Application of aspergillus flavus in prevention and treatment of epidemic blight bacteria of Eucommia wilsonii

Abstract

The invention discloses a new application of aspergillus flavus in prevention and treatment of epidemic blight of Eupin, and relates to the technical field of microbial prevention and treatment, the aspergillus flavus is CZB-13, the taxonomic name of the aspergillus flavus is Aspergillus flavus, the strain is preserved in China Center for Type Culture Collection on July 8, 2024, the preservation address is Wuhan University, Wuhan, China, and the preservation number is CCTCC NO: M 20241494. The aspergillus flavus CZB-13 provided by the invention has a remarkable inhibition effect on the epidemic blight of the epidemic blight of the epidemic blight of the epidemic blight of the epidemic blight of the epidemic blight of the epidemic blight of the epidemic blight of the epidemic blight of the epidemic blight of the epidemic blight of the epidemic blight of the epidemic blight of the epidemic blight of the epidemic blight of the epidemic blight of the epidemic blight of the epidemic blight of the epidemic blight of the epidemic blight of the epidemic blight of the epidemic blight. The method is also of great significance in protecting the ecological environment and promoting green prevention and control of epidemic diseases of the elaeagnus plants.

Description

Technical Field

[0001] The invention relates to the technical field of microbial control, and in particular to a new use of Aspergillus flavus in the control of Elaeocarp blight. Background Art

[0002] Elaeocarpus is mainly used for greening and landscaping. After being infected with the disease, its vascular system is destroyed, and the leaves and branches turn yellow and wilt. Most diseased trees will die within 5 years, seriously affecting their ornamental value. Huang et al. conducted a disease survey on Elaeocarpus apiculatus and E. hainanensis in 7 areas of Guangdong Province from 2020 to 2022. The results showed that the incidence of Elaeocarpus blight was as high as 85% or more (Huang et al., 2022). It can be seen that the prevention and control of Elaeocarpus blight is urgent. Pseudocryphonectria elaeocarpicola is the pathogen that Huang et al. (2022) first isolated from the bark of newly infected Elaeocarpus blight and confirmed by Koch's postulates. Through morphological methods and ITS, LSU, tef1, rpb2 multi-gene sequencing and phylogenetic cluster analysis, the fungus was identified as the only species of the genus Pseudocryphonectria in July 2022. The fungus can produce dimorphic spores that are different from other genera in the family Pseudocryphonectria in morphology, and has strong pathogenicity and transmissibility. It harms the branches and stems of Elaeocarpus elaeocarpus, causing them to wilt and rot, and the bark is covered with orange-yellow tendrils.

[0003] The genus Pseudocryphonectria belongs to the order Diaporthale and the family Cryphonectriaceae. The order Diaporthale is rich in taxa, and the family Cryphonectriaceae is an important pathogenic family. The family Cryphonectriaceae contains many important pathogens, such as the chestnut blight pathogen Crysopora parasitica, which has been a global forest pathogen and a quarantine pathogen in my country since the 1970s. Jiang et al. (2020) re-evaluated the family Cryphonectriaceae based on the morphological and molecular biological data of the previous strains and accepted two branches of 21 genera and 55 species in the family; subsequently, Wang et al. (2020) and Huang et al. (2022) added three new genera, Capillaureum, Parvosmorbus, and Pseudocryphonectria, based on further morphological and phylogenetic arguments. Pathogenic fungi of the family Echinochloa often have host specialization and only cause disease to plants of the same species or genus under natural conditions. Therefore, many genera include fewer species. This is one of the reasons why Echinochloa pseudocryptochloa is the only species in the genus Echinochloa pseudocryptochloa.

[0004] At present, only relevant research on the taxonomy, pathogenicity and bioassay targets of Elaeocarp blight pathogens can be found, and no relevant reports on its biocontrol antagonistic bacteria have been found. Under the policy of "double reduction" of fertilizers and pesticides, the screening of candidate biosource bacteria for Elaeocarp blight pathogens is also of great significance for protecting the ecological environment and promoting the green prevention and control of Elaeocarp plant diseases. Summary of the invention

[0005] The purpose of the present invention is to provide a new use of Aspergillus flavus in the prevention and treatment of Elaeocarp blight, which is also of great significance in reducing the residue of chemical drugs, protecting the ecological environment and promoting green prevention and control of Elaeocarp blight.

[0006] In order to achieve the above-mentioned purpose, the present invention provides an application of Aspergillus flavus in the prevention and control of Elaeocarp blight, wherein the Aspergillus flavus is CZB-13, which has been deposited in the China Center for Type Culture Collection on July 8, 2024, with the preservation address being Wuhan University, Wuhan, China, and the preservation number being CCTCC NO: M 20241494.

[0007] Preferably, the application comprises the prevention and treatment of Elaeocarp blight pathogen, wherein the Elaeocarp blight pathogen is caused by infection of Elaeocarp pseudocryptocephalus.

[0008] The present invention also provides an application of a flavus inoculant in the field of Elaeocarp cultivation. The inoculant is prepared from the above-mentioned Aspergillus flavus CZB-13 or its fermentation liquid. The application includes the prevention and treatment of Elaeocarp blight pathogen, and is particularly useful for the prevention and treatment of Elaeocarp blight pathogen caused by infection of Elaeocarp pseudocryptoplexiae.

[0009] The present invention has the following advantages:

[0010] The present invention provides a new use of Aspergillus flavus CZB-13, which broadens the application value of microorganisms. The strain or its fermentation liquid can be used to prepare a bacterial agent for the prevention and treatment of Elaeocarpus blight, which can effectively reduce the residue of chemical drugs, and is also of great significance for protecting the ecological environment and promoting the green prevention and control of Elaeocarpus plant diseases. At the same time, it also has potential application value in environmental protection and ecological civilization construction. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is a colony morphology diagram of Aspergillus flavus CZB-13 in the present invention.

[0012] Figure 2 This is the result of the confrontation between Aspergillus flavus CZB-13 and Elaeocarpium edulis in the present invention.

[0013] Figure 3 The present invention shows the inhibitory effect of the fermentation liquid of Aspergillus flavus CZB-13 on Elaeocarpium edulis.

[0014] Figure 4 This is the phylogenetic tree result of Aspergillus flavus CZB-13 in the present invention. DETAILED DESCRIPTION

[0015] 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.

[0016] Note: The experimental methods in the following examples are conventional methods unless otherwise specified, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0017] Experimental Example 1 Isolation and screening of strains

[0018] Unde-soaped tea seed cake was selected as the experimental material. After the unde-soaped tea seed cake was crushed into powder, 10 g of tea seed cake powder was accurately weighed and transferred to a glass fermentation bottle with a solid-liquid ratio of 1:1.6. 16 mL of sterile water was added, stirred evenly, and left overnight. A high-pressure steam sterilizer was used to sterilize at 121°C and 20 min to obtain a sterile tea seed cake fermentation sample.

[0019] The experimental sample sites are located in the Qilin South Student Dormitory Area of ​​South China Agricultural University and the Arboretum of South China Agricultural University. Both experimental sites are located in Tianhe District, Guangzhou City, Guangdong Province. The average annual temperature in the urban area is 21℃~23℃, and the relative humidity is moderate, which is conducive to the growth of microorganisms and suitable for fermentation experiments. The fermentation samples were placed in the experimental sample site and brought back to the laboratory after 20 days of natural fermentation. After the surface of the fermentation bottle was disinfected with 75% alcohol, it was placed in the clean bench, and the fermented tea seed cake was picked and inoculated in PDA medium (containing 500g / mL streptomycin sulfate). The inoculated PDA medium was dark cultured at 28℃, and the growth of the strains on the tea seed cake on the PDA medium was observed every day. At the same time, the fungi with obviously different colony morphology were separated, purified and preserved. A total of 16 strains of bacteria were purified and marked as CZB-1, CZB-2,..., CZB-15, and CZB-16 in turn.

[0020] Experimental Example 2 Determination of antibacterial effect of strains

[0021] 1. Initial screening of antagonistic fungi

[0022] The plate confrontation method was used for the initial screening of antagonistic fungi. The pathogenic bacteria for the test were Pseudocryphonectria elaeocarpicola, and the fungi separated from tea seed cakes were used as antagonistic bacteria for antagonistic experiments. The method was as follows: a 5mm puncher was used to punch the bacterial cakes at the edge of the colonies of the activated pathogenic bacteria and antagonistic fungi for use, and then the bacterial cakes of the pathogenic fungi and the antagonistic fungi were inoculated on both sides of the same PDA culture medium. The distance between the two bacterial cakes was greater than 20mm, and the center line of the two bacterial cakes passed through the center of the PDA culture medium. At the same time, the pathogenic fungal bacterial cakes were inoculated in the center of another separate PDA culture medium as a blank control, and each group was repeated 3 times. After inoculation, it was placed in an incubator at 28°C for dark culture for 5 days, and the growth status of each group was observed and counted, and the strains that had an inhibitory effect on the pathogenic fungi were selected for rescreening.

[0023] Through confrontation culture experiments, it was found that strain CZB-13 had a significant inhibitory effect on Elaeocarp. Figure 1 As shown in the figure, the initial screening results of the plate confrontation with Elaeocarp pathogen are as follows Figure 2 As shown, A in the figure shows the confrontation situation, and B in the figure shows the flat plate growth situation of the red shell of Eurya pseudocryptoclonus.

[0024] 2. Rescreening of antagonistic fungi

[0025] According to the initial screening results, the strain CZB-13 with antibacterial effect was selected and cultured on a PDA plate for 5 days. After that, a 5mm punch was used to punch out the bacterial cake and inoculated into a conical flask containing PDB medium. Five bacterial cakes were inoculated into each flask and cultured at 28°C and 160r / min for 15 days to obtain the antagonistic strain fermentation liquid. After filtering the above fermentation liquid through sterile gauze, centrifuge at 8000r / min for 10 minutes, collect the supernatant in the centrifuge tube, filter it with a 0.45μm filter membrane to obtain a sterile fermentation liquid, and transfer it to a 10mL sterile centrifuge tube. In the clean bench, the filtered sterile fermentation broth and the PDA medium that was heated and melted and then cooled to about 55°C were mixed in a volume ratio of 1:5 to prepare a PDA medium containing antagonistic fungal fermentation broth. At the same time, a PDA medium with sterile water was poured in the same proportion as a blank control; after the plate solidified, a 5mm punch was used to punch out the fungal cakes of the pathogenic fungi, which were inoculated on the culture medium of the control group and the experimental group, respectively. Each group was repeated 3 times and cultured in the dark at 28°C. Observe daily, and when the mycelium of the pathogenic fungi in the control group grows to the edge of the culture dish, use the cross method to count the colony sizes of the pathogenic fungi in the control group and the pathogenic fungi in the experimental group. The calculation formula for the inhibition rate of antagonistic fungal fermentation broth on pathogenic fungi is as follows:

[0026] Inhibition rate = (pathogen control colony radius - treated colony radius) / pathogen control colony diameter × 100%.

[0027] The experimental data were subjected to analysis of variance (ANOVA) using Excel and SPSS26.0 and data tables were drawn. The results were tested using the LSD method (P < 0.05).

[0028] The inhibition of the fermentation liquid of CZB-13 on Elaeocarpium edulis was as follows: Figure 3 As shown, Figure 3 A in the figure is the control group of Elaeocarp, and B in the figure is the growth of the Elaeocarp treatment group added with CZB-13 fermentation liquid. At the same time, the inhibition of the fermentation liquid of the strain on Elaeocarp is shown in Table 1. It can be seen that the fermentation liquid of CZB-13 has a good inhibitory effect on Elaeocarp, and the inhibition rate is (98.41±1.71)%.

[0029] Table 1 Inhibition rate of fermentation liquid on Elaeocarp

[0030]

[0031] It can be seen that the fermentation liquid of this strain has the effect of inhibiting the blight of Elaeocarpus edulis. The strain can be prepared into a bacterial agent for biological control of Elaeocarpus edulis. It can effectively reduce the residues of chemical drugs. It is also of great significance to protecting the ecological environment and promoting the green prevention and control of Elaeocarpus plant diseases. It has application prospects and value in the landscaping industry.

[0032] Experimental Example 3 Identification of antagonistic strain CZB-13

[0033] The isolated strain CZB-13 was inoculated on a new PDA medium, and an appropriate amount of mycelium of the strain grown on the PDA medium for 4-5 days was picked and placed in a 1.5mL centrifuge tube containing 50μL DNA extract. After being placed in a 100℃ water bath for 15min, the water droplets on the surface of the centrifuge tube were wiped dry. After the centrifuge tube was cooled to room temperature, it was placed in a centrifuge and centrifuged at 15000rpm for 3min to separate the mycelium and the extract. The supernatant in the centrifuge tube was aspirated with a pipette and transferred to another clean 1.5mL centrifuge tube to obtain the strain DNA template for PCR reaction.

[0034] The fungal universal primers ITS4 and ITS5 were used to amplify the ITS region sequence of the DNA template obtained above. The PCR reaction used a 50μL reaction system: 2×Taq PCRMasterMix 25μL, ddH2O21μL, 1μL of each of the upper and lower primers, and 2μL of the obtained DNA template. After mixing evenly, PCR amplification was performed. The amplification conditions were 95℃ pre-denaturation for 2min; 94℃ denaturation for 40s, 56℃ annealing for 40s, 72℃ extension for 1min20s, 35 cycles of amplification; 72℃ repair extension for 10min, and 30℃ storage for 5min to obtain the PCR amplification product. The amplified product was sent to Shanghai Bioengineering Co., Ltd. for bidirectional sequencing to obtain the corresponding gene sequence. The DNAMAN software was used to complete the sequence complementation. After the complete sequence was spliced, the obtained rDNA-ITS sequence was submitted to the NCBI database (http: / / www.ncbi.nlm.nih.gov), and its accession number was obtained and BLAST was performed. A homology search was performed in the GenBank database, and the sequences with higher similarity scores were downloaded. After sequence alignment on MAFFT Version 7, MEGA 7.0.26 software was used to use the neighbor-joining method in the Maximum Composite Likelihood mode. The number of repeated sampling of the Bootstrap method was set to 1000 to construct the phylogenetic tree of the strain.

[0035] The phylogenetic tree of CZB-13 constructed based on rDNA-ITS gene is shown in Figure 4 As shown, it can be known that the strain is Aspergillus flavus. The strain was preserved and deposited in the China Center for Type Culture Collection on July 8, 2024. The preservation address is Wuhan University, Wuhan, China, and the preservation number is CCTCC NO: M 20241494.

[0036] Although the content of the present invention has been described in detail through the above preferred embodiments, it should be appreciated that the above description should not be considered as a limitation of the present invention. After reading the above content, it will be apparent to those skilled in the art that various modifications and substitutions of the present invention will occur. Therefore, the protection scope of the present invention should be limited by the appended claims.

Claims

1. An application of Aspergillus flavus in the prevention and treatment of Elaeocarp disease, It is characterized in that The Aspergillus flavus described is CZB-13, which was deposited in the China Center for Type Culture Collection on July 8, 2024. The deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M 20241494.

2. The use according to claim 1, It is characterized in that The elora blight pathogenic bacteria include Elaeocarpus pseudocryptoclonus.

3. Application of a flavus fungicide in the field of Elaeocarpus plant cultivation, It is characterized in that The bacterial agent is prepared from Aspergillus flavus CZB-13 or its fermentation broth, wherein the Aspergillus flavus is CZB-13, which has been deposited in the China Center for Type Culture Collection on July 8, 2024, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being CCTCC NO: M20241494.

4. The use according to claim 3, It is characterized in that The application includes the prevention and treatment of Elaeocarp blight bacteria.

5. The use according to claim 4, It is characterized in that The blight pathogen of Elaeocarpus elaeocarpus is Elaeocarpus pseudocryptoclonus.

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