Secondary Metabolites with Herbicidal and Antifungal Activities from Marine-Derived Fungus Alternaria iridiaustralis

Abstract

Weed and soil-borne pathogens could synergistically affect vegetable growth and result in serious losses. Investigation of agricultural bioactive metabolites from marine-derived fungus Alternaria iridiaustralis yielded polyketides (1–4), benzopyrones (5–7), meroterpenoid derivatives (8), and alkaloid (9). The structures and absolute configurations of new 1, 3, 5–6, and 8 were elucidated by extensive spectroscopic analyses, as well as comparisons between measured and calculated ECD and ¹³C NMR data. Compounds 1–4, 6, and 9 showed herbicidal potentials against the radicle growth of Echinochloa crusgalli seedlings. Especially 9 exhibited inhibition rates over 90% at concentrations of 20 and 40 μg/mL, even better than the commonly used chemical herbicide acetochlor. Furthermore, 9 also performed a wide herbicidal spectrum against the malignant weeds Digitaria sanguinalis, Portulaca oleracea, and Descurainia sophia. Compounds 5–8 showed antifungal activities against carbendazim-resistant strains of Botrytis cinerea, with minimum inhibitory concentration (MIC) values ranging from 32 to 128 μg/mL, which were better than those of carbendazim (MIC = 256 μg/mL). Especially 6 exhibited integrated effects against both soil-borne pathogens and weed. Overall, marine-derived fungus A. iridiaustralis, which produces herbicidal and antifungal metabolites 1–9, showed the potential for use as a microbial pesticide to control both weed and soil-borne pathogens.

1. Introduction

Weed seeds widely distributed in soil could compete for nutrients, moisture, and light with vegetables, while soil-borne pathogens could directly invade vegetable roots and further reinforce weed harm [1,2]. For example, Echinochloa crusgalli is the most destructive malignant weed in the rice field [3], while Botrytis cinerea and Fusarium oxysporum are seriously damaging soil-borne pathogens that cause gray mold and wilt diseases of vegetables, respectively [4]. Due to ongoing unrestricted applications of chemical pesticides, weed and soil-borne pathogens have gradually developed multiple resistances, and, especially, no chemical pesticides could control both weed and soil-borne pathogens [5,6]. Therefore, the search for integrated biocontrol alternatives is always in demand.

Suaeda glauca, a kind of salt-tolerant plant, mainly grows in coastal or intertidal zones [7]. Due to its internal and external high-salinity environments, S. glauca has been considered a potential source for various bioactive endophytes, which could produce different interesting secondary metabolites [8,9,10,11,12]. The endophytic genus of Alternaria is a ubiquitous group growing in diverse ecosystems, producing a broad array of secondary metabolites. These metabolites mainly include polyketides, nitrogen-containing compounds, quinones, terpenes, and so on [13,14]. Research on their bioactive potentials mainly focused on the pharmacological applications, such as anticancer, antibacterial, antioxidant, and enzyme inhibitory effects, but there were few reports on their agricultural bioactive potential [13,14,15,16].

During our ongoing search for biocontrol agents in agriculture [8,9,10,17,18], S. glauca-derived endophytic fungus of A. iridiaustralis has attracted our attention because of its integrated potential against both weed and soil-borne pathogens. Our search for agricultural bioactive metabolites obtained nine ones (1–9), including four polyketides (1–4), three benzopyrones (5–7), one meroterpenoid derivative (8), and one alkaloid (9) (Figure 1). The isolation, structural elucidation, and agricultural bioactive evaluation of isolated metabolites are discussed herein.

2. Results and Discussion

2.1. Structure Elucidations

The molecular formula of compound 1 was obtained as C₁₉H₂₆O₄ by HRESIMS (Figure S1 in the Supporting Information, SI), implying seven degrees of unsaturation. The one and two-dimensional NMR data (

Table 1. ¹H (500 MHz) and ¹³C (125 MHz) NMR data of 1 and 3 (CD₃OD, δ: ppm).

	Compound 1		Compound 3
No.	δC (type)	δH (Mult., J in Hz)	δC (type)	δH (Mult., J in Hz)
1	47.7, CH	2.63, t (10.9)	47.7, CH	2.80, dd (11.7, 10.0)
2	36.5, CH	2.55, m	36.5, CH	2.71, m
3	131.6, CH	5.47, dd (10.5, 2.0)	131.6, CH	5.64, dd (10.0, 1.7)
4	132.7, CH	5.69, dd (10.5, 2.5)	132.7, CH	5.85, dd (10.0, 2.6)
5	38.5, CH	2.16, m	38.5, CH	2.32, m
6	30.9, CH2	1.71, m; 1.25, m	30.9, CH2	1.87, m; 1.41, m
7	29.5, CH2	1.38, m	29.5, CH2	1.56, m
8	27.3, CH2	1.73, m; 1.22, m	27.3, CH2	1.91, m; 1.36, m
9	21.9, CH2	1.45, m	21.9, CH2	1.62, m
10	37.6, CH	2.24, m	37.6, CH	2.40, m
11	170.8, C		170.6, C
12	98.6, CH	6.67, s	98.7, CH	6.84, s
13	171.5, C		171.1, C
13-OCH3	57.9, CH3	3.99, s	57.9, CH3	4.15, s
14	101.4, C		102.1, C
15	167.5, CO		167.8, CO
16	64.1, CH2	4.31, s	61.3, CH2	4.68, d (10.4); 4.52, d (10.4)
16-OCH3	58.3, CH3	3.33, s
17	20.5, CH3	0.96, d (7.0)	20.5, CH3	1.12, d (7.0)
1′			15.6, CH3	1.29, d (6.3)
2′			81.0, CH	3.48, ov
3′			71.6, CH	3.79, m
4′			18.4, CH3	1.26, d (6.4)

ov: overlapped ¹H NMR signals.

and Figure 2) exhibited one carbonyl carbon (δ_C 167.5 CO) and three double bonds (δ_C 171.5 C, 170.8 C, 132.7 CH, 131.6 CH, 101.4 C, 98.6 CH), totally accounting for four degrees of unsaturation. Therefore, the remaining three degrees indicate the presence of three rings in the structure of 1.

The decalin ring system, requiring two degrees of unsaturation, was deduced by the consecutive COSY cross-peaks from H-1 to H-10 and from H-2 to H₃-17 (Figure 2), while the presence of one pyrone ring was confirmed by the observed HMBC correlations shown in Figure 2. Detailed analyses of its NMR data suggested that the structure of compound 1 was similar to that of solanapyrone B (compound 2) [19], except the signals of one more OCH₃ group (δ_H 3.33 and δ_C 58.3) were observed in ¹H and ¹³C NMR spectra of 1. The key HMBC correlation between H-16 and this OCH₃ carbon further confirmed the linkage between C-16 and the OCH₃ group (Figure 2).

Solanapyrone S (compound 3) was confirmed to have the molecular formula C₂₂H₃₂O₅ by its HRESIMS data, requiring seven degrees of unsaturation (Figure S9). Its one-dimensional NMR and HSQC data (Table 1 and Figure S13) exhibited marked similarities to those of solanapyrone B (compound 2) [19], except the presence of 2′,3′-butanediol residue (CH₃-1′ δ_H 1.29/δ_C 15.6, OCH-2′ δ_H 3.48/δ_C 81.0, OCH-3′ δ_H 3.79/δ_C 71.6, CH₃-4′ δ_H 1.26/δ_C 18.4) was observed in ¹H and ¹³C NMR spectra of 3. Finally, the consecutive COSY cross-peaks from H₃-1′ to H₃-4′ and the key HMBC correlation between H-16 and OCH-2′ confirmed the connection between C-16 and 2′,3′-butanediol residue (Figure 2).

The NMR signals of 1 and 3 associated with the decalin unit were almost identical to those of solanapyrone B (2) [19], indicating their same relative configurations, which were confirmed by the key NOE correlations from H-10 to H-2 and H-5, as well as from H-1 to H-12 and H₃-17 (Figure 2). The absolute configurations of decalin fragments of 1 and 3 were determined as 1R, 2S, 5R, and 10R via the agreement between the experimental and calculated ECD spectra, showing the same positive Cotton Effect (CE) around 210 nm and the negative CE near 295 nm (Figure 3). While the calculated ECD spectra of (1S, 2R, 5S and 10S)-1 and 3 exhibited mirror-corresponding CEs. The same CEs of 1 and 3 should be related to their common pyrone and cis-decalin ring systems, while the 2′,3′-butanediol residue of 3 was far from the chromophore center and therefore did not exert the obvious effect of its CEs.

The DFT re-optimization of initial MMFF conformers of 1 and 3 at the B3LYP/6-311++g(d, p) level afforded three low-energy conformers above 1% population, respectively (Figures S36 and S37). Their further ¹³C NMR calculations could support the absolute configurations of the decalin fragments of 1 and 3 assigned by the ECD calculations and also confirm the 2′,3′-butanediol residue of 3 as 2′R and 3′R [20,21]. The correlation coefficients (R²) of 1 and 3 from linear regression analyses between calculated and experimental ¹³C NMR data were 0.9982 and 0.9979, respectively (Figure S39).

The HRESIMS data for compound 5 demonstrated its molecular formula to be C₁₃H₁₄O₆S, indicating seven degrees of unsaturation (Figure S17). The one- and two-dimensional NMR data (

Table 2. ¹H (500 MHz) and ¹³C (125 MHz) NMR data of 5–6 and 8 (δ: ppm).

	Compound 5		Compound 6			Compound 8
No.	δC	δH	δC	δH	No.	δC	δH
1	167.3, C		165.7, C		1	203.0, CO
2	109.3, CH	6.10, s	111.5, CH	5.97, s	2	126.1, CH	5.88, s
3	182.5, C		182.4, C		3	161.0, C
4	105.2, C		115.1, C		4	32.0, CH2	2.63, dd (18.3, 3.7) 2.53, dd (18.3, 6.0)
5	159.9, C		140.8, C		5	73.1, CH	4.02, m
6	101.2, C		124.7, C		6	49.9, C
7	164.0, C		162.3, C		7	18.5, CH3	1.09, s
8	90.6, CH	6.45, s	100.7, CH	6.65, s	8	23.4, CH2	1.70, q (7.5)
9	158.6, C		159.4, C		9	7.7, CH3	0.86, t (7.5)
10	20.7, CH3	2.39, s	19.7, CH3	2.30, s	1′	15.5, CH3	1.08, ov
11	49.4, CH2	4.45, s	17.6, CH3	2.75, s	2′	49.6, CH	2.30, m
12			11.6, CH3	2.17, s	3′	70.2, CH	3.80, dt (12.9, 6.0)
5-OH		13.35, s			4′	21.4, CH3	1.25, d (6.0)
SCH3	41.5	2.91, s
7-OCH3	56.6	3.95, s

Compounds 5 and 8 were determined using CDCl₃, while 6 using CD₃OD (¹H of 500 MHz and ¹³C of 125 MHz), respectively; ov: overlapped ¹H NMR signals.

and Figure 2) exhibited one carbonyl carbon (δ_C 182.5 CO), six aromatic carbons (δ_C 164.0 C, 159.9 C, 158.6 C, 105.2 C, 101.2 C, 90.6 CH), and one double bond (δ_C 167.3 C, 109.3 CH), totally accounting for five degrees of unsaturation. Therefore, the remaining two degrees should be related to the cyclic ring systems.

Detailed analysis of the HMBC spectrum deduces the presence of a benzopyrone skeleton with CH₃ (δ_H 2.39), OH (δ_H 13.35), and OCH₃ (δ_H 3.95) groups substituted at C-1, C-5, and C-7, respectively (Figure 2). Considering its molecular formula and the representative fragmentation [M—SO₂CH₃]⁺ (219.06537), compound 5 should contain a SO₂ group, which is rarely found in Alternaria metabolites (Figure S17). The one-dimensional NMR spectra of 5 showed remarkable similarities to those of chaetoquadrin D [22], except that signals of N-ethyl acetamide residue in chaetoquadrin D (SCH₂ δ_H 3.20/δ_C 52.7, NCH₂ δ_H 3.78/δ_C 32.8, CO δ_C 170.2, CH₃ δ_H 1.94/δ_C 23.2) were absent from the NMR spectra of 5. Instead, SCH₃ signals (δ_H 2.91/δ_C 41.5) were observed, which was also confirmed by the key HMBC correlation between H-SCH₃ to C-11 (Figure 2).

The molecular formula C₁₂H₁₂O₃ of compound 6 was assigned on the basis of its HRESIMS data, indicating seven degrees of unsaturation (Figure S23). Detailed analyses of ¹H-¹H COSY and HMBC spectra confirmed the presence of a benzopyrone skeleton with three CH₃ groups (δ_H 2.30, 2.75, and 2.17) substituted at C-1, C-5, and C-6, respectively (Figure 2). One-dimensional NMR spectra of 6 were almost identical to those of chaetosemin D (7) [23], except that signals of 2′-hydroxy propyl residue in 7 (CH₂-1′ δ_H 2.69/δ_C 44.1, OCH-2′ δ_H 4.20/δ_C 66.9, CH₃-3′ δ_H 1.29/δ_C 23.5) were absent from NMR spectra of 6. Instead, CH₃ signals (δ_H 2.30/δ_C 19.7) were observed in 6.

Compound 8 showed the molecular formula C₁₃H₂₂O₃ as determined by HRESIMS, suggesting three degrees of unsaturation (Figure S28). Its NMR data (Table 2 and Figure 2) exhibited one carbonyl carbon (δ_C 203.0 CO) and one double bond (δ_C 161.0 C, 126.1 CH), which indicated two degrees of unsaturation. Therefore, the remaining one degree should be related to the presence of one cyclic ring, which was also confirmed by the ¹H-¹H COSY and HMBC spectra (Figure 2). The ethyl and 3′-hydroxy butyl residues were deduced by the consecutive COSY cross-peaks from H₂-8 to H₃-9 and from H₃-1′ to H₃-4′. The key HMBC correlations from H₃-7 to C-1, 6 and 8, as well as from H-2′ to C-2, 3 and 4, finally connected the ethyl and 3′-hydroxy butyl residues to C-6 and C-3, respectively.

The relative configuration of the cyclohexanone skeleton in compound 8 was deduced by the key NOE correlation from H-5 to H₃-7 (Figure 2). The agreement of experimental and calculated ECD spectra of 8, showing the same positive CE around 240 nm and negative CEs near 210 and 330 nm, confirmed the absolute configurations of the cyclohexanone fragment as 5R and 6R (Figure 3). Its further ¹³C NMR calculation deduced the absolute configuration of 3′-hydroxy butyl group as 2′S and 3′S [20,21]. The correlation coefficient (R²) of 8 from linear regression analysis between calculated and experimental ¹³C NMR data was 0.9948 (Figures S38 and S39).

The isolation of bioactive fractions from the culture extract also resulted in other known metabolites, including polyketides (2 and 4), benzopyrone (7), and alkaloids (9). Their structures were determined by detailed analyses of their spectroscopic data and comparisons with previously published reports as follows: solanapyrone B (2) [19], probetaenone I (4) [24], chaetosemin D (7) [23], and tenuazonic acid (9) [25]. All known metabolites (2, 4, 7, and 9) were first isolated from the species of A. iridiaustralis.

2.2. Herbicidal and Antifungal Evaluations

The isolated metabolites (1–9) were evaluated for their herbicidal and antifungal activities. The herbicidal potential was assessed using the representative malignant weed E. crusgalli, while the antifungal activity was assessed using two groups of representative soil-borne pathogens: carbendazim-resistant isolates of B. cinerea from grape (BCG) and strawberry (BCS), as well as F. oxysporum strains of F. oxysporum f. sp. cucumerinum (FOC) and F. oxysporum f. sp. Lycopersici (FOL).

The polyketides 1–4, benzopyrone 6, and alkaloid 9 showed herbicidal potentials against the radicle growth of E. crusgalli seedlings with a dose-dependent relationship (

Table 3. Herbicidal (Inhibition rates: %) and antifungal (MIC: μg/mL) potentials of the isolated metabolites 1–9.

No.	Herbicidal Activities			Antifungal Activities
	40 μg/mL	20	10	BCG	BCS	FOC	FOL
1	57.1 ± 3.1c	45.4 ± 2.9d	<20	—	—	—	—
2	61.3 ± 2.4c	50.4 ± 1.2d	23.7 ± 2.6c	—	—	—	—
3	43.1 ± 1.7d	28.9 ± 3.0f	n.d.	—	—	—	—
4	50.4 ± 2.3cd	36.2 ± 3.5e	n.d.	—	—	—	—
5	—	n.d.	n.d.	64	64	—	—
6	72.6 ± 1.9b	60.3 ± 2.4c	30.1 ± 2.2b	64	64	128	256
7	—	n.d.	n.d.	128	128	256	256
8	—	n.d.	n.d.	32	32	128	256
9	98.3 ± 0.3a	90.2 ± 1.5a	67.3 ± 2.7a	—	—	—	—
CK	91.7 ± 3.4a	80.4 ± 2.1b	74.2 ± 1.5a	256	256	8	8

CK of herbicidal and antifungal bioassays were commonly used chemical pesticides acetochlor and carbendazim, respectively; “—”: no activity; n.d.: not detected. Different lowercase letters in a column indicated the means were significantly different at p < 0.05.

). Especially, 9 exhibited significant inhibition rates over 90% at concentrations of 20 and 40 μg/mL, even better than the commonly used chemical herbicide acetochlor, while 6 showed moderate inhibition rates of 60.3% and 72.6%, respectively (Table 3 and Figure 4). The further bioassay of the herbicidal spectrum of 9 suggested that it performed significant herbicidal potential against the malignant weed Digitaria sanguinalis, almost identical to that of acetochlor (Figure S40), while 9 also exhibited moderate activities against Portulaca oleracea and Descurainia sophia (Table S1). The preliminary structure-activity analysis of solanapyrone polyketides 1–3 indicated that the substituted group at C-16 should be related to their herbicidal activities.

Benzopyrones 5–6 and meroterpenoid derivative 8 showed antifungal potentials against two carbendazim-resistant strains of B. cinerea with MIC values ranging from 32 to 64 μg/mL, significantly better than those of carbendazim (MIC = 256 μg/mL) (Table 3). B. cinerea could widely invade various crops and vegetables during both the pre- and post-harvest stages. More seriously, its resistance to commonly used fungicides was developing year by year, also resulting in higher pesticide residue [4]. The antifungal target of carbendazim was related to β-tubulin proteins [26], suggesting that the antifungal mechanisms of 5–6 and 8 should be different from that of carbendazim. Furthermore, 6–8 also exhibited moderate antifungal activities against two F. oxysporum strains.

Alkaloid 9, possessing a relatively simple skeleton and a wide herbicidal spectrum, showed the potential for use as a bio-herbicide. Although the antifungal and herbicidal activity of 6 was weaker than that of 8 and 9, respectively, its integrated agricultural potential against both soil-borne pathogens and weeds indicated its application in the development of bio-pesticides.

3. Materials and Methods

3.1. General Procedures

NMR spectra were recorded at 500 and 125 MHz for ¹H and ¹³C, respectively, on a Bruker Avance III spectrometer (Bruker, Rheinstetten, Germany). HRESIMS data were determined on a mass spectrometer of Thermo Scientific Orbitrap Fusion Lumos Tribrid (Thermo Scientific, MA, USA) and analyzed using Thermo Xcalibur 4.2 SP1. The circular dichroism (CD) spectrum was acquired on a JASCO J-810 CD spectrometer (JASCO, Tokyo, Japan). Column chromatography (CC) was performed with Silica gel (200–300 mesh; Qingdao Haiyang Chemical Co., Qingdao, China), Lobar LiChroprep RP-18 (40–63 μm; Merck, Kenilworth, NJ, USA), and Sephadex LH–20 (18–110 μm; Merck, Kenilworth, NJ, USA). Semi-preparative HPLC (semi-pHPLC) was performed using a Dionex HPLC system equipped with a P680 pump (flow rate: 3 mL/min), an ASI-100 automated sample injector, and a UVD340U multiple wavelength detector (Detection wavelength: 230 nm) controlled using Chromeleon software, version 6.80 (Dionex Corporation, Sunnyvale, CA, USA).

3.2. Fungal Strain and Weed Seeds

The fungal strain of A. iridiaustralis was isolated from the root of S. glauca, which was collected from the intertidal zone of the Yellow River Delta, Dongying, China, in October 2021. The fungus was identified on the basis of morphological characteristics and molecular analyses of the ITS (Internal Transcribed Spacer)-5.8S rDNA region sequence [10]. The strain was deposited in the Green Pesticide Development Laboratory, Qingdao Agricultural University. F. oxysporum strains, as well as weed seeds of E. crusgalli, D. sanguinalis, P. oleracea, and D. sophia, were provided by the College of Plant Disease, Qingdao Agricultural University, while carbendazim-resistant strains of B. cinerea were isolated and identified by the Green Pesticide Development Laboratory.

3.3. Fermentation, Extraction, and Isolation

The fungus A. iridiaustralis was transferred to PDA medium and cultured at 28 °C for 7 days. Then pieces of fresh mycelia were inoculated and statically fermented at 28 °C for 30 days on the solid rice medium, which was conducted in 40 × 1 L conical flasks containing rice (100 g/flask), peptone (0.6 g/flask), and natural seawater (100 mL/flask).

The fungal culture was exhaustively extracted using ethyl acetate (EtOAc) to obtain a crude extract (12.6 g), which was fractionated via silica gel vacuum liquid chromatography with the eluting gradient of petroleum ether/EtOAc (40:1, 20:1, 10:1, 5:1, and 1:1) and then dichloromethane (CH₂Cl₂)/methanol (MeOH) (20:1, 10:1, 5:1, and 1:1) to yield nine fractions [Fractions (Frs.) 1–9].

Antagonistic Fr. 5 was purified via CC over RP-C18 eluting with a MeOH−H₂O gradient (from 1:9 to 1:0) to obtain six subfractions (Fr.5-1 to 5-6). Fr.5-2 was isolated via CC over Sephadex LH-20 (MeOH) to yield two subfractions; one was purified using semi-pHPLC (35% MeOH−H₂O) to obtain compounds 5 (8.2 mg, t_R 11.2 min), 6 (12.3 mg, t_R 14.5 min), and 7 (9.1 mg, t_R 17.9 min), while the other used semi-pHPLC (40% MeOH−H₂O) to obtain compound 8 (4.4 mg, t_R 13.7 min). Fr.5-3 was first separated via CC over Sephadex LH-20 (MeOH) and then purified using semi-pHPLC (53% MeOH−H₂O) to obtain compounds 9 (21.7 mg, t_R 16.3 min) and 4 (13.8 mg, t_R 19.7 min). Fr.5-4 was first isolated via semi-pHPLC (70% MeOH−H₂O) and then via CC over Sephadex LH-20 (acetone) to obtain compounds 1 (6.6 mg), 2 (9.2 mg), and 3 (6.8 mg).

16-methoxy solanapyrone B (1): White powder. [α${]}_{\mathrm{D}}^{24}$ = –42.4, c 1.15, CHCl₃; UV (CH₃OH) λ_max (log ε) 205 (2.31), 302 (1.15) nm; ECD (CH₃OH) λ_max ([θ]) 298 (–30.64), 207 (+59.15) nm; ¹H and ¹³C NMR data, see Table 1; HRESIMS m/z 319.19006 [M + H]⁺ (calcd for C₁₉H₂₇O₄, 319.19039; Mass error: –3.26 ppm).

Solanapyrone S (3): White powder. [α${]}_{\mathrm{D}}^{24}$ = –53.7, c 1.22, CHCl₃; UV (CH₃OH) λ_max (log ε) 206 (2.29), 304 (1.08) nm; ECD (CH₃OH) λ_max ([θ]) 298 (–21.92), 206 (+88.03) nm; ¹H and ¹³C NMR data, see Table 1; HRESIMS m/z 399.21359 [M + Na]⁺ (calcd for NaC₂₂H₃₂O₅, 399.21420; Mass error: –6.05 ppm).

Alternanone A (5): White powder. [α${]}_{\mathrm{D}}^{24}$ = +11.4, c 0.94, CH₃OH; UV (CH₃OH) λ_max (log ε) 206 (4.37), 238 (3.81), 255 (4.02), 298 (3.06) nm; ¹H and ¹³C NMR data, see Table 2; HRESIMS m/z 299.05847 [M + H]⁺ (calcd for C₁₃H₁₅O₆S, 299.05839; Mass error: 0.85 ppm), 219.06537 [M—SO₂CH₃]⁺.

Alternanone B (6): White powder. [α${]}_{\mathrm{D}}^{24}$ = +9.8, c 0.93, CH₃OH; UV (CH₃OH) λ_max (log ε) 205 (4.47), 235 (3.72), 254 (4.06), 296 (3.11) nm; ¹H and ¹³C NMR data, see Table 2; HRESIMS m/z 205.08658 [M + H]⁺ (calcd for C₁₂H₁₃O₃, 205.08592; Mass error: 6.59 ppm).

Alternanone C (8): White powder. [α${]}_{\mathrm{D}}^{24}$ = +8.4, c 1.07, CH₃OH; UV (CH₃OH) λ_max (log ε) 208 (2.31), 242 (2.04), 328 (1.03) nm; ECD (CH₃OH) λ_max ([θ]) 330 (–2.45), 240 (+17.12), 212 (–3.93) nm; ¹H and ¹³C NMR data, see Table 2; HRESIMS m/z 227.16408 [M + H]⁺ (calcd for C₁₃H₂₃O₃, 227.16417; Mass error: –0.91 ppm).

3.4. Calculations of ECD and ¹³C NMR Data

Conformational searches were carried out by means of the Merck Molecular Force Field (MMFF) using Spartan’s 10 software. The conformers with a Boltzmann population over 1% were chosen for ECD and ¹³C NMR data calculations. The optimized geometries of predominant conformers (weighting factors) for compounds 1, 3, and 8 at the B3LYP/6-311++g(d, p) level above 1% population were shown in Figures S36–S38, respectively. Further calculations of their ECD and ¹³C NMR data were performed as described previously [10,17,21].

3.5. Herbicidal and Antifungal Evaluations

Herbicidal bioassays of compounds 1–9 against E. crusgalli were performed using the grinded plant tissue powders mixed with agar method as described previously [9,10]. Briefly, weed seeds were pretreated with sodium hypochlorite (0.2%) for 15 min and then soaked with flowing water for 4–6 h. Wet seeds were germinated for 12 h on the moist filter paper under 28 °C in a dark condition. Isolated compounds were dissolved with methanol to obtain sample solutions of different concentrations. 1 mL sample solution and 99 mL water (containing 0.5 g agar) were mixed to yield the agar solution (1% methanol), which was further divided into three beakers. Subsequently, germinated seeds with the same radicle lengths were planted into beakers and then cultivated in the artificial climate box under a 28 °C light-avoidance condition. After 3 days, the stem and root lengths of weed seedlings were measured and compared to the untreated control. The inhibition rate was calculated using the formula as follows:

Inhibition rate (%) = [(L_control − L_treatment)/L_control] × 100

Due to the significant herbicidal potential of 9, its herbicidal spectrum was further evaluated using malignant weeds D. sanguinalis, P. oleracea, and D. Sophia, which were widely distributed in North China, resulting in serious economic losses.

Antifungal bioassays of isolated metabolites (1–9) against two groups of representative soil-borne phytopathogens, including carbendazim-resistant isolates of B. cinerea from grape (BCG) and strawberry (BCS), as well as F. oxysporum strains of F. oxysporum f. sp. cucumerinum (FOC) and F. oxysporum f. sp. Lycopersici (FOL), were performed using the broth microdilution method in 96-well plates [17,18]. Briefly, isolated compounds, dissolving in 50% DMSO aqueous solution, were added 5 μL per well to 95 μL Potato Dextrose Broth (PDB). 96-well plates with B. cinerea and F. oxysporum strains were cultivated at 25 °C and 28 °C for three days, respectively.

4. Conclusions

An investigation of agricultural bioactive metabolites from the marine-derived fungus A. iridiaustralis obtained nine metabolites (1–9), including five novel ones (1, 3, 5–6, and 8). Their structures and absolute configurations were elucidated by extensive spectroscopic analyses as well as comparisons between measured and calculated ECD and ¹³C NMR data. Compounds 1–4, 6, and 9 showed herbicidal potential against the radicle growth of E. crusgalli seedlings. Especially 9 exhibited inhibition rates over 90% at concentrations of 20 and 40 μg/mL, even better than the chemical herbicide acetochlor. Furthermore, 9 also performed a wide herbicidal spectrum against malignant weeds D. sanguinalis, P. oleracea, and D. Sophia. Compounds 5–8 showed antifungal activities against carbendazim-resistant strains of B. cinerea that were better than those of carbendazim. Although the antifungal activity of 6 was weaker than that of 8, its integrated agricultural potential against both weeds and soil-borne pathogens indicated its application in the development of bio-pesticides. Overall, the marine-derived fungus A. iridiaustralis, which produces herbicidal and antifungal metabolites 1–9, showed the potential for use as a microbial pesticide to control both weed and soil-borne pathogens.