The Lignicolous Fungus Coniochaeta pulveracea and Its Interactions with Syntrophic Yeasts from the Woody Phylloplane

Abstract

The yeast-like fungus Coniochaeta pulveracea was studied with regard to its novel lignocellulolytic activities and the possible effect thereof on yeasts from the woody phylloplane. An enrichment procedure was used to isolate C. pulveracea from a decaying Acacia tree, and the identity of the isolate was confirmed using morphology, as well as molecular and phylogenetic techniques. This isolate, as well as strains representing C. pulveracea from different geographical regions, were compared with regard to optimum growth temperature and enzyme activity to representatives of closely related species. These include strains of Coniochaeta boothii, Coniochaeta rhopalochaeta, and Coniochaeta subcorticalis. Plate assays for cellulase and xylanase activity indicated that all representatives of the above-mentioned species were able to produce extracellular hydrolytic enzymes and were also able to degrade birchwood toothpicks during a 50-day incubation period at 30°C. To test the ability of these fungi and their enzymes to release simple sugars from complex cellulosic substrates, filtrates obtained from liquid cultures of Coniochaeta, cultivated on carboxymethyl cellulose (CMC) as sole carbon source, were analyzed using high-performance liquid chromatography analysis. Consequently, the presence of mono- and disaccharides such as glucose and cellobiose was confirmed in these culture filtrates. Two subsequent experiments were conducted to determine whether these simple sugars released from woody material by Coniochaeta may enhance growth of phylloplane yeasts. In the first experiment, representatives of Coniochaeta were co-cultured with selected yeasts suspended in agar plates containing birchwood toothpicks, followed by examination of plates for colony formation. Results indicated that Coniochaeta growth on the toothpicks enhanced growth of nearby yeast colonies in the agar plates. In the second experiment, representatives of selected yeasts and Coniochaeta species were co-cultured on CMC and xylan-containing plates where after yeast colony formation was recorded on the plates. Saccharomyces cerevisiae strains, engineered to utilize specific wood degradation products, i.e., cellobiose or xylose, as sole carbon source were used as positive controls. While it was found that cellobiose released from CMC was assimilated by the yeasts, no evidence could be obtained that xylose released from xylan was used as carbon source by the yeasts. These ambiguous results could be ascribed to secretion of nutritious metabolic end products, other than the products of fungal xylanases.

The microcyclic conidial stage of Coniochaeta pulveracea and its effect on selected biological interactions

“…The filamentous nature of C. pulveracea on a solid growth medium incubated at its optimum growth temperature of 30°C (Van Heerden et al 2011) was confirmed by studying the morphology of C. pulveracea CAB683, CBS110471 and CBS114628 on oatmeal agar (OA) using light-and scanning electron microscopy. Material for scanning electron microscopy was fixed with 3 % (v/v) glutardialdehyde (Merck, Darmstadt, Germany) dissolved in 0.1 mol/L sodium phosphate buffer at pH 7.0 for 4 h. The fixed material was subsequently rinsed twice for 10 min with the phosphate buffer.…”

“…These strains, together with the yeast strains used in the co-culture study, are listed in Table 1. The strain CAB683 was isolated from an Acacia tree in the Northern Cape of South Africa using an enrichment broth containing molasses, chopped wheat straw and chloramphenicol (Van Heerden et al 2011). Cryptococcus neoformans CAB935 originated from a nearby tree, while Cryptococcus neoformans CAB858 was originally isolated from a clinical sample taken from a patient suffering from HIV/AIDS.…”

“…Recent studies showed that C. pulveracea CAB683, CBS114252 and CBS114628 in their hyphal growth phase may enhance growth of yeasts such as Meyerozyma guilliermondii and Cryptococcus neoformans (Van Heerden et al 2011) which may naturally be associated with wood degrading fungi (Van Heerden et al 2008) or decaying wood (Hiremath et al 2008). In addition, it was found that hyphal growth of the abovementioned C. pulveracea strains enabled growth of the recombinant strain Saccharomyces cerevisiae SIGMA [SSFI] whilst suspended within an agar medium containing carboxymethylcellulose (CMC) as sole carbon source.…”

“…However, since its discovery more than two centuries ago (Monod et al 2002) relatively little work was conducted on the physiology and interactions of C. pulveracea (Romero et al 1999;Weber 2002;García et al 2006). It has recently been reported that C. pulveracea and its closest relatives produce extracellular hydrolytic enzymes for degradation of proteins and woody materials (Van Heerden et al 2011). Similar to well-known filamentous cellulolytic fungi, i.e.…”

“…In contrast, growth of S. cerevisiae SIGMA [SSFI] was found to be inhibited by C. pulveracea CBS110471 within a medium containing glucose as carbon source. Since it is known that other members of Coniochaeta may produce antifungal agents such as coniosetin and coniochaetones (Asgari et al 2007) it was speculated that this growth inhibition may have been the result of such antifungal metabolites produced by C. pulveracea (Van Heerden et al 2011). These interactions of C. pulveracea occurred whilst the fungus showed filamentous growth on a solid substrate.…”