Synthesis of N-Methylmorpholinium Derivatives Possessing a 1,3,4-Oxadiazole Core as Feasible Antibacterial Agents against Plant Bacterial Diseases

To develop a kind of quaternary ammonium compounds that can safely apply in agriculture for managing the plant bacterial diseases, herein, a series of N-methylmorpholinium derivatives possessing a classical 1,3,4-oxadiazole core were prepared and the antibacterial activities both in vitro and in vivo were screened. Bioassay results revealed that compounds 3l and 3i showed the strongest antibacterial activity toward pathogens Xanthomonas oryzae pv. oryzae and X. axonopodis pv. citri with the lowest EC50 values of 1.40 and 0.90 μg/mL, respectively. Phytotoxicity test trials indicated that target compounds bearing a bulky Nmethylmorpholinium pendant are safe for plants. +e following in vivo bioassays showed that compound 3l could control the rice bacterial blight disease, thereby affording good control efficiencies of 55.95% (curative activity) and 53.09% (protective activity) at the dose of 200 μg/mL. Preliminary antibacterial mechanism studies suggested that target compounds had strong interactions with the cell membrane of bacteria via scanning electron microscopy imaging. Additionally, this kind of framework also displayed certain antifungal activity toward Fusarium oxysporum and Phytophthora cinnamomi. Given the above privileged characteristics, this kind of 1,3,4-oxadiazole-tailored N-methylmorpholinium derivatives could stimulate the design of safe quaternary ammonium bactericides for controlling plant bacterial diseases.


Introduction
Phytopathogens are a group of aggressive microorganisms that can invade all sorts of plants for nutrient competition and/or self-reproduction, thereby seriously threatening the quality and yield of agricultural products [1][2][3][4]. e representative notorious bacterial strains, namely, Xanthomonas oryzae pv. oryzae (Xoo) and X. axonopodis pv. citri (Xac), are the widely distributed pathogens worldwide and can lead to the development of severe diseases on major crops, exemplified by the bacterial leaf blight on rice and canker on citrus [5][6][7][8][9]. As for the bacterial blight disease, it can occur in all stages of rice growth, thereby reducing approximately 10 to 50 percent rice production annually in rice-growing countries [10][11][12][13][14]. is outcome has strongly strained the demand for food supply. Currently, the long-term use and/ or abuse of traditional bactericides have contributed to the development of immeasurable microorganic resistance and ineffective control efficiency toward plant infections [15][16][17][18][19]. erefore, novel and safe frameworks with highly efficient bioactivity should be urgently excavated and developed [20][21][22][23].
In the development of bactericides, quaternary ammonium compounds possessing the typical positive charge on the nitrogen atom have been intensively highlighted in consideration of their versatile biological profiles, especially in the antibacterial and antifungal aspects [24][25][26][27][28][29][30]. Investigations found that these kinds of quaternary ammonium salts (QASs) displayed strong interactions with the cell membrane of microorganisms, thereby leading to cell membrane destruction and finally bacterial death [31][32][33][34][35][36][37][38]. Given this feature, many QASs were commercialized and exploited to sterilize medical instruments, hospital protective clothing, medical implants, wound dressings, food packaging materials, and daily consumer goods [39]. Although this kind of substrates have many prominent properties, most of which are only used in industries. To date, rare QASs are applied in agriculture due to the possible phytotoxicity. us, exploration and development of a kind of quaternary ammonium compounds that can safely apply in agriculture for managing the plant bacterial diseases is highly pursued and approved.
On the other hand, morpholinium salts, owning a large steric hindrance scaffold, as a member of QASs, were used in medicine and pesticides ( Figure 1). e representative structure of N,N-dimethylmorpholine chloride was used as a type of plant growth regulator that can increase the yield of cotton [40][41][42]. Another morpholinium salt, namely, pinaverium bromide has been applied as a gastrointestinal crystallizer that can inhibit the flow of calcium ions into intestinal smooth muscle cells. Based on this, the associated pinaverium bromide tablets are safely used for intestinalrelated pain [43][44][45]. Meanwhile, novel biological ingredients possessing the typical morpholinium moiety were constantly prepared and evaluated. For instance, Bakhite et al. screened the insecticidal activity of a kind of morpholinium derivatives and found that compound 1 showed good insecticidal activities against nymphs of cowpea aphid, which was superior to that of the insecticide acetamiprid [46]. Yang et al. prepared the morpholinium-tailored substrate soyaethyl morpholinium ethosulfate (SME) that could self-assemble into micelles with excellent antibacterial effects toward both the Staphylococcus aureus and methicillin-resistant S. aureus. Additionally, SME presented a relatively low toxicity to mammalian cells [47]. Given the unique superiority of this morpholinium scaffold, it should be introduced into the final target framework for the development of safe agricultural chemicals.
In our previous work, we developed and evaluated the antibacterial functions of an array of pyridinium-tailored compounds and found that they showed excellent antibacterial activities but high phytotoxicity toward rice leaves [15,48]. To sequentially develop a kind of safe quaternary ammonium compounds that can safely apply in agriculture for managing the plant bacterial diseases, herein, the morpholinium scaffold was used to replace the planar pyridinium group to yield 1,3,4-oxadiazole-tailored Nmethylmorpholinium derivatives. e following in vitro and in vivo bioassays against Xoo and Xac and the relevant phytotoxicity test were evaluated. Simultaneously, the antifungal activity toward Fusarium oxysporum (F. oxysporum) and Phytophthora cinnamomi (P. cinnamomi) was also screened.

Instruments and Chemicals.
e NMR spectra of synthesized compounds were measured using the Bruker Biospin-AG-500 apparatus (BRUKEROPTICS, Switzerland). DMSO and TMS were used as the solvent and internal standard, respectively. All chemicals were purchased from Energy Chemical and used without further purification. All solvents meet the standard of analytical purity. e reaction process was monitored by using thin-layer chromatography.

In Vitro Antibacterial Bioassays.
e antibacterial activity against pathogens Xoo and Xac was evaluated by the turbidimeter test [3,22,48]. All the synthesized compounds were tested under different concentrations (such as 50.0, 25.0, 12.5, 6.25, 3.125, and 1.5625 μg/mL). Bismerthiazol and thiadiazole copper served as positive controls, whereas dimethyl sulfoxide (DMSO) in sterile distilled water was the blank control. Absorbing 40 μL bacteria solution (OD 595 � 0.8, logarithmic phase of growth) was added into a 5 mL nutrient broth medium (components: 3 g beef extract, 5 g peptone, 1 g yeast powder, 10 g glucose, 18 g agar in 1 L of distilled water, pH 7.0-7.2) containing different dosages of target compounds and controls. en, these samples were incubated in a constant shaker (180 rpm) at 28 ± 1°C for about 24-48 h until the DMSO control reached the logarithmic growth phase. After that, 200 μL samples were tested the optical density at 595 nm (OD 595 ) through a microplate reader.
e turbidity corrected values � OD bacterial wilt − OD no bacterial wilt , and the inhibition rate I was calculated by I � (C − T)/C × 100%. C is the corrected turbidity value of bacterial growth on untreated NB (blank control), and T is the corrected turbidity value of bacterial growth on treated NB. By using SPSS 17.0 software and the obtained inhibition rates at different concentrations, a regression equation was provided. e results of antibacterial activities (expressed by EC 50 ) against Xoo and Xac were calculated from the equation. e experiment was repeated three times.

In Vivo Bioassays against Rice Bacterial Blight.
Compound 3l, thiadiazole copper (TC, 20% suspending agent) as the positive control, and an equivalent DMSO as a blank control in sterile distilled water were used to test the bioactivity against rice bacterial blight [22]. e rice variety "Fengyouxiangzhan" was planted for about 8 weeks before use. For the curative activity, an aseptic scissor dipped with Xoo cells was used to infect rice leaves. One day later, compound 3l and TC with a concentration of 200 μg/mL were evenly sprayed on leaves. Meanwhile, the drug-free solution was used for the negative group. All the treated plants were cultured in the constant temperature (28°C) and humidity (90% RH) incubator for 14 days before testing the disease index. For the protective effects, the difference was that the drug solution with the same dosage was firstly sprayed before inoculation with Xoo cells. e related disease index could be obtained after 14 days. e corresponding control efficiencies I were provided according to the formula: I � (CK − T)/ CK × 100%. CK and T represent the disease index of negative and drug-treated controls, respectively.

Scanning Electron Microscopy (SEM) Imaging.
Scanning electron microscopy (SEM) imaging of Xoo cells triggered by compound 3l. 1.5 mL Xoo cells incubated at the logarithmic phase were centrifuged and washed with PBS (pH � 7.2) and resuspended in 1.5 mL of PBS buffer (pH � 7.2). en, these Xoo cells were incubated with compound 3l at a dose of 14.0 μg/mL and an equivalent volume of DMSO (solvent control) for 8 h in a shaker under 180 rpm at 28°C. After incubation, these samples were washed 3 times with PBS (pH � 7.2). Subsequently, the bacterial cells were fixed for 8 h at 4°C with 2.5% glutaraldehyde and then dehydrated with graded ethanol series and pure tert-butanol (2 times with 10 min/time). Following dehydration, samples were freeze-dried and coated with gold and visualized using Nova Nano SEM 450 [3,22].

2.5.
In Vitro Antifungal Bioassay against F. oxysporum and P. cinnamomi. All target molecules were dissolved in DMSO (1.0 mL) and then added into 9.0 mL sterilized water containing Tween 20 (volume ratio 0.1%) before mixing with potato dextrose agar (PDA, 90.0 mL). ese compounds were tested at a concentration of 25 μg/mL. e stock solution was transferred into three 9 cm diameter Petri dishes evenly.
en, mycelia dishes of approximately 4 mm diameter were cut from the culture medium and inoculated in the middle of the PDA plate aseptically. e inoculated plates were incubated at 28 ± 1°C for 3-5 days. DMSO in sterile distilled water was used as the negative control, whereas hymexazol served as the positive control. Each treatment condition contained three replicates. Radial growth of the fungal colonies was measured, and the data were statistically analyzed. Inhibitory effects toward these fungi were calculated by the formula I � [(C − T)/(C − 0.4)] × 100%, where C represents the diameter of fungal growth on untreated PDA, T represents the diameter of fungi on treated PDA, and I represents the inhibition rate [49].

General Procedures for Preparing Intermediate 2.
Intermediate 1 was prepared by following our previous works [48,49]. en, intermediate 1 (1 mmol) was dissolved in 8 mL DMF containing K 2 CO 3 (1.3 mmol) and 1,8dibromooctane, 1,10-dibromodecane, or 1,12-dibromododecane (1.3 mmol). After stirring the mixture for 2 h at 25°C, the organic matter was extracted with EtOAc, washed twice with purified water, and dried with anhydrous sodium sulfate. After that, the solvent was removed under a reduced pressure. Finally, intermediate 2 was obtained by silica gel column chromatography using petroleum ether/EtOAc (10/ 1, V/V) as the eluent.

Phytotoxicity Study, In Vivo Antibacterial Activity, and
Antibacterial Mechanism. Phytotoxicity test trials at 200 µg/ mL showed that the bioactive compound 3l gave a lower phytotoxicity against rice leaves (Figure 3), indicating that this kind of compounds bearing a bulky N-methylmorpholinium pendant are safe for rice plants.
e following in vivo bioassays showed that compound 3l could control the rice bacterial leaf blight disease and thus providing good control efficiencies of 55.95% (curative activity) and 53.09% (protective activity) at the dose of 200 μg/mL (Table 2 and Figure 4). ose data were superior to those of TC (37.53% and 36.68%), suggesting that this kind of 1,3,4oxadiazole-tailored N-methylmorpholinium derivatives can serve as safe quaternary ammonium bactericides for controlling plant bacterial diseases. e preliminary antibacterial mechanism was investigated via scanning electron microscopy imaging. As displayed in Figure 5, the morphology of Xoo cells were transformed from well shaped to corrugated and/or broken after treating Xoo with compound 3l (14 μg/mL, 10 × EC 50 ), suggesting that target compounds had strong interactions with the cell membrane of bacteria.

Antifungal Activities.
e antifungal activity against two kinds of plant fungal pathogens was screened by the mycelium growth rate method [49], while hymexazol was coassayed for comparison. Table 3 shows that some target compounds were identified with good antifungal activities at 25.0 µg/mL. Among them, the inhibition rate of compounds   is outcome manifests that this kind of 1,3,4-oxadiazole-tailored N-methylmorpholinium derivatives also can serve as antifungal leads for the future design of novel fungicides.  Negative control. b Statistical analysis was conducted using ANOVA under the condition of equal variances assumed (P > 0.05) and equal variances not assumed (P < 0.05). Different uppercase letters indicate the values of bioactivity with significant differences among different treatment groups at P < 0.05.

Conclusions
In summary, a novel type of N-methylmorpholinium derivatives bearing a typical 1,3,4-oxadiazole scaffold was prepared to explore a kind of quaternary ammonium compounds that could safely apply in agriculture for controlling the plant bacterial diseases. In vitro bioassay screening results showed that compounds 3l and 3i displayed the best antibacterial activity toward pathogens Xoo and Xac with the minimal EC 50 values of 1.40 and 0.90 μg/mL, respectively. Interestingly, this kind of Nmethylmorpholinium salts gave the lower phytotoxicity toward rice plants. In vivo bioassays revealed that the bioactive compound 3l could control the rice bacterial blight disease and thus providing good curative activity and protective activity of 55.95% and 53.09% at the dose of 200 μg/mL, respectively. Given the above superiority, 1,3,4-oxadiazole-tailored N-methylmorpholinium compounds could be further investigated as safe quaternary ammonium bactericides for managing plant bacterial diseases.
Data Availability e data supporting these results are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that they have no conflicts of interest.

Authors' Contributions
Xinxin Tuo contributed to investigation, data curation, formal analysis, and methodology and prepared the original draft. Jie Yang and Yedong Zhang contributed to data curation, formal analysis, and methodology. Peiyi Wang contributed to data curation, supervision, and formal analysis and reviewed and edit the manuscript.