- Email: [email protected]
Study of the in vivo antiviral activity against TMV treated with novel 1-(t-butyl)-5-amino-4-pyrazole derivatives containing a 1,3,4-oxadiazole sulfide moiety
Pesticide Biochemistry and Physiology xxx (xxxx) xxx
Contents lists available at ScienceDirect
Pesticide Biochemistry and Physiology journal homepage: www.elsevier.com/locate/pest
Study of the in vivo antiviral activity against TMV treated with novel 1-(t-butyl)-5-amino-4-pyrazole derivatives containing a 1,3,4-oxadiazole sulfide moiety Guangqian Yang, Huanlin Zheng, Wubin Shao, Liwei Liu, Zhibing Wu * State Key Laboratory Breeding Base of Green Pesticide and Agricultural Bioengineering, Key Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of Education, Center for R & D of Fine Chemicals of Guizhou University, Guiyang 550025, China
A R T I C L E I N F O
A B S T R A C T
Keywords: 1,3,4-oxadiazole sulfide Tobacco mosaic virus Defense enzyme activity Green fluorescent protein Insecticidal activity
A series of new 1-tert-butyl-5-amino-4-pyrazole bioxadiazole sulfide derivatives containing a 1,3,4-oxadiazole moiety were designed and synthesized. The bioactivity results showed that some title compounds exhibited excellent protective activity against TMV and certain insecticidal activity. Among the tested compounds, the EC50 values of 5d, 5j, 5k and 5l were 165.8, 163.2, 159.7 and 193.1 mg/L, respectively, which are better than the EC50 value of ningnanmycin (271.3 mg/L). The chlorophyll contents and the defense enzyme activities of the tobacco leaves after treatment with 5j were significantly increased, which indicated that this series of title compounds may induce the systemic acquired resistance of host to defend against diseases. Further in vivo protective activity research on 5j using TMV with a GFP gene tag found that it can effectively inhibit the spread of TMV in inoculated tobacco. A morphological study with TEM revealed that title compound 5h can cause a distinct break of the rod-shaped TMV. Moreover, the insecticidal activity revealed that the fatality rates of 5a, 5b and 5m against aphidoidea were 85%, 83% and 87%, respectively, which indicated that the title compounds can effectively block the common carrier of plant viruses, thereby effectively reducing the TMV infection risk of tobacco. This series of synergistic effects provide key information for the research and development of antiviral agents.
1. Introduction Plant viral diseases can cause serious effects on the growth of food crops and cash crops. The economic losses caused by plant viral diseases in agricultural and forestry production worldwide are approximately $60 billion each year (Zhao et al., 2015). Tobacco mosaic virus (TMV), a typical plant virus, can infect a series of Solanaceae plants that are rep resented by tobacco. TMV first parasitizes plant cells and then uses their energy and enzyme systems to replicate and synthesize its own coat protein in the ribosomes of the cells (Buck, 1999; Beachy and Heinlein, 2000). Due to the differences in the immune system between plants and animals, it is difficult to prevent and control diseases caused by TMV infection (Fedorkin et al., 2001; Lv et al., 2020). Due to the different action mechanisms and obvious defects of the
traditional commercialized agents ningnanmycin and ribavirin, they did not exhibit the desired prevention and controlling effect (Han et al., 2014; Wang et al., 2014). Therefore, highly efficient anti-TMV agents must be energetically developed and pursued to control viral diseases. Different from traditional agrochemicals, plant activators are novel kinds of agrochemicals that prevent disease by inducing systemic ac quired resistance of the plant’s own defense system (Kessmann et al., ¨rlach et al., 1996; Kunz et al., 1997; Noutoshi et al., 2012). To 1994; Go date, some plant activators have been reported with highly efficient, environmentally friendly and unique modes of action (Du et al., 2012; Du et al., 2013; Friedrich et al., 1996; Silverman et al., 2005a, 2005b, 2005c). To obtain high antiviral activity lead compounds with novel action mechanisms, a series of new 1-tert-butyl-5-amino-4-pyrazole
Abbreviations: TMV, tobacco mosaic virus; 1H NMR, 1H nuclear magnetic resonance; 13C NMR, 13C nuclear magnetic resonance; HRMS, high-resolution mass spectrometry; EC50, median effective concentration; SOD, superoxide dismutase; POD, peroxidase; CAT, catalase; PAL, phenylalanine ammonia-lyase; CK, blank control; LNT, lentinan; GFP, green fluorescent protein; PBS, phosphate buffer saline; TEM, transmission electron microscopy;; dpi, days post-inoculation. * Corresponding author. E-mail address: [email protected] (Z. Wu). https://doi.org/10.1016/j.pestbp.2020.104740 Received 29 September 2020; Received in revised form 2 November 2020; Accepted 2 November 2020 Available online 7 November 2020 0048-3575/© 2020 Published by Elsevier Inc.
Please cite this article as: Guangqian Yang, Pesticide Biochemistry and Physiology, https://doi.org/10.1016/j.pestbp.2020.104740
G. Yang et al.
Pesticide Biochemistry and Physiology xxx (xxxx) xxx
bioxadiazole sulfide derivatives containing a 1,3,4-oxadiazole moiety were designed and synthesized based on our previous work (Fig. 1) (Wu et al., 2016). In vivo antiviral bioassays revealed that some title com pounds exhibited excellent protective activity against TMV. The chlo rophyll content and defense enzyme activities of the tobacco leaves treated with 5j significantly increased, demonstrating that this series of title compounds may induce the host to obtain systemic acquired resistance to enhance its ability to defend against diseases. Further antiviral activity studies using Nicotiana benthamiana leaves infected with TMV that labeled with the GFP gene tag (Chen et al., 2018) and treated with 5j found that it can inhibit the spreading of TMV in inoc ulated tobacco leaves. A morphological study with TEM revealed that 5h can significantly shorten the polymerization length of TMV particles and form a distinct break on the rod-shaped TMV. Moreover, the insecticidal activity revealed that some title compounds have certain activity against aphids, which are common carriers of plant viruses.
hydrate (30 mL), reacted at 100 ◦ C for 4 h, cooled and extracted with dichloromethane. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to obtain 3 as a yellow solid, with a yield of 74%. To a solution of KOH (166.0 mg, 29.7 mmol) in 50 mL ethanol was added 3 (3.9 g, 19.8 mmol), and CS2 (4.5 g, 59.3 mmol) was slowly added dropwise into the solution, reacted at r.t. for 10 h and then reacted at 80 ◦ C for 8 h. The solution was concentrated in vacuo. The pH value was adjusted to 6 with 1 M hydrochloric acid solution, and the solution was filtered, washed with water and dried to obtain 4, which was used in the next reaction without further purification. 2.2.3. General synthesis procedure for title compounds 5a–5m (Wang et al., 2019a, 2019b) To a 50 mL round bottom flask, 4 (500.0 mg, 2.1 mmol), potassium hydroxide (175.8 mg, 3.1 mmol), and H2O (15.0 mL) were added and stirred for 10 min. Then, 2-methylchlorobenzyl (323.2 mg, 2.3 mmol) was added and stirred at room temperature for approximately 8 h. The mixture was extracted with ethyl acetate and washed with water 2–3 times. The organic layer was dried with anhydrous sodium sulfate and filtered. The solvent was concentrated in vacuo to obtain the crude product and purified by column chromatography (petroleum ether/ ethyl acetate 20/1) to obtain 5a as a yellow solid, with a yield of 44% and a m.p. of 116–117 ◦ C. 1H NMR (400 MHz, DMSO‑d6) δ 7.56 (s, 1H, pyrazole H), 6.05 (s, 2H, NH2), 5.99–5.88 (m, 2H, SCH2), 5.34–5.09 (m, 3H, CH3), 1.56 (s, 9H, tert-butyl).13C NMR (100 MHz, DMSO‑d6) δ 162.3, 159.5, 146.7, 133.3, 119.6, 89.5, 59.1, 35.4, 28.8. HRMS (ESI): m/z calcd for C11H18ON5S [M + H]+, 268.12266; found, 268.12210. The synthesis procedure of the other title compounds was similar to that of 5a, and the physical and spectral data of 5a–5m are provided in the supporting information.
2. Materials and methods 2.1. Instruments and chemicals 1 H and 13C NMR spectra were obtained with a JEOL-400 NMR spectrometer (JEOL CO., LTD., Japan) with tetramethylsilane (TMS) as the internal standard and DMSO‑d6 as the solvent. High-resolution mass spectrometry (HRMS) was conducted with a Thermo Scientific Q Exac tive mass spectrometer (Thermo Scientific, Missouri, USA). Melting points were measured with an XT-4 binocular microscope melting point apparatus without correction. The morphology of the TMV was observed by transmission electron microscopy (TEM). TEM experiments were recorded with an FEI Talos F200C microscope (Thermo Fisher Scientific, Massachusetts, USA). Cytation™5 multimode readers (BioTek In struments, Inc. USA) and ultraviolet light (Blak-Ray B-100AP, Upland, CA, USA) were also used. All reagents and solvents are commercially available as chemically or analytically pure.
2.3. Antiviral activity in vivo TMV was purified by Gooding’s method (Gooding et al., 1967). The leaves of Nicotiana glutinosa inoculated with TMV were selected, ground in phosphate buffer, and filtered through a double-layer pledget. The filtrate was centrifuged at 10000g, treated twice with poly(ethylene glycol), and centrifuged again. The supernatant was extracted and stored in a refrigerator at 4 ◦ C. The whole experiment was conducted at 4 ◦ C. Model plants (Nicotiana tabacum cv. K326) were used for in vivo antiviral activity testing against TMV for the title compounds with the half-leaf method (Ma et al., 2014; Gan et al., 2015). Tobacco plants of the same age with 5 to 6 leaves were selected as the host and used to test systemic TMV infection in vivo. There were three in vivo activity modes: curative, protective and inactivating. The preliminary antiviral activity screening concentration of the title compounds was 500 mg/L. The median effective concentrations (EC50 values) of the highly active compounds were further determined and calculated with SPSS software 20. The commercialized antiviral agent ningnanmycin was used as a positive control. Standard deviation (SD) values are calculated based on the data of three repetitions for each test compound.
2.2. Synthesis 2.2.1. General synthesis procedure for intermediate 2 (Xu et al., 2011) A mixture of 1 (10.0 g, 59.1 mmol), tert-butyl hydrazine (8.8 g, 70.9 mmol) and ethanol (25 mL) was stirred at 80 ◦ C for 8 h. Then, the solvent was concentrated in vacuo, and the residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate 20/1) to obtain 2 as light yellow oil, with a yield of 76%. 1H NMR (400 MHz, DMSO‑d6) δ 7.42 (s, 1H, pyrazole H), 6.08 (s, 2H, NH2), 4.17 (q, J = 7.1 Hz, 2H, OCH2), 1.54 (s, 9H, tert-butyl), 1.24 (t, J = 7.1 Hz, 3H, CH3). 13C NMR (101 MHz, DMSO‑d6) δ 164.6, 149.9, 137.0, 95.7, 59.2, 58.8, 28.7, 14.9. HRMS (ESI): m/z calcd for C10H18O2N3 [M + H]+, 212.13935; found, 212.13861. 2.2.2. General synthesis procedure for intermediates 3 and 4 (Xu et al., 2012) Intermediate 2 (7.8 g, 37.1 mmol) was dissolved in 80% hydrazine
Fig. 1. Design and optimization of the title compounds. 2
G. Yang et al.
Pesticide Biochemistry and Physiology xxx (xxxx) xxx
2.3.1. Protective activity against TMV in vivo First, the test compound solution was smeared on the left side of the leaf, while the solvent as the blank control was smeared on the right side. Then, the entire leaves were inoculated with the virus after 12 h. The leaves were washed with water after 30 min and cultivated in a light incubator at a temperature of 27 ± 1 ◦ C and a light intensity of 10,000 Lux. The local lesion numbers appearing 3–4 days after inoculation were counted.
selected, and the surface of the leaves was rubbed with quartz sand to form microwounds. In addition, 1000 μL supernatant containing the virus was added to each leaf. Then, these plants were continuously observed under ultraviolet light for one week, and photographs of the progress of the viral infection were recorded. The commercialized antiviral agent ribavirin was used as a positive control.
2.3.2. Inactivating activity against TMV in vivo The virus was mixed with the test compound solution at an equal volume for 30 min. Then, the mixture was inoculated on the left side of the leaves, while a mixture of solvent and virus was smeared on the right side of the leaves as the blank control. Then, the leaves were cultivated in a light incubator at a temperature of 27 ± 1 ◦ C and a light intensity of 10,000 Lux. The local lesion numbers were recorded 3–4 days after inoculation.
To observe the effects of the high inactivation activity of the title compounds on the morphology of TMV, the morphology of the TMV after treatment with 5h was observed by TEM. The research method refers to that reported in the literature (Wang et al., 2019a, 2019b). A total of 101 μL of a solution of the testing compound mixed with TMV was prepared, and the concentration was 500 mg/L. After mixing for 30 min, the mixture was adsorbed using a 200-mesh carbon membrane support (copper grid) and subjected to negative staining with 1% phosphotungstic acid at a pH of 7.4. The morphology of the TMV par ticles was observed by TEM at 200 kV. The blank control (CK) was TMV particles in PBS and 1% DMSO without any other agents. The commercialized antiviral agent ningnanmycin was used as a positive control.
2.7. Morphological study with TMV
2.3.3. Curative activity against TMV in vivo First, TMV was used to infect whole leaves. Then, the leaves were washed with water and dried after 30 min. The test compound solution was smeared on the left side of the leaves, while the solvent as the blank control was smeared on the right side. Then, the leaves were cultivated in a light incubator at a temperature of 27 ± 1 ◦ C and a light intensity of 10,000 Lux. The local lesion numbers were counted and recorded 3–4 days after inoculation.
2.8. Insecticidal activity The insecticidal activity of title compounds 5a–5m against aphidoi dea was investigated using reported methods (Tian et al., 2018; Luo and Yang, 2007). Aphidoidea was cultivated in a greenhouse at a temperature of 25 ± 1 ◦ C, a humidity of 75 ± 5%, and under a 14:10 h light/dark photoperiod. The concentration of the test compounds was 500 mg/L. The fatality rate was evaluated after 48 h by recording the number of dead pests. The commercialized pesticide chlorpyrifos was used as the positive control. Three repetitions were conducted for each test compound.
2.4. Determination of the chlorophyll content The chlorophyll content of the tobacco leaves treated with 5j at a concentration of 500 mg/L was measured using a previously reported method with minor revisions (McDowell and Dangl, 2000). The tobacco leaves were dried with filter paper, and 0.5 mm small discs were ob tained with a puncher. Fifty milligrams of discs and 5.0 mL of extract (85% acetone: 85% ethanol = 1: 1) were mixed and ground. The ho mogenate was transferred to a 15 mL centrifuge tube, mixed for 1 h in the dark at 35 ◦ C, and centrifuged at 4000 rpm for 5 min at room tem perature. The extract was used to measure the absorbance at the wavelengths of 663 nm and 645 nm.
3. Results and discussion 3.1. Synthesis The synthesis route of the title compounds is shown in Fig. 2. As initial materials, ethyl (ethoxymethylene) cyanoacetate and tertbutylhydrazine were used to synthesize intermediate ester 2. Then, key intermediate 3 was obtained from 2 by a hydrazinolysis reaction in the presence of 80% hydrazine hydrate and reacted with CS2 under base conditions to form intermediate 4. Title compounds 5a–5m were syn thesized from 4 with different halogenated substituted benzyl groups. All the title compounds were characterized by 1H nuclear magnetic resonance (NMR), 13C NMR and high-resolution mass spectrometry (HRMS).
2.5. Defense enzyme (SOD, POD, CAT and PAL) activities in vivo Nicotiana tabacum cv. K326 with 5 to 6 leaves were selected as the host and cultivated at a temperature of 23 ± 1 ◦ C and a light intensity of 10,000 Lux. The tobacco leaves treated with the title compounds were chosen for defense activity testing on days 1, 3, 5 and 7, respectively. The title compound 5j was selected for further mechanistic study, and the commercialized agents ningnanmycin and lentinan (LNT) were used as a positive control. The concentration of the test compounds was 500 mg/L. The SOD, POD, CAT and PAL enzyme activities were determined by assay kits BC0175, BC0095, BC0205 and BC0215, respectively, which were purchased from Beijing Solarbio Science & Technology Co., Ltd., China (Wang et al., 2020). The details of the testing procedure and calculation method are provided in the supporting information.
3.2. Antiviral activity As indicated in Table 1, the antiviral activities of this series of title compounds exhibited obvious protective and inactivation effects against TMV in vivo at 500 mg/L. Among them, the protective activities of 5c (61.4%), 5d (72.5%), 5h (68.2%), 5i (65.2%), 5j (70.7%), 5k (78.0%), 5l (65.1%) and 5m (65.3%) were similar to that of the commercialized agent ningnanmycin (71.5%). Compounds 5c (63.5%), 5d (64.5%), 5f (64.1%), 5 h (84.4%), 5j (82.8%), 5k (76.2%) and 5l (59.1%) had inactivation activities similar to those of ningnanmycin (80.5%). The EC50 values in Table 2 indicate that compounds 5d, 5j, 5k and 5l exhibited excellent protective activity, and the EC50 values were 165.8, 163.2, 159.7 and 193.1 mg/L, respectively, which are better than that of ningnanmycin (EC50 = 271.3 mg/L). As shown in Table 3, the inacti vation activity EC50 value of 5h was 83.8 mg/L, which is similar to that of ningnanmycin (78.5 mg/L).
2.6. The progression of TMV in leaves marked by a GFP tag and treated with the title compounds To further study the effect in tobacco plants and TMV treated with compound 5j, TMV with a green fluorescent protein (GFP) gene tag was used for the experiment (Sheen et al., 1995; Chen et al., 2010; Casper and Holt, 1996; Itaya et al., 1997). As reported in the literature (Xiang et al., 2019; Weststeijn, 1981), 2 mL phosphate buffered saline (PBS) was added to 0.1 g of infected tobacco leaves, ground into a homoge nate, and centrifuged at 5000 rpm for 5 min, and then the supernatant was removed. The fourth or fifth leaves of Nicotiana benthamiana were 3
G. Yang et al.
Pesticide Biochemistry and Physiology xxx (xxxx) xxx
Fig. 2. Synthetic route of the title compounds 5a–5m.
3.3. Mechanistic study of the protective activity of 5j
Table 1 In vivo antiviral activities of the title compounds at 500 mg/L against TMVa. Compound no. 5a 5b 5c 5d 5e 5f 5g 5h 5i 5j 5k 5l 5m Ningnanmycin a
3.3.1. Chlorophyll content Chlorophyll is the major component in chloroplasts; it plays an important role in photosynthesis and can provide energy for plant growth. As depicted in Fig. 3, the chlorophyll a, chlorophyll b, chloro phyll a/chlorophyll b and total chlorophyll contents of the tobacco leaves treated with 5j were continuously measured after inoculation with TMV. As shown in Fig. 4A, B and D, the contents of chlorophyll a, b and total chlorophyll increased obviously from day 1 to day 3 and remained stable from day 3 to day 7, reaching the highest values of 8.0, 3.5 and 12.5 mg/L on the seventh day, respectively. The results demonstrated that title compound 5j can increase the chlorophyll con tent in the leaves, promote photosynthesis ability, and improve the disease resistance of the host.
Inhibition (%) Protective activity
Curative activity
Inactivation activity
54.0 ± 8.0 57.1 ± 3.6 61.4 ± 3.2 72.5 ± 9.5 43.2 ± 3.8 18.5 ± 7.8 24.3 ± 5.7 68.2 ± 5.7 65.2 ± 6.3 70.7 ± 2.6 78.0 ± 7.3 65.1 ± 1.6 65.3 ± 9.9 71.5 ± 9.1
58.9 ± 7.5 45.9 ± 5.1 40.4 ± 6.4 59.5 ± 9.5 40.0 ± 12.1 66.1 ± 15.2 64.3 ± 1.5 50.0 ± 9.4 44.5 ± 7.2 35.7 ± 8.6 59.0 ± 9.0 45.8 ± 6.0 49.0 ± 1.8 76.6 ± 8.8
28.1 ± 8.6 38.2 ± 3.5 63.5 ± 4.1 64.5 ± 7.2 0 64.1 ± 6.2 21.6 ± 1.8 84.4 ± 6.1 53.1 ± 7.2 82.8 ± 5.8 76.2 ± 9.0 59.1 ± 4.1 37.1 ± 3.0 80.5 ± 1.8
3.3.2. SOD activity analysis As depicted in Fig. 4A, the SOD activity of the leaves after treatment with 5% DMSO (CK) did not exhibit obvious differences from day 1 to day 7. In contrast, the SOD activity of tobacco leaves treated with 5j obviously increased on the first day and reached a peak value of 1261.2 U/g, which is much better than that of ningnanmycin (798.3 U/g) and LNT (768.5 U/g). Although the SOD activity of the leaves treated with 5j decreased from the third day, it was obviously better than that of the CK and the positive control groups (ningnanmycin and LNT). We speculated that title compound 5j may enhance the ability of the host to scavenge oxygen free radicals in vivo.
Average of three replicates.
Table 2 In vivo protective activity EC50 values of the title compounds against TMVa. Compound no.
EC50 (mg/L)
toxic regression equation
R2
5c 5d 5h 5i 5j 5k 5l 5m Ningnanmycin
272.0 ± 165.8 ± 350.1 ± 257.5 ± 163.2 ± 159.7 ± 193.1 ± 333.7 ± 271.3 ±
y = 0.5842× + 3.5777 y = 0.826× + 3.1666 y = 0.9738× + 2.5225 y = 0.912× + 2.8013 y = 0.707× + 3.4357 y = 0.7565× + 3.3333 y = 0.9314× + 2.8711 y = 1.3318× + 1.6394 y = 0.8823× + 2.853
0.951 0.973 0.939 0.979 0.990 0.992 0.940 0.992 0.951
a
2.4 1.0 7.0 5.8 5.0 6.9 2.5 3.2 5.8
3.3.3. POD activity analysis As shown in Fig. 4B, the POD activity of the treated leaves gradually increased from the first day. On the third day, the POD activity treated with ningnanmycin and LNT reached the highest value of 33,403.3 U/g and 17,600.0 U/g, respectively. For leaves treated with 5j, the POD activity reached the highest value (31,540.0.0 U/g) on the fifth day, which is better than that of ningnanmycin (24,056.7 U/g) and LNT (13,063.3 U/g). We speculated that the POD system is induced by title compound 5j to remove excess oxygen free radicals and hydrogen peroxide (Gozzo, 2003).
Average of three replicates.
Table 3 In vivo inactivation activity EC50 values of the title compounds against TMVa. Compound no.
EC50 (mg/L)
toxic regression equation
R2
5d 5f 5h 5j 5k Ningnanmycin
220.0 ± 2.4 252.5 ± 4.2 83.8 ± 5.6 163.6 ± 5.2 100.3 ± 2.8 78.5 ± 3.1
y = 1.3292× y = 1.2697× y = 1.1047× y = 1.8488× y = 2.2779× y = 1.5844×
0.965 0.953 0.924 0.978 0.920 0.925
a
+ 1.8865 + 1.9498 + 2.8755 + 0.9071 + 0.4412 + 1.9974
3.3.4. CAT activity analysis As depicted in Fig. 4C, the CAT activity of the leaves treated with ningnanmycin and 5j quickly increased and reached the highest value of 52.4 U/g and 78.3 U/g, respectively, on the first day, better than CK (54.56 U/g). The CAT activity of leaves treated with LNT reached the highest value (47.2 U/g) on the third day. CAT is an important hydrogen
Average of three replicates.
4
G. Yang et al.
Pesticide Biochemistry and Physiology xxx (xxxx) xxx
Fig. 3. The effect of tobacco leaves treated with 5j on the chlorophyll content. Contents of chlorophyll a (A), chlorophyll b (B), chlorophyll a/chlorophyll b (C), and total chlorophyll (D). (Bars with different letters are significantly different and values are mean ± S.D. (standard deviation); p < 0.05)
peroxide scavenging enzyme. The immune response of the host will produce too much hydrogen peroxide after infection with the virus. However, excessive hydrogen peroxide is not helpful for the growth of the host. The CAT enzyme can remove excess hydrogen peroxide and improve the ability of plants to defend against diseases (Dat et al., 2010). The results revealed that the title compound 5j can obviously improve the CAT activity of the tobacco leaves.
obviously infected by TMV at 2 dpi, and the TMV proliferated gradually from 2 dpi to 4 dpi. At 5 dpi, the virus was distinctly spread to the top leaves of the tobacco treated with CK and ribavirin (Fig. 5A and C), except for those treated with 5j (Fig. 5B). This result demonstrated that 5j can effectively inhibit the spreading of TMV in inoculated tobacco leaves. 3.5. Morphological study of TMV with TEM
3.3.5. PAL activity analysis PAL is a kind of catalytic enzyme in the plant defense system. It not only regulates the synthesis of phytoalexins but also promotes the con version rate of phenylalanine to cinnamic acid, which is a process that produces salicylic acid to achieve systemic resistance of the host to resist the infection and replication of the virus (Gozzo, 2003). As shown in Fig. 4D, the PAL activity of the leaves treated with the tested compounds gradually decreased from day 1 to day 5. However, the PAL activity of leaves treated with 5j had the highest value of 12.8 U/g on the seventh day, which is much better than that of the CK group (8.0 U/g). The re sults indicated that the title compound 5j can improve the PAL activity of the host, which could enhance its ability to resist TMV infection. With TMV infection, the plant itself will produce a series of immune responses to achieve systemic resistance (McDowell and Dangl, 2000; Yatskou et al., 2001). The results of the experiments revealed that the chlorophyll content and the defense enzyme activities of the tobacco leaves treated with 5j were significantly improved, which was beneficial to fight against viral infection.
Morphological observations of TMV treated with compound 5h with high inactivation activity were performed with transmission electron microscopy (TEM). As depicted in Fig. 6A, the TMV in the blank control showed a rod-shaped structure. Further analysis of Fig. 6B and C found that rod-shaped TMV formed a distinct break after treatment with ningnanmycin and 5h at a concentration of 500 mg/L for 30 min (break points indicated with red arrows in the figures). As shown in Fig. C4, the details reveal that TMV treated with 5h broke into small rods, as shown with a ruler length of 20 nm. 3.6. Insecticidal activity analysis Aphids are common carriers of plant viruses and can indirectly transmit viruses from infected plants to normal plants. This is an important transmission route for TMV (Yu et al., 2016; Yang et al., 2020). As indicated in Table 4, compounds 5a, 5b and 5m exhibited certain insecticidal activities against aphidoidea, and the fatality rates were 85%, 83% and 87%, respectively. The insecticidal activity results revealed that some title compounds can block the spread of TMV by killing the carrier aphid. A series of new 1-tert-butyl-5-amino-4-pyrazole bioxadiazole sulfide derivatives containing a 1,3,4-oxadiazole group were designed and
3.4. Analysis of the protective activity in vivo using tobacco labeled with GFP As shown in Fig. 5, the leaves of Nicotiana benthamiana were 5
G. Yang et al.
Pesticide Biochemistry and Physiology xxx (xxxx) xxx
Fig. 4. The effect of tobacco leaves treated with 5j on defense enzyme activities. The effect on SOD (A), POD (B), CAT (C), and PAL (D). (Bars with different letters are significantly different and values are mean ± S.D. (standard deviation); p < 0.05)
Fig. 5. Tobacco marked with GFP used to study the protective activity mechanism in vivo. Analysis of the transmission efficiency of TMV in leaves treated with CK (A), those treated with 5j (B), and those treated with ribavirin (C).
synthesized. The bioactivity results showed that some compounds exhibited excellent protective activity against TMV. The EC50 values of 5d, 5j, 5k and 5l were 165.8, 163.2, 159.7 and 193.1 mg/L, respec tively, which are better than that of ningnanmycin (271.3 mg/L). The
inactivation activity EC50 value of 5h against TMV was 83.8 mg/L, similar to that of ningnanmycin (78.5 mg/L). Further study found that the chlorophyll contents and the defensive enzyme activities of the to bacco leaves after treatment with 5j were significantly improved. 6
G. Yang et al.
Pesticide Biochemistry and Physiology xxx (xxxx) xxx
Fig. 6. TEM images of TMV after treatment with CK (A), ningnanmycin (B), and 5h (C) at a concentration of 500 μg/mL. The ruler lengths are 200 nm (1), 100 nm (2), 50 nm (3), and 20 nm (4).
TMV that reducing the infection effect of the virus. Simultaneously, the title compounds can effectively block the common carrier for plant viruses, thereby blocking infection with the viruses. This series of syn ergistic effects provide key information for the research and develop ment of antiviral agents.
Table 4 Insecticidal activity of the title compounds at 500 mg/L against aphidoideaa. Compound no.
Fatality rate (%)
Compound no.
Fatality rate (%)
5a 5b 5c 5d 5e 5f 5g
85 83 0 0 47 38 57
5h 5i 5j 5k 5l 5m Chlorpyrifos
0 0 0 0 0 87 100
a
Funding This work was supported by the National Natural Science Foundation of China (No. 21662008) and the Science and Technology Project of Guizhou Province (No. [2017]5788).
Average of three replicates.
Declaration of Competing Interest
Further antiviral activity research on 5j using TMV with a GFP gene tag found that it can inhibit the spread of TMV in inoculated tobacco leaves. A morphological study with TEM revealed that title compound 5h treatment can significantly shorten the polymerization length of TMV particles and cause a distinct break of the rod-shaped TMV. Moreover, the insecticidal activity revealed that the fatality rates of 5a, 5b and 5m against aphidoidea were 85%, 83% and 87%, respectively. These results reveal that this series of title compounds not only can induce the host to obtain systemic acquired resistance and enhance its ability to defend against diseases, but also can cause a distinct break of the rod-shaped
The authors declare that there are no competing financial interests. Acknowledgments The authors acknowledge Jian Zhang and Zhengjun Liu, Guizhou University, for testing the insecticidal activity against aphids.
7
G. Yang et al.
Pesticide Biochemistry and Physiology xxx (xxxx) xxx
Appendix A. Supplementary data
Ma, J., Li, P., Li, X.Y., Shi, Q.C., Wan, Z.H., Hu, D.Y., Jin, L.H., Song, B.A., 2014. Synthesis and antiviral bioactivity of novel 3-((2-((1E,4E)-3-Oxo-5-arylpenta-1,4dien-1-yl)phenoxy) methyl)-4(3H)-quinazolinone derivatives. J. Agric. Food Chem. 62 (36), 8928–8934. McDowell, J.M., Dangl, J.L., 2000. Signal transduction in the plant immune response. Trends Biochem. Sci. 25 (2), 79–82. https://doi.org/10.1016/S0968-0004(99) 01532-7. Noutoshi, Y., Okazaki, M., Shirasu, K., 2012. Imprimatins A and B: novel plant activators targeting salicylic acid metabolism in Arabidopsis thaliana. Plant Signal. Behav. 7 (12), 1715–1717. https://pubmed.ncbi.nlm.nih.gov/23073003/. Sheen, J., Hwang, S., Niwa, Y., Kobayashi, H., Galbraith, D.W., 1995. Green-fluorescent protein as a new vital marker in plant cells. Plant J. 8 (5), 777–784. https://doi.org/ 10.1046/j.1365-313X.1995.08050777.x. Silverman, F.P., Petracek, P.D., Heiman, D.F., Fledderman, C.M., Warrior, P., 2005a. Salicylate activity. 3. Structure relationship to systemic acquired resistance. J. Agric. Food Chem. 53 (25), 9775–9780. https://doi.org/10.1021/jf051383t. Silverman, F.P., Petracek, P.D., Ju, Z., Fledderman, C.M., Heiman, D.F., Warrior, P., 2005b. Salicylate activity. 1. Protection of plants from paraquat injury. J. Agric. Food Chem. 53 (25), 9764–9768. https://doi.org/10.1021/jf0513819. Silverman, F.P., Petracek, P.D., Ju, Z., Fledderman, C.M., Heiman, D.F., Warrior, P., 2005c. Salicylate activity. 2. Potentiation of atrazine. J. Agric. Food Chem. 53 (25), 9769–9774. https://doi.org/10.1021/jf0513821. Tian, P.Y., Liu, D.Y., Liu, Z.J., Shi, J., He, W.J., Qi, P.Y., Chen, J.X., Song, B.A., 2018. Design, synthesis, and insecticidal activity evaluation of novel 4-(N, Ndiarylmethylamines)furan-2(5H)-one derivatives as potential acetylcholine receptor insecticides. Pest Manag. Sci. 75, 427–437. https://doi.org/10.1002/ps.5132. Wang, Z.W., Wei, P., Liu, Y.X., Wang, Q.M., 2014. D and E rings may not be indispensable for antofine: discovery of phenanthrene and Alkylamine chain containing antofine derivatives as novel antiviral agents against tobacco mosaic virus (TMV) based on interaction of antofine and TMV RNA. J. Agric. Food Chem. 62, 10393–10404. https://doi.org/10.1021/jf5028894. Wang, H.J., Chen, Y., Zhang, W.K., 2019a. A single-molecule atomic force microscopy study reveals the antiviral mechanism of tannin and its derivatives. Nanoscale. 11, 16368–16376. https://doi.org/10.1039/C9NR05410C. Wang, M.W., Zhu, H.H., Wang, P.Y., Zeng, D., Wu, Y.Y., Liu, L.W., Wu, Z.B., Li, Z., Yang, S., 2019b. Synthesis of thiazolium-labeled 1,3,4-oxadiazole thioethers as prospective antimicrobials: in vitro and in vivo bioactivity and mechanism of action. J. Agric. Food Chem. 67 (46), 12696–12708. https://doi.org/10.1021/acs. jafc.9b03952. Wang, P.Y., Xiang, M., Luo, M., Liu, H.W., Zhou, X., Wu, Z.B., Liu, L.W., Li, Z., Yang, S., 2020. Novel piperazine-tailored ursolic acid hybrids as significant antibacterial agents targeting phytopathogens Xanthomonas oryzae pv. Oryzae and X. axonopodis pv. Citri probably directed by activation of apoptosis. Pest Manag. Sci. 76, 2746–2754. https://doi.org/10.1002/ps.5822. Weststeijn, E.A., 1981. Lesion growth and virus localization in leaves of Nicotiana tabacum cv. Xanthi nc. after inoculation with tobacco mosaic virus and incubation alternately at 22 ◦ C and 32 ◦ C. Physiol. Mol. Plant P. 18 (3), 357–368. https://doi. org/10.1016/S0048-4059(81)80086-0. Wu, Z.B., Li, X.Y., Zhou, X., Yang, W.Q., Ye, Y.Q., Song, B.A., Yang, S., 2016. Synthesis and anti-TMV Activitie of 1-(3-chloropyridin-2-yl)-5-N-substituted-4-(5(methylthio)-1,3,4-oxadiazol-2-yl)-pyrazole derivatives. Sci. Sin. Chim. 46, 1195–1203. https://doi.org/10.1360/N032016-00122. Xiang, S.Y., Lv, X., He, L.H., Shi, H., Liao, S.Y., Liu, C.Y., Huang, Q.Q., Li, X.Y., He, X.T., Chen, H.T., Wang, D.B., Sun, X.C., 2019. Dual-action pesticide carrier that continuously induces plant resistance, enhances plant anti-tobacco mosaic virus activity, and promotes plant growth. J. Agric. Food Chem. 67 (36), 10000–10009. https://doi.org/10.1021/acs.jafc.9b03484. Xu, W.M., Yang, S., Bhadury, P., He, J., He, M., Gao, L.L., Hu, D.Y., Song, B.A., 2011. Synthesis and bioactivity of novel sulfone derivatives containing 2,4-dichlorophenyl substituted 1,3,4-oxadiazole/thiadiazole moiety as chitinase inhibitors. Pestic. Biochem. Physiol. 101, 6–15. https://doi.org/10.1016/j.pestbp.2011.05.006. Xu, W.M., Han, F.F., He, M., Hu, D.Y., He, J., Yang, S., Song, B.A., 2012. Inhibition of tobacco bacterial wilt with sulfone derivatives containing an 1,3,4-oxadiazole moiety. J. Agric. Food Chem. 60 (4), 1036–1041. https://doi.org/10.1021/ jf203772d. Yang, Z.B., Li, P., He, Y.J., Luo, J., Zhou, J., Wu, Y.H., Chen, L.T., 2020. Novel pyrethrin derivatives containing an 1,3,4-oxadiazole thioether moiety: design, synthesis, and insecticidal activity. J. Heterocyclic Chem. 57 (1), 81–88. https://doi.org/10.1002/ jhet.3750. Yatskou, M.M., Koehorst, R.B.M., Hoek, A.V., Donker, H., Schaafsma, T.J., 2001. Spectroscopic properties of a self-assembled zinc porphyrin tetramer II. Timeresolved fluorescence spectroscopy. J. Phy. Chem. A. 105 (51), 11432–11440. https://doi.org/10.1021/jp010411h. Yu, X.D., Liu, Z.C., Huang, S.L., Chen, Z.Q., Sun, Y.W., Duan, P.F., Ma, Y.Z., Xia, L.Q., 2016. RNAi-mediated plant protection against aphids. Pest Manag. Sci. 72 (6), 1090–1098. https://doi.org/10.1002/ps.4258. Zhao, L., Feng, C.H., Wu, K., Chen, W.B., Chen, Y.J., Hao, X.A., Wu, Y.F., 2015. Advances and prospects in biogenic substances against plant virus: a review. Pestic. Biochem. Physiol. 135, 15–26. https://doi.org/10.1016/j.pestbp.2016.07.003.
Supporting information includes the experimental characterization data, testing procedure and calculation method for the chlorophyll content and defensive enzyme activities. Supplementary data to this article can be found online at https://doi.org/10.1016/j.pestbp.2020.10 4740. References Beachy, R.N., Heinlein, M., 2000. Role of P30 in replication and spread of TMV. Traffic 1 (7), 540–544. https://doi.org/10.1034/j.1600-0854.2000.010703.x. Buck, K.W., 1999. Replication of tobacco mosaic virus RNA. Philos. Trans. R. Soc. Lond. B 354, 613–627. https://doi.org/10.1098/rstb.1999.0413. Casper, S.J., Holt, C.A., 1996. Expression of the green fluorescent protein-encoding gene from a tobacco mosaic virus-based vector. Gene 173 (1), 69–73. https://doi.org/ 10.1016/0378-1119(95)00782-2. Chen, Q.J., Dong, L., Ouyang, M.A., Xie, L.H., Lin, Q.Y., 2010. Detection of anti-TMV activity of Bruceine D by green fluorescent protein labeling. Acta Laser Biol. Sin. 06, 36–41. https://kns.cnki.net/kns8/defaultresult/index. Chen, L.J., Zou, W.S., Wu, G., Lin, H.H., Xi, D.H., 2018. Tobacco alpha-expansin EXPA4 plays a role in Nicotiana benthamiana defence against Tobacco mosaic virus. Planta 247 (2), 355–368. https://doi.org/10.1007/s00425-017-2785-6. Dat, J.F., Pellinen, R., Beeckman, T., Cotte, B.V.D., Langebartels, C., Kangasjarvi, J., lnze, D., Breusegem, F.V., 2010. Changes in hydrogen peroxide homeostasis trigger an active cell death process in tobacco. Plant J. 33 (4), 621–632. https://doi.org/ 10.1046/j.1365-313X.2003.01655.x. Du, Q.S., Zhu, W.P., Zhao, Z.J., Qian, X.H., Xu, Y.F., 2012. Novel benzo-1,2,3thiadiazole-7-carboxylate derivatives as plant activators and the development of their agricultural applications. J. Agric. Food Chem. 60 (1), 346–353. https://doi. org/10.1021/jf203974p. Du, Q.S., Shi, Y.X., Li, P.F., Zhao, Z.J., Zhu, W.P., Qian, X.H., Li, B.J., Xu, Y.F., 2013. Novel plant activators with thieno[2,3-d]-1,2,3-thiadiazole-6-carboxylate scaffold: synthesis and bioactivity. Chin. Chem. Lett. 24 (11), 967–969. https://doi.org/ 10.1016/j.cclet.2013.07.003. Fedorkin, O.N., Solovyev, A.G., Yelina, N.E., Zamyatnin, A.A., Zinovkin, R.A., Makinen, K., Schiemann, J., Morozov, S.Y., 2001. Cell-to-cell movement of potato virus X involves distinct functions of the coat protein. J. Gen. Virol. 82, 449–458. https://doi.org/10.1099/0022-1317-82-2-449. Friedrich, L., Lawton, K., Ruess, W., Masner, P., Specker, N., Rella, M.G., Meier, B., Dincher, S., Staub, T., Uknes, S., Metraux, J.P., Kessmann, H., Ryals, J., 1996. A benzothiadiazole derivative induces systemic acquired resistance in tobacco. The Plant J. 10 (1), 61–70. https://doi.org/10.1046/j.1365-313x.1996.10010061.x. Gan, X.H., Hu, D.Y., Li, P., Wu, J., Chen, X.W., Xue, W., Song, B.A., 2015. Design, synthesis, antiviral activity and three-dimensional quantitative structure-activity relationship study of novel 1,4-pentadien-3-one derivatives containing the 1,3,4oxadiazole moiety. Pest Manag. Sci. 72, 534–543. https://doi.org/10.1021/ jf502162y. Gooding Jr., G.V., Hebert, T.T., 1967. A simple technique for purification of tobacco mosaic virus in large quantities. Phytopathology. 57, 1285–1287. https://doi.org/ 10.1098/rstl.1767.0047. G¨ orlach, J., Volrath, S., Knauf-Beiter, G., Hengy, G., Beckhove, U., Kogel, K.H., Oostendorp, M., Staub, T., Ward, E., Kessmann, H., Ryals, J., 1996. Benzothiadiazole, a novel class of inducers of systemic acquired resistance, activates gene expression and disease resistance in wheat. Plant Cell 8 (4), 629–643. https:// www.jstor.org/stable/3870340. Gozzo, F., 2003. Systemic acquired resistance in crop protection: from nature to a chemical approach. J. Agric. Food Chem. 51 (16), 4487–4503. https://doi.org/ 10.1016/j.bmc.2006.12.002. Han, Y.G., Luo, Y., Qin, S.R., Xi, L., Wan, B., Du, L.F., 2014. Induction of systemic resistance against tobacco mosaic virus by Ningnanmycin in tobacco. Pestic. Biochem. Physiol. 111 (1), 14–18. https://doi.org/10.1016/j.pestbp.2014.04.008. Itaya, A., Hickman, H., Bao, Y., Richard, N., Ding, B., 1997. Cell-to-cell trafficking of cucumber mosaic virus movement protein:green fluorescent protein fusion produced by biolistic gene bombardment in tobacco. Plant J. 12, 1223–1230. https://doi.org/ 10.1046/j.1365-313X.1997.12051223.x. Kessmann, H., Staub, T., Ligon, J., Oostendorp, M., Ryals, J., 1994. Activation of systemic acquired disease resistance in plants. Eur. J. Plant Pathol. 100 (6), 359–369. https:// doi.org/10.1007/BF01874804. Kunz, W., Schurter, R., Maetzke, T., 1997. The chemistry of benzothiadiazole plant activators. J. Pestic. Sci. 50 (4), 275–282. https://doi.org/10.1002/(SICI)1096-9063 (199708)50:4<275::AID-PS593>3.0.CO;2-7. Luo, Y.P., Yang, G.F., 2007. Discovery of a new insecticide lead by optimizing a targetdiverse scaffold: Tetrazolinone derivatives. Bioorg. Med. Chem. 15 (4), 1716–1724. Lv, X., Xiang, S.Y., Wang, X.C., Wu, L., Liu, C.Y., Yuan, M.T., Gong, W.W., Win, H., Hao, C.Y., Xue, Y., Ma, L.S., Cheng, D.Q., Sun, X.C., 2020. Synthetic chloroinconazide compound exhibits highly efficient antiviral activity against tobacco mosaic virus. Pest Manag. Sci. https://doi.org/10.1002/ps.5910.
8
Contents lists available at ScienceDirect
Pesticide Biochemistry and Physiology journal homepage: www.elsevier.com/locate/pest
Study of the in vivo antiviral activity against TMV treated with novel 1-(t-butyl)-5-amino-4-pyrazole derivatives containing a 1,3,4-oxadiazole sulfide moiety Guangqian Yang, Huanlin Zheng, Wubin Shao, Liwei Liu, Zhibing Wu * State Key Laboratory Breeding Base of Green Pesticide and Agricultural Bioengineering, Key Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of Education, Center for R & D of Fine Chemicals of Guizhou University, Guiyang 550025, China
A R T I C L E I N F O
A B S T R A C T
Keywords: 1,3,4-oxadiazole sulfide Tobacco mosaic virus Defense enzyme activity Green fluorescent protein Insecticidal activity
A series of new 1-tert-butyl-5-amino-4-pyrazole bioxadiazole sulfide derivatives containing a 1,3,4-oxadiazole moiety were designed and synthesized. The bioactivity results showed that some title compounds exhibited excellent protective activity against TMV and certain insecticidal activity. Among the tested compounds, the EC50 values of 5d, 5j, 5k and 5l were 165.8, 163.2, 159.7 and 193.1 mg/L, respectively, which are better than the EC50 value of ningnanmycin (271.3 mg/L). The chlorophyll contents and the defense enzyme activities of the tobacco leaves after treatment with 5j were significantly increased, which indicated that this series of title compounds may induce the systemic acquired resistance of host to defend against diseases. Further in vivo protective activity research on 5j using TMV with a GFP gene tag found that it can effectively inhibit the spread of TMV in inoculated tobacco. A morphological study with TEM revealed that title compound 5h can cause a distinct break of the rod-shaped TMV. Moreover, the insecticidal activity revealed that the fatality rates of 5a, 5b and 5m against aphidoidea were 85%, 83% and 87%, respectively, which indicated that the title compounds can effectively block the common carrier of plant viruses, thereby effectively reducing the TMV infection risk of tobacco. This series of synergistic effects provide key information for the research and development of antiviral agents.
1. Introduction Plant viral diseases can cause serious effects on the growth of food crops and cash crops. The economic losses caused by plant viral diseases in agricultural and forestry production worldwide are approximately $60 billion each year (Zhao et al., 2015). Tobacco mosaic virus (TMV), a typical plant virus, can infect a series of Solanaceae plants that are rep resented by tobacco. TMV first parasitizes plant cells and then uses their energy and enzyme systems to replicate and synthesize its own coat protein in the ribosomes of the cells (Buck, 1999; Beachy and Heinlein, 2000). Due to the differences in the immune system between plants and animals, it is difficult to prevent and control diseases caused by TMV infection (Fedorkin et al., 2001; Lv et al., 2020). Due to the different action mechanisms and obvious defects of the
traditional commercialized agents ningnanmycin and ribavirin, they did not exhibit the desired prevention and controlling effect (Han et al., 2014; Wang et al., 2014). Therefore, highly efficient anti-TMV agents must be energetically developed and pursued to control viral diseases. Different from traditional agrochemicals, plant activators are novel kinds of agrochemicals that prevent disease by inducing systemic ac quired resistance of the plant’s own defense system (Kessmann et al., ¨rlach et al., 1996; Kunz et al., 1997; Noutoshi et al., 2012). To 1994; Go date, some plant activators have been reported with highly efficient, environmentally friendly and unique modes of action (Du et al., 2012; Du et al., 2013; Friedrich et al., 1996; Silverman et al., 2005a, 2005b, 2005c). To obtain high antiviral activity lead compounds with novel action mechanisms, a series of new 1-tert-butyl-5-amino-4-pyrazole
Abbreviations: TMV, tobacco mosaic virus; 1H NMR, 1H nuclear magnetic resonance; 13C NMR, 13C nuclear magnetic resonance; HRMS, high-resolution mass spectrometry; EC50, median effective concentration; SOD, superoxide dismutase; POD, peroxidase; CAT, catalase; PAL, phenylalanine ammonia-lyase; CK, blank control; LNT, lentinan; GFP, green fluorescent protein; PBS, phosphate buffer saline; TEM, transmission electron microscopy;; dpi, days post-inoculation. * Corresponding author. E-mail address: [email protected] (Z. Wu). https://doi.org/10.1016/j.pestbp.2020.104740 Received 29 September 2020; Received in revised form 2 November 2020; Accepted 2 November 2020 Available online 7 November 2020 0048-3575/© 2020 Published by Elsevier Inc.
Please cite this article as: Guangqian Yang, Pesticide Biochemistry and Physiology, https://doi.org/10.1016/j.pestbp.2020.104740
G. Yang et al.
Pesticide Biochemistry and Physiology xxx (xxxx) xxx
bioxadiazole sulfide derivatives containing a 1,3,4-oxadiazole moiety were designed and synthesized based on our previous work (Fig. 1) (Wu et al., 2016). In vivo antiviral bioassays revealed that some title com pounds exhibited excellent protective activity against TMV. The chlo rophyll content and defense enzyme activities of the tobacco leaves treated with 5j significantly increased, demonstrating that this series of title compounds may induce the host to obtain systemic acquired resistance to enhance its ability to defend against diseases. Further antiviral activity studies using Nicotiana benthamiana leaves infected with TMV that labeled with the GFP gene tag (Chen et al., 2018) and treated with 5j found that it can inhibit the spreading of TMV in inoc ulated tobacco leaves. A morphological study with TEM revealed that 5h can significantly shorten the polymerization length of TMV particles and form a distinct break on the rod-shaped TMV. Moreover, the insecticidal activity revealed that some title compounds have certain activity against aphids, which are common carriers of plant viruses.
hydrate (30 mL), reacted at 100 ◦ C for 4 h, cooled and extracted with dichloromethane. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to obtain 3 as a yellow solid, with a yield of 74%. To a solution of KOH (166.0 mg, 29.7 mmol) in 50 mL ethanol was added 3 (3.9 g, 19.8 mmol), and CS2 (4.5 g, 59.3 mmol) was slowly added dropwise into the solution, reacted at r.t. for 10 h and then reacted at 80 ◦ C for 8 h. The solution was concentrated in vacuo. The pH value was adjusted to 6 with 1 M hydrochloric acid solution, and the solution was filtered, washed with water and dried to obtain 4, which was used in the next reaction without further purification. 2.2.3. General synthesis procedure for title compounds 5a–5m (Wang et al., 2019a, 2019b) To a 50 mL round bottom flask, 4 (500.0 mg, 2.1 mmol), potassium hydroxide (175.8 mg, 3.1 mmol), and H2O (15.0 mL) were added and stirred for 10 min. Then, 2-methylchlorobenzyl (323.2 mg, 2.3 mmol) was added and stirred at room temperature for approximately 8 h. The mixture was extracted with ethyl acetate and washed with water 2–3 times. The organic layer was dried with anhydrous sodium sulfate and filtered. The solvent was concentrated in vacuo to obtain the crude product and purified by column chromatography (petroleum ether/ ethyl acetate 20/1) to obtain 5a as a yellow solid, with a yield of 44% and a m.p. of 116–117 ◦ C. 1H NMR (400 MHz, DMSO‑d6) δ 7.56 (s, 1H, pyrazole H), 6.05 (s, 2H, NH2), 5.99–5.88 (m, 2H, SCH2), 5.34–5.09 (m, 3H, CH3), 1.56 (s, 9H, tert-butyl).13C NMR (100 MHz, DMSO‑d6) δ 162.3, 159.5, 146.7, 133.3, 119.6, 89.5, 59.1, 35.4, 28.8. HRMS (ESI): m/z calcd for C11H18ON5S [M + H]+, 268.12266; found, 268.12210. The synthesis procedure of the other title compounds was similar to that of 5a, and the physical and spectral data of 5a–5m are provided in the supporting information.
2. Materials and methods 2.1. Instruments and chemicals 1 H and 13C NMR spectra were obtained with a JEOL-400 NMR spectrometer (JEOL CO., LTD., Japan) with tetramethylsilane (TMS) as the internal standard and DMSO‑d6 as the solvent. High-resolution mass spectrometry (HRMS) was conducted with a Thermo Scientific Q Exac tive mass spectrometer (Thermo Scientific, Missouri, USA). Melting points were measured with an XT-4 binocular microscope melting point apparatus without correction. The morphology of the TMV was observed by transmission electron microscopy (TEM). TEM experiments were recorded with an FEI Talos F200C microscope (Thermo Fisher Scientific, Massachusetts, USA). Cytation™5 multimode readers (BioTek In struments, Inc. USA) and ultraviolet light (Blak-Ray B-100AP, Upland, CA, USA) were also used. All reagents and solvents are commercially available as chemically or analytically pure.
2.3. Antiviral activity in vivo TMV was purified by Gooding’s method (Gooding et al., 1967). The leaves of Nicotiana glutinosa inoculated with TMV were selected, ground in phosphate buffer, and filtered through a double-layer pledget. The filtrate was centrifuged at 10000g, treated twice with poly(ethylene glycol), and centrifuged again. The supernatant was extracted and stored in a refrigerator at 4 ◦ C. The whole experiment was conducted at 4 ◦ C. Model plants (Nicotiana tabacum cv. K326) were used for in vivo antiviral activity testing against TMV for the title compounds with the half-leaf method (Ma et al., 2014; Gan et al., 2015). Tobacco plants of the same age with 5 to 6 leaves were selected as the host and used to test systemic TMV infection in vivo. There were three in vivo activity modes: curative, protective and inactivating. The preliminary antiviral activity screening concentration of the title compounds was 500 mg/L. The median effective concentrations (EC50 values) of the highly active compounds were further determined and calculated with SPSS software 20. The commercialized antiviral agent ningnanmycin was used as a positive control. Standard deviation (SD) values are calculated based on the data of three repetitions for each test compound.
2.2. Synthesis 2.2.1. General synthesis procedure for intermediate 2 (Xu et al., 2011) A mixture of 1 (10.0 g, 59.1 mmol), tert-butyl hydrazine (8.8 g, 70.9 mmol) and ethanol (25 mL) was stirred at 80 ◦ C for 8 h. Then, the solvent was concentrated in vacuo, and the residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate 20/1) to obtain 2 as light yellow oil, with a yield of 76%. 1H NMR (400 MHz, DMSO‑d6) δ 7.42 (s, 1H, pyrazole H), 6.08 (s, 2H, NH2), 4.17 (q, J = 7.1 Hz, 2H, OCH2), 1.54 (s, 9H, tert-butyl), 1.24 (t, J = 7.1 Hz, 3H, CH3). 13C NMR (101 MHz, DMSO‑d6) δ 164.6, 149.9, 137.0, 95.7, 59.2, 58.8, 28.7, 14.9. HRMS (ESI): m/z calcd for C10H18O2N3 [M + H]+, 212.13935; found, 212.13861. 2.2.2. General synthesis procedure for intermediates 3 and 4 (Xu et al., 2012) Intermediate 2 (7.8 g, 37.1 mmol) was dissolved in 80% hydrazine
Fig. 1. Design and optimization of the title compounds. 2
G. Yang et al.
Pesticide Biochemistry and Physiology xxx (xxxx) xxx
2.3.1. Protective activity against TMV in vivo First, the test compound solution was smeared on the left side of the leaf, while the solvent as the blank control was smeared on the right side. Then, the entire leaves were inoculated with the virus after 12 h. The leaves were washed with water after 30 min and cultivated in a light incubator at a temperature of 27 ± 1 ◦ C and a light intensity of 10,000 Lux. The local lesion numbers appearing 3–4 days after inoculation were counted.
selected, and the surface of the leaves was rubbed with quartz sand to form microwounds. In addition, 1000 μL supernatant containing the virus was added to each leaf. Then, these plants were continuously observed under ultraviolet light for one week, and photographs of the progress of the viral infection were recorded. The commercialized antiviral agent ribavirin was used as a positive control.
2.3.2. Inactivating activity against TMV in vivo The virus was mixed with the test compound solution at an equal volume for 30 min. Then, the mixture was inoculated on the left side of the leaves, while a mixture of solvent and virus was smeared on the right side of the leaves as the blank control. Then, the leaves were cultivated in a light incubator at a temperature of 27 ± 1 ◦ C and a light intensity of 10,000 Lux. The local lesion numbers were recorded 3–4 days after inoculation.
To observe the effects of the high inactivation activity of the title compounds on the morphology of TMV, the morphology of the TMV after treatment with 5h was observed by TEM. The research method refers to that reported in the literature (Wang et al., 2019a, 2019b). A total of 101 μL of a solution of the testing compound mixed with TMV was prepared, and the concentration was 500 mg/L. After mixing for 30 min, the mixture was adsorbed using a 200-mesh carbon membrane support (copper grid) and subjected to negative staining with 1% phosphotungstic acid at a pH of 7.4. The morphology of the TMV par ticles was observed by TEM at 200 kV. The blank control (CK) was TMV particles in PBS and 1% DMSO without any other agents. The commercialized antiviral agent ningnanmycin was used as a positive control.
2.7. Morphological study with TMV
2.3.3. Curative activity against TMV in vivo First, TMV was used to infect whole leaves. Then, the leaves were washed with water and dried after 30 min. The test compound solution was smeared on the left side of the leaves, while the solvent as the blank control was smeared on the right side. Then, the leaves were cultivated in a light incubator at a temperature of 27 ± 1 ◦ C and a light intensity of 10,000 Lux. The local lesion numbers were counted and recorded 3–4 days after inoculation.
2.8. Insecticidal activity The insecticidal activity of title compounds 5a–5m against aphidoi dea was investigated using reported methods (Tian et al., 2018; Luo and Yang, 2007). Aphidoidea was cultivated in a greenhouse at a temperature of 25 ± 1 ◦ C, a humidity of 75 ± 5%, and under a 14:10 h light/dark photoperiod. The concentration of the test compounds was 500 mg/L. The fatality rate was evaluated after 48 h by recording the number of dead pests. The commercialized pesticide chlorpyrifos was used as the positive control. Three repetitions were conducted for each test compound.
2.4. Determination of the chlorophyll content The chlorophyll content of the tobacco leaves treated with 5j at a concentration of 500 mg/L was measured using a previously reported method with minor revisions (McDowell and Dangl, 2000). The tobacco leaves were dried with filter paper, and 0.5 mm small discs were ob tained with a puncher. Fifty milligrams of discs and 5.0 mL of extract (85% acetone: 85% ethanol = 1: 1) were mixed and ground. The ho mogenate was transferred to a 15 mL centrifuge tube, mixed for 1 h in the dark at 35 ◦ C, and centrifuged at 4000 rpm for 5 min at room tem perature. The extract was used to measure the absorbance at the wavelengths of 663 nm and 645 nm.
3. Results and discussion 3.1. Synthesis The synthesis route of the title compounds is shown in Fig. 2. As initial materials, ethyl (ethoxymethylene) cyanoacetate and tertbutylhydrazine were used to synthesize intermediate ester 2. Then, key intermediate 3 was obtained from 2 by a hydrazinolysis reaction in the presence of 80% hydrazine hydrate and reacted with CS2 under base conditions to form intermediate 4. Title compounds 5a–5m were syn thesized from 4 with different halogenated substituted benzyl groups. All the title compounds were characterized by 1H nuclear magnetic resonance (NMR), 13C NMR and high-resolution mass spectrometry (HRMS).
2.5. Defense enzyme (SOD, POD, CAT and PAL) activities in vivo Nicotiana tabacum cv. K326 with 5 to 6 leaves were selected as the host and cultivated at a temperature of 23 ± 1 ◦ C and a light intensity of 10,000 Lux. The tobacco leaves treated with the title compounds were chosen for defense activity testing on days 1, 3, 5 and 7, respectively. The title compound 5j was selected for further mechanistic study, and the commercialized agents ningnanmycin and lentinan (LNT) were used as a positive control. The concentration of the test compounds was 500 mg/L. The SOD, POD, CAT and PAL enzyme activities were determined by assay kits BC0175, BC0095, BC0205 and BC0215, respectively, which were purchased from Beijing Solarbio Science & Technology Co., Ltd., China (Wang et al., 2020). The details of the testing procedure and calculation method are provided in the supporting information.
3.2. Antiviral activity As indicated in Table 1, the antiviral activities of this series of title compounds exhibited obvious protective and inactivation effects against TMV in vivo at 500 mg/L. Among them, the protective activities of 5c (61.4%), 5d (72.5%), 5h (68.2%), 5i (65.2%), 5j (70.7%), 5k (78.0%), 5l (65.1%) and 5m (65.3%) were similar to that of the commercialized agent ningnanmycin (71.5%). Compounds 5c (63.5%), 5d (64.5%), 5f (64.1%), 5 h (84.4%), 5j (82.8%), 5k (76.2%) and 5l (59.1%) had inactivation activities similar to those of ningnanmycin (80.5%). The EC50 values in Table 2 indicate that compounds 5d, 5j, 5k and 5l exhibited excellent protective activity, and the EC50 values were 165.8, 163.2, 159.7 and 193.1 mg/L, respectively, which are better than that of ningnanmycin (EC50 = 271.3 mg/L). As shown in Table 3, the inacti vation activity EC50 value of 5h was 83.8 mg/L, which is similar to that of ningnanmycin (78.5 mg/L).
2.6. The progression of TMV in leaves marked by a GFP tag and treated with the title compounds To further study the effect in tobacco plants and TMV treated with compound 5j, TMV with a green fluorescent protein (GFP) gene tag was used for the experiment (Sheen et al., 1995; Chen et al., 2010; Casper and Holt, 1996; Itaya et al., 1997). As reported in the literature (Xiang et al., 2019; Weststeijn, 1981), 2 mL phosphate buffered saline (PBS) was added to 0.1 g of infected tobacco leaves, ground into a homoge nate, and centrifuged at 5000 rpm for 5 min, and then the supernatant was removed. The fourth or fifth leaves of Nicotiana benthamiana were 3
G. Yang et al.
Pesticide Biochemistry and Physiology xxx (xxxx) xxx
Fig. 2. Synthetic route of the title compounds 5a–5m.
3.3. Mechanistic study of the protective activity of 5j
Table 1 In vivo antiviral activities of the title compounds at 500 mg/L against TMVa. Compound no. 5a 5b 5c 5d 5e 5f 5g 5h 5i 5j 5k 5l 5m Ningnanmycin a
3.3.1. Chlorophyll content Chlorophyll is the major component in chloroplasts; it plays an important role in photosynthesis and can provide energy for plant growth. As depicted in Fig. 3, the chlorophyll a, chlorophyll b, chloro phyll a/chlorophyll b and total chlorophyll contents of the tobacco leaves treated with 5j were continuously measured after inoculation with TMV. As shown in Fig. 4A, B and D, the contents of chlorophyll a, b and total chlorophyll increased obviously from day 1 to day 3 and remained stable from day 3 to day 7, reaching the highest values of 8.0, 3.5 and 12.5 mg/L on the seventh day, respectively. The results demonstrated that title compound 5j can increase the chlorophyll con tent in the leaves, promote photosynthesis ability, and improve the disease resistance of the host.
Inhibition (%) Protective activity
Curative activity
Inactivation activity
54.0 ± 8.0 57.1 ± 3.6 61.4 ± 3.2 72.5 ± 9.5 43.2 ± 3.8 18.5 ± 7.8 24.3 ± 5.7 68.2 ± 5.7 65.2 ± 6.3 70.7 ± 2.6 78.0 ± 7.3 65.1 ± 1.6 65.3 ± 9.9 71.5 ± 9.1
58.9 ± 7.5 45.9 ± 5.1 40.4 ± 6.4 59.5 ± 9.5 40.0 ± 12.1 66.1 ± 15.2 64.3 ± 1.5 50.0 ± 9.4 44.5 ± 7.2 35.7 ± 8.6 59.0 ± 9.0 45.8 ± 6.0 49.0 ± 1.8 76.6 ± 8.8
28.1 ± 8.6 38.2 ± 3.5 63.5 ± 4.1 64.5 ± 7.2 0 64.1 ± 6.2 21.6 ± 1.8 84.4 ± 6.1 53.1 ± 7.2 82.8 ± 5.8 76.2 ± 9.0 59.1 ± 4.1 37.1 ± 3.0 80.5 ± 1.8
3.3.2. SOD activity analysis As depicted in Fig. 4A, the SOD activity of the leaves after treatment with 5% DMSO (CK) did not exhibit obvious differences from day 1 to day 7. In contrast, the SOD activity of tobacco leaves treated with 5j obviously increased on the first day and reached a peak value of 1261.2 U/g, which is much better than that of ningnanmycin (798.3 U/g) and LNT (768.5 U/g). Although the SOD activity of the leaves treated with 5j decreased from the third day, it was obviously better than that of the CK and the positive control groups (ningnanmycin and LNT). We speculated that title compound 5j may enhance the ability of the host to scavenge oxygen free radicals in vivo.
Average of three replicates.
Table 2 In vivo protective activity EC50 values of the title compounds against TMVa. Compound no.
EC50 (mg/L)
toxic regression equation
R2
5c 5d 5h 5i 5j 5k 5l 5m Ningnanmycin
272.0 ± 165.8 ± 350.1 ± 257.5 ± 163.2 ± 159.7 ± 193.1 ± 333.7 ± 271.3 ±
y = 0.5842× + 3.5777 y = 0.826× + 3.1666 y = 0.9738× + 2.5225 y = 0.912× + 2.8013 y = 0.707× + 3.4357 y = 0.7565× + 3.3333 y = 0.9314× + 2.8711 y = 1.3318× + 1.6394 y = 0.8823× + 2.853
0.951 0.973 0.939 0.979 0.990 0.992 0.940 0.992 0.951
a
2.4 1.0 7.0 5.8 5.0 6.9 2.5 3.2 5.8
3.3.3. POD activity analysis As shown in Fig. 4B, the POD activity of the treated leaves gradually increased from the first day. On the third day, the POD activity treated with ningnanmycin and LNT reached the highest value of 33,403.3 U/g and 17,600.0 U/g, respectively. For leaves treated with 5j, the POD activity reached the highest value (31,540.0.0 U/g) on the fifth day, which is better than that of ningnanmycin (24,056.7 U/g) and LNT (13,063.3 U/g). We speculated that the POD system is induced by title compound 5j to remove excess oxygen free radicals and hydrogen peroxide (Gozzo, 2003).
Average of three replicates.
Table 3 In vivo inactivation activity EC50 values of the title compounds against TMVa. Compound no.
EC50 (mg/L)
toxic regression equation
R2
5d 5f 5h 5j 5k Ningnanmycin
220.0 ± 2.4 252.5 ± 4.2 83.8 ± 5.6 163.6 ± 5.2 100.3 ± 2.8 78.5 ± 3.1
y = 1.3292× y = 1.2697× y = 1.1047× y = 1.8488× y = 2.2779× y = 1.5844×
0.965 0.953 0.924 0.978 0.920 0.925
a
+ 1.8865 + 1.9498 + 2.8755 + 0.9071 + 0.4412 + 1.9974
3.3.4. CAT activity analysis As depicted in Fig. 4C, the CAT activity of the leaves treated with ningnanmycin and 5j quickly increased and reached the highest value of 52.4 U/g and 78.3 U/g, respectively, on the first day, better than CK (54.56 U/g). The CAT activity of leaves treated with LNT reached the highest value (47.2 U/g) on the third day. CAT is an important hydrogen
Average of three replicates.
4
G. Yang et al.
Pesticide Biochemistry and Physiology xxx (xxxx) xxx
Fig. 3. The effect of tobacco leaves treated with 5j on the chlorophyll content. Contents of chlorophyll a (A), chlorophyll b (B), chlorophyll a/chlorophyll b (C), and total chlorophyll (D). (Bars with different letters are significantly different and values are mean ± S.D. (standard deviation); p < 0.05)
peroxide scavenging enzyme. The immune response of the host will produce too much hydrogen peroxide after infection with the virus. However, excessive hydrogen peroxide is not helpful for the growth of the host. The CAT enzyme can remove excess hydrogen peroxide and improve the ability of plants to defend against diseases (Dat et al., 2010). The results revealed that the title compound 5j can obviously improve the CAT activity of the tobacco leaves.
obviously infected by TMV at 2 dpi, and the TMV proliferated gradually from 2 dpi to 4 dpi. At 5 dpi, the virus was distinctly spread to the top leaves of the tobacco treated with CK and ribavirin (Fig. 5A and C), except for those treated with 5j (Fig. 5B). This result demonstrated that 5j can effectively inhibit the spreading of TMV in inoculated tobacco leaves. 3.5. Morphological study of TMV with TEM
3.3.5. PAL activity analysis PAL is a kind of catalytic enzyme in the plant defense system. It not only regulates the synthesis of phytoalexins but also promotes the con version rate of phenylalanine to cinnamic acid, which is a process that produces salicylic acid to achieve systemic resistance of the host to resist the infection and replication of the virus (Gozzo, 2003). As shown in Fig. 4D, the PAL activity of the leaves treated with the tested compounds gradually decreased from day 1 to day 5. However, the PAL activity of leaves treated with 5j had the highest value of 12.8 U/g on the seventh day, which is much better than that of the CK group (8.0 U/g). The re sults indicated that the title compound 5j can improve the PAL activity of the host, which could enhance its ability to resist TMV infection. With TMV infection, the plant itself will produce a series of immune responses to achieve systemic resistance (McDowell and Dangl, 2000; Yatskou et al., 2001). The results of the experiments revealed that the chlorophyll content and the defense enzyme activities of the tobacco leaves treated with 5j were significantly improved, which was beneficial to fight against viral infection.
Morphological observations of TMV treated with compound 5h with high inactivation activity were performed with transmission electron microscopy (TEM). As depicted in Fig. 6A, the TMV in the blank control showed a rod-shaped structure. Further analysis of Fig. 6B and C found that rod-shaped TMV formed a distinct break after treatment with ningnanmycin and 5h at a concentration of 500 mg/L for 30 min (break points indicated with red arrows in the figures). As shown in Fig. C4, the details reveal that TMV treated with 5h broke into small rods, as shown with a ruler length of 20 nm. 3.6. Insecticidal activity analysis Aphids are common carriers of plant viruses and can indirectly transmit viruses from infected plants to normal plants. This is an important transmission route for TMV (Yu et al., 2016; Yang et al., 2020). As indicated in Table 4, compounds 5a, 5b and 5m exhibited certain insecticidal activities against aphidoidea, and the fatality rates were 85%, 83% and 87%, respectively. The insecticidal activity results revealed that some title compounds can block the spread of TMV by killing the carrier aphid. A series of new 1-tert-butyl-5-amino-4-pyrazole bioxadiazole sulfide derivatives containing a 1,3,4-oxadiazole group were designed and
3.4. Analysis of the protective activity in vivo using tobacco labeled with GFP As shown in Fig. 5, the leaves of Nicotiana benthamiana were 5
G. Yang et al.
Pesticide Biochemistry and Physiology xxx (xxxx) xxx
Fig. 4. The effect of tobacco leaves treated with 5j on defense enzyme activities. The effect on SOD (A), POD (B), CAT (C), and PAL (D). (Bars with different letters are significantly different and values are mean ± S.D. (standard deviation); p < 0.05)
Fig. 5. Tobacco marked with GFP used to study the protective activity mechanism in vivo. Analysis of the transmission efficiency of TMV in leaves treated with CK (A), those treated with 5j (B), and those treated with ribavirin (C).
synthesized. The bioactivity results showed that some compounds exhibited excellent protective activity against TMV. The EC50 values of 5d, 5j, 5k and 5l were 165.8, 163.2, 159.7 and 193.1 mg/L, respec tively, which are better than that of ningnanmycin (271.3 mg/L). The
inactivation activity EC50 value of 5h against TMV was 83.8 mg/L, similar to that of ningnanmycin (78.5 mg/L). Further study found that the chlorophyll contents and the defensive enzyme activities of the to bacco leaves after treatment with 5j were significantly improved. 6
G. Yang et al.
Pesticide Biochemistry and Physiology xxx (xxxx) xxx
Fig. 6. TEM images of TMV after treatment with CK (A), ningnanmycin (B), and 5h (C) at a concentration of 500 μg/mL. The ruler lengths are 200 nm (1), 100 nm (2), 50 nm (3), and 20 nm (4).
TMV that reducing the infection effect of the virus. Simultaneously, the title compounds can effectively block the common carrier for plant viruses, thereby blocking infection with the viruses. This series of syn ergistic effects provide key information for the research and develop ment of antiviral agents.
Table 4 Insecticidal activity of the title compounds at 500 mg/L against aphidoideaa. Compound no.
Fatality rate (%)
Compound no.
Fatality rate (%)
5a 5b 5c 5d 5e 5f 5g
85 83 0 0 47 38 57
5h 5i 5j 5k 5l 5m Chlorpyrifos
0 0 0 0 0 87 100
a
Funding This work was supported by the National Natural Science Foundation of China (No. 21662008) and the Science and Technology Project of Guizhou Province (No. [2017]5788).
Average of three replicates.
Declaration of Competing Interest
Further antiviral activity research on 5j using TMV with a GFP gene tag found that it can inhibit the spread of TMV in inoculated tobacco leaves. A morphological study with TEM revealed that title compound 5h treatment can significantly shorten the polymerization length of TMV particles and cause a distinct break of the rod-shaped TMV. Moreover, the insecticidal activity revealed that the fatality rates of 5a, 5b and 5m against aphidoidea were 85%, 83% and 87%, respectively. These results reveal that this series of title compounds not only can induce the host to obtain systemic acquired resistance and enhance its ability to defend against diseases, but also can cause a distinct break of the rod-shaped
The authors declare that there are no competing financial interests. Acknowledgments The authors acknowledge Jian Zhang and Zhengjun Liu, Guizhou University, for testing the insecticidal activity against aphids.
7
G. Yang et al.
Pesticide Biochemistry and Physiology xxx (xxxx) xxx
Appendix A. Supplementary data
Ma, J., Li, P., Li, X.Y., Shi, Q.C., Wan, Z.H., Hu, D.Y., Jin, L.H., Song, B.A., 2014. Synthesis and antiviral bioactivity of novel 3-((2-((1E,4E)-3-Oxo-5-arylpenta-1,4dien-1-yl)phenoxy) methyl)-4(3H)-quinazolinone derivatives. J. Agric. Food Chem. 62 (36), 8928–8934. McDowell, J.M., Dangl, J.L., 2000. Signal transduction in the plant immune response. Trends Biochem. Sci. 25 (2), 79–82. https://doi.org/10.1016/S0968-0004(99) 01532-7. Noutoshi, Y., Okazaki, M., Shirasu, K., 2012. Imprimatins A and B: novel plant activators targeting salicylic acid metabolism in Arabidopsis thaliana. Plant Signal. Behav. 7 (12), 1715–1717. https://pubmed.ncbi.nlm.nih.gov/23073003/. Sheen, J., Hwang, S., Niwa, Y., Kobayashi, H., Galbraith, D.W., 1995. Green-fluorescent protein as a new vital marker in plant cells. Plant J. 8 (5), 777–784. https://doi.org/ 10.1046/j.1365-313X.1995.08050777.x. Silverman, F.P., Petracek, P.D., Heiman, D.F., Fledderman, C.M., Warrior, P., 2005a. Salicylate activity. 3. Structure relationship to systemic acquired resistance. J. Agric. Food Chem. 53 (25), 9775–9780. https://doi.org/10.1021/jf051383t. Silverman, F.P., Petracek, P.D., Ju, Z., Fledderman, C.M., Heiman, D.F., Warrior, P., 2005b. Salicylate activity. 1. Protection of plants from paraquat injury. J. Agric. Food Chem. 53 (25), 9764–9768. https://doi.org/10.1021/jf0513819. Silverman, F.P., Petracek, P.D., Ju, Z., Fledderman, C.M., Heiman, D.F., Warrior, P., 2005c. Salicylate activity. 2. Potentiation of atrazine. J. Agric. Food Chem. 53 (25), 9769–9774. https://doi.org/10.1021/jf0513821. Tian, P.Y., Liu, D.Y., Liu, Z.J., Shi, J., He, W.J., Qi, P.Y., Chen, J.X., Song, B.A., 2018. Design, synthesis, and insecticidal activity evaluation of novel 4-(N, Ndiarylmethylamines)furan-2(5H)-one derivatives as potential acetylcholine receptor insecticides. Pest Manag. Sci. 75, 427–437. https://doi.org/10.1002/ps.5132. Wang, Z.W., Wei, P., Liu, Y.X., Wang, Q.M., 2014. D and E rings may not be indispensable for antofine: discovery of phenanthrene and Alkylamine chain containing antofine derivatives as novel antiviral agents against tobacco mosaic virus (TMV) based on interaction of antofine and TMV RNA. J. Agric. Food Chem. 62, 10393–10404. https://doi.org/10.1021/jf5028894. Wang, H.J., Chen, Y., Zhang, W.K., 2019a. A single-molecule atomic force microscopy study reveals the antiviral mechanism of tannin and its derivatives. Nanoscale. 11, 16368–16376. https://doi.org/10.1039/C9NR05410C. Wang, M.W., Zhu, H.H., Wang, P.Y., Zeng, D., Wu, Y.Y., Liu, L.W., Wu, Z.B., Li, Z., Yang, S., 2019b. Synthesis of thiazolium-labeled 1,3,4-oxadiazole thioethers as prospective antimicrobials: in vitro and in vivo bioactivity and mechanism of action. J. Agric. Food Chem. 67 (46), 12696–12708. https://doi.org/10.1021/acs. jafc.9b03952. Wang, P.Y., Xiang, M., Luo, M., Liu, H.W., Zhou, X., Wu, Z.B., Liu, L.W., Li, Z., Yang, S., 2020. Novel piperazine-tailored ursolic acid hybrids as significant antibacterial agents targeting phytopathogens Xanthomonas oryzae pv. Oryzae and X. axonopodis pv. Citri probably directed by activation of apoptosis. Pest Manag. Sci. 76, 2746–2754. https://doi.org/10.1002/ps.5822. Weststeijn, E.A., 1981. Lesion growth and virus localization in leaves of Nicotiana tabacum cv. Xanthi nc. after inoculation with tobacco mosaic virus and incubation alternately at 22 ◦ C and 32 ◦ C. Physiol. Mol. Plant P. 18 (3), 357–368. https://doi. org/10.1016/S0048-4059(81)80086-0. Wu, Z.B., Li, X.Y., Zhou, X., Yang, W.Q., Ye, Y.Q., Song, B.A., Yang, S., 2016. Synthesis and anti-TMV Activitie of 1-(3-chloropyridin-2-yl)-5-N-substituted-4-(5(methylthio)-1,3,4-oxadiazol-2-yl)-pyrazole derivatives. Sci. Sin. Chim. 46, 1195–1203. https://doi.org/10.1360/N032016-00122. Xiang, S.Y., Lv, X., He, L.H., Shi, H., Liao, S.Y., Liu, C.Y., Huang, Q.Q., Li, X.Y., He, X.T., Chen, H.T., Wang, D.B., Sun, X.C., 2019. Dual-action pesticide carrier that continuously induces plant resistance, enhances plant anti-tobacco mosaic virus activity, and promotes plant growth. J. Agric. Food Chem. 67 (36), 10000–10009. https://doi.org/10.1021/acs.jafc.9b03484. Xu, W.M., Yang, S., Bhadury, P., He, J., He, M., Gao, L.L., Hu, D.Y., Song, B.A., 2011. Synthesis and bioactivity of novel sulfone derivatives containing 2,4-dichlorophenyl substituted 1,3,4-oxadiazole/thiadiazole moiety as chitinase inhibitors. Pestic. Biochem. Physiol. 101, 6–15. https://doi.org/10.1016/j.pestbp.2011.05.006. Xu, W.M., Han, F.F., He, M., Hu, D.Y., He, J., Yang, S., Song, B.A., 2012. Inhibition of tobacco bacterial wilt with sulfone derivatives containing an 1,3,4-oxadiazole moiety. J. Agric. Food Chem. 60 (4), 1036–1041. https://doi.org/10.1021/ jf203772d. Yang, Z.B., Li, P., He, Y.J., Luo, J., Zhou, J., Wu, Y.H., Chen, L.T., 2020. Novel pyrethrin derivatives containing an 1,3,4-oxadiazole thioether moiety: design, synthesis, and insecticidal activity. J. Heterocyclic Chem. 57 (1), 81–88. https://doi.org/10.1002/ jhet.3750. Yatskou, M.M., Koehorst, R.B.M., Hoek, A.V., Donker, H., Schaafsma, T.J., 2001. Spectroscopic properties of a self-assembled zinc porphyrin tetramer II. Timeresolved fluorescence spectroscopy. J. Phy. Chem. A. 105 (51), 11432–11440. https://doi.org/10.1021/jp010411h. Yu, X.D., Liu, Z.C., Huang, S.L., Chen, Z.Q., Sun, Y.W., Duan, P.F., Ma, Y.Z., Xia, L.Q., 2016. RNAi-mediated plant protection against aphids. Pest Manag. Sci. 72 (6), 1090–1098. https://doi.org/10.1002/ps.4258. Zhao, L., Feng, C.H., Wu, K., Chen, W.B., Chen, Y.J., Hao, X.A., Wu, Y.F., 2015. Advances and prospects in biogenic substances against plant virus: a review. Pestic. Biochem. Physiol. 135, 15–26. https://doi.org/10.1016/j.pestbp.2016.07.003.
Supporting information includes the experimental characterization data, testing procedure and calculation method for the chlorophyll content and defensive enzyme activities. Supplementary data to this article can be found online at https://doi.org/10.1016/j.pestbp.2020.10 4740. References Beachy, R.N., Heinlein, M., 2000. Role of P30 in replication and spread of TMV. Traffic 1 (7), 540–544. https://doi.org/10.1034/j.1600-0854.2000.010703.x. Buck, K.W., 1999. Replication of tobacco mosaic virus RNA. Philos. Trans. R. Soc. Lond. B 354, 613–627. https://doi.org/10.1098/rstb.1999.0413. Casper, S.J., Holt, C.A., 1996. Expression of the green fluorescent protein-encoding gene from a tobacco mosaic virus-based vector. Gene 173 (1), 69–73. https://doi.org/ 10.1016/0378-1119(95)00782-2. Chen, Q.J., Dong, L., Ouyang, M.A., Xie, L.H., Lin, Q.Y., 2010. Detection of anti-TMV activity of Bruceine D by green fluorescent protein labeling. Acta Laser Biol. Sin. 06, 36–41. https://kns.cnki.net/kns8/defaultresult/index. Chen, L.J., Zou, W.S., Wu, G., Lin, H.H., Xi, D.H., 2018. Tobacco alpha-expansin EXPA4 plays a role in Nicotiana benthamiana defence against Tobacco mosaic virus. Planta 247 (2), 355–368. https://doi.org/10.1007/s00425-017-2785-6. Dat, J.F., Pellinen, R., Beeckman, T., Cotte, B.V.D., Langebartels, C., Kangasjarvi, J., lnze, D., Breusegem, F.V., 2010. Changes in hydrogen peroxide homeostasis trigger an active cell death process in tobacco. Plant J. 33 (4), 621–632. https://doi.org/ 10.1046/j.1365-313X.2003.01655.x. Du, Q.S., Zhu, W.P., Zhao, Z.J., Qian, X.H., Xu, Y.F., 2012. Novel benzo-1,2,3thiadiazole-7-carboxylate derivatives as plant activators and the development of their agricultural applications. J. Agric. Food Chem. 60 (1), 346–353. https://doi. org/10.1021/jf203974p. Du, Q.S., Shi, Y.X., Li, P.F., Zhao, Z.J., Zhu, W.P., Qian, X.H., Li, B.J., Xu, Y.F., 2013. Novel plant activators with thieno[2,3-d]-1,2,3-thiadiazole-6-carboxylate scaffold: synthesis and bioactivity. Chin. Chem. Lett. 24 (11), 967–969. https://doi.org/ 10.1016/j.cclet.2013.07.003. Fedorkin, O.N., Solovyev, A.G., Yelina, N.E., Zamyatnin, A.A., Zinovkin, R.A., Makinen, K., Schiemann, J., Morozov, S.Y., 2001. Cell-to-cell movement of potato virus X involves distinct functions of the coat protein. J. Gen. Virol. 82, 449–458. https://doi.org/10.1099/0022-1317-82-2-449. Friedrich, L., Lawton, K., Ruess, W., Masner, P., Specker, N., Rella, M.G., Meier, B., Dincher, S., Staub, T., Uknes, S., Metraux, J.P., Kessmann, H., Ryals, J., 1996. A benzothiadiazole derivative induces systemic acquired resistance in tobacco. The Plant J. 10 (1), 61–70. https://doi.org/10.1046/j.1365-313x.1996.10010061.x. Gan, X.H., Hu, D.Y., Li, P., Wu, J., Chen, X.W., Xue, W., Song, B.A., 2015. Design, synthesis, antiviral activity and three-dimensional quantitative structure-activity relationship study of novel 1,4-pentadien-3-one derivatives containing the 1,3,4oxadiazole moiety. Pest Manag. Sci. 72, 534–543. https://doi.org/10.1021/ jf502162y. Gooding Jr., G.V., Hebert, T.T., 1967. A simple technique for purification of tobacco mosaic virus in large quantities. Phytopathology. 57, 1285–1287. https://doi.org/ 10.1098/rstl.1767.0047. G¨ orlach, J., Volrath, S., Knauf-Beiter, G., Hengy, G., Beckhove, U., Kogel, K.H., Oostendorp, M., Staub, T., Ward, E., Kessmann, H., Ryals, J., 1996. Benzothiadiazole, a novel class of inducers of systemic acquired resistance, activates gene expression and disease resistance in wheat. Plant Cell 8 (4), 629–643. https:// www.jstor.org/stable/3870340. Gozzo, F., 2003. Systemic acquired resistance in crop protection: from nature to a chemical approach. J. Agric. Food Chem. 51 (16), 4487–4503. https://doi.org/ 10.1016/j.bmc.2006.12.002. Han, Y.G., Luo, Y., Qin, S.R., Xi, L., Wan, B., Du, L.F., 2014. Induction of systemic resistance against tobacco mosaic virus by Ningnanmycin in tobacco. Pestic. Biochem. Physiol. 111 (1), 14–18. https://doi.org/10.1016/j.pestbp.2014.04.008. Itaya, A., Hickman, H., Bao, Y., Richard, N., Ding, B., 1997. Cell-to-cell trafficking of cucumber mosaic virus movement protein:green fluorescent protein fusion produced by biolistic gene bombardment in tobacco. Plant J. 12, 1223–1230. https://doi.org/ 10.1046/j.1365-313X.1997.12051223.x. Kessmann, H., Staub, T., Ligon, J., Oostendorp, M., Ryals, J., 1994. Activation of systemic acquired disease resistance in plants. Eur. J. Plant Pathol. 100 (6), 359–369. https:// doi.org/10.1007/BF01874804. Kunz, W., Schurter, R., Maetzke, T., 1997. The chemistry of benzothiadiazole plant activators. J. Pestic. Sci. 50 (4), 275–282. https://doi.org/10.1002/(SICI)1096-9063 (199708)50:4<275::AID-PS593>3.0.CO;2-7. Luo, Y.P., Yang, G.F., 2007. Discovery of a new insecticide lead by optimizing a targetdiverse scaffold: Tetrazolinone derivatives. Bioorg. Med. Chem. 15 (4), 1716–1724. Lv, X., Xiang, S.Y., Wang, X.C., Wu, L., Liu, C.Y., Yuan, M.T., Gong, W.W., Win, H., Hao, C.Y., Xue, Y., Ma, L.S., Cheng, D.Q., Sun, X.C., 2020. Synthetic chloroinconazide compound exhibits highly efficient antiviral activity against tobacco mosaic virus. Pest Manag. Sci. https://doi.org/10.1002/ps.5910.
8