Application of aluminum chloride in increasing the number of predatory organs produced by oligosporic nodes

Abstract

The present invention relates to the use of aluminum chloride to increase the number of predatory organs produced by oligosporoid spores. This invention relates to the field of microorganisms, particularly the reproduction of microbial biocides. The present invention is the first to discover that aluminum chloride can increase the number of predatory organs produced by oligosporoid spores, thereby improving the predation efficiency of oligosporoid spores on nematodes, thereby reducing costs.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, in particular to the propagation of microbial biocides. Background Art

[0002] Plant parasitic nematodes can parasitize the roots, stems, leaves and other parts of crops. The wounds they cause are easily infected by pathogens. They are the main cause of plant diseases around the world, causing huge losses to the global economy every year and seriously threatening food crop safety.

[0003] Currently, nematode-trapping fungi are widely used as an effective biocontrol resource for plant-parasitic nematodes. Compared with traditional chemical pesticides, microbial agents have the advantages of being safe, pollution-free, and leaving no residue, but they also have the problem of low efficacy.

[0004] Oligosporon Arthrobotrys oligospora ) is a type of nematode-predating fungus that captures and kills nematodes by producing feeding organs, or three-dimensional nets. The more feeding organs a oligosporon produces from its spores, the more efficient it is at capturing nematodes. However, the number of feeding organs produced by oligosporon spores is affected by various factors. Summary of the Invention

[0005] One aspect of the present invention provides the use of aluminum chloride in increasing the number of predatory organs produced from spores of oligosporic nodes or the yield of molecular spores.

[0006] In a specific embodiment, the amount of aluminum chloride used is 2.5 μmol / L or more.

[0007] In a specific embodiment, the amount of aluminum chloride in the culture medium is 2.5 μmol / L or more.

[0008] In a specific embodiment, the culture medium may be, for example, WA solid medium.

[0009] In one embodiment, the amount of aluminum chloride used is 2.5 μmol / L to 30 μmol / L.

[0010] In one embodiment, the amount of aluminum chloride in the culture medium is 2.5 μM / L to 30 μM / L.

[0011] In one embodiment, the amount of aluminum chloride used is 2.5 μmol / L to 10 μmol / L.

[0012] In one embodiment, the amount of aluminum chloride in the culture medium is 2.5 μM / L to 10 μM / L.

[0013] In one embodiment, the amount of aluminum chloride is 10 μmol / L to 30 μmol / L.

[0014] In one embodiment, the amount of aluminum chloride in the culture medium is 10 μM / L to 30 μM / L.

[0015] In one embodiment, when aluminum chloride (calculated as anhydrous aluminum chloride) is applied to a field, the application rate is 0.75 kg / ha to 9 kg / ha, wherein the field application rate is converted to 2,250,000 kg of topsoil mass per hectare.

[0016] In one embodiment, when aluminum chloride is applied to a field, the application rate is 0.75 kg / hectare to 7.5 kg / hectare.

[0017] In one embodiment, when aluminum chloride is applied to a field, the application rate is 0.75 kg / hectare to 5.25 kg / hectare.

[0018] In one embodiment, when aluminum chloride is applied to a field, the application rate is 2.25 kg / hectare to 5.25 kg / hectare.

[0019] In one embodiment, when aluminum chloride is applied to a field, the application rate is 0.75 kg / hectare to 2.25 kg / hectare.

[0020] In a specific embodiment, the oligosporic spores are YMF1.01435 strain, Δ414 strains and Δ415 At least one of the strains; wherein the YMF1.01435 strain is deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms with the deposit number CGMCC No. 3591; Δ414 The strain was obtained by knocking out the AOL_s00215g414 gene from the spore YMF1.01435 via the oligosporic node; Δ415 The strain was obtained by knocking out AOL_s00215g415 from the oligosporic node YMF1.01435.

[0021] The second aspect of the present invention provides a method for increasing the number of predatory organs produced by oligosporic nodes from spores, which comprises the following steps:

[0022] 1) adding aluminum chloride to the culture medium to obtain a culture medium containing aluminum chloride;

[0023] 2) inoculating the oligosporic node spores onto the medium containing aluminum chloride, culturing, and obtaining the feeding organs produced by the oligosporic node spores.

[0024] In a specific embodiment, the concentration of aluminum chloride in the culture medium is above 10 μM / L.

[0025] In a specific embodiment, the concentration of aluminum chloride in the culture medium is 10 μM / L to 30 μM / L.

[0026] In a specific embodiment, the culture medium is WA solid medium.

[0027] The present invention has the following beneficial effects: It has been discovered for the first time that aluminum chloride can increase the number of predatory organs produced by oligosporic spores and the yield of molecular spores. Based on this discovery, aluminum chloride can be added to culture media indoors to induce the production of large numbers of predatory organs for scientific research. In the field, aluminum chloride can be applied to the soil, causing the applied oligosporic spores to produce large numbers of predatory organs in the soil, thereby improving the insecticidal effect against nematodes. Furthermore, aluminum chloride has stable chemical properties and is inexpensive, which can reduce production costs. It is also a soil conditioner that can improve soil structure, regulate soil pH, form a more stable soil aggregate structure, and enhance soil water and fertilizer retention. It can also provide aluminum to crops, promoting root development and increasing leaf photosynthesis efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Microscopic photographs of the feeding organs produced by the wild-type strain YMF1.01435 of A. oligosporus on various culture media are shown.

[0029] Figure 2 The numbers of predatory organs produced by the wild-type strain YMF1.01435 of A. oligosporus on each culture medium are shown.

[0030] Figure 3 The insecticidal activity of the wild strain YMF1.01435 of A. oligosporus against Caenorhabditis elegans on various culture media and the insecticidal activity of the aluminum chloride culture medium control against Caenorhabditis elegans are shown.

[0031] Figure 4 shows oligosporon mutant strains Δ414 Microscopic photographs of the feeding organs produced on each culture medium.

[0032] Figure 5 shows oligosporon mutant strains Δ414 The number of predatory organs produced on each culture medium.

[0033] Figure 6 shows oligosporon mutant strains Δ414 Insecticidal activity against Caenorhabditis elegans on various media.

[0034] Figure 7 shows oligosporon mutant strains Δ415 Microscopic photographs of the feeding organs produced on each culture medium.

[0035] Figure 8 shows oligosporon mutant strains Δ415 The number of predatory organs produced on each culture medium.

[0036] Figure 9 shows oligosporon mutant strains Δ415 Insecticidal activity against Caenorhabditis elegans on various media.

[0037] Figure 10 Shown are photos of the colonization of each strain in each seedling tray in Example 4.

[0038] Figure 11 A photograph of the colonization in Example 4 is shown after circling the soil surface area and the fungal growth area.

[0039] Figure 12 A bar graph of colonization rates in Example 4 is shown.

[0040] Figure 13 Conidia under a microscope in Example 4 are shown. DETAILED DESCRIPTION

[0041] The above contents of the present invention are further described in detail below in the form of preferred implementation cases, but they do not constitute a limitation of the present invention.

[0042] Unless otherwise specified, the reagents in the embodiments of the present invention can be purchased through commercial channels.

[0043] The wild strain YMF1.01435 of A. oligosporum was deposited in the General Microbiology Center of China Culture Collection Administration under the deposit number CGMCC No. 3591 on January 18, 2010 at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Arthrobotrys oligospora It is also preserved in the Yunnan University State Key Laboratory of Bioresource Conservation and Utilization's Fungus Bank - Southwest China Wild Germplasm Resources Bank, with the accession number YMF1.01435.

[0044] Arthrodes oligosporus mutant strain Δ414A strain of YMF1.01435 was obtained by knocking out the AOL_s00215g414 gene (GeneID: 22898840, encoding L-ornithine oxidase, the first step in siderophore biosynthesis in A. oligosporus). (For details on the knockout procedure, refer to the dissertation "Functional Study of Siderophore Biosynthesis Genes in the 215g Gene Cluster in A. oligosporus," by Jiang Yang, Yunnan University, 2022.) This strain can spontaneously produce feeding organs. It is deposited in the Southwest China Wild Germplasm Resource Bank, State Key Laboratory of Conservation and Utilization of Biological Resources, Yunnan University, under the accession number YMF1.07673.

[0045] Arthrodes oligosporus mutant strain Δ415: A strain of Arthrospora oligospora, YMF1.01435, was obtained by knocking out AOL_s00215g415 (GeneID: 22898841, encoding a non-ribosomal polypeptide synthetase and a core gene for siderophore biosynthesis) (for details on this gene knockout, refer to the dissertation "Functional Study of Siderophore Biosynthesis Genes in the 215g Gene Cluster in Arthrospora oligospora," by Jiang Yang, Yunnan University, 2022). This strain has a slightly stronger ability to produce feeding organs than the wild-type strain YMF1.01435. It is deposited in the Southwest China Wild Germplasm Resource Bank, State Key Laboratory of Conservation and Utilization of Biological Resources, Yunnan University, under the accession number YMF1.07674.

[0046] CMA solid medium: Mix 20 g cornstarch with 1 L deionized water, boil for 30 min, and filter repeatedly through 8 layers of gauze until no obvious residue is visible. Add 2 g yeast extract and make up to 1 L with deionized water. Sterilize at 121°C.

[0047] WA solid medium: 15 g agar, dilute to 1000 mL with ultrapure water, and sterilize at 121 degrees Celsius.

[0048] Oatmeal culture medium: 100 g oatmeal, add 100 mL ultrapure water to soak the oatmeal, and sterilize at 121 degrees Celsius.

[0049] 10 mM aluminum chloride aqueous solution: Weigh 241 mg of aluminum chloride hexahydrate and dissolve it in 100 mL of ultrapure water, dissolve, and filter sterilize.

[0050] The WA solid culture medium was heated to melt, and then cooled to an unsolidified state at 50 to 60 degrees Celsius. A 10 mM aqueous solution of aluminum chloride was added thereto so that the final concentrations of aluminum chloride in the culture medium were 10 micromoles / liter and 30 micromoles / liter, respectively. The mixture was mixed evenly to obtain culture medium containing aluminum chloride at various concentrations. The culture medium containing aluminum chloride was then quickly poured into a culture dish and used to culture oligosporic arthrospores after solidification. Example 1

[0051] The wild strain YMF1.01435 from the oligosporic arthrospore was inoculated onto CMA solid culture medium and cultured at 28 degrees Celsius for 14 days. The large number of conidia produced were washed with sterile water and filtered through six layers of sterile lens paper. The filtrate was collected, and 2 microliters of the filtrate was aspirated and counted on a hemocytometer. The concentration of conidia was diluted to 100 / microliter to obtain a spore suspension.

[0052] 100 microliters of spore suspension was added to WA solid culture medium with a final aluminum chloride concentration of 10 micromol / L and a final aluminum chloride concentration of 30 micromol / L, respectively, spread, and cultured at 28 degrees Celsius for 72 hours. Each culture dish was used as a replicate, and there were 3 replicates in total. WA solid culture medium without aluminum chloride was used as the control culture medium. Five 1 cm areas were selected on each culture dish using the five-point sampling method, and the number of predatory organs produced was observed and counted under a microscope, and the average value was calculated. The number of predatory organs produced on the control culture medium, WA solid culture medium with an aluminum chloride content of 10 micromol / L, and WA solid culture medium with an aluminum chloride content of 30 micromol / L was statistically analyzed. The photos observed under the microscope are as follows. Figure 1 The results of the number of predatory organs are shown in Figure 2 .

[0053] C. elegans ( Caenorhabditis elegans ), cultured at 20 degrees Celsius for 15 days, filtered using a Behmann funnel method, centrifuged at 2000 rpm for 2 minutes, discarded the supernatant, washed with sterile deionized water, and centrifuged, repeated three times, and finally diluted to 15 nematodes / μl to obtain a nematode suspension.

[0054] 100 μL of spore suspension was added to WA solid medium with a final aluminum chloride concentration of 10 μM / L and 30 μM / L, respectively, and the culture was spread. After incubation at 28°C for 72 h, 400 Caenorhabditis elegans were added to each culture dish. 2 h after the addition of nematodes, the dead nematodes were counted under a microscope, and the nematode mortality rate was calculated based on the following formula (1). Each culture dish was used as a replicate, for a total of 3 replicates. WA solid medium without aluminum chloride was inoculated with YMF1.01435 spore suspension and then incubated with nematodes as a control medium; WA solid medium with a final aluminum chloride concentration of 10 μM / L and a final aluminum chloride concentration of 30 μM / L was incubated without YMF1.01435 spore suspension and then incubated with nematodes as an aluminum chloride medium control.

[0055] Nematode mortality = number of dead nematodes / total number of nematodes × 100% (1).

[0056] The results of the nematode mortality bar graph are shown in Figure 3 . Example 2

[0057] The wild strain YMF1.01435 of Arthrospora oligospora was replaced with the mutant strain of Arthrospora oligospora Δ414 WA solid medium with a final aluminum chloride concentration of 10 μM / L and a final aluminum chloride concentration of 30 μM / L was not inoculated with the YMF1.01435 spore suspension and then incubated with nematodes as an aluminum chloride medium control. Other conditions were the same as in Example 1.

[0058] Microscopic photos of Figure 4 The results of the number of predatory organs are shown in Figure 5 The results of the nematode mortality bar chart are shown in Figure 6 . Example 3

[0059] The wild strain YMF1.01435 of Arthrospora oligospora was replaced with the mutant strain of Arthrospora oligospora Δ415 WA solid medium with a final aluminum chloride concentration of 10 μM / L and a final aluminum chloride concentration of 30 μM / L was not inoculated with the YMF1.01435 spore suspension and then incubated with nematodes as an aluminum chloride medium control. Other conditions were the same as in Example 1.

[0060] Microscopic photos of Figure 7 The results of the number of predatory organs are shown in Figure 8 The results of the nematode mortality bar chart are shown in Figure 9 .

[0061] Figure 1 、 Figure 2 、 Figure 4 、 Figure 5 、 Figure 7 and Figure 8 The results showed that aluminum chloride can effectively induce oligosporangial nodes to produce more predatory organs from spores. Figure 3 、 Figure 6 and Figure 9 The results showed that oligosporic spores that produced more predatory organs had better insecticidal activity against Caenorhabditis elegans. Example 4

[0062] In the soil, the predatory organs of fungi grow close to the soil. Since the soil is opaque, the predatory organs growing in the soil medium cannot be directly observed using a microscope. However, the prerequisite for fungi to produce predatory organs in the soil is that they can colonize in the soil. The higher the colonization rate of fungi in the soil, the better their growth in the soil and the more conducive it is to the formation of their predatory organs in the soil. In addition, conidia represent the reproductive capacity of fungi. The more conidia a fungus produces, the stronger its ability to adapt to the environment. Therefore, the ability of fungi to produce predatory organs in the soil can be reflected by the colonization rate of fungi in the soil. In other words, the colonization of fungi in the soil reflects their survival competitiveness in the soil. A strong survival competitiveness indicates that they can grow better in the soil and produce more predatory organs.

[0063] YMF1.01435, Δ414 and Δ415 Each strain was activated on a 9 cm CMA solid medium plate for 7 days. One-quarter of the bacterial mass was then inoculated into a 500 mL Erlenmeyer flask containing 250 mL of PDB medium. The culture was statically incubated at 28°C for 24 hours and then incubated at 180 rpm for 3 days at 28°C to obtain the fermentation broth of each strain. The fermentation broth of each strain was diluted 2-fold with an equal volume of tap water to obtain a dilution of each strain. A 100 μM aluminum chloride solution was added to the dilution until the final concentration of aluminum chloride in each dilution reached 10 μM, 30 μM, 70 μM, and 100 μM, respectively, to obtain the aluminum chloride-containing dilution of each strain.

[0064] Soil treatment: 60 g of soil collected from under the Yunshan camphor tree at Yunnan University was placed in each seedling tray. The seedling tray was placed in a flower pot tray of appropriate size, and 15 mL of aluminum chloride dilutions of each strain was evenly sprayed on the surface of the soil in the seedling tray. The soil was kept warm at 20 degrees Celsius for 5 days, and 10 mL of water was added to each seedling tray every day to keep the soil moist. Three replicates were set for each treated soil. The dilution without aluminum chloride was used as a control. Among them, for the addition of 15 mL of 10 μmol / L aluminum chloride, the content of aluminum chloride in the soil was 0.0025 μmol / g soil. After conversion, the content of aluminum chloride in the soil was 3.334×10 -7 g / g soil; for adding 15 mL of 30 μmol / L aluminum chloride, the content of aluminum chloride in the soil is 0.0075 μmol / g soil. After conversion, the content of aluminum chloride in the soil is 1.000×10 -6 g / g soil; for adding 15mL 70μmol / L aluminum chloride, the content of aluminum chloride in the soil is 0.0175μmol / g soil. After conversion, the content of aluminum chloride in the soil is 2.334×10 -6g / g soil; for adding 15 mL of 100 μmol / L aluminum chloride, the content of aluminum chloride in the soil is 0.025 μmol / g soil. After conversion, the content of aluminum chloride in the soil is 3.334×10 -6 g / g soil.

[0065] The colonization of fungi in the soil was observed every day. On the 5th day, the colonization photos of each strain in each seedling tray were as follows: Figure 10 As shown; using Image J software, use green lines to circle the boundaries of the soil along each seedling tray to calculate the surface area of ​​the soil in the seedling tray; use red lines to circle the areas where fungi grow along the whitened areas of the soil to calculate the area of ​​fungal growth; the planting photos after circling the soil surface area and fungal growth area are shown in Figure 11 As shown. Then the colonization rate is calculated based on the soil surface area and the fungal growth area, that is, colonization rate = fungal growth area / soil surface area × 100%. The colonization rate bar chart is shown as Figure 12 shown.

[0066] In addition, on the 5th day, soil with a depth of 3 mm where fungi were colonized was selected and conidia were observed under a microscope. Figure 13 The results showed that aluminum chloride can promote the increase of conidia production.

[0067] The above results indicate that aluminum chloride can promote the colonization of oligosporic spores and increase the conidia production, thereby facilitating the oligosporic spores to produce more predatory organs and provide nematicidal effects under the same environment.

Claims

1. The use of aluminum chloride in increasing the number of predatory organs or conidia produced by oligosporic spores, wherein the amount of aluminum chloride used in the culture medium is 2.5 μmol / L to 30 μmol / L, and the oligosporic spores are strains YMF1.01435, Δ 414 strains and Δ415 At least one of the strains; wherein, The YMF1.01435 strain is deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with the deposit number CGMCC No. 3591; Δ414 The strain was obtained by knocking out the AOL_s00215g414 gene from the spore YMF1.01435 via the oligosporic node; Δ415 The strain was obtained by knocking out AOL_s00215g415 from the oligosporic node YMF1.01435.

2. The use according to claim 1, characterized in that The amount of aluminum chloride used in the culture medium is 10 micromoles / liter to 30 micromoles / liter.

3. The application of aluminum chloride in increasing the number of predatory organs or conidia produced by oligosporic spores, when aluminum chloride is applied to the field, the application rate is 0.75 kg / hectare to 9 kg / hectare, the oligosporic spores are YMF1.01435 strain, Δ414 strains and Δ415 At least one of the strains; wherein, The YMF1.01435 strain is deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with the deposit number CGMCC No. 3591; Δ414 The strain was obtained by knocking out the AOL_s00215g414 gene from the spore YMF1.01435 via the oligosporic node; Δ415 The strain was obtained by knocking out AOL_s00215g415 from the oligosporic node YMF1.01435.

4. The use according to claim 3, characterized in that When aluminum chloride is applied to the field, the application rate is 0.75 kg / ha to 7.5 kg / ha.

5. The use according to claim 3, characterized in that When aluminum chloride is applied to the field, the application rate is 0.75 kg / ha to 5.25 kg / ha.

6. The use according to claim 3, characterized in that When aluminum chloride is applied to the field, the application rate is 2.25 kg / ha to 5.25 kg / ha.

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