Arthrobacter sp. ZJUHUYJH1 and application thereof in preventing and treating gramineae meloidogyne

Abstract

The invention provides arthrobacter ZJUHUYJH1 and application thereof in preventing and treating gramineous meloidogyne, and belongs to the technical field of prevention and treatment of plant meloidogyne. The arthrobacter ZJUHUYJH1 provided by the invention has the preservation number of CCTCC NO: M 2025922, is separated from rice root soil infected by the gramineous meloidogyne, and has the advantages that the preservation number is CCTCC NO: M 2025922; the composition has a good prevention and treatment effect on the gramineous meloidogyne; the correction fatality rate of second-stage larvae of the meloidogyne reaches 91.4%, and the root knot number and the ratio of the root knot number to the dry weight of rice infected by the pseudogramineous meloidogyne can be reduced; the method has potential and practical application value in research of the Meloidogyne pseudoaceae.

Description

Technical Field

[0001] The invention belongs to the field of preventing and controlling plant root-knot nematodes, and in particular relates to an Arthrobacter ZJUHUYJH1 strain and an application thereof in preventing and controlling root-knot nematodes of the Poaceae family. Background Art

[0002] Rice is one of the world's most important food crops, providing the staple food for nearly half of the world's population. Its stable production is crucial for food security. However, the root-knot nematode (Meloidogyne graminicola), an obligate parasitic nematode primarily found in major rice-producing regions of South and Southeast Asia, severely undermines rice production and causes substantial economic losses annually. The root-knot nematode's stages are divided into egg (egg), larval stages (J2, J3, and J4), and adult stages (adult). Under conditions of persistent flooding in rice fields, eggs can survive in the soil for up to 14 months. Throughout its life cycle, the root-knot nematode feeds only during the J2 stage. The J2 larvae use their stylet to penetrate the root system, bypassing the endodermal barrier and migrating to the phloem near the root apical meristem to establish a feeding site. The esophageal gland cells secrete various effectors through the stylet to induce the formation of giant cells, which appear as root knots in the plant, typically with a hooked shape. When the rice root system is infected by root-knot nematodes, the main and lateral roots will be short, the fibrous roots will be increased, and the root structure will be deformed. At the same time, the above-ground part of the rice will show short plants and chlorotic leaves. When the number of root knots is too large, the root system of the plant will lose its original function in the later growth stages. If the nematode population density in the paddy field soil is high and the pest is serious, it will cause the plants to flower and mature early, and the rice ears will have many barren grains, which will seriously affect the rice yield. Because the root-knot nematodes of the Poaceae family have a short life cycle, even if the initial nematode population density in the soil is low, they will complete multiple life cycles, causing the nematode population to become rampant throughout the entire life cycle of the rice.

[0003] In agricultural production practices, chemical methods are often used to control plant nematodes, combined with physical methods such as field water and fertilizer management and crop rotation to limit the survival of root-knot nematode populations in the soil. Chemical control primarily relies on various highly toxic phosphorus-containing chemical pesticides. Long-term use can damage soil structure and disrupt soil microbial communities, and residues in the soil can pose a threat to human health through biomagnification effects within the food chain. Currently, biological control is gaining widespread attention due to its environmental friendliness, safety, and non-toxicity. Leveraging plant-microbe interactions and fully utilizing rhizosphere microbial resources to develop biofertilizers and biocontrol agents can reduce reliance on chemical fertilizers and pesticides and maintain a healthy soil ecosystem. The potential of biocontrol bacteria in controlling root-knot nematodes provides a research basis for the development of novel biocontrol strategies. In the future, the discovery and application of more biocontrol bacteria are expected to provide more effective solutions for the sustainable management of root-knot nematodes. Summary of the Invention

[0004] In view of this, the object of the present invention is to provide an Arthrobacter ZJUHUYJH1 and its application in preventing and controlling root-knot nematodes of the Poaceae family.

[0005] The invention provides an Arthrobacter sp. ZJUHUYJH1, with a preservation number of CCTCC NO: M2025922.

[0006] The present invention provides the use of the Arthrobacter ZJUHUYJH1 in preventing and controlling root-knot nematodes of the Poaceae family.

[0007] Preferably, the Arthrobacter ZJUHUYJH1 kills root-knot nematodes of the Poaceae family.

[0008] The present invention provides the use of the Arthrobacter ZJUHUYJH1 in improving the resistance of rice to root-knot nematodes of the Poaceae family.

[0009] Preferably, the Arthrobacter ZJUHUYJH1 can increase the content of defense hormones in rice roots.

[0010] Preferably, the defense hormone comprises 12-oxophytodienoic acid.

[0011] The invention provides a biocontrol agent for preventing and controlling root-knot nematodes of the Poaceae family, comprising the Arthrobacter ZJUHUYJH1.

[0012] Preferably, the concentration of Arthrobacter ZJUHUYJH1 is 1 to 10×10 8 CFU / mL.

[0013] Preferably, the method of using the biocontrol agent includes root irrigation treatment.

[0014] Preferably, the dosage of the root irrigation treatment is 5 to 15 mL per plant.

[0015] Compared with the existing technology, the present invention has the following beneficial effects: the Arthrobacter ZJUHUYJH1 provided by the present invention is isolated from the soil of the rice root system infected by the root-knot nematode of the Poaceae family, and this strain has a good control effect on the root-knot nematode of the Poaceae family. Experiments show that the cell suspension of Arthrobacter ZJUHUYJH1 has a corrected mortality rate of 91.4% against the second-instar larvae of the root-knot nematode, and the fermentation supernatant of Arthrobacter ZJUHUYJH1 has a corrected mortality rate of 22.3%%; in a sterilized soil system, ZJUHUYJH1 can significantly improve the resistance of rice to the root-knot nematode of the Poaceae family, reducing the number of root knots by 43%. It has potential and practical application value in the research of the root-knot nematode of the Poaceae family. The application of the biocontrol agent provided by the present invention in the control of root-knot nematodes can reduce the excessive dependence of traditional agriculture on chemical fertilizers and pesticides, provide a research basis for the development of new biological control strategies, and provide a guarantee of biocontrol resources for the general direction of sustainable agricultural development. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the plate colony observation result of Arthrobacter ZJUHUYJH1;

[0017] Figure 2 This is the phylogenetic analysis result of Arthrobacter ZJUHUYJH1;

[0018] Figure 3 The effects of different components of Arthrobacter on the mortality of root-knot nematodes in the Poaceae family;

[0019] Figure 4 The root knot number and root knot specific dry weight of rice roots infected by root-knot nematodes were measured after applying Arthrobacter in sterilized soil;

[0020] Figure 5 This is the effect of applying Arthrobacter on rice root hormones.

[0021] Biological Deposit Description

[0022] Arthrobacter sp. ZJUHUYJH1 was deposited in the China Center for Type Culture Collection with the deposit number CCTCCNO: M 2025922. The deposit date was April 27, 2025. The deposit address is Wuhan University, Wuhan, China. DETAILED DESCRIPTION

[0023] The present invention provides an Arthrobacter sp. ZJUHUYJH1, with a deposit number of CCTCC NO: M2025922. The Arthrobacter sp. ZJUHUYJH1 provided by the present invention is isolated and purified from soil of rice roots infected by root-knot nematodes of the Poaceae family. The Arthrobacter sp. ZJUHUYJH1 of the present invention has the following biological characteristics: after culturing on an LB medium plate at 30°C for 2 days, single colonies of the strain are milky white, with a smooth surface and regular edges. The bacteria are rod-shaped, aggregate together in a beaded arrangement, and are Gram-positive. The 16S rDNA sequence of the Arthrobacter sp. ZJUHUYJH1 is 1419 bp in length, and the specific sequence is shown in SEQ ID NO.1.

[0024] The present invention provides the use of the Arthrobacter ZJUHUYJH1 in preventing and controlling root-knot nematodes of the Poaceae family.

[0025] In the present invention, the Arthrobacter ZJUHUYJH1 is capable of killing root-knot nematodes of the Poaceae family. Both the bacterial suspension and the fermentation supernatant of the Arthrobacter ZJUHUYJH1 are lethal to the Poaceae root-knot nematodes, with the bacterial suspension having a greater lethal effect than the fermentation supernatant. In the present invention, the Arthrobacter ZJUHUYJH1 is capable of reducing the number of root knots and the specific dry weight of the root knots in rice infected with the Poaceae root-knot nematodes.

[0026] The present invention also provides the use of the Arthrobacter ZJUHUYJH1 in improving the resistance of rice to root-knot nematodes of the Poaceae family. In the present invention, the Arthrobacter ZJUHUYJH1 can increase the content of defense hormones in rice roots; the defense hormones preferably include 12-oxophytodienoic acid.

[0027] The present invention provides a biocontrol agent for controlling root-knot nematodes of the Poaceae family, comprising the Arthrobacter ZJUHUYJH1. In the present invention, the concentration of the Arthrobacter ZJUHUYJH1 is preferably 1 to 10×10 8 CFU / mL. The present invention does not specifically limit the specific composition of the biocontrol agent, and may also include other components such as a carrier. In the present invention, the biocontrol agent is preferably a liquid agent, and the preferred method of application is root irrigation. The dosage for root irrigation is preferably 5-15 mL / plant, more preferably 8-12 mL / plant, and most preferably 10 mL / plant. The present invention does not specifically limit the timing of application of the biocontrol agent.

[0028] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0029] Example 1

[0030] Isolation and identification of Arthrobacter ZJUHUYJH1

[0031] 1. Isolation of Arthrobacter ZJUHUYJH1

[0032] The test strain was isolated from soil containing rice roots infected with root-knot nematodes of the graminaceous family. Strain ZJUHUYJH1 was isolated using the plate dilution method on LB medium. The specific steps are as follows:

[0033] 1) Collecting rhizosphere soil: 21 days after the rice roots were inoculated with root-knot nematodes of the family Poaceae, the rice root soil was collected, the loose soil on the surface was shaken off, and the remaining soil attached to the roots was collected as the rhizosphere soil.

[0034] 2) Prepare soil dilution: Weigh 1g of soil and place it in a conical flask containing 10mL of sterile water; shake it on a shaker at 200r / min for 30 minutes to fully mix the soil and water and completely disperse the bacteria. Pipette 100μL of soil suspension into a centrifuge tube containing 900μL of sterile water, dilute it 10 times, and shake to mix. Repeat this process to make 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 and 10 -7 Dilute the soil solution and set aside.

[0035] 3) Spreading: Spread soil bacteria suspensions of different dilutions on corresponding culture medium plates.

[0036] 4) Cultivation: Place the plate upside down in a 30°C constant temperature incubator and culture the bacteria for 48 hours.

[0037] 5) Purification: Pick a single colony and perform streak separation.

[0038] The entire operation process must strictly follow the principles of aseptic operation to avoid contamination by miscellaneous bacteria.

[0039] 2. Identification of Arthrobacter ZJUHUYJH1

[0040] A single purified colony was picked and dissolved in a PCR tube containing 100 μL of sterile water. The tube was incubated at 98°C for 5 minutes in a PCR instrument to serve as a template. The template was amplified using the universal 16S rRNA primers 27F and 1492R. The PCR conditions were as follows: 98°C pre-denaturation for 45 seconds, 98°C denaturation for 10 seconds, 60°C annealing for 5 seconds, and 72°C extension for 2 minutes for 31 cycles, followed by a final extension at 72°C for 2 minutes. The PCR product was purified using 1% agarose gel electrophoresis and sent to Zhejiang Shangya Biotechnology Co., Ltd. for sequencing.

[0041] The sequencing results showed that the 16S rRNA sequence amplified from strain ZJUHUYJH1 was 1419 bp long, and its nucleotide sequence was shown in SEQ ID NO. 1, as follows:

[0042]

[0043] The PCR product sequencing results were compared with the NCBI database by BLAST analysis. The phylogenetic tree was constructed using MEGA11 to determine the taxonomic status. Figure 2 shown.

[0044] The morphological observation results of the strain showed that the single colonies of the strain were milky white, with smooth surface and regular edges, and were aggregated together in a beaded arrangement. Gram staining was positive.

[0045] Example 2

[0046] Effects of different components of Arthrobacter on the mortality rate of root-knot nematodes in Poaceae

[0047] 1. Preparation of ZJUHUYJH1 Fermentation Supernatant and Bacterial Suspension

[0048] Use an inoculating loop to pick a loop of bacterial liquid stored in a 30% glycerol cryovial, streak it on an LB agar plate, invert and culture for 48 hours, and then observe the growth of the colonies on the plate to ensure that the strain is not contaminated. Pick the single colony obtained after separation and inoculate it into a shaking tube containing 4ml LB liquid culture medium. Place it in a 30℃ shaker and shake and culture at 180r / min for 24 hours until the liquid becomes turbid and the OD value of the bacterial liquid is between 1.0 and 1.5. Pipette 2ml of the shaking cultured bacterial liquid into a 2ml sterilized centrifuge tube and centrifuge it at 6000r / min for 4min. Collect the supernatant and filter it through a 0.22μm filter to obtain the fermentation supernatant; after removing the supernatant, wash it three times with sterile water, resuspend the bacteria with sterile water, and adjust the bacterial liquid OD to 1.0 to obtain a bacterial suspension.

[0049] 2. Preparation of Second-instar Root-knot Nematode Larvae Suspension

[0050] Rice seedlings were transplanted into diseased soil and incubated in a greenhouse for 25-40 days. The rice was then pulled out, the roots rinsed, chopped, and placed in a collection device with water. Incubated at room temperature for one week to obtain root-knot nematodes of the Poaceae family. Second-instar larvae were isolated using the Baermann funnel method. The larvae were then sterilized by immersion in a solution containing 200 mg / L streptomycin sulfate for 4 hours. The sterilized root-knot nematodes were then washed with sterile 0.085% NaCl to obtain sterile nematodes.

[0051] 3. Contact toxicity of fermentation supernatant and bacterial suspension against second-instar larvae

[0052] The collected second-instar larvae were diluted with sterile water to a concentration of 500 larvae / mL to obtain a root-knot nematode suspension;

[0053] Different treatment groups and control groups were set up as follows: treatment group 1: bacterial suspension, treatment group 2: fermentation supernatant; control group 1: sterile water; control group 2: LB culture medium.

[0054] The test on root-knot nematodes of the Poaceae family was conducted and observed in 96-well plates. 100 μL of the root-knot nematode suspension was transferred to a 96-well plate, with 30 to 50 nematodes per well. 100 μL of the fermentation supernatant / bacterial suspension was added. Four replicates were set up per group. An equal volume of LB broth / sterile water was added to the control group.

[0055] For the treatment group with added fermentation supernatant, the number of dead nematodes was observed using an inverted phase contrast microscope at 0 h, 3 h, 6 h, 9 h, and 12 h after the addition of the sample.

[0056] For the treatment group with bacterial suspension added, the number of dead nematodes was observed using an inverted phase contrast microscope at 0 h, 3 h, 6 h, 12 h, 18 h, and 24 h after the addition of the sample, and the mortality rate and corrected mortality rate were calculated.

[0057] Mortality rate (%) = number of dead nematodes / total number of nematodes × 100

[0058] Corrected mortality rate (%) = (nematode mortality rate in the treatment group - mortality rate in the control group) / (1 - mortality rate in the control group) × 100

[0059] 4. Test results

[0060] The results are as follows Figure 3 As shown, the bacterial suspension of strain ZJUHUYJH1 has a significant lethal effect on root-knot nematodes of the Poaceae family. It was observed that at 24 hours, most of the nematodes in the bacterial suspension treatment group were in a rigor mortis, and only 2-3 nematodes survived, while the nematodes in the control group were still active, with a corrected mortality rate of 91.4%; the fermentation supernatant of strain ZJUHUYJH1 also had a lethal effect on root-knot nematodes of the Poaceae family. It was observed that at 9 hours, the nematodes in the treatment group with added fermentation supernatant moved slowly, with discontinuous movement trajectories and even stagnation. At 12 hours, 11-18 nematodes were in a rigor mortis, while only 5-6 nematodes in the control group were in a rigor mortis, with a corrected mortality rate of 28.8%.

[0061] Table 1 Number of surviving root-knot nematodes of Poaceae treated with bacterial suspension

[0062]

[0063] Table 2 Number of surviving root-knot nematodes of Poaceae treated with sterile supernatant

[0064]

[0065] Example 3

[0066] Greenhouse potted plant experiment

[0067] The soil used in the experiment was selected from the Zijingang Campus of Zhejiang University. The soil was sterilized in sterile bags at 121°C for 30 minutes to eliminate most microbial influences, resulting in sterile soil that was evenly distributed into 200 mL rectangular cups. The rice variety used was Nipponbare (a nematode-susceptible variety). Before germination, the rice seeds were disinfected with a 1 / 1000 concentration of prochloraz. The seeds were placed in a 30°C light incubator for 10 days, with the water changed daily. After 10 days of germination, the rice was transferred to a seedling box and, after reaching the seedling stage, transplanted into the rectangular cups. After 14 days of growth, the rice was irrigated with a bacterial suspension at an OD of 1.0 per pot. (The fermentation medium contains high concentrations of salt and sugar, which may damage plant roots. Therefore, the fermentation supernatant was removed, and the cells were resuspended in sterile water and adjusted to an OD of 1.0 to obtain a bacterial suspension.) An equal volume of sterile water was used as a control treatment. Forty-eight hours after root irrigation with the bacterial solution, the rhizosphere soil of each rice plant was perforated and inoculated with 200 J2 root-knot nematodes of the family Poaceae. Seven days after inoculation, the rice roots were harvested and the number of root knots was calculated. The roots were then oven-dried at 65°C for 48 hours. The dry weight of the underground portion of each rice plant was then weighed to calculate the root knot number to dry weight ratio.

[0068] The results are as follows Figure 4 As shown, the root knot number of the treatment group inoculated with the bacterial suspension of strain ZJUHUYJH1 was reduced by 43% compared with the control group, and the root knot number to dry weight ratio of the treatment group was significantly lower than that of the control group.

[0069] Example 4

[0070] Effects of Arthrobacter on Rice Root Hormones

[0071] Rice was treated according to the method in Example 3. 24 hours after nematode inoculation, the rice roots were collected, placed in liquid nitrogen for rapid freezing for 30 minutes, and ground to obtain rice root powder. An internal standard solution was prepared in advance. The internal standard solution used 100% methanol as the solvent. The final internal standard solution contained deuterated jasmonic acid-D5 (d5-JA). 13 C-labeled jasmonic acid-isoleucine ( 13The samples were mixed with 100 ng / mL of each of deuterated salicylic acid (d4-SA), deuterated abscisic acid (d6-ABA), and deuterated indoleacetic acid (d5-IAA). A standard mix was then prepared, containing 990 μL of ethyl acetate as the extract and 10 μL of internal standard per 1 mL. Weigh 0.1g of rice root powder, add 1mL of mixed standard to every 0.1g of rice root powder, vortex on a vortexer for 10min, centrifuge at 4℃ and 12000rpm for 20min, draw the supernatant into a new 2mL centrifuge tube, and evaporate it on a rotary evaporator for 35min to concentrate the plant hormones; finally, add 200μL of 70% methanol to each concentrated centrifuge tube, vortex at 4℃ for 5min, centrifuge at 13000rpm for 10min, and draw 100μL of supernatant into a sample bottle (2mL) with an inner tube (250μL) placed to extract indoleacetic acid, salicylic acid, abscisic acid, 12-oxophytodienoic acid, jasmonic acid, and jasmonic acid isoleucine complex from the rice root powder, and analyze them by UHPLC MS / MS to obtain the effect of SC-R-1 bacterial suspension treatment on the hormone content in rice under nematode infection, with 6-8 replicates per treatment. The results are as follows Figure 5 shown.

[0072] The results showed that treatment with the bacterial suspension of Arthrobacter ZJUHUYJH1 could promote the synthesis of defense-related hormones in rice roots under nematode infection. Compared with the control group without the addition of bacterial agents, the content of 12-oxophytodienoic acid in the rice roots after 24 hours of ZJUHUYJH1 treatment was significantly increased, indicating that ZJUHUYJH1 can inhibit the infection of root-knot nematodes of the Poaceae family by increasing the hormone content in the rice roots.

[0073] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A strain of Arthrobacter sp. ZJUHUYJH1, characterized in that: The deposit number is CCTCC NO:M2025922.

2. Use of the Arthrobacter ZJUHUYJH1 according to claim 1 in preventing and controlling root-knot nematodes of the Poaceae family.

3. The use according to claim 2, characterized in that The Arthrobacter ZJUHUYJH1 kills root-knot nematodes of the Poaceae family.

4. Use of the Arthrobacter ZJUHUYJH1 according to claim 1 in improving the resistance of rice to root-knot nematodes of the Poaceae family.

5. The use according to claim 4, characterized in that The Arthrobacter ZJUHUYJH1 can increase the content of defense hormones in rice roots.

6. The use according to claim 5, characterized in that The defense hormones include 12-oxophytodienoic acid.

7. A biocontrol agent for controlling root-knot nematodes of the Poaceae family, characterized in that: Including the Arthrobacter ZJUHUYJH1 described in claim 1.

8. The biocontrol agent according to claim 7, characterized in that The concentration of Arthrobacter ZJUHUYJH1 is 1 to 10×10 8 CFU / mL.

9. The biocontrol agent according to claim 8, characterized in that The method for using the biocontrol agent includes root irrigation treatment.

10. The biocontrol agent according to claim 9, characterized in that The dosage of the root irrigation treatment is 5 to 15 mL per plant.

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