Application of Pinus massoniana in the prevention and treatment of tobacco black shank

Abstract

The application discloses application of pinocembrin in prevention and treatment of tobacco black shank, and relates to pinocembrin and agriculturally acceptable derivatives thereof. The compound can significantly inhibit hypha growth and sporangium formation of the tobacco black shank fungus; when the concentration of the pinocembrin is 25 mg / L, the hypha inhibition rate of the pinocembrin is 77.6%; the pinocembrin can dose-dependently inhibit sporangium formation in the range of 0.78125-12.5 mg / L, the pinocembrin can completely inhibit sporangium formation when the concentration of the pinocembrin is 3.125 mg / L or higher, and the EC 50 The pinocembrin is a natural plant source component, is green, easy to degrade, and low in residue, can be used for green prevention and treatment of the tobacco black shank, and provides a new direction for development of a new type of plant source fungicide.

Description

Technical Field

[0001] This invention belongs to the field of bio-agriculture and relates to the application of pine resin in the prevention and control of tobacco black shank disease. Background Technology

[0002] Tobacco black shank is a devastating root and stem disease of tobacco caused by the parasitic fungus *Phytophthora nicotianae*. It causes severe economic losses to tobacco production worldwide and is difficult to control once it occurs, making it one of the most destructive diseases affecting tobacco production today. Currently, production relies mainly on chemical control, but long-term use of chemical fungicides easily leads to increased pathogen resistance, excessive residues in agricultural products, and environmental pollution. Therefore, there is an urgent need to develop new, green control agents that are low in toxicity, residue-free, and environmentally friendly.

[0003] Dihydroflavonoids (also known as flavanones) are an important branch of flavonoids, derived from 2-phenyldihydrochromone as the parent nucleus. They are saturated with double bonds at the C2-C3 positions, are mostly colorless, and often contain chiral carbon atoms, exhibiting optical activity. In plants, they mostly exist as hydroxyl derivatives. The parent nucleus may contain methoxy or other substituents. Treatment with alkali readily leads to ring-opening to chalcone, while acidification converts them back to dihydroflavonoids. Both often coexist in plants. As plant-derived secondary metabolites, these compounds possess advantages such as being green, natural, easily degradable, and exhibiting diverse biological activities. They have been proven to have inhibitory effects on various pathogens. Among them, arbutin, a dihydroflavonoid isolated from propolis, has clear antibacterial activity. Naringenin, a common dihydroflavonoid, has also been shown to have antibacterial and anti-inflammatory effects. Sennaol, also a dihydroflavonoid, has a certain inhibitory effect on common pathogens. While there are some reports on the use of dihydroflavonoids for the control of plant diseases in the prior art, there are no reports on the application of pine resin in the control of tobacco black shank fungus. There is also a lack of research on the specific activity of pine resin on mycelial growth and sporangium formation of tobacco black shank fungus, as well as a lack of standardized experimental methods and clear concentration-activity data. This invention aims to fill this technological gap. Summary of the Invention

[0004] The purpose of this invention is to provide the application of pine resin in the control of tobacco black shank disease. By targeting and inhibiting the mycelial growth and sporangium formation of the tobacco black shank fungus, it achieves efficient control of the disease. At the same time, it clarifies standardized experimental methods to ensure that the technology can be repeatedly implemented, thus providing support for the research and development of new plant-derived pesticides.

[0005] The technical solution of the present invention is as follows:

[0006] Application of jugain in the prevention and treatment of tobacco black shank disease.

[0007] As a preferred embodiment of the present invention, the arborine is in the form of an aglycone or glycoside of arborine, or an agriculturally acceptable salt, ester, solvate, or isomer thereof.

[0008] As a preferred embodiment of the present invention, the concentration of the pine resin is greater than or equal to 3 mg / L.

[0009] As a preferred embodiment of the present invention, the pine resin can inhibit the formation of sporangia and the growth of mycelia of *Tobacco Black Shank*.

[0010] Application of jugain in the preparation of formulations for the prevention and treatment of tobacco black shank.

[0011] As a preferred embodiment of the present invention, the arborine is in the form of an aglycone or glycoside of arborine, or an agriculturally acceptable salt, ester, solvate, or isomer thereof.

[0012] As a preferred embodiment of the present invention, the concentration of terpineol in the preparation is greater than or equal to 3 mg / L.

[0013] As a preferred embodiment of the present invention, the concentration of berberine in the preparation is greater than or equal to 25 mg / L.

[0014] Beneficial effects

[0015] This invention discovers that pine resin can inhibit the mycelial growth and sporangium formation of *Hypericum spp.*, the causal agent of tobacco black shank, through dual targeting, filling the application gap of pine resin in the control of tobacco black shank. Pine resin exhibits excellent antibacterial activity; at a concentration of 25 mg / L, it significantly inhibits mycelial growth with an inhibition rate as high as 77.6%. Regarding sporangium inhibition, pine resin can completely inhibit growth at a concentration of 3.125 mg / L, with an inhibitory effect far superior to naringin and senna. Pine resin is derived from the natural substance propolis, leaving no residue and being easily degradable. It fundamentally solves the problems of drug resistance and environmental pollution caused by traditional chemical fungicides, aligning with the needs of green plant protection development. Furthermore, its wide availability and low production cost facilitate large-scale production and application. Attached Figure Description

[0016] Figure 1 Colony morphology of *Tobacco Black Shank* mycelial growth at different concentrations of naringenin, jugol, sennaol, and a blank control.

[0017] Figure 2 Microscopic observation of sporangia of the control (DMSO) fungus *Diospyros nicotine* (210 μm, 39 sporangia).

[0018] Figure 3 Microscopic images (210 μm) showing the inhibition of sporangium formation by naringenin, jugol, and sennaol at five different concentrations of *Bacillus thuringiensis*. Detailed Implementation

[0019] Test materials

[0020] Test reagents: Naringenin (purity ≥98%), Pinocemrin (purity ≥98%), and Eriodictyol (purity ≥98%).

[0021] Test pathogen: Phytophthora nicotianae (Tobacco black shank pathogen);

[0022] Culture media: V8 liquid medium, V8 solid medium (V8 liquid medium with 16g / L agar added).

[0023] Solvent: Dimethyl sulfoxide (DMSO, analytical grade);

[0024] Instruments: ultrasonic cleaner, sterile petri dishes (9cm in diameter), hole punch (6mm in diameter), sterile toothpicks, constant temperature incubator, microscope.

[0025] Example 1: Mycelial Plate Inhibition Test (25 mg / L Concentration)

[0026] (1) Preparation of mother liquor: Take 10 mg of naringenin, juglone and sennaol respectively, dissolve them in 100 μl of DMSO solvent, and sonicate until completely dissolved to obtain a mother liquor of each product with a concentration of 100 g / L. The remaining concentrations are diluted accordingly, and then stored in the dark and refrigerated for later use.

[0027] (2) Preparation of drug administration plates: Blank control group: 100 μl of DMSO was added to 100 mL of melted V8 solid medium, shaken thoroughly and evenly distributed to 3 sterile culture dishes, and cooled and solidified to obtain CK plates; Drug treatment group: The mother liquor was added to melted V8 solid medium according to the ratio to prepare a series of drug-containing mediums with final concentrations of 12.5, 25, 50 and 100 mg / L, shaken thoroughly and poured into plates, with 3 replicates for each concentration.

[0028] (3) Inoculation and culture: Using a sterile punch with a diameter of 6 mm, collect fungal cakes from the edge of the colony of Tobacco Blackleg Fungus that has been cultured to the logarithmic phase; use a sterile toothpick to pick up the fungal cakes, place them upside down in the center of each drug administration plate, seal the plate and place it in a constant temperature incubator at 28℃; first, culture upright until the hyphae germinate and grow, then switch to upside-down culture until the hyphae of the control group grow to the vicinity of the edge of the plate.

[0029] (4) Index determination: After opening the plates, the growth diameter of the mycelium in each plate was determined using the cross-hatching method. The inhibition rate was calculated according to the formula "Inhibition rate (%) = (Control mycelium diameter - Treatment mycelium diameter) / Control mycelium diameter × 100%". The growth status of the colonies was recorded by taking pictures at the same time. The average value of the results of 3 plates for each treatment was taken, and the EC was calculated by concentration. 50 value.

[0030] The mycelial plate inhibition results are shown in Table 1. The inhibitory effects of three dihydroflavonoids—naringenin, jucin, and sennaol—on the mycelial growth of *Tobacco Black Shank* fungus increased with increasing concentration, exhibiting a clear dose-dependent relationship. Jucin showed the best inhibitory activity, with EC50 at [value missing]. 50 The value was 14.417 mg / L, and the inhibition rate reached 84.03% at a concentration of 100 mg / L; naringenin was the second most effective, with EC... 50 The value was 30.528 mg / L, and the inhibition rate at a concentration of 100 mg / L was 77.23%; the activity of sennaol was relatively weak, EC 10. 50 The concentration was 73.053 mg / L, and the inhibition rate at a concentration of 100 mg / L was 60.33%. The control group showed normal mycelial growth without inhibition, indicating that the experimental method was stable and reliable. It is evident that even among dihydroflavonoids, the inhibitory effects on the mycelial growth of *Tobacco Black Shank* vary greatly, with arbutin showing significantly better inhibitory effects than naringenin and sennaol.

[0031] Table 1. Inhibitory effects of different concentrations of dihydroflavonoids on the mycelial growth of *Tobacco Black Shank* fungus.

[0032] Naringin 0 36.61 45.08 51.39 77.23 30.528 Qiao Songsu 0 41.78 68.78 78.85 84.03 14.417 Sacred herbol 0 10.80 23.94 36.37 60.33 73.053

[0033] Example 2 Sporangium Inhibition Test

[0034] (1) Preparation of antibacterial agents: Naringenin, styracin and styracin were dissolved in DMSO and then diluted in half to prepare a series of antibacterial agent solutions with concentrations of 1.25 mg / mL, 0.625 mg / mL, 0.3125 mg / mL, 0.15625 mg / mL and 0.078125 mg / mL. The solutions were stored in the dark and refrigerated for later use. Mixed antibacterial agent solutions were prepared according to the same concentration gradient, and the concentrations of the three compounds were kept consistent.

[0035] (2) Induction of mycelial formation: The tobacco black shank pathogen was transferred to V8 solid medium and cultured continuously in the dark at 28℃ for 14 days; mycelial cakes were punched at the edge of the colony using a sterile punch with a diameter of 6 mm, and 7-10 mycelial cakes were placed in each sterile culture dish. 15 mL of V8 liquid medium was added, and each group of 3 culture dishes was placed in the dark at 28℃ for 48 h to induce mycelial formation.

[0036] (3) Induction of sporangium formation: After the mycelium forms, the V8 liquid culture medium in the culture dish is poured off, and the mycelium is rinsed 3 times with sterile water; 50 mL of sterile water is taken, and 0.5 mL of antibacterial agent solution of different concentrations is added to prepare sterile water containing drugs with final concentrations of 12.5 mg / L, 6.25 mg / L, 3.125 mg / L, 1.5625 mg / L, and 0.78125 mg / L, respectively, and each is added to the culture dish of the corresponding group; 0.5 mL of DMSO is added to 50 mL of sterile water in the control group; all culture dishes are placed in a 28℃ light environment for 72 h to induce sporangium formation.

[0037] (4) Index determination: The formation of sporangia in each culture dish was observed under a microscope. The number of sporangia was counted in three randomly selected fields of view (39 sporangia in the control group). The inhibition rate was calculated according to the formula "inhibition rate (%) = (39 - number of sporangia in the treatment group) / 39 × 100%" (a negative inhibition rate indicates that sporangia formation is promoted). Each concentration was repeated 4 times, and EC was calculated through the concentration-effect relationship. 50 value.

[0038] The sporangium inhibition results are shown in Tables 2 and 3. Using the control group of 39 sporangia as a baseline, the three dihydroflavonoids showed significant differences in their effects on sporangium formation of *Tobacco Black Shank*: jugazolinone showed the best performance, with an inhibition rate of 76.92% at 0.78125 mg / L, rising to 97.44% at 1.5625 mg / L, and completely inhibiting sporangium formation at concentrations of 3.125 mg / L and above. (EC50) 50 The concentration was 0.52 mg / L; naringenin showed an inhibition rate of 43.59% at 0.78125 mg / L and 89.74% at 1.5625 mg / L. Concentrations of 6.25 mg / L and above completely inhibited sporangium formation. EC 50 The concentration was 0.832 mg / L; sennaol exhibited a promoting effect at low concentrations and an inhibitory effect at high concentrations: at 0.78125 mg / L, the number of sporangia was 53, with an inhibition rate of -36.9%, showing a growth-promoting effect; at 1.5625 mg / L, it turned into an inhibitory effect (inhibition rate 23.08%), and the inhibitory effect increased with increasing concentration, reaching an inhibition rate of 82.05% at a concentration of 12.5 mg / L, EC 0.05%. 50 The value was 3.083 mg / L.

[0039] Table 2. Inhibition rate of compounds on sporangium formation of *Tobacco Black Shank* (control group sporangium count = 39)

[0040] Naringin 43.59 89.74 97.44 100 100 0.832 Qiao Songsu 76.92 97.44 100 100 100 0.520 Sacred herbol -36.90 23.08 58.97 74.36 82.05 3.083

[0041] Table 3. Number of sporangia of *Tobacco Black Shank* under compound treatment (control group = 39 sporangia)

[0042] Naringin 22 4 1 0 0 Qiao Songsu 9 1 0 0 0 Sacred herbol 53 30 16 10 7

Claims

1. Application of jugazosin in the prevention and treatment of tobacco black shank disease.

2. The application according to claim 1, characterized in that, The geraniol is in the form of aglycone or glycoside of geraniol, or in the form of an agriculturally acceptable salt, ester, solvate, or isomer thereof.

3. The application according to claim 1, characterized in that, The concentration of berberine is greater than or equal to 3 mg / L.

4. The application according to claim 1, characterized in that, The arborine can inhibit the formation of sporangia and the growth of mycelium of *Tobacco Blackleg*.

5. Application of jugazosin in the preparation of formulations for the prevention and treatment of tobacco black shank.

6. The application according to claim 5, characterized in that, The geraniol is in the form of aglycone or glycoside of geraniol, or in the form of an agriculturally acceptable salt, ester, solvate, or isomer thereof.

7. The application according to claim 5, characterized in that, The concentration of berberine in the preparation is greater than or equal to 3 mg / L.

8. The application according to claim 5, characterized in that, The concentration of berberine in the preparation is greater than or equal to 25 mg / L.

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