A kind of Mortierella oligosporida and its application

Abstract

The present invention discloses a Mortierella oligospora and its application, wherein the strain number of the Mortierella oligospora is JSAFC 3583, which was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration Committee on May 20, 2024, and the deposit number is CGMCC No.41310. The biocontrol bacteria Mortierella oligospora JSAFC 3583 provided by the present invention can effectively inhibit the growth of Botrytis cinerea, and the inhibition rate of plate confrontation culture is 58.19%. At the same time, the extracellular metabolites secreted by it can also significantly reduce the growth of Botrytis cinerea. When the concentration of JSNL 3583 extracellular metabolites is 10mL / 100mL, the inhibition rate of B. cinerea reaches 39.78%, and when the concentration of JSNL 3583 extracellular metabolites is 100mL / 100mL, the inhibition rate of B. cinerea reaches 51.33%. At the same time, there is a growth-promoting effect on blueberry plants.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and relates to Mortierella oligosporida and applications thereof. Background Art

[0002] Blueberry gray mold is a common and difficult-to-control fungal disease of open-field and protected crops. It is mainly caused by infection with Botrytis cinerea, which can infect flowers, fruits, leaves, and stems, seriously affecting the healthy growth of plants and causing huge economic losses. Blueberry gray mold caused by Botrytis cinerea has become one of the main diseases of blueberries in recent years, causing great harm to the healthy growth and industrial development of blueberries. The symptoms of blueberry gray mold in the field are mainly manifested as lesions on leaves and flower organs at the beginning of infection. As the infection time increases, the lesions spread in a yellow-brown water-soaked state, with a fast speed and a wide range, until the flower organs and stems turn black and rot, the leaves die, and the underground part of the infected plants stops growing. Blueberries in facilities are susceptible to gray mold due to high humidity, poor ventilation, and poor variety resistance, resulting in reduced yields. In severe cases, it can cause a significant reduction in yield, making gray mold one of the diseases that affects facility blueberry potted plants in general and with a serious degree of harm.

[0003] For gray mold, we should thoroughly clean the orchard in spring and strengthen orchard management to reduce the initial infection source. At the same time, we should remove the diseased flowers, fruits and leaves in time before gray mold grows on the diseased plants, put them in plastic bags, take them out of the greenhouse orchard and bury or burn them. However, there are some disadvantages in the conventional prevention and control methods: the disease is treated only when the symptoms are found. At present, most blueberry growers do not have the habit of cleaning the orchard, lack disease prevention awareness, and do not have the concept that prevention is better than treatment; and habitual use of drugs leads to strong drug resistance of pathogens; often using drugs that have been used in the past and have had good effects can easily lead to the continuous use of a limited number of pesticides, making the pathogens develop strong drug resistance, which not only makes the effect worse and worse, but also increases costs and reduces economic benefits.

[0004] At present, agricultural control cannot completely eliminate the pathogen and is prone to recurrence; chemical control is prone to produce pesticide residues and may also cause disease resistance; biological control can effectively kill pathogens without damaging the environment, and is the future direction and trend of disease control. The control methods for blueberry gray mold caused by Botrytis cinerea mostly use agricultural control and chemical control, and biological control methods are relatively rare. As an effective means of green control, microorganisms are increasingly important in my country's agriculture.

[0005] Therefore, finding an environmentally friendly and effective prevention and control measure for gray mold is an urgent problem to be solved today. The antibacterial substances produced by biocontrol bacteria usually act directly on the corresponding pathogens and have strong specificity, so they will not have an adverse effect on other beneficial microorganisms in the agricultural ecosystem, which is conducive to maintaining ecological balance.

[0006] In summary, the prior art still lacks a green and highly effective probiotic for preventing blueberry gray mold. Summary of the invention

[0007] Purpose of the invention: The purpose of the present invention is to provide a Mortierella oligospora and its application in inhibiting the growth of Botrytis cinerea and promoting the growth of blueberries.

[0008] Technical solution: The present invention provides a Mortierella oligospora, the strain number of the Mortierella oligospora is JSAFC 3583, which was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on May 20, 2024, with a deposit number of CGMCC No.41310.

[0009] Furthermore, the nucleotide sequence of the ITS gene of Mortierella oligospora is shown in SEQ ID NO.1.

[0010] The present invention also provides a biocontrol fungus agent, wherein the active ingredient of the biocontrol fungus agent is the above-mentioned Mortierella oligospora.

[0011] The present invention also provides the use of the above-mentioned Mortierella oligospora or its extracellular metabolites and biocontrol fungi in inhibiting the growth of gray mold fungi (Botrytis cinerea).

[0012] The present invention also provides the use of the above-mentioned Mortierella oligospora and biocontrol fungi in promoting the growth of blueberries.

[0013] Furthermore, the method comprises planting blueberries in peat soil containing mycelium of Mortierella oligospora JSAFC 3583, thereby promoting plant height and root growth of blueberries.

[0014] The present invention also provides the use of the above-mentioned Mortierella oligospora and the biocontrol fungus agent in preventing and controlling blueberry gray mold.

[0015] Furthermore, the blueberry gray mold is caused by Botrytis cinerea.

[0016] Beneficial effects: Compared with the prior art, the present invention has the following outstanding advantages: the biocontrol bacteria Mortierella oligospora JSAFC 3583 provided by the present invention can effectively inhibit the growth of Botrytis cinerea, and the inhibition rate of plate confrontation culture is 58.19%. At the same time, the extracellular metabolites secreted by the present invention can also significantly reduce the growth of blueberry gray mold. When the concentration of JSNL 3583 extracellular metabolites is 10mL / 100mL, the inhibition rate of B. cinerea reaches 39.78%, and when the concentration of JSNL 3583 extracellular metabolites is 100mL / 100mL, the inhibition rate of B. cinerea reaches 51.33%. At the same time, it has a growth-promoting effect on blueberry plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 : Phylogenetic tree of Mortierella oligospora JSAFC 3583.

[0018] Figure 2 : The biocontrol bacteria Mortierella oligospora JSAFC 3583 of the present invention and Botrytis cinerea are cultured in confrontation.

[0019] Figure 3 : The effects of the extracellular metabolites secreted by the biocontrol bacterium Mortierella oligospora JSAFC 3583 of the present invention on Botrytis cinerea.

[0020] Figure 4 : The effect of the biocontrol bacteria Mortierella oligospora JSAFC 3583 of the present invention on the plant height of blueberry plants.

[0021] Figure 5 : The effect of the biocontrol bacteria Mortierella oligospora JSAFC 3583 of the present invention on the root system of blueberry. DETAILED DESCRIPTION

[0022] The following detailed descriptions are all illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0023] As used herein, the term "biocontrol bacteria" refers to beneficial microorganisms that can control plant diseases.

[0024] As described in the present invention, the term "blueberry gray mold (Botrytis cinerea)" is classified as: Ascomycota, Leotiomycetes, Helotiales, Sclerotiniaceae, Botrytis.

[0025] Example 1: Isolation and identification of Mortierella oligospora JSAFC 3583

[0026] 1. Isolation of Mortierella oligospora JSAFC 3583

[0027] Healthy blueberry plants with good growth were selected from the fields in Zhejiang, China, and the root tissues were taken to isolate JSAFC 3583. The colony characteristics were as follows: when cultured on PDA flat culture medium, the colony grew rapidly and initially took on a felt-like shape with flocculent hairs attached to the surface of the culture medium.

[0028] 2. Molecular biological identification of Mortierella oligospora JSAFC 3583

[0029] (1) The genomic DNA of strain JSAFC 3583 was extracted using a kit from TIANGEN.

[0030] (2) Amplification of ITS gene from genomic DNA. DNA amplification was performed in a 30 μL reaction volume, including 15 μL 2×EasyTaq PCR SuperMix (+dye), 1 μL (10 μM) of primer pair ITS1 (SEQ ID NO.2: 5'-CTTGGTCATTTAGAGGAAGTAA-3') & ITS4 (SEQ ID NO.3: 5'-TCCTCCGCTTATTGATATGC-3'), 2 μL template DNA and 11 μL ddH2O. The PCR amplification program conditions were as follows: 94°C pre-denaturation for 5 min; 94°C denaturation for 30 s, 54°C annealing for 45 s, 72°C extension for 1 min, for a total of 35 cycles; and finally 72°C extension for 10 min.

[0031] (3) The amplified PCR products were subjected to 1% agarose gel electrophoresis and observed under ultraviolet light. The PCR products with target bands were sent to Nanjing Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results showed that the ITS gene sequence of strain JSAFC 3583 was SEQ ID NO. 1 (TCATCTTGGGGGGGAAAAAAAAAAAAAGGACAGGTTTCCGTAGGTGAACCTGC).

[0032] (4) Compare SEQ ID NO.1 on the NCBI database website. The comparison results are shown in Table 1. A phylogenetic tree is established based on SEQ ID NO.1. Figure 1 .according to Figure 1From the results in Table 1, it can be inferred that the biocontrol bacteria JSAFC3583 of the present invention is Mortierella oligospora, named Mortierella oligospora JSAFC 3583, and the strain was deposited in the General Microbiological Center of China Microorganism Culture Collection Administration on May 20, 2024, with a deposit number of CGMCC NO.41310, and classified as Mortierella oligospora JSAFC 3583, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0033] Table 1 Sequence alignment results of SEQ ID NO.1

[0034]

[0035] Example 2: Inhibitory effect of Mortierella oligospora JSAFC 3583 on Botrytis cinerea

[0036] (1) On a clean bench, a 4 mm diameter bacterial cake of B. cinerea JSAFC 2091 was inoculated on one side of a PDA culture medium plate as a control group, and a 4 mm diameter bacterial cake of Mortierella oligospora JSAFC 3583 was inoculated on the symmetrical side of the PDA culture medium as an experimental group; and a culture medium plate inoculated with only B. cinerea was used as a control group.

[0037] (2) The experimental group and control group cultures of step (1) were inverted and cultured in a constant temperature incubator at 25°C under the conditions of 8 hours of light and then 16 hours of dark culture.

[0038] (3) After the experimental group and control group culture media of step (2) were artificially cultured under the above culture conditions for 7 days, the colony radius of B. cinerea was measured by the plate confrontation method, and the inhibitory effect of Mortierella oligospora JSAFC 3583 on B. cinerea was observed.

[0039] The results are as follows Figure 2 As shown, the B. cinerea colony radius of the blank control group was 4.52 cm; in the experimental group, the average radius of the B. cinerea pathogen was only 1.89 cm as determined by the plate confrontation method, and the inhibition rate was 58.19%, indicating that Mortierella oligospora JSAFC 3583 had a significant inhibitory effect on B. cinerea.

[0040] Example 3: Effects of Mortierella oligospora JSAFC 3583 extracellular metabolites on B. cinerea

[0041] (1) Use a cork puncher to take 5 mm diameter mycelium blocks of B. cinerea JSAFC 2091 and Mortierella oligospora JSAFC 3583 and inoculate them into 9 cm diameter culture dishes containing 15 mL of PDA medium, and place them in a 25°C incubator in the dark for 5 days. When the B. cinerea strains have grown all over the culture dish, use a cork puncher (5 mm diameter) to punch holes at the edge of the colony to prepare bacterial plates.

[0042] (2) Cut a 3 mm × 3 mm mycelium block at the edge of a 5-day-old JSAFC 3583 colony, transfer 30 mycelium blocks into 200 ml PDB liquid culture medium under sterile conditions, and culture at 25°C, dark, and 160 rpm for 7 days. First, filter out the mycelium blocks in the culture medium with two layers of sterile gauze, centrifuge at 10,000 rpm for 30 min at room temperature, and collect the supernatant; filter the supernatant once with a 0.45 μm bacterial filter and then filter it again with a 0.22 μm bacterial filter to completely remove the JSAFC 3583 bacteria; obtain a sterile filtrate.

[0043] (3) Take 10 mL and 100 mL of the above sterile filtrate respectively, pour each into 100 mL of PDA medium (cooled to about 50°C), and mix quickly to prepare plates containing JSAFC 3583 sterile filtrate at concentrations of 10 mL / 100 mL and 100 mL / 100 mL. For control, sterile water was used instead of sterile filtrate to prepare sterile level plates of corresponding concentrations.

[0044] (4) In an ultra-clean workbench, use an inoculation needle to pick up a B. cinerea bacterial disc and inoculate it into the above-prepared plate, with the mycelium side facing downward. Set up 3 replicates for each concentration (sterile filtrate and sterile water).

[0045] (5) Place the inoculated culture dish in a 25°C incubator, incubate in the dark for 3-5 days, and measure the colony diameter. The inhibition rate is calculated according to the formula: inhibition rate = (colony diameter of the control group - colony diameter of the treatment group) / colony diameter of the control group × 100%.

[0046] The results are as follows Figure 3 As shown in Table 2

[0047] Table 2 Inhibition rate of extracellular metabolites secreted by strain JSAFC 3583 on B. cinerea

[0048]

[0049]

[0050] The numerical expression of colony diameter in the table is: mean ± standard error.

[0051] from Figure 3 As shown in Table 2, the extracellular metabolites secreted by strain JSAFC 3583 have an inhibitory effect on B. cinerea, and the inhibitory effect tends to increase with the increase of the concentration of extracellular metabolites. When the concentration of JSAFC 3583 extracellular metabolites is 10mL / 100mL, the inhibition rate of B. cinerea reaches 39.78%, and when the concentration of JSAFC 3583 extracellular metabolites is 100mL / 100mL, the inhibition rate of B. cinerea reaches 51.33%.

[0052] Example 4: Effect of Mortierella oligospora JSAFC 3583 on the growth of blueberries

[0053] (1) A 5 mm diameter mycelium block of Mortierella oligospora JSAFC 3583 was taken out using a borer and inoculated into a 9 cm diameter culture dish (containing 15 ml of PDA medium) and cultured at 25°C in the dark for 7 days.

[0054] (2) Select a pot with a diameter of 15 cm, and cut the culture medium of Mortierella oligospora JSAFC 3583 that has been cultured for 7 days into 0.5 cm × 0.5 cm flat blocks. The experiment was set up with two groups. The control group was: 4 0.5 cm × 0.5 cm PDA flat blocks were added to each pot; the experimental group was: 4 0.5 cm × 0.5 cm Mortierella oligospora JSAFC 3583 flat blocks were added to each pot. Use sterilized peat soil to mix various flat blocks evenly, add to the pot to 1 / 3, transplant a healthy blueberry seedling with consistent growth into each pot, and then cover it with peat soil. Each treatment was set up with 3 replicates.

[0055] (3) Water each group of blueberries with sterile water and count the plant heights of the blueberries after 30 days.

[0056] The results are shown in Table 3 and Figure 4 shown.

[0057] Table 3 Statistical results of blueberry plant height

[0058]

[0059] The numerical expression of plant height in the table is mean ± standard error.

[0060] From the data in Table 3, we can see that the plant height of the experimental group is significantly different from that of the control group. The above results show that Mortierella oligospora JSAFC 3583 has a growth-promoting effect on blueberry plants. Figure 5 This is a comparison of the roots of the experimental group and the control group, indicating that Mortierella oligospora JSAFC 3583 also has a growth-promoting effect on the blueberry root system.

Claims

1. A Mortierella oligospora, characterized in that The strain number of the Mortierella oligospora is JSAFC 3583, which was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on May 20, 2024, with the deposit number CGMCC No. 41310.

2. A biocontrol agent, characterized in that: The active ingredient of the biocontrol fungus agent is the Mortierella oligospora described in claim 1.

3. Use of the Mortierella oligospora according to claim 1 and the biocontrol agent according to claim 2 in inhibiting the growth of blueberry gray mold (Botrytis cinerea).

4. Use of the Mortierella oligospora according to claim 1 and the biocontrol agent according to claim 2 in promoting the growth of blueberries.

5. The use according to claim 4, characterized in that: The application method comprises planting blueberries in peat soil containing mycelium of Mortierella oligospora JSAFC 3583, thereby promoting plant height and root growth of the blueberries.

6. Use of the Mortierella oligospora according to claim 1 and the biocontrol fungus agent according to claim 2 in controlling blueberry gray mold.

Images