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

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.


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 J. Fungi 2023, 9, 716 2 of 10 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].

Structure Elucidations
The molecular formula of compound 1 was obtained as C19H26O4 by HRESIM ure S1 in the Supporting Information, SI), implying seven degrees of unsaturat one and two-dimensional NMR data (Table 1 and Figure 2) exhibited one carbony (δC 167.5 CO) and three double bonds (δC 171.5 C, 170.8 C, 132.7 CH, 131.6 CH, 98.6 CH), totally accounting for four degrees of unsaturation. Therefore, the re three degrees indicate the presence of three rings in the structure of 1.
The decalin ring system, requiring two degrees of unsaturation, was deduce consecutive COSY cross-peaks from H-1 to H-10 and from H-2 to H3-17 ( Figure 2 the presence of one pyrone ring was confirmed by the observed HMBC correlation in Figure 2. Detailed analyses of its NMR data suggested that the structure of com 1 was similar to that of solanapyrone B (compound 2) [19], except the signals of o OCH3 group (δH 3.33 and δC 58.3) were observed in 1 H and 13 C NMR spectra of 1. HMBC correlation between H-16 and this OCH3 carbon further confirmed the link tween C-16 and the OCH3 group ( Figure 2).

Structure Elucidations
The molecular formula of compound 1 was obtained as C 19 H 26 O 4 by HRESIMS ( Figure  S1 in the Supporting Information, SI), implying seven degrees of unsaturation. The one and two-dimensional NMR data (Table 1 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 H 3 -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 OCH 3 group (δ H 3.33 and δ C 58.3) were observed in 1 H and 13 C NMR spectra of 1. The key HMBC correlation between H-16 and this OCH 3 carbon further confirmed the linkage between C-16 and the OCH 3 group (Figure 2). Solanapyrone S (compound 3) was confirmed to have the molecular formula C 22 H 32 O 5 by its HRESIMS data, requiring seven degrees of unsaturation ( Figure S9). Its onedimensional 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 (CH 3 -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, CH 3 -4 δ H 1.26/δ C 18.4) was observed in 1 H and 13 C NMR spectra of 3. Finally, the consecutive COSY cross-peaks from H 3 -1 to H 3 -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). peaks from H3-1′ to H3-4′ and the key HMBC correlation between H-16 and OC firmed 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 H 3 -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.
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 13 C 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 (R 2 ) of 1 and 3 from linear regression analyses between calculated and experimental 13 C 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 and Figure 2) exhibited one carbonyl carbon (δC 182.5 CO), six aromatic carbons (δC 164.0 C, 159.9 C, 158.6 C, 105.2 C, 101.2 C, 90.6 CH), and one double bond (δC 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.
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 1 H-1 H COSY and HMBC spectra confirmed the presence of a benzopyrone skeleton with three CH3 groups (δH 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 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 13 C 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 (R 2 ) of 1 and 3 from linear regression analyses between calculated and experimental 13 C NMR data were 0.9982 and 0.9979, respectively ( Figure S39).
The HRESIMS data for compound 5 demonstrated its molecular formula to be C 13 H 14 O 6 S, indicating seven degrees of unsaturation ( Figure S17). The one-and twodimensional NMR data (Table 2 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.
The molecular formula C 12 H 12 O 3 of compound 6 was assigned on the basis of its HRESIMS data, indicating seven degrees of unsaturation ( Figure S23). Detailed analyses of 1 H-1 H COSY and HMBC spectra confirmed the presence of a benzopyrone skeleton with three CH 3 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 (CH 2 -1 δ H 2.69/δ C 44.1, OCH-2 δ H 4.20/δ C 66.9, CH 3 -3 δ H 1.29/δ C 23.5) were absent from NMR spectra of 6. Instead, CH 3 signals (δ H 2.30/δ C 19.7) were observed in 6.  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 1 H-1 H COSY and HMBC spectra (Figure 2). The ethyl and 3 -hydroxy butyl residues were deduced by the consecutive COSY cross-peaks from H 2 -8 to H 3 -9 and from H 3 -1 to H 3 -4 . The key HMBC correlations from H 3 -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 H 3 -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 13 C NMR calculation deduced the absolute configuration of 3 -hydroxy butyl group as 2 S and 3 S [20,21]. The correlation coefficient (R 2 ) of 8 from linear regression analysis between calculated and experimental 13 C NMR data was 0.9948 ( Figures S38 and S39).

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). 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 structureactivity analysis of solanapyrone polyketides 1-3 indicated that the substituted group at C-16 should be related to their herbicidal activities. 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.
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). 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. 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. 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- 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.

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.

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).

Calculations of ECD and 13 C 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 13 C 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 13 C NMR data were performed as described previously [10,17,21].

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: 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.

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 13 C 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 carbendazimresistant 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.
Author Contributions: J.F. and F.G. both performed the experiments for the isolation and bioactivity evaluation of the isolated compounds; C.Z., H.L. and T.Q. contributed to the antagonistic evaluation; F.D. determined the structures of the isolated compounds; L.X. and F.D. supervised the research and also prepared the manuscript. All authors have read and agreed to the published version of the manuscript.