Golden yellow butterfly-shaped basidiospora zeng-LT01 and application thereof

Abstract

The invention discloses golden yellow butterfly-shaped basidiospora zeng-LT01 and application thereof, and belongs to the technical field of microorganisms. The golden yellow butterfly-shaped basidiospora zeng-LT01 is separated from citrus fruits, is named as Papiliotrema aurea zeng-LT01, and is preserved in the China Center for Type Culture Collection on December 18, 2024, and the preservation number is CCTCC NO: M 20242841. The golden yellow butterfly-shaped basidiospora zeng-LT01 can be used for preparing the golden yellow butterfly-shaped basidiospora zeng-LT01. The golden yellow butterfly-shaped basidiospora zeng-LT01 has a remarkable antagonistic effect on penicillium digitatum and has a remarkable control effect on the postharvest penicilliosis of the citrus fruits, and a new solution is provided for preventing and treating the postharvest penicilliosis of the citrus fruits.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to a golden yellow butterfly basidiosporic yeast ( Papiliotrema aurea ) zeng-LT01 and its applications. Background Art

[0002] Citrus is a plant of the genus Citrus in the Rutaceae family. It is one of the most widely grown fruits in the world. After harvest, it is very easy to rot due to environmental changes during storage and transportation, resulting in a decline in fruit quality and huge economic losses. Citrus is a non-climacteric fruit, and its rot is mainly caused by infectious fungal diseases. More than 20 types of post-harvest fungal diseases of citrus have been reported, the main diseases are citrus penicillium, acid rot, and green mold, which are caused by Penicillium italicum, Penicillium odoratum, and Penicillium odoratum. Penicillium italicum Geotrichum candidum Geotrichum citri-aurantii , Penicillium digitatum Penicillium digitatum These three pathogens infect the fruit through wounds on the peel surface. Citrus peels are easily injured and infected during storage and transportation. Therefore, post-harvest disease prevention and control is extremely important.

[0003] At present, chemical fungicides are mainly used for citrus preservation. Chemical fungicides for the prevention and control of green mold include imazalil, prochloraz, fludioxonil, pyrimethanil, thiabendazole, etc. However, they have problems such as environmental pollution, drug resistance and residues, so biological control has gradually received widespread attention. Using antagonistic yeast to control post-harvest fruit diseases is an effective biological control method. It can not only reduce the infection of harmful microorganisms on fruits and vegetables, but also has no harm to the environment and human health. It is an effective method to achieve safe production.

[0004] With the continuous isolation and screening of antagonistic yeasts, people have gradually discovered more antagonistic yeast species, which are mainly isolated from fruit surfaces, rhizosphere soil, leaves, seawater, etc. Antagonistic yeasts can effectively inhibit the growth and pathogenesis of pathogens. The biocontrol mechanisms reported in existing studies mainly include: (1) Nutrition and space competition, such as antagonistic bacteria Candida Oleophila (2) Direct parasitism, by secreting hydrolytic enzymes such as chitinase and β-1,3-glucanase, which hydrolyze the cell wall or mycelium of pathogens, such as antagonists Kloeckera apiculata , Pseudocyma antarctica ; (3) Produce antibacterial substances, such as antagonistic yeast Pichia galeiformis ; (4) Inducing host resistance, e.g. Yarrowia Lipolytica It can induce the activity of citrus defense enzymes, the production of flavonoids and total phenolic compounds, and improve the resistance of citrus to green mold disease (Liu Shuqi et al. Research progress on the main post-harvest fungal diseases of citrus and the application of antagonistic yeast. Botanical Research, 2020, 9(6): 561-572.).

[0005] Therefore, it is of great significance to develop more antagonistic yeasts that can effectively prevent and control citrus green mold diseases. In the research of the present invention, a new golden butterfly basidiosporic yeast ( Papiliotrema aurea ) strain, golden butterfly basidiospore yeast belongs to the phylum Basidiomycetes and the class Tremella. Existing studies have shown that this type of yeast is a potential source of extracellular polysaccharide EPS production (Zhao Yingjie et al., Kinetics and structural differences of polysaccharide fermentation synthesis by golden butterfly basidiospore yeast under different carbon conditions. Journal of Nuclear Agricultural Sciences, 2021, 35(2): 0384-0395.). So far, there has been no research report on the application of golden butterfly basidiospore yeast in the prevention and control of green mold diseases in citrus post-harvest fruits. Summary of the invention

[0006] The purpose of the present invention is to provide a yeast with a significant control effect on post-harvest green mold disease of citrus fruits, so as to broaden the application scope of yeast in the field of biological control of fruits and vegetables and provide a new solution for the prevention and control of post-harvest green mold disease of citrus fruits.

[0007] To achieve the above object, the present invention adopts the following technical solution: The present invention isolates a new golden yellow butterfly basidiosporic yeast ( Papiliotrema aurea ) strain, whose biological characteristics are: the colonies in YEPD medium are cheese-like, milky white, smooth, slightly shiny, and have neat edges. The ITS sequence of the strain is shown in SEQ ID NO.1. By constructing a phylogenetic tree, it was identified that the strain belongs to Papiliotrema aurea Therefore, the strain was named as golden yellow butterfly basidiospora yeast ( Papiliotrema aurea ) zeng-LT01, 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: M 20242841.

[0008] Furthermore, the culture conditions of the golden butterfly basidiosporic yeast zeng-LT01 are: cultured on YEPD medium at 25-28°C, and the colony morphology is cheese-like, milky white, with a smooth surface, slightly shiny, and neat edges.

[0009] The present invention combines the golden yellow butterfly basidiosporic yeast zeng-LT01 with the digitate Penicillium ( Penicillium digitatum ) were used for plate confrontation experiment, and the results showed that the golden butterfly basidiosporic yeast zeng-LT01 had a significant antagonistic effect on digitate Penicillium.

[0010] Therefore, the present invention provides the use of the golden yellow butterfly basidiosporium yeast zeng-LT01 in preventing and controlling green mold diseases of plants.

[0011] Further, the green mold disease is caused by Penicillium digitatum ( Penicillium digitatum ) caused by infection.

[0012] Furthermore, the application is to prevent and treat green mold disease in citrus fruits. Studies have shown that the golden yellow butterfly basidiosporium yeast Zeng-LT01 can significantly control the post-harvest rot of citrus fruits caused by infection of digitate Penicillium.

[0013] Furthermore, the application includes: applying the golden yellow butterfly basidiosporic yeast zeng-LT01 to the epidermis of citrus fruits.

[0014] Furthermore, the cells of the golden yellow butterfly basidiospora yeast zeng-LT01 were collected after subculture and resuspended in sterile water to a concentration of 1×10 8 cells / mL of cell suspension, and then inoculated on the citrus fruit epidermis and allowed to stand until the cell suspension was completely absorbed.

[0015] Furthermore, the conditions for the subculture are as follows: inoculating the golden yellow butterfly basidiosporium yeast zeng-LT01 into the YEPD medium, culturing at 28°C and 200 rpm for 18-24 h, and subculturing twice continuously.

[0016] The present invention also provides a microbial agent for preventing and treating citrus green mold disease, wherein the active ingredient comprises the golden yellow butterfly basidiosporium yeast ( Papiliotrema aurea )zeng-LT01.

[0017] The present invention has the following beneficial effects: The present invention provides a new golden yellow butterfly basidiosporic yeast ( Papiliotrema aurea )zeng-LT01, 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

[0018] Figure 1 This is the colony morphology of the golden yellow butterfly basidiosporic yeast zeng-LT01 growing on YEPD medium.

[0019] Figure 2 The phylogenetic tree constructed by neighbor-joining analysis of ITS sequences depicts the relationship between zeng-LT01 and its related taxa.

[0020] Figure 3 Depiction of a phylogenetic tree constructed by neighbor-joining analysis of ITS sequences P. digitatumRelationship to its related taxa.

[0021] Figure 4 For zeng-LT01 and P. digitatum Flat panel confrontation.

[0022] Figure 5 For zeng-LT01 and P. digitatum Statistical graph of pathogen colony diameter in plate confrontation experiment.

[0023] Figure 6 This is a diagram showing the control effect of zeng-LT01 on post-harvest green mold disease in citrus fruits.

[0024] Figure 7 This is a statistical chart showing the incidence of postharvest green mold disease on citrus fruits controlled by zeng-LT01.

[0025] Figure 8 This is a statistical chart of the lesion diameter of zeng-LT01 in controlling postharvest green mold disease on citrus fruits.

[0026] 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

[0027] 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.

[0028] 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.

[0029] Example 1: Screening and identification of yeast zeng-LT01 1. Isolation and screening of yeast zeng-LT01 Citrus fruits 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-LT01.

[0030] YEPD medium: yeast extract powder (10 g / L), peptone (20 g / L), glucose (20 g / L), and agar (20 g / L).

[0031] 2. Identification of yeast zeng-LT01 2.1 Colony morphology like Figure 1 As shown in the figure, the colonies of yeast zeng-LT01 after being cultured on YEPD medium at 28 ℃ for 2-3 days were cheese-like, milky white, with a smooth surface, slightly shiny, and neat edges.

[0032] 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.

[0033] 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.

[0034] Primer ITS1: 5′-TCCGTAGGTGAACCTGCGG-3′; Primer ITS4: 5′-TCCTCCGCTTATTGATATGC-3′.

[0035] 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.

[0036] 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.

[0037] 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-LT01.

[0038] Sequence analysis: The sequencing results of the tested yeast strains were aligned with the ITS region sequences of known yeast species in the GenBank database using ClustalX software (Alignment). A phylogenetic tree was constructed using the neighbour-joining method, and the evolutionary distance was calculated according to the Kimura-2-Parameter Distance model in MEGA 11.0 software. The reliability of the phylogenetic tree topology was evaluated using the Bootstrap method (1000 replicates).

[0039] The results are as Figure 2 shown. The phylogenetic tree shows that strain zeng-LT01 clusters together with the known yeast Papiliotrema aurea (GenBank accession number: MG251417.1:1-473), with a very close genetic relationship and 100% homology. There is a 1.91% base difference in the 26S ITS region sequences between the two (9 out of 469 common nucleotides). It was identified that strain zeng-LT01 belongs to Papiliotrema aurea .

[0040] 3. Analysis of taxonomic status Combining the morphological characteristics and phylogenetic analysis of strain zeng-LT01, it was identified that strain zeng-LT01 belongs to the kingdom Fungi ( Fungi ), phylum Basidiomycota ( Basidiomycota ), class Tremellomycetes ( Tremellomycetes ), order Tremellales ( Tremellales ), family ( Rhynchogastremaceae ), genus ( Papiliotrema ), and species Cryptococcus aureus ( Papiliotrema aurea ).

[0041] Therefore, strain zeng-LT01 was named Papiliotrema aurea zeng-LT01. It was deposited in the China Center for Type Culture Collection on December 18, 2024. The deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M 20242841. It was detected as viable on December 25, 2024.

[0042] Example 2: Plate confrontation between strain zeng-LT01 and P. digitatum Using a sterile inoculation loop, streak the center of a PDA plate with a 1×10 8 cells / mL zeng-LT01 bacterial suspension. Use sterile water as the control group, and repeat each treatment three times. After culturing at 25 °C for 48 h, use a pipette to aspirate 10 μL of a 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.

[0043] The method used in this embodiment P. digitatum Isolate from citrus fruit and identify the strain. Phylogenetic tree constructed by neighbor-joining analysis of ITS sequences is shown in Figure 3 shown.

[0044] The results are as follows Figure 4 and Figure 5 As shown, the zeng-LT01 group P. digitatum The colony diameter of P <0.05), indicating that zeng-LT01 has P. digitatum Mycelial growth inhibition effect.

[0045] Example 3: Control effect of strain zeng-LT01 on postharvest green mold disease in citrus fruits Zeng-LT01 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.

[0046] YEPD medium: yeast extract powder (10 g / L), peptone (20 g / L), glucose (20 g / L).

[0047] Citrus fruits (summer oranges) 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. digitatum After 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.

[0048] The calculation formula is as follows: Disease incidence (%) = (number of diseased holes in the fruit / total number of holes in the fruit) × 100; Lesion diamete = (lateral diameter of the lesion hole + longitudinal diameter of the lesion hole) / 2.

[0049] The results are as follows Figure 6-Figure 8 Zeng-LT01 could 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 in the control group. On the 5th day of storage, the incidence rate and lesion diameter of zeng-LT01 were 30% and 18.5 mm, which were 38% and 38.1 mm lower than those in the control group, respectively. This indicates that zeng-LT01 can significantly control postharvest green mold disease in citrus fruit.

Claims

1. A golden yellow butterfly basidiosporic yeast zeng-LT01, characterized in that The strain was isolated from citrus fruit and named Papiliotrema aurea zeng-LT01 is deposited in China Center for Type Culture Collection with the deposit number of CCTCCNO: M 20242841.

2. The golden yellow butterfly basidiosporic yeast zeng-LT01 according to claim 1, characterized in that The culture conditions of the golden yellow butterfly basidiosporic yeast zeng-LT01 are as follows: culture on a YEPD medium at 25-28° C., and the colony morphology is cheese-like, milky white, with a smooth surface, slightly shiny, and neat edges.

3. Use of the golden yellow butterfly basidiosporic yeast zeng-LT01 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 ( Penicillium digitalatum ) infection, the golden yellow butterfly basidiosporic yeast zeng-LT01 has an antagonistic effect on digitate Penicillium.

5. The use according to claim 3, 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 the golden yellow butterfly basidiosporic yeast zeng-LT01 to the epidermis of citrus fruits.

7. The use according to claim 6, characterized in that The cells of the golden yellow butterfly basidiospora yeast zeng-LT01 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.

8. The use according to claim 7, characterized in that The conditions for the subculture are as follows: inoculating the golden yellow butterfly basidiosporium yeast zeng-LT01 into the YEPD medium, culturing for 18-24 h at 28° C. and 200 rpm, and subculturing for 2 consecutive times.

9. A microbial agent for preventing and treating citrus green mold disease, characterized in that: The active ingredient comprises the golden yellow butterfly basidiospora yeast zeng-LT01 as claimed in claim 1 or 2.

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