1. Introduction
As a kind of soil worm, plant-parasitic nematode (PPN) is one of the global pathogens affecting agricultural production and causing economic losses of up to USD 157 billion annually [
1]. Its species are diverse, among which root-knot nematodes (RKNs; i.e.,
Meloidogyne spp.) are the most ubiquitous and notorious [
2]. RKN can infect over 2000 plant species [
3], especially in fruit and vegetable crop. The control of these pests mainly depends on chemical nematicides. However, nematicide products account for only 2.5% of the global pesticides. The proportion is low compared to the great losses due to PPN [
4]. More importantly, many chemical nematicides have been banned or restricted due to safety or environmental problems. Therefore, it is urgent to develop novel and eco-friendly nematicides.
Currently, biocontrol agents based on the biomolecules themselves or their derivatives have received much attention. These agents pose less risk to humans and animals than their synthetic predecessor did, have a selective mode of action, and avoid the emergence of resistant races of pest species [
5]. Therefore, they can be applied to integrated pest management (IPM) programs, which tend to use natural or green methods to control crop diseases below the threshold. Chitosan oligosaccharide (COS) is an alternative biological macromolecule worthy of deep research in pest control.
COS, containing 2–20 D-glucosamine units, is produced by the degradation of chitin or chitosan, which is the only cationic compound found in nature. Due to its excellent biocompatibility and unique physiological and biological activity, COS has been widely used as a biostimulant, antifungal agent, seed treatment agent, soil conditioner, and fertilizer in agriculture. As biostimulants, COS can enhance the crop disease resistance by stimulating the secretion of immune enzymes and compounds (such as salicylic acid and jasmonic acid) [
6], cell wall reinforcement [
7], production of reactive oxygen species [
8], and hypersensitive response-mediated cell death [
7]. These immune responses are also effective against RKN [
9]. Based on this, being endowed with nematicidal activity, COS can act on both nematodes and hosts, thus exerting a desirable control effect in IPM. However, there are few studies on nematicidal COS.
Modification is an effective way to improve the application of natural polymer compounds, including chitin and chitosan [
10]. COS can obtain ideal nematicidal activity by grafting active groups. Fluoroalkenyl groups are a kind of active group that are capable of selection. Compounds obtained by combining fluoroalkenyl groups with thiazole, oxazole, or other heterocyclic structures can perform a high nematicidal activity [
11,
12]. Meanwhile, the fluoroalkenyl compounds also possess lower toxicity to vertebrates than organophosphate- or carbamate-based nematicides [
12]. However, these compounds have a risk of phytotoxicity [
13], a defect that is expected to be avoidable by combination with COS. In the previous study, we grafted the structure of trifluoroethylene and thiadiazole onto the nitrogen position of COS to obtain a derivative with high nematicidal activity and low phytotoxicity [
14], which confirmed the feasibility of fluoroalkenyl COS derivative on nematode control. However, the effect of derivatization based on other microstructures or grafting sites on activity and the action mechanism needs to be studied.
In this paper, one 6-O-(trifluoroebutylene-oxadiazol)-COS derivative was synthesized. Meanwhile, its control effect on Meloidogyne incognita was comprehensively evaluated by analyzing the hatching rate of eggs and mortality, and the mechanism of action was explored by analyzing the morphology of second-stage juveniles (J2s) and plant resistance. Finally, the phytotoxicity was estimated in vitro. This study was to offer novel fluoroalkenyl COS derivatives and more data support on nematode control by chitin biomacromolecule modification.
3. Discussion
Plant, nematode, and soil environment interact to form a complete ecosystem. Strengthening infection barriers, reducing the number of living bodies, and changing survival environments are efficient ways to control nematode diseases. The combination of the three would play a more efficient nature control function, which conforms to the strategy of IPM. COS derivative may control nematode from the above three pathways at the same time. Chitin or chitosan can reduce the nematode reproduction factor and its population in the soil by mediated chitinolytic microorganisms [
18,
19,
20]. COS and its derivatives may possess similar efficiency. However, as the soil microenvironment is complex, it will not be discussed here.
Grafting exogenous active groups can undoubtedly improve the nematicidal activity of COS, however, there is no report on how derivatives affect the physiology of nematodes. In this study, derivative
6 did not significantly influence the cuticle, therefore, it was likely to be swallowed by nematodes into the intestine to work. The intestinal autofluorescence in nematode is caused by lipofuscin, a marker of aging and oxidative stress induced by reactive oxygen species (ROS) [
21]. Exposure to toxins can increase ROS levels, leading to lipid peroxidation, which eventually produces lipofuscin. The lipofuscin will deposit on the cells and accelerate aging [
22]. In this study, autofluorescence of nematode was significantly enhanced after being treated (
Figure 4D), indicating that the derivative
6 may lead to lipofuscin accumulation and accelerate aging by affecting ROS metabolism. ROS also change the architecture of cell membranes and damage tissue and cellular components [
16]. In this study, ROS may be involved in the intestinal cell damage accompanied by aging, including nuclear depolymerization, cell shrink and rupture, and lipid vacuoles forming et ac.
The inducible activity of derivatives may attribute to the COS skeleton. As an elicitor of plant innate immunity, COS can trigger plant responses against fungal and bacterial infection, including Ca
2+ spiking, NO and ROS accumulation, activation of the MAPK cascade, upregulation of defense gene expression, activation of the SA and JA-mediated signaling pathways, callose deposition, and molecular flux via plasmodesmata [
23,
24]. Although nematode and fungi or bacteria are completely different organisms in both lifestyle and infection modes, the immune responses of plants to the two diseases are similar. For example, ROS plays a major role in plant-nematode interactions. ROS stimulates genes of the phenylpropanoid pathway and enhances the synthesis of phenols by L-Phenylalanine ammonla-lyase [
8], resulting in formatting lignin and suberin that strengthen the physical barrier of the cell wall [
25]. ROS accumulation can lead to defense-related cell death or a hypersensitive response, which helps to isolate the pest [
26]. ROS also amplify and propagate intra- and intercellular defense signals, activating the long-lasting resistance in non-infected tissues as well (termed systemic acquired resistance) [
27]. Besides, ROS largely relies on the accumulation of salicylic acid (SA) at the site of infection [
27]. SA or jasmonic acid, which mediate systemic acquired resistance, lead to the induction of pathogenesis-related proteins, which are important signaling molecules in plant immunity [
28]. Boosting the SA pathway, which was stimulated by the pathogen or exogenous inducer, might play a defensive role against newly hatched J2s during the early stages of nematode invasion [
29]. In this study, the target derivative improved SOD activity, an antioxidant enzyme with an elevation in the level of ROS. Therefore, the above immune substance may be involved in the resistance of cucumber to
M. incognita improved by derivative
6.
Usually, prevention is the main control method of root-knot nematodes, because once nematode larvae invade plant tissues, ordinary nematicides will find it difficult to produce a marked control effect as an in vitro assay. Therefore, the derivative with nematicidal activity and inducing activity will have a greater advantage in dealing with crops in the disease stage. Enhancing resistance and improving growth will weaken the damage caused by nematode invasion and slow down the treatment threshold in IPM. In addition, compared with fluoroalkenyl compounds, the derivative avoided the risk of phytotoxicity and will have safer application methods.
4. Experimental
4.1. Materials and Methods
Chitosan oligosaccharide (Mw < 3000 Da, DD > 85%) was purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). The 4-Bromo-1,1,2-trifluoro-1-butene (98%) (BTF) was purchased from Aladdin Reagent (Shanghai, China) Co., Ltd. Hydrazine hydrate (80%), Hydrochloric acid, and other analytical reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). SOD activity kit was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).
FTIR spectra were performed ranging from 4000 cm−1 to 400 cm−1 using a Thermo Scientific Nicolet iS10 FT-IR spectrometer (Waltham, MA, USA). The 1H NMR, 13C NMR, and 19F NMR were recorded with a JEOL JNM-ECP600 spectrometer (Tokyo, Japan), using D2O as solvents. A METTLER TGA-DSC 1 SF/1382 (Zurich, Swiss Confederation) was used to record the TG/DTG curves of the polymers from 25 to 500 °C in nitrogen, and the heating rate was 10 °C/min.
4.2. Synthesis of COS Derivatives
Synthesis of N-(4-methoxybenzylidene) chitosan oligosaccharide derivative (COSMBA).
In order to ensure the water solubility of the target derivative, the amino group of COS needs to be protected with Schiff base (
Scheme 1). Briefly, COS (10 g) and sodium hydroxide (4 g) were dissolved in distilled water (100 mL), then p-methoxybenzaldehyde (9.1 mL) was added to react for 10 h at room temperature. After filtration, the filter cake was washed with distilled water and ethanol successively and dried to obtain COSMBA. The yield was 82.5%.
Synthesis of 6-(2-ethoxy-2-oxoethoxy)-N-(4-methoxybenzylidene) chitosan oligosaccharide derivative (1).
For product derivative 1, COSMBA (6 g) and pyridine (4 mL) were mixed in N, N-Dimethylformamide (DMF) (40 mL), then ethyl chloroacetate/DMF solution (6.75/10 mL) was dripped in an ice bath. After reaction for 12 h at room temperature, acetone was added to the mixture to precipitate. The sediment was washed with absolute ethanol and dried at 60 °C to obtain a tawny solid. The yield was 62.5%.
Synthesis of 6-(2-hydrazinyl-2-oxoethoxy)-N-(4-methoxybenzylidene) chitosan oligosaccharide derivative (2).
To produce derivative 2, derivative 1 (5.9 g) and hydrazine hydrate (6 mL) were added in methanol (50 mL) and refluxed for 10 h. After filtration, the filter cake was washed with absolute ethanol and dried at 60 °C, and a yellow-white solid was obtained. The yield was 47.1%.
Synthesis of 6-((5-mercapto-1,3,4-oxadiazol-2-yl)methoxy) chitosan oligosaccharide derivative (3).
A mixture of derivative 2 (2 g), sodium hydroxide (0.6 g), and carbon disulfide (1.6 mL) in ethanol (20 mL) was heated to 60 °C for 7 h. After the reaction, the ethanol was removed by vacuum distillation. The residue was dissolved in water, adjusted to pH 3–4 with 3% hydrochloric acid, and stirred for 1 h. Derivative 3 was obtained by concentrating, adding ethanol to precipitate, filtering, washing filter cake with anhydrous ethanol, and drying at 60 °C. The yield was 85.0%.
Synthesis of 6-((5-mercapto-1,3,4-oxadiazol-2-yl) methoxy)-N-(4-methoxybenzylidene) chitosan oligosaccharide derivative (4).
The synthesis of derivative 4 was similar to COSMBA. Briefly, derivative 3 (1.6 g) and sodium hydroxide (0.8 g) were dissolved in distilled water (20 mL), then p-methoxybenzaldehyde (1.45 mL) was added to react for 10 h at room temperature. After filtration, the filter cake was washed with distilled water and ethanol successively and dried to obtain the Schiff base derivative. The yield was 93.1%.
Synthesis of 6- ((5-((3,4,4-trifluorobut-3-en-1-yl) thio)-1,3,4-oxadiazol-2-yl)methoxy) -N-(4-methoxybenzylidene) chitosan oligosaccharide derivative (5).
Derivative 4 (1.3 g), pyridine (2 mL), and BTF (1.12 mL) with 1.1 times molar mass were added in DMF (20 mL) successively and raised to 70 °C for reaction overnight. After filtering at room temperature, the filter cake was washed with anhydrous ethanol and dried to obtain a yellowish-brown solid. The yield was 31.7%.
Synthesis of 6- ((5-((3,4,4-trifluorobut-3-en-1-yl) thio)-1,3,4-oxadiazol-2-yl)methoxy) chitosan oligosaccharide derivative (6).
A mixture of derivative 5 (0.4 g) with acetone (10 mL) was adjusted to have a pH of 3–4 by diluted hydrochloric acid (3 M) and stirred for 1 h. Finally, the precipitate was collected, washed with ethanol, and dried to obtain the target derivative. The yield was 60.6%.
4.3. Nematicidal Assay In Vitro
4.3.1. Nematode Populations Collection
M. incognita eggs were collected from infected tomato roots cultured in the laboratory. Briefly, the roots were cut into 1 cm-long pieces and shaken in 0.6% (w/v) sodium hypochlorite solution for 4 min. The segments were successively sieved by 200 mesh and 500 mesh screens, then washed with plenty of water. The eggs were collected with sterile water and the suspension was prepared (2500 eggs/mL). For obtaining J2s, the eggs were put into Baermann funnels to hatch for 3–5 days at 28 °C. J2s collected at 1–2 days were eliminated. The eggs and J2s were observed with a stereo-microscope, optical microscope, and fluorescence microscope (excitation: 488 nm, emission: 510 nm) as required.
4.3.2. Egg Hatching Assay
Egg suspension (40 μL) was placed into a 48-well culture plate, and the number of eggs was counted. Then, 200 μL of derivative solution (2, 1, 0.5, 0.25, 0.125 mg/mL) was added into each well. The plates were placed in the dark environment at 25 °C and J2 numbers were counted at 7th day. Sterile water was used as blank control, fluensulfone and avermectin (Av) in 1% Tween20 solution (0.01 and 0.025 mg/mL) were used as positive controls, and each experiment was repeated three times. The hatching inhibition activity is expressed by the corrected hatching inhibition rate (CHI):
4.3.3. Nematicidal Activity
The evaluated method of nematicidal activity was similar to the egg hatching assay. Briefly, 40 μL of J2s suspension and 200 μL of derivative solution (2, 1, 0.5, 0.25, 0.125 mg/mL) were added into 48-well culture plate. After being cultured for 48 h, the number of dead J2s (rigidity nematode) was counted. In this experiment, fosthiazate (Ft) and fluensulfone were used as positive controls, and each experiment was repeated three times. The nematicidal activity was evaluated by CM, which was calculated as the followed equation:
4.4. In Vivo Control Effect Assay
The control effect of COS derivative on M. incognita was estimated by the greenhouse test tube method. Briefly, the river sand was sifted by a 20-mesh sieve, sterilized, then placed in a glass tube (2 × 10 cm). Next, cucumber seeds that had had their germination accelerated were dibbled and then placed in a light incubator. The culture condition was 12 h, 26 °C, and light intensity 15,000 Lux in the daytime; 12 h, 22 °C at night; the humidity was 70–80%. After seven days, 0.5 mL of sample solution (1.0 and 0.5 mg/mL) and fluensulfone solution (0.025 mg/mL) were applied. On the next day, 100 eggs were inoculated. There were left to continue to culture for 15 days, and the root knots were counted in water for disease classification (DC). In this assay, the water was blank control, and every experiment was replicated three times. The DC standard was described as follows:
Level 0: no visible root knot. Level 1, level 2, level 3, and level 4 represent root caking rates of 1–25%, 26–50%, 51–75%, and 76–100%, respectively.
The effect of derivative on root-knot nematode disease was estimated by control effect rate (CE), which was calculated as the following formula:
4.5. Scanning Electron Microscopy and Transmission Electron Microscopy
For SEM, 1000 J2s were immersed in 1 mg/mL of derivative 6 solutions overnight, then collected by centrifugation (5000 rpm/min, 4 min). After washing with water three times, the J2s were fixed with 2.5% glutaraldehyde at 4 °C overnight. Next, the J2s were washed with 0.1 M of pH 7.0 phosphate buffer, dehydrated in an ethanol gradient (30–100% ethanol; v/v), and sprayed with gold ion sputter for SEM using a Hitachi SU8020 field-emission scanning electron microscope (Hitachi, Japan).
For TEM, J2s were fixed with 2.5% glutaraldehyde overnight and 1% osmic acid for 1–2 h, respectively. Then, the specimens were dehydrated in an ethanol gradient (30–100% ethanol; v/v) for 15 min and acetone for 20 min, and treated with different ratios of resin/acetone solution (1:1 for 1 h, 3:1 for 3 h, and 1:0 at 70 °C overnight; v/v). The embedded materials were cut in Leica EM UC7 Ultramicrotome to obtain 70–90 nm slices. The slices were stained with uranyl acetate and lead citrate staining solution for 5 min, respectively, and then imaged with a JEM-1200ex transmission electron microscope (JEOL, Japan).
4.6. Induced Resistance Assay
The germinating cucumber seeds were soaked in 0.2 mg/mL of derivative
6 solutions and distilled water until the radicle grew to 1 cm long (about one night). Then, two seeds of two groups were washed with water and placed symmetrically on the edge of a φ9 cm petri dish covered with 5 mL of Pluronic F-127 gel (23%) at room temperature, respectively. The gel was prepared according to the method described by Zhan et al. [
30]. Next, 100 J2s were placed in the center of the dish and incubated in a dark environment for 48 h. The seedlings of the two groups were washed with distilled water and cultured in a humid environment for 7 days. Finally, the average number of root-knots (AR) was counted, and every experiment was replicated three times.
4.7. Root SOD Activity Assay
As above assay, cucumber seeds were cultured in a humid environment until roots grew to 4 cm long. A total of eight seedlings were selected and treated with 1 mg/mL of derivative 6 or water (blank control) for 48 h. Then, every two roots were washed with distilled water, ground into pulp with quartz sands, and diluted to 10 mL with phosphoric acid buffer (50 mM, pH 7.8). After centrifugation at 8000 rpm for 10 min at 4 °C, the supernatant was collected to detect SOD activity according to kit instructions. The SOD content was represented as U/g fresh root.
4.8. Phytotoxicity Assay
In this assay, 10 uniform and plump cucumber seeds were chosen and put in a φ9 cm petri dish lined with filter paper. Then, 4 mL of derivative solution (0.5 and 1.0 mg/mL) and positive control solution (0.025 mg/mL) were added. The seeds were cultured in the dark at 25.0 °C. After 48 h, the germination rate and radicle elongation were determined. Each treatment was repeated three times, and distilled water was used as blank control. GI was calculated by the following formula:
where the letter s and c represent the results of the sample treatment group and the blank control group, respectively; RL and GS denote the length of radicle and the number of germinating seeds, respectively. When GI exceeds 1, the derivative is considered to have no phytotoxicity; conversely, GI lower than 0.8 is considered to be phytotoxic.
4.9. Data Analysis
Statistics were performed using SPSS 24 statistical software. Except for data of SOD activity analyzed using Tukey’s HSD test, all data were determined using Duncan’s multiple range test. In all histograms, different letters (a, b, c, etc.) or symbols (*) were used to represent statistical significance at the p ≤ 0.05 level.