Cystobasidium silofanense zeng-LT02 and application thereof
Through the isolated and identified Slofibaspermia zeng-LT02 from citrus leaves, the problem of the poor prevention and control of existing antagonistic yeast species on post-harvest green mold disease in citrus was solved, and the significant disease control effect on citrus fruits was achieved.
Patent Information
- Application Number
- CN202510276462.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-10
- Publication Date
- 2025-05-09
AI Technical Summary
The existing antagonistic yeast species have poor effect on the prevention and control of post-harvest green mold in citrus, and the effect is unstable, which limits its widespread use in commercial applications.
A new strain of Cystobasidium slooffiae was isolated from citrus leaves, named zeng-LT02, and was identified by constructing a phylogenetic tree. The strain grew at acidic pH, tolerated high salt environments, and had a significant antagonistic effect on Penicillium fingers.
Slofibaspermia zeng-LT02 significantly controls post-harvest green mold disease caused by Penicillium fingers, reduces the incidence of diseases and lesions diameter, and provides an effective biological control method.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of microbial technology, and in particular to a Sloephyra Cystobasidium slooffiae ) zeng-LT02 and its applications. Background Art
[0002] Green mold is the most common infectious disease during citrus storage. The pathogen is Penicillium digitatum. Penicillium digitatum This pathogen can grow saprophytically on various organic substances and produce a large number of conidia that spread into the air and spread through air currents. The pathogen of citrus green mold must penetrate through wounds on the peel to cause damage, causing the fruit to rot. In the early stage, small water-soaked light brown round spots appear on the fruit surface, the diseased tissue is soft and rotten, and it quickly expands and shrinks. White mold gradually grows in the center, and then the mold continues to thicken and spread to the surrounding area, and the color gradually turns to gray-green or dark gray. The peel of citrus fruit is very easy to be injured and infected during storage and transportation. Therefore, it is extremely important to prevent and control post-harvest diseases.
[0003] In order to effectively prevent and control citrus green mold disease, chemical fungicides are often used in production to control the occurrence of the disease. However, the large-scale use of chemical fungicides will endanger human health, pollute the environment and cause pathogens to develop drug resistance, and biological control has gradually received widespread attention. Antagonistic yeast (also known as biocontrol yeast) is a promising alternative to chemical fungicides for controlling postharvest rot due to its wide distribution, antagonistic ability, environmental friendliness and safety to humans. In the past few decades, the biological control mechanisms of antagonistic yeasts have been widely studied, such as nutrient and space competition, fungal parasitism and induction of host resistance (Xiaokang Z. et al . Antagonistic Yeasts: A Promising Alternative to ChemicalFungicides for Controlling Postharvest Decay of Fruit. Journal of Fungi , 2020,6(3):158.).
[0004] Currently, there are few known antagonistic yeast species that can prevent and control citrus post-harvest diseases, and the effects are not good. The unstable effect of disease prevention and control has become the main obstacle to the commercial application of antagonistic yeast. Generally, the biological control mechanisms of antagonistic yeasts of different genera are quite different. Therefore, finding new and excellent antagonistic yeast species is of great significance to promote the development of efficient biocontrol agents. In the research of the present invention, a new strain of Sloephidae ( Cystobasidium slooffiae), existing research reports show that S. slovii can utilize most sugars, organic acids and amino acids, but cannot grow with formic acid, acetic acid and arginine. In addition, the strain can also grow at acidic pH and tolerate up to 8% (W / V) NaCl. Sucrose is the best carbon source for this strain (Moradian, JM, et al. Efficient biohydrogen and bioelectricity production from xylose by microbial fuel cell with newly isolated yeast of Cystobasidium slooffiae. INTERNATIONAL JOURNAL OF ENERGYRESEARCH , 2020. 44(1): 325-333.), mainly used in microbial fuel cells to improve the utilization of xylose in MFCs and achieve simultaneous power generation and hydrogen production (Moradian, JM, et al . Enhancement of bioelectricity and hydrogen production from xylose by a nanofiber polyanilinemodified anode with yeast microbial fuel cell. Fuel , 2022. 326: 125056.). There is no research on the control of postharvest diseases of fruits and vegetables using Aspergillus slovakia. Summary of the invention
[0005] The purpose of the present invention is to provide a yeast having antagonistic effect on pathogenic bacteria of citrus green mold disease, develop the yeast into a biological agent, and apply it to the biological control of plant green mold disease.
[0006] To achieve the above object, the present invention adopts the following technical solution: The present invention isolates a new strain of Aspergillus sloeffyi from citrus leaves. Cystobasidium slooffiae ) strain, whose biological characteristics are: the colonies in YEPD medium are cheese-like, orange-pink, smooth, slightly shiny, and have neat edges. The ITS region sequence of the strain is shown in SEQ ID NO.1. By constructing a phylogenetic tree, it was identified that the strain belongs to Aspergillus slophii ( Cystobasidium slooffiae ), therefore, the strain was named Cystobasidium slooffiae zeng-LT02 was deposited in China Center for Type Culture Collection on December 18, 2024, with the deposit address: Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M20242842.
[0007] Furthermore, the culture conditions of Capsella slovii zeng-LT02 are: cultured on YEPD medium at 25-28°C.
[0008] The present invention combines Sloephidae Capsulatum zeng-LT02 with Penicillium digitatum ( Penicillium digitatum ) were used for plate confrontation experiment, and the results showed that S. slovii zeng-LT02 had a significant antagonistic effect on Penicillium digitatum.
[0009] Therefore, the present invention provides the described Capsulatus slophii ( Cystobasidium slooffiae ) Application of zeng-LT02 in preventing and controlling green mold diseases of plants.
[0010] Further, the green mold disease is caused by Penicillium digitatum ( Cystobasidium slooffiae ) caused by infection.
[0011] Furthermore, the application is to prevent and treat green mold disease in citrus fruits. Studies have shown that Aspergillus sloeffyi zeng-LT02 can significantly control post-harvest rot of citrus fruits caused by infection of Penicillium digitatum.
[0012] Furthermore, the application includes: applying the Aspergillus slovii zeng-LT02 to the epidermis of citrus fruit.
[0013] Furthermore, the cells of Aspergillus slophii zeng-LT02 were collected after subculture and resuspended in sterile water to a concentration of 1×10 8 cells / mL of cell suspension was then inoculated on the citrus fruit epidermis and allowed to stand until the cell suspension was completely absorbed.
[0014] Furthermore, the conditions for the subculture are as follows: inoculating the Aspergillus slovii zeng-LT02 into the YEPD medium, incubating at 28°C and 200 rpm for 18-24 h, and subculturing twice.
[0015] The present invention also provides a microbial agent for preventing and treating citrus green mold disease, wherein the active ingredient comprises the aforementioned Aspergillus sloeffyi ( Cystobasidium slooffiae )zeng-LT02.
[0016] The present invention has the following beneficial effects: The present invention provides a new strain of Capsella slophii ( Cystobasidium slooffiae )zeng-LT02, which has a significant antagonistic effect on Penicillium digitatum and has a significant control effect on post-harvest green mold disease of citrus fruits. The present invention provides a new solution for the prevention and treatment of post-harvest green mold disease of citrus fruits. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Figure 1 The colony morphology of zeng-LT02 grown on YEPD medium.
[0018] Figure 2 The phylogenetic tree constructed by neighbor-joining analysis of ITS sequences depicts the relationship between zeng-LT02 and its related taxa.
[0019] Figure 3 Depiction of a phylogenetic tree constructed by neighbor-joining analysis of ITS sequences P. digitatum Relationship to its related taxa.
[0020] Figure 4 For zeng-LT02 and P. digitatum Flat panel confrontation.
[0021] Figure 5 For zeng-LT02 and P. digitatum Statistical graph of pathogen colony diameter in plate confrontation experiment.
[0022] Figure 6 This is a diagram showing the control effect of zeng-LT02 on post-harvest green mold disease in citrus fruits.
[0023] Figure 7 This is a statistical chart showing the incidence of postharvest green mold disease in citrus fruits controlled by zeng-LT02.
[0024] Figure 8 This is a statistical chart of the lesion diameter of zeng-LT02 in controlling postharvest green mold disease on citrus fruits.
[0025] Note: The columns and vertical lines in the figure represent the mean and standard deviation of biological replicates, respectively. Different letters at the same time point indicate significant differences ( P <0.05), Control: control group (sterile water). DETAILED DESCRIPTION
[0026] The present invention will be further described below in conjunction with specific embodiments. The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of application of the present invention. Without departing from the spirit and essence of the present invention, modifications or replacements made to the inventive method, steps or conditions all belong to the scope of the present invention.
[0027] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.
[0028] Example 1: Screening and identification of yeast zeng-LT02 1. Isolation and screening of yeast zeng-LT02 Citrus leaves were collected from citrus orchards in Meishan City, Sichuan Province as samples. The samples were completely immersed in sterile distilled water and shaken at 150 r / min for 10-30 min to prepare a sample suspension. The suspension was diluted 10 times in a gradient manner, and then 100 μL was pipetted and spread on YEPD medium. After culturing at 28°C for 2-3 days, a single colony with a yeast-like appearance was selected for purification and culture, and named zeng-LT02.
[0029] YEPD medium: yeast extract powder (10 g / L), peptone (20 g / L), glucose (20 g / L), agar (20 g / L).
[0030] 2. Identification of yeast zeng-LT02 2.1 Colony morphology like Figure 1 As shown in the figure, the colonies of zeng-LT02 cultured on YEPD medium at 28°C for 2-3 days were cheese-like, orange-pink in color, with a smooth, slightly shiny surface and neat edges.
[0031] 2.2 Molecular identification DNA extraction: The purified strain was cultured in liquid culture, and the culture solution was taken to extract bacterial DNA using a genomic DNA extraction kit.
[0032] PCR reaction system: 12.5 µL PCR supermix, 8.5 µL ultrapure water, 1 µL each of primers ITS1 and ITS4, and 2 µL bacterial solution.
[0033] Primer ITS1: 5′-TCCGTAGGTGAACCTGCGG-3′; Primer ITS4: 5′-TCCTCCGCTTATTGATATGC-3′.
[0034] PCR reaction conditions: 95 °C, 5 min; 95 °C, 1 min; 54 °C, 2 min; 72 °C, 1 min 30 s, 36 cycles; 72 °C, 10 min.
[0035] Gel electrophoresis detection of PCR products: Take 2.5 µL of PCR amplification solution and spot it on 1% agarose gel (containing nucleic acid dye) for electrophoresis for 20-30 min. After the end, preliminarily determine whether the target fragment is obtained under the UV gel imaging system.
[0036] Sequencing: After confirming that the PCR experiment was successful, the PCR product was immediately sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. After strict verification of the sequencing results, a DNA fragment with a nucleotide sequence as described in SEQ ID NO.1 was obtained, i.e., the ITS region sequence of strain zeng-LT02.
[0037] Sequence analysis: ClustalX software was used to align the sequencing results of the test yeast strains with the ITS region sequences of known yeast species in the GenBank database. The phylogenetic tree was constructed using the neighbor-joining method, and the evolutionary distance was calculated based on the Kimura-2-Parameter Distance model in MEGA 11.0 software. The Bootstrap method (1000 repetitions) was used to test and evaluate the reliability of the topological structure of the phylogenetic tree.
[0038] The results are as follows Figure 2 As shown, the phylogenetic tree shows that strain zeng-LT02 is related to known yeast Cystobasidium slooffiae (GenBank accession number: MN788428.1:3-515) are clustered together and have a very close relationship with a homology of 100%. There is a 2.67% base difference in the ITS region sequence between the two (9 out of 526 common nucleotides). Identification of strain zeng-LT02 belongs to Cystobasidium slooffiae .
[0039] 3. Taxonomic status analysis Combined with the morphological characteristics and phylogenetic analysis of strain zeng-LT02, strain zeng-LT02 was identified as belonging to the kingdom Fungi ( Fungi )、Basidiomycota( Basidiomycota )、Capsulatum( Cystobasidiomycetes ), Capsididiomycetes ( Cystobasidiales )、Mycosaceae( Cystobasidiaceae ), Cystobasidiomyces ( Cystobasidium ), Slophidium species ( Cystobasidium slooffiae ).
[0040] Therefore, strain zeng-LT02 was named Cystobasidium slooffiae zeng-LT02 was deposited in China Center for Type Culture Collection on December 18, 2024, with the deposit address: Wuhan University, Wuhan, China, with the deposit number CCTCCNO: M 20242842, and was detected as alive on December 25, 2024.
[0041] Example 2: strain zeng-LT02 and P. digitatum Tablet confrontation Use a sterile inoculating loop to pick up 1×10 8 The zeng-LT02 bacterial suspension of 1 × 10 cells / mL was streaked in the center of the PDA plate, and sterile water was used as the control group. Each treatment was repeated three times. After culturing at 25°C for 48 h, 10 μL of 1 × 10 5 cells / mL P. digitatum The spore suspension was inoculated at a distance of 2.5 cm from the streaked cells. After 6 days of continuous culture at 25°C, the radial growth of pathogenic hyphae on both sides of the antagonist was recorded using the cross method.
[0042] The method used in this embodiment P. digitatum The phylogenetic tree constructed by neighbor-joining analysis of ITS sequences was as follows: Figure 3 shown.
[0043] The results are as follows Figure 4 and Figure 5 As shown, the zeng-LT02 group P. digitatum The colony diameter of P <0.05), indicating that zeng-LT02 has P. digitatum Mycelial growth inhibition effect.
[0044] Example 3: Control effect of strain zeng-LT02 on postharvest green mold disease in citrus fruits Zeng-LT02 was subcultured twice in YEPD medium, with a growth time of 18-24 h (28°C, 200 rpm) for each generation. The cultured bacterial liquid was centrifuged (5000×g, 5 min) to collect the bacteria, discard the supernatant, wash with an appropriate amount of sterile water, resuspend the yeast cells, and then adjust the concentration of the yeast cell suspension to 1×10 8 cells / mL for future use.
[0045] YEPD medium: yeast extract powder (10 g / L), peptone (20 g / L), glucose (20 g / L).
[0046] Citrus (summer orange) fruits were first soaked in a 2% sodium hypochlorite solution for 2 min, rinsed with clean water, and dried naturally at room temperature. The fruits were randomly grouped and two holes (3 mm in diameter and 3 mm in depth) were punched symmetrically at the equator of each fruit using a sterile hole puncher. 20 μL of (1) sterile water and (2) yeast cell suspension were added to each wound. 10 μL of the following was inoculated into each wound 4 h later. P. digitatumAfter the fruit has fully absorbed the spore suspension, the fruit is packaged with PE film and stored at 25°C and 85% to 90% relative humidity. The fruit lesion diameter and incidence rate are measured every 24 hours. Each treatment contains 3 parallels, and each parallel contains 10 fruits.
[0047] The calculation formula is as follows: Disease incidence (%) = (number of diseased holes in the fruit / total number of holes in the fruit) × 100; Lesion diameter = (lateral diameter of the lesion hole + longitudinal diameter of the lesion hole) / 2.
[0048] The results are as follows Figures 6 - 8 As shown in the data, zeng-LT02 can control the development of green mold disease in citrus fruit. During the entire storage period, the incidence rate and lesion diameter of the treated group were significantly lower than those of the control group. On the 5th day of storage, the incidence rate and lesion diameter of zeng-LT02 were 48% and 35.25 mm, which were 20% and 24.8 mm lower than those of the control group, respectively. This indicates that zeng-LT02 can significantly control postharvest green mold disease in citrus fruit.
Claims
1. A Slophidium zeng-LT02, characterized in that The Slophidium zeng-LT02 was isolated from citrus leaves and named Cystobasidium slooffiae zeng-LT02 is deposited in China Center for Type Culture Collection with the deposit number of CCTCC NO: M 20242842.
2. The Aspergillus slovii zeng-LT02 according to claim 1, characterized in that The culture conditions of the Aspergillus sluifensis zeng-LT02 are: cultured on a YEPD medium at 25-28°C.
3. Use of the Aspergillus slovii zeng-LT02 as claimed in claim 1 or 2 in preventing and controlling green mold diseases of plants.
4. The use according to claim 3, characterized in that The green mold disease is caused by Penicillium digitatum ( Cystobasidium slooffiae ) infection, and the Sloughiella zeng-LT02 has an antagonistic effect on Penicillium digitatum.
5. The use according to claim 2, characterized in that The application is to prevent and control green mold disease of citrus fruits.
6. The use according to claim 5, characterized in that The application comprises: applying Aspergillus sluifensis zeng-LT02 to the epidermis of citrus fruits.
7. The use according to claim 6, characterized in that After subculturing, the cells of Aspergillus slovii zeng-LT02 were collected and resuspended in sterile water to a concentration of 1×10 8 cells / mL of cell suspension was then inoculated on the citrus fruit epidermis and allowed to stand until the cell suspension was completely absorbed.
8. The use according to claim 7, characterized in that The conditions for the subculture are as follows: inoculating the Aspergillus sluifensis zeng-LT02 into the YEPD medium, culturing at 28°C and 200 rpm for 18-24 h, and subculturing twice continuously.
9. A microbial agent for preventing and treating citrus green mold disease, characterized in that: The active ingredient includes the Capsella slovakia zeng-LT02 as described in claim 1 or 2.