Biocontrol bacterium for preventing and controlling soybean phomopsis seed rot and application of biocontrol bacterium

Abstract

The invention discloses a biocontrol bacterium for preventing and treating soybean phomopsis longicolla caused by the phomopsis longicolla and application of the biocontrol bacterium, the biocontrol bacterium is named as Aspergillus qilianyuensis JSNL-ZJ66, the preservation unit is China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC NO.41157. The invention further discloses a preparation method of the biocontrol bacterium for preventing and treating the soybean phomopsis longicolla caused by the phomopsis longicolla. The strain is mainly applied to prevention and control of soybean pith seed rot caused by P.lon gilcola, the severity of the soybean pith seed rot can be remarkably reduced, and growth indexes such as the budding rate, the plant height and the fresh weight of the soybean pith seed rot are remarkably improved.

Description

Technical Field

[0001] The invention belongs to the technical field of biocontrol of soybean phomopsis seed rot and relates to a biocontrol bacterium for preventing and controlling soybean phomopsis seed rot and an application thereof. Background Art

[0002] Phomopsis / Diaporthe longicolla is an important plant pathogen that can infect soybean, mung bean, pea, lima bean, clover, peanut, garlic, onion, tomato, pepper, Xanthium sibiricum, Rumex longicolla and other plants. This pathogen can cause seed rot and stem / pod blight in soybean, and has been reported in the United States, Canada, Argentina, Italy, South Korea and other places. In 2009, stem blight caused by Phomopsis / Diaporthe longicolla was first reported in Jiamusi City, Heilongjiang Province, my country, and then the disease was found in Taiwan, Guangdong, Jiangxi, Hubei and Huanghuaihai region.

[0003] The main damage period of soybean phomopsid seed rot is the seed storage period and seedling stage. The infected part of soybean seeds does not show symptoms, while some seeds are shrunken, cracked, chalky, and covered with hyphae on the seed coat. Infected seeds usually germinate slowly or not at all, and the plants are short, stunted, and even rot. The diseased fields generally cause a 10%-30% reduction in soybean production, and in severe cases, even a total loss of harvest. The pathogen can also co-infect with pathogenic fungi of the same genus, causing soybean stem canker and leaf spot diseases, causing huge losses to soybean production.

[0004] In the prior art, the prevention and control of soybean phomopsis seed rot mainly includes the following methods: 1. Planting disease-resistant varieties. This method is the most economical and effective way to prevent and control diseases in soybean production. However, due to the complex field environment and the rapid variation of pathogen virulence, soybean varieties are prone to lose resistance. 2. Chemical control. Although this method can prevent and control soybean phomopsis seed rot to a certain extent, the large-scale use of chemical agents poses a great threat to the ecological environment and food safety, and will also lead to the continuous enhancement of pathogen resistance.

[0005] Endophytic fungi can help plants obtain nutrients, induce resistance in plants, and improve their ability to survive under biotic or abiotic stress conditions. Since endophytic fungi come from plants, they are safe for plants, environmentally friendly, and not prone to drug resistance. Using these microorganisms to control plant diseases is beneficial to maintaining ecological balance. Therefore, endophytic fungi are a source of new compounds that promote organic agriculture.

[0006] In summary, the prior art still lacks a green and efficient probiotic for preventing soybean phomopsis seed rot. Summary of the invention

[0007] In view of the above problems, the present invention provides a biocontrol strain Aspergillus qilianyuensis JSNL-ZJ66 capable of effectively preventing and controlling soybean phomopsis seed rot and applications thereof.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] In the first aspect, the present invention provides a plant endophytic fungus JSNL-ZJ66, which is classified and named Aspergillus qilianyuensis and isolated from the roots of healthy strawberries in Biancheng Town, Jurong City, Zhenjiang City. The strain has been deposited in the General Microbiological Center of China Microbiological Culture Collection Administration (CGMCC for short) on April 15, 2024, with a deposit number of CGMCCNO.41157, and the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0010] In a second aspect, the present invention further provides a biocontrol agent for soybean phomopsis seed rot, wherein the active ingredient of the biocontrol agent includes hyphae or conidia, or extracellular metabolites of the endophytic fungus JSNL-ZJ66.

[0011] Furthermore, the extracellular metabolites are obtained by culturing the plant endophytic fungus JSNL-ZJ66 in a PDA medium for several days and then removing hyphae and conidia.

[0012] In a third aspect, the present invention also provides a method for preparing the biocontrol agent, comprising inoculating the plant endophytic fungus JSNL-ZJ66 with a preservation number of CGMCC NO.41157 into a sterile culture medium and culturing it under dark conditions at 22-28°C for several days.

[0013] In a fourth aspect, the present invention further provides a microbial pesticide for controlling soybean phomopsis seed rot, wherein the active ingredient of the microbial pesticide comprises the endophytic fungus JSNL-ZJ66 described in the first aspect or the biocontrol agent described in the second aspect.

[0014] In a fifth aspect, the present invention further provides the use of the plant endophytic fungus JSNL-ZJ66 described in the first aspect, the biocontrol agent described in the second aspect, or the microbial pesticide described in the fourth aspect in preventing and controlling soybean phomopsis seed rot.

[0015] In a sixth aspect, the present invention further provides a method for preventing and controlling soybean phomopsid seed rot, the method comprising applying the endophytic fungus JSNL-ZJ66 described in the first aspect, the biocontrol agent described in the second aspect, or the microbial pesticide described in the fourth aspect into the plant growth environment.

[0016] In certain embodiments, the plant growth environment is a plant root.

[0017] In certain embodiments, the plant is soybean.

[0018] Compared with the prior art, the present invention has the following technical effects:

[0019] The biocontrol bacterium Aspergillus qilianyuensis JSNL-ZJ66 provided by the present invention has an inhibition rate of 61.20% on soybean phomopsis longicolla, and can significantly inhibit the mycelial growth of the pathogenic bacterium soybean phomopsis longicolla. At the same time, the biocontrol bacterium Aspergillus qilianyuensis JSNL-ZJ66 provided by the present invention can significantly reduce the disease severity of soybean phomopsis longicolla, and significantly improve growth indicators such as soybean germination rate, plant height and fresh weight. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the specific implementation manner of the present invention or the technical solution in the prior art, the drawings required for describing the specific implementation manner are briefly introduced below.

[0021] Figure 1 This is a diagram showing the effect of Aspergillus qilianyuensis JSNL-ZJ66 in Example 1 on inhibiting soybean phomopsis seed rot on a plate;

[0022] Figure 2 The inhibitory activity of Aspergillus qilianyuensis JSNL-ZJ66 against soybean phomopsis seed rot pathogen in Example 1;

[0023] Figure 3 The effect of the extracellular metabolites of Aspergillus qilianyuensis JSNL-ZJ66 on soybean phomopsis seed rot pathogen in Example 2;

[0024] Figure 4 The effect of Aspergillus qilianyuensis JSNL-ZJ66 on soybean plant growth in Example 3;

[0025] Figure 5 The effect of Aspergillus qilianyuensis JSNL-ZJ66 on soybean phomopsis longicolla caused by Phomopsis longicolla in Example 4;

[0026] Figure 6This is the effect of Aspergillus qilianyuensis JSNL-ZJ66 in Example 4 on soybean phomopsis longicolla caused by Phomopsis longicolla. DETAILED DESCRIPTION

[0027] The embodiments of the technical solution of the present invention will be described in detail below in conjunction with the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and are therefore only used as examples, and the protection scope of the present invention cannot be limited by this. It should be noted that, unless otherwise specified, the technical terms or scientific terms used in this application should be the usual meanings understood by those skilled in the art to which the present invention belongs.

[0028] As described in the present invention, the term "biocontrol bacteria" refers to beneficial microorganisms that can prevent and control plant diseases, mainly bacteria, fungi and actinomycetes.

[0029] As described in the present invention, the term "Phomopsis / Diaporthe longicolla" is classified as Fungi, Ascomycota, Sordariomycete, Diaporthales, Diaporthaceae, and Diaporthe. It has a wide host range and can infect plants of the Leguminosae, Solanaceae, Asteraceae, Liliaceae, and Polygonaceae. It is widely distributed worldwide and causes serious damage in some major soybean producing countries such as the United States, Canada, and Argentina.

[0030] Example 1 Screening of Aspergillus qilianyuensis JSNL-ZJ66

[0031] 1. Isolation of potential biocontrol bacteria

[0032] 1.1. Obtaining experimental materials: Select healthy strawberry plants with good growth from the field and take their root tissues.

[0033] 1.2. Isolation and culture of endophytic fungi in strawberry roots: Cut the strawberry root tissue, mix well, weigh 2g, and disinfect the surface. Add 100mL sterile water and 2 drops of Tween 20, shake at 25℃ and 220rpm for 20min, treat with sterile water for 20s, soak in 70% (v / v) ethanol for 30s, soak in 2.5% (v / v) sodium hypochlorite solution for 2min, and finally rinse with sterile water 3-4 times, and dry the surface moisture with sterile absorbent paper. Under sterile conditions, the roots are further cut into smaller segments (about 0.3cm), and 5 segments are randomly selected for each sample and placed on the PDA flat plate culture medium. Seal the culture dish and culture it at 25℃ in the dark for 1-2d. When the mycelium emerges from the root tissue, carefully transfer the small piece of culture medium growing on the edge of the culture medium together with the mycelium to a new PDA plate.

[0034] 2. Screening of antagonistic bacteria by plate confrontation method

[0035] The PDA plate confrontation method was used. The soybean phomopsis seed rot pathogen cake (5 mm in diameter) and the endophytic fungus cake (5 mm in diameter) cultured for 3-5 days were symmetrically inoculated 2 cm from the edge of a 9 cm diameter plate containing 15 mL of PDA medium. The plate inoculated with only the soybean phomopsis seed rot pathogen cake was used as the control. The plates were cultured at 25°C in the dark. After 7 days, the colony radius of the soybean phomopsis seed rot pathogen was measured and the inhibition rate was calculated. The inhibition rate was calculated according to the formula: inhibition rate = (colony radius of the control group - colony radius of the treatment group) / colony radius of the control group × 100%.

[0036] Finally, a strain with good antagonistic activity against soybean phomopsis seed rot on the plate was screened out, which was specifically manifested by strong inhibition of the mycelial growth of the pathogen, and was numbered JSNL-ZJ66.

[0037] like Figure 1 As shown, compared with the control, the colonies of the pathogenic bacteria in the experimental group were significantly smaller, and the inhibition rate of JSNL-ZJ66 on soybean phomopsis seed rot pathogen (P. longicolla) was 61.20%, indicating that the hyphae growth of the soybean phomopsis seed rot pathogen was inhibited.

[0038] like Figure 2 As shown, the results of optical microscopic observation showed that the mycelium of the pathogen in the control group had regular shape and grew straight; after being cultured against JSNL-ZJ66, the mycelium of P. longicolla was chaotically distributed, intertwined, and could not grow normally.

[0039] 3. Molecular biological identification of the strains obtained

[0040] 3.1. The genomic DNA of strain JSNL-ZJ66 was extracted using the kit (DP320-03) from TIANGEN.

[0041] 3.2. Amplify the ITS gene and BenA gene of genomic DNA respectively. DNA amplification uses a 30 μL reaction volume, including 15 μL 2×EasyTaq PCR SuperMix (+dye), 1 μL of primer F / R (10 μM), 2 μL template DNA and 11 μL ddH2O. The PCR amplification program conditions are as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 54℃ annealing for 45 s, 72℃ extension for 60 s, a total of 35 cycles; and finally 72℃ extension for 10 min.

[0042] 3.3. 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 Shanghai Sangon Biotechnology Co., Ltd. for sequencing. The sequencing results were as follows: the ITS gene sequence of the strain JSNL-ZJ66 screened in step 2 was SEQ ID NO.1, and the BenA gene sequence was SEQ ID NO.2.

[0043] 3.4. Compare the two sequences of SEQ ID NO.1 and SEQ ID NO.2 on the NCBI database website. The comparison results are shown in Table 1. According to the results in Table 1, it can be inferred that the biocontrol bacteria JSNL-ZJ 66 of the present invention is Aspergillus qilianyuensis, and is named Aspergillus qilianyuensis JSNL-ZJ66.

[0044] Table 1 Sequence alignment results of SEQ ID NO.1 and SEQ ID NO.2

[0045]

[0046] Example 2 Effect of extracellular metabolites of Aspergillus qilianyuensis JSNL-ZJ66 on the pathogen of soybean phomopsis seed rot

[0047] 1. Inoculate soybean stem rot pathogen (P.longicolla) and Aspergillus qilianyuensis JSNL-ZJ66 into 9 cm diameter culture dishes containing 15 mL PDA medium, place in a 25°C incubator, and culture in the dark for 3-5 days. Use a cork puncher (5 mm in diameter) to punch holes at the edge of the colony to prepare bacterial plates.

[0048] 2. Acquisition of extracellular metabolites secreted by strain JSNL-ZJ66

[0049] A water filter membrane (pore size 0.22 μm, diameter 5 cm) was covered on a 6 cm diameter PDA medium plate, and the filter membrane was closely attached to the surface of the PDA plate. Then, a Aspergillus qilianyuensis JSNL-ZJ66 bacterial plate (diameter 5 mm) was inoculated on the filter membrane, and a blank PDA medium was used instead of the JSNL-ZJ66 bacterial plate (diameter 5 mm) for control, and cultured at 25°C in the dark.

[0050] 3. After culturing for 2 days, use tweezers to carefully remove the filter membrane together with the Aspergillus qilianyuensis JSNL-ZJ66 culture on it. For the control, tear off the filter membrane and the blank PDA block to prepare a plate containing JSNL-ZJ66 extracellular metabolites and a blank PDA plate.

[0051] 4. In an ultra-clean workbench, use an inoculation needle to pick up each pathogenic bacteria disc (5 mm in diameter) and inoculate it into the above-prepared flat plate, with the mycelium facing downward. Set up 3 replicates for each.

[0052] 5. Place the inoculated plate in a 25°C incubator, incubate in dark for 2-3 days, and measure the colony size using the cross method. 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%.

[0053] The results are as follows Figure 3 As shown, compared with the control, the colonies of the pathogenic bacteria in the experimental group were significantly smaller, and the inhibition rate of the extracellular metabolites secreted by the strain JSNL-ZJ66 on P. longicolla reached 76.25%, indicating that the extracellular metabolites secreted by JSNL-ZJ66 had a certain inhibitory effect on the hyphae growth of soybean phomopsis seed rot fungus.

[0054] Example 3 Effect of Aspergillus qilianyuensis JSNL-ZJ66 on Plant Growth

[0055] 1. Inoculate the strain JSNL-ZJ66 in a 9 cm diameter culture dish (containing 15 ml PDA medium) and culture at 25°C in the dark for 5 days. Cut a 3 mm × 3 mm mycelium block from the edge of the JSNL-ZJ66 colony, transfer 10 mycelium blocks to 400 mL PDB liquid medium under sterile conditions, and culture at 25°C with shaking or static conditions for 4 days.

[0056] 2. Select pots with a diameter of 12 cm. Set up two groups for the experiment. The experimental group is: add 400mL of JSNL-ZJ66 bacterial suspension to each pot; the control group is: add 400mL of PDB liquid culture medium to each pot. Mix each treatment evenly with sterilized vermiculite, add it to 2 / 3 of the pot, evenly spread 10 soybean seeds (Dongsheng No. 1) in each pot, and then cover it with a thin layer of mycelium block and vermiculite mixture. Set up 3 replicates for each treatment.

[0057] 3. Water each group of soybeans with sterile water every other day. After 14 days, calculate the germination rate, plant height and fresh weight.

[0058] The results are as follows Figure 4 And as shown in Table 2.

[0059] Table 2 Statistical results of soybean emergence rate, plant height and fresh weight

[0060]

[0061] Among them, the numerical expressions of germination rate, plant height and fresh weight in the table are: mean ± standard error.

[0062] from Figure 4 From the data in Table 2, we can see that the plant height and fresh weight of the experimental group are better than those of the control group, but the difference is not significant. It can be seen that Aspergillus qilianyuensis JSNL-ZJ66 has a growth-promoting effect on soybean plants, but it is not obvious.

[0063] Example 4 Effect of Aspergillus qilianyuensis JSNL-ZJ66 on Soybean Phomopsis Seed Rot Caused by P. longicolla

[0064] 1. Inoculate Aspergillus qilianyuensis JSNL-ZJ66 and P. longicolla into 9-cm diameter culture dishes containing 15 mL of PDA medium, place them in a 25°C incubator and culture in the dark for 5 days.

[0065] 2. Cut 3 mm × 3 mm mycelium blocks from the edge of the JSNL-ZJ66 colony, transfer 10 mycelium blocks to 400 mL PDB liquid culture medium under sterile conditions, and culture at 25°C with shaking for 6 days. Use a blade to cut the P. longicolla culture medium into small pieces of 0.5 cm × 0.5 cm.

[0066] 3. Select pots with a diameter of 12 cm and set up two groups for the experiment. The experimental group is: 400 mL of JSNL-ZJ66 bacterial suspension and 2 P. longicolla flat plates were added to each pot; the control group is: 400 mL of PDB liquid culture medium and 2 P. longicolla flat plates were added to each pot, and 3 replicates were set for each treatment.

[0067] 3.4. Mix the above treatments with sterilized vermiculite evenly, add to 2 / 3 of the pot, put 10 soybeans (variety: Dongsheng No. 1) in each pot, and then cover them with a thin layer of vermiculite mixture.

[0068] 3.5. Water each group of soybeans with sterile water every other day. Count the germination rate, plant height and fresh weight of soybeans 14 days after sowing.

[0069] The results are as follows Figure 5 , Figure 6 And as shown in Table 3.

[0070] Table 4 Statistical results of soybean emergence rate, plant height and fresh weight

[0071]

[0072] from Figure 5 and Figure 6 It can be seen that the plant height, number of sprouts, root length and luxuriance of the experimental group were significantly better than those of the control group. It can be seen that Aspergillus qilianyuensis JSNL-ZJ66 has a preventive or control effect on soybean phomopsis seed rot caused by P. longicolla.

[0073] Unless otherwise specifically stated, the numerical value set forth in these embodiments does not limit the scope of the present invention. In all examples shown and described here, unless otherwise specified, any specific value should be interpreted as merely exemplary, rather than as restriction, and therefore, other examples of exemplary embodiments may have different values.

Claims

1. A biocontrol bacterium for preventing and controlling soybean phomopsis seed rot caused by Phomopsis longicolla, characterized in that: The biocontrol bacteria is Aspergillus qilianyuensis JSNL-ZJ66, with a preservation number of CGMCC NO.41157, and the preservation unit is the General Microbiology Center of China Microbiological Culture Collection Administration.

2. A biocontrol agent for soybean phomopsis seed rot, characterized in that: The active ingredients of the biocontrol agent include hyphae or conidia, or extracellular metabolites of the endophytic fungus JSNL-ZJ66 according to claim 1.

3. The biocontrol agent according to claim 2, characterized in that The extracellular metabolites are obtained by culturing the plant endophytic fungus JSNL-ZJ66 in a PDA medium for several days and then removing hyphae and conidia.

4. A method for preparing the biocontrol agent according to claim 2 or 3, characterized in that: The preparation method comprises inoculating the plant endophytic fungus JSNL-ZJ66 with the deposit number of CGMCC NO.41157 as claimed in claim 1 into a sterile culture medium and culturing it under dark conditions at 22-28°C for several days.

5. A microbial pesticide for preventing and treating soybean phomopsis seed rot, characterized in that: The active ingredient of the microbial pesticide comprises the endophytic fungus JSNL-ZJ66 according to claim 1 or the biocontrol agent according to any one of claims 2-3.

6. Use of the plant endophytic fungus JSNL-ZJ66 according to claim 1, the biocontrol agent according to any one of claims 2 to 3, or the microbial pesticide according to claim 5 in preventing and controlling soybean phomopsid seed rot.

7. The use according to claim 6, characterized in that: The soybean phomopsis seed rot is soybean phomopsis seed rot caused by P. longicolla.

8. A method for preventing and treating soybean phomopsis seed rot, characterized in that: The method comprises applying the plant endophytic fungus JSNL-ZJ66 according to claim 1, the biocontrol agent according to any one of claims 2 to 3, or the microbial pesticide according to claim 5 to the plant growth environment.

Images