Antifungal Metabolites From Alternaria atrans : An Endophytic Fungus in Psidium guajava

Abstract

Six metabolites (1–6), including 1 new fusaric acid derivative, designated atransfusarin (1), and 5 known compounds, (3R,6R)-3-benzyl-6-isopropyl-4-methylmorpholine-2,5-dione (2), daucoterol (3), adenosine (4), cerebroside B (5), and 2,3-dihydroxypropyl (Z,Z)-9,12-octadecadienate (6), were isolated from the solid culture of an endophytic Alternaria atrans MP-7, associated with the medicinal plant Psidium guajava. The structure of the new compound was elucidated by spectral data. The antifungal test indicated that (3R,6R)-3-benzyl-6-isopropyl-4-methylmorpholine-2,5-dione (2) showed obvious inhibitory activity against Alternaria solani, Colletotrichum gloeosporioides, and Phyricularia grisea. Compared to fusaric acid (1a), even though the antifungal activity of atransfusarin (1) against A. solani, C. gloeosporioides, and P. grisea was greatly decreased, its inhibitory effect against Botrytis cinerea was still retained.

Endophytes are defined as microorganisms that inhabit internal tissues of plants without causing any apparent disease symptoms. ^1–4 They live with their host in a mutualistic relationship during at least a part of their life span and are thought to protect their host against pathogens. ⁵ Endophytic fungi are also considered to be a pivotal and prolific source of bioactive secondary metabolites with promising medicinal, agricultural, or industrial applications. ⁶

Psidium guajava (family Myrtacea) is a fruit tree wildly distributed throughout the tropical and subtropical areas. Its leaves are used as antidiabetic, hepatoprotective, antioxidant, hemostatic, and anti-inflammatory agents in folk medicine. ⁷ Chemical investigations on the leaves of P. guajava led to the isolation of several sesquiterpene- and monoterpene-based meroterpenoids with unprecedented skeletons. ⁸ However, little is known about secondary metabolites of endophytes harbored inside the healthy tissues of P. guajava.

During our ongoing search for new biologically active compounds from fungi, ^9–12 we isolated the endophyte Alternaria atrans MP-7 from the leaves of P. guajava. In the present study, a new fusaric acid derivative, atransfusarin (1), along with 5 known compounds (2–6), was isolated from a methanol extract derived from a solid rice medium of A. atrans. In this study, we reported the structure elucidation of a new natural compound 1 along with 5 known compounds. In addition, their antifungal activity against four phytopathogenic fungi including Alternaria solani, Colletotrichum gloeosporioides, Phyricularia grisea, and Botrytis cinerea was tested.

Fractionation of the chloroform soluble extracts of culture of A. atrans MP-7 grown on solid rice medium by multiple Sephadex LH-20 and MCI column chromatography (CC) yielded one new fusaric acid (1a) derivative, atransfusarin (1), along with 5 known compounds, (3R,6R)−3-benzyl-6-isopropyl-4-methylmorpholine-2,5-dione (2) ¹³ (Figure 1(a)), daucoterol (3), ¹⁴ adenosine (4), ⁴ cerebroside B (5), ¹⁴ and 2,3-dihydroxypropyl (Z,Z)−9,12-octadecadienate (6). ¹⁵ Compound 2 was isolated from genus Alternaria for the first time.

Figure 1

(a) Structures of compounds 1, fusaric acid (1a), (S)-(+)-fusarinolic acid (1b), and 2; (b) Key HMBC and COSY correlations of compound 1.

Compound 1 was isolated as a colorless crystal. Its molecular formula was determined to be C₁₃H₁₈N₂O₄ by high-resolution electrospray ionization–mass spectrometry (HRESIMS) at m/z 267.1325 [M + H]⁺ (calcd for 267.1339), indicating 6 degrees of unsaturation. The ultraviolet (UV) spectrum indicated the presence of aromatic ring at 245 nm. The infrared (IR) spectrum showed absorption bands of hydroxyl and amide –NH– groups (overlap 3442 cm^–1) and carbonyl groups (1663 cm^–1). The ¹³C NMR data coupled with the heteronuclear single quantum correlation spectrum of 1 showed 13 carbon signals corresponding to 2 methyl groups at δ_C 24.0 (C-11) and 52.5 (13-OMe); 3 methylene groups at δ_C 29.4 (C-8), 40.2 (C-9), and 41.4 (C-12); 4 methine carbons at δ_C 67.2 (C-10), 122.3 (C-3), 137.2 (C-4), and 148.6 (C-6); and four quaternary carbons at δ_C 141.1 (C-5), 147.3 (C-2), 164.9 (C-7), and 170.4 (C-13). In the ¹H NMR spectrum, there were three aromatic protons at δ_H 7.68 (1H, d, J = 7.9 Hz, H-4), 8.09 (1H, d, J = 7.9 Hz, H-3), and 8.43 (1H, s, H-6). These data suggested the presence of a 2,5-disubstituted pyridine ring, which was confirmed by the key heteronuclear multiple bond correlation (HMBC) cross-peaks from H-3 to C-5, from H-4 to C-2 and C-6, and from H-6 to C-2 and C-4, as well as by the correlation spectroscopy (COSY) correlations of H-3/H-4 (Figure 1(b)).

Furthermore, 1 oxygenated methine at δ_H 3.83 (1H, m, H-10); 2 methyl groups at δ_H 1.25 (3H, d, J = 5.3 Hz, H₃-11) and 3.79 (3H, s, 14-OMe); and 3 methylene groups at δ_H 1.77 (2H, d, J = 6.4 Hz, H₂-9), 2.73–2.86 (2H, m, H₂-8), and 4.26 (2H, d, J = 5.3 Hz, H₂-12) were observed (Table 1). The COSY correlations of H₂-8/H₂-9/H-10/H₃-11 (Figure 1(b)), together with the HMBC correlations from H₂-8/C-5, C-10, H₂-9/C-8 and C-10, and H₃-11/C-10, and H-4 and H-6/C-8 indicated a hydroxy-containing butyl moiety at C-5 and the OH group attached to C-10. Further HMBC correlations of H₂-12/C-7 and C-13, H₃-14/C-13 suggested a methyl acetate moiety linked to C-7 via an NH group (Figure 1(b)). From the above data, the structure of 1 was identified as a new fusaric acid derivative containing a glycine methyl ester. Attempts to determine the absolute configuration of C-10 by the modified Mosher’s method were unsuccessful due to the insufficient amount of this sample. Notably, the mycotoxin fusaric acid (1a) is produced by a large number of species of the Fusarium genus. ¹⁶

Table 1

1D and 2D NMR Data of Compound 1 (CDCl₃).

No	δH	δC	HMBC	COSY
2		147.3
3	8.09 (1H, d, J = 7.9 Hz)	122.3	5, 7	4
4	7.68 (1H, d, J = 7.9 Hz)	137.2	2, 6, 8	3
5		141.1
6	8.43 (s, H)	148.6	2, 4, 5, 8
7		164.9
8	2.73–2.86 (2H, m)	29.4	4, 5, 6, 9, 10	9
9	1.77 (2H, d, J = 6.4 Hz)	40.2	5, 8, 10, 11	8, 10
10	3.83 (1H, m)	67.2	8	9, 11
11	1.25 (3H, d, J = 5.3 Hz)	24.0	8, 9, 10	10
12	4.26 (2H, d, J = 5.3 Hz)	41.4	7, 13
13		170.4
14	3.79 (3H, s)	52.5	13

The absolute configuration of the chiral center C-10 in 1 was established to be S, as indicated by its specific optical rotation value ([α]_D ²⁵ +5.2, c 0.10 in MeOH), which has the same positive value as that of the related synthetic compound, (S)-(+)-fusarinolic acid (1b) ([α]_D ²⁵ +19.5, c 1.5 in MeOH). ¹⁶ This lower value is due to a possibility that a small amount of enantiomer is present in compound 1. Thus, the structure of 1 was identified as, and named, (+)-atransfusarin (Figure 1).

All isolated compounds (1–6) were tested for their toxic and antifungal activity. Only 2 exhibited toxic activity against the brine shrimp (Artemia salina) with a median lethal concentration (LC₅₀) value of 25.5 µg/mL, while the others showed no activity (Table S1). In addition, the antifungal activity of 1– 6 was also tested against several phytopathogenic fungi including B. cinerea, A. solani, C. gloeosporioides, and P. grisea (Cooke) Sacc. As shown in Table 2, compound 2 exhibited remarkable antifungal activity against A. solani, C. gloeosporioides, and P. grisea with minimum inhibitory concentrations (MICs) of all 6.25 µM, which were much better than that of the positive control, carbendazim and fusaric acid (1a). What is more, (+)-atransfusarin (1) exerted weak activity against B. cinerea and A. solani (MIC = 50 µM), but none of the remaining compounds proved to be active against the test pathogens (MIC >100 µM).

Table 2

Antifungal Activity of Compounds (1–6).

Compound	Phytopathogenic fungi (MIC/μM)
	Botrytis cinerea	Alternaria solant	Colletotrichum gloeosporioides	Phyricularia grisea
1	50	50	100	100
2	25	6.25	6.25	6.25
3	200	100	100	＞200
4	>200	>200	>200	>200
5	>200	>200	200	>200
6	200	100	200	>200
Carbendazim	25	12.5	50	50
Fusaric acid	50	12.5	50	50

In this study, 6 compounds were purified from an endophytic A. atrans MP-7, in which compound 1 was new and compound 2 was isolated from genus Alternaria for the first time. As the main constituent, compound 2 showed remarkable antifungal activity against all the 4 test phytopathogenic fungi including B. cinerea, A. solani, C. gloeosporioides, and P. grisea, better than that of a common broad-spectrum fungicide carbendazim. As a result, it could be exploited as a new natural resource of antifungal agent.

Experimental

General

Optical rotations were measured on a Rudolph Autopol III automatic polarimeter (Rudolph Research Analytical, Hackettstown, New Jersey). UV spectra were obtained on an UV−vis Thermo Evolution 300 spectrometer (Thermo Scientific, Waltham, Massachusetts). IR spectra were measured with a Bruker TENSOR 27 spectrophotometer (BrukerOptics, Ettlingen, Germany) in KBr pellets. ESIMS data were obtained on a Thermo Fisher LTQ Fleet instrument spectrometer (Thermo Scientific, MA, USA). HRESIMS was performed on a VG Autospec-3000 spectrometer (VG, Manchester, UK). NMR spectra were recorded on a Bruker Avance Ⅲ 500 spectrometer (Bruker BioSpin, Rheinstetten, Germany), δ in ppm rel. to trimethylsilane as internal standard, J in Hz. High performance liquid chromatography analysis and semipreparation was performed on a Waters 1525 instrument (Waters Corp., USA). Column chromatographywas conducted on silica gel (SiO2: 200–300 mesh, Qingdao Marine Chemical Group, Co., Qingdao, China), Sephadex LH-20 (Amersham Pharmacia Biotech, Uppsala, Sweden), MCI Gel (Mitsubishi Chemical Corp., Tokyo, Japan), and Lichroprep RP-18 gel (40, 63 µm, Merck, Germany). GF₂₅₄ plates (Qingdao Marine Chemical Inc., Qingdao, China) were used for thin-layer chromatography (TLC). The fractions were monitored by TLC, and spots were visualized by heating silica gel plates sprayed with 5% H₂SO₄ in EtOH.

Fungal Material

The stems and leaves of P. guajava were collected from Nanning city, Guangxi Province of China, in August 2011, and authenticated by Mr Z.-H. Wu. A voucher specimen (0095460) was deposited at the herbarium of the Northwest A&F University. The endophytic fungal strain MP-7 was isolated under aseptic conditions from the healthy leaves of P. guajava.

The leaves were washed twice with sterile, refined water and dried, followed by surface sterilization using 70% ethanol (2 × 1 min, each) under the flow hood. After drying, the disinfected leaves were dissected into small pieces of 0.5 cm length and placed on a potato dextrose agar (PDA) plate (containing 0.005% Rose bengal and 0.01% kanamycin) followed by incubation for several days at 25°C. Strain MP-7, grown on the plate, was isolated and maintained on another PDA plate.

The strain was identified according to the phylogenetic taxonomy with sequence alignment of internal transcribed spacer and had a genetic closeness of 100% to the A. atrans strain. The strain was thus defined as A. atrans MP-7 and kept in one of the authors’ lab.

Extraction and Isolation

The fungal strain was cultivated axenically on solid rice medium, prepared by autoclaving 40 g of rice and 60 mL of water in a 500 mL Erlenmeyer flask. Fermentation was carried out in 200 Erlenmeyer flasks for 28 days at 25°C under static conditions. After 28 days of fermentation, the fungal culture in each flask was exhaustively extracted with MeOH overnight (3 × 500 mL) followed by filtration and solvent evaporation under reduced pressure. The obtained crude extract was dissolved in 90% MeOH and defatted with petroleum ether to give a crude MeOH extract, which was dissolved in 50% aq. MeOH and partitioned with chloroform. The chloroform soluble portion (45.3 g) was subjected to a silica gel column eluted with CHCl₃-MeOH (100:0, 50:1, 20:1, 10:1, 0:100, v/v), providing 5 fractions 1–5.

Fraction 1 (2.3 g) was subjected to MCI gel (30% MeOH-H₂O), followed by purification with Sephadex LH-20 (MeOH) CC, to yield compounds 2 (184 mg), 3 (7 mg), and 6 (7 mg). Fraction 2 (3.9 g) was subjected to MCI gel (50% MeOH-H₂O), followed by purification with Sephadex LH-20 (MeOH) CC to yield 2 (80 mg). Fraction 3 (5 g) was subjected to MCI gel (70% MeOH-H₂O), followed by purification with Sephadex LH-20 (MeOH) CC, and silica gel CC to give 1 (4.6 mg). Fraction 4 (2.8 g) was subjected to MCI gel (90% MeOH-H₂O), followed by purification with Sephadex LH-20 (MeOH) CC, and silica gel CC to give 4 (14 mg) and 5 (23.7 mg).

Brine Shrimp Bioassay

Metabolites were tested in vitro for the brine shrimp (A. salina) bioassay, processed according to the previously identified method. ^17,18 The final concentrations of the tested compounds were 10, 50, and 100 µg/mL. Toosendanin was used as the positive control and DMSO as the negative control. The lethality of each concentration was recorded.

Antifungal Bioassay

The test phytopathogenic fungi used in this study were B. cinerea, A. solani, C. gloeosporioides, and P. grisea (Cooke) Sacc. All the fungi were isolated from infected plant organs at the Northwest A&F University.

Antifungal activity was assessed by the microbroth dilution method in 96-well culture plates using a PD medium. ^3,19,20 The test compounds were made up to 2 mg/mL in DMSO. Carbendazim (Aladdin Chemistry Co., Ltd, Shanghai, China), a commercial fungicide, was used as a positive control, and the solution of equal concentration of DMSO was used as a negative control. The tested fungi were incubated in the PD medium for 18 hours at 28°C ± 0.5°C at 150 rpm, and spores of different microorganism concentrations were diluted to approximately 1 × 10⁶ CFU with PD medium. Test compounds (10 µL) were added to 96-well microplates, and 90 µL of PD medium was added. Serial dilutions were made in the 96-well round-bottom sterile plates in triplicate in 50 µL of PD medium, and then 50 µL of the fungal suspension was added. After incubation for 48 hours at 28°C ± 0.5°C, MIC was taken as the lowest concentration of the test compounds in the wells of the 96-well plate in which no microbial growth could be observed.

Atransfusarin (1)

Colorless crystal.

[α]_D ²⁵ +5.2 (c 0.10, MeOH).

UV (MeOH) λ_max: 245 nm.

IR (KBr) υ_max: 3442, 1744, 1663, 1530 cm^–1.

¹H (500 MHz) and ¹³C NMR (125 MHz): Table 1.

HRESIMS (positive): m/z 267.1325 [M + H]⁺ (calcd for C₁₃H₁₉N₂O₄, 267.1339), 289.1155 [M + Na]⁺, 555.2447 [2M + Na]⁺.