Phytotoxic and Antifungal Metabolites from Curvularia crepinii QTYC-1 Isolated from the Gut of Pantala flavescens

Four metabolites (1–4), including a new macrolide, O-demethylated-zeaenol (2), and three known compounds, zeaenol (1), adenosine (3), and ergosta-5,7,22-trien-3b-ol (4) were isolated and purified from Curvularia crepinii QTYC-1, a fungus residing in the gut of Pantala flavescens. The structures of isolated compounds were identified on the basis of extensive spectroscopic analysis and by comparison of the corresponding data with those reported in the literature previously. The new compound 2 showed good phytotoxic activity against Echinochloa crusgalli with an IC50 value of less than 5 µg/mL, which was comparable to that of positive 2,4-dichlorophenoxyacetic acid (2,4-D). Compound 1 exhibited moderate herbicidal activity against E. crusgalli with an IC50 value of 28.8 μg/mL. Furthermore, the new metabolite 2 was found to possess moderate antifungal activity against Valsa mali at the concentration of 100 µg/mL, with the inhibition rate of 50%. These results suggest that the new macrolide 2 and the known compound 1 have potential to be used as biocontrol agents in agriculture.


Introduction
Both weeds and plant fungal diseases have always been thought as big agricultural and environmental problems [1][2][3].Organic synthetic herbicides and fungicides are widely used to control these issues.However, these synthetic chemicals may create serious problems to human health and lead to environmental pollution due to their slow biodegradation.Moreover, concerns of toxicity makes it necessary to discover more effective and safer herbicides and fungicides.The study of microbial secondary metabolites offers useful evidence in the investigation of new types of natural herbicides and fungicides that could be less harmful and more specific than the synthetic chemical agents currently used in agriculture [4][5][6].Therefore, microbial natural products have attracted attention as reliable alternatives to synthetic agricultural chemicals [7].
Insect gut microorganisms constitute a rich and complex microbial population, and have gained attention as a resource of new bioactive metabolites [8][9][10].In the proceeding of our ongoing efforts to find new natural herbicides and fungicides from insect gut microbes [11], we found that the extract from the solid fermentation of the fungal strain Curvularia crepinii QTYC-1, isolated from the gut of Pantala flavescens, exhibited potent phytotoxic activities against the radical growth of Echinochloa crusgalli and Amaranthus retroflexus, as well as antifungal activity against plant pathogens.Further examination of the crude extract led to the isolation of one new macrolide along with three known compounds.Here, we reported the details of the isolation, structure characterization, and bioactivities of these metabolites.

Identification of the Fungus
Colonies of QTYC-1 on malt-extract agar (MEA) grew quickly at 28 • C, covering the whole plate (9 cm in diameter) in 10 days.The morphological features of the strain QTYC-1 were similar to those of Curvularia sp [2], which produced four-celled ellipsoidal conidia.Phylogenetic taxonomy with the sequence alignment of ITS (Internal Transcribed Spacer) rDNA of the fungus was achieved with MEGA 5.0 software (http://www.megasoftware.net).The phylogenetic tree (Figure 1) indicated that the title fungus was closely related to Curvularia crepinii (KU877619), with the ITS sequence similarity of 100%.Combined with the morphological characteristics, the fungus was identified as C. crepinii.Echinochloa crusgalli and Amaranthus retroflexus, as well as antifungal activity against plant pathogens.Further examination of the crude extract led to the isolation of one new macrolide along with three known compounds.Here, we reported the details of the isolation, structure characterization, and bioactivities of these metabolites.

Identification of the Fungus
Colonies of QTYC-1 on malt-extract agar (MEA) grew quickly at 28 °C, covering the whole plate (9 cm in diameter) in 10 days.The morphological features of the strain QTYC-1 were similar to those of Curvularia sp [2], which produced four-celled ellipsoidal conidia.Phylogenetic taxonomy with the sequence alignment of ITS (Internal Transcribed Spacer) rDNA of the fungus was achieved with MEGA 5.0 software (http://www.megasoftware.net).The phylogenetic tree (Figure 1) indicated that the title fungus was closely related to Curvularia crepinii (KU877619), with the ITS sequence similarity of 100%.Combined with the morphological characteristics, the fungus was identified as C. crepinii.

Identification of Active Compounds
Bioassay-guided fractionation of the constituents in the ethyl acetate extract of C. crepinii QTYC-1 yielded a novel macrolide derivate 2 and three known active metabolites (Figure 2).

Identification of Active Compounds
Bioassay-guided fractionation of the constituents in the ethyl acetate extract of C. crepinii QTYC-1 yielded a novel macrolide derivate 2 and three known active metabolites (Figure 2).Compound 2 was obtained as a white powder.Its molecular formula C18H22O7 was deduced from ESI-MS m/z 373 [M + Na] + , 701 [2M + H] + , which was consistent with the 1 H-NMR and 13 C-NMR data (Supplementary Materials, Figures S1-S6).The 1 H-NMR (Table 1) of 2 indicated the presence of 1,3-disubstituted benzene ring (δH 6.45, J = 2.4 Hz; δH 6.27, J = 2.4 Hz), one methyl (δH 1.47), and two methylenes.Five proton signals at δH 3.93, 3.96, 3.80, 9.37, and 11.89 were assigned to hydroxy groups because no HMQC correlation was observed.The 1 H-and 13 C-NMR data were similar to those of zeaenol (1), except the O-demethylation at position 15 in 1 appeared to be OCH3 [12], which corresponded to the decrease in molecular weight of 2 by 14 amu compared to 1.This was further confirmed by the HMBC correlation of HO-15 to C-14 (δC 108.Compound 2 was obtained as a white powder.Its molecular formula C 18 H 22 O 7 was deduced from ESI-MS m/z 373 [M + Na] + , 701 [2M + H] + , which was consistent with the 1 H-NMR and 13 C-NMR data (Supplementary Materials, Figures S1-S6).The 1 H-NMR (Table 1) of 2 indicated the presence of 1,3-disubstituted benzene ring (δ H 6.45, J = 2.4 Hz; δ H 6.27, J = 2.4 Hz), one methyl (δ H 1.47), and two methylenes.Five proton signals at δ H 3.93, 3.96, 3.80, 9.37, and 11.89 were assigned to hydroxy groups because no HMQC correlation was observed.The 1 H-and 13 C-NMR data were similar to those of zeaenol (1), except the O-demethylation at position 15 in 1 appeared to be OCH 3 [12], which corresponded to the decrease in molecular weight of 2 by 14 amu compared to 1.This was further confirmed by the HMBC correlation of HO-15 to C-14 (δ C 108. oxacyclotetradecin-1-one.The planar structure of compound 2 has been described previously as an intermediate in the synthesis of derivative 1 [13].However, it had not been described from nature and the relative configuration of C-8 in 2 has not been previously assigned.The other secondary metabolites were identified as zeaenol (1) [12], adenosine (3) [14], and ergosta-5,7,22-trien-3b-ol (4) [15], by comparing their MS and NMR spectroscopic data with those reported in the literatures.
The genus of Curvularia species was well-known to produce a variety of secondary metabolites.Previous investigations led to the isolation of several compounds such as antimicrobial curvulone A-B and curvularides A-E [16,17], motility inhibitor murranofuran A [18], cytotoxic pentanorlanostane, and pyrenocine J [19,20].However, to the best of our knowledge, this is the first report of the new macrolide 2 isolated from the title strain C. crepinii QTYC-1, a fungus residing in the P. flavescens gut.

Phytotoxic Activity of the Bioactive Metabolites
Compounds 1-4 were assayed for their ability to inhibit radicle growth of E. crusgalli and A. retroflexus using a Petri dish bioassay.The result (Figure 3) showed that compounds 1 and 2 were very active in reducing radicle growth of E. crusgalli with inhibition rates of 85.5% and 85.6%, respectively, which was comparable to the activity of positive 2,4-dichlorophenoxyacetic acid (2,4-D) with an inhibition rate of 97.5% under the concentration of 100 µg/mL.Compounds 1-3 showed potent phytotoxic activity against A. retroflexus with inhibition rates of 66~76% at the concentration of 100 µg/mL.However, compound 4 showed weak inhibitory effect against A. retroflexus and no obvious inhibitory effects were displayed by compounds 3 and 4 against E. crusgalli in this bioassay.The other secondary metabolites were identified as zeaenol (1) [12], adenosine (3) [14], and ergosta-5,7,22-trien-3b-ol (4) [15], by comparing their MS and NMR spectroscopic data with those reported in the literatures.
The genus of Curvularia species was well-known to produce a variety of secondary metabolites.Previous investigations led to the isolation of several compounds such as antimicrobial curvulone A-B and curvularides A-E [16,17], motility inhibitor murranofuran A [18], cytotoxic pentanorlanostane, and pyrenocine J [19,20].However, to the best of our knowledge, this is the first report of the new macrolide 2 isolated from the title strain C. crepinii QTYC-1, a fungus residing in the P. flavescens gut.

Phytotoxic Activity of the Bioactive Metabolites
Compounds 1-4 were assayed for their ability to inhibit radicle growth of E. crusgalli and A. retroflexus using a Petri dish bioassay.The result (Figure 3) showed that compounds 1 and 2 were very active in reducing radicle growth of E. crusgalli with inhibition rates of 85.5% and 85.6%, respectively, which was comparable to the activity of positive 2,4-dichlorophenoxyacetic acid (2,4-D) with an inhibition rate of 97.5% under the concentration of 100 µg/mL.Compounds 1-3 showed potent phytotoxic activity against A. retroflexus with inhibition rates of 66~76% at the concentration of 100 µg/mL.However, compound 4 showed weak inhibitory effect against A. retroflexus and no obvious inhibitory effects were displayed by compounds 3 and 4 against E. crusgalli in this bioassay.Compounds 1 and 2 were further tested to elucidate their phytotoxic activity at different concentrations against E. crusgalli, compared to 2,4-D co-assayed as a positive reference.The result (Figure 4) showed that compounds 1 and 2 exhibited phytotoxic activity in a dose-dependent Compounds 1 and 2 were further tested to elucidate their phytotoxic activity at different concentrations against E. crusgalli, compared to 2,4-D co-assayed as a positive reference.The result (Figure 4) showed that compounds 1 and 2 exhibited phytotoxic activity in a dose-dependent manner (5-50 µg/mL).Furthermore, the new compound 2 exhibited higher phytotoxic activity than compound 1 at the same concentration.The 50% inhibitory concentration (IC 50 value) of the new compound 2 was less than 5 µg/mL, which was comparable to that of positive 2,4-D.Compound 1 exhibited moderate phytotoxic activity against E. crusgalli with an IC 50 value of 28.8 µg/mL.
Molecules 2018, 23, x FOR PEER REVIEW 5 of 9 manner (5-50 µg/mL).Furthermore, the new compound 2 exhibited higher phytotoxic activity than compound 1 at the same concentration.The 50% inhibitory concentration (IC50 value) of the new compound 2 was less than 5 µg/mL, which was comparable to that of positive 2,4-D.Compound 1 exhibited moderate phytotoxic activity against E. crusgalli with an IC50 value of 28.8 µg/mL.

In Vitro Effect on Mycelial Growth of Phytopathogenic Fungi
The effects of metabolites 1-4 against the mycelial growth of five phytopathogenic fungi were evaluated in vitro under the concentration of 100 µg/mL (Table 2).The results showed that metabolite 2 possessed moderate antifungal activity against Valsa mali with an inhibition rate of 50% when compared with that of positive cycloheximide.However, it weakly inhibited the growth of Gibberella sanbinetti, Alternaria solani, and Fusarium oxysporum f.sp.mornordicae with an inhibition rate of less than 45%.In general, compounds 1, 3 and 4 exhibited weak activities against all tested phytopathogenic fungi with inhibition rates of less than 42%.In summary, we identified one new macrolide, together with three known compounds, from C. crepinii QTYC-1, a fungus residing in the gut of Pantala flavescens.The new macrolide 2 and the known compound 1 attenuated the radicle growth of E. crusgalli and A. retroflexus, and 2 possessed moderate antifungal activity against V. mali in vitro.These results suggested that the compounds 1

In Vitro Effect on Mycelial Growth of Phytopathogenic Fungi
The effects of metabolites 1-4 against the mycelial growth of five phytopathogenic fungi were evaluated in vitro under the concentration of 100 µg/mL (Table 2).The results showed that metabolite 2 possessed moderate antifungal activity against Valsa mali with an inhibition rate of 50% when compared with that of positive cycloheximide.However, it weakly inhibited the growth of Gibberella sanbinetti, Alternaria solani, and Fusarium oxysporum f.sp.mornordicae with an inhibition rate of less than 45%.In general, compounds 1, 3 and 4 exhibited weak activities against all tested phytopathogenic fungi with inhibition rates of less than 42%.In summary, we identified one new macrolide, together with three known compounds, from C. crepinii QTYC-1, a fungus residing in the gut of Pantala flavescens.The new macrolide 2 and the known compound 1 attenuated the radicle growth of E. crusgalli and A. retroflexus, and 2 possessed moderate antifungal activity against V. mali in vitro.These results suggested that the compounds 1 and 2 have some potential as agents for weeds or pathogenic fungal control.Further studies will be carried out to better understand the mechanism of action associated with phytotoxic and antifungal effects.In addition, the discovery of our study provided additional evidence that the special microorganisms in uninvestigated habitats, just like the title strain, may inspire the discovery of chemical agents with interesting biological activity.

Isolation and Identification of Strain QTYC-1
The strain was isolated based on methods described previously [21].P. flavescens were collected from the suburb of Jinhua, Zhejiang Province, PR China during the growing season.The insects were brought to the lab and starved for 24 h.The insects were surface-sterilized in 75% ethanol for 3 min, followed by rinsing in sterilized water three times (30 s each).They were then degutted using sterile forceps.The guts were lightly homogenized, and dilution series (10 −1 , 10 −2 , 10 −3 ) were spread-plated on malt-extract agar (MEA) medium (consisting of 20 g malt extract, 1 g peptone, 20 g sucrose, 20 g agar in 1 L of distilled water) containing antibacterial antibiotics (chloramphenicol and penicillin, 100 µg/mL each) to isolate the fungal strain.All of the plates were incubated aerobically in a chamber for 72 h at 28 ± 0.5 • C and any fungal colonies that formed were sub-cultured on new MEA medium to obtain pure cultures.The isolated strain was preserved on MEA slants at 4 • C until use.The title strain was deposited at the China Center for Type Culture Collection (CCTCC) as CCTCC M 2014308, and identified by comparing the morphological character and ITS sequence to those of standard records.

Microbial Fermentation
Based on the methods detailed elsewhere with trivial changes [22], the fungal strain was cultured on MEA medium at 28 ± 0.5 • C for four days.Then, pieces of fresh mycelium were inoculated into 250-mL Erlenmeyer flasks, each containing 100 mL of malt-extract (ME) liquid medium.After three days of incubation at 28 ± 0.5 • C on rotary shakers at 170 rpm, 10 mL cultural liquid was transferred as a seed into 1-L Erlenmeyer flasks, each containing 160 g of rice and 200 mL distilled H 2 O, and incubated at 28 ± 0.5 • C for 40 days.
Structural identifications of the secondary metabolites were made according to the spectroscopic analysis.The electrospray ionization mass spectrometry (ESI-MS) spectra were collected on a Time-of-Flight Mass Spectrometer G6230AA (Agilent Technologies, Santa Clara, CA, USA). 1 H nuclear magnetic resonance (NMR), 13 C-NMR and distortionless enhancement by polarization transfer (DEPT) spectra were acquired on a Bruker AVANCE-600 (Bruker, Fällanden, Switzerland), and chemical shifts were obtained in δ (ppm) by referring to the tetramethylsilane (TMS) and solvent signals as internal standards.HMQC and HMBC experiments were optimized for 145.0 and 8.0 Hz, respectively.

Figure 1 .
Figure 1.The phylogenetic tree of QTYC-1 based on the 5.8S rDNA sequences.

Figure 1 .
Figure 1.The phylogenetic tree of QTYC-1 based on the 5.8S rDNA sequences.

Figure 4 .
Figure 4.The phytotoxic effects of compounds 1 and 2 with different concentrations on radicle growth of E. crusgalli.

Figure 4 .
Figure 4.The phytotoxic effects of compounds 1 and 2 with different concentrations on radicle growth of E. crusgalli.

Table 2 .
Inhibition rate of the compounds of QTYC-1 against phytopathogenic fungi (in %) a .
bCycloheximide was used as the positive control.NI = not inhibited.

Table 2 .
Inhibition rate of the compounds of QTYC-1 against phytopathogenic fungi (in %) a .The concentration of compounds was 100 µg/mL; results were presented as the mean ± standard deviation for triplicate experiments.b Cycloheximide was used as the positive control.NI = not inhibited. a