhibit the growth of Aspergillus flavus in peanuts.
[0057] f. Fermentation broth of biocontrol strain W2 prevents Aspergillus flavus from infecting stored peanuts: Take 20 peanuts and put them into 2 sterilized culture dishes, numbered A and B. Spray 3 mL of sterilized PDB medium on the surface of peanuts in A as the control group; spray 3 mL of filtered and sterilized fermentation broth on the surface of peanuts in B as the treatment group. Three parallel experiments were set up for each group of experiments, and the experiment was repeated 3 times. After the above groups of peanuts were dried in the shade, they were placed at room temperature and stored in a ventilated and dry place. Samples were taken every 30 days to detect the content of Aspergillus flavus in peanuts for a total of 90 days. The results are shown in Table 4.
[0058] Table 4 Aflatoxin content (cfu / g)
[0059] Days 30d 60d 90d Control group <![CDATA[5.3×10 3 ]]> <![CDATA[6.4×10 4 ]]> <![CDATA[8.9×10 5 ]]> Treatment Group Not detected 20 <![CDATA[1.3×10 2 ]]>
[0060] As shown in the data in Table 4, as time goes by, the content of Aspergillus flavus in the treatment group is much less than that in the control group; spraying the fermentation liquid of Penicillium bilaminarium W2 can effectively prevent the contamination of Aspergillus flavus in peanuts and prolong the storage period of peanuts.
[0061] Example 4 Experiment on promoting peanut seed germination and growth by Penicillium bile W2 fermentation liquid
[0062] a. Experiment on promoting seed germination by Penicillium birei W2 fermentation liquid: Take 20 plump peanut seeds freshly harvested that year and place them in a sterile culture dish. Spray 10 ml of 15% W2 fermentation liquid evenly on the surface of the peanut seeds, cover the culture dish, and culture in a 28°C incubator away from light for 48 hours. Count the seed germination rate every 6 hours, and use the whitening of peanut seeds as the germination standard, and count for a total of 60 hours. Use sterile water as a control and do a blank test. Set up 3 parallels for each group of experiments, and repeat the experiment 3 times. The results are shown in Table 5.
[0063] Table 5 Effect of Penicillium bilai W2 fermentation liquid on peanut seed germination
[0064]
[0065] From the data in Table 5 and Figure 5 As shown in the figure, the seeds of the treatment group started to germinate at 24h and almost all germinated at 48h, while the seeds of the control group started to germinate at 30h and all germinated at 54h. This shows that W2 fermentation liquid can significantly promote the germination of peanut seeds.
[0066] b. Experiment on promoting the growth of peanut seedlings with Penicillium bilainum W2 fermentation liquid: Take the plump peanut seeds freshly harvested that year, disinfect them with 75% alcohol for 2 minutes, rinse them repeatedly 2-3 times, and disinfect the surface of the peanut seeds. Plant them in a hole-sowing manner, place the nutrient soil in a seedling tray, irrigate it with sterile water, and transplant the germinated peanut seedlings after 3 days in a 25°C greenhouse. Place the nutrient soil in another seedling tray, irrigate it with 15% concentration of W2 fermentation liquid as the treatment group, and irrigate the control group with sterile water, and ensure that the amount of watering is consistent. Transplant the peanut seedlings into the seedling trays of the above-mentioned treatment group and control group, respectively, place them in an artificial 25°C constant temperature growth room for cultivation, harvest and test them after 30 days of cultivation, and use biological indicators such as plant fresh weight, root length, and plant height as indicators for judging the growth-promoting effect of Penicillium bilainum W2 fermentation liquid. Three parallels are set for each group of experiments, and the experiment is repeated 3 times. The results are shown in Table 6.
[0067] Table 6 Effect of Penicillium bilainum W2 fermentation liquid on peanut seedlings
[0068] Experimental treatment Fresh weight(g) Root length (cm) Plant height (cm) Control group 13.26±6.58 18.79±5.98 32.23±9.76 Treatment Group 17.56±5.78 22.78±7.87 34.45±7.49
[0069] From the data in Table 6, we can see that compared with the control, the plant height of the treatment group was significantly higher than that of the control group, with a growth rate of 6.89%. The root length also increased compared with the control group, with a growth rate of 21.23%. The fresh weight of the whole plant in the treatment group was 4.3g / plant heavier than that of the control group, with a growth rate of 32.43%. The results show that the fermentation liquid of Penicillium bilainum W2 can not only promote the germination and growth of peanut seeds, but also may produce some growth-promoting substances during the fermentation process, which further promotes the germination and growth of seeds.
Claims
1. Penicillium bilaiae W2, characterized in that It was deposited in the General Microbiology Center of China Culture Collection Administration on July 12, 2024, with the deposit number CGMCCNO: 41463.
2. Penicillium bilaiae W2 according to claim 1, characterized in that Its nucleotide sequence is shown in SEQ ID NO:
1.
3. Use of Penicillium bilaiae W2 according to claim 1 in inhibiting spore germination and / or hyphae growth of Aspergillus flavus.
4. The use according to claim 3, characterized in that: When the concentration of Penicillium bilaiae W2 fermentation broth was 50%, the spores and / or hyphae of Aspergillus flavus almost stopped germinating and / or growing.
5. Use of Penicillium bilaiae W2 according to claim 1 in preventing Aspergillus flavus from infecting stored peanuts / extending the storage period of peanuts.
6. The use according to claim 5, characterized in that: The concentration of Penicillium bilaiae W2 fermentation broth is 50%.
7. Use of Penicillium bilaiae W2 according to claim 1 in promoting peanut seed germination and / or seedling growth.
8. The use according to claim 7, characterized in that: The concentration of Penicillium bilaiae W2 fermentation broth is 15%.
9. Use of Penicillium bilaiae W2 according to claim 1 in promoting peanut seed germination and / or seedling growth while antagonizing Aspergillus flavus in peanut.
10. A peanut biocontrol agent, characterized in that: The method comprises the Penicillium bilaiaeW2 described in claim 1 as a main raw material.