Molecular Design and Synthesis of Novel Salicyl Glycoconjugates as Elicitors against Plant Diseases

A new series of salicyl glycoconjugates containing hydrazide and hydrazone moieties were designed and synthesized. The bioassay indicated that the novel compounds had no in vitro fungicidal activity but showed significant in vivo antifungal activity against the tested fungal pathogens. Some compounds even had superior activity than the commercial fungicides in greenhouse trial. The results of RT-PCR analysis showed that the designed salicyl glycoconjugates could induce the expression of LOX1 and Cs-AOS2, which are the specific marker genes of jasmonate signaling pathway, to trigger the plant defense resistance.


Introduction
In the past two decades, the goal of sustainable and green agriculture had been inspiring researchers to explore the feasibility of restricting toxic agrochemical usage to reduce their impact on environment and food chains. One of the alternatives, which had been studied intensively in recent years, was to make use of plant defense potentials. Induction of plant defense resistance in crops by chemical or biological elicitors had drawn increasing attentions and was considered as a prospective strategy for disease control [1,2].
During the long process of co-evolution, plants had evolved lots of defense mechanisms to deal with pests and pathogens. Following plant-pathogen interaction, a number of plant defense responses could be induced (e.g., callus deposition, PR-protein accumulation, et al.) at the site of infection, and also in uninfected tissues, activated by signal molecules associated with defense responses, which resulted in increased resistance to subsequent infections. The systemic acquired resistance is a ''whole-plant'' defense response that occurred following an earlier localized exposure to a pathogen. Activation of systemic acquired resistance required the accumulation of endogenous salicylic acid [3][4][5]. Besides the salicylic acid dependent defense signaling pathway, the others had also been reported. For example, endogenous jasmonic acid and methyl jasmonate were also the potent signaling molecules which could induce a large set of defense responses [6]. Systemic acquired resistance possessed low specificity, was not easily overcome by new pathogens which emerged frequently.
Chemical elicitors are agrochemicals which do not show a direct effect on pathogens and lacked fungicidal activity themselves but induce defense mechanisms, which clearly distinguish them from conventional pesticides [7]. Some of these agrochemicals are known to have signaling functions in planta, such as benzothiadiazole [8][9][10][11][12][13], which is a functional analog of salicylic acid, while others may mimic the attack of a pathogen, such as harpin [14] or flagellin [15].
Saccharides are known as potent elicitors [16]. The fragments of chitin and chitosan, which act as elicitors in many plants, could induce the production of nitric oxide and hydrogen peroxide in some plant epidermal cells [17][18][19][20]. Even neutral saccharides, such as b-glucans derived from cellulose or laminarin [21,22], are capable of enhancing plant resistance. The accumulation of phytoalexins could be induced by branched hexa (b-D-glucopyranosyl)-D-glucitols in soybean [23,24]. Oligoglucans with polymerization between 8 and 17 could induce the chitinase activity in tobacco BY-2 suspension cells [25,26]. The phenolic pathway could be rapidly induced by the mannose and glucose disaccharides in Rubus cells [27]. It is evident that saccharides have the ability to trigger defense responses in plants, enhance resistance toward infection, and even support plant growth [28,29].

Instruments
All the melting points were determined with a Cole-Parmer melting point apparatus (Cole-Parmer, Vernon Hills, Illinois, USA) while the thermometer was uncorrected. Optical rotation data were recorded on a KRUSS P8000 instrument (KRUSS, Karlsruhe, Germany). IR spectra were recorded on a Nicolet NEXUS-470 FTIR spectrometer (International Equipment Trading Ltd., Vernon Hills, Illinois, USA) with KBr pellets. 1 H NMR spectra were recorded with Bruker DPX300 (Bruker, Fallanden, Switzerland) and JEOL JNM-ECS400 (JEOL Ltd., Tokyo, Japan), while tetramethylsilane was used as an internal standard. Analytical thin-layer chromatography was carried out on silica gel 60 F254 plates, and spots were visualized with ultraviolet light. Elemental analysis was carried out with a Flash EA 1112 elemental analyzer (Thermo Finnigan, Bremen, Germany). Mass spectra were measured on a Bruker APEX IV spectrometer (Bruker, Fallanden, Switzerland).

Synthetic procedures
General synthetic procedure for hydrazides 2a and 2b. Preparation of hydrazides 2a and 2b: Esters 1a and 1b (30 mmol) was suspended in 100 mL methanol and reacted with 98% hydrazine monohydrate (60 mmol, 2.9 mL) under reflux for 12 h. The solid was filtered, washed with methanol and dried to afford hydrazides 2a and 2b.
2-mercaptobenzohydrazide (2a  General synthetic procedure for hydrazides 5a-d and hydrazones 7a-g. The key intermediates hydrazides 5a-d were obtained almost quantitatively by the hydrazinolysis of compounds 4a,d in alcohol. Compounds 5a,d were condensed with 5substituted phenyl-2-furfural to form the glycosyl hydrazones 7ag. All the chemical characterization was given in reference [35].

Bioassays
In vitro fungicidal activity. In vitro fungicidal activity of the salicylic glycoconjugates against Colletotrichum orbiculare, Fusarium oxysporum, Rhizoctonia solanii, and Phytophthora capsici were evaluated using mycelium growth rate test [43][44][45]. The tested compounds were dissolved in DMSO (dimethyl sulfoxide) and mixed with sterile molten potato dextrose agar to a final concentration of 50 mg/mL. In vitro fungicidal activity of the salicylic glycoconjugates against Sphaerotheca fuliginea was evaluated using colonized detached leaves method [43][44][45]. The conidial suspensions were prepared by seeding about 2610 5 spores mL 21 conidia in a 0.05% Tween 80 solution, and the DMSO solution of compounds (5000 mg/mL) was diluted with conidial suspension to a final concentration of 50 mg/mL. The solution was sprayed with a hand sprayer on the surface of the detached leaves which were inoculated with S. fuliginea. P. capsici was maintained on oat medium at 17uC. C. orbiculare, F. oxysporum, and R. solanii were maintained on potato dextrose agar medium at 4uC. Five commercial fungicides: thiophanate-methyl, benomyl, chlorothalonil, validamycin, and dimethomorph were used as controls against the above mentioned fungal pathogens under the same conditions. Three replicates were performed. The relative inhibition rate of the synthetic compounds compared to blank control was calculated via the following equation:

I~C{T ð Þ=C|100%
In which, I stands for the rate of inhibition (%), C is the diameter of mycelia in the blank control test (in mm), and T is the diameter of mycelia in the presence of tested compounds (in mm).
The culture plates were cultivated at 2461uC. Germination was conducted by soaking cucumber seeds in water for 2 h at 50uC and then keeping the seeds moist for 24 h at 28uC in an incubator. When the radicles were 0.5 cm, the seeds were grown in plastic pots containing a 1:1 (v/v) mixture of vermiculite and peat. Cucumber plants used for inoculations were at the stage of two seed leaves. Ten plants were used for each treatment.
Tested compounds were confected to 2.5% EC (emulsifiable cocentration) formulations, in which pesticide emulsifier 500 (0.375%) and pesticide emulsifier 600 (2.125%) were the additives, Novel Salicyl Glycoconjugates as Elicitors against Plant Diseases PLOS ONE | www.plosone.org DMSO (0.1%) was the solvent, and xylene was the co-solvent. The formulation was diluted to a concentration of 500 mg/mL with water. The solution was sprayed with a hand sprayer on the surface of seed leaves which were then inoculated with C. orbiculare, S. fuliginea, and R. solanii, respectively. Tested compounds and commercial fungicides were applied by irrigation at seedling stage, which were then inoculated with F. oxysporum and P. capsici, respectively. Three replicates for each treatment were applied.
Inoculations of C. orbiculare and S. fuliginea were carried out by spraying conidial suspension, and inoculation of R. solanii was carried out by spraying a mycelial suspension. F. oxysporum assay was carried out by embryo root inoculation, and P. capsici assay was carried out by irrigation inoculation.
Three replicates for each treatment were applied. After inoculation, the plants were maintained at 2461uC and above 80% relative humidity.
The fungicidal activity was evaluated when the untreated cucumber plant (blank control) fully developed symptoms. The area of inoculated leaves covered by disease symptoms was assessed and compared to that of untreated ones to determine the average disease index. The relative control efficacy of compounds compared to the blank assay was calculated via the following equation: where I is relative control efficacy, CK is the average disease index during the blank assay and PT is the average disease index after treatment during testing.
RT-PCR for detection of pathogenesis-related gene expression. Tested compounds (500 mg/mL) were sprayed with a hand sprayer on the surface of the cucumber (Cucumis sativus) seed leaves, which were collected after 24 h, 48 h, and 72 h. The leaves were treated by liquid nitrogen. RNA isolation was performed with the RNAiso Plus Kit (Takara Bio). First-strand cDNA was synthesized from 100 mg/mL total RNA, which was quantified with QuantiT RNA Assay Kit (Invitrogen), by reverse transcription using the QuantiTect Reverse Transcription Kit (QIEGEN). Gene-specific primers (Table S1 in File S1) were designed and actin was used as the housekeeping gene [47,48]. Each reaction mixture (30 mL) contained 1 mL of the cDNA template, 100 pmol of each primer, 10 mL of Premix Ex Taq HS (Takara Bio), and 20 mL reaction buffer. The thermal cycling conditions were as follows: initial denaturation (94uC, 5 min), followed by 40 cycles of denaturation (94uC, 30 s), annealing (30 s) and extension (72uC, 30 s), and one final cycle of extension (72uC, 5 min). Finally, RT-PCR products were separated by electrophoresis and visualized in 1% agarose gel.

Ethics statement
No specific permits were required for the described field studies. No specific permissions were required for these locations. We confirm that the location is not privately-owned or protected in any way. We confirm that the field studies did not involve endangered or protected species.

Synthesis
The synthetic routes of 2-mercaptobenzohydrazide 2a, 2hydroxybenzohydrazide 2b and glycosyl hydrazides 5a-d were shown in Figure 1. The hydrazides 5a-d were obtained almost quantitatively by hydrazinolysis of the esters 4a-d in alcohol.  Finally, the hydrazides 5a-d were reacted with aldehyde 6 by condensation to form the glycosyl hydrazones 7a-g.

Fungicidal activity
The in vitro fungicidal results were shown in Table 1. The hydrazides 2a and 2b showed excellent activity against the tested fungi (Figures 2 and 3). For example, the inhibitory rates of the hydrazides 2a and 2b against C. orbiculare were 97.3% and 95.4%, which were better than thiophanate-methyl (91.0%). After modification of sugars, the in vitro activity of all the derivatives was decreased and they exhibited poor inhibitory rates. Although the in vitro activity of these glycosides was not encouraging, the in vivo tests gave promising results (Table 2), with all the carbohydrate derivatives showing considerable activity, especially against F. oxysporum (Table 2), C. orbiculare (Figure 4), and S. fuliginea ( Figure 5). Among them, hydrazide 5d and hydrazone 7f had activity of 71.0% and 74.9% on F. oxysporum, respectively, which is similar to the control benomyl (74.5%) against the same pathogen. 5d also showed good activity of 68.6% and 73.9% against C. orbiculare and S. fuliginea, respectively. Some hydrazones 7 exhibited promising activity against P. capsici. For examples, 7c showed an inhibitory rate of 83.5%, and the inhibitory rates of 7a, 7d and 7e were more than 75%.
The bioassay results showed that the tested compounds had in vivo antifungal activity against pathogenic fungi of Ascomycota (C. orbiculare, F. oxysporum and S. fuliginea), Basidiomycota (R. solanii), and Oomycete (P. capsici). The observed in vivo antifungal activity also had some association with the issue of pathogen biology. The tested compounds exhibited activity not only against the obligatory parasite pathogen (S. fuliginea), but also against the facultative parasite pathogens (C. orbiculare, F. oxysporum, R. solanii and P. capsici). The tested compounds also showed good activity against the soil-borne fungal disease (F. oxysporum, R. solanii and P. capsici). Also, we confirmed that all of these test compounds were safe for the host plants.

Defense activity of designed compound in plant
There are two important defense signaling pathways in plant system. One is mediated by salicylic acid and the other is mediated by jasmonic acid. In each defense pathway, there are specific marker genes which expression could be influenced by corresponding signaling molecules. In order to unveil the mode of action of our designed compounds, RT-PCR was performed to check the expression patterns of pathogenesis-related genes (PR1a, PR8, LOX1, Cs-AOS2) ( Figure 6). Among them, PR1a and PR8 were the specific marker genes mediated by salicylic acid, whereas LOX1 and Cs-AOS2 were the specific marker genes mediated by jasmonic acid. Our results showed that expressions of the LOX1 and Cs-AOS2 genes were significantly induced by hydrazide 5d, and the expression level was comparable with that mediated by BTH (S-methyl benzo [1,2,3]thiadiazole-7-carbothioate). However, hydrazide 5d had no obvious effect on the expressions of PR1a and PR8.

Conclusions
A new series of glycosyl hydrazines and hydrozone derivatives were designed and synthesized. Their antifungal tests indicated that most of the salicylic glycoconjugates had no in vitro fungicidal activity but showed considerable in vivo antifungal activity. The plant defense activity showed that expressions of the LOX1 and Cs-AOS2 genes were significantly induced by hydrazide 5d, but the compound had no effect on the expressions of PR1a and PR8. Intriguingly, although the designed compounds were the derivatives of salicylic acid, they did not mimic the mode of action of salicylic acid, but seem to follow the jasmonic acid mediated pathway to induce the plant defense resistance.