Nicosulfuron pseudomonas and application thereof in prevention and treatment of pepper meloidogyne incognita
Through the root irrigation treatment of Pseudomonas nicosulfuron MiC45 strain, the prevention and control problem of root knot nematode disease in southern chili was solved, and the effect of efficient prevention and control and promoting pepper growth was achieved, while environmentally friendly.
Patent Information
- Application Number
- CN202311838429.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-12-28
- Publication Date
- 2025-07-01
AI Technical Summary
The prior art is difficult to effectively prevent and control the root knot nematode disease of chili pepper, and the conventional methods are inefficient, costly and prone to environmental pollution.
The MiC45 strain of Pseudomonas nicosulfuron was used to prevent and control the pepper plants through root irrigation treatment, and a fermentation broth of OD600 was prepared with OD600 not less than 1 was processed the next day, with no less than 5mL of each plant, and no less than 3 times.
Pseudomonas nicosulfuron has high linear killing activity on southern root knot nematodes, which can effectively reduce nematodes' infestation on the root system of pepper, alleviate diseases and promote pepper growth, improve root system and above ground growth, and is pollution-free to the environment.
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Figure CN120230660A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of preventing and controlling Meloidogyne incognita, and relates to Pseudomonas nicosulfuron and its application in preventing and controlling Meloidogyne incognita on chili peppers, specifically to Pseudomonas nicosulfuron MiC45 and its application in preventing and controlling Meloidogyne incognita on chili peppers. Background Art
[0002] Root-knot nematodes ( Meloidogyne spp.) are a type of pathogenic nematodes with specialization characteristics. They form soil-borne diseases by parasitizing on plant roots, and the plant diseases caused by them are difficult to control and prone to recurrence. The host range of plant nematodes is very wide, and they can damage a variety of crops, such as cucumbers, tomatoes, Chinese cabbages, radishes, peanuts, etc. It is estimated that plant parasitic nematodes cause economic losses of up to $157 billion to global agriculture every year. Among them, Meloidogyne incognita ( Meloidogyne incognita ) has strong adaptability, is most widely distributed in tropical and subtropical regions of the world, and causes the most serious crop diseases.
[0003] China is the largest producer and consumer of chili peppers in the world, and is also one of the countries with the largest chili pepper exports in the world, occupying a dominant position in the international chili pepper industry. Chili peppers are a common cash crop with both edible and medicinal values, but they are extremely susceptible to a variety of plant diseases, and root-knot nematode disease is one of the main diseases. Compared with other types of root-knot nematodes, Meloidogyne incognita is more common in damaging chili peppers. Its larvae infect the roots of chili peppers and form root knots, which hinder the absorption of water and nutrients by chili peppers, resulting in stunted plants, yellowing leaves, fewer tillers, and small fruits, etc., seriously reducing the yield and quality of chili peppers.
[0004] There are already various physical or chemical means for preventing and controlling Meloidogyne incognita diseases in agricultural production. However, due to reasons such as low efficiency, high cost, and easy environmental pollution, their control effects have always been unsatisfactory. Therefore, developing green and economic biological control technologies, such as screening biocontrol strains with direct killing effects on Meloidogyne incognita, has become a current research hotspot. Previous studies have shown that bacteria of the genus Pseudomonas can prevent and control chili pepper susceptible pathogenic bacteria such as Phytophthora infestans, Colletotrichum gloeosporioides, and Botrytis cinerea, and have a certain control effect on Meloidogyne incognita on tomatoes. However, there is no research on bacteria of the genus Pseudomonas derived from plant rhizosphere soil and colonizing in plant rhizosphere for preventing and controlling Meloidogyne incognita on chili peppers. Summary of the Invention
[0005] The technical problem to be solved by the present invention is to overcome the deficiencies of the prior art and provide a Pseudomonas nicosulfuron with high nematicidal activity, which can reduce the infection of Meloidogyne incognita on chili pepper roots, effectively alleviate the Meloidogyne incognita diseases suffered by chili peppers, and can also promote the growth of chili peppers, and its application in preventing and controlling Meloidogyne incognita on chili peppers.
[0006] To solve the above technical problems, the present invention adopts the following technical solutions.
[0007] A strain of Pseudomonas nicosulfuron, and the name of the Pseudomonas nicosulfuron strain is Pseudomonas nicosulfuron MiC45 ( Pseudomonas nicosulfuronedens MiC45), its preservation number in the China Center for Type Culture Collection is CCTCC NO: M 20232540, the address of the preservation unit is located at the China Center for Type Culture Collection (Wuhan University), No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, and the preservation date is December 11, 2023.
[0008] The Pseudomonas nicosulfuron MiC45 strain has the following obvious characteristics: the strain is a Gram-negative bacterium, identified as Pseudomonas by molecular biology, the cells are rod-shaped under the microscope, cultured on an LB agar plate (pH 7.0) for 24 h, forming a light yellow translucent colony, the surface and edge of the colony are smooth, easy to pick, grow well under aerobic conditions, cultured on a PKO modified inorganic phosphorus (tricalcium phosphate) agar plate (pH 7.4 ± 0.2) for 48 h, and a transparent circle is formed around the colony; the optimal growth temperature is 28°C. The 16S rDNA sequence of the strain is as shown in SEQ ID NO.1.
[0009] As a general technical concept, the present invention also provides an application of the above-mentioned Pseudomonas nicosulfuron in controlling Meloidogyne incognita of peppers.
[0010] For the above application, preferably, the application includes the following steps: Prepare a bacterial fermentation broth with an OD 600 not less than 1, perform root irrigation treatment on diseased pepper plants, not less than 5 mL per plant, treat once every other day, and the number of treatments is not less than 3 times.
[0011] For the above application, preferably, the preparation method of the Pseudomonas nicosulfuron fermentation broth includes the following steps: (1) Inoculate Pseudomonas nicosulfuron into an LB liquid medium for activation; (2) Centrifuge the activated bacterial suspension, discard the supernatant, and resuspend with sterile water to obtain the Pseudomonas nicosulfuron fermentation broth.
[0012] For the above application, preferably, in step (1), the inoculation amount of the Pseudomonas nicosulfuron is 1% - 3%, and the activation conditions are: temperature 26°C - 32°C, rotation speed 160 rpm - 220 rpm, constant temperature shaking culture for 24 h - 48 h.
[0013] In the above application, preferably, in step (1), in the LB liquid medium, the concentration of peptone is 10 g / L, the concentration of NaCl is 5 g / L to 10 g / L, the concentration of yeast extract is 5 g / L, the pH is 7.0, and the solvent is water.
[0014] Compared with the prior art, the advantages of the present invention are as follows: The Pseudomonas nicosulfuronis MiC45 of the present invention is derived from the rhizosphere soil of tobacco infected with Meloidogyne incognita. The obtaining method is simple and it is easy to culture. Pseudomonas nicosulfuronis has previously been commonly used as a herbicide for the bioremediation of nicosulfuron-contaminated soil, and its effect on Meloidogyne incognita has not been reported. The Pseudomonas nicosulfuronis MiC45 of the present invention not only has high nematicidal activity against Meloidogyne incognita, can reduce the infection of pepper roots by Meloidogyne incognita, effectively alleviate the Meloidogyne incognita disease suffered by peppers, but also can promote the growth of peppers and improve the growth conditions of roots and above-ground parts. This strain has no adverse effects on plants, animals, humans, etc., and will not cause environmental pollution, and is suitable for popularization and use.
[0015] A Pseudomonas nicosulfuronis, the name of the Pseudomonas nicosulfuronis strain is Pseudomonas nicosulfuronis MiC45 ( Pseudomonas nicosulfuronedens MiC45), its preservation number in the China Center for Type Culture Collection is CCTCC NO: M 20232540, the address of the preservation unit is located at the China Center for Type Culture Collection (Wuhan University), No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, and the preservation date is December 11, 2023. Description of the Drawings
[0016] Figure 1 It is the microscopic morphology diagram of Pseudomonas nicosulfuronis MiC45 in Example 1 of the present invention.
[0017] Figure 2 It is the colony morphology diagram of Pseudomonas nicosulfuronis MiC45 on the LB agar plate in Example 1 of the present invention.
[0018] Figure 3 It is the transparent circle formed by Pseudomonas nicosulfuronis MiC45 on the PKO agar plate in Example 1 of the present invention.
[0019] Figure 4 It is the phylogenetic tree of Pseudomonas nicosulfuronis MiC45 in Example 1 of the present invention.
[0020] Figure 5The nematicidal activity of Pseudomonas nicosulfuronis MiC45 against Meloidogyne incognita (J2s) in Example 1 of the present invention. Among them, A is the morphological diagram of nematodes treated with sterile water, B is the morphological diagram of nematodes treated with 200-fold diluted avermectin solution, and C is the morphological diagram of nematodes treated with MiC45.
[0021] Figure 6 The control effect diagram of Pseudomonas nicosulfuronis MiC45 against Meloidogyne incognita on peppers in Example 2 of the present invention. Among them, A is the phenotypic diagram of pepper roots treated with sterile water (left) and MiC45 (right), and the swollen part is the root knot. B is the columnar statistical chart of the number of root knots of peppers treated with sterile water and MiC45.
[0022] Figure 7 The growth promotion effect diagram of Pseudomonas nicosulfuronis MiC45 on peppers in Example 2 of the present invention. Among them, A is the phenotypic diagram of the above-ground part of peppers treated with sterile water, and B is the phenotypic diagram of the above-ground part of peppers treated with MiC45. Detailed implementation manners
[0023] The present invention will be further described below in conjunction with the accompanying drawings of the specification and specific preferred embodiments, but the protection scope of the present invention is not limited thereby. The materials and instruments used in the following examples are all commercially available.
[0024] Example 1 A Pseudomonas nicosulfuronis of the present invention, and the name of the Pseudomonas nicosulfuronis strain is Pseudomonas nicosulfuronis MiC45 ( Pseudomonas nicosulfuronedens MiC45), and its preservation number in the China Center for Type Culture Collection is CCTCC NO: M 20232540. The address of the preservation unit is located at the China Center for Type Culture Collection (Wuhan University), No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, and the preservation date is December 11, 2023.
[0025] This strain is a Gram-negative bacterium and is identified as Pseudomonas by molecular biology. Its main biological characteristics are as follows: Figure 1 As shown, the bacterial cells are rod-shaped under the microscope; as Figure 2 shown, when cultured on an LB agar plate (pH 7.0) for 24 h, a light yellow semi-transparent colony is formed. The surface and edge of the colony are smooth and easy to pick, and it grows well under aerobic conditions; as Figure 3 shown, when cultured on a PKO modified inorganic phosphorus (tricalcium phosphate) agar plate (pH 7.4 ± 0.2) for 48 h, a transparent circle is formed around the colony; the optimal growth temperature is 28°C. The 16S rDNA sequence of the strain is as shown in SEQ ID NO.1.
[0026] The method for isolation and purification of the Pseudomonas nicosulfuronis strain in this example includes the following steps: (1)Sampling: Soil samples were taken from the rhizosphere of tobacco (but not limited to) in Yuelu District, Changsha City, Hunan Province, and transported back to the laboratory in a timely manner. The soil samples were placed in 50 mL sterile centrifuge tubes and placed in a foam ice box. The temperature of the soil samples was maintained at about 4 °C during transportation.
[0027] (2)Isolation of plant rhizosphere soil microorganisms: Add 30 mL of PBS buffer containing surfactant TWEEN-20 (final concentration 1‰) to a 50 mL centrifuge tube containing plant roots and rhizosphere soil. After tightening the tube cap, vortex for 2 min. Transfer the vortexed soil homogenate to a 15 mL sterile centrifuge tube (10 mL / tube), centrifuge at 3900 rpm at room temperature for 5 min, discard the supernatant, add 2 mL of PBS buffer without TWEEN-20 to the tube, resuspend by pipetting, and serially dilute the resuspended solution to 10 -4 times. Take 50 μL of the diluted solution and spread it on a PKO agar plate, and incubate at 28 °C for 3 d.
[0028] (3)Purification of strains: Select colonies with clear zones and perform purification operations on an LB agar plate by streaking in zones, and incubate at 28 °C for 24 h until single colonies grow.
[0029] (4)Re-purification and qualitative detection of phosphorus-solubilizing characteristics: Pick the purified single colonies and streak them in zones on a PKO agar plate for re-purification, and incubate at 28 °C for 48 h. The appearance of obvious clear zones around the colonies indicates that the strain has phosphorus-solubilizing characteristics.
[0030] (5)Sequencing and construction of phylogenetic tree: Pick single colonies on the PKO plate for colony PCR, amplify the full length of the 16S rDNA of the strain using universal primers (27F / 1492R), send the original PCR solution to Tsingke Biotechnology Co., Ltd. for sequencing; perform sequence alignment of the sequenced DNA sequences on the NCBI website, and use MEGA 11 to construct a phylogenetic tree. As Figure 4 shown, the strain of the present invention is Pseudomonas nicosulfuron.
[0031] (6)Cryopreservation: Pick an appropriate amount of purified strain and inoculate it into LB liquid medium at an inoculation amount of 2%, incubate at 28 °C and 180 rpm for 24 h with constant shaking. Take a 2 mL sterile centrifuge tube, add 800 μL of the bacterial suspension and mix it evenly with 50% glycerol (protective agent) at a ratio of 1:1, and cryopreserve it in a -80 °C ultra-low temperature freezer.
[0032] In the above steps, the formulations of the medium and buffer are as follows: LB liquid medium: Peptone 10 g / L, NaCl 10 g / L, Yeast extract 5 g / L, pH 7.0, and the solvent is pure water.
[0033] LB agar medium: On the basis of LB liquid medium, add agar powder with a content of 20 g / L. PKO modified inorganic phosphorus dissolving agar medium: Glucose 10 g / L, (NH4)2SO4 0.5 g / L, NaCl 0.3 g / L, MgSO4·7H2O 0.3 g / L, MnSO4·H2O 0.03 g / L, KCl 0.3 g / L, FeSO4·7H2O 0.03 g / L, Ca3(PO4)2 5 g / L, yeast extract 0.5 g / L, agar powder 15 g / L, pH 7.4 ± 0.2, and the solvent is pure water. PBS buffer: NaH2PO4 6.33 g / L, Na2HPO4 8.5 g / L, pH 6.5, and the solvent is pure water; The above media and buffers are autoclaved at 121 °C under high temperature and high pressure for 15 min.
[0034] The PCR system and procedure in the above steps are as follows: (a) 50 μL system: Template (i.e., pick a small amount of bacterial cells), 1 μL of 27F, 1 μL of 1492R, 23 μL of sterile water, 2× FineTaq TM PCR SuperMix (+dye) 25 μL; (b) Procedure: Pre-denaturation at 94 °C for 10 min, (denaturation at 94 °C for 30 sec, annealing at 52 °C for 30 sec, extension at 72 °C for 1.5 min, 30 - 35 cycles), sufficient extension at 72 °C for 5 min, and constant temperature at 4 °C.
[0035] The in vitro screening method for the Pseudomonas nicosulfuronis strain to control Meloidogyne incognita in the above example of the present invention includes the following steps: (1) Activation: Take an appropriate amount of bacterial liquid from the cryopreservation tube containing the Pseudomonas nicosulfuronis strain (such as the strain cryopreserved in Example 1), inoculate it into LB liquid medium (20 mL of medium is contained in a 50 mL conical flask), the inoculation amount is 2%, at 28 °C, 180 rpm, and incubate with constant shaking for 24 h.
[0036] (2) Preparation of the bacterial strain fermentation broth: Centrifuge the cultured bacterial liquid (12000 rpm, 4 °C, 2 min), discard the supernatant, and resuspend the bacterial cells with sterile water to OD 600 = 1.5 to obtain the bacterial strain fermentation broth.
[0037] (3)Obtaining second-stage juveniles (J2s) of Meloidogyne incognita: Rinse the roots of tobacco plants grown in this laboratory. Use forceps to pick up nematode egg masses, surface disinfect them with 1.0% NaClO, place them in a 90 mm glass petri dish, add sterile water, and incubate at room temperature for 3 days. Then collect the hatched J2s of Meloidogyne incognita. Observe and count the concentration under a microscope, and adjust it to 50 nematodes / 20 μL.
[0038] (4)In vitro screening test: Add 80 μL of the strain fermentation broth and 20 μL of the Meloidogyne incognita suspension (containing 50 J2s) to a 96-well cell culture plate. Wrap it with tin foil and place it in an incubator at 28°C for 12 h. Place the 96-well plate on the stage of an inverted microscope to observe and record the death of J2s, and calculate the mortality rate. Judgment criterion: Nematodes that are rigid and motionless are dead, and those that are curved and soft are alive. In this experiment, sterile water was used as the negative control, and a 200-fold dilution of 1.8% abamectin EC (Zhiqingsong brand) was used as the positive control. Each treatment was repeated 3 times.
[0039]
[0040] Results: As Figure 5 shown, after treating Meloidogyne incognita with the fermentation broth of MiC45, compared with the treatment with sterile water, J2s showed obvious lethality. After co-incubating the fermentation broth of MiC45 and J2s for 12 h, the corrected mortality rate was as high as 91%, and there was no significant difference in the nematicidal activity compared with the abamectin dilution. The specific data are shown in Table 1, indicating that the fermentation broth of MiC45 has good nematicidal activity.
[0041] Table 1 Corrected mortality rate of MiC45 against J2s of Meloidogyne incognita
[0042] Note: Different lowercase letters in the same column of the table indicate significant differences between treatments at the 0.05 level (P < 0.05).
[0043] Example 2 An application of the Pseudomonas sulfometuron-methyl of the present invention in controlling Meloidogyne incognita on chili peppers, specifically a pot experiment method for using the Pseudomonas sulfometuron-methyl strain to control Meloidogyne incognita on chili pepper crops, using the Pseudomonas sulfometuron-methyl strain of Example 1. Meloidogyne incognita can infect chili peppers and cause serious diseases. In this pot experiment, Qiemen chili peppers (a disease-susceptible variety) were used as the model crop for research. The steps include: (1) Activation: Take an appropriate amount of bacterial liquid from the cryopreservation tube containing the Pseudomonas strain with nicosulfuron (such as the strain cryopreserved in Example 1), and inoculate it into LB liquid medium (20 mL of medium in a 50 mL conical flask). The inoculation amount is 2%, and it is cultured at 28°C with constant shaking at 180 rpm for 24 h.
[0044] (2) Preparation of the bacterial fermentation broth: Centrifuge the cultured bacterial liquid (12000 rpm, 4°C, 2 min), discard the supernatant, and resuspend the bacterial cells with sterile water to an OD 600 = 1.5 to obtain the bacterial fermentation broth.
[0045] (3) Obtaining second-stage juveniles (J2s) of Meloidogyne incognita: Rinse the roots of the tobacco plants grown in this laboratory, pick the nematode egg masses with forceps, surface disinfect them with 1.0% NaClO, place them in a 90 mm glass petri dish, add sterile water, and incubate at room temperature for 3 d. Then collect the hatched J2s of Meloidogyne incognita. Observe and count the concentration under a microscope, and adjust it to 250 individuals / mL.
[0046] (4) When the Qiemen pepper is grown to the four true-leaf stage, irrigate the roots with the MiC45 fermentation broth (OD 600 = 1.5), 10 mL per plant, and treat it once every other day for a total of 3 times. Use sterile water as the negative control and a 200-fold dilution of avermectin as the positive control. Two days after the third treatment, inoculate a suspension of J2s of Meloidogyne incognita (250 individuals / mL) at a depth of 2 cm near the roots, 4 mL per plant. Each treatment is repeated 3 times. After 30 d, observe the growth phenotype of the plants and record the fresh weight of the roots to evaluate the growth-promoting effect of the MiC45 fermentation broth on Qiemen pepper; through fuchsin staining, observe and record the number of root knots, calculate the reduction rate of root knots, and evaluate the control effect of the MiC45 fermentation broth on Meloidogyne incognita of pepper.
[0047]
[0048] The results are as shown in Figure 6 and Figure 7 The MiC45 fermentation broth can effectively prevent the occurrence of Meloidogyne incognita disease in peppers, and the reduction rate of root knots is 47.16%. After inoculating nematodes, the MiC45 fermentation broth alleviates the damage of nematodes to peppers, thereby promoting the growth of peppers. The leaves in the above-ground part are larger and plumper than those treated with sterile water and a 200-fold dilution of avermectin. Treating with the MiC45 fermentation broth can significantly promote the root development of crops, the roots are more developed, and the fresh root weight is also significantly higher than that treated with sterile water and a 200-fold dilution of avermectin. For specific data, see Table 2.
[0049] Table 2 Control effect of MiC45 on Meloidogyne incognita disease of pepper
[0050] Note: Different lowercase letters in the same column of the table indicate significant differences between treatments at the 0.05 level (P < 0.05).
[0051] In summary, the present invention provides Pseudomonas nicosulfuron MiC45 and its application in controlling Meloidogyne incognita on peppers. This strain was deposited at the China Center for Type Culture Collection (Wuhan University) on December 11, 2023, with the deposit number CCTCC NO: M 20232540, and the deposit address is the China Center for Type Culture Collection (Wuhan University), No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China. After co-incubating the MiC45 strain with Meloidogyne incognita (J2s) for 12 h in the present invention, it has an obvious lethal effect, up to 91%, indicating that the MiC45 strain has high nematicidal activity against Meloidogyne incognita; the pot experiment shows that this strain can effectively alleviate the damage of Meloidogyne incognita to peppers, reduce the infection of nematodes on the plant roots, and thus promote the growth of peppers. In addition, in agriculture, the applied phosphate fertilizer is easily fixed in alkaline soil (pH > 7.5) and converted into calcium phosphate (Ca-P). The present invention qualitatively detects the effect of the MiC45 strain on dissolving inorganic phosphorus, and an obvious transparent circle is formed around the colony, indicating that MiC45 has a certain effect on dissolving phosphorus and can degrade the insoluble phosphorus in alkaline soil for plant absorption and utilization. It can be seen that Pseudomonas nicosulfuron MiC45 is a highly effective biocontrol bacterium with both growth-promoting effects, and has profound practical significance and broad application prospects in controlling Meloidogyne incognita on peppers.
[0052] The above is only a preferred embodiment of the present invention, and does not impose any form of limitation on the present invention. Although the present invention has been disclosed as above with preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make many possible changes and modifications to the technical solution of the present invention by using the above-disclosed methods and technical contents, or modify it into equivalent embodiments with equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments based on the technical essence of the present invention without departing from the technical solution of the present invention still fall within the scope of the protection of the technical solution of the present invention.
Claims
1. A strain of Pseudomonas nicosulfuron, characterized in that, The name of the said Pseudomonas nicosulfuron strain is Pseudomonas nicosulfuron MiC45 ( Pseudomonas nicosulfuronedens MiC45), and its deposit number at the China Center for Type Culture Collection is CCTCC NO: M 20232540.
2. Use of Pseudomonas nicosulfuronedens according to claim 1 in controlling Meloidogyne incognita on peppers.
3. The application according to claim 2, characterized in that, The said use comprises the following steps: Preparation of OD 600 Use the fermentation broth of Pseudomonas nicosulfuron with a content not less than 1 to perform root irrigation on the diseased pepper plants, with each plant receiving no less than 5 mL, treating once every other day, and the number of treatments being no less than 3 times.
4. The application according to claim 3, wherein The method for preparing the fermentation broth of Pseudomonas nicosulfuronedens comprises the following steps: (1) Inoculate Pseudomonas nicosulfuronedens into LB liquid medium for activation; (2) Centrifuge the activated bacterial suspension, discard the supernatant, and resuspend with sterile water to obtain the fermentation broth of Pseudomonas nicosulfuronedens.
5. The application according to claim 4, wherein In step (1), the inoculation amount of Pseudomonas nicosulfuronedens is 1% - 3%, and the conditions for activation are: temperature 26°C - 32°C, rotation speed 160 rpm - 220 rpm, constant temperature shaking culture for 24 h - 48 h.
6. The application according to claim 4 or 5, characterized in that In step (1), in the LB liquid medium, the concentration of peptone is 10 g / L, the concentration of NaCl is 5 g / L - 10 g / L, the concentration of yeast extract is 5 g / L, the pH is 7.0, and the solvent is water.