Application of chlorambucil in disease control and growth promotion of panax notoginseng

Abstract

The invention relates to application of chlorambucil in prevention and control and growth promotion of panax notoginseng diseases. It is found that chlorambucil has a good pseudo-ginseng rust rot fungus inhibiting effect and a good growth promoting effect on beneficial bacteria such as mortierella and trichoderma and can be used for promoting growth of pseudo-ginseng, and therefore chlorambucil can be widely applied to pseudo-ginseng disease control and growth promoting.

Description

Technical Field

[0001] This invention relates to the field of plant disease control technology, and in particular to the application of phenylbutyric acid mustard in the control of diseases and growth promotion of Panax notoginseng. Background Technology

[0002] Yunnan Province, as a traditional high-quality production area of ​​Panax notoginseng, is currently facing a continuous increase in market demand for Panax notoginseng, which has led to the continuous expansion of the planting area. However, in the actual production process, a significant problem has gradually emerged: the soil obstacles caused by continuous cropping of Panax notoginseng have made it impossible to use these soils for cultivation again, which has led to a shortage of land resources for Panax notoginseng cultivation and seriously hindered the further development of the Panax notoginseng industry. The specific manifestations of the obstacles of continuous cropping of Panax notoginseng are particularly prominent, including: (1) the germination rate of Panax notoginseng seeds is negatively affected and has declined significantly; (2) the overall quality and growth status of Panax notoginseng plants show a declining trend; (3) root rot can directly lead to a sharp reduction in Panax notoginseng yield or even complete crop failure in severe cases; (4) in order to restore soil suitability, land that has been planted with Panax notoginseng needs to be rotated for a long time, with a rotation interval of up to 10 to 30 years. This measure is difficult to implement on a large scale in practice. Although the application of chemical pesticides can temporarily control pests and diseases, their effectiveness is unstable and often accompanied by severe environmental pollution. They cannot fundamentally solve the problem of continuous cropping obstacles and may even exacerbate soil acidification and salinization. Furthermore, while soil fumigation and other treatments have some effect, they severely damage the quality of medicinal materials, disrupt soil structure, and inhibit soil microbial activity. Given the increasingly stringent quality requirements for Panax notoginseng, the complexity of root rot, and the limitations of existing control measures, improving the quality of Panax notoginseng and mitigating diseases have become critical issues that urgently need to be addressed in Panax notoginseng production.

[0003] Currently, there is a lack of effective methods to promote the growth of Panax notoginseng and to prevent root rot. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides the application of chlorambucil in the prevention and control of diseases and the promotion of growth in Panax notoginseng; in particular, it provides the application of chlorambucil or its derivatives or pharmaceutically acceptable salts in promoting beneficial microorganisms of the genus *Morchella* and *Trichoderma* in the rhizosphere of Panax notoginseng to prevent root rot caused by rust fungi.

[0005] In a first aspect, the present invention provides at least one of the following applications of chlorambucil or a pharmaceutically acceptable salt thereof:

[0006] (1) As a bacteriostatic agent for plant pathogens;

[0007] (2) Inhibits plant pathogens;

[0008] (3) Prevention and control of root rot in Panax notoginseng;

[0009] (4) Promotes the growth of beneficial bacteria;

[0010] (5) Promotes the growth of Panax notoginseng.

[0011] Preferably, the plant pathogen is a rust-rot fungus. The antibacterial agent of the present invention can prevent and control root rot of Panax notoginseng caused by rust-rot fungus.

[0012] Preferably, the beneficial bacteria include *Morchella* and *Trichoderma*.

[0013] Preferably, the application comprises administering a pharmaceutically acceptable salt of chlorambucil or a derivative thereof to Panax notoginseng. More preferably, the application is a root irrigation.

[0014] In a second aspect, the present invention also provides at least one of the following methods, said method comprising administering chlorambucil or a pharmaceutically acceptable salt thereof to Panax notoginseng:

[0015] (1) Methods to inhibit plant pathogens;

[0016] (2) Methods for preventing and controlling root rot in Panax notoginseng;

[0017] (3) Methods to promote the growth of beneficial bacteria;

[0018] (4) Methods to promote the growth of Panax notoginseng.

[0019] Preferably, the application is a root irrigation.

[0020] In this invention, when inhibiting rust-rot fungi, the concentration of chlorambucil or its pharmaceutically acceptable salt is not less than 10 ppm. Preferably, the optimal concentration is 10 ppm to 100 ppm.

[0021] This invention demonstrates, through antibacterial activity assays, that chlorinated mustard exhibits excellent inhibitory activity against *Panax notoginseng* rust-rot fungus. Chlorinated mustard, as a novel fungicide, is suitable for the chemical control of plant diseases.

[0022] In this invention, when promoting the growth of *Morchella*, the concentration of chlorambucil or a pharmaceutically acceptable salt thereof is not less than 0.1 ppm. Preferably, the concentration is 0.1 ppm to 100 ppm, more preferably 10 ppm to 100 ppm.

[0023] In this invention, when promoting the growth of Trichoderma, the concentration of chlorambucil or its pharmaceutically acceptable salt does not exceed 0.1 ppm. Preferably, the concentration is 0.005 ppm to 0.1 ppm, more preferably 0.01 ppm to 0.05 ppm. The promoting effect is stronger at lower concentrations.

[0024] In this invention, a concentration of 10 ppm effectively promotes the growth of Panax notoginseng. Therefore, the concentration of chlorambucil or its pharmaceutically acceptable salt is 1-50 ppm; more preferably, the concentration is 5-15 ppm; and most preferably, the concentration is 10 ppm.

[0025] Preferably, promoting the growth of Panax notoginseng includes increasing the survival rate of one-year-old Panax notoginseng and / or increasing the dry weight of Panax notoginseng above ground and / or increasing the dry weight of Panax notoginseng below ground and / or promoting the growth of Panax notoginseng fibrous roots.

[0026] Preferably, promoting the growth of Panax notoginseng fibrous roots includes increasing the root length and / or increasing the surface area and / or increasing the root volume of Panax notoginseng fibrous roots.

[0027] In this invention, the CAS No. of the chlorinated nitrogen mustard Chlorambucil is 305-03-3, and its molecular formula is C. 14 H 19 C l2 NO2, molecular weight: 304.21, comes from pine root secretions.

[0028] In this invention, the ppm concentration of a pharmaceutically acceptable salt of chlorambucil or its derivatives refers to 10 -6 (g / ml).

[0029] In this invention, pharmaceutically acceptable salts of m-chlorobenzoic acid include pharmaceutically acceptable acid addition salts or base addition salts of m-chlorobenzoic acid. The term "pharmaceutically acceptable salt" refers to a salt formed with an inorganic or organic acid, or an inorganic or organic base, that maintains the bioavailability of the free form without other side effects. Inorganic acids include hydrochloric acid, bromic acid, nitric acid, sulfuric acid, perchloric acid, and phosphoric acid; organic acids include citric acid, acetic acid, lactic acid, maleic acid, fumaric acid, mesylic acid, succinic acid, tartaric acid, galacturonic acid, pamoic acid, glutamic acid, aspartic acid, oxalic acid, (D) or (L) malic acid, mesylic acid, ethanesulfonic acid, 4-toluenesulfonic acid, salicylic acid, citric acid, benzoic acid, and malonic acid. Salts derived from inorganic bases include, but are not limited to, sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, and aluminum salts. Salts derived from organic bases include, but are not limited to, the following: primary amines, secondary amines, and tertiary amines; substituted amines, including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, triethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, betaine, ethylenediamine, glucosamine, methylglucosamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, etc.

[0030] The beneficial effects of this invention are:

[0031] 1) The chlorinated mustard or its pharmaceutically acceptable salt described in this invention has important applications in the prevention and control of root rot caused by Panax notoginseng rust fungus. It can effectively inhibit the growth of Panax notoginseng rust fungus, thereby effectively preventing and controlling root rot of Panax notoginseng and providing a new control agent for the prevention and control of Panax notoginseng diseases.

[0032] 2) The chlorambucil or its pharmaceutically acceptable salt described in this invention can promote the growth of beneficial bacteria such as *Morchella spp.* and *Trichoderma spp.*, which helps in the prevention and control of diseases of Panax notoginseng.

[0033] 3) The chlorinated mustard or its pharmaceutically acceptable salt described in this invention can promote the survival of one-year-old Panax notoginseng seedlings, increase the dry weight of individual Panax notoginseng plants above and below ground, and promote the growth of Panax notoginseng fibrous roots, which meets the requirements of sustainable development and has broad prospects for research and market application. Attached Figure Description

[0034] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0035] Figure 1 Example 1 of the present invention: the inhibitory effect of chlorinated mustard on rust-rot fungi.

[0036] Figure 2 This is a photograph of a plate containing chlorambucil against rust-rot fungi, as shown in Example 1 of the present invention.

[0037] Figure 3 Example 2 of the present invention: the growth-promoting effect of chlorinated nitrogen mustard on *Morchella spp.*

[0038] Figure 4 Example 2 of the present invention: a plate photograph of chlorambucil against *Morchella spp.*

[0039] Figure 5 Example 3 of the present invention: the growth-promoting effect of phenylbutyric acid nitrogen mustard on Trichoderma.

[0040] Figure 6 Example 3 of the present invention: a plate photograph of Trichoderma with chlorinated mustard.

[0041] Figure 7 Example 4 of the present invention: Effect of chlorinated mustard on the survival rate of Panax notoginseng seedlings.

[0042] Figure 8 Example 4 of the present invention: Effect of phenylbutyric acid nitrogen mustard on the aboveground dry weight of Panax notoginseng single plant.

[0043] Figure 9 This is a diagram illustrating the effect of phenylbutyric acid mustard on the underground dry weight of Panax notoginseng potted plants in Example 4 of the present invention.

[0044] Figure 10 This is a diagram illustrating the effect of phenylbutyric acid mustard on the root growth of Panax notoginseng fibrous roots in Example 4 of the present invention.

[0045] Figure 11 Example 4 of the present invention: Effect of phenylbutyric acid mustard on the surface area of ​​Panax notoginseng rootlets.

[0046] Figure 12 This is a diagram illustrating the effect of phenylbutyric acid mustard on the root volume of Panax notoginseng fibrous roots in Example 4 of the present invention.

[0047] Figure 13 This is the palmitic acid-treated plate for rust-rot fungi provided in Comparative Example 1 of the present invention.

[0048] Figure 14 This is a diagram showing the inhibitory effect of palmitic acid on rust-rot fungi, provided in Comparative Example 1 of this invention.

[0049] Figure 15 This invention provides Comparative Example 2, which describes the effect of palmitic acid on the survival rate of Panax notoginseng seedlings.

[0050] Figure 16 This is the effect of palmitic acid on the dry weight of Panax notoginseng above ground provided in Comparative Example 2 of the present invention.

[0051] Figure 17 This is the effect of palmitic acid on the dry weight of Panax notoginseng underground single plant provided in Comparative Example 2 of the present invention;

[0052] In the figure, CK is the control group, and the others are treatments with different concentrations of the drug; different letters in the figure indicate significant differences between treatments (P<0.05), * indicates significant differences (p<0.05), ** indicates extremely significant differences (p<0.01), and ns indicates no difference. Detailed Implementation

[0053] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0054] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0055] Example 1

[0056] This embodiment verifies the inhibitory effect of chlorinated mustard on rust-rot fungi. The specific experimental steps are as follows:

[0057] 1. Place 200 ml of PDA culture medium (40 g potato; 3 g agar; 4 g glucose; 200 ml water) in a high-pressure steam sterilizer for 15 min at 121℃. After sterilization, place it in a clean bench for UV sterilization for 20 min.

[0058] 2. Prepare a solution from the compound: Dissolve 10 mg of chlorambucil in 10 ml of methanol to prepare a stock solution ①

[0059] Take 1 ml of ① and add 9 ml of methanol → ②

[0060] Take 1ml ② and add 9ml of methanol → ③

[0061] Take 1ml ③ and add 9ml of methanol → ④

[0062] The control is methanol → ⑤

[0063] 3. After sterilization, use a pipette to add 1 ml of each of ④-① to 99 ml of culture medium to obtain culture media with concentrations of 0.01 ppm, 0.1 ppm, 1 ppm, and 10 ppm, respectively. Dissolve 100 mg of chlorambucil in 10 ml of methanol, and add 1 ml of the solution to 99 ml of culture medium to obtain a culture medium with a concentration of 100 ppm. For the control group, add 1 ml of the methanol solution to 99 ml of culture medium. After mixing, pour the solution into plates and cool. Use a 5 mm diameter punch to break up the rust-rot fungus into mycelial cakes of approximately 5 mm in size. Use an inoculation needle to inoculate the mycelial cakes into the culture medium containing the compound solution. Incubate at 26-28℃ for 5 days. After 5 days, observe the colony morphology and measure the colony diameter.

[0064] like Figure 1 , Figure 2 As shown, the results indicate that when the concentration of chlorambucil was 10 ppm, the mycelial growth of rust-rot fungi was significantly lower than that of the control group, and the mycelia could not extend outward. This indicates that chlorambucil at a concentration of 10 ppm has a significant inhibitory effect on rust-rot fungi, and the antibacterial effect is enhanced with the increase of concentration. Therefore, the application range of chlorambucil is 10 ppm-100 ppm.

[0065] Example 2

[0066] This embodiment verifies the growth-promoting effect of chlorambucil on *Morchella*. The specific experimental steps are as follows:

[0067] 1. Prepare a solution from the compound: Dissolve 10 mg of chlorambucil in 10 ml of methanol to prepare a stock solution ①

[0068] Take 1 ml of ① and add 9 ml of methanol → ②

[0069] Take 1ml ② and add 9ml of methanol → ③

[0070] Take 1ml ③ and add 9ml of methanol → ④

[0071] The control is methanol → ⑤

[0072] 2. After sterilization, use a pipette to add 1 ml of each of ④-① to 99 ml of culture medium, obtaining culture media with concentrations of 0.01 ppm, 0.1 ppm, 1 ppm, and 10 ppm, respectively. For the control group, add 1 ml of methanol solution to 99 ml of culture medium. Shake well, pour into plates, and cool. Use a 5 mm diameter punch to break the *Epipremnum aureum* into mycelial cakes of approximately 5 mm in size. Use an inoculation needle to inoculate the mycelial cakes into the culture medium containing the compound solution. Incubate at 26-28℃ for 7 days. After 7 days, observe the colony morphology and measure the colony diameter.

[0073] like Figure 3 , Figure 4 As shown, the results indicate that when the concentration of chlorambucil was 0.1 ppm, the mycelial growth of *Morchella spp.* was slightly higher than that of the control group. When the concentration of chlorambucil was 10 ppm, the mycelial growth of *Morchella spp.* was significantly higher than that of the control group. This suggests that chlorambucil at a concentration of 10 ppm has a significant growth-promoting effect on *Morchella spp.* mycelia, and the growth-promoting effect is enhanced with increasing concentration. Therefore, the application range of chlorambucil is 0.1 ppm to 10 ppm.

[0074] Example 3

[0075] This embodiment verifies the growth-promoting effect of chlorinated mustard on Trichoderma. The specific experimental steps are as follows:

[0076] 1. Prepare a solution from the compound: Dissolve 10 mg of chlorambucil in 10 ml of methanol to prepare a stock solution ①

[0077] Take 1 ml of ① and add 9 ml of methanol → ②

[0078] Take 1ml ② and add 9ml of methanol → ③

[0079] Take 1ml ③ and add 9ml of methanol → ④

[0080] The control is methanol → ⑤

[0081] 2. After sterilization, use a pipette to add 1 ml of each of ④-① to 99 ml of culture medium, obtaining culture media with concentrations of 0.01 ppm, 0.1 ppm, 1 ppm, and 10 ppm, respectively. For the control group, add 1 ml of methanol solution to 99 ml of culture medium. Shake well, pour into plates, and cool. Use a 5 mm diameter punch to break the *Epipremnum aureum* into mycelial cakes approximately 5 mm in size. Use an inoculation needle to inoculate the mycelial cakes into the culture medium containing the compound solution. Place the plates in an incubator at 26-28℃ and incubate for 2 days. After 2 days, observe the colony morphology and measure the colony diameter.

[0082] like Figure 5 , Figure 6 As shown, the results indicate that when the concentration of chlorambucil was 0.01 ppm, the colony diameter of Trichoderma was significantly higher than that of the control group. When the concentration of chlorambucil continued to increase beyond 0.1 ppm, the promoting effect gradually weakened, indicating that chlorambucil has a significant growth-promoting effect on Trichoderma. Moreover, as the concentration increases, the growth-promoting effect first increases and then weakens. Therefore, the application range of chlorambucil is 0.01 ppm-10 ppm, and the effect is best at low concentrations.

[0083] Example 4

[0084] This embodiment verifies the effects of chlorinated nitrogen mustard on improving the survival rate, single-plant dry weight, and root growth of one-year-old Panax notoginseng. The specific experimental steps are as follows:

[0085] 1. Prepare the compound into a solution: Based on the plate effect, the concentration of chlorambucil was selected in the Panax notoginseng pot experiment. 1 mg of chlorambucil was added to 10 ml of methanol to prepare the stock solution.

[0086] 2. Take 10 ml of the mother liquor and add it to 1 L of water to make a concentration of 1 ppm. Mix well and then add 50 ml of the compound to each pot according to the concentration. Take 10 ml of methanol and add it to 1 L of water for the control. Then, the root irrigation experiment continued for 6 weeks (once a week). After the Panax notoginseng continued to grow for 2 months, the potted Panax notoginseng was collected, cleaned, and the number of Panax notoginseng plants in each compound concentration gradient treatment was counted. The Panax notoginseng fibrous roots were scanned, and the above-ground and underground parts were placed in envelopes. The plants were blanched at 115℃ for 30 minutes and dried at 60℃ for 3 days. The dry weight was weighed and calculated.

[0087] Figure 7 The results showed that root irrigation with chlorambucil at a concentration of 10 ppm significantly increased the survival rate of Panax notoginseng, indicating that chlorambucil is beneficial to the growth of Panax notoginseng. Figure 8 and Figure 9 The results showed that root irrigation with chlorambucil at a concentration of 10 ppm significantly increased the dry weight of individual plants in both the aboveground and underground parts. Figure 10 , Figure 11 and Figure 12Applying chlorambucil to the roots at a concentration of 10 ppm can significantly increase the growth of Panax notoginseng fibrous roots. Overall results show that 10 ppm can significantly improve the growth of Panax notoginseng plants.

[0088] Comparative Example 1

[0089] This comparative example provides another substance detected in root exudates—palmitic acid (CAS: 10025-99-7)—and tests its inhibitory effect on rust-rot fungi. The only difference in the experimental procedure from Example 1 is that chlorambucil was replaced with palmitic acid.

[0090] The results are as follows Figure 13 , Figure 14 As shown, after the addition of palmitic acid, the diameter of the bacterial cake on the plate was significantly higher than that of the control at concentrations above 0.01 ppm, indicating that palmitic acid promoted the growth of rust-rot fungi at concentrations of 0.01 ppm and above.

[0091] Comparative Example 2

[0092] This comparative example verified the effect of another substance detected in root exudates—palmitic acid (CAS: 10025-99-7)—on the growth of Panax notoginseng potted plants through bottle cultivation experiments. The only difference in the experimental procedure from Example 2 was that chlorinated mustard was replaced with m-chlorobenzoic acid.

[0093] The results are as follows Figure 15 , Figure 16 and Figure 17 As shown, the results indicate that m-chlorobenzoic acid does not increase the dry weight of the aboveground parts of Panax notoginseng or the growth of its fibrous roots.

[0094] In addition to the compounds exemplified in the comparative examples above, this invention has also verified many other compounds obtained from pine root exudates. It was found that many other compounds do not have the effects of inhibiting rust-rot fungi, promoting the growth of beneficial bacteria, or promoting the growth of Panax notoginseng, and in particular, they cannot have the above effects simultaneously. These will not be listed here.

[0095] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. At least one of the following applications of chlorambucil or a pharmaceutically acceptable salt thereof: (1) As a bacteriostatic agent for plant pathogens; (2) Inhibits plant pathogens; (3) Prevention and control of root rot in Panax notoginseng; (4) Promotes the growth of beneficial bacteria; (5) Promotes the growth of Panax notoginseng.

2. The application according to claim 1, characterized in that, The plant pathogen is a rust-rot fungus; the root rot of Panax notoginseng is caused by the rust-rot fungus.

3. The application according to claim 1, characterized in that, The beneficial bacteria include *Morchella* and *Trichoderma*.

4. The application according to claim 1, characterized in that, The application includes administering pharmaceutically acceptable salts of isobutylamine mustard or its derivatives to Panax notoginseng.

5. At least one of the following methods, wherein the method comprises administering chlorambucil or a pharmaceutically acceptable salt thereof to Panax notoginseng: (1) Methods to inhibit plant pathogens; (2) Methods for preventing and controlling root rot in Panax notoginseng; (3) Methods to promote the growth of beneficial bacteria; (4) Methods to promote the growth of Panax notoginseng.

6. The application or method according to any one of claims 1-5, characterized in that, When inhibiting rust-rot fungi, the concentration of chlorambucil or a pharmaceutically acceptable salt thereof is not less than 10 ppm; preferably, the concentration is 10 ppm to 100 ppm.

7. The application or method according to any one of claims 1-5, characterized in that, When promoting the growth of beneficial bacteria such as *Morchella*, the concentration of chlorambucil or a pharmaceutically acceptable salt thereof is not less than 0.1 ppm; preferably, the concentration is 0.1 ppm to 100 ppm, more preferably 10 ppm to 100 ppm.

8. The application or method according to any one of claims 1-5, characterized in that, When promoting the growth of Trichoderma in beneficial bacteria, the concentration of chlorambucil or its pharmaceutically acceptable salt shall not exceed 0.1 ppm; preferably, the concentration is 0.005 ppm to 0.1 ppm, more preferably 0.01 ppm to 0.05 ppm.

9. The application or method according to any one of claims 1-5, characterized in that, When promoting the growth of Panax notoginseng, the concentration of chlorambucil or its pharmaceutically acceptable salt is 1-50 ppm; more preferably, the concentration is 5-15 ppm; and most preferably, the concentration is 10 ppm.

10. The application or method according to any one of claims 1-5, characterized in that, The promotion of Panax notoginseng growth includes increasing the survival rate of one-year-old Panax notoginseng and / or increasing the dry weight of Panax notoginseng above ground and / or increasing the dry weight of Panax notoginseng below ground and / or promoting the growth of Panax notoginseng fibrous roots.

Images