Pesticide Biochemistry and Physiology 146 (2018) 13–18

Contents lists available at ScienceDirect

Pesticide Biochemistry and Physiology

journal homepage: www.elsevier.com/locate/pest

Munronin O, a potential activator for plant resistance T

a,b b b b c c
Ying Yan , Lei Tang , Jiaqi Hu , Jianta Wang , Tiwalade Adegoke Adelakun , Dongqiong Yang ,
⁎
Yingtong Dic, Yu Zhangc, Xiaojiang Haoa,
a
State Key Laboratory of Functions and Applications of Medicinal Plants, Guizhou Medical University, Guiyang 550014, People's Republic of China
b
Guizhou Chemical Drug Research and Development Engineering Technical Center, Guiyang 550004, People's Republic of China
c
State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, Yunnan 650201,
People's Republic of China

A R T I C L E I N F O A B S T R A C T

Keywords: A series of limonoids (1–8) were isolated from the whole plant of Munronia henryi and antiviral activities of the
Limonoids compounds were evaluated. The bioassay results demonstrated that Munronin O (1) showed remarkable pro-
Munronia henryi harms tective activity and compounds 7 and 8 showed significant inactivating, protective, and curative activities
Tobacco mosaic virus against tobacco mosaic virus (TMV). With a 50% effective concentration (EC50) value of 91.5 μg/mL, compound
Antiviral activity
1 exhibited the best protective activity compared with ningnanmycin (192.3 μg/mL). The potential for these
Plant resistance
compound of inducing systemic acquired resistance (SAR) was also evaluated, and compound 1 showed ex-
cellent induction activities. Furthermore, it was found that potentiation of defense-related enzyme activity and
the contents of SA was increased. Compound 1 could also inhibit the expression of TMV CP and up-regulate the
expression of defense-related genes. This work revealed that compound 1 can induce resistance and enhance
plant tolerance to TMV infection. Hence, compound 1 can be considered as a potential activator for inducing
plant resistance.

1. Introduction cinchnaglycoside C [13], 7-deoxy-trans -dihydronarciclasine [14], 3-

acetonyl-3-hydroxyoxindole [15], β-carbolines [16], quassinoids [17],
Tobacco mosaic virus (TMV) is a well-studied plant virus worldwide limonoids [18], and eudesmanolides [19]. This property has been
and causes significant crop loss. This virus infects > 400 plant species widely exploited in China to prevent the spread of plant viral diseases.
of 36 families, including tobacco, tomato, potato, pepper, cucumber, Therefore, finding antiviral agents from natural products to protect
and other plants [1]. Viral infection is extremely difficult to control plants has proven useful.
under field conditions. Ningnanmycin is the most effective plant virus Up till now, only limited compounds with obvious activity inducing
inhibitor and is used to prevent TMV disease; however, this antiviral is system acquired resistance (SAR) in plants have been reported, and the
not widely used in crops due to photosensitivity and difficulty in ap- agricultural applications of plant inducers are far from being developed.
plying the product [2]. Thus far, no chemical treatment can completely Limonoids are a class of triterpenoids that exhibit wide range of
inhibit TMV once it has infected a plant [3]. Thus, experts face the bioactivities including anti-virus, antifeedant and have attracted a great
challenge of fully protecting plants from TMV infection. deal of interesting for their potential in agriculture application [20–25].
Resistance induced by activators, including salicylic acid (SA) and The present work investigated plant defense response mechanisms of
its derivatives [4–6], benzo[d][1,2,3]-thiadiazole-7-carboxylic acid compound 1; such mechanisms include defense-related enzyme ac-
(CGA210007) and its derivatives [7,8], and jasmonic acid (JA) [9] can tivity, salicylic acid content, expression of deence-related gene and
provide defense against pathogens and these activators have been TMV CP gene. To the best of our knowledge, this study first demon-
widely used agriculturally. Plant metabolic products are highly efficient strated that compound 1 can enhance resistance against TMV as po-
and environment-friendly and involve unique modes of action [10,11]. tential inducers of plant systemic resistance induction.
Thus, these metabolic products are considered a rich source of plant
activators that help protect plants from TMV infection. Some metabolic
products exhibit good inhibitory activities against TMV, and these
metabolic products include seco-pregnane steroids [12],

⁎
Corresponding author.
E-mail address: [email protected] (X. Hao).

https://doi.org/10.1016/j.pestbp.2018.02.001
Received 30 November 2017; Received in revised form 1 February 2018; Accepted 4 February 2018
Available online 06 February 2018
0048-3575/ © 2018 Published by Elsevier Inc.
Y. Yan et al. Pesticide Biochemistry and Physiology 146 (2018) 13–18

2. Materials and methods 2.5. Gene expression analysis by real-quantitative PCR

2.1. Antiviral biological assay Tobacco leaf samples were collected and frozen immediately in li-
quid nitrogen until use. Total RNA was extracted using the Takara
Nicotiana tabacum cv. K326 and Nicotiana tabacum L. plants were Trizol Reagent Kit. This was followed by DNase digestion and sepera-
cultivated in a greenhouse. N. tabacum cv. K326 was used to determine tion through adsorption columns to eliminate possible contamination
systemic TMV infection and N. tabacum L. was used as local lesion host with DNA. The first strand cDNA was synthesised from 500 ng of total
when the plants grew to the 5–6 leaf stage. TMV was purified by DNA-free RNA with a PrimeScript® RT reagent Kit (TaKaRa, Japan)
Gooding method [26], and in vivo actions of compounds were de- according to the manufacturer's instructions. SYBR® Green realtime RT-
termined through a reported technique [27]. The commercial antiviral PCR analysis was carried out with a real time PCR detection system
agent ningnanmycin was used as a positive control. Measurements were using primers 5′-GACATGAAGGAGGAGCTTGC-3′ and 5′-ATCATGGAT
performed in triplicate. GGCTGGAAGAG-3′ for β-actin, 5′-GCTTCCCCTTTTATGCCTT C-3′ and
5′-CCTGGGTTCACGTTAA TGCT-3′ for PR-5, 5′-CAATACGGCGAAAAC
CTAGCTGA-3′ and 5′-CCTAGCACA TCCAACACGAA-3′ for PR-1a. 5′-
2.2. Plant growth and compound treatments ATT AGACCCGCTAGTCACAGCAC-3′ and 5′-GTGGGGTTCGCCTGAT
TTT-3′ for TMV CP. The PCR thermal cycling program was set at 95 °C
N. tabacum cv. K326 was grown to the 7-leaf stage and 200 μg/mL of for 5 min, followed by 45 cycles of 95 °C for 5 s, 55 °C for 30 s, 72 °C for
compound 1 solution was smeared on whole leaves. Healthy plants 1 min. After the cycles, dissociation melt curve analysis (65 °C–95 °C
pretreated with 0.05% DMSO was used as a mock infection (Mock), every 0.5 °C for 1 s) was carried-out. The qRT-PCR reactions were
TMV-inoculated tobacco leaves treated with 0.05% DMSO was used as performed in triplicate for each sample. Quantitative analysis was
the negative control group (CK), TMV-inoculated tobacco leaves treated performed using the 2−ΔΔCT method with β-actin used as an internal
with ningnanmycin as the positive control group (N + TMV), and TMV- control. The intensities of PCR-generated fragments were analyzed and
inoculated tobacco leaves treated with 1 as the treatment group quantified using ABI ViiA 7 real time PCR system.
(1 + TMV). Plant leaves were inoculated with the virus after 12 h and
cultivated in a greenhouse. Four groups were adopted: Mock, CK,
N + TMV, and 1 + TMV. Tissue samples were collected at 1, 3, 5, and 2.6. Determination of defense-related enzyme activities
7 days after inoculation treatment to assay for defense-related enzyme
activity, salicylic acid content, expression of defense-related gene and Activities of superoxide dismutase (SOD), peroxidase (POD),
TMV CP gene. Measurements were performed in triplicate. Polyphenol oxidase (PPO), and phenylalanine ammonia lyase (PAL)
were measured and calculated with enzyme assay reagent kits in ac-
cordance with manufacturer's instructions (Suzhou Comin
2.3. Screening of systemic acquired resistance Bioengineering Institute, China).

Three pots of N. tabacum cv. K326 plants at the 5 to 6-leaf stage

were chosen for the screening of each compound. A 200 μL amount of 2.7. Quantification of salicylic acid (SA) accumulation
the target compound or 200 μL of solvent (control solution) was gently
smeared on the lower leaves (the third to fourth leaves from the top) of With slight modifications of a described method [29], the samples
each plant. After 12 h, the treated leaves were inoculated with TMV. were tested for salicylic acid content every two days. Test samples in
The plants were cultured in the green house for 3 days of further cul- triplicate of each treatment were weighed 1.0 g and immediately frozen
tivation, and then 10 leaf disks with a diameter of 1 cm and without in liquid nitrogen and then stored at −80 °C. After centrifugation at
major veins were cut from the treated leaves surface. A week later, a 7500 ×g for 10 min, the precipitate was re-extracted with 2.5 mL of
further 10 leaf disks were cut from the newly grown leaves of the in- methanol (100%) and re-centrifuged. The supernatants from both ex-
oculated plants. The leaf disks were kept at −80 °C before Western blot tractions were combined and air dried in a water bath (60 °C). The dried
analysis. samples were resuspended in 1.25 mL of 5% trichloroacetic acid (TCA),
and centrifuged at 7500 ×g for 10 min. The supernatants were col-
lected and extracted in 4.0 mL of ethylacetate/cyclopentane (1:1, v:v)
2.4. SDS-PAGE and western blot analysis of TMV coat protein by vigorous vortexing for 30 min and centrifuged at 7500 ×g for
10 min. The top organic phase (including SA) was collected and air
Using a modified reported method [28], leaf disks were ground in dried in a water bath (60 °C). The TCA phase was hydrolyzed using
protein-loading buffer (40 g/L SDS, 10 mL/L β-ME, 200 mL/L glycerin, 1.5 mL 4 M HCl in a water bath (80 °C) for 1 h, and added 4.0 mL of 1/1
2 g/L bromophenol blue, 0.1 mol/L Tris-HCl, pH 6.8), and then 5 μL of (vol) ethylacetate/cyclopentane, incubated for 30 min by vigorously
sample and 3 μL of marker (Blue Plus Protein Marker, 16–94 kDa, vortexing and then centrifuged at 7500 ×g for 10 min. The top organic
Beijing, China) were loaded on a polyacrylamide gel (5% stacking gel, phase (including SAG) was combined and dried in a water bath (60 °C).
12.5% separating gel). Samples were run in duplicate. After SDS-PAGE, The residues were dissolved for SA were measured and calculated with
TMV protein bands were transferred at 200 mA for 45 min onto ni- salicylic acid assay reagent kits in accordance with manufacturer's in-
trocellulose membrane (0.2 μm) using an electrotransfer system structions (Suzhou Comin Bioengineering Institute, China).
(BioeRad, Hercules, CA). The membrane was washed in TBST (1 mol/L
Tris-HCl, pH 7.5; 1 mol/L NaCl; 0.05% Tween-20) and blocked with 5%
nonfat milk powder in TBST for 1 h at 37 °C. The membrane was wa- 2.8. Statistical analysis
shed three times for 15 min with TBST and reacted with a mixture of
1:5000 alkaline phosphatase-conjugated antirabbit IgG (Sigma, St. Each measurement was performed in triplicate and arranged with
Louis, MO) and 1:8000 polyclonal antibodies of TMV for 1 h at 37 °C. completely randomized design. Data are expressed as mean ± SD.
Thereafter, the membrane was incubated in substrate buffer (1 mol/L STATISTICA version 12.0 for Windows was used to perform the sta-
Tris-HCl, pH 9.5; 1 mol/L NaCl; 1 mol/L MgCl) with 330 μL/mL NBT tistical analysis. Experimental data were analyzed using SPSS version
and 165 μL/mL BCIP for 3–5 min in the dark until the bands of the CP 16.0 (SPSS, Chicago. IL, USA). The significance levels for tests were
were clear. p < .05.

14
Y. Yan et al. Pesticide Biochemistry and Physiology 146 (2018) 13–18

Fig. 1. Structures of compounds 1–8 from Munronia henryi.

3. Results (R = furan) exhibited greater inactivating, protective and curative ac-

tivities than ningnanmycin. These findings implied that furan-2(5H)-
3.1. Antiviral activity against TMV in vivo one and furan groups can enhance antiviral activity. Notably, 1
(R = ethynl) exhibited excellent protective activity with values of
Antiviral activities of compounds 1–8 (Fig. 1) against TMV were 81.0%, which was superior to ningnanmycin. However, 2–6 demon-
tested and are reported in Table 1 [30,31]. Some compounds exhibited strated lower antiviral activity against TMV compared with ningnan-
remarkable antiviral activities against TMV at 32 μg/mL. 1, 7, and 8 mycin. These results reveal that substituted moieties do not favour for
exhibited superior inactivation activities with values of 57.4%, 94.1% antiviral activity. EC50 values of protective activities suggest that
and 93.3%, respectively, compared with ningnanmycin (58.9%). 1, 7, compounds 1, 7, and 8 hold remarkable protective activities against
and 8 exhibited significantly greater protective activities against TMV TMV. EC50 values ranged from 91.5 to 198.4 μg/mL. In particular,
with values of 81.0%, 55.4% and 66.7%, respectively, than ningnan- compound 1 displayed the best protective activity with EC50 value of
mycin (62.8%). 7 and 8 showed superior curative activities with values 91.5 μg/mL. This value was superior to that of ningnanmycin
of 60.7% and 57.7%, respectively, compared with ningnanmycin (192.3 μg/mL). Results also revealed that limonoid containing the
(40.7%). Among these compounds, 7 (R = furan-2(5H)-one), and 8 ethynyl moiety hold greater protective activity against TMV than those
of other groups. The results indicated that pretreatment with 1 could
increase the resistance of the host plant to TMV infection.
Table 1
In vivo antiviral activities of limonoids against TMV.a

3.2. Effect on induction of resistance to TMV in tobacco plant

Compound Inactivation Protective Curative EC50 of
activityb (%) activityc (%) activityc (%) protective
activity (μg/ Following the protective treatment, to assess whether the protective
mL) effects of compounds 1–8 resulted from inducing SAR in the tobacco
plants, the SAR screening of these three compounds in the systemic
1 57.4 ± 6.6b 81.0 ± 3.6a 29.9 ± 4.0c 91.5 ± 3.1h
2 49.1 ± 4.9c 40.3 ± 6.7e 30.7 ± 4.2c 226.1 ± 2.4d infection host N. tabacum cv. K326 along with Western blot analysis of
3 45.4 ± 3.5c 42.5 ± 6.1d,e 29.1 ± 4.0c 211.9 ± 2.8e TMV coat protein (CP) was carried out (Fig. 2). The bands of CP in
4 36.8 ± 3.6d 49.7 ± 3.8c,d 29.8 ± 3.9c 262.7 ± 5.1b Fig. 2A showed that on the third day after inoculation accumulation of
5 30.9 ± 5.3d 35.6 ± 3.9e 25.2 ± 4.8c 248.9 ± 3.6c TMV in the treated leaves (with compounds 1, 7, and 8) were less than
6 32.5 ± 3.5d 30.5 ± 5.3e 27.3 ± 4.5c 348.2 ± 2.7a
7 94.1 ± 4.6a 55.4 ± 5.3c 60.7 ± 3.4a 190.5 ± 5.1g
that of ningnanmycin, while leaves treated with compounds 2–6
8 93.3 ± 5.3a 66.7 ± 3.1b 57.7 ± 5.2a 198.4 ± 4.8f showed strong bands of CP, which indicated low activities. Never-
Ningnanmycind 58.9 ± 4.6b 50.2 ± 5.2c,d 40.7 ± 2.9b 192.3 ± 2.4fg theless, the bands of CP in Fig. 3B showed different evidence of virus
accumulation in the newly grown leaves. With the treatment on the
Different letters marked in the same column represent significantly different among dif-
lower leaves with compounds 1, 7, and 8, the CP bands of the newly
ferent compounds, (p < .05). Data are means ± standard deviation (n = 3).
a grown leaves were undetected (1) or relatively weaker (7 and 8) than
Average of three replicates.
b
Concentration of compounds is 32 μg/mL. that of ningnanmycin. It was possible that compound 1 induced SAR in
c
Concentration of compounds is 200 μg/mL. the plants to inhibit virus accumulation and movement. Among the
d
Commercial antiviral agent ribavirin and ningnanmycin as positive control. tested compounds, 1 showed higher induction activity on both treated

15
Y. Yan et al. Pesticide Biochemistry and Physiology 146 (2018) 13–18

5 (Fig. 4B). As a defense gene, POD can induce biosynthetic pathway of

SA, lignin, and phytoalexins, which can activate SAR46 and promote
cell-wall reinforcement and pathogen inhibition [36]. Antioxidant de-
fense machinery protects plant cells from oxidative damage induced by
ROS. Furthermore, ROS generation can reflect the state of photo-
synthesis [34]. Therefore, we analyzed SOD activities of 1-treated to-
bacco. SOD activity of 1 + TMV treatment group increased from day 1
to day 3 and reached the highest value on the third day. SOD activity of
1 + TMV treatment group was higher than that of TMV-inoculated
group. Then, activity decreased from day 3 to day 7 (Fig. 4C). PPO is
formed by catalyzing lignin and quinone compounds, to form a pro-
tective shield that protects cells from pathogens and can also directly
exert disease-resistant effects by forming quinone [37]. PPO activities
Fig. 2. Western blot analysis. (A) Accumulation of TMV coat protein in the lower leaves increased significantly in N + TMV and 1 + TMV treatment groups and
treated with compounds 1–8 (200 μg/mL) on the third day after inoculation; M, marker; reached their maximum values at day 3 (Fig. 4D). Activities (54.9% and
N, ningnanmycin; CK, negative control. (B) Accumulation of TMV coat protein in newly
55.8%, respectively) were higher than that of Mock. Results of defen-
grown leaves on the seventh day after inoculation.
sive enzyme activity assay demonstrated that 1 can improve disease
resistance of tobacco through induction defensive responses in the form
of enzymes.

3.5. Effect on defense-related genes expression in tobacco plant

Expression of the two defense-related genes was retained at very

low level in TMV-inoculated tobacco leaves, while pathogen challenge
triggered accelerated gene expression at different time points with the
exception of defense-related genes. Transcript levels of all two genes in
TMV-inoculated tobacco leaves treated with 1 increased faster and to
greater extents than after the other groups at day 3 (Fig. 5). A priming
effect of 2.76- and 3.03-fold, was observed in TMV-inoculated tobacco
leaves treated with 1 for an up-regulated expression of PR-5 and PR-1a,
Fig. 3. Effects of compound 1 on relative TMV CP gene of expression, The x-axis indicates respectively, in compared to that in the control at day 1, 1 induced
the time after inoculation (days). Vertical bars refer to mean ± SD (n = 3). stronger expression of PR-5 and PR-1a in tobacco plant when chal-
lenged with TMV at day 3. Moreover, the change in expression level of
leaves and newly grown leaves. PR-5 and PR-1a exhibited a similar tendency in pathogen inoculated
fruit that reached a higher level than after other treatments at day 3.
The results indicated that compound 1 can up-regulate the expression
3.3. Effect on TMV CP gene expression
of defense-related genes in the tobacco plant to enhance disease re-
sistance.
TMV CP transcript levels in the inoculated leaves by qRT-PCR assay.
TMV CP transcript levels in the TMV-inoculated systemic leaves treated
3.6. Effect on salicylic acid contents
with 1 and ningnanmycin were significantly less than TMV-inoculated
group, and transcript levels in TMV-inoculated tobacco leaves treated
Salicylic acid is a signaling molecule in tobacco and plays an im-
with 1 was lower than that in TMV-inoculated tobacco leaves treated
portant role in tobacco defense against diseases. It is an endogenous
with ningnanmycin (Fig. 3). At 7 days after incolation, the expression of
signaling molecule that activates tobacco allergy and system-acquired
TMV CP decreased to the lowest point in TMV-inoculated tobacco
resistance and enhances plant abiotic stress resistance [38]. In our
leaves treated with 1, which indicated that 1 could induce anti-TMV
study, salicylic acid content of tobacco plant (Fig. 6) decreased gra-
activity that could be transported to systemic leaves by interfering with
dually with TMV inoculation. However, in TMV-inoculated tobacco
the expression of TMV CP. TMV CP had been reported to be involved in
leaves treated with 1, salicylic acid content increased from day 1 to day
long distance movement [32]. Thus, the results show that 1 did also
3 and reached the highest value at day 3. Variation in trends of
disturb the long distance movement of TMV, and did down regulate the
1 + TMV treatment surpassed that of N + TMV treatment. Salicylic
expression and accumulation of TMV CP.
acid content of 1-treated tobacco decreased gradually. Hence, 1 may
increase salicylic acid content and thereby enhance plant host re-
3.4. Effect on defense-related enzyme activities sistance to diseases.

Induced resistance is correlated with enhanced activities of defen- 4. Disscusion

sive enzymes, such as PAL, POD, SOD and PPO [33,34]. In TMV-in-
oculated tobacco leaves treated with 1, PAL activity of tobacco leaves To further confirm the anti-TMV activities of compounds 1–8, the
increased from day 1 to day 5, reached maximum on the fifth day, and relative content of TMV in inoculated and systematic leaves was mea-
then dropped from day 5 to day 7 (Fig. 4A). PAL activity of 1 + TMV sured. With the treatment on lower leaves by compounds 1, the CP
treatment group was higher (54.4.0%) than that of CK group on the bands of newly grown leaves were undetected. It was possible that
fifth day. PAL is a catalytic enzyme involved in biosynthesis of phe- compounds 1 induced systemic acquired resistance in the plants to in-
nylpropanoids to cinnamic acid. Such process can produce SA for de- hibit virus accumulation and movement. Among the tested compound 1
fense against pathogens [35]. POD activities of CK, N + TMV, and showed higher induction activities on both treated leaves and newly
1 + TMV treatment groups were higher than that of Mock. Notably, grown leaves. The physiological and biochemical activities and changes
POD activity of 1 + TMV treatment group was visibly higher than that of disease-resistance genes in TMV-inoculated tobacco leaves treated
of Mock treatment group and reached the highest value (41.9%) at day with 1 were analyzed. The results showed that compound 1 can

16
Y. Yan et al. Pesticide Biochemistry and Physiology 146 (2018) 13–18

Fig. 4. Effects of compound 1 on PAL (A), POD (B), SOD (C), and PPO (D) activity in tobacco leaves, The x-axis indicates the time after inoculation (days). Vertical bars refer to
mean ± SD (n = 3).

significantly enhance activities of defensive enzymes, increase salicylic

acid content and up-regulate the expression of disease-resistance genes
in TMV-inoculated tobacco leaves. Based on these comprehensive ef-
fects, compound 1 showed better inhibitory activity against TMV than
that of ningnanmycin. Related studies have shown that the increase of
defensive enzyme activities in plants is closely related to salicylic acid
signaling pathway [39]. Hahlbrock and Scheel showed that PAL as a
key enzyme in the metabolism of polyphenols can increase the sec-
ondary metabolites responsible for plant defense response [40]. Ras-
mussen et al. also showed that salicylic acid can increase PAL, POD
activity. PR-1a and PR-5 genes play an important role as downstream
regulatory proteins of salicylic acid signaling pathway in plant anti- Fig. 6. Effects of compound 1 on salicylic acid in tobacco leaves, The x-axis indicates the
bacterial, antiviral and injury repair [41]. The present study showed time after inoculation (days). Vertical bars refer to mean ± SD (n = 3).
that the content of salicylic acid reached the maximum on the third day,
and the expression of PR-1a and PR-5 genes were up-regulated at var-
of ningnanmycin. Accordingly, compound 1 exhibited potency as a
ious stage in TMV-inoculated tobacco leaves treated with 1 from day 1
control against plant viruses.
to day 3. Meantime, PAL, POD, SOD and PPO activity increased in
different degrees from day 3 to day 5. These results indicate that
compound 1 may be closely related to the increase of salicylic acid 5. Conclusion
content, the up-regulation of PR-1a and PR-5 genes expression and the
increase of defense enzyme activity in inhibition mechanism against In summary, eight limonoids, containing different substituent
TMV. Our results suggested that 1 tested bio-agent have the ability to groups at C-17 were isolated from the whole plant of Munronia henryi
control viral infections, and its protective effect was stronger than that Harms. In vivo studies indicated that compounds 1, 7, and 8 exhibited
better curative, protective, and inactivating activities than that of

Fig. 5. Expression analysis of PR-5 and PR-1a genes in tobacco plant by qRT-PCR, The x-axis indicates the time after inoculation (days). Vertical bars refer to mean ± SD (n = 3).

17
Y. Yan et al. Pesticide Biochemistry and Physiology 146 (2018) 13–18

ningnanmycin, especially compound 1, whose ethynyl moiety at C-17 [17] X.H. Yan, J. Chen, X.J. Hao, et al., Anti-tobacco mosaic virus (TMV) quassinoids
exhibited the best protective activity among these compounds. This from Brucea javanica (L.) Merr, J. Agric. Food Chem. 58 (2010) 1572–1577.
[18] Y.H. Ge, K.X. Liu, X.J. Hao, et al., The limonoids and their antitobacco mosaic virus
protective ability was associated with potentiation of defensive enzyme (TMV) activities from Munronia unifoliolata Oliv, J. Agric. Food Chem. 60 (2012)
activity, salicylic acid content of tobacco after treatment with 1. This 4289–4295.
finding was confirmed by up-regulated expression of defense-related [19] Z.W. Wang, P. Wei, Q.M. Wang, Design, synthesis, and anti-tobacco mosaic virus
(TMV) activity of phenanthroindolizidines and their analogues, J. Agric. Food
genes. This study demonstrated that 1 can induce resistance and en- Chem. 60 (2012) 10212–10219.
hance plant tolerance to TMV infection. Compound 1 can also be con- [20] Y.Y. Zhang, H. Xu, Recent progress in the chemistry and biology of limonoids, RSC
sidered as a potential activator in the field of plant protection. Adv. 7 (2017) 35191–35220.
[21] Y. Guo, R.G. Yang, H. Xu, Semisynthesis of esters and oxime esters/sulfonates from
furyl-ring-based acetylation derivaives of fraxinellone as insecticidal agents, RSC
Acknowledgment Adv. 7 (2017) 28009–28015.
[22] X. Yu, D.F. Shi, H. Xu, et al., Synthesis and quantitative structure–activity re-
lationship (QSAR) study of C7-oxime ester derivatives of obacunone as insecticidal
The work was supported financially by the National Natural Science
agents, RSC Adv. 5 (2015) 31700–31707.
Foundation of China (No. 31160374), the Scientific and Technological [23] Y. Guo, R.G. Yang, H. Xu, New insecticidal agents from halogenation/acylation of
Planning Project of Guizhou (No. QKH-JC [2017]1141), and initial the furyl-ring of fraxinellone, Sci. Rep. 6 (2016) 35321.
funding of PhD (No. YBH-J [2016-7]). [24] Q. Li, X.B. Huang, H. Xu, et al., Semisynthesis of esters of fraxinellone C4/10-oxime
and their pesticidal activities, J. Agric. Food Chem. 64 (2016) 5472–5478.
[25] Y. Guo, H. Qu, H. Xu, et al., Semisynthesis and insecticidal activity of some frax-
References inellone derivatives modified in the B ring, J. Agric. Food Chem. 61 (2013)
11937–11944.
[26] G.V. Gooding Jr., T.T. Hebert, A simple technique for purification of tobacco mosaic
[1] A.N. Creager, K.B. Scholthof, V. Citovsky, et al., Tobacco mosaic virus: pioneering virus in large quantities, Phytopathology 57 (1967) 1285–1287.
research for a century, Plant Cell 11 (1999) 301–308. [27] X.H. Gan, D.Y. Hu, B.A. Song, et al., Novel trans-Ferulic acid derivatives containing
[2] M.H. Chen, Z. Chen, B.A. Song, et al., Synthesis and antiviral activities of chiral a chalcone moiety as potential activator for plant resistance induction, J. Agric.
thiourea derivatives containing an R-aminophosphonatemoiety, J. Agric. Food Food Chem. 65 (2017) 4367−4377.
Chem. 57 (2009) 1383–1388. [28] W. Yang, X.N. Xu, Y. Li, et al., Rutin-mediated priming of plant resistance to three
[3] M. Reichman, Y. Devash, R.J. Suhadolnik, et al., Human leukocyte interferon and bacterial pathogens initiating the early SA signal pathway, PLoS One 11 (2016)
the antiviral factor (AVF) from virus-infected plants stimulate plant tissues to (e0146910/1–e0146910/15).
produce nucleotides with antiviral activity, Virology 128 (1983) 240–244. [29] Y. Yan, J.X. Zhang, X.J. Hao, et al., Seco-pregnane steroidal glycosides from the
[4] Y. Kim, E. Komoda, M. Miyashita, et al., Continuous stimulation of the plant im- roots of Cynanchum atratum and their anti-TMV activity, Fitoterapia 97 (2014)
mune system by the peptide elicitor PIP-1 is required for phytoalexin biosynthesis 50–63.
in tobacco cells, J. Agric. Food Chem. 62 (2014) 5781–5788. [30] Y. Yan, C.M. Yuan, X.J. Hao, et al., Limonoids from Munronia henryi and their anti-
[5] F.P. Silverman, P.D. Petracek, C.M. Fledderman, et al., Salicylate activity. 2. tobacco mosaic virus activity, Fitoterapia 107 (2015) 29–35.
Potentiation of atrazine, J. Agric. Food Chem. 53 (2005) 9769–9774. [31] Y. Yan, J.X. Zhang, X.J. Hao, et al., Bioactive limonoid constituents of Munronia
[6] F.P. Silverman, P.D. Petracek, D.F. Heiman, et al., Salicylate activity. 3. Structure henryi, J. Nat. Prod. 78 (2015) 811–821.
relationship to systemic acquired resistance, J. Agric. Food Chem. 53 (2005) [32] M.E. Hilf, W.O. Dawson, The tobamovirus capsid protein functions as a host-specific
9775–9780. determinant of long-distance movement, Virology 193 (1993) 106–114.
[7] Q.S. Du, W.P. Zhu, Y.F. Xu, et al., Novel benzo-1,2,3-thiadiazole-7-carboxylate [33] H.T. Fan, B.A. Song, S. Yang, et al., Antiviral activity and mechanism of action of
derivatives as plant activators and the development of their agricultural applica- novel thiourea containing chiral phosphonate on tobacco mosaic virus, Int. J. Mol.
tions, J. Agric. Food Chem. 60 (2012) 346–353. Sci. 12 (2011) 4522–4535.
[8] Z.J. Fan, Z.G. Shi, H.K. Zhang, et al., Synthesis and biological activity evaluation of [34] K.J. Dietz, I. Turkan, A. Krieger-Liszkay, Redox- and reactive oxygen species-de-
1,2,3-thiadiazole derivatives as potential elicitors with highly systemic acquired pendent signaling into and out of the photosynthesizing chloroplast, Plant Physiol.
resistance, J. Agric. Food Chem. 57 (2009) 4279–4286. 171 (2016) 1541–1550.
[9] H. Kauss, K. Krause, W. Jeblick, Methyl jasmonate conditions parsley suspension [35] R.L. Nicholson, R. Hammerschmidt, Phenolic compounds and their role in disease
cells for increased elicitation of phenylpropanoid defense responses, Biochem. resistance, Annu. Rev. Phytopathol. 30 (1992) 369–389.
Biophys. Res. Commun. 189 (1992) 304–308. [36] F. Gozzo, Systemic acquired resistance in crop protection: from nature to a chemical
[10] X.H. Qian, P.W. Lee, S. Cao, China: forward to the green pesticides via a basic approach, J. Agric. Food Chem. 51 (2003) 4487–4503.
research program, J. Agric. Food Chem. 58 (2010) 2613–2623. [37] L.H. Zhao, J.H. Dong, H.R. Chen, et al., Anti-TMV activity and functional me-
[11] J.N. Seiber, Sustainability and agricultural and food chemistry, J. Agric. Food chanisms of two sesquiterpenoids isolated from Tithonia diversifolia, Pestic.
Chem. 59 (2011) 1–21. Biochem. Physiol. 140 (2017) 24–29.
[12] Y.M. Li, L.H. Wang, X.J. Hao, et al., Seco-pregnane steroids target the subgenomic [38] J. Malamy, J.P. Carr, D.F. Klessig, et al., Salicylic acid: a likely endogenous signal in
RNA of alphaviruslike RNA viruses, Proc. Natl. Acad. Sci. U. S. A. 104 (2007) the resistance response of tobacco to viral infection, Science 250 (1990)
8083–8088. 1002–1004.
[13] Y.M. Li, Y.T. Jia, X.J. Hao, et al., Purification and characterization of a new ribo- [39] P. Keller, B.E. Galiana, L. Pruvot, et al., Salicylic acid mediates elicitin-induced
some inactivating protein from cinchonaglycoside C-treated tobacco leaves, J. systemic acquired resistance, but not Nectosisin tobacco, Mol. Plant Microbe
Integr. Plant Biol. 49 (2007) 1327–1333. Ineract. 9 (1996) 696–703.
[14] Y.H. Wang, Z.K. Zhang, X.J. Hao, et al., Benzyl phenethylamine alkaloids from [40] K. Hahlbrock, D. Scheel, Physiology and molecular bi-ology of phenylpropanoid
Hosta plantaginea with inhibitory activity against tobacco mosaic virus and acet- metabolism, Annu. Rev. Plant Physiol. Plant Mol. Biol. 40 (1989) 347–369.
ylcholinesterase, J. Nat. Prod. 70 (2007) 1458–1461. [41] J.B. Rusmussen, R. Hammersschmidt, M.N. Zook, Systemic induction of SA accu-
[15] Y.M. Li, Z.K. Zhang, X.J. Hao, et al., 3-Acetonyl-3-hydroxyoxindole: a new inducer mulation in cucumber after inoculation with pseudomonas Syringae pv. Syringae,
of systemic acquired resistance in plants, Plant Biotechnol. J. 6 (2008) 301–308. Plant Physiol. 97 (1991) 1342–1347.
[16] J. Chen, X.H. Yan, X.J. Hao, et al., Tobacco mosaic virus (TMV) inhibitors from
Picrasma quassioides Benn, J. Agric. Food Chem. 57 (2009) 6590–6595.

18