Two nematicidal furocoumarins from Ficus carica L. leaves and their physiological effects on pine wood nematode (Bursaphelenchus xylophilus)

Abstract The ethanol extract of the Ficus carica L. leaves was tested to show strong nematicidal activity against pine wood nematode (PWN), Bursaphelenchus xylophilus, causing 90.93% corrected mortality within 72 h at 1.0 mg/mL. From the ethyl acetate soluble fraction of the F. carica L. leaves extract, the main nematicidal constituents were obtained by bioassay-guided isolation and identified as linear furocoumarins bergapten (1) and psoralen (2) by mass and NMR spectral data analysis. Bergapten and psoralen had significant nematicidal activity against PWN with the LC50 values of 97.08 aKSnd 115.03 μg/mL within 72 h, respectively. The two furocoumarins could inhibit the activities of amylase, cellulase and acetylcholinesterase (AchE) from PWN. The morphologies of PWNs changed much after they were treated by bergapten and psoralen. The physiological effects of bergapten and psoralen on PWN might provide helpful clues to elucidate their nematicidal mechanisms. Graphical abstract


Introduction
Pine wood nematode (PWN), Bursaphelenchus xylophilus, is considered the causal pathogen of pine wilt disease which has greatly damaged some pine species (Mamiya 1983) and become a severe worldwide threat to forest resources (Guo et al. 2000). At present, synthetic pesticides are mainly used to control the disease (Takai et al. 2000(Takai et al. , 2003Wu et al. 2004), which causes more and more concerns about possible drug resistance, environmental pollution and human health. Therefore, alternative ecofriendly nematicides are urgently needed. Phytochemicals as natural products are considered good candidates because they can be used not only as nematicides but also as model compounds to chemically synthesised derivatives with enhanced nematicidal activity. So far, there have been many reports about natural nematicidal phytochemicals from various genera of plants (Kogiso et al. 1976;Matsuda et al. 1989Matsuda et al. , 1991Zhao 1996;Choi et al. 2006Choi et al. , 2007Choi et al. , 2008Zhang et al. 2011;Wang et al. 2012).
According to our survey on a series of medicinal plants, we found that F. carica L. leaves showed strong nematicidal activity and was worthy of thorough study on its nematicidal constituents. In this paper, two nematicidal linear furocoumarins, bergapten (1) and psoralen (2), were identified. The nematicidal activity of bergapten (1) was reported for the first time although psoralen (2) was mentioned in the previous report . Influences of the furocoumarins on the several enzymes and morphology of PWN were also studied. This study will offer some clues to exploit natural products in the management of PWD.

Nematicidal activity
The ethanol extract of the F. carica L. leaves had strong nematicidal activity against PWN in vitro (Table 1). Its corrected mortality reached 90.93% within 72 h at the concentration of 1.0 mg/mL. The two fractions derived from the ethanol extract had different nematicidal activities. The ethyl acetate soluble fraction was shown to have much stronger activity with the corrected mortality of 95.27% in 72 h at the concentration of 1.0 mg/mL than the aqueous fraction only with the corrected mortality of 18.27% under the same conditions. By tracing nematicidal activity, the isolated compounds bergapten and psoralen from more active fractions, F3 and F4, both showed nematicidal activity against PWN with LC 50 value in 72 h of 97.08 and 115.03 μg/mL, respectively (Table 2).
Bergapten was found a little more active than psoralen, and the group 5-OCH 3 in bergapten might contribute to its higher nematicidal activity. Since both of the isolated nematicidal compounds belong to coumarins, we hypothesise that the core molecular structure of coumarins is closely related to the nematicidal activity against PWN. Therefore, we will pay close attention to the nematicidal activity against PWN of other coumarins and find more clues to clarify the relationship between their molecular structures and the nematicidal activity in our following research.

Effect of bergapten and psoralen on enzymes from PWN
The results indicated that both bergapten and psoralen could inhibit the activities of the two digestive enzymes from PWN. Bergapten showed much better inhibitory effect on amylase with IC 50 value of 372.45 μg/mL than psoralen with IC 50 value of 1072.82 μg/mL (Table S2). Bergapten and psoralen exhibited similar inhibiting effect on cellulase with IC 50 value of 450.40 and 458.92 μg/mL, respectively (Table S2). Amylase and cellulase are important digestive enzymes in nematodes (Yan & Yang 1997;Palomares-Rius et al. 2014), and inhibition of the two coumarins on amylase and cellulase may also be responsible for their nematicidal activities by weakening digestive ability of PWN. Bergapten was proved to be more active on the amylase from PWN than psoralen (Table S2), which coincides to the stronger nematicidal activity of bergapten compared with psoralen (Table 2).
In addition, bergapten and psoralen were both proved to inhibit the activity of AchE from PWN with IC 50 value of 493.11 and 564.59 μg/mL, respectively (Table S2). Inhibition on AchE is possible to disorder nervous system of PWN and threaten their lives, since AchE plays an important role in terminating nerve impulse by hydrolysing acetylcholine in most vertebrates, insects and nematodes (Massoulié et al. 1993), and is an important target for the action of organophosphate and carbamate pesticides (Kang et al. 2011).

Effects of bergapten and psoralen on morphology of PWN
The morphology of nematodes changed much after they were treated by the purified bergapten and psoralen. Observation by scanning electron microscope (SEM) indicated that the controlled nematodes were smooth and full, while the surface of the treated nematodes was rough and some nematodes even partially autolysed and cavitated ( Figure S2). Whether this phenomenon is the cause or result of PWN death needs for further research.

Conclusions
Two strongly nematicidal coumarins against PWN, bergapten and psoralen, were isolated from F. carica L. leaves. Our results indicated the coumarins were the main nematicidal components of F. carica L. leaves. According to assays of bergapten and psoralen on enzymes from PWN, the two nematicidal coumarins could inhibit the activities of amylase, cellulase and AchE. The morphologies of PWNs treated by bergapten and psoralen changed greatly compared with the control.

Supplementary material
The experimental section, nematicidal activities of column chromatography fractions from the F. carica L. leaves extract (Table S1), IC 50 values of bergapten and psoralen on amylase, cellulase and AchE (Table S2), chemical structures of bergapten and psoralen ( Figure S1) and observation by SEM on morphologies of PWNs treated by bergapten and psoralen ( Figure S2) are available as supplementary online material.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This work was supported by the National Natural Science Foundation of China [grant number 31070575].