Use of benzyl methyl sulfide in inhibiting plant pathogens

Abstract

The application discloses application of benzyl methyl sulfide in inhibition of plant pathogenic bacteria and belongs to the technical field of plant disease prevention and treatment. The application discloses the application of benzyl methyl sulfide in inhibition of plant pathogenic bacteria, and the inhibiting effect of benzyl methyl sulfide on different pathogenic bacteria is determined through indoor toxicity experiments, wherein the inhibiting effect on alternaria alternata and alternaria macrospora is the best, and when 10 muL / L of the benzyl methyl sulfide is used, the inhibition rate of the benzyl methyl sulfide on the alternaria alternata and the alternaria macrospora can reach 100%. The EC 50 concentration of the benzyl methyl sulfide on the alternaria alternata is 0.63 muL / L, the EC 50 concentration of the benzyl methyl sulfide on the alternaria macrospora is 1.03 muL / L. In addition, the benzyl methyl sulfide also has a good inhibiting effect on phoma exigua, and the EC 50 concentration of the benzyl methyl sulfide on the phoma exigua is 3.98 muL / L. The methyl benzyl sulfide has the advantages of good prevention and treatment effect and low residue, and can be used as a potential biocontrol agent for plant pathogenic bacteria.

Description

Technical Field

[0001] This invention relates to the field of plant disease control technology, and more specifically to the application of benzyl methyl sulfide in inhibiting plant pathogens. Background Technology

[0002] With the continuous development of society and science and technology, and in today's advocacy of green consumption and ecological environmental protection, green, efficient, and low-toxicity pesticides have become a research hotspot in the field of plant protection in recent years. Studies have found that antibacterial substances in volatile compounds can easily diffuse and penetrate into the soil and air, increasing the inhibition rate against target microorganisms. This has important guiding significance for the development of efficient and novel biological fungicides for fruits and vegetables.

[0003] Benzyl methyl sulfide, also known as methyl benzyl sulfide, is a volatile compound used as a food-grade flavoring. However, there are currently no reports on the inhibitory effects of benzyl methyl sulfide's volatile gases on plant pathogens.

[0004] Alternaria is a globally distributed plant pathogen with a wide range of host species and readily forms conidia on its hosts. Black spot disease of jujube is a fungal disease caused by *Alternaria alternate (Fr.) Keissler*, a highly pathogenic and rapidly spreading fungus. Infected fruits develop dark brown, circular lesions surrounded by halos of different colors, or may lack a fruit peel, or have slightly sunken or rotten parts of the flesh, resulting in reduced yield and quality, severely impacting the marketability of the fruit and causing significant economic losses to jujube farmers. The pathogen of *Alternaria heveae* leaf spot disease of rubber trees belongs to the Deuteromycetes, Hyphomycetes, Hyphomyales, Dictamniaceae, and *Alternaria* genus. Infected leaves develop numerous black, circular, slightly sunken dot-like lesions, 0.1–0.4 mm in diameter, surrounded by a yellow halo. Young leaves of rubber trees are susceptible to disease. During the rainy season, or when management is poor or the plants are not growing well, latex production is reduced, resulting in economic losses.

[0005] Diaporthepassifloricola is the pathogen of citrus black spot disease. This pathogen belongs to the family Diaporthepassiaceae (asexual form, genus Diaporthepassifloricola) and occurs in citrus-producing areas in China, including Jiangxi, Guangxi, Guangdong, Shaanxi, Fujian, Hunan, Chongqing, and Shanghai.

[0006] *Plasmodium microsporum* is a well-known plant pathogen that can cause leaf blight in macadamia trees, olive fruit rot, gray spot disease in short-spike palms, leaf blight in oil palm seedlings, leaf blight in tea buds, and leaf blight in jatropha curcas. In macadamia nut plantations in Yunnan, diseases such as leaf blight and leaf drop caused by *Plasmodium microsporum* are frequently found, leading to gradual decline in tree growth, reduced fruit set, and consequently, decreased orchard yield, severely impacting the orchard's economic value.

[0007] Powdery mildew is one of the most serious foliar diseases affecting rubber trees, caused by the fungus *Oidium heveae* Steinmann. This disease primarily damages young leaves (bronze leaves, pale green leaves), shoots, and inflorescences. It is highly contagious, often causing significant leaf drop, leading to reduced latex and fruit yields, even crop failure, slowed growth, and sometimes plant death, resulting in substantial economic losses to rubber production. Therefore, it has long been a major concern for rubber-growing countries worldwide.

[0008] Corynespora cassiicola is a plant pathogenic fungus with a wide host range and worldwide distribution, capable of infecting 530 species of plants in 380 genera, including Cucurbitaceae, Sapindaceae, Euphorbiaceae, Caricaceae, Malvaceae, Asteraceae, Solanaceae, Convolvulaceae, Amaranthaceae, Fabaceae, Araceae, Oleaceae, and Meliaceae. The pathogen causing rubber tree leaf drop is Corynespora cassiicola (Berk and curt.) Wei, belonging to the class Hyphomycetes, order Hyphomycetales, family Dematiaceae, and genus Corynespora. It infects the seedlings, old leaves, young trees, and tender leaves of mature rubber trees, leading to sparse canopies and dwarfing of the plant.

[0009] Anthracnose is a group of globally distributed plant pathogens. In tropical and subtropical regions, anthracnose also threatens various crops such as grains, fruits, vegetables, and rubber, causing serious economic losses. Anthracnose is an important disease of rubber trees, occurring in rubber-growing areas worldwide. Anthracnose affects rubber seedlings, young trees in the field, and tapping trees, infecting multiple parts including young leaves, petioles, shoots, and fruits. It causes leaf drop, shoot dieback, and fruit rot. In severe cases, it leads to repeated leaf drop and shoot dieback, delaying tapping and reducing latex yield.

[0010] Therefore, providing the application of benzyl methyl sulfide in inhibiting plant pathogens is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0011] In view of this, the present invention provides the application of benzyl methyl sulfide in inhibiting plant pathogens.

[0012] To achieve the above objectives, the present invention adopts the following technical solution:

[0013] The application of benzyl methyl sulfide in inhibiting plant pathogens, wherein the plant pathogens are Colletotrichum gloeosporioides, Pestalotiopsis microspora, Corynespora cassiicola, Alternaria alternate, Colletotrichum alienum, Diaporthepassifloricola, Colletotrichum siamense, Alternaria heveae, and Oidium heveae Steinmann.

[0014] Benzyl methyl sulfide can inhibit the mycelial growth of plant pathogens through gaseous action in a closed environment without contact with the pathogens themselves. It is particularly effective against *Alternaria heveae*, the pathogen of *Alternaria* leaf spot, and *Alternaria alternate*, the pathogen of brown spot disease in jujube.

[0015] Furthermore, the hosts of the plant pathogen are rubber, nuts, jujubes, and citrus.

[0016] Furthermore, when the plant pathogen is *Colletotrichum gloeosporioides*, the concentration of benzyl methyl sulfide used is 21 μL / L-126 μL / L (2 μL / plate-12 μL / plate). Furthermore, when the plant pathogen is *Pestalotiopsis microspora*, the concentration of benzyl methyl sulfide used is 21 μL / L-158 μL / L (2 μL / plate-15 μL / plate).

[0017] Furthermore, when the plant pathogen is *Corynespora cassiicola*, the concentration of benzyl methyl sulfide used is 5 μL / L-42 μL / L (0.5 μL / plate-4 μL / plate).

[0018] Furthermore, when the plant pathogen is *Alternaria alternate*, the concentration of benzyl methyl sulfide used is 2 μL / L-52 μL / L (0.2 μL / plate-5 μL / plate).

[0019] Furthermore, when the plant pathogen is *Colletotrichum alienum*, the concentration of benzyl methyl sulfide used is 21 μL / L-158 μL / L (2 μL / plate-15 μL / plate).

[0020] Furthermore, when the plant pathogen is Diaporthepassifloricola, the concentration of benzyl methyl sulfide used is 21 μL / L-210 μL / L (2 μL / plate-20 μL / plate).

[0021] Furthermore, when the plant pathogen is *Colletotrichum siamense*, the concentration of benzyl methyl sulfide used is 21 μL / L-126 μL / L (2 μL / plate-12 μL / plate).

[0022] Furthermore, when the plant pathogen is *Alternaria heveae*, the concentration of benzyl methyl sulfide used is 2 μL / L-31 μL / L (0.2 μL / plate-3 μL / plate).

[0023] Furthermore, when the plant pathogen is Oidium heveae Steinmann, the concentration of benzyl methyl sulfide used shall not be less than 50% (by volume).

[0024] This invention demonstrates, through indoor toxicity testing, that benzyl methyl sulfide has a significant inhibitory effect on plant pathogens.

[0025] As can be seen from the above technical solution, compared with the prior art, this invention discloses the application of benzyl methyl sulfide in inhibiting plant pathogens. It utilizes benzyl methyl sulfide to fumigate pathogens in a sealed environment, inhibiting mycelial growth through the action of gases without direct contact with the plant pathogens. Indoor toxicity tests confirmed that benzyl methyl sulfide exhibits varying degrees of inhibitory effects against different pathogens, with the best inhibitory effect against *Alternaria rubberata* and *Alternaria alternata*, followed by *Anthracnose heterotrophus* and *Corynebacterium* leaf fall pathogens. Benzyl methyl sulfide is volatile and is a food flavoring agent permitted for use under national regulations. Therefore, this invention determines the optimal concentration of benzyl methyl sulfide for controlling nine pathogens. Compared with existing agents, it has the advantages of good disease prevention effect and low residue, thus possessing strong development and application prospects. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present 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 only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0027] Figure 1 The attached figure shows the colony morphology of *Colletotrichum gloeosporioides*, the pathogen of the rubber anthrax of the present invention, after treatment with benzyl methyl sulfide.

[0028] The amounts of benzyl methyl sulfide added for 1-6 are 0 μl, 2 μl, 3 μl, 4 μl, 5 μl, and 12 μl, respectively.

[0029] Figure 2 The attached figure shows the colony morphology of *Pestalotiopsis microspora* after treatment with benzyl methyl sulfide according to the present invention.

[0030] The amounts of benzyl methyl sulfide added for 1-6 are 0 μl, 2 μl, 3 μl, 5 μl, 10 μl, and 15 μl, respectively.

[0031] Figure 3 The attached figure shows the colony morphology of *Corynespora cassiicola*, the leaf-bearing pathogen of this invention, after treatment with benzyl methyl sulfide.

[0032] The amounts of benzyl methyl sulfide added for 1-6 are 0 μl, 0.5 μl, 1 μl, 2 μl, 3 μl, and 4 μl, respectively.

[0033] Figure 4 The attached figure shows the colony morphology of *Colletotrichum alienum* of the present invention after treatment with benzyl methyl sulfide.

[0034] The amounts of benzyl methyl sulfide added for 1-6 are 0 μl, 2 μl, 3 μl, 7 μl, 10 μl, and 15 μl, respectively.

[0035] Figure 5 The attached figure shows the colony morphology of Diaporthepassifloricola, a citrus black spot disease of the present invention, after treatment with benzyl methyl sulfide.

[0036] The amounts of benzyl methyl sulfide added for 1-6 are 0 μl, 2 μl, 3 μl, 5 μl, 10 μl, and 15 μl, respectively.

[0037] Figure 6 The attached figure shows the colony morphology of *Colletotrichum siamense* (the present invention) after treatment with benzyl methyl sulfide.

[0038] The amounts of benzyl methyl sulfide added for 1-6 are 0 μl, 2 μl, 3 μl, 5 μl, 10 μl, and 12 μl, respectively.

[0039] Figure 7 The attached figure shows the colony morphology of *Alternaria heveae* after treatment with benzyl methyl sulfide.

[0040] The amounts of benzyl methyl sulfide added for 1-6 are 0 μl, 0.2 μl, 0.5 μl, 1 μl, 2 μl, and 3 μl, respectively.

[0041] Figure 8 The attached figure shows the colony morphology of *Alternaria alternate* after treatment with benzyl methyl sulfide according to the present invention;

[0042] The amounts of benzyl methyl sulfide added for 1-6 are 0 μl, 0.2 μl, 0.7 μl, 1 μl, 3 μl, and 5 μl, respectively.

[0043] Figure 9 The attached figure shows the spore morphology of *Oidium heveae Steinmann* after treatment with benzyl methyl sulfide (magnification 200; scale bar 20 μm).

[0044] Among them, 1 is the control group treated with water; 2 is the spores treated with benzyl methyl sulfide. Detailed Implementation

[0045] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0046] Compound: benzyl methyl sulfide (>98.0% (GC), liquid), purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0047] The fungi *Colletotrichum gloeosporioides*, *Pestalotiopsis microspora*, *Corynespora cassiicola*, *Alternaria alternate*, *Colletotrichum alienum*, *Diaporthe passifloricola*, *Colletotrichum siamense*, and *Alternaria heveae* were all preserved by the Plant Protection and Microbial Utilization Research Center of the Yunnan Provincial Institute of Tropical Crops. The tested pathogen of powdery mildew on rubber trees, *Oidium heveae Steinmann*, was collected from naturally occurring rubber tree leaves with fresh lesions in Xishuangbanna, Yunnan Province.

[0048] Example 1: Application of benzyl methyl sulfide in inhibiting plant pathogens

[0049] (1) Eight plant pathogens (Alternaria glomerata, Alternaria microsporidium, Alternaria glomerata leaf drop, Alternaria alternata, Alternaria heterophylla, Alternaria citrinum, Alternaria sicca, Alternaria rubra) were cultured on PDA solid medium at 28°C for 4-7 days, and then holes were punched along the outer edge of the colony using a 4 mm diameter punch.

[0050] (2) Eight pathogenic bacterial blocks were inoculated into the bottom of a petri dish (9 cm in diameter, 1.5 cm in height, and 95 mL in volume) containing PDA medium. Different volumes of benzyl methyl sulfide (the amount of benzyl methyl sulfide added to each dish for each strain is shown in Table 1) were added to the lid of the petri dish containing PDA medium. The bottom and lid of the petri dish were then inverted and sealed with sealing film, and incubated at 28°C. Plates inoculated only with pathogenic bacteria and without benzyl methyl sulfide served as controls. When the mycelia in the control group reached the edge of the petri dish, the colony morphology was as follows: Figure 1-8 At this point, the colony diameter was measured using a cross-sectional method. The inhibition rate was calculated using the formula, with each treatment repeated three times. The inhibition rate (%) was calculated as follows: [(Control pathogen hyphae diameter - Treated pathogen hyphae diameter) / (Control pathogen hyphae diameter - Disease diameter)] × 100. The inhibition rate results are shown in Table 1.

[0051] Table 1. Inhibition rates of different concentrations of benzyl methyl sulfide against eight plant pathogens.

[0052]

[0053]

[0054] The results in Table 1 show that when using 0.2 μL / plate of benzyl methyl sulfide, the inhibition rate of *Alternaria alternate* and *Alternaria heveae* can reach over 80%. When using 1 μL / plate of benzyl methyl sulfide, the inhibition rate of *Alternaria alternate* and *Alternaria heveae* can reach 100%. Furthermore, 2 μL / plate of benzyl methyl sulfide can also achieve an inhibition rate of over 50% against six plant pathogens: *Colletotrichum gloeosporioides*, *Pestalotiopsis microspora*, *Corynespora cassiicola*, *Colletotrichum alienum*, *Diaporthepassifloricola*, and *Colletotrichum siamense*. 10 μL / plate of benzyl methyl sulfide completely inhibited the growth of Diaporthe passifloricola, the causal agent of citrus black spot; 12 μL / plate of benzyl methyl sulfide completely inhibited the growth of Colletotrichum siamense and Colletotrichum gloeosporioides; and 15 μL / plate of benzyl methyl sulfide completely inhibited the growth of Pestalotiopsis microspora and Colletotrichum alienum.

[0055] After calculating the inhibition rates of benzyl methyl sulfide against various plant pathogens, the virulence regression equation and EC were obtained. 50 Values ​​are shown in Table 2.

[0056] Table 2. Virulence regression equations and EC50 values ​​of benzyl methyl sulfide against eight plant pathogens. 50 value

[0057]

[0058] The calculated data in Table 2 show that benzyl methyl sulfide has an EC50 effect on Alternaria alternanthematosus. 50 A concentration of 0.63 μL / L was used to inhibit the EC50 of Alternaria heveae. 50The concentration was 1.03 μL / L. Furthermore, EC50 was used to treat *Corynespora cassiicola*, the leaf-bearing causal agent of *Corynespora cassiicola*. 50 The concentration was 3.98 μL / L.

[0059] Example 2: Inhibitory effect of benzyl methyl sulfide on powdery mildew of rubber trees

[0060] Preparation of conidia suspension: Rubber tree pollen fungi produced on rubber tree leaves were washed off with sterile water, filtered through three layers of lens paper, and finally obtained a concentration of approximately 2 × 10⁻⁶. 4 A spore suspension of 1 spore per mL.

[0061] Spore germination inhibition assay: 300 μL of spore suspension was added to two 1.5 mL sterile centrifuge tubes. The treatment group received 300 μL of benzyl methyl sulfide, while the blank control group received 300 μL of sterile water. All samples were incubated at 28℃ for 6 hours, followed by microscopic examination of spore germination. Results are shown below. Figure 9 .

[0062] Figure 9 The results showed that, under an optical microscope, the germination rate of *Rhizoctonia solani* spores without benzyl methyl sulfide was 57.8% after 6 hours of cultivation, while no germination was observed in the samples treated with benzyl methyl sulfide. The results demonstrate that benzyl methyl sulfide has a significant inhibitory effect on the growth of *Rhizoctonia solani* spores.

[0063] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The application of benzyl methyl sulfide in inhibiting plant pathogens, characterized in that, The plant pathogens mentioned are Colletotrichum gloeosporioides, Pestalotiopsis microspora, Corynespora cassiicola, Alternaria alternate, Colletotrichum alienum, Diaporthepassifloricola, Colletotrichum siamense, Alternaria heveae, and Oidium heveae Steinmann.

2. The application of benzyl methyl sulfide according to claim 1 in inhibiting plant pathogens, characterized in that, The hosts of the plant pathogen are rubber, nuts, jujubes, and citrus.

3. The application of benzyl methyl sulfide according to claim 1 in inhibiting plant pathogens, characterized in that, When the plant pathogen is Colletotrichum gloeosporioides, the concentration of benzyl methyl sulfide used is 21 μL / L-126 μL / L; When the plant pathogen is Pestalotiopsis microspora, the concentration of benzyl methyl sulfide used is 21 μL / L-158 μL / L. When the plant pathogen is *Corynespora cassiicola*, the concentration of benzyl methyl sulfide used is 5 μL / L-42 μL / L. When the plant pathogen is Alternaria alternate, the concentration of benzyl methyl sulfide used is 2 μL / L-52 μL / L; When the plant pathogen is Colletotrichum alienum, the concentration of benzyl methyl sulfide used is 21 μL / L-158 μL / L; When the plant pathogen is Diaporthepassifloricola, the concentration of benzyl methyl sulfide used is 21 μL / L-210 μL / L; When the plant pathogen is Colletotrichum siamense, the concentration of benzyl methyl sulfide used is 21 μL / L-126 μL / L; When the plant pathogen is *Alternaria heveae*, the concentration of benzyl methyl sulfide used is 2 μL / L-31 μL / L; When the plant pathogen is Oidium heveae Steinmann, the concentration of benzyl methyl sulfide used shall not be less than 50%.

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