2.2. Structure Elucidation
The fractionation of the extract from the culture broth of endolichenic fungi
Xylaria sp fermented in malt liquid medium led to the isolation and structural identification of two new piliformic derivatives, (+)-9-hydroxypiliformic acid (
1) and (+)-8-hydroxypiliformic acid (
2) together with the known compounds, (+)-piliformic acid (
3) as the main metabolite of extract, hexylaconitic acid A anhydride (
4), 2-hydroxyphenylacetic acid (
5) and 4-hydroxyphenylacetic acid (
6) (
Figure 2).
Compound
1 was isolated as colourless oil. Its molecular formula was determined as C
11H
18O
5 by the positive ion mode HRESIMS data at
m/z 253.1059 [M+Na]
+ (calcd. for C
11H
18O
5Na, 253.1052), indicating three degrees of unsaturation. The IR spectrum showed absorption bands at v
max 3355, 1689 and 1637 cm
-1, which indicated the presence of a hydroxyl group, carboxylic acid and double bound, respectively. In the
1H RMN spectrum (
Table 1 and
Figure S1) was observed resonances of one methyl group at δ 1.31 (d, J=7.1 Hz, H-11) coupled with a methine at δ 3.64 (dq, J=7.1 Hz, H-2) and one olefinic proton at δ 6.85 (t, J=7.6Hz). Other signals observed were multiplets attributed to the protons of three overlapped methylenes of an aliphatic chain at δ 1.42-1.66 and the signal at δ 3.55 (t, J=6.6Hz, H-9) of hydroxymethylene group. Analysis the
13C NMR (
Table 1 and
Figure S2) data in combination with gHSQC spectrum (
Figure S4) showed eleven carbon signals, two carbonyl groups at δ 178.0 (C-1) and 170.3 (C-10), two olefinic carbons at δ 134.4. (C-3) and 144.7 (C-4) of a trisubstituted double bond, one oxygenated methylene at δ 62.8 (C-9), one methine at δ 39.0 (C-2), one methyl group at δ 16.4 (C-11), as well as four methylene carbons at δ 29.4 (C-5), 29.6 (C-6), 26.7 (C-7) and 33.4 (C-8) in aliphatic region. The
1H-
1H COSY NMR spectrum of
1 (
Figure S3) showed coupling between the H-2 and H-11 and also established a partial structure from the olefinic proton at δ 6.85 (H-4) along the aliphatic chain with a spin system of methylenic protons H-5/ H-6/H-7/H-8/H-9.
The low field resonance of a methine proton at δ 3.64 (H-2) in the CH-CH3 grouping indicated deshielding effects due to carboxyl groups in the molecule. The carboxyl groups are located on C-1 and C-10, as supported by the HMBC correlations between H-2 and C-1/ C-3/ C-4/C-10/C-11 and between H-11 methyl with C-1/C-2/C-3 (
Figure 3). The position of the double bond was confirmed by the correlations previously indicated for H-2 and those observed between H-4 and C-2/ C-3/ C-5/ C-10. The HMBC experiment (
Figure S5) showed also connectivities of H-5 with C-2, C-3, C-4, C-6 and C-7, and H-9 with C-7 and C-8, which confirmed the structure of compound
1 closely related to the known fungal metabolite (+) piliformic acid (
3) [
26,
27]
, also isolated from this fungal strain and whose structure has been obtained by chemical synthesis [
28]. The configuration of double bound was assigned
E based on the NOESY between H-3 and H-5 (
Figure S6), however with the available spectroscopic data the configuration at C-2 in compound
1 could not be defined. Thus, this new compound was identified as (+)-(
E)-9-hydroxypiliformic acid (
1).
Compound
2 exhibited an ion peak in its HRMS (ESI+) at
m/z 253.1058 [M+Na]
+, corresponding to the molecular formula C
11H
18O
5Na, which was identical to that compound
1. The main difference observed in its
1H-NMR (
Table 1 and
Figure S8) is the absence of signal of hydroxymethylene group at C-9 in
1 that was replaced by a new signal due to hydroxyl group at δ H 3.74 coupled with a new doublet methyl signal at δ 1.16 (3H, d, J=6.2 Hz). These data suggested that these two compounds differ in the position of hydroxyl group in
2. Furthermore, the
13C NMR spectrum showed an additional methyl carbon at δ 23.5.8 (C-9) and a methine group at 68.2 (C-8) (
Figure S9).
The location of hydroxyl group at C-8 was confirmed by the
1H-
1H COSY crosspeak of H-9 (δ 1.16)/ H-8 (δ 3.74) together the HMBC correlations of H-9 methyl group with C-7 and C-8 (
Figure 3 and
Figure S12). A detailed analysis of the
13C NMR (
Table 1 and
Figure S9) showed double signals for C-5, C-6, C-7 and C-8, which indicated that compound
2 was the mixture of two epimers at C-8 with a ratio 2:1 according to the height of double peaks. These observations, together with a careful analysis of the COSY, HMBC and NOESY experiments, led to the identification compound
2 as a mixture (ratio 2:1) of two epimers of (+)-(
E)-8-hydroxypiliformic acid.
The known compound
4 was identified as 2-carboxymethyl-3-n-hexyl-maleic acid anhydride and named hexylaconitic acid A anhydride. Its molecular formula was determined as C
12H
16O
5 in accordance with its HRESIMS (
m/z 239.0920 [M-H]
-, calculated for C
12H
15O
5 239.0919) (
Figure S18). The
1H NMR of compound
4 revealed signals representative of one n-alkyl chain (n-hexyl moiety) and a deshielded singlet signal that integrated for two protons at δ 3.55 (H-11). The
13C NMR spectrum (
Figure S17) revealed the presence of three carboxyl carbons δ 173.2 (C-12), 165.3 (C-1), 165.2 (C-4), two olefinic quaternary carbons at δ 148.3 (C-2), 135.6 (C-3) and one methyl group at δ 14.1 (C-10) alongside with the presence of six methylenes δ 31.4 (C-8), 27.6 (C-6), 25.1 (C-5), 22.5 (C-9) and two overlapped carbons at δ 29.3 (C-7, C-11). The
1H and
13C NMR spectral data are consistent with those reported for 2-carboxymethyl-3-n-hexyl-maleic acid anhydride, which was first isolated as a natural product from
Aspergillus niger [
29] and later obtained by chemical synthesis [
30,
31]. Now in this work, we have completed the assignments of the proton and carbon resonances in the
1H and
13C spectra using 2D-NMR experiments (COSY, HSQC and HMBC), which had not been previously assigned. The configuration
Z of double bound was also confirmed by the crosspeak of H
2-5/H
2-11 in the NOESY spectrum (
Figure S27).
Another known compounds isolated from this fungus were identified as (+)-piliformic acid (
3) [
26,
27,
28], a fungal metabolite isolated previously from different fungi belonging to the
Xylaria genus [
32], and the phenolic compounds, 2-hydroxyphenylacetic acid (
5) [
33] and 4-hydroxyphenylacetic acid (
6) [
34]. In the experimental part we provide complementary NMR and MS data of these compounds.
2.3. Biopesticide Activity
The EtOAc extract and compounds obtained from the fermentation of HYP6 were tested for their biopesticide properties against insect pest (
Myzus persicae,
Rhopalosiphum padi and
Spodoptera littoralis), the plant parasitic nematode
Meloidogyne javanica and fungal phytopathogens (
Alternaria alternata,
Botrytis cinerea and
Fusarium oxysporum).The extract showed strong antifeedant effects against
M. persicae (EC
50 value of 10.9 µg/cm
2) followed by a moderate activity against
R. padi and it was not active on
S. littorallis. This extract was also antifungal to
B. cinerea and moderately active on
F. oxysporum and
A. alternata but it did not have any nematicidal effect (
Table 2).
When tested the compounds on insect pests (
Table 2),
4 showed a strong antifeedant activity against
M. persicae (EC
50 value of 1.6 µg/cm
2) and
R. padi (EC
50 value of 8.9 µg/cm
2), being more effective than the extract. Additionally, compounds
5 and
6 showed a selective antifeedant activity against
M. persicae with EC
50 value of 4.5 µg/cm
2 and EC
50 value of 15.5 µg/cm
2, respectively. None of the tested compounds showed any antifeedant effects against
S. littoralis. Compound
4 also exhibited a strong inhibition of mycelial growth of
B. cinerea (EC
50 value of 0.12 mg/ml) and moderate against
A. alternata (EC
50 value of 0.24 mg/ml), whereas compound
3 was moderately active on
B. cinerea (EC
50 value of 0.36 mg/ml). When tested against
M. javanica, compound
4 was very active with an LD
50 value of 0.10 mg/ml.
The extract and compounds were also tested for phytotoxic effects on seeds of mono- and dicotyledoneous plant species (
L. perenne and
L. sativa) (
Figure 4). The extract of
Xylaria sp. strongly inhibited the germination of
L. perenne with 88.2% inhibition after 7 days compared to the control. The extract also exhibited phytotoxic effects on
L. perenne, with 89.3% and 100% inhibition of leaf and root growth, respectively and affected the root growth of
L. sativa (66.8% inhibition). Among the compounds tested, compound
3 affected the germination of
L. sativa (86.7% inhibition), decreased moderately root growth of
L. perenne (56.8% inhibition) and affected the root growth of
L. sativa (72.6% inhibition). Compound
4 reduced significantly the germination of
L. perenne and
L. sativa (72.2% and 94.7% inhibition, respectively) with a strong inhibition of root growth (100% and 97.9% inhibition, respectively). Additionally, this compound also decreased the leaf growth of
L. perenne (73.1% inhibition). Compound
5, with reported phytotoxic effects [
35,
36], showed significant effect on root growth on
L. perenne and
L. sativa root growth (85.4% and 91.3% inhibition, respectively) (
Figure 4). The most phytotoxic compounds (
3,
4) were further tested in dose-response experiments against the two plant species. At lower concentrations, both compounds stimulated the root growth of
L. sativa. Compound
3 increased the root length up to 170% at a concentration of 0.1 mg/ ml and
4 up to 200% at 0.05 mg/ml.
In this work, (+)-piliformic acid (
3) showed moderate antifungal effects against
B. cinerea. Similarly, previous reports showed moderate activity of
3 against
Colletotrichum gloeosporioides, one of the phytopathogenic fungus responsible for the anthracnose disease [
37]. Compound
4, with aphid antifeedant, antifungal and nematicidal effects was identified as hexylaconitic A anhydride. This compound (
4) isolated from
Aspergillus niger[
29,
38] and
Aspergillus tubigensis [
39] has been previously reported for its fungicidal activity against
Neurospora crassa [
39], but it was found inactive against other fungal plant pathogens (
Gaeumannomyces graminis var
. tritici,
Rhizoctonia solani, and
Phytophthora cinnamomic) [
38]. This may indicate a species-dependent antifungal effects that could be used to target specific fungal pathogens. However, this is the first report on the aphid antifeedant and nematicidal effects of compound
4. Phenolic acids related to
5 and
6, are known for their insecticidal activities [
40] and play an important role in the plant resistance against insect pest [
41]. For example, previous reports showed that phenylacetic compounds isolated from
Streptomyces gramineus have insecticidal activity against
Thrips palmi, also known as melon thrips, a sap-sucking phytophagous insect [
42].
The phytotoxic effects observed are consistent with those of the analogous compounds of alkylitaconic acid derivatives, which promoted radicle growth at low doses, while inhibited the seedlings growth at higher doses particularly in dicotyledonous species [
35,
43]. Additionally, Mondal et al. [
44] reported that compound
4 stimulates germination and seedling growth in cauliflower at low ppm concentrations. This behaviour could be explained by hormetic effects of toxic agent showing a biphasic response, promoting early seedling development (and potentially later plant growth) at low concentrations, while inhibiting growth at high concentrations [
45,
46]. This work has shown for the first time the potent phytotoxic effects of (+)-piliformic acid (
3) and hexylaconitic A anhydride (
4) on mono and dicotyledonous plant (
L. perenne and
L. sativa) and stimulating effect the growth of
L. sativa root at lower dose.