Arthrobacter ZJUHUYJH1 and application thereof in prevention and treatment of meloidogyne chitwoodi

Abstract

The application provides a strain of Arthrobacter sp. ZJUHUYJH1 and application thereof in prevention and treatment of paratyphaceae root-knot nematodes, and belongs to the technical field of prevention and treatment of plant root-knot nematodes. The Arthrobacter sp. ZJUHUYJH1 provided by the application has a preservation number of CCTCC NO: M 2025922, is isolated from rice rhizosphere soil infected by paratyphaceae root-knot nematodes, has a good prevention and treatment effect on paratyphaceae root-knot nematodes, a rectification mortality of 91.4% on second instar larvae of root-knot nematodes, can reduce the number of root-knots and the ratio of the number of root-knots to dry weight of rice infected by paratyphaceae root-knot nematodes, and has potential and practical application value in the research of paratyphaceae root-knot nematodes.

Description

Technical Field

[0001] This invention belongs to the field of plant root-knot nematode control, and particularly relates to a strain of Arthrobacter ZJUHUYJH1 and its application in the control of root-knot nematodes of the Poaceae family. Background Technology

[0002] Rice is one of the world's most important food crops, providing staple food for nearly half the global population, and its stable production is crucial for food security. However, the root-knot nematode (Meloidogyne graminicola), an obligate parasitic nematode, is mainly distributed in several major rice-producing areas in South and Southeast Asia, severely disrupting rice production and causing substantial economic losses annually. The life cycle of the root-knot nematode is mainly divided into egg stage, larval stage (J2, J3, J4), and adult stage. Under conditions of continuous flooding in paddy fields, eggs can survive in the soil for up to 14 months. Throughout its life cycle, the root-knot nematode only exhibits feeding behavior during the J2 stage. The J2 larvae use their stylets to invade the root system, bypass the endoderm barrier, and migrate to a location near the phloem in the root apical meristem to establish a feeding site. Their esophageal gland cells secrete various effectors through the stylet, inducing the formation of giant cells, which appear as root knots on the plant's surface, generally hook-shaped. When rice roots are infected by root-knot nematodes, they exhibit symptoms such as short main and lateral roots, increased fibrous roots, and deformed root structure. Simultaneously, the above-ground parts of the rice plant show stunted growth and chlorotic leaves. When the number of root knots is excessive, the root system loses its original function in the later stages of growth. High nematode population density in paddy fields leads to severe infestation, causing premature flowering and maturity, and resulting in a high proportion of empty grains on the panicles, significantly impacting rice yield. Due to their short life cycle, even low initial nematode populations in the soil can cause widespread infestation throughout the rice's life cycle as root-knot nematodes of the Poaceae family complete multiple life cycles.

[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 inhibit the survival of root-knot nematode populations in the soil. Chemical control mainly relies on various highly toxic phosphorus-containing chemical pesticides, which, with long-term use, can damage soil structure and soil microbial communities, and remain in the soil, posing a threat to human health through the biomagnification effect of the food chain. Currently, biological control is receiving widespread attention due to its green, environmentally friendly, safe, and non-toxic characteristics. Based on plant-microbe interactions, fully utilizing rhizosphere microbial resources to develop biofertilizers and biocontrol agents can reduce dependence on chemical fertilizers and pesticides and maintain a healthy soil ecological environment. The potential of biocontrol bacteria in root-knot nematode control provides a research basis for developing new biological control strategies. In the future, with the discovery and application of more biocontrol bacteria, it is expected to provide more effective solutions for the sustainable management of root-knot nematodes. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a strain of Arthrobacter ZJUHUYJH1 and its application in the control of root-knot nematodes of the Poaceae family.

[0005] This invention provides a strain of Arthrobacter p. ZJUHUYJH1, with accession number CCTCC NO:M2025922.

[0006] This invention provides the application of the aforementioned Arthrobacter ZJUHUYJH1 in the control of root-knot nematodes of the Poaceae family.

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

[0008] This invention provides the application of the aforementioned 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 defensive hormones include 12-oxophytadienoic acid.

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

[0012] Preferably, the concentration of Arthrobacter ZJUHUYJH1 used is 1–10 × 10⁻⁶. 8 CFU / mL.

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

[0014] Preferably, the amount of root drenching treatment used is 5-15 mL per plant.

[0015] Compared with existing technologies, this invention has the following beneficial effects: The *Arthrobacter* ZJUHUYJH1 strain provided by this invention was isolated from the root soil of rice plants infected with *Poaceae* root-knot nematodes. This strain exhibits good control effects against *Poaceae* root-knot nematodes. Experiments show that the corrected lethality rate of *Arthrobacter* ZJUHUYJH1 bacterial suspension against second-instar larvae of root-knot nematodes reaches 91.4%, and the corrected lethality rate of *Arthrobacter* ZJUHUYJH1 fermentation supernatant is 22.3%. In a sterilized soil system, ZJUHUYJH1 can significantly improve the resistance of rice to *Poaceae* root-knot nematodes, reducing the number of root knots by 43%. It has potential and practical application value in the research of *Poaceae* root-knot nematodes. The application of the biocontrol agent provided by this invention in the control of root-knot nematodes can reduce the excessive reliance of traditional agriculture on chemical fertilizers and pesticides, provide a research basis for developing new biological control strategies, and provide a guarantee of biological control resources for the general direction of sustainable agricultural development. Attached Figure Description

[0016] Figure 1 The results of plate colony observation for Arthrobacter ZJUHUYJH1;

[0017] Figure 2 The results of phylogenetic analysis of Arthrobacter ZJUHUYJH1;

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

[0019] Figure 4 The number of root knots and the root knot-to-dry weight of rice roots infected by root-knot nematodes were measured after applying Arthrobacter to sterilized soil.

[0020] Figure 5 To investigate the effects of Arthrobacter on root hormones in rice.

[0021] Biological Preservation Instructions

[0022] Arthrobacters p. ZJUHUYJH1 is deposited at the China Center for Type Culture Collection, accession number CCTCCNO:M 2025922, date of deposit on April 27, 2025, at Wuhan University, Wuhan, China. Detailed Implementation

[0023] This invention provides a strain of *Arthrobacter* p. ZJUHUYJH1, with accession number CCTCC NO: M2025922. The *Arthrobacter* p. ZJUHUYJH1 provided by this invention was isolated and purified from soil containing rice roots infested by root-knot nematodes (a type of grass). The *Arthrobacter* p. ZJUHUYJH1 of this invention has the following biological characteristics: after culturing on LB agar plates at 30°C for 2 days, single colonies of this strain are milky white, with a smooth surface and regular edges. The bacteria are rod-shaped, aggregated together in a beaded arrangement, and show Gram-positive staining. The 16S rDNA sequence of *Arthrobacter* p. ZJUHUYJH1 is 1419 bp in length, and the specific sequence is shown in SEQ ID NO. 1.

[0024] This invention provides the application of the aforementioned Arthrobacter ZJUHUYJH1 in the control of root-knot nematodes of the Poaceae family.

[0025] In this invention, *Arthrobacter ZJUHUYJH1* is lethal to *Poaceae* root-knot nematodes. Both the bacterial suspension and fermentation supernatant of *Arthrobacter ZJUHUYJH1* are lethal to *Poaceae* root-knot nematodes, with the bacterial suspension showing a stronger lethal effect than the fermentation supernatant. In this invention, *Arthrobacter ZJUHUYJH1* can reduce the number of root knots and the root knot-to-dry weight ratio in rice infected with *Poaceae* root-knot nematodes.

[0026] This invention also provides the application of *Arthrobacter ZJUHUYJH1* in improving the resistance of rice to root-knot nematodes of the Poaceae family. In this invention, *Arthrobacter ZJUHUYJH1* can increase the content of defensive hormones in rice roots; the defensive hormones preferably include 12-oxophytic dienoic acid.

[0027] This invention provides a biocontrol agent for controlling root-knot nematodes of the Gramineae family, comprising *Arthrobacter* ZJUHUYJH1. In this invention, the preferred concentration of *Arthrobacter* ZJUHUYJH1 is 1–10 × 10⁻⁶. 8 CFU / mL. This invention does not specifically limit the composition of the biocontrol agent, and it may also include other components such as a carrier. In this invention, the biocontrol agent is preferably a liquid agent, and the preferred method of application is root drenching; the preferred dosage for root drenching is 5–15 mL / plant, more preferably 8–12 mL / plant, and most preferably 10 mL / plant. This invention does not specifically limit the timing of application of the biocontrol agent.

[0028] The technical solutions provided by the present invention will be 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 tested strain was isolated from rice root soil infected with root-knot nematodes (a species of grass). Strain ZJUHUYJH1 was isolated on LB agar using the plate dilution method. The specific steps are as follows:

[0033] 1) Collect rhizosphere soil: 21 days after inoculating the roots of Nipponbare rice with root-knot nematodes of the Poaceae family, collect the rice root soil, shake off the loose surface soil, and collect the remaining soil attached to the roots as rhizosphere soil.

[0034] 2) Preparation of soil dilution: Weigh 1g of soil and place it in an Erlenmeyer flask containing 10mL of sterile water; shake on a shaker at 200r / min for 30 minutes to thoroughly mix the soil and water and completely disperse the bacteria. Add 100μL of the soil suspension to a centrifuge tube containing 900μL of sterile water, dilute 10 times, and mix thoroughly by shaking. Repeat this process to prepare 10... -1 10 -2 10 -3 10 -4 10 -5 10 -6 and 10 -7 The diluted soil solution is ready for use.

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

[0036] 4) Incubation: Invert the plate in a 30℃ constant temperature incubator and incubate the bacteria for 48 hours.

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

[0038] The entire operation must strictly adhere to aseptic principles to avoid contamination by other microorganisms.

[0039] 2. Identification of Arthrobacter ZJUHUYJH1

[0040] Purified single colonies were picked and dissolved in 100 μL of sterile water in a PCR tube, and incubated at 98°C for 5 min in a PCR instrument as templates. The templates were amplified using universal 16S rRNA primers 27F and 1492R. PCR conditions were: 98°C pre-denaturation for 45 s, 98°C denaturation for 10 s, 60°C annealing for 5 s, 72°C extension for 2 min, 31 cycles, followed by a final extension at 72°C for 2 min. The PCR products were purified and recovered by 1% agarose gel electrophoresis and sent to Zhejiang Shangya Biotechnology Co., Ltd. for sequencing.

[0041] Sequencing results showed that the 16S rRNA sequence amplified by strain ZJUHUYJH1 was 1419 bp in length, and its nucleotide sequence is shown in SEQ ID NO.1, as follows:

[0042]

[0043] PCR product sequencing results were analyzed using BLAST alignment in the NCBI database. A phylogenetic tree was constructed using MEGA11 to determine taxonomic positions. Results are as follows: Figure 2 As shown.

[0044] Morphological observation of the strain showed that single colonies of the strain were milky white, with smooth surfaces and regular edges, and were arranged in a beaded pattern when aggregated together. Gram staining was positive.

[0045] Example 2

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

[0047] 1. Preparation of fermentation supernatant and cell suspension of strain ZJUHUYJH1

[0048] Pick a loopful of bacterial culture from a cryopreserved tube containing 30% glycerol using an inoculation loop, streak it onto an LB agar plate, and incubate upside down for 48 hours. Observe the colony growth on the plate to ensure the strain is not contaminated. Pick a single colony obtained after isolation and inoculate it into a shaker tube containing 4 ml of LB liquid medium. Place the tube in a shaker at 30°C and shake at 180 rpm for 24 hours until the liquid becomes turbid and the OD value of the bacterial culture is between 1.0 and 1.5. Transfer 2 ml of the shaker culture to a 2 ml sterile centrifuge tube and centrifuge at 6000 rpm for 4 minutes. Collect the supernatant, filter it through a 0.22 μm filter to obtain the fermentation supernatant. After removing the supernatant, wash the tube three times with sterile water, resuspend the bacterial culture in sterile water, and adjust the OD value of the bacterial culture to 1.0 to obtain the bacterial suspension.

[0049] 2. Preparation of suspension of second-instar larvae of root-knot nematodes

[0050] Rice seedlings were transplanted into diseased soil and cultured in a greenhouse for 25–40 days. The rice seedlings were then pulled out, their roots thoroughly rinsed, chopped, and placed in a collection device with water. They were incubated at room temperature for one week to obtain root-knot nematodes (Pleistocene nematodes). Second-instar larvae were isolated using the Bellman funnel method. The nematodes were then sterilized by soaking in a solution containing 200 mg / L streptomycin sulfate for 4 hours, followed by washing 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 effects on root-knot nematodes of the Poaceae family were investigated and observed in 96-well plates. 100 μL of root-knot nematode suspension was added to each 96-well plate, with 30–50 nematodes per well. 100 μL of fermentation supernatant / cell suspension was added. Each group was replicated in 4 steps. The control group was treated with an equal volume of LB medium / sterile water.

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

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

[0057] Mortality rate (%) = Number of nematode deaths / 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 had a significant lethal effect on root-knot nematodes of the Poaceae family. At 24 h, most of the nematodes in the bacterial suspension treatment group were in a rigid state, with only 2-3 nematodes surviving, while the nematodes in the control group remained 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. At 9 h, the nematodes in the treatment group with added fermentation supernatant moved slowly, with discontinuous movement trajectories, and even showed signs of stagnation. At 12 h, 11-18 nematodes were in a rigid state, while only 5-6 nematodes in the control group were in a rigid state, with a corrected mortality rate of 28.8%.

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

[0062]

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

[0064]

[0065] Example 3

[0066] Greenhouse pot 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℃ for 30 minutes to eliminate most microbial influences, resulting in sterile soil, which was then evenly distributed into 200mL cylindrical cups. The rice variety used was Nipponbare (a nematode-sensitive variety). Before germination, the rice seeds were disinfected by soaking in a 1 / 1000 concentration of prochloraz and then germinated in a 30℃ light incubator for 10 days, with the water changed daily. After 10 days of germination, the rice was transferred to seedling trays, and once it reached the seedling stage, it was transplanted into the cylindrical cups. After 14 days of growth, a bacterial suspension was applied to the roots, using 10ml of a bacterial suspension with an OD of 1.0 per pot (the fermentation medium contains high concentrations of salt and sugar, which may damage plant roots; therefore, the supernatant of the fermentation broth was removed before application, and the bacterial suspension was resuspended in sterile water and adjusted to OD 1.0 to obtain the bacterial suspension). The control treatment was inoculated with an equal volume of sterile water. Forty-eight hours after drenching the roots with the bacterial solution, holes were made around the rhizosphere soil of each rice plant, and 200 *Poaceae* root-knot nematodes J2 were inoculated into each pot. Seven days after inoculation, rice roots were collected to determine the number of root knots. The rice roots were then dried in a 65℃ oven for 48 hours, and the corresponding dry weight of the underground portion of each rice plant was measured to obtain the root knot number-to-dry weight ratio.

[0068] The results are as follows Figure 4 As shown, the number of root knots in the treatment group inoculated with the bacterial suspension of strain ZJUHUYJH1 decreased by 43% compared with the control group, and the number of root knots in the treatment group was significantly lower than the dry weight compared with 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. After inoculation with nematodes for 24 hours, rice roots were collected, rapidly frozen in liquid nitrogen for 30 minutes, and then ground to obtain rice root powder. An internal standard solution was prepared in advance, using 100% methanol as the solvent. The final internal standard solution contained deuterated jasmonic acid-D5 (d5-JA). 13 C-labeled jasmonic acid-isoleucine ( 13C6-JA-Ile), deuterated salicylic acid-D4 (d4-SA), deuterated abscisic acid-D6 (d6-ABA), and deuterated indoleacetic acid-D5 (d5-IAA) were each 100 ng / mL. A mixed standard was then prepared, with each 1 mL of the mixed standard containing 990 μL of ethyl acetate as the extraction buffer and 10 μL of internal standard solution. Weigh 0.1g of rice root powder, add 1mL of mixed standard to each 0.1g of rice root powder, vortex for 10min, centrifuge at 12000rpm for 20min at 4℃, and transfer the supernatant to a new 2mL centrifuge tube. Concentrate the plant hormones by rotary evaporation for 35min. Finally, add 200μL of 70% methanol to each concentrated centrifuge tube, vortex for 5min at 4℃, centrifuge at 13000rpm for 10min, and transfer 100μL of the supernatant to a 2mL sample vial containing a 250μL inner tube. This method is used to extract indoleacetic acid, salicylic acid, abscisic acid, 12-oxophytic dienoic acid, jasmonic acid, and jasmonic acid-isoleucine complex from the rice root powder. The results are analyzed by UHPLC MS / MS to obtain the effect of SC-R-1 bacterial suspension treatment on hormone content in rice under nematode infection conditions. Each treatment was replicated 6-8 times. Results are as follows: Figure 5 As shown.

[0072] The results showed that treatment with Arthrobacter ZJUHUYJH1 bacterial suspension under nematode infection conditions could promote the synthesis of hormones related to root defense in rice. Compared with the control group without bacterial agent, the content of 12-oxophytic dienoic acid in rice roots after 24 h 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 rice roots.

[0073] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Arthrobacter sp. (ZJUHUYJH1), characterized in that, Arthrobacter sp. The accession number is CCTCC NO: M2025922. ​ 2. The application of Arthrobacter ZJUHUYJH1 as described in claim 1 in the control of root-knot nematodes of the Poaceae family.

3. Use according to claim 2, characterized in that, The Arthrobacter ZJUHUYJH1 strain is lethal to root-knot nematodes of the Poaceae family.

4. The application of Arthrobacter ZJUHUYJH1 as described in claim 1 in improving the resistance of rice to root-knot nematodes of the Poaceae family.

5. Use according to claim 4, characterized in that, The *Arthrobacter* ZJUHUYJH1 strain can increase the content of 12-oxophytic acid, a root defense hormone, in rice roots infected with *Poaceae* root-knot nematodes.

6. A biocontrol agent for controlling root-knot nematodes of the family Longidoridae, characterized by comprising the bacteria of claim 1. Includes Arthrobacter ZJUHUYJH1 as described in claim 1.

7. The biocontrol agent of claim 6, wherein, The Arthrobacter ZJUHUYJH1 is used at a concentration of 1-10 x 10 8 CFU / mL.

8. The biocontrol agent of claim 7, characterized in that, The method of using the biocontrol agent includes root irrigation.

9. The biocontrol agent of claim 8, characterized in that, The dosage for root drenching is 5-15 mL per plant.

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