Phenolic compounds and bioactive extract produced by endophytic fungus Coriolopsis rigida

Abstract Coriolopsis rigida was isolated as an endophytic fungus from the medicinal plant Cochlospermum regium, and their production of secondary metabolites has not yet been investigated. Thus, the endophyte was cultivated on rice solid media to evaluate its ability to produce bioactive compounds and then the chloroform extract was obtained. Two phenolic compounds, tyrosol (1) and a new natural product p-hydroxyphenylacetamide (2), were isolated from the extract. The structures of the compounds were elucidated mainly by NMR. The extract showed potent antioxidant activity with an efficient concentration (EC50) value of 0.33 mg mL−1. Additionally, demonstrated allelopathic activity inhibited the seedling growth of Lactuca sativa L. and Raphanus sativus L. by 63% and 55%, respectively. Graphical Abstract


Introduction
Microorganisms that live in symbiotic associations with other organisms, such as endophytic fungi, are perceived as an alternative source for new biologically active molecules (Gupta et al. 2020;Chapla et al. 2013). The bioactive compounds isolated from endophytes are structurally diverse and exhibit biological properties, including antioxidant, antitumor and antifungal, however, many endophytic fungi remain unknown (Manganyi and Ateba 2020).
The fungus Coriolopsis rigida (Berk. & Mont.) Murrill (Polyporales, Basidiomycota) is one of the 21 species that constitute the genus Coriolopsis (Pires et al. 2017;Saparrat et al. 2014). It is a saprotrophic organism and is mainly found on decaying wood (Saparrat et al. 2014). It belongs to a group of white-rot fungi that are considered model organisms for the study and production of laccases (Saparrat et al. 2014D ıaz et al. 2010). However, there are no known studies for the production of secondary metabolites from C. rigida. Studies from the genera include the Coriolopsis sp. J5 where were identified six new furan derivatives and two new tremulan sesquiterpenes from the fungus (Chen et al. 2017a(Chen et al. , 2017b, in addition nine new acetylenic acids were isolated from a culture of C. gallica (Zhou et al. 2008).
Cochlospermum regium (Bixaceae) is a medicinal plant, native from Brazil, the roots are used in the preparation of decoctions, infusions and bottles for the treatment of uterine, intestinal and ovarian inflammations (Camillo et al. 2016). However there are few studies from the microbiota of this plant (Arruda et al. 2021, Montel et al. 2021. The aim of this work was to evaluate the metabolism and biotechnological potential from endophytic fungi associated with C. regium, thus the plant was submitted to endophytic isolation and Coriolopsis rigida was obtained. Herein we describe the antioxidant and allelochemical activities of the extract produced by the endophyte and the production of phenolic compounds by C. rigida in solid medium.

Identification of the endophytic fungus
Molecular identification up to the species level was based on the ITS region of the DNA, which revealed it to be the endophytic fungus Coriolopsis rigida. Molecular analysis showed that the endophytic fungus Cr-1 had 99% genetic similarity to the species Coriolopsis rigida and allowed us to build a phylogenetic tree of clear kinship with a clade (phylogenetic base) that supports separation at the species level. The nucleotide sequence was deposited in the GenBank database under the accession number MN991225. The fungus demonstrated, in previous studies, antagonistic activity against Bipolaris oryzae and Curvularia lunata, in addition showing VOCs allelochemical activity inhibiting the development of lettuce seedlings (data not published).

Compounds produced by Coriolopsis r ıgida
The evaluation of the presence of secondary metabolites class indicated the presence of flavones, flavonols and xanthones in crude extract due the changing in color observed in the solutions. After fractionation and chromatographic separation of crude extract produced by C. rigida, two compounds were identified (Figure 1). Compound 1, tyrosol (1) (Christophoridou and Dais 2009), is a fungal signaling (quorum-sensing) molecule that is found in a variety of natural sources, such as an endophytic fungus named Diaporthe helianthin (Specian et al. 2014). And p-hydroxyphenylacetamide (2) (Wagh et al. 1991;NMR 2021) is reported here, for the first time, as a natural product. Their chemical structures were determined using spectroscopic techniques and by comparing the data in literature (Table S1, see supplementary material). This is the first report of phenolic compounds being produced by the fungus C. rigida.
The presence of flavones, flavonols and xanthones in the extract showed the potential of the production by the fungus for aromatic compounds, especially phenolic. Studies by Pedroso et al. (2019), reported the presence of these metabolic classes in the host species (C. regium), and organic extracts from the plant showed high levels of phenolic compounds, flavones, and flavonols. In addition, the phenolic compound gallic acid, rutin, myricetin, morin and kaempferol were produced by C. regium (Arunachalam et al. 2019). These results reinforce the concept that endophytes may be able to biosynthesize classes of substances similar to those found in their host plant.

Antioxidant and allelopathic properties
The highest % AA (antioxidant activity) was associated with an extract concentration of 1000 lg mL À1 (78.87%) ( Figure S1, see supplementary material). The extract obtained from endophyte showed potent antioxidant activity, with EC 50 of 0.33 mg mL À1 and demonstrated the potential to sequester DPPH free radicals. The antioxidant activity has been correlated with the presence of flavones, flavonols, and xanthones, described with antioxidant properties (Rice-Evans 2012), which were identified in the extract. This activity also may be attributed to tyrosol (1), which has antioxidant properties (Ramos 2013). Thus far, there has been no report on the antioxidant activity of the fungus C. rigida, although studies in the literature suggest that fungi belonging to the family have antioxidant properties (Al-Fatimi et al. 2005).
The extract showed an inhibitory allelopathic effect on the germination of L. sativa (lettuce) seeds at the highest concentrations (4.5% for 2000 mg L À1 , and 17.8% for 3000 mg L À1 ). The extracts showed no inhibitory effect on the germination of R. sativus (radish) seeds, for all concentrations. There was a decrease in the mean root length of R. sativus ( Figure S2), the strongest effect (63%) was observed at the highest concentration of the extract (4000 mg L À1 ). There was a 55% reduction in the mean length of R. sativus seedlings exposed to C. rigida extracts at 4000 mg L À1 . The extract concentration at 3000 mg L À1 inhibited the development of L. sativa roots by 67%. Furthermore, at 3000 mg L À1 , there was a 63% reduction in the mean length of L. sativa seedlings ( Figure S2).
Our results for L. sativa and R. sativus seedlings were indicative of an allelochemical inhibitory action of the extract on seedling development, with the highest inhibitory effects being associated with the highest crude extract concentrations. However, there was a difference in the inhibition of the two species of seedlings. The crude extract inhibited the growth of L. sativa seedlings to a greater extent, as compared to R. sativus seedlings, which indicates that the crude extract had selective allelochemical activity. Phenolic compounds, tannins, and flavonoids are agents that are most commonly associated with allelopathic effects (Fiorenza et al. 2016). Some of these compounds were identified in the extract, after analysis of the presence of the secondary metabolites.
Studies have reported the advantages of allelopathic effects in agricultural systems, such as weed control, inhibition of pests, disease, improvement of soil nutrition, microbial interactions and suitable substitute for synthetic herbicides because allelochemicals do not have residual or toxic effects (Cheng and Cheng 2015;Xie et al. 2021).
In conclusion, this work highlights the biotechnological potential of the endophytic fungus C. rigida due the potent antioxidant activity and allelopathy properties obtained from the extract of the endophyte. The phenolic compounds are related here for the first time from C. rigida, this production may have been associated with the antioxidant and allelochemical activity. The results of the present study showed the chemical potential and the technological application of Coriolopsis rigida.