High-temperature-resistant nematode predatory fungus arthrobotrys oligospora K302 strain and application thereof
By screening and identifying the Orbilia oligospora K302 strain, the strain maintains the ability to prey nematodes at high temperatures, solving the problem of insufficient stability of nematode predation fungi in high temperature environments in the prior art, and has the potential for application in hot summers and high temperature environments.
Patent Information
- Application Number
- CN202411336404.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-24
- Publication Date
- 2024-12-13
AI Technical Summary
The existing nematode predation fungi have insufficient stability in playing a role in high temperature environments, especially in hot summers, which makes it difficult to effectively control agricultural nematode diseases.
A fungal strain called Orbilia oligospora K302 was screened and identified. The strain can still grow normally at high temperatures of 40°C and maintains efficient predation of nematodes, and has high temperature tolerance of 40-60°C.
The strain maintains significant predation of nematodes under high temperature conditions, fills the gap in the prior art, has great application prospects in hot summers and a variety of high temperature environments, and reduces the negative impact of climate change on crop growth.
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Figure CN119120215A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of biotechnology, and relates to a fungus Orbilia oligospora K302 with nematode predation ability and a use method thereof. Background Art
[0002] Plant parasitic nematodes are common plant infectious diseases worldwide. They have a wide host range and are very harmful, causing huge losses to crops around the world every year. This poses a very serious threat to agricultural production, affecting fruits, vegetables and field crops, causing a sharp drop in their harvests. In the past few decades, chemical pesticides have remained the main means of controlling plant parasitic nematodes due to their simplicity, good prevention effect and rapid effect. However, chemical pesticides have many safety hazards, such as: while killing nematodes, they are also toxic to non-target organisms, and are easy to remain in the soil environment and crops, which seriously affects food safety and environmental safety. With the rapid development of the economy and society, there are higher requirements for ecological environment protection, people's living needs, and agricultural product quality. "Green agriculture" has become more and more the focus of public attention. Exploring and applying efficient and non-toxic green management methods such as biological control and RNA pesticides is the only way to modernize agriculture.
[0003] Compared with traditional chemical pesticides, microbial preparations have the advantages of safety, pollution-free and no residue, but also have the problem of low prevention effect. Nematode predatory fungi are a large and diverse group of natural enemies of parasitic nematodes in the soil, and are considered to be a powerful weapon for effectively controlling nematode diseases. Nematode predatory fungi can transform their morphology through rapid response to carnivorous growth, forming various fungal capture devices, such as sticky nets, sticky nodes, sticky branches, contractile and non-contractile rings, etc. In the presence of nematodes, nematode predatory fungi will use these special structures to capture and devour nematodes. Among them, Orbilia oligospora is the most typical nematode predatory fungus, and researchers have conducted some research on this. Invention patent application 201710178114.9 discloses "a method for inducing oligospora to produce traps in water using urea in large quantities", which can obtain a large amount of mycelium at low cost and quickly. Invention patent application 202311826101.X discloses "a genetically engineered strain of oligosporon and its application", which improves the control rate of the engineered strain against southern root-knot nematodes through gene editing.
[0004] However, the application conditions of oligosporon fungus described in the above studies are all room temperature. But in actual application, the environmental factors of nematode predation fungi vary greatly, and high temperature often leads to insufficient stability in their function. According to relevant research, due to climate change, the temperature curve has shifted by one standard deviation interval, which means that extreme summer high temperatures occur more and more often. This not only affects human comfort, but also has an important impact on the growth of crops. Screening a high-temperature resistant nematode predation fungus will help improve the fungus's ability to kill nematodes during the high incidence period of nematode diseases in hot summers, which is in line with the needs of current social development.
[0005] At present, there are no reports on nematode-trapping fungi that can withstand high temperature stress and related applications. Summary of the invention
[0006] Based on the current status of the application of nematode-trapping fungi, the present invention provides a strain of nematode-trapping fungi Orbilia oligospora K302. The nematode-trapping fungi Orbilia oligospora K302 can still grow normally under 40°C high temperature stress, and its ability to kill nematodes has not been significantly reduced, which is of great significance for field application in hot summer.
[0007] The technical solution of the present invention:
[0008] A strain of Orbilia oligospora that preys on nematodes, the strain is named Orbilia oligospora K302 strain, and is deposited in the China Center for Type Culture Collection with a deposit number of CCTCCNO: M 20241227 and a deposit date of June 14, 2024. The strain K302 has high temperature tolerance, and the high temperature is 40-60°C. Therefore, the K302 strain still has a strong ability to prey on nematodes under conditions as high as 40°C, filling the gap in the prior art and having great application prospects in hot summer fields and various high temperature environments.
[0009] A bacterial agent comprising the above-mentioned Arthrospora oligosporida strain.
[0010] The application of the above-mentioned Arthrodial Synechococcus oligosporus strain in preventing and controlling agricultural nematode diseases. Wherein, the prevention and control of agricultural nematode diseases is to prey on agricultural nematodes and reduce the infection rate of agricultural nematodes. The agricultural nematodes are one or more of the group consisting of Archaeopterygii, Peanut Root-Knot Nematode, Northern Root-Knot Nematode, Southern Root-Knot Nematode and Corn Cyst Nematode.
[0011] Preferably, the temperature condition for preventing and controlling agricultural nematode diseases is 25-40°C.
[0012] A biological agent for preventing and controlling agricultural nematode diseases, the biological agent comprising the above-mentioned Arthrosporum oligosporum K302 strain or a bacterial agent containing the K302 strain.
[0013] Technical effects of the present invention:
[0014] (1) The present application first provides a strain of Arthrospora oligosporida K302 with high temperature resistance. The strain still has a strong ability to prey on nematodes under conditions as high as 40°C, filling the gap in the prior art.
[0015] (2) The Arthrospora oligosporida K302 strain described in the present application can reduce the negative impact of climate change on crop growth to a certain extent, and has great application prospects in hot summer fields and various high-temperature environments.
[0016] (3) The Arthrospora oligosporida K302 strain described in the present application is expected to be used to prepare biological agents and expand their application prospects in preventing agricultural nematode diseases. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Attached Figure 1 Screened pictures of fungi preying on nematodes; A: Caenorhabditis elegans, B: nematodes preying on fungi.
[0018] Attached Figure 2 The colony size and mycelium density of the different nematode predatory fungi screened were cultured at 40°C for 7 days.
[0019] Attached Figure 3 Phylogenetic tree diagram of the nematode-trapping fungus Orbilia oligospora K302 strain of the present invention;
[0020] Attached Figure 4 Germination of conidia of the nematophagous fungus Orbilia oligospora K302 strain after heat shock at different temperatures and for different periods of time
[0021] Attached Figure 5 Colony growth and hyphae density of the nematode-feeding fungus Orbilia oligospora K302 strain at different temperatures.
[0022] Attached Figure 6 Average mycelial biomass of the nematode-feeding fungus Orbilia oligospora K302 strain under liquid culture conditions at different temperatures.
[0023] Attached Figure 7The mortality rate of nematodes after co-culture with the nematode Orbilia oligospora K302 strain at different temperatures for 48 hours.
[0024] Attached Figure 8 The predatory ability of the nematode-trapping fungus Orbilia oligospora K302 strain on different plant-parasitic nematodes. DETAILED DESCRIPTION
[0025] The present invention will be further described below in conjunction with the embodiments.
[0026] Example 1: Isolation and screening of strains
[0027] Take rotten plant root samples from the green belt beside Shandong Expressway, grind them and stir them evenly with a homogenizer to obtain slurry. Evenly spread an appropriate amount of slurry on YPSSA culture medium, add 25μg / ml chloramphenicol to each culture medium to inhibit the growth of bacteria, and obtain screening culture medium. Add about 100 healthy experimental animal model Caenorhabditis elegans to each screening culture medium as bait, seal the plate and culture at 25℃ for 2 weeks. Observe the plate under a dissecting microscope and record the fungi that separate and capture the nematodes. The process is as follows Figure 1 As shown. Single spores were isolated on YPSSA medium, and pure cultures were obtained after 2-3 subcultures. In order to obtain nematode predatory fungi that can withstand high temperatures, the pure cultures obtained above were cut into mycelial agar discs with a sterilized puncher, and inoculated into the center of a new YPSSA medium and cultured at 40°C for 7 days. By observing and comparing the growth area of mycelium in the culture medium and the density of mycelium, a strain with significantly greater colony and mycelium density at 40°C than other nematode predatory fungi was screened and named K302. Figure 2 shown.
[0028] Example 2: Identification of K302 strain
[0029] The PCR amplification method was used to amplify the ITS rDNA sequence of the K302 strain genome, and the primers are shown in Table 1.
[0030] Table 1 Universal PCR primers
[0031]
[0032] After the PCR products were sequenced, the sequencing results were compared with the known fungal ITS sequences using Blastn on the NCBI website, and the ITS sequences of the standard strains in the comparison results were selected to construct an ITS sequence evolution tree. Figure 3It can be seen that the ITS sequence of the K302 strain and the currently known typical Orbilia oligospora ITS sequence are clustered in the same cluster of the evolutionary tree, thus determining that the species of the K302 strain is Orbilia oligospora, named Orbilia oligospora K302 strain, which is deposited in the China Center for Type Culture Collection, CCTCC NO: M 20241227, and the preservation date is June 14, 2024.
[0033] Example 3: High temperature tolerance of conidia of K302 strain
[0034] Conidia is the main asexual reproduction mode of oligosporon, and its heat resistance determines the amount of hyphae produced by the strain under high temperature environment. Therefore, this example firstly measured the heat resistance of conidia of K302 strain.
[0035] Specific operation: Inoculate the purified strain into YPSSA medium and culture it in a 25°C incubator for 2 weeks. When a large number of conidia are produced, add an appropriate amount of sterile 0.2% Tween 80 to the medium, and use a sterile spreading stick to gently scrape the conidia for later use. Take three centrifuge tubes, add 500μl of the above conidia suspension to each, and then place them in 40°C, 50°C and 60°C water baths, respectively, and heat shock for 10min, 30min and 60min respectively. Use a pipette to draw up the conidia suspension in the centrifuge tube and inoculate it into the YPSSA medium by multi-point spraying; after culturing at 25°C for 72 hours, observe the germination of conidia under a microscope. The results are shown in Table 2 and Figure 4 shown.
[0036] Table 2 Germination of conidia at different times under heat shock at different temperatures
[0037]
[0038] According to Table 2, (1) after heat shock at 40℃ for 10 minutes, the germination rate of conidia was 92.8%; after heat shock at 30 minutes, the germination rate decreased to 85.3%; after heat shock at 60 minutes, the germination rate decreased to 67.6%. (2) after heat shock at 50℃ for 10 minutes, the germination rate of conidia was 88.6%; after heat shock at 30 minutes, the germination rate decreased to 77.6%; after heat shock at 60 minutes, the germination rate decreased to 59.7%. (3) after heat shock at 60℃ for 10 minutes, the germination rate of conidia was 84.3%; after heat shock at 30 minutes, the germination rate decreased to 65.3%; after heat shock at 60 minutes, the germination rate decreased to 52.9%.
[0039] In summary, with the increase of heat shock temperature and the extension of heat shock time, the germination rate of conidia decreased significantly, which is consistent with Figure 4However, even after 60 minutes of heat shock at 60°C, the germination rate of conidia can still reach 52.9%, which shows that the conidia of K302 strain have a strong tolerance to high temperature, so it can produce enough hyphae in high temperature environment and has the possibility of high temperature application.
[0040] Example 4: High temperature tolerance of K302 strain
[0041] The hyphae of A. oligosporus can quickly form fungus-catching devices in the presence of nematodes, and the heat resistance of A. oligosporus determines the nematode-catching ability of A. oligosporus under high temperature conditions. Therefore, this example measured the heat resistance of the hyphae of strain K302.
[0042] (1) Effect of temperature on conidia in solid culture medium.
[0043] Specific operation: Spread the conidia of K302 strain evenly on YPSSA solid medium and culture it at 25℃ for 2 weeks. Cut the above-mentioned K302 strain culture medium into multiple mycelium agar discs with a sterilized puncher and inoculate them into the center of the new YPSSA medium; place each group of newly inoculated culture medium in a constant temperature incubator at 25℃, 30℃, 35℃ and 40℃ respectively, and observe the colony growth after 2 weeks of culture. For details, see Figure 5 Since this example is carried out in a solid culture medium, a high temperature for a long time will dehydrate the culture medium and make the strain unable to grow, so the maximum temperature is selected to be 40°C.
[0044] like Figure 5 As shown in the figure, after 2 weeks of cultivation, the colony size and hyphae density of the K302 strain in the 30℃, 35℃ and 40℃ incubators were not significantly different from those at 25℃ under naked eye observation; this shows that the K302 strain has the ability to withstand high temperatures of 40℃. In addition, the strains cultured at 35℃ and 40℃ produced significantly more conidia (the yellow-brown part of the colony). This is consistent with the results reported in previous literature that filamentous fungi produce conidia faster under adverse environments such as high temperature and strong ultraviolet light.
[0045] (2) Effect of temperature on mycelium production in liquid culture medium
[0046] In order to further quantify and compare the growth of K302 strain at different temperatures, the same size mycelium discs cut from YPSSA solid medium were inoculated into YPSSA liquid medium and then cultured at 180 rpm in a constant temperature shaker at 25°C, 30°C, 35°C and 40°C for 7 days. The obtained culture was centrifuged at 5000r / min for 10 minutes, the upper liquid was discarded, the obtained mycelium was taken out of the agar block, poured into a weighing bottle weighed in advance, placed in a 60°C oven to dry to constant weight, recorded, and calculated the weight of K302 mycelium after 7 days of culture at different temperatures. For details, see Figure 6 Among them, after 7 days of cultivation in 25℃ liquid culture medium, the mycelium weight was 206.5mg / 100ml; after 7 days of cultivation in 30℃ liquid culture medium, the mycelium weight was 212.7mg / 100ml; after 7 days of cultivation in 35℃ liquid culture medium, the mycelium weight was 209.0mg / 100ml; after 7 days of cultivation in 40℃ liquid culture medium, the mycelium weight was 196.2.0mg / 100ml.
[0047] This shows that, similar to the results observed on solid culture medium, there is no significant difference in the biomass of K302 strain under liquid culture conditions of 25°C, 30°C, 35°C and 40°C. This once again proves that K302 strain has the ability to withstand high temperatures.
[0048] Example 5: The ability of K302 strain to kill nematodes at different temperatures
[0049] In order to clarify the ability of K302 strain to prey on nematodes under high temperature conditions, the conidia of K302 strain were evenly spread on YPSSA solid culture medium with a diameter of 9 cm and placed in a constant temperature culture at 25°C until the hyphae covered the entire culture dish. Healthy Caenorhabditis elegans from which the surface bacteria had been removed were added to the culture dish and placed in incubators at 25°C, 30°C, 35°C and 40°C, respectively, with three replicates in each group. The typical oligosporic spores (CGMCC 3.6770) preserved in the China General Microbiological Culture Collection was used as a control. After 48 hours, 50 worm bodies were randomly sampled from the culture medium for microscopic observation.
[0050] like Figure 7As shown, after the control group strain was infected for 48 hours, the mortality rates of nematodes in the incubators at 25°C, 30°C, 35°C and 40°C were 24.4%, 15.7%, 6.9% and 0%, respectively; this shows that the mortality rate of nematodes in the control group gradually decreased with the increase of the culture temperature. When it reached 40°C, the oligosporic spores of the control group could not prey on nematodes. After the K302 strain was infected for 48 hours, the mortality rates of nematodes in the incubators at 25°C, 30°C, 35°C and 40°C were 27.0%, 25.4%, 23.8% and 23.4%, respectively, with no significant difference under different temperature conditions. This shows that the K302 strain described in this application has the characteristics of high temperature resistance and still has a strong ability to prey on nematodes under conditions as high as 40°C. This not only fills the gap in the prior art, but also has great application potential in hot summer fields and various high temperature environments.
[0051] Example 6: Predation ability of K302 strain on different plant parasitic nematodes
[0052] The nematodes used in Examples 1 and 5 are all experimental animal models, Caenorhabditis elegans. In order to further clarify the broad spectrum of nematode predation by the K302 strain, the conidia of the K302 strain were evenly spread on a YPSSA solid culture medium with a diameter of 9 cm and cultured at a constant temperature of 25°C until the hyphae covered the entire culture dish. Five typical plant parasitic nematodes, namely, the root-knot nematode Archaea, the root-knot nematode Peanut, the northern root-knot nematode, the southern root-knot nematode, and the corn cyst nematode, which had been free of surface bacteria, were added to culture dishes and cultured in a 25°C incubator. After 48 hours, 50 worms of each nematode were randomly sampled for microscopic observation. For details, see Figure 8 .like Figure 8 As shown, the mortality rates of Archaea root-knot nematode, Peanut root-knot nematode, Northern root-knot nematode, Southern root-knot nematode and Corn cyst nematode were 32.6%, 29.1%, 30.1%, 27.0% and 30.0%, respectively; this indicates that the K302 strain has a strong predation ability on the above five nematodes and has no significant difference with Caenorhabditis elegans.
[0053] In summary, the Orbilia oligospora K302 strain described in the present application can tolerate high temperature stress of up to 40°C compared with existing plant parasitic nematode predating fungi, and its ability to prey on nematodes is almost unaffected under such high temperature conditions, filling the gap in the prior art and having important application prospects.
Claims
1. A strain of Arthrospora oligosporida that preys on nematodes, characterized in that: The Orbilia oligospora strain is named Orbilia oligospora K302 strain, which is deposited in the China Center for Type Culture Collection with a deposit number of CCTCCNO:M 20241227 and a deposit date of June 14, 2024.
2. The oligosporon strain according to claim 1, characterized in that: The strain has high temperature tolerance.
3. The oligosporon strain according to claim 2, characterized in that: The high temperature is 40-60°C.
4. A bacterial agent comprising the Arthrospora oligosporida strain according to claim 1.
5. Use of the Arthrospora oligosporida strain as claimed in claim 1 in preventing and controlling agricultural nematode diseases.
6. The use according to claim 5, characterized in that: The method for preventing and controlling agricultural nematode diseases is to prey on agricultural nematodes and reduce the infection rate of agricultural nematodes.
7. The use according to claim 6, characterized in that: The agricultural nematodes are one or more of the group consisting of the root-knot nematode Archaea, the root-knot nematode Peanut, the root-knot nematode Northern, the root-knot nematode Southern and the corn cyst nematode.
8. The use according to claim 5 or 6, characterized in that: The temperature condition for preventing and controlling agricultural nematode diseases is 25-40°C.
9. A biological agent for preventing and controlling agricultural nematode diseases, characterized in that: The biological agent includes the Arthrospora oligosporida K302 strain as described in claim 1 or the bacterial agent as described in claim 4.