Chlamyphilone, a Novel Pochonia chlamydosporia Metabolite with Insecticidal Activity

Metabolites from a collection of selected fungal isolates have been screened for insecticidal activity against the aphid Acyrthosiphon pisum. Crude organic extracts of culture filtrates from six fungal isolates (Paecilomyces lilacinus, Pochonia chlamydosporia, Penicillium griseofulvum, Beauveria bassiana, Metarhizium anisopliae and Talaromyces pinophilus) caused mortality of aphids within 72 h after treatment. In this work, bioassay-guided fractionation has been used to characterize the main bioactive metabolites accumulated in fungal extracts. Leucinostatins A, B and D represent the bioactive compounds produced by P. lilacinus. From P. griseofulvum and B. bassiana extracts, griseofulvin and beauvericin have been isolated, respectively; 3-O-Methylfunicone and a mixture of destruxins have been found in the active fractions of T. pinophilum and M. anisopliae, respectively. A novel azaphilone compound, we named chlamyphilone, with significant insecticidal activity, has been isolated from the culture filtrate of P. chlamydosporia. Its structure has been determined using extensive spectroscopic methods and chemical derivatization.


Introduction
Alarm over the impact of pesticides on the environment and human health is increasing year after year, and rigorous pesticide registration procedures have been introduced. Through Directive 2009/128/EC, the European Community has severely restricted the use of synthetic pesticides in plant protection, and the new regulations have reduced the number of chemicals available in agriculture [1]. Novel pesticides, including natural product-based formulations, have been developed to counteract the evolution of resistance among plant pathogen and pest populations [2].
All fungi have been grown in static conditions and the culture filtrates were extracted exhaustively with ethyl acetate. The crude extracts were fractionated by column chromatography to provide a total of 79 organic fractions. The extracts and all the fractions were tested at different concentrations on Acyrthosiphon pisum. Table 1 reports the percentage of mortality at 72h of the active extracts/fractions obtained from different fungal isolates. Only extracts obtained from P. lilacinus, P. griseofulvum, P. chlamydosporia, B. bassiana, M. anisopliae and T. pinophilus showed significant insecticidal activity (data obtained from the other fungal strains are not showed). Moreover, the bioassay-guided fractionation indicated that only one fraction per strain was responsible of a significant aphid mortality after 72 h exposure (Table 1).
Chemical characterization of the active fractions was obtained using NMR and/or LC-MS qTOF analyses. Fraction No. 3 of P. lilacinus (showing 30% aphid mortality; Table 1) was constituted by a  Figure 1), with the molecular weights (MW) 1218.634 g/mol, 1204.607 g/mol and 1104.4799 g/mol, respectively ( Figure S1). Table 1. Insecticidal activity of fungal organic extracts and active fractions as mortality (%) of the pea aphid Acyrthosiphon pisum at 72 h after exposure. Different lower letters refer to significant differences (p < 0.05) among treatments at the same incubation time. Extracts from T. tomentosum F19, T. asperellum CINO1, T. harzianum M10, T. harzianum F53, T. velutinum F28, P. chrysogenum F5, P. decumbens F29, P. restrictum F55 and C. virescens F57 did not show significant insecticidal activity. The mycotoxin griseofulvin (4 in Figure 1) was determined as the main metabolite of P. griseofulvum and was isolated in the active fraction number 7 (showing 73,3% aphid mortality; Table  1). Figure S3 reports the mass spectrum of the isolated compound.

Microbes
The residue recovered after the organic extraction (350 mg) of P. chlamydosporia culture filtrate was subjected to flash column chromatography, eluting with CH2Cl2/MeOH (90:10 v/v). Fractions showing similar thin-layer chromatography (TLC) profiles were combined and further purified by using preparative TLC separation (Si gel; CH2Cl2/MeOH 90:10 v/v). Both fractions and pure The mycotoxin griseofulvin (4 in Figure 1) was determined as the main metabolite of P. griseofulvum and was isolated in the active fraction number 7 (showing 73,3% aphid mortality; Table 1). Figure S3 reports the mass spectrum of the isolated compound.
The residue recovered after the organic extraction (350 mg) of P. chlamydosporia culture filtrate was subjected to flash column chromatography, eluting with CH 2 Cl 2 /MeOH (90:10 v/v). Fractions showing similar thin-layer chromatography (TLC) profiles were combined and further purified by using preparative TLC separation (Si gel; CH 2 Cl 2 /MeOH 90:10 v/v). Both fractions and pure compounds were tested for insecticidal activity. Twelve milligrams of a novel compound, named chlamyphilone (5 in Figure 1; 5 mg/L), were obtained as a yellow amorphous solid in pure form after TLC (Rf in CH 2 Cl 2 /MeOH 90:10 v/v = 0.8) and has: [α] 25 D −21.9 • (c 1; CH 2 Cl 2 ); UV (CH 2 Cl 2 ) λ max (log ε) 219 (3.57), 332 (4.23). 1 H and 13 C NMR spectral data of chlamyphilone (in CDCl 3 ) are presented in Table 2 Figure S4). The results obtained by 13 C NMR (Table 2 and Figure S6) and LC-MS qTOF analyses ( Figure S4) were consistent with a compound having a molecular weight of 220.0814, corresponding to the molecular formula C 12 H 12 O 4 with seven unsaturations. The UV spectrum showed absorption peaks characteristic of the azaphilone family, which are natural products containing a 6H-isochromene-6,8(7H)-dione. Six carbon signals at 102.9, 114.4, 115.3, 145.2, 151.7, and 155.1 ppm revealed three double bonds, whereas signals at 196.3 and 196.5 ppm indicated the presence of two ketones. The other four signals in the 13 C spectrum were all shifted upfield in the 12.7-83.11 ppm range. The DEPT data demonstrated that one of the protons in the molecule is bound to oxygen, and that in the molecule there are three CH 3 , two CH, and seven fully substituted C atoms. The 1 H-1 H COSY, HSQC analysis, and the chemical shift evaluation (Table 2) allowed the identification of the structural fragment. In Table 2 all the compound signals are reported.
The connectivity of the spin systems was deduced by a long-range 1 H-13 C heterocorrelated experiment that was obtained with the HMBC (Table 2). In particular, the most relevant HMBC correlations are reported in Figure 2, which implied that the structure of the metabolite 5 is 7-hydroxy-3,4,7-trimethyl-isochromene-6,8-dione. The configuration of chlamyphilone was evident from NOESY experiment. The MM-2 energy calculation (16.0 Kcal/mol) was run to find the most stable conformational model ( Figure S12). The connectivity of the spin systems was deduced by a long-range 1 H-13 C heterocorrelated experiment that was obtained with the HMBC (Table 2). In particular, the most relevant HMBC correlations are reported in Figure 2, which implied that the structure of the metabolite 5 is 7hydroxy-3,4,7-trimethyl-isochromene-6,8-dione. The configuration of chlamyphilone was evident from NOESY experiment. The MM-2 energy calculation (16.0 Kcal/mol) was run to find the most stable conformational model ( Figure S12). To confirm the proposed structure, a sample of chlamyphilone was acetylated using acetic anhydride/pyridine (6 in Figure 1). The 1 H-NMR spectrum of the product (acetic acid 3,4,7-trimethyl-6,8-dioxo-7,8-dihydro-6H-isochromen-7-yl ester) contained one acetate resonance, confirming the presence of the hydroxyl group bound to the carbon at 83.1. The 13 C-NMR spectrum contained an additional signal for the acetyl carbonyl resonance at 170.8 and for the acetate methyl carbon at 18.4 ppm. To confirm the proposed structure, a sample of chlamyphilone was acetylated using acetic anhydride/pyridine (6 in Figure 1).
The 1 H-NMR spectrum of the product (acetic acid 3,4,7-trimethyl-6,8-dioxo-7,8-dihydro-6H-isochromen-7-yl ester) contained one acetate resonance, confirming the presence of the hydroxyl group bound to the carbon at 83.1. The 13 C-NMR spectrum contained an additional signal for the acetyl carbonyl resonance at 170.8 and for the acetate methyl carbon at 18.4 ppm.
Chlamyphilone (5 in Figure 1) showed the highest insecticidal activity against Acyrthosiphon pisum with a median Lethal Dose (LD50) of 175 µg/mL and a minimal inhibitory concentration (MIC) of 150 µg/mL (Table 3). The insecticidal activity of the B. bassiana strain used in the present work was mainly dependent on the presence of beauvericin (7 in Figure 1). This metabolite is a cyclohexadepsipeptide mycotoxin with the molecular formula C 45 H 57 N 3 O 9 ( Figure S13).
From M. anisopliae extract, destruxin B2 has been characterized (8 in Figure 1). Destruxins are cyclic hexadepsipeptides composed of an α-hydroxy acid and five amino acid residues. Individual destruxins differ in terms of the hydroxy acid, N-methylation, and R group of the amino acid residues. Destruxin B2 has the molecular formula C 29 H 49 N 5 O 7 , as revealed by the MS spectrum ( Figure S14).

Discussion
In this work, a functional screening of fungal strains, preliminarily selected because of their ability to synthetize active metabolites, has been carried out. Bioassay-guided fractionation has been used to isolate microbial metabolites with insecticidal activity against the pea aphid A. pisum (Hemiptera: Aphididae), long used as a model to study plant-insect interactions [9]. This aphid is an important pest of several plants, is a phloem-feeding insect determining direct negative effects on plants in terms of nutritive subtraction and injection of toxic saliva. Moreover, it has been involved in the transmission of diverse plant viruses [9].
The active fraction obtained from P. lilacinus was constituted by a mixture of leucinostatins A, B, and D. Leucinostatins are peptides containing an unsaturated fatty acid, nine amino acid residues, and a basic component joined together by amide linkages [10]. These antibiotic metabolites also showed antitumor and nematocidal activities and an uncoupling effect on rat liver mitochondrial function [10,11]. To the best of our knowledge, our work is the first report on the insecticidal activity of leucinostatins against A. pisum.
Griseofulvin was determined to be the main metabolite of P. griseofulvum. The insecticidal activity of this metabolite has been previously demonstrated in the corn earworm, Helicoverpa zea Boddie, and the fall armyworm, Spodoptera frugiperda (J. E. Smith), by oral administration in an artificial diet (250 ppm) [12][13][14]. In the case of soft-tegument aphids like A. pisum, the activity is probably topical as oral administration may be excluded because of their peculiar feeding pattern. In fact, aphids use their piercing, sucking mouthparts to feed on plant fluid [9].
The extract of P. chlamydosporia culture filtrate yielded 12 mg of a novel compound named chlamyphilone. This metabolite belongs to the class of azaphilones, which contain a 6H-isochromene-6,8(7H)-dione or an isoquinoline-6,8(2H,7H)-dione skeleton (and its substituted derivatives thereof). This is the first report on the isolation of an azaphilone metabolite from P. chlamydosporia. A similar azaphilone, named myxostiol, with plant growth regulating activity, has been previously isolated from Myxotrichum stipitatum [15]. Over 170 different azaphilone compounds, classified into 10 structural groups, have been isolated from fungi belonging to 23 genera [16]. Azaphilones display a wide range of biological activities, such as antimicrobial, antifungal, antiviral, antioxidant, cytotoxic, nematocidal, and anti-inflammatory [16]. Many of these properties may be explained by the reactions of azaphilones with amino groups, such as those found in amino acids, proteins, and nucleic acids, resulting in the formation of vinylogous c-pyridones [16][17][18]. To the best of our knowledge, this is the first evidence of the insecticidal activity of a metabolite belonging to the class of azaphilones. Recently, the involvement of small molecules (e.g., aurovertins) in the interaction between nematodes and P. chlamydosporia has been demonstrated [19].
An active beauvericin has been extracted from culture filtrate of B. bassiana. This metabolite is a mycotoxin whose insecticidal properties have been previously reported (e.g., against the wheat aphid Schizaphis graminum at 0.5 mg/mL) [20,21]. This molecule contains three residues, each with D-2-hydroxyisovaleric acid (Hiv) and L-N-methylphenylalanine linked alternately [21].
Destruxin B2 is the active molecule isolated from M. anisopliae extract. Destruxins exhibited a wide variety of biological activities, but are best known for their insecticidal and phytotoxic properties [22,23]. However, the effect of destruxin B2 against Acyrthosiphon pisum is reported here for the first time.
T. pinophilus strain F36CF produced 3-O-methylfunicone. This compound was downregulated in the presence of T. harzianum M10 [8], exhibited notable antibiotic and antitumor properties, and recently exhibited an insecticidal effect, thus expanding the biological activities of this compound [24]. Recently, talarodiolide, a new 12-membered macrodiolide, was isolated and characterized from the culture filtrate of a T. pinophilus strain [25]. This metabolite did not show insecticidal activity, highlighting that the strain collection is important to select the best producers of bioactive compounds.

Materials and Methods
Fungal strains. The microbes used in the present study were present in the fungal collection at Department of Agricultural Sciences, University of Naples Federico II (UNINA Collection) or isolated from different sources (Table 4). The fungi were identified according to morphological features and molecular analyses. Briefly, an amount of 10 6 spores/mL was inoculated in a 250-mL Erlenmeyer Flasks containing 100 mL of sterile Potato Dextrose Broth (PDB, HIMEDIA, Mumbai, India). Each flask was incubated at 25 • C in an orbital shaker (150 r.p.m.) and fungi were left to grow for seven days. Mycelium was recovered, ground to a powder in liquid nitrogen, and used to perform the DNA extraction. Genetic analysis was carried out through PCR and sequencing of the rDNA Internal Transcribed Spacer (ITS) and β-tubulin gene, using the most common primers to identify fungal strains: ITS1 and ITS4 [26], as well as tub2 and BenA [27]. The PCR products were subjected to gel electrophoresis, excised, and sequenced [26,27]. Analysis of the ITS gave 99% of identity with GenBank sequences of the fungi reported in Table 4, confirming the identity of the microbes.
Secondary metabolites production. The fungi were maintained on potato dextrose agar (PDA, HIMEDIA) at room temperature and sub-cultured bimonthly. Liquid cultures were prepared in 5000 L-Erlenmeyer flasks containing 1 L of PDB and inoculated with five mycelial plugs (7 mm 2 ) from a fresh PDA culture of each fungal strain. After 30 days of incubation at 25 • C in static conditions, the cultures were filtered through filter paper (Whatman No. 4) and the filtrate was analyzed by LC-MS qTOF [28].
Monodimensional 1 H and 13 C acquisitions were conducted as follows: proton spectra were acquired with 2 s of thermal equilibrium delay (number of scans = 64), a 90 • pulse length 7.7 µs, 50 transients and 16 ppm (6410.2 Hz) as spectral widths, whereas proton-decoupled carbon acquisitions were executed by both inverse-gated and DEPT 135 • pulse sequences, adopting 7 and 5 s of equilibrium delay, 12,500 and 2400 transients, respectively, and a spectral width of 250 ppm (25.152 KHz). A time domain of 32,768 points was adopted for all cited mono-dimensional experiments. Homo-nuclear 1 H-1 H COSY (COrrelation SpectroscopY), TOCSY (TOtal Correlation SpectroscopY), NOESY (Nuclear Overhauser Enhancement SpectroscopY), and hetero-nuclear 1 H-13 C HSQC (Hetero-nuclear Single-Quantum Correlation) and HMBC (Hetero-nuclear Multiple Bond Coherence) experiments (2D) were used for structural identification of metabolites. 2D homo-and heteronuclear spectra experiments were acquired with 48 and 80 scans, respectively, 16 dummy scans, a time domain of 2k points (F2) and 256 experiments (F1). TOCSY and NOESY experiments were conducted with a mixing time of 80 and 1000 ms, respectively, while HSQC and HMBC experiments were optimized for 145 Hz short and 6.5 Hz long range J CH couplings, respectively. All executed 2D experiments were gradient enhanced, except for the TOCSY acquisition. A Qsine weighting function associated to a magnitude mode was used to process NOESY spectrum with the purpose to emphasize the weak cross-peaks and minimize the noise artefacts. The free induction decay (FID) of mono-dimensional spectra was multiplied by an exponential factor corresponding to 0.1 Hz, for 1 H and 13 C acquisitions, and to 1 Hz for DEPT 135 • experiment. All above mentioned spectra were baseline corrected and processed by using Bruker Topspin Software (v.4.0.2).
LC-MS/MS Q-TOF analysis were done on an Agilent HP 1260 Infinity Series liquid chromatograph equipped with a DAD system (Agilent Technologies, Santa Clara, CA, USA) coupled to a Q-TOF mass spectrometer model G6540B (Agilent Technologies). Separations were performed on a Zorbax Eclips Plus C18 column, 4.6 × 100 mm, with 3.5 µm particles (Agilent Technologies). The analyses were done at a constant temperature of 37 • C and using a linear gradient system composed of A: 0.1% (v/v) formic acid in water, and B: 0.1% (v/v) formic acid in acetonitrile. The flow was 0.6 mL/min, 95% A graduating to 100% B in 12 min, 100% B 12-15 min, 95% A 15-17 and equilibrating 95% A 17-20 min. The UV spectra were collected by DAD every 0.4 s from 190 to 750 nm with a resolution of Molecules 2019, 24, 750 9 of 11 2 nm. The MS system was equipped with a Dual Electrospray Ionization (ESI) source and operated with Agilent MassHunter Data Acquisition Software, rev. B.05.01 in the positive or negative mode. Mass spectra were recorded in the range m/z 100-1600 as centroid spectra, with 3 scans per second. Two reference mass compounds were used to perform the real-time lock mass correction, purine (C 5 H 4 N 4 at m/z 121.050873, 10 µmol/L) and hexakis (1H,1H, 3H-tetrafluoropentoxy)-phosphazene (C 18 H 18 O 6 N 3 P 3 F 24 at m/z 922.009798, 2 µmol/L). The capillary was maintained at 4000 V, fragmentor voltage at 180 V, cone 1 (skimmer 1) at 45 V, Oct RFV at 750 V. Gas temperature was 350 • C during the run at 11 L/min, and the nebulizer was set at 45 psig. The injected sample volume was 5 µL.
MS/MS spectra were simultaneously recorded for confirmation purposes of new compounds, using the operating parameters described above, unless otherwise stated. The instrument was operated in the range m/z 100-1000, recording two spectra per second in targeted acquisition mode (targeted mass: 244.1197, Z = 1, RT 5.88 ±0.5 min). The sample collision energy was set to 20 V.
LC-MS data were evaluated using MassHunter Qualitative Analysis Software B.06.00 and compared to known compounds included in an in-house database. The database contains information of about 4000 known secondary metabolites isolated from more than 80 different fungal genera, and recorded according to their name, molecular formula, monoisotopic mass and producing organism. Positive identifications of fungal metabolites were reported if the compound was detected with a mass error below 10 ppm and with a sufficient score. Standards were used to confirm the chemical identifications.
UV spectra were recorded with a V-730 UV-Visible Spectrophotometer JASCO (Mary's Court, Easton, MD, USA). Column chromatography was performed using silica gel (Merck silica gel 60 GF 254 ; Merck, Darmstadt, Germany), and TLC with glass pre-coated silica gel GF 254 plates (Merck Kieselgel 60 GF 254 , 0.25 mm). The compounds were detected on TLC plates using UV light (254 or 366 nm) and/or by dipping the plates in a 5% (v/v) H 2 SO 4 solution in ethanol followed by heating at 110 • C for 10 min [28,29].
In vivo insecticidal assay. Insecticidal activity of organic extracts, each column fraction and pure metabolites were tested against the pea aphid Acyrthosiphon pisum. Aphids were reared on Vicia faba var. aguadulce in a growth chamber (20 ± 1 • C, 70% RH, 18 h light/6 h dark photoperiod). Small populations were synchronized to obtain newborn nymphs every 24 h. Third-instar nymphs were dipped in the assay solution for 10 s and put on a paper towel to dry. Then, each insect was carefully transferred by a soft paintbrush on a leaf plug [30,31]. Twenty treated (or untreated-solvent control) 3rd-instar nymphs were placed on two circular (35 mm diameter) leaf plugs on 2% water agar layer in a Petri dish, in order to keep the leaf turgid. Plates were incubated at the climatic conditions described above and the number of dead aphids was assessed 24, 48, and 72 h after treatments. Extracts, fractions, and pure compounds were tested at 500, 400, 300, 200, 100, and 50 µg/mL. Each metabolite solution was also tested for phytotoxicity on V. faba. The aphid mortality has been calculated in each treatment as the mean value ± standard deviation, in three replicates for each time point (24,48, and 72 h). The experiments were repeated at least twice.
Statistical analysis. Data analysis was performed with SPSS 11.0 software (Statistics for Windows Version 24.0, IBM Corp., Armonk, NY, USA), and statistical analysis was done using one-way analysis of variance (ANOVA). The Least Significant Difference (LSD) post hoc test with p < 0.05 was used to analyse the multiple comparisons. The mean values between different treatments at the same time point and the mean values between the same treatments at different time points were compared.

Conclusions
In this work six known metabolites with activity against the pea aphid A. pisum have been isolated from culture filtrates of selected fungal strains; for some of them this is the first report of insecticidal activity (Table S1). Moreover, a novel anti-aphid natural product, named chlamyphilone, has been isolated form the culture filtrate of P. chlamydosporia and fully characterized. The isolation of natural products with beneficial effects for plants may help to formulate novel secondary metabolites-based biopesticides, which represent promising alternatives to synthetic chemicals in agriculture. Further investigations should optimize extraction protocols and define environmental parameters that can affect the commercial formulations based on these natural compounds.
Supplementary Materials: The following are available online, Figures S1-S11 and S13-S14: NMR and MS spectra of compounds. Figure S12: Conformational model of the new compound named chlamyphilone. Table S1: Biological activity of the isolated compounds.