Application of phenazine-1-formamide in prevention and treatment of phytopathogen and meloidogyne incognita mixed diseases

Abstract

The invention discloses application of phenazine-1-formamide to prevention and treatment of phytopathogen and meloidogyne incognita mixed diseases, and relates to the technical field of agricultural biology. The phenazine-1-formamide has a good inhibition effect on main plant pathogenic bacteria including bacterial wilt pathogenic bacteria, citrus canker pathogenic bacteria, cassava fusarium wilt pathogenic bacteria, pseudomonas syringae, tomato pathogenic variety pathogenic bacteria, rice bacterial leaf blight pathogenic bacteria and bacterial stripe pathogenic bacteria through indoor medicament inhibition determination; the phenazine-1-formamide is emphasized to be capable of effectively preventing and treating mixed diseases of phytopathogens and meloidogyne incognita. The phenazine-1-formamide provided by the invention can be applied to prevention and control of common plant diseases in agricultural production, and has a wide market application prospect.

Description

Technical Field

[0001] The present invention relates to the field of agricultural biotechnology, and particularly to the application of phenazine-1-carboxamide in controlling mixed diseases caused by plant pathogens and Meloidogyne incognita. Background Art

[0002] China is a large populated country with a great demand for fruits and vegetables. The problem of fruit and vegetable yield caused by pests and diseases is very serious. As an agricultural production material, pesticides play an important and indispensable role in maintaining human life, living standards, environmental quality, and the sustainable utilization of land and biological resources. However, due to the extensive use of chemical pesticides and fertilizers by humans, pests and diseases have become rampant, soil fertility has declined, the extinction rate of wild biological species has accelerated, the quality of cultivated species has deteriorated, and the natural adjustment ability has decreased.

[0003] Therefore, using biotechnology innovation to develop highly effective and low-toxic microbial pesticides is an effective way to achieve efficient and sustainable agricultural development.

[0004] The mixed diseases of plants caused by plant pathogens and Meloidogyne incognita are major obstacles in agricultural production. However, no effective solution has been found for the control of such mixed diseases caused by plant pathogens and Meloidogyne incognita. Therefore, developing the application of phenazine-1-carboxamide in controlling such mixed diseases not only has important theoretical significance but also has broad application prospects. Summary of the Invention

[0005] In view of the above-mentioned defects of the prior art, the present invention proposes the application of phenazine-1-carboxamide in controlling mixed diseases caused by plant pathogens and Meloidogyne incognita to solve the problems raised in the above background art.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] The application of phenazine-1-carboxamide in controlling mixed diseases of plant pathogens and plant nematodes, wherein the plant pathogens include Ralstonia solanacearum, Xanthomonas campestris, Xanthomonas manihotis, Pseudomonas syringae pv. tomato DC3000, Xanthomonas oryzae pv. oryzae, and Xanthomonas oryzae pv. oryzicola. The chemical structural formula of the phenazine-1-carboxamide is shown in the following figure:

[0008]

[0009] Preferably, the plant nematodes include root-knot nematodes.

[0010] Preferably, the root-knot nematodes include Meloidogyne incognita.

[0011] Preferably, the minimum use concentration of phenazine-1-carboxamide for inhibiting the activity against the pathogen of bacterial wilt (Ralstonia solanacearum) is 7.81 μg / mL.

[0012] Preferably, the inhibitory concentration of phenazine-1-carboxamide against plant pathogens and Meloidogyne incognita is 250 μg / mL.

[0013] Preferably, the application is to apply phenazine-1-carboxamide to the surface or roots of diseased plants.

[0014] Preferably, the treatment method for the roots is root irrigation.

[0015] Preferably, the concentration of the root irrigation is 100 μg / mL.

[0016] Preferably, the prevention and control of plant nematode diseases is to inhibit the hatching of plant nematode eggs and / or kill plant nematodes.

[0017] Preferably, the plants include chili peppers, tobacco, citrus, cassava, tomatoes, rice, cucumbers, barley, and areca nuts.

[0018] Preferably, when phenazine-1-carboxamide simultaneously controls the mixed diseases of the pathogen of bacterial wilt (Ralstonia solanacearum) and Meloidogyne incognita, the bactericidal inhibition rate against the pathogen of bacterial wilt (Ralstonia solanacearum) is 51.41 ± 3.35%, and the inhibition rate against Meloidogyne incognita is 39.80 ± 0.90%.

[0019] Preferably, the use concentration of phenazine-1-carboxamide for inhibiting the activity against the pathogen of bacterial wilt (Ralstonia solanacearum) is 7.81 - 8.00 μg / mL.

[0020] Preferably, the bactericidal inhibition rate of phenazine-1-carboxamide against the pathogen of bacterial wilt (Ralstonia solanacearum) is 87.88%.

[0021] Preferably, the bactericidal inhibition rate of phenazine-1-carboxamide against the pathogen of citrus canker (Xanthomonas Campestris) is 71.46%.

[0022] Preferably, the bactericidal inhibition rate of phenazine-1-carboxamide against the pathogen of cassava wilt disease (Xanthomonas Maniihotis) is 31.53%.

[0023] Preferably, the bactericidal inhibition rate of phenazine-1-carboxamide against the pathogen of Pseudomonas syringae pv. tomato DC3000 is 45.23%.

[0024] Preferably, the bactericidal inhibition rate of phenazine-1-carboxamide against the pathogen of rice bacterial blight (Xanthomonas oryzae pv. Oryzae) is 54.25%.

[0025] Preferably, the bactericidal inhibition rate of phenazine-1-carboxamide against the pathogen of bacterial leaf streak (Xanthomonas oryzae pv. Oryzicola) is 40.86%.

[0026] Preferably, the lethality rate of phenazine-1-carboxamide against Meloidogyne incognita is 83.68%.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0028] (1) In the present invention, the endophytic bacterium biocontrol bacterium W-126 is screened and isolated from the medicinal plant Waltheria indica, and the active substance phenazine-1-carboxamide (PCN) is isolated from the fermentation metabolites of the biocontrol bacterium W-126. After being applied to the indoor pharmaceutical inhibitory assay, it is not only found that phenazine-1-carboxamide has good inhibitory effects on the main plant pathogens including the pathogen of bacterial wilt, the pathogen of citrus canker, the pathogen of cassava wilt disease, the pathogen of Pseudomonas syringae pv. tomato DC3000, the pathogen of rice bacterial blight, and the pathogen of bacterial leaf streak, but also found that phenazine-1-carboxamide can effectively control the mixed diseases of plant pathogens and Meloidogyne incognita, and its inhibitory effect is better than that of the traditional fungicides streptomycin sulfate, abamectin, and Zhuorun fungicide.

[0029] (2) It is proposed in this application that phenazine-1-carboxamide can be applied to the prevention and control of common plant diseases in agricultural production, which can effectively solve the problem that plant pathogenic fungi such as the pathogen of bacterial wilt and the pathogen of citrus canker develop resistance to existing fungicides, resulting in poor control effects. Phenazine-1-carboxamide is a microbial source pesticide isolated from endophytic bacteria of medicinal plants, which is environmentally friendly, green and pollution-free, meets the pesticide residue toxicity standards of the World Health Organization, and has broad market application prospects. Description of the Drawings

[0030] Figure 1For the inhibitory effect of Pseudomonas aeruginosa W-126 on Ralstonia solanacearum, where A represents the control; B represents the confrontation between W-126 and Ralstonia solanacearum; C represents the treatment with 20 μg / mL streptomycin sulfate;

[0031] Figure 2 For the morphological observation of the antagonistic bacterium W-126, where A represents the colony morphology of W-126 on the LB plate; B represents the result of Gram staining of the bacteria;

[0032] Figure 3 For the phylogenetic evolution tree of the antagonistic bacterium W-126 based on the 16S rDNA gene sequence;

[0033] Figure 4 For the antibacterial effects of various organic extracts of the fermentation broth of the antagonistic bacterium W-126, where A is the petroleum ether phase, B is the ethyl acetate phase, C is the n-butanol phase, D is the methanol phase, and E is the control;

[0034] Figure 5 For the identification spectrum of phenazine-1-carboxamide, where A is the structural diagram, B is the mass spectrum, C is the carbon spectrum, and D is the hydrogen spectrum;

[0035] Figure 6 For the inhibitory effect of phenazine-1-carboxamide on Ralstonia solanacearum, where A is the determination of the inhibition of Ralstonia solanacearum by phenazine-1-carboxamide; B is the CK;

[0036] Figure 7 For the antibacterial effects of different concentrations of phenazine-1-carboxamide on Ralstonia solanacearum. Detailed implementation manners

[0037] The technical solutions of the present invention will be further described in detail below with reference to the accompanying drawings and specific implementation manners.

[0038] Example 1 Discovery process and identification of phenazine-1-carboxamide

[0039] 1.1 Isolation and screening of antagonistic bacteria

[0040] Waltheria indica is a traditional medicinal plant. We isolated 209 strains of endophytic bacteria from Waltheria indica at 31 locations in Hainan Province, and then isolated, activated and screened to obtain the biocontrol bacterium Pseudomonas aeruginosa W-126 (see details in Figure 1 ). Using Ralstonia solanacearum as the indicator bacterium, its antibacterial effect was determined by the filter paper diffusion method. The diameter of the antibacterial circle of W-126 against Ralstonia solanacearum was 27 mm. One strain with good antagonistic effect against Ralstonia solanacearum was screened out, named W-126, and subsequent research was carried out with it as the target strain.

[0041] 1.2 Identification of the antagonistic bacterium Pseudomonas aeruginosa W-126

[0042] 1.2.1 Morphological characteristics: The antagonistic bacterium W-126 grew normally on the LB solid plate. Through morphological observation, it was found that its colonies were slightly yellowish and greenish, with a moist surface, slightly raised, relatively viscous texture, and were easy to pick up (see Figure 2 A). After Gram staining, the bacteria were short rod-shaped and red (see Figure 2 B), and were Gram-negative bacteria.

[0043] 1.2.2 Physiological and biochemical characteristics

[0044] The physiological and biochemical test results of the antagonistic bacterium W-126 are shown in Table 1. Pseudomonas aeruginosa is a Gram-negative bacillus, positive for oxidase, capable of producing pyocyanin, liquefying gelatin, reducing nitrate, and growing at 42 °C.

[0045] Table 1 Physiological and biochemical test results

[0046]

[0047] Note: "+" indicates a positive reaction, and "-" indicates a negative reaction

[0048] 1.2.3 Molecular biology identification

[0049] After the sequencing results were spliced and processed by the DNAman software, the antagonistic bacterium W-126 was subjected to BLAST sequence alignment on the NCBI website, and a phylogenetic evolution tree was constructed based on the 16S rDNA gene sequence (see Figure 3 ). It was found that the strain W-126 had a very high homology with Pseudomonas aeruginosa. Considering the morphological characteristics, physiological and biochemical characteristics of the biocontrol bacterium and the analysis results of the phylogenetic tree, the strain W-126 was confirmed to be Pseudomonas aeruginosa.

[0050] 1.3 Preparation of the metabolites of the fermentation broth of the antagonistic bacterium W-126 and determination of its antibacterial effect

[0051] After the antagonistic bacterium W-126 was extracted with petroleum ether, ethyl acetate, n-butanol, and methanol respectively, the antibacterial effects of the secondary metabolites in each phase were determined. When the concentration was 0.3 mg / mL, the pathogens of bacterial wilt were inhibited to varying degrees, showing obvious inhibition zones. Among them, the inhibition diameter of the ethyl acetate phase was 10 mm (see Figure 4 B). The inhibition rates of the ethyl acetate extract on the bacterial wilt pathogens indicated that there were indeed many active substances with antibacterial functions in the metabolites of the fermentation broth in each phase.

[0052] 1.4 Identification of the compound phenazine-1-carboxamide (PCN) and its antagonistic effect on the bacterial wilt pathogens

[0053] According to the principle of tracking active compounds, we isolated highly active compounds from dozens of components. After identification, the highly active compound was confirmed to be phenazine-1-carboxamide (see details in Figure 5 ).

[0054] Phenazine-1-carboxamide (PCN): C 13 H 9 N 3 O, yellow solid, m.p. 201 - 203 °C, 1H NMR (400 MHz, Chloroform-d) δ 9.02 (dd, J = 7.1, 1.5 Hz, 1H), 8.46 (dd, J = 8.6, 1.5 Hz, 1H), 8.36 - 8.29 (m, 1H), 8.29 - 8.20 (m, 1H), 8.03 - 7.88 (m, 3H). 13C NMR (101 MHz, Chloroform-d) δ 166.66, 143.41, 143.08, 141.57, 140.85, 136.06, 134.35, 131.86, 131.22, 130.00, 129.71, 129.14, 128.76. ESI-MS: m / z 224.08 [M+H] - (calcd C 13 H 9 N 3 O for 223.07).

[0055] When the concentration of the compound phenazine-1-carboxamide was 1 mg / mL, the inhibition diameter reached 25 mm (see details in Figure 6 ), and an obvious inhibition zone appeared, indicating an obvious inhibitory effect.

[0056] Example 2 Determination of the Activity of Compound Phenazine-1-carboxamide (PCN) against 6 Kinds of Phytopathogenic Bacteria and Root-knot Nematodes

[0057] 2.1 Experimental Method for Controlling Phytopathogenic Bacteria with Phenazine-1-carboxamide

[0058] The drug sensitivity of phenazine-1-carboxamide to phytopathogenic bacteria was determined by the colony growth rate inhibition method. The growth rate method, also known as the poisoned medium method, is particularly suitable for the test strains cultured in liquid medium and meets the requirements of this experiment.

[0059] The specific operation steps are as follows:

[0060] ① Activation of pathogens: inoculate the six plant pathogens, including bacterial wilt pathogen, citrus canker pathogen, cassava wilt pathogen, Pseudomonas syringae pv. tomato pathogen, rice white leaf blight pathogen and bacterial leaf streak pathogen in the storage tube into LB liquid culture medium, culture for 10 to 12 hours, and then place in a 4°C constant temperature refrigerator for use.

[0061] ② Prepare the mother solution: Use the solvent ethyl acetate to prepare phenazine-1-carboxamide into a mother solution with a concentration of 1000 μg / mL.

[0062] ③Prepare 96-well culture plates with virus: Add LB liquid culture medium to the mother solution to prepare culture medium containing 1.95μg / mL, 3.91μg / mL, 7.81μg / mL, 15.63μg / mL, 31.25μg / mL, 125μg / mL, 250μg / mL, 500μg / mL, and 1000μg / mL of drug solution. Add the corresponding volume of ethyl acetate to the control group, mix well, and pour into the culture dishes to cool. Set up 3 replicates for each treatment.

[0063] ④ Inoculation of pathogens: Use a pipette to add pathogens to the pre-culture, inoculate into a 96-well culture plate, seal it and place it in a 25°C constant temperature incubator for culture.

[0064] Data processing: When the edge of the colony in the control group was close to the dish wall, the turbidity of the bacterial solution at each treatment concentration was measured using an enzyme marker, and the growth inhibition rate was calculated.

[0065]

[0066] 2.2 The experimental method of using phenazine-1-carboxamide to control root-knot nematodes refers to patent CN110301441B.

[0067] 2.3 Test Results

[0068] The results are shown in Tables 2, 3, 4 and Figure 7 As shown:

[0069] Table 2 EC values ​​of phenazine-1-carboxamide against 6 plant pathogenic bacteria 50 and LC against incognita 50

[0070]

[0071]

[0072] Table 3 Effects of phenazine-1-carboxamide on the hatching of southern root-knot nematode eggs

[0073]

[0074] Note: Data are presented as mean ± standard deviation (P<0.05)

[0075] Table 4 Nematicidal activity of phenazine-1-carboxamide

[0076]

[0077] Note: Data are presented as mean ± standard deviation (P<0.05)

[0078] Tables 2, 3, 4 and Figure 7 showed the antibacterial activities of PCN against six phytopathogenic bacteria and Meloidogyne incognita. The results showed that phenazine-1-carboxamide (PCN) had a high inhibitory activity against Ralstonia solanacearum, with an EC 50 of 64.16 μg / mL (Table 2). When the concentration of PCN was 7.81 μg / mL, PCN also showed an inhibitory activity against Ralstonia solanacearum. In addition, phenazine-1-carboxamide (PCN) showed a certain inhibitory activity against Xanthomonas citri subsp. citri, with an EC 50 of 138.70 μg / mL, and PCN showed a certain activity against Meloidogyne incognita, with a median lethal concentration LC50 of 118.63 μg / mL at 72 h (Table 2).

[0079] Example 3 Determination of the activity of phenazine compounds against mixed pests of Ralstonia solanacearum and Meloidogyne incognita

[0080] 3.1 The results are shown in Table 5 as follows:

[0081] Table 5 Inhibitory effects of phenazine compounds and positive control against Ralstonia solanacearum and Meloidogyne incognita

[0082]

[0083] The treatment concentrations of phenazine compounds were all 125 μg / mL, and only 3-Amino-phenazin-2-ol, PCN, Phenazine, Phenazine methosulfate and Neutral Red had an inhibition rate against Ralstonia solanacearum exceeding 60%. The inhibition rate of phenazine against Ralstonia solanacearum was 66.33%, indicating that the phenazine structure had good inhibitory activity against Ralstonia solanacearum. Although PCN was not the most active among these compounds, it had the second highest inhibition rate against Ralstonia solanacearum among all PCN analogues, at 68.18%. Analysis together with the activity of phenazine showed that the formamide structure on PCN enhanced the activity of phenazine. Against Meloidogyne incognita, only the inhibition rates of Phenazin-1-ylamine, PCN, Phenazine and 1-Hydroxyphenazine exceeded 50%.

[0084] Activity Assay of Compound Phenazine-1-carboxamide (PCN) against Mixed Pests of Ralstonia solanacearum and Meloidogyne incognita

[0085] 4.1 The experiment was divided into 7 treatment groups: a pepper group without Ralstonia solanacearum and Meloidogyne incognita (CK-); a pepper group with Ralstonia solanacearum and Meloidogyne incognita (CK); a pepper group with Ralstonia solanacearum and Meloidogyne incognita under PCN treatment (PCN); a pepper group with Ralstonia solanacearum and Meloidogyne incognita under the treatment of bacterial solution W-126 (W-126); a pepper group with Ralstonia solanacearum and Meloidogyne incognita under the treatment of streptomycin sulfate, as a positive control for Ralstonia solanacearum; a pepper group with Ralstonia solanacearum and Meloidogyne incognita under the treatment of abamectin (Abamectin), as a positive control for nematode diseases; a pepper group with Ralstonia solanacearum and Meloidogyne incognita under the treatment of Zhuorun microbial agent (Zhuorun), as a positive control for mixed diseases. PCN or streptomycin sulfate was irrigated at a dose of 5 mL with a concentration of 100 μg / mL; abamectin was irrigated at 50 mL with a concentration of 10 7 CFU / mL; the antagonistic bacterium W-126 was irrigated at 50 mL with a concentration of 10 7 CFU / mL. Nematodes were inoculated on the second day after the irrigation treatment, and Ralstonia solanacearum was inoculated on the third day.

[0086] 4.2 The results are shown in Table 6

[0087] Table 6 Control of Mixed Diseases of Ralstonia solanacearum and Meloidogyne incognita by Phenazine-1-carboxamide

[0088]

[0089] It was found that there was no difference in plant height among the 7 treatment groups 35 days after nematode inoculation. The plant weight of CK- was significantly higher than that of other treatments, indicating that Ralstonia solanacearum and Meloidogyne incognita affected the weight of peppers. The number of root knots in CK was significantly higher than that in the plants treated with PCN, Abamectin, bacterial solution W-126 and Zhuorun microbial agent, indicating that PCN, bacterial solution W-126, Zhuorun microbial agent and abamectin all had inhibitory effects on Meloidogyne incognita. The Ralstonia solanacearum disease index of CK was significantly higher than that in the plants treated with PCN, streptomycin sulfate, bacterial solution W-126 and Zhuorun microbial agent. The inhibition rates of PCN, streptomycin sulfate, bacterial solution W-126 and Zhuorun were 51.41%, 37.57% and 30.58% respectively. The inhibition rates of PCN and streptomycin sulfate against Ralstonia solanacearum both exceeded 50% and there was no significant difference, indicating that in the presence of Meloidogyne incognita, the control effects of PCN and streptomycin sulfate on Ralstonia solanacearum were not much different.

[0090] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. The use of phenazine-1-carboxamide in preventing and controlling mixed diseases of plant pathogens and plant nematodes, characterized in that: The plant pathogens include Ralstonia solanacearum, Xanthomonas Campestris, Xanthomonas Manihotis, Pseudomonas Syringae DC3000, Xanthomonas oryzae pv. Oryzae, and Xanthomonas oryzae pv Oryzicola. The chemical structure of phenazine-1-carboxamide is as follows:

2. The use according to claim 1, characterized in that: The plant nematodes include root-knot nematodes.

3. The use according to claim 2, characterized in that: The root-knot nematodes include the southern root-knot nematode.

4. The use according to claim 1, characterized in that: The minimum concentration of the phenazine-1-carboxamide for inhibiting the bacterial wilt pathogen (Ralstonia solanacearum) is 7.81 μg / mL.

5. The use according to claim 3, characterized in that: The inhibitory concentration of the phenazine-1-carboxamide on plant pathogens and southern root-knot nematodes is 250 μg / mL.

6. The use according to claim 1, characterized in that: The application is to apply phenazine-1-carboxamide to the surface or root of diseased plants.

7. The use according to claim 6, characterized in that: The root treatment method is root irrigation.

8. The use according to claim 7, characterized in that: The root irrigation concentration is 100 μg / mL.

9. The use according to claim 1, characterized in that: Controlling plant nematode diseases is to inhibit the hatching of plant nematode eggs and / or kill the plant nematodes.

10. The use according to claim 1, characterized in that: The plants include pepper, tobacco, citrus, cassava, tomato, rice, cucumber, barley and betel nut.

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