﻿Cladosporium spp. (Cladosporiaceae) isolated from Eucommiaulmoides in China

﻿Abstract Eucommiaulmoides is a rare tree species in China with high medicinal and gum value. Nine strains of hyphomycetous fungi were isolated from the leaf litter of E.ulmoides in Guizhou Province. Preliminary identifications based on ITS indicated that they belong to the genus Cladosporium. Morphology and phylogenetic analyses based on the internal transcribed spacer regions (ITS) of the nrDNA, the partial translation elongation factor 1-α (tef1) gene and partial of actin (act) gene confirmed that the strains represent four species, including two novel taxa, viz., Cladosporiumeucommiae and C.guizhouense and two new substrate records for known species.


Introduction
Eucommia ulmoides Oliver ('du-zhong' in Chinese), the single extant species of Eucommiaceae (related to Ulmaceae), is a dioecious, wind-pollinated tree evenly distributed in mixed mesophytic forest habitats of valleys, hills, and low mountains in central and eastern China (Cronquist 1981;Zhang 2016). E. ulmoides is widely cultivated in China and other countries owing to its high medicinal and gum value.
The fungal genus Cladosporium was established by Link (1816). Cladosporium (Cladosporiaceae) is a ubiquitous genus in Dothideomycetes (Abdollahzadeh et al. 2020). This genus is widely distributed throughout the world and isolated from various sources such as air, soil, plants, food, debris, cloth, paint and other organic materials (Ellis 1977;Bensch et al. 2010Bensch et al. , 2012Bensch et al. , 2018Temperini et al. 2018;Chung et al. 2019). Most Cladosporium species are saprobic (Bensch et al. 2010), and they occur on various senescing and dead leaves and stems of herbaceous and woody plants (Brown et al. 1998;El-Morsy 2000). The morphology of Cladosporium is mainly characterized by its asexual morph, which comprises differentiated conidiophores producing acropetal chains of conidia from mono-or polyblastic conidiogenous cells (Isabel et al. 2021). Both the conidiogenous cells and conidia show conidiogenous loci (scars) with a distinctive coronal structure, which is composed of a central convex dome surrounded by a raised periphery, usually thickened, refractive and dark (David 1997;Isabel et al. 2021). A molecular approach combined with morphological features has recognized more than 230 species in Cladosporium, which are grouped into three species complexes, i.e., the C. cladosporioides, C. herbarum and C. sphaerospermum complex (Schubert et al. 2007;Bensch et al. 2010Bensch et al. , 2012Bensch et al. , 2015Bensch et al. , 2018Sandoval-Denis et al. 2016;Marin-Felix et al. 2017).
In a recent research program, we have carried out a survey of micro-fungi associated with E. ulmoides in a forest in China. In this study, four Cladosporium taxa were isolated from fallen leaves of this plant species in Guizhou Province, including two new species, namely C. eucommiae and C. guizhouense spp. nov., which are introduced based on morphology and phylogenetic analyses. Newly generated molecular data, descriptions and illustrations of C. tenuissimum and C. perangustum are also provided herein.

Sample collection and fungal strains isolation
Fallen leaves of E. ulmoides were collected in a forest plantation of Guizhou University, Guiyang, Guizhou Province, China, in January 2021. The samples were stored in envelopes and several topsoil samples from the forest were stored in self-sealing bags, then taken back to the laboratory and photographed. Before isolation, collected leaves samples were sprayed two to three times with 75% ethanol to disinfect the leaf surface. Pure cultures of the fungi were obtained by single spore isolation (Chomnunti et al. 2014). Fungi in the soil samples were isolated by the dilution plate method (Zhang et al. 2015). A small amount of soil (1 g) per sample was collected and added to 9 mL of sterile water in a 15 mL sterile glass test tube. It was manually mixed and then the suspension was diluted to a series of concentrations (10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 and 10 -6 ), and 100 µL from each concentration was spread onto 90-mm-diam Petri dishes containing Synthesis of low nutrient Agar (SNA), Potato Dextrose Agar (PDA), Malt Extract Agar (MEA) and Oatmeal Agar (OA) (Zhang et al. 2017). These SNA, PDA, MEA and OA plates were incubated at constant temperature (25 °C) in a controlled temperature light incubator. Holotype specimens of the new species were conserved in the Herbarium of the Department of Plant Pathology, Agricultural College, Guizhou University (HGUP). Ex-type cultures were conserved in the Culture Collection at the Department of Plant Pathology, Agriculture College, Guizhou University, P.R. China (GUCC).

Morphological description
Pure cultures were grown on SNA, PDA, MEA and OA media in a constant temperature incubator (25 °C). Culture characteristics were recorded and examined using a dissecting microscope (LEICA S9i, Germany). The morphological observations and measurements on SNA were made using a Zeiss Scope 5 compound microscope (Axioscope 5, China) with an attached camera AxioCam 208 color (ZEN 3.0) and measurements were made using ZEN 3.0. Taxonomic information for the two new taxa were deposited in MycoBank (www.mycobank.org).

DNA extraction, PCR amplification and sequencing
Fresh mycelia were scraped from the PDA plates with a sterilized scalpel. Genomic DNA was extracted using Fungal gDNA Kit (Biomiga #GD2416, San Diego, California, USA) in accordance with the manufacturer's instructions. PCR amplification was performed in a 25 µL reaction volume following Liang et al. (2018). Primer pairs ITS4/ITS5 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999) and ACT-512F/ACT-783R (Carbone and Kohn 1999) were used for ITS, tef1 and act, respectively. The amplification procedures were performed using the method described by Halo et al. (2019). Purification and sequencing of these three gene loci were carried out by the SinoGenoMax company (Beijing, China).

Phylogeny
Sequences used in this study (Table 1) were assembled based on the closest matches from the BLASTn search results (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and previous publications (Sandoval-Denis et al. 2016;Bensch et al. 2018;Halo et al. 2019). Alignments were conducted with the online version of MAFFT v. 7.307 (Katoh and Standley 2016), checked visually and improved manually where necessary using BioEdit v. 7.1.3.0 (Hall 1999). SequenceMatrix v. 1.7.8 (Vaidya et al. 2011) was used to concatenate the aligned sequences of the different loci. Ambiguous areas were excluded from the analysis using AliView (Larsson 2014) and gaps were coded as missing data. The Maximum Likelihood (ML) analyses were carried out at the CIPRES web portal (Miller et al. 2010) using RAxML (Stamatakis 2006). The tree search included 1,000 non-parametric bootstrap replicates and the best scoring tree was selected from suboptimal trees under the GTRGAMMA substitution model. The resulting replicates were plotted on to the best scoring tree obtained previously. Non-parametric bootstrap analysis was implemented with 1,000 duplicates. Maximum Parsimony (MP) analyses were performed with PAUP v. 4.0a (Swofford 2003), using the heuristic search option with 1,000 random sequence addition replicates and tree bisection-reconnection (TBR) with reconnection limit (=8) as the branch swapping algorithm. Maxtrees was set at 5,000. Branches collapsed (creating polytomies) if maximum branch length is zero. The Tree Length (TL), Consistency Indices (CI), Retention Indices (RI), Rescaled Consistency Indices (RC) and Homoplasy Index (HI) were calculated for each tree generated. Bayesian Inference (BI) analyses were performed in MrBayes v. 3.2.7a (Ronquist et al. 2012). Six Markov chain Monte Carlo runs were started, and the random start trees were calculated for 50,000,000 generations and sampled every 1,000 generations. 25% of the trees initially produced were discarded as burn-in. ML bootstrap support (MLBS) and MP bootstrap support (PBS) equal or greater than 70% (Hillis and Bull 1993) and Bayesian posterior probabilities (PP) equal or greater than 0.95 (Hespanhol et al. 2019) are displayed on the edited phylogenetic tree. The phylogenetic tree was drawn with FigTree v. 1.4.4 (Rambaut 2009).

Genealogical Phylogenetic Species Recognition (GCPSR) analysis
Morphological and phylogenetically related species were analyzed using the genealogical consistency phylogenetic species identification (GCPSR) model as described by Taylor et al. (2000) by pin-pair homogeneity index test (PHI) (Bruen et al. 2006). The PHI tests were performed in SplitsTree v. 4.17.1 (Huson 1998;Huson and Bryant 2006) as described by Quaedvlieg et al. (2014) to determine the level of recombination within phylogenetically closely related species. The results can be visualized by constructing a split graph using LogDet conversion and the Splits options. The hypothesis of this analysis is if the PHI value is below 0.05 (Фw < 0.05), there is significant evidence for the presence of recombination.
Culture characteristics: Colonies on SNA 45-55 mm diam, after 2 weeks at 25 °C, pale olive, flat, velvety, margin regularly, reverse light olive. Colonies on PDA 40-50 mm diam, after 2 weeks at 25 °C, smoke-gray to light olive-gray, reverse leaden-gray, gray-olive at edge both surface and reverse, woolly or felty, broad edge, regular, growth low convex, without protuberant exudates, reverse formed cracks in the middle small circle. Colonies on MEA 30-40 mm diam, after 2 weeks at 25 °C, smoke-gray to light olive-gray, woolly or felty, fluffy, with a whitish narrow final edge; reverse olive-yellow or olive-brown, radially furrowed, irregularly folded, with a whitish narrow final edge. Colonies on OA 30-45 mm diam, after 2 weeks at 25 °C, gray-green or olive, granular and fluffy mycelium, woolly and felty edge, with an irregularly folded whitish and olive final edge; reverse olive-yellow or olive-brown, with a whitish narrow final edge.

Cladosporium perangustum
Culture characteristics. Colonies on SNA 30-40 mm diam, after 2 weeks at 25 °C, pale olive to pale whitish, flat, velvety, with a regular edge, reverse light olive to light white. Colonies on PDA 30-40 mm diam, after 2 weeks at 25 °C, olive-gray to olive-green or olive-brown, powdery or flocculent, fluffy, regular, radially furrowed, lacerated or feathery, and often forming a gray-white or olive bulge in the colony kernel; reverse dark olive or dull green to black. Colonies on MEA 35-45 mm diam, after 2 weeks at 25 °C, gray-green to white or gray-white, fluffy, radially furrowed, with a whitish final edge; reverse olive-yellow to olive-gray to olive-green, with a whitish final edge. Colonies on OA 35-45 mm diam, after 2 weeks at 25°C, white to olive-green, with a pale gray final edge, velvety or fluffy, margins colorless or pale gray, glabrous, regular; reverse olive-green to dark green.

Cladosporium tenuissimum
Culture characteristics: Colonies on SNA 50-55 mm diam, after 2 weeks at 25 °C, pale olive to pale white, flat, velvety, with a regular edge, reverse light olive to light white. Colonies on PDA 40-55 mm diam, after 2 weeks at 25 °C, smoke-gray to light olive-gray or olive to light olive-gray, reverse leaden-gray, gray-olive at edge both surface and reverse, woolly or felty, broad edge, regular, growth low convex, without protuberant exudates, occasionally reverse formed a sunflower like shape in the middle. Colonies on MEA 40-50 mm diam, after 2 weeks at 25 °C, olive-gray or gray, fluffy; reverse olive-green to dark olive, with an olive-yellow to gray-white edge, radially furrowed. Colonies on OA 40-60 mm diam, after 2 weeks at 25 °C, gray-white or irongray to gray-olive, fluffy to felty; reverse olive-brown to olive.
Our five strains pertain to two known species, viz., C. perangustum and C. tenuissimum, but with E. ulmoides as new substrate records for these species. The main focus of this study was the exploration of the diversity of microfungi associated with a E. ulmoides plantation forest. In previous studies, Cladosporium parapenidielloides was found on Eucalyptus sp. in Australia, C. perangustum on Magnolia sp. in the USA, and C. pini-ponderosae on Pinus ponderosa in Argentina. So far, Cladosporium species have never been isolated from fallen leaves of E. ulmoides, the only species of the genus Eucommia.