Sugarcane: an unexpected habitat for black yeasts in Chaetothyriales

Abstract

Sugarcane (Saccharum officinarum, Poaceae) is cultivated on a large scale in (sub)tropical regions such as Brazil and has considerable economic value for sugar and biofuel production. The plant is a rich substrate for endo- and epiphytic fungi. Black yeasts in the family Herpotrichiellaceae (Chaetothyriales) are colonizers of human-dominated habitats, particularly those rich in toxins and hydrocarbon pollutants, and may cause severe infections in susceptible human hosts. The present study assessed the diversity of Herpotrichiellaceae associated with sugarcane, using in silico identification and selective isolation. Using metagenomics, we identified 5833 fungal sequences, while 639 black yeast-like isolates were recovered in vitro. In both strategies, the latter fungi were identified as members of the genera Cladophialophora, Exophiala, and Rhinocladiella (Herpotrichiellaceae), Cyphellophora (Cyphellophoraceae), and Knufia (Trichomeriaceae). In addition, we discovered new species of Cladophialophora and Exophiala from sugarcane and its rhizosphere. The first environmental isolation of Cladophialophora bantiana is particularly noteworthy, because this species up to now is exclusively known from the human host where it mostly causes fatal brain disease in otherwise healthy patients.

Keywords: Cladophialophora bantiana; Metagenomics; Selective isolation.

Fig. 1

Distribution of the fungi in Herpotrichiellaceae on sugarcane, based on the specific molecular markers applied in the metagenome database. I. Evaluated metagenomic samples from different parts of sugarcanes from São Paulo, Brazil. II. Percentage of metagenomic sequences from BioProject PRJNA319259: A in leaf endophytic, B in leaf exophytic, C in stalk endophytic, D in stalk exophytic, and E rhizosphere samples

Fig. 2

Phylogeny of a representative selection of species in Chaetothyriales based on LSU rDNA sequences, constructed by Bayesian analysis and maximum likelihood. Values of > 70% for Bayesian probability (left) and Bootstrap values > 70% for maximum likelihood (right) from 1000 resampled datasets are shown with branches. Novel species are indicated in bold. The outgroup was Knufia epidermidis (CBS 120353). ^T = Ex-type strain

Fig. 2

Phylogeny of a representative selection of species in Chaetothyriales based on LSU rDNA sequences, constructed by Bayesian analysis and maximum likelihood. Values of > 70% for Bayesian probability (left) and Bootstrap values > 70% for maximum likelihood (right) from 1000 resampled datasets are shown with branches. Novel species are indicated in bold. The outgroup was Knufia epidermidis (CBS 120353). ^T = Ex-type strain

Fig. 3

Phylogenetic tree of Herpotrichiellaceae based on the alignment of ITS1-5.8S-ITS2 and β-tubulin sequences, constructed with Maximum likelihood implemented in MEGA7 with Tamura-Nei model. Capronia kleinmondensis (CBS 122671) was chosen as outgroup. Bootstrap values (1000 replicates) above 70% are added to supported branches. Novel species and isolates of Cladophialophora bantiana and Exophiala cancerae are indicated in bold. ^T = Ex-type strain

Fig. 3

Phylogenetic tree of Herpotrichiellaceae based on the alignment of ITS1-5.8S-ITS2 and β-tubulin sequences, constructed with Maximum likelihood implemented in MEGA7 with Tamura-Nei model. Capronia kleinmondensis (CBS 122671) was chosen as outgroup. Bootstrap values (1000 replicates) above 70% are added to supported branches. Novel species and isolates of Cladophialophora bantiana and Exophiala cancerae are indicated in bold. ^T = Ex-type strain

Fig. 4

Cladophialophora rhizosphaerae microscopic morphology (UPCB 96826). A Colony on MEA; B–C Long conidial chains; D–E Conidial chains arranged sinuously; G Conidial clusters; H Coherent conidial chain with budding cells; I–K Conidial chains with ellipsoid to ovoid conidia. Bars = 10 μm

Fig. 5

Cladophialophora griseolivacea, microscopic morphology (UPCB 96824). A Colony on SGA; B–G Conidiophores with conidia produced sympodially; E–F Proliferating rachis on conidiogenous cells, presenting slightly prominent and unpigmented scars; H–J Conidia; K Hyphae with chlamydospore-like cells; L Anastomosis. Bars = 10 μm

Fig. 6

Cladophialophora molassis microscopic morphology (UPCB 96823). A Colony on MEA; B-D Conidia; E Fertile hyphae; F Conidiophore; G Hyphae; H Anastomosis; I Spirally twisted hyphae. Bars = 10 μm

Fig. 7

Exophiala sacchari microscopic morphology (UPCB 96825). A Colony on SGA; B–E, G–H Oval to lemon-shaped conidia; F Conidiophore. Bars = 10 μm

Fig. 8

Temperature relations of Cladophialophora and Exophiala species after 3 wk incubation at temperatures ranging from 18 to 42 °C