Use of tryptophan in the control of plant diseases

Abstract

The present application relates to the technical field of pharmaceutical chemistry, and particularly relates to application of tryptophanone in prevention and treatment of plant bacteria and part of plant fungi. The present application discloses that natural product tryptophanone can effectively prevent and treat plant bacterial and part of plant fungal diseases. The application of tryptophanone as an agricultural bactericide in plant bacterial diseases has excellent inhibitory effect on rice bacterial leaf blight, rice bacterial leaf streak, citrus canker, cabbage black rot, tomato bacterial spot, kiwi canker, tobacco bacterial wilt, cucumber bacterial angular spot, potato bacterial wilt, soybean bacterial spot, tomato canker and the like. The tryptophanone also shows good inhibitory activity on plant pathogenic fungi such as grapevine Eutypa lata, tobacco alternaria alternata, eggplant verticillium wilt, pepper fusarium wilt, colletotrichum gloeosporioides, tea tree colletotrichum gloeosporioides and sorghum zylosporium.

Description

Technical Field

[0001] This invention relates to the field of medicinal chemistry, specifically to the application of tryptophan in the prevention and control of plant bacterial and some fungal diseases. Background Technology

[0002] The rapid and widespread spread of plant diseases causes enormous yield and economic losses to agricultural production every year. In China, plant diseases caused by pathogenic fungi and bacteria rank first and second. Studies show that more than 10,000 plant diseases caused by pathogenic fungi account for 10-30% of total crop losses annually. Rice bacterial wilt, caused by the fungus *Bacillus thuringiensis*, is considered one of the world's deadliest rice diseases, having reduced rice yields by 50% in some Asian countries. However, the declining efficacy of antimicrobial agents due to pathogen resistance is one of the challenges facing chemical pesticides. Long-term use may also lead to side effects such as unsatisfactory field efficacy and harm to the environment and plant health. Therefore, developing highly effective, low-risk, and environmentally friendly antimicrobial agents is an urgent priority.

[0003] Natural products, with their unique chemical skeletons, good biological activity, and environmental compatibility, provide many ideal molecules for the development of new drugs and pesticides. Tryptophanone belongs to the indolequinazoline alkaloid class, with the chemical name indolo[2,1-b]quinazolin-6,12-dione. It is one of the main components of the traditional Chinese medicines Indigofera tinctoria and Isatis tinctoria, as well as the medicinal plants Strobilanthes cusia, Polygonum tinctoria, and Isatis indigotica. In 1879, Sommaruga first obtained tryptophanone by sublimation of indigo under reduced pressure. In 1902, Seidel determined the structure of tryptophanone. In 1971, Schindiels et al. isolated tryptophanone from the metabolites of Candida lipolytica cultured with tryptophan, hence the name. In 1977, Bergman et al. isolated tryptophanone from Candida albicans, thus initiating research on the pharmacological activity of tryptophanone. The results showed that tryptamine and its derivatives possessed good anti-inflammatory, antibacterial, antitumor, antituberculosis, immunomodulatory, and antileishmaniasis activities, providing important research and development prospects for the search of lead compounds. In this study, we found that commercially available natural tryptamine exhibited good inhibitory activity against some plant pathogens, which is expected to provide an important scientific basis for the discovery and creation of new plant-derived pesticides. Summary of the Invention

[0004] This invention provides the application of tryptophan in the prevention and control of plant diseases.

[0005] Furthermore, the plant diseases specifically include diseases caused by bacteria and diseases caused by fungi.

[0006] Furthermore, the diseases caused by the aforementioned bacteria include those caused by pathogens of rice bacterial blight, *Xanthomonas oryzae* (rice leaf spot pathogen), *Xanthomonas carpetii*, *Xanthomonas rapae* (rapeseed pathogen), *Pseudomonas syringae* (tomato leaf spot pathogen), *Pseudomonas syringae* (kiwifruit pathogen), *Ralstonia solanacearum*, *Pseudomonas syringae* (cucumber angular leaf spot pathogen), *Ralstonia solanacearum*, *Xanthomonas rapae* (soybean pathogen), or *Corynebacterium micranthum* (tomato canker pathogen).

[0007] Furthermore, the diseases caused by the fungi include those caused by Verticillium wilt of eggplant, Fusarium wilt of pepper, Ficus pumila of grape, Alternaria alternata of tobacco, Colletotrichum gloeosporioides, Anthracnose of tea, and Colletotrichum sorghum.

[0008] The present invention also provides the application of tryptophanone in the preparation of drugs for the prevention and treatment of plant diseases.

[0009] The present invention has the following beneficial effects:

[0010] This invention discloses that the natural product tryptophanone can effectively control bacterial diseases in plants, especially showing excellent inhibitory activity against plant pathogenic bacteria such as *Bacillus thuringiensis* (rice bacterial blight) and *Citrus canker* (citrus canker). Simultaneously, it exhibits excellent inhibitory activity against the plant pathogenic fungus *Verticillium wilt* (eggplant wilt). This type of natural framework has extremely high research value in the discovery of novel pesticides against plant pathogenic fungi. Detailed Implementation

[0011] This document details various exemplary embodiments of the present invention. Unless otherwise specified, the methods used in these embodiments employ conventional methods, and the reagents used are commercially available or prepared using conventional methods. This detailed description should not be construed as a limitation of the invention, but rather as a more detailed description of certain aspects, characteristics, and embodiments of the invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, regarding numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as any smaller range between any other stated value or intermediate value within said range, are also included within the scope of this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0012] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0013] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0014] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0015] Example

[0016] In this invention, tryptophan (98%) was purchased from Shanghai Bide Pharmaceutical Technology Co., Ltd. The natural product tryptophan exhibits good bioactivity against plant bacteria. The following examples illustrate its activity against rice bacterial blight, citrus canker, and kiwifruit canker. The in vitro EC50 of tryptophan against these three plant pathogens was tested using the turbidimetric method. 50 Value testing. Citrus canker pathogens, kiwifruit canker pathogens, and rice bacterial blight pathogens were activated using NA solid medium (10g glucose, 5g peptone, 1g yeast, 3g beef extract, 15g agar, and 1000mL deionized water) and placed in a 28℃ incubator until single colonies appeared. A suitable amount of yellow single colonies were selected using an inoculation loop and placed in an Erlenmeyer flask containing NB medium (10g glucose, 5g peptone, 1g yeast, 3g beef extract, and 1000mL deionized water). The flasks were then incubated in a shaker until the logarithmic growth phase for later use.

[0017] Prepare the required test drugs to the specified concentrations, and add 1 mL of each to a test tube containing 4 mL of NB medium. Then, add 40 μL of NB medium containing *Rhizoctonia solani*, *Citrus canker*, and *Rhizoctonia solani* to the test tubes. Incubate at 26–28°C with shaking on a shaker at 180 rpm. Observe the OD values ​​of the blank control group's NB liquid medium. 595 The values ​​ranged from 0.6 to 0.8. The OD values ​​of the bacterial solutions at various concentrations were measured using a spectrophotometer. 595 value.

[0018] Correcting OD595 Value = OD of bacterial culture medium 595 - Sterile culture medium OD 595

[0019] Inhibition rate (%) = (OD595 of the corrected control culture medium - OD595 of the corrected drug-containing culture medium) 595 ) / OD value of bacterial suspension in the corrected control culture medium × 100%.

[0020] Table 1. EC5 of the natural product tryptophan ketone against plant pathogenic bacteria. 50

[0021]

[0022] As shown in Table 1, tryptophan exhibits excellent in vitro antibacterial activity against *Bacillus thuringiensis*, with an EC50 of [missing information]. 50 =0.10 mg / L, its in vitro antibacterial activity was significantly better than that of the commercial control drug, tebuconazole (EC). 50 =31.1 mg / L) and thiabendazole copper (EC) 50 >50 mg / L); Tryptophan exhibits excellent in vitro antibacterial activity against *Citrus canker*, with its EC50 concentration exceeding 50 mg / L. 50 =13.9 mg / L, its in vitro antibacterial activity was significantly better than that of the commercial control drug tebuconazole (EC). 50 >50 mg / L) and thiamethoxam (EC) 50 >50 mg / L); Tryptophan showed good in vitro antibacterial activity against *Actinidia kiwifruit* causal agent, with its EC50 concentration exceeding 50 mg / L. 50 =159.5 mg / L, its in vitro antibacterial activity is similar to that of the commercial control drug, tebuconazole (EC). 50 >50 mg / L) and thiamethoxam (EC) 50 (equivalent to >50mg / L).

[0023] The natural product tryptophan also showed good antifungal activity against some fungi. The following are examples of its activity against Verticillium wilt of eggplant, Fusarium wilt of pepper, clematis of grape, Alternaria solani of tobacco, Colletotrichum gloeosporioides, Anthracnose of tea, and Colletotrichum sorghum.

[0024] The mycelial growth rate method, also known as the toxic medium method, is one of the routine methods for determining the toxicity of fungicides. The main principle is to mix the test agent with a culture medium and measure the toxicity of the agent by the rate at which colonies grow on the toxic medium. This example uses *Rhizoctonia solani*, *Botrytis cinerea*, *Fusarium graminearum*, *Fusarium wilt* of pepper, and *Verticillium wilt* of eggplant as test subjects, and DMSO (dimethyl sulfoxide) as a blank control.

[0025] The specific procedures are as follows: 1) Weigh an appropriate amount of drug according to the test concentration, dissolve it in DMSO (the amount should not exceed 1% of the final toxic medium), then add 0.1% Tween 20 aqueous solution to make up to 10 mL, pour it into 90 mL of melted PDA medium, mix well, and then pour it into 9 petri dishes for later use; 2) Sterilize the punch (with an inner diameter of 5 mm) by flame, and after it cools, punch holes in the hyphae near the edge of the pre-activated strain, and use an inoculation needle to place the hyphae facet to the center of the toxic medium. After treatment, incubate them all at 25℃; 3) After the colony diameter of the control group grows to 5.5-6.0 cm, use the cross-cross method to determine the colony diameter of the control group and each drug treatment group; 4) Calculate the inhibition rate (%) using the following formula:

[0026] Inhibition rate % = (CT) / (C-0.5)×100%;

[0027] Where C is the colony diameter of the control group, T is the colony diameter of the drug-treated group, and 0.5 is the diameter of the inoculated mycelium.

[0028] EC 50 Median effective concentration (MEC) is an important indicator for evaluating the sensitivity of plant pathogens to compounds, and it is also a crucial parameter for setting compound concentrations when studying the mechanism of action of compounds. In concentration gradient experiments, five appropriate concentrations were set using the two-fold dilution method. Finally, the inhibition rate of the agent against the plant pathogen and the agent concentration were converted into logarithmic values, and the toxicity curve was obtained through regression analysis using SPSS software to calculate the EC50. 50 .

[0029] The embodiments of this invention are provided to illustrate the technical solutions of this invention, but the content of the embodiments is not limited thereto. The experimental results of the target compound are shown in Tables 2 and 3.

[0030] Table 2. Inhibitory activity of the natural product tryptophan against some plant pathogenic fungi (50 μg / mL)

[0031]

[0032] Table 3. EC5 activity of the natural product tryptophan against Verticillium wilt, the causal agent of eggplant wilt. 50 value

[0033]

[0034] As shown in Tables 2 and 3, in in vitro experiments, the natural product tryptamine exhibited certain inhibitory activity against plant pathogenic fungi (such as *Botrytis cinerea*, *Tobacco Star*, *Verticillium wilt*, *Fusarium wilt*, *Colletotrichum candida*, *Colletotrichum tea*, and *Colletotrichum sorghum*). It showed excellent inhibitory activity against the plant pathogenic fungus *Verticillium wilt*, with an EC50 of [missing value]. 50 The effective concentration was 7.4 μg / mL, which was superior to the control drug thiabendazole. It also showed good inhibitory activity against *Colletotrichum sacchari*, but lower than the positive control drug thiabendazole. Therefore, this type of natural skeleton has extremely high research value in the discovery of novel pesticides against plant pathogenic fungi.

[0035] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Application of tryptophan in inhibiting Verticillium wilt of eggplant.

2. Application of tryptophan in the preparation of pesticides for controlling Verticillium wilt in eggplant.

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