Antimicrobial metabolites produced by endophytic fungi associated with the leaves of Vochysia divergens

Abstract Investigation of the endophytic fungi Nigrospora sphaerica, Nigrospora oryzae, and Pseudofusicoccum stromaticum MeOH fractions isolated from the leaves of Vochysia divergens, a medicinal species from the Brazilian Pantanal, led to the identification of five compounds, namely a new compound (1E,8Z)-10,11-dihydroxy-5,5,8-trimethyl-4-oxocycloundeca-1,8-diene-1-carbaldehyde (1) and four known compounds: 5-methylmellein (2), sclerone (3), daldinone A (4), and lasiodiplodin (5). All compounds were identified using spectroscopic methods, and 1 was corroborated with mass spectrometry, while the known compounds were compared with data in the literature. The relative configuration of compound 1 was determined based on theoretical conformational studies as well as the J experimental values between the hydroxymethyne hydrogens. The antimicrobial activity of the compounds was evaluated. Promising results were obtained for compounds 2, 4, and 5 since they inhibited the bacterium Pseudomonas aeruginosa, an opportunistic pathogen, suggesting the potential of these microorganisms as a source of new antibacterial agents. Graphical Abstract


Introduction
Vochysia divergens POHL (Vochysieaceae), popularly known as cambará, is a medicinal plant distributed in Brazil and Bolivia's Pantanal region (Pott et al. 2011).Its leaves and bark are used to treat respiratory infections such as flu, coughs, pneumonia, colds, and asthma.The flora, fauna, and microbial community of this floodplain are influenced by the surrounding biomes (Nunes and Junk 2004).
Living asymptomatically in plant tissues, endophytic fungi are natural sources of antibiotics that can inhibit or kill various disease-causing agents in humans and animals, such as bacteria, other fungi, and viruses (Newman and Cragg 2020).Moreover, they can be used to produce high-value-added compounds that enable the production of new drugs (González-Medina et al. 2017).
Endophytic fungi isolated from V. divergens were able to produce antimicrobial secondary metabolites (Hokama et al. 2016).Therefore, considering that the increasing emergence of drug-resistant pathogenic bacterial and yeast strains presents a threat to global public health and motivates the search and development of new antimicrobial agents (Silva et al. 2022), the microorganisms of this plant and biome are an important niche to be explored.
We are interested in revisiting the chemistry of previously investigated biological species using contemporary approaches for the analysis of prefractionated extracts.The aim is the detection of compounds that may constitute either novel bioactive chemical scaffolds or putative biosynthetic intermediates that may clarify the biosynthesis of metabolites.In the context of the continuous search for new antimicrobial agents, this paper presents the study of extracts obtained from endophytes of these genera, resulting in the isolation and identification of five compounds, namely the n e w ( 1 E , 8 Z ) -1 0 , 1 1 -d i h y d r o x y -5 , 5 , 8 -t r i m e t h y l -4 -o x o c y c l o u n d e c a -1,8-diene-1-carbaldehyde (1) from N. sphaerica, the known compounds 5-methylmellein (2), sclerone (3) and daldinone (4) from N. oryzae, and lasiodiplodin (5) from P. stromaticum (Figure 1).Furthermore, their antimicrobial activity against human pathogens was evaluated.
Analysis of the 13 C NMR spectra revealed the presence of two carbonyls, one with a characteristic formyl group at δ The assignment of carbon to the respective hydrogens was based on the correlations observed in the HSQC-dEPT experiment (Figure S8).The 1E configuration was inferred based on the chemical shifts of C-2 (δ 153.00 ppm) and H-2 (δ 6.51 ppm) (Traore et al. 2007), and the 8Z configuration was supported based on the observed chemical shift for the C13 methyl group (δ 21.18 ppm), revealing the absence of the γ-gauche effect (Zhang et al. 2018).
The stereochemistry of 1 was determined based on theoretical calculations of conformational analysis and on the value observed for the coupling constant between H-10 and H-11 (J ~ 9 Hz).For both diastereomers of 1, quantum chemical calculations showed that only one conformer predominates in the gas phase [Tables S2 and S3; Figures 2 and 3].Vicinal coupling constants (J) are affected by various intrinsic structural effects, mainly by the dihedral angle, with the magnitude of J generally being smallest when the torsion angle (θ) is close to 90°.The dihedral angle between H-10 and H-11 in 1a was found to be ~99°, whereas in 1b, it was ~130°.The experimental J value was compatible only with the θ found for diastereomer 1b, and therefore a relative configuration of 10R*, 11S* was proposed for 1.
Compound 2 was isolated as a white amorphous solid.Based on NMR data analysis, it was identified as 5-methylmellein (Zhao et al. 2012), previously reported to exhibit cytotoxic (Arora et al. 2016) and phytotoxic activity (Meepagala et al. 2018).Compound 3 was isolated as a red amorphous solid, and it was identified as 4,5-dihydroxy-3,4-dihydronaphthalen-1(2H)-one, known as sclerone, with prior reports of mild nematocidal activity (Zhu et al. 2008).Compound 4 was isolated as an amorphous solid.Analysis of uni and two-dimensional NMR spectra and comparison with literature data allowed the identification of daldinone A (Quang et al. 2002).To the best of our knowledge, there are no previous reports of biological activity for this compound.Compound 5 was isolated as a white solid, identified as lasiodiplodin (Wang et al. 2009), and reported to have antifungal, antibacterial (Yang et al. 2006), and antileukemic activity (Sultan et al. 2014). 1 H and 13 C NMR δ values of compounds 2, 3, 4, and 5 are available in the Supplementary Material.Multidrug-resistant bacteria are now considered a world health threat by the World Health Organization (WHO).WHO listed ESKAPE pathogens in a list of 12 bacteria against which new antibiotics are urgently needed.The term 'ESKAPE' encompasses six of these pathogens with increasing multidrug resistance: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.
These pathogens are responsible for most nosocomial infections and can 'escape' the action of available antibiotics (Freire et al. 2022).In the present investigation, Table S4 shows the results of the antimicrobial evaluation of isolated compounds.5-Methylmellein (2), daldinone (4), and lasiodiplodin (5) exhibited antibacterial activity against P. aeruginosa with promising inhibition halos (10, 20, and 13 mm, respectively).This bacterium is ubiquitous and particularly prevalent among patients with burn wounds, cystic fibrosis, organ transplants (Azam and Khan 2019), and acute leukaemia (Zhao et al. 2020).Compounds 1 and 3 did not exhibit antimicrobial activity against the tested microorganisms.

Conclusion
The study of endophytic fungi associated with the species V. divergens resulted in the isolation and identification of five compounds.One of them was new, namely (1E,8Z)-10,11-dihydroxy-5,5,8-trimethyl-4-oxocycloundeca-1,8-diene-1-carbaldehyde (1), from the endophytic fungus N. sphaerica.This is also the first report of the isolation of daldinone A (4) and lasiodiplodin (5) from the endophytic fungi N. oryzae and P. stromaticum, respectively, and the first report of bioactivity for daldinone A (4).Compounds 2, 4, and 5 exhibited promising antimicrobial potential against the bacterium P. aeruginosa.Further studies are needed to assess the potential application of these bioactive compounds and the bioactivity of the new compound.The absence of reports in the literature on the antimicrobial activity of these compounds supports the importance of endophytic fungi associated with V. divergens as potential sources of new antibacterial agents to be explored.Our results demonstrate the unambiguous identification of a new compound, constituting a reliable strategy to unveil metabolites that can be of chemical and biological interest for further investigations.