Rhodococcus ZJUHULJT1 and application thereof in prevention and treatment of paratrichoder root knot nematode

Abstract

The application provides a strain of Rhodococcus ZJUHULJT1 and application of the strain in prevention and treatment of paratyphaceae root-knot nematodes, and belongs to the technical field of biological prevention and treatment of root-knot nematodes. Rhodococcus The Rhodococcus (sp.) ZJUHULJT1 is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO: M 20251182; the Rhodococcus ZJUHULJT1 can significantly inhibit the infection and reproduction of root-knot nematodes, reduce the number of rice root knots, promote the synthesis of defense-related hormones of rice roots, and improve the disease resistance of rice; through the multiple inhibition of paratyphaceae root-knot nematodes, the Rhodococcus ZJUHULJT1 can be applied to the prevention and treatment of rice root-knot nematodes, has good prevention and treatment effect, and provides a new technical means for the pollution-free reduction of paratyphaceae root-knot nematode diseases.

Description

Technical Field

[0001] This invention belongs to the field of biological control technology of root-knot nematodes, and particularly relates to a strain of Rhodococcus ZJUHULJT1 and its application in controlling root-knot nematodes of the Poaceae family. Background Technology

[0002] Root-knot nematodes ( Meloidogyne Root-knot nematodes (Spp.) are serious plant pathogenic nematodes that widely damage various crops such as rice, corn, and vegetables. Especially in rice-growing areas of Asia, Africa, and Latin America, root-knot nematode disease has become a significant factor restricting food production. Root-knot nematodes infect plant roots, inducing the formation of root knots, disrupting the root's absorption function, leading to stunted plant growth, reduced nutrient absorption capacity, and ultimately causing stunted growth, yellowing leaves, significantly reduced yield, and even crop failure. Traditional control methods for root-knot nematodes mainly rely on chemical pesticides, such as fumigants and non-fumigant nematicides. Furthermore, the overuse of chemical pesticides has led to increasingly serious problems of pesticide resistance in root-knot nematodes. The emergence of resistant populations has significantly reduced the effectiveness of chemical control, further exacerbating the difficulty of disease control. Therefore, developing green, environmentally friendly, efficient, and sustainable biological control methods has become a research hotspot and urgent need in the field of agricultural disease control.

[0003] In recent years, research on the use of beneficial microorganisms for plant disease control has received widespread attention. Studies have shown that certain indigenous microorganisms, such as Bacillus, Actinomycetes, and fungi, can effectively inhibit pathogen infection by secreting antimicrobial substances (such as chitinases, proteases, and lipopeptide compounds), competitively inhibiting the growth of pathogenic microorganisms, and inducing systemically acquired resistance (SAR) or locally acquired resistance (LOA) in plants. For example, Bacillus subtilis , Pseudomonas fluorescens Strains have been shown to have good antagonistic effects against a variety of plant pathogens and have demonstrated certain application potential in agricultural production. However, despite the progress made in the application of microorganisms in plant disease control, many challenges remain in the research on the biological control of root-knot nematodes. First, the current screening of microbial strains with anti-root-knot nematode activity mainly relies on in vitro antimicrobial tests and field trials, which have low screening efficiency and the field effects of strains are often unstable. Second, the mechanisms of action of microorganisms are not yet fully understood, and the insect-resistant active ingredients and their targets of action of many strains still require further research. In addition, there is limited exploration and research on the rice rhizosphere microbial community, especially in its application research in the control of grass-like root-knot nematodes, where there is still a significant gap.

[0004] Especially for root-knot nematodes of the Poaceae family ( Meloidogyne graminicolaResearch on the biological control of root-knot nematodes in rice still lacks efficient, stable, and easily scalable microbial control solutions. Existing studies have shown that although some microbial strains exhibit good anti-root-knot nematode activity under greenhouse conditions, their control effects often fall short of expectations in practical applications due to the complexity of environmental conditions and the diversity of pathogens. Therefore, screening strains with highly efficient anti-root-knot nematode activity from the rice rhizosphere and conducting in-depth research on their mechanisms of action is of significant theoretical and practical value for developing novel biological control agents and achieving sustainable green agriculture. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a strain of Rhodococcus ZJUHULJT1 and its application in the control of root-knot nematodes of the Poaceae family; the Rhodococcus ZJUHULJT1 can significantly inhibit the infection and reproduction of root-knot nematodes, reduce the number of root knots in rice, and improve the disease resistance of rice.

[0006] This invention provides a strain of Rhodococcus ( Rhodococcus sp.) ZJUHULJT1, deposited at the China Center for Type Culture Collection, accession number CCTCC NO: M 20251182.

[0007] This invention provides the application of Rhodococcus ZJUHULJT1 in the control of root-knot nematodes of the Poaceae family.

[0008] Preferably, the Rhodococcus ZJUHULJT1 bacterium has a significant lethal effect on root-knot nematodes of the Poaceae family.

[0009] Preferably, the Rhodococcus ZJUHULJT1 can promote the synthesis of 12-oxophytic dienoic acid and jasmonic acid in crop roots.

[0010] Preferably, the Rhodococcus ZJUHULJT1 significantly inhibits the invasion of root-knot nematodes of the Poaceae family and reduces the number of root knots.

[0011] This invention provides a rice biocontrol agent, comprising the aforementioned Rhodococcus ZJUHULJT1.

[0012] Preferably, the concentration of Rhodococcus ZJUHULJT1 used is 1×10⁻⁶. 5 ~1×10 8 CFU / mL.

[0013] This invention provides a method for controlling rice root-knot nematodes using the aforementioned biocontrol agent, wherein the biocontrol agent is applied to the roots of rice plants.

[0014] Preferably, the method of action includes root irrigation, and the amount of biocontrol agent used is 5-15 ml / plant / time.

[0015] This invention provides the application of Rhodococcus ZJUHULJT1 in promoting the synthesis of rice root defense-related hormones, which include 12-oxophytic dienoic acid and jasmonic acid.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The present invention isolates and purifies Rhodococcus ZJUHULJT1 from rice root soil infested with Gramineae root-knot nematodes; the bacterial suspension of this strain has a significant direct inhibitory effect on Gramineae root-knot nematodes; infection experiments show that after inoculation with Rhodococcus ZJUHULJT1 bacterial suspension, the number of root knots per unit dry weight of roots decreased by 82.3% compared with the control, which can significantly inhibit the invasion of Gramineae root-knot nematodes; compared with the control group, the treatment group with Rhodococcus ZJUHULJT1 bacterial suspension, after 24 h of nematode suspension addition, can promote the synthesis of rice root defense-related hormones 12-oxophytic dienoic acid (OPDA) and jasmonic acid (JA), indicating that Rhodococcus ZJUHULJT1 can also inhibit Gramineae root-knot nematode infection by increasing the content of rice root defense-related hormones. In summary, the Rhodococcus ZJUHULJT1 strain exhibits multiple inhibitory effects against root-knot nematodes of the Poaceae family and can be applied to the control of rice root-knot nematodes with good control efficacy. Attached Figure Description

[0017] Figure 1 The colony morphology of Rhodococcus ZJUHULJT1 on R2A solid medium is shown.

[0018] Figure 2 Phylogenetic tree of Rhodococcus ZJUHULJT1;

[0019] Figure 3 Corrected lethality of Rhodococcus ZJUHULJT1 bacterial suspension against nematodes;

[0020] Figure 4 The inhibitory effect of Rhodococcus ZJUHULJT1 on root knot infection;

[0021] Figure 5 To analyze the relative fold increase of hormone expression in rice roots induced by Rhodococcus ZJUHULJT1 and its correlation with infection inhibition rate.

[0022] Biological Preservation Instructions

[0023] Rhodococcus ( Rhodococcus sp.) ZJUHULJT1 is deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, on May 26, 2025, with accession number CCTCC NO: M 20251182. Detailed Implementation

[0024] This invention provides a strain of Rhodococcus ( Rhodococcus sp.) ZJUHULJT1, deposited at the China Center for Type Culture Collection, accession number CCTCC NO: M 20251182.

[0025] The Rhodococcus ZJUHULJT1 strain described in this invention was isolated and screened from the rhizosphere soil of infected rice plants. It exhibits activity against root-knot nematodes. The Rhodococcus ZJUHULJT1 strain possesses the following biological characteristics: after culturing on oligotrophic R2A medium at 30°C for one day, it forms orange-yellow circular colonies with a flat, moist surface and a mucous interior, measuring approximately 0.8–1.6 mm in diameter, and is Gram-positive. The Rhodococcus ZJUHULJT1 strain significantly inhibits the infection and reproduction of root-knot nematodes, reduces the number of root knots in rice, and improves the disease resistance of rice. The Rhodococcus ZJUHULJT1 strain can be used to prepare biological mitigation agents, which are environmentally friendly, highly efficient, and safe, providing a new approach for the pollution-free reduction of root-knot nematode disease in rice plants.

[0026] The present invention also provides the application of the aforementioned Rhodococcus ZJUHULJT1 in the control of root-knot nematodes of the Poaceae family.

[0027] In this invention, the mechanism of action of Rhodococcus ZJUHULJT1 in controlling root-knot nematodes of the Poaceae family is as follows: 1. Rhodococcus ZJUHULJT1 has a significant direct lethal effect on root-knot nematodes of the Poaceae family; 2. Rhodococcus ZJUHULJT1 can promote the synthesis of rice root defense hormones 12-oxophytic acid and jasmonic acid, thereby increasing rice resistance; In this invention, Rhodococcus ZJUHULJT1 significantly inhibits the invasion of root-knot nematodes of the Poaceae family and reduces the number of root knots in rice.

[0028] This invention provides a rice biocontrol agent, comprising the aforementioned Rhodococcus ZJUHULJT1.

[0029] In this invention, the rice biocontrol agent is a liquid or solid inoculant, and the preferred concentration of Rhodococcus ZJUHULJT1 is 1×10⁻⁶. 5 ~1×10 8 CFU / mL. This invention does not specify a particular method for preparing the rice biocontrol agent; any preparation method known in the art may be used.

[0030] The present invention also provides a method for controlling rice root-knot nematodes using the aforementioned biocontrol agent, wherein the biocontrol agent is applied to the roots of rice.

[0031] In this invention, the preferred method of action includes root irrigation, and the preferred time for root irrigation is during the sowing period and the seedling period; the preferred number of times for root irrigation is to apply 1 to 2 times each during the sowing period and the seedling period, with an interval of 7 to 10 days between each application; the amount of biocontrol agent used is 5 to 15 mL / plant / application.

[0032] This invention also provides the application of Rhodococcus ZJUHULJT1 in promoting the synthesis of rice root defense-related hormones, which include 12-oxophytedienoic acid and jasmonic acid. Rhodococcus ZJUHULJT1 can increase the content of rice root defense-related hormones, thereby enhancing rice resistance and inhibiting the infection of root-knot nematodes of the Poaceae family.

[0033] 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.

[0034] Example 1

[0035] Isolation and identification of target strains

[0036] 1.1 Isolation of strains

[0037] During the vigorous growth period of rice infected with root-knot nematodes, the soil around the roots of rice plants was slowly excavated to a depth of 0-10 cm, ensuring the roots were intact and undamaged. After collection, loose soil particles on the root surface were immediately shaken off to reduce interference from external microorganisms. The shaken rice roots were then placed in a 50 mL sterile centrifuge tube containing 25 mL of phosphate-buffered saline (PBS). The tube was shaken at 2000 rpm for 1 min to fully release microorganisms from the root surface into the PBS buffer. The roots were then carefully removed, and the washing process was repeated once to ensure thorough elution of microorganisms from the root surface. The PBS buffers from both washings were combined. The combined PBS buffer was centrifuged at 5000 rpm for 5 min at 4°C, and the supernatant was removed to obtain the rice rhizosphere soil. 1 g of the treated rhizosphere soil was weighed and transferred to a 50 mL sterile centrifuge tube. 10 mL of sterile water was added, and the mixture was vortexed until homogeneous to prepare a soil suspension. This suspension was then serially diluted to a final concentration of 10%. -6 The diluted soil suspension was evenly spread onto R2A oligotrophic medium plates using a sterile spreader. After the surface of the medium dried, it was incubated at a constant temperature of 30°C until single colonies with clearly visible morphological characteristics appeared. Using a sterile inoculation loop, the picked single colonies were purified by streak plating. This process was repeated 2-3 times to ensure the acquisition of pure cultures of single colonies with the following morphology: Figure 1As shown, the surface is flat and moist, the interior of the colony is mucous, and the diameter is about 0.8 ~ 1.2 mm.

[0038] 1.2 Identification of the target strain

[0039] Single colonies obtained in the above steps were picked and dissolved in 100 μL of sterile water in a PCR tube. The mixture was incubated at 95°C for 10 min in a PCR instrument as a template. PCR amplification was performed on the template using full-length 16S primers 27F and 1492R. The PCR reaction was performed using the Takara Primer Star kit (purchased from Baori Biotechnology (Beijing) Co., Ltd.). The 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; 72°C extension for 2 min. The PCR amplification products were sent to Zhejiang Shangya Biotechnology Co., Ltd. for sequencing. The sequencing results showed that the 16S RNA nucleotide sequence of this strain is as follows (SEQ ID NO.1, length 1388 bp):

[0040]

[0041] Based on the 16S rRNA Blast identification and comparison results of the strain (Table 1), the strain described in this invention was identified by bioinformatics analysis as... Rhodococcus Genus. BLAST analysis showed that the amino acid sequence of this strain is similar to that known in the NCBI database. Rhodococcus aetherivorans and Rhodococcus ruber The species exhibits high similarity, with sequence alignment percentages ranging from 99.353% to 99.927%, alignment lengths from 1332 to 1391, and E-values ​​all less than 0.001, indicating highly significant matching results. Therefore, there is a >99% probability that this strain belongs to the genus *Rhodococcus*. Rhodococcus ), and named it Rhodococcus sp. ZJUHULJT1, phylogenetic tree as follows Figure 2 As shown.

[0042] Table 1. Results of 16S rRNA Blast identification and comparison of strains.

[0043]

[0044] Example 2

[0045] Rhodococcus Determination of the direct effect of sp. ZJUHULJT1 on nematodes

[0046] 2.1 Isolation of sterile nematodes

[0047] Using a sterile inoculating loop, pick a loopful of bacterial culture from a cryopreserved tube containing 30% glycerol and inoculate it into a sterile centrifuge tube containing 4 mL of R2A liquid medium. Incubate the centrifuge tube at 30°C and 200 rpm for 24 h, until the OD of the bacterial culture reaches its maximum. 600 The concentration reached 1.5. The cultured bacterial suspension was centrifuged at 5000 rpm for 5 min, and the supernatant was removed. The cells were washed twice with sterile PBS buffer, followed by three washes with sterile 0.085% NaCl solution. Finally, the bacterial concentration was adjusted to OD0.05. 600 =1.0, get 10 8 CFU / mL bacterial suspension. Rice roots infected with *Poaceae* root-knot nematodes were selected, chopped, and placed in a sterile collection device. The nematodes were incubated at 28°C. Freshly hatched second-instar larvae (J2s) of the *Poaceae* root-knot nematodes were collected daily and stored at 4°C for later use. The collected nematode suspension was soaked in a sterile solution containing 200 mg / L streptomycin sulfate for 4 h, followed by washing three times with sterile 0.085% NaCl solution to obtain sterile nematodes.

[0048] 2.2 Corrected lethality determination

[0049] Sterile nematodes were dissolved in sterile water and adjusted to a concentration of 1000 nematodes / mL to prepare a root-knot nematode suspension. 50 µL of the root-knot nematode suspension was added to each well of a sterile 96-well plate. Subsequently, 50 µL of sterile water (as a control) or 50 µL of bacterial suspension (OD200) was added, respectively. 600 =1 (as the treatment group), ensuring a total volume of 100 µL per well. After sample addition, the number of dead nematodes was observed and recorded at 0 h and 24 h using an inverted phase-contrast microscope. The nematode survival rate after 24 h was calculated, and the corrected lethality calculation formula is as follows:

[0050] Corrected mortality rate = ×100%

[0051] Microscopic observation revealed that the addition of bacteria significantly reduced the number of surviving root-knot nematodes (results shown in Figure 1). Figure 3 As shown in the figure, the average lethality rate reached 17.73%. The experimental results show that the bacteria have a significant lethal effect on root-knot nematodes. The experimental results verify that the screened strains have significant anti-root-knot nematode activity, providing a scientific basis for further development of biological nematode control agents.

[0052] Example 3

[0053] Rhodococcus Effects of sp. ZJUHULJT1 on rice's resistance to root-knot nematodes (Poaceae)

[0054] 3.1 Rice seedling stage treatment

[0055] The rice variety Nipponbare (Japan) was used to control root-knot nematodes of the Poaceae family. Oryza sativa Using *C. Nipponbare* as the experimental material, rice seeds were soaked in sterile water and placed in a 30°C light incubator for germination. The sterile water was changed daily for 10 days until germination. After germination, the germinated rice seeds were transplanted into sterile seedling trays containing sterile Hoagland's nutrient solution and placed in a greenhouse for cultivation. Greenhouse cultivation conditions were: a 14 h / 10 h light / dark cycle, a temperature of 28°C / 25°C, and approximately 60% humidity. Once the rice seedlings reached a suitable transplanting stage, they were transplanted into black flowerpots (10 cm long, 10 cm wide, and 20 cm high) containing sterile sand. After transplanting, regular watering and replenishment with sterile Hoagland's nutrient solution were maintained to keep the soil moist and ensure normal rice growth. Cultivation conditions were: a 14 h / 10 h light / dark cycle, a temperature of 28°C / 25°C, and approximately 60% humidity.

[0056] 3.2 Root irrigation with bacterial solution and nematode inoculation

[0057] Will Rhodococcus sp. ZJUHULJT1 strain was inoculated into sterile liquid culture medium and cultured until the bacterial cell concentration reached OD500. 600 =1.0. Using a sterile pipette, 10 mL of the bacterial suspension was evenly sprinkled onto the surface of the rice rhizosphere soil, ensuring full contact between the bacterial solution and the roots. Forty-eight hours after root irrigation, holes were made around the rhizosphere soil of each rice plant using a sterile pipette, and 200 pre-infection second-instar larvae (J2s) of the grass-like root-knot nematode were inoculated into each pot. After inoculation, the plants were kept in a greenhouse under the same light, temperature, and humidity conditions. Seven days after nematode inoculation, the rice plants were carefully removed, and the roots were washed to remove sand and impurities, collecting the rice roots. Subsequently, the number of root knots on the rice roots was counted (results are shown in Figure 1). Figure 4 As shown in the figure, sterile water was applied as a control. The infection inhibition rate was calculated using the following formula:

[0058] Infection inhibition rate = ×100%

[0059] By comparing the ratio of root knot number to root dry weight, it was found that... Rhodococcus The sp. ZJUHULJT1 strain achieved an average infection inhibition rate of 82.3% against root-knot nematodes, indicating that this strain, screened from rice rhizosphere soil, has an anti-root-knot nematode effect, providing a new technical means for the pollution-free reduction of root-knot nematode diseases in the Poaceae family.

[0060] Example 4

[0061] application Rhodococcus Effects of sp. ZJUHULJT1 on rice root hormones

[0062] Following the method described in Example 3, rice inoculation and cultivation were completed. Rice roots were rapidly collected 24 h after inoculation with a suspension of *Poaceae* root-knot nematodes. The collected roots were immediately flash-frozen in liquid nitrogen and then transferred to a -80°C freezer for storage. Two treatment groups were set up in the experiment: one group received the bacterial suspension treatment (adding...). Rhodococcus One group received a bacterial suspension of *Sp. ZJUHULJT1*, while the other group served as a control (treated with sterile water only). One hour after treatment, root samples were taken from both the treated and untreated sides to determine hormone content. Rice roots were rapidly frozen in liquid nitrogen for 30 minutes, then ground to obtain root powder. An internal standard solution containing deuterated jasmonic acid was prepared using 100% methanol. D5 (d5) JA), carbon-13 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) The concentrations of jasmonic acid (IAA), etc., were all 100 ng / mL. Each 1 mL of the mixed standard contained 990 μL of ethyl acetate and 10 μL of internal standard solution. 0.1 g of rice root powder was weighed, added to 1 mL of the mixed standard, vortexed for 10 min, centrifuged at 12000 rpm for 20 min at 4℃, and the supernatant was collected and concentrated in a rotary evaporator for 35 min. 200 μL of 70% methanol was added and vortexed for 5 min. The mixture was centrifuged at 13000 rpm for 10 min at 4℃, and 100 μL of the supernatant was transferred to a sample vial. The contents of hormones such as jasmonic acid, jasmonic acid isoleucine, and abscisic acid in the sample were determined using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-QqQ-MS / MS). Eight replicates were performed for each treatment. The relative folds of each hormone were calculated, and the results are shown below. Figure 5 As shown.

[0063] The results showed that the relative fold increases of OPDA and JA in the roots on the side of the fungicide application were significantly increased (P < 0.05), 1.42-fold and 1.46-fold respectively compared to the control group. The expression levels of OPDA and JA were significantly positively correlated with the infection inhibition rate (number of root knots / root dry weight) (P < 0.05), with Mantel correlation coefficients of 0.185 and 0.358, respectively. This strain significantly enhanced resistance to root-knot nematodes of the Poaceae family by inducing the expression of OPDA and JA in rice roots. The upregulation of these two hormones was significantly positively correlated with the increase in root knot number and root dry weight, indicating that the strain can effectively inhibit root-knot nematode infection by activating the jasmonic acid signaling pathway and improving plant defense capabilities.

[0064] As can be seen from the above embodiments, the Rhodococcus ZJUHULJT1 provided by the present invention can significantly inhibit the infection and reproduction of root-knot nematodes, reduce the number of root knots in rice, and improve the disease resistance of rice. Through multiple inhibitory effects on root-knot nematodes of the Poaceae family, Rhodococcus ZJUHULJT1 can be applied to control rice root-knot nematodes and has a good control effect.

[0065] 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. A strain of Rhodococcus sp. ZJUHULJT1, characterized in that, Rhodococcus sp. ) ZJUHULJT1, characterized in that, The Rhodococcus is preserved in China Center for Type Culture Collection, and the preservation number is CCTCC NO: M 20251182.

2. The Rhodococcus ZJUHULJT1 in claim 1 is applied to control Meloidogyne graminicola.

3. Use according to claim 2, characterized in that, The Rhodococcus ZJUHULJT1 has a significant lethal effect on Meloidogyne graminicola.

4. The use according to claim 2, characterized in that, The Rhodococcus ZJUHULJT1 can promote the synthesis of 12-oxophytodienoic acid and jasmonic acid in rice roots.

5. The use according to any one of claims 2 to 4, characterized in that, The Rhodococcus ZJUHULJT1 significantly inhibits the invasion of Meloidogyne graminicola and reduces the number of rice root nodules.

6. A biocontrol agent for rice, characterized by comprising the bacterium according to any one of claims 1 to 5. The Rhodococcus ZJUHULJT1 in claim 1 is included.

7. The biocontrol agent for rice according to claim 6, characterized by, The concentration of the Rhodococcus ZJUHULJT1 used is 1 x 10 5 CFU / mL. 8 CFU / mL.

8. The method for controlling the infection of rice by Meloidogyne graminis with the biocontrol agent according to claim 6 or 7, characterized in that, The biocontrol agent is applied to the rice roots.

9. The method of claim 8, wherein, The application mode includes root irrigation, and the use amount of the biocontrol agent is 5-15 mL / plant / time.

10. The method of claim 8, wherein, The biocontrol agent promotes the synthesis of defense-related hormones in rice roots infected by Meloidogyne graminicola, and the defense-related hormones in rice roots are 12-oxophytodienoic acid and jasmonic acid.

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