Secondary Metabolites with Agricultural Antagonistic Potential from Aspergillus sp. ITBBc1, a Coral-Associated Marine Fungus

A marine-derived fungal strain, Aspergillus sp. ITBBc1, was isolated from coral collected from the South China Sea in Hainan province. Intensive chemical investigation of the fermentation extract of this strain afforded four new secondary metabolites (1–4), named megastigmanones A–C and prenylterphenyllin H, along with four known compounds (5–8). Their structures were elucidated by extensive spectroscopic analysis including one-and two-dimensional (1D and 2D) NMR spectroscopy and high-resolution electrospray ionization mass spectrometry (HR-ESI-MS). The modified Mosher’s method was undertaken to determine the absolute configurations of new compounds. The phytotoxic activity test showed that compounds 6–8 exhibited significant antagonistic activity against the germination of Triticum aestivum L. and Oryza sativa L. seeds with a dose-dependent relationship.


Introduction
Fungi are ubiquitous in nature, with a wide variety and large quantities.Many fungi are beneficial to the environment, humans and animals, while some are harmful due to the production of toxic metabolites (mycotoxins) [1].More than 300 mycotoxins have been identified so far [2], and most of them are produced by well-known genera including Aspergillus, Fusarium and Penicillium [3-7].Mycotoxins not only carry the potential to cause contaminations and result in serious annual yield and quality losses but also pose life-threatening risks to human and animal health [8].Therefore, they have become a major issue in the sciences [9,10].With synthetic agrochemicals becoming increasingly controversial due to their environmental and toxicological problems [11], mycotoxins have drawn great attention because of their potential as natural herbicides [12][13][14].Modern agriculture heavily relies on natural herbicides for weed control due to their high efficiency and low cost [15].Thus, herbicidal mycotoxins are considered environmentally friendly and sustainable alternative agents for controlling weeds.However, little is known about the occurrence and mechanisms of biosynthesis under various environmental conditions [16].Therefore, excavating these mycotoxins has a vital impact on exploring the great potential of new natural herbicides.
With the expansion of research scope, more and more fungal mycotoxins with herbicidal potential have been discovered from marine fungi [17,18].Many fungal mycotoxins can also be used as lead compounds, based on their structure, to obtain more active toxic compounds through chemical synthesis.The synthesis of Cinnacidin analogues is a typical case, and the synthesized analogues have better phytotoxic activity against weeds, demonstrating their potential application value as herbicides [19].
As part of our continuing exploration of structurally novel and biologically interesting secondary metabolites from marine-derived microorganisms [20][21][22], a coral-associated fungus, Aspergillus sp.ITBBc1, has drawn our attention.Furthermore, intensive chemical investigation of this fungus led to the identification of four new compounds (1)(2)(3)(4) and four known compounds (5-8) (Figure 1).Herein, we report the isolation, structural elucidation, and biological activity evaluation of these compounds.With the expansion of research scope, more and more fungal mycotoxins with herbicidal potential have been discovered from marine fungi [17,18].Many fungal mycotoxins can also be used as lead compounds, based on their structure, to obtain more active toxic compounds through chemical synthesis.The synthesis of Cinnacidin analogues is a typical case, and the synthesized analogues have better phytotoxic activity against weeds, demonstrating their potential application value as herbicides [19].
As part of our continuing exploration of structurally novel and biologically interesting secondary metabolites from marine-derived microorganisms [20][21][22], a coral-associated fungus, Aspergillus sp.ITBBc1, has drawn our attention.Furthermore, intensive chemical investigation of this fungus led to the identification of four new compounds (1-4) and four known compounds (5-8) (Figure 1).Herein, we report the isolation, structural elucidation, and biological activity evaluation of these compounds.

Antagonistic Evaluation
The phytotoxic activity of the isolated compounds was tested using Triticum aestivum L. (monocots), Oryza sativa L. (monocots) and Amaranthus retroflexus L. (dicotyledon) seeds.As a result, p-terphenyl derivatives of compounds 6-8 exhibited significant antagonistic activities against the germination of Triticum aestivum L. and Oryza sativa L. seeds as well as the root and shoot lengths of seedlings with a dose-dependent relationship (Figures 4 and 5).Compound 7 exhibited almost identical phytotoxic activity, indicating that the hydroxyl group at position C-5 has no impact on their phytotoxicity.However, they have no significant effects against Amaranthus retroflexus L.
Combining the germination morphology and inhibition rate, as shown in Figure 4, it was found that there was a significant difference between Triticum aestivum L. seeds treated with 20 µg/L of compounds 6-8 and below 20 µg/L and the control.Shoot growth inhibition is more obvious than that of roots.The germination rate of Oryza sativa L. seeds treated with higher concentrations (50-200 µg/L) was significantly lower than the control or even no germination, and the development of roots and shoots was significantly inhibited compared to the control.The germination rate as well as the root and shoot lengths of seedlings of Amaranthus retroflexus L. seeds treated with higher concentrations (20-150 µg/L) were not affected.By comparing the inhibitory effects of compound 7 on Triticum aestivum L. and Oryza sativa L. seeds, it was found that Oryza sativa L. seeds are more sensitive to the treatment and can be effectively inhibited by compound 7 at a low concentration (20 µg/L).To sum up, the results indicated that the minimum inhibitory concentration for three germination inhibitors with significant inhibitory effects against Triticum aestivum L. is 20 µg/L.However, they have no significant inhibitory effects against Amaranthus retroflexus L., even at concentrations up to 150 µg/L.Therefore, it can be concluded that these compounds have significant selectivity for monocotyledonous and dicotyledonous plants seeds, and it is implied that they are more possibly only phytotoxic against monocots.Amaranthus retroflexus L., even at concentrations up to 150 μg/L.Therefore, it can be concluded that these compounds have significant selectivity for monocotyledonous and dicotyledonous plants seeds, and it is implied that they are more possibly only phytotoxic against monocots.

Fungal Material and Fermentation
The symbiotic epiphytic fungal strain of Aspergillus sp.ITBBc1 was previously isolated from coral collected from the South China Sea in Hainan and identified as Aspergillus sp. by internal transcribed spacer (ITS) sequencing (GenBank accession number OP614945) [20].The strain was cultivated on ME medium (malt extract 10.0 g/L, sucrose 10.0 g/L, tryptone 1.0 g/L) cultured on a rotary shaker (180 rpm) at 28 • C for 3 days to afford a seed culture, which was then inoculated into rice solid media (rice 90.0 g and 0.1 L water) in 1L-Erlenmeyer flasks and fermented at 28 • C for 45 d under static conditions.

Antagonistic Bioassay
The Triticum aestivum L. (monocots), Oryza sativa L. (monocots) and Amaranthus retroflexus L. (dicotyledon) seeds were disinfected with 8% sodium hypochlorite solution for 15 min and then washed three times with sterilized water.Then, Triticum aestivum L. seeds were placed in a 15 mL tube containing 5 mL of 1/2 MS medium with four concentrations (0, 5, 10, 20, and 50 µg/mL).Oryza sativa L. and A. retroflexus seeds were evenly placed on sterilized filter paper in separate 120 mm Petri dishes containing 2 mL of compound 7 with four concentrations (0, 20, 50, 100, 150, and 200 µg/mL).Seeds cultivated at a 25 °C growth chamber for upright growth for 5 d (16 h/8 h light/dark cycle).Then, the shoot length and root length of each treatment were measured, and the germination inhibition rates were calculated.The commonly used synthetic herbicide 2, 4-dichlorophenoxyacetic acid (2, 4-D) was used as a positive control.The inhibition percentage was calculated using the formula as follows: inhibition percentage (%) = (L control − L treatment )/L control × 100

Conclusions
In summary, this study focused on the isolation and structural elucidation of four new compounds and four known compounds from the marine fungus Aspergillus sp.ITBBc1.The identification of these compounds was accomplished through the application of diverse spectroscopic techniques and comparison with published data.Compounds 1-3 have a similar structure to compounds reported by González Coloma et al., obtained from Stemphylium solani [30], which have biocidal activity simultaneously against more than one category of harmful organisms affecting plants.Compound 4 belongs to p-terphenyl, possessing the same skeleton with prenylterphenyllin G [31] and modified by hydroxy group at C-2 ′′′ .Compound 5 is an anazaphilone derivative that was first reported by Zhang et al. [23].Compounds 6 and 7 are p-terphenyl derivatives and have the ability to inhibit wheat coleoptile growth significantly according to the previous reports [32].Compound 7 has also been reported as an important anti-proliferative and pro-apoptotic agent for ovarian cancer [33].Compound 8 is a terphenyllin derivative.Our results showed that compounds 6-8 exhibited remarkable antagonistic activities against the germination Mar. Drugs 2024, 22, x FOR PEER REVIEW 2 of 10

Figure 2 .
Figure 2. Key HMBC, 1 H-1 H COSY and ROESY correlations of new compounds 1-4.Megastigmanone B (2) was isolated as colorless oil.Its molecular formula, C13H22O4 was determined based on the HR-ESI-MS ion at m/z 265.1447 [M + Na] + (calcd, C13H22NaO4 265.1410), indicating three degrees of unsaturation.Comparison of its 1D and 2D NMR data with those for compound 1 revealed that compound 2 shared the same skeleton with compound 1, except for the different configurations of H-5, revealed by the ROESY corre lation between CH3-6 and H-5 in 2. The absolute configuration of C-5 and C-2′ in com pound 2 was also determined by the modified Mosher's method and has been determined to be (5S, 6R)-5, 6-dihydroxy-3-((S)-2′-hydroxypentyl)-2, 6-dimethylcyclohex-2-en-1-one according to the ΔδRS value analysis of the 1 H NMR data of (S) and (R)-MTPA esters o compound 2 (Figure 3; Figures S20-S23).Megastigmanone C (3) was also obtained as colorless oil.Its molecular formula C13H22O4, was determined based on the HR-ESI-MS ion at m/z 243.1595 [M + H] + (calcd C13H23O4, 243.1591), indicating three degrees of unsaturation.The 1D and 2D NMR data for compound 3 (Figures S26-S31) were very similar to those of compound 1, allowing the