Cladosporium cladosporioides LPSC 1088 Produces the 1,8-Dihydroxynaphthalene-Melanin-Like Compound and Carries a Putative pks Gene

Abstract

Cladosporium cladosporioides is a dematiaceous fungus with coloured mycelia and conidia due to the presence of dark pigments. The purpose of this study was to characterize the dark pigments synthetized by Cladosporium sp. LPSC no. 1088 and also to identify the putative polyketide synthase (pks) gene that might be involved in the pigment biosynthesis. Morphological as well as molecular features like the ITS sequence confirmed that LPSC 1088 is Cladosporium cladosporioides. UV-visible, Fourier Transform Infrared (FTIR) and Electron Spin Resonance (ESR) spectroscopy analysis as well as melanin inhibitors suggest that the main dark pigment of the isolate was 1,8 dihydroxynaphthalene (DHN)-melanin-type compound. Two commercial fungicides, Difenoconazole and Chlorothalonil, inhibited fungal growth as well as increased pigmentation of the colonies suggesting that melanin might protect the fungus against chemical stress. The pigment is most probably synthetized by means of a pentaketide pathway since the sequence of a 651 bp fragment, coding for a putative polyketide synthase, is highly homologous to pks sequences from other fungi.

Response of the fungus Pseudocercospora griseola f. mesoamericana to Tricyclazole

“…Recently, Saparrat et al (2009) described that representatives of P. griseola from both gene pools synthesize 1,8-dihydroxynaphthalene (DHN) melanin. These same pigments have already been found in most Ascomycota, such as in Alternaria alternata (Carzaniga et al 2002), Bipolaris oryzae (Moriwaki et al 2004), Bipolaris sorokiniana (Kumar et al 2015), Cladosporium cladosporioides (Llorente et al 2012), Colletotrichum lagenarium, Magnaporthe grisea (Moriwaki et al 2004), Pseudocercospora fijiensis (Beltrán-García et al 2014) and Sporothrix schenckii (Romero-Martinez et al 2000). However, information regarding the pathway of pigment synthesis as well as its localization at the ultrastructural level in P. griseola is lacking.…”

“…It was kept at −20°C on filter paper impregnated with a conidia-mycelium suspension and as a culture grown for 7 days on a potato dextrose agar (PDA) medium at 24± 2°C and maintained at 4°C. Inoculation was carried out by using a conidial suspension obtained according to Llorente et al (2012), and unless specifically stated, cultures on PDA (control) and PDA supplemented with tricyclazole (Ultra Scientific Analytical Solutions, United States) were incubated in the dark at 24±2°C for 21 days.…”

“…However, there is no information regarding the effect of these types of fungicides on melanin synthesis and/or their biological impact in this fungus. However, Llorente et al (2012), while studying melanin synthesis on Cladosporium cladosporioides, found that Difenoconazole (Bogard 25 %, Syngenta Agro S.A., Argentina) and Clorotalonil (Daconil 72F, Syngenta Agro S.A., Argentina) generate a drastic reduction in fungal growth and conidiation. Concomitantly, cultures turned darker, most probably due to melanin deposition that seems to be a quencher of stress.…”

“…1a) is a molecule that affects the DHN-melanin biosynthesis pathway through the inhibition of reductases that catalyze the transformation of 1, 3,6,3,6, to scytalone and of 1,3,8-trihydroxynaphthalene (1, 3, 8-THN) to vermelone (Bell and Wheeler 1986;Nobre and Ferreiro 2004). Saparrat et al (2009) and Llorente et al (2012) demonstrated that this fungicide reduced growth and melanization both on P. griseola and C. cladosporioides. Furthermore, this compound is used to control plant pathogens such as Magnaporthe grisea, Bipolaris oryzae, B. sorokiniana, Blumeria graminis, Colletotrichum lagenarium, C. lindemuthianum and Rhynchosporium secalis (Mares et al 2004;Kunova et al 2012;Kumar and Mukherjee 2013;Kumar et al 2015).…”

“…Identifying biosynthetic intermediates provides an opportunity to investigate the role of the melanin in the causative agent of ALS and the potential use of tricyclazole to control it. Furthermore, we studied the localization of these dark pigments in fungal cells by Fourier Transform Infrared (FTIR) and microscopical analysis, methods that are diagnostic of melanin (Kayatz et al 2001;Tavzes et al 2009;Llorente et al 2012). …”