Molecular identification of Pseudozyma aphidis ( Henninger & Windisch ) Boekhout : first record from a Brazilian mangrove swamp

(Molecular identification of Pseudozyma aphidis (Henninger & Windisch) Boekhout: first record from a Brazilian mangrove swamp). Pseudozyma aphidis (Henninger & Windisch) Boekhout is an anamorph yeast-like basidiomycete commonly found as saprotroph in vegetable debris, such as leaves and flowers. However, there are also reports that this species is pathogenic in humans, usually occurring in patients with some type of immunosuppression, which predisposes to opportunistic infections. Specimens of P. aphidis were collected from water samples and Rhizophora mangle L. leaves at different salinities along the Perequê River, located in a mangrove swamp area at Parque Estadual da Ilha do Cardoso, Cananéia municipality, São Paulo State, Brazil. Aliquots of these samples were spread in Petri dishes. The samples baited with cataphylls of Allium cepa L. were incubated for seven days at 21 oC. After incubation, the baits were observed under microscope and the specimens were isolated and purified. The identification of the specimens was made through the phylogenetic analysis of the ITS rDNA region. This is the first record of P. aphidis in São Paulo State.


Introduction
Pseudozyma Bandoni emend.Boekhout pro tempore comprises approximately 25 distinct anamorphic yeast species from the Ustilaginaceae, Basidiomycota (Index Fungorum 2016).Although a reliable connection with their teleomorph stages has not been established for many of these species (Boekhout 2011), recent studies have proposed to transfer anamorphic yeast species to their corresponding teleomorphic genera based on strongly supported phylogenetic affinities (Piatek et al. 2015, Wang et al. 2015).From these studies, many species of the genus Pseudozyma have been transferred to the genus Moesziomyces, as is the case of Pseudozyma aphidis (= Moesziomyces aphidis) and the use of the "pro tempore" was suggested by the authors to indicate that in Pseudozyma species names are temporarily remained.The results of these studies allowed the proposition of 16 new combinations for Pseudozyma (Index Fungorum 2016), however, the taxonomic status of five species still remains to be determined because of their uncertain phylogenetic positions (Bandoni 1985, Boekhout 1995, Sugita et al. 2003, Wang et al. 2006, Liou et al. 2009).
In Latin America, the first report of P. aphidis was in Brazil when Costa (2006) reported the presence of this species, through molecular identification of the region D1/D2 of 26S rDNA, in semi-arid soil from Mucugê municipality, Bahia State.Tristão et al. (2012) reported an association of P. aphidis with pineapple fruits sampled from the Central Region of Tocantins State.As a human pathogen, the first case was reported from a public pediatric oncology center in Recife, Pernambuco State, causing pulmonary infection in a patient with Burkitt lymphoma (Parahym et al. 2013).
The occurrence and activity of yeasts in marine waters have been observed worldwide since first reported by Fischer & Brebeck (1894) and is well established due to their role in the decomposition of organic substrates, nutrient cycling, hydrocarbon biodegradation, and as pathogenic for a variety of marine organisms (Vogel et al. 2007, Kutty & Philip 2008).Species of Pseudozyma, including P. aphidis, have commonly been reported in marine and mangrove environments (Gadanho et al. 2003, Statzell-Tallman et al. 2010, Fell et al. 2011).
Several papers have also shown the potential of this species for inducing systemic resistance and as a biocontrol agent against plant pathogens of different crops (Tristão et al. 2012, Buxdorf et al. 2013a, b, Gafni et al. 2015).They are also reported as a potential source of squalene (Chang et al. 2008), lipase (Dimitrijevic et al. 2011) and biosurfactant mannosylerythritol lipids (Morita et al. 2007, Fan et al. 2014, Lorenz et al. 2014, Gunther et al. 2015).
Molecular methods are useful tools for the accurate positioning of yeasts within the Kingdom Fungi, being the classic methods of limited value due to the low morphological variation between species and the existence of a significant yet undescribed diversity (Hoog et al. 1998).The use of nucleotide sequences of internal transcribed spacer (ITS) of the rDNA region is considered the best tool for rapid and accurate identification of yeast isolates (White et al. 1990).
During a survey at the Parque Estadual da Ilha do Cardoso (PEIC), we identified four isolates of P. aphidis based on the phylogenetic analysis of the ITS rDNA region.Thus, the aim of this study is to present the phylogeny of the genus with the inclusion of specimens collected in this mangrove area of Brazilian Atlantic Rainforest.

Material and methods
The Pseudozyma specimens used in this study were isolated from water samples and Rhizophora mangle L. leaves collected (in sterile plastic bottle) in the months of August and November 2012 at different salinities (1.5 to 3%) along the Perequê River.The study area is located in a mangrove swamp from Parque Estadual da Ilha do Cardoso (PEIC), 25°03'05"-25°18'18"S; 47°53'48"-48°05'42"W, situated in Cananéia municipality, São Paulo State, Brazil.This island is a preserved fragment of Atlantic Rainforest that contains different types of vegetation, including tropical forest, "restinga", dunes and mangrove swamps, with several water bodies like waterfalls, streams and rivers (Barros et al. 1991).During the sampling, the surface water temperature of the estuary was lower in August, about 20 ºC and higher in November, about 27 ºC.
Aliquots (30 mL) of water samples and disks (1 cm diameter) of R. mangle leaves (washed in sterilized seawater) together with 30 mL of a dilution of 50% reverse osmosis and 50% seawater, were placed separately in Petri dishes.These samples were baited with cataphylls of Allium cepa L. (Sparrow 1960, Milanez 1989) and incubated for seven days at 21 ºC.After incubation, the baits were observed under microscope and the specimens were isolated and purified onto PYGs culture medium (Fuller & Jaworski 1987) prepared with a dilution of 50% sterile seawater, penicillin G 0.4 g L -1 and streptomycin sulphate 0.4 g L -1 .Pseudozyma aphidis IMUFRJ51941 FN424100
* Taxa, strain numbers and GenBank accession numbers of the isolates of Pseudozyma aphidis sequenced in this study.The specimens were preserved by the Castellani's method (50% sterile reverse osmosis water and 50% sterile seawater) and posteriorly deposited at the CCIBt culture collection (Coleção de Culturas de Algas, Cianobactérias e Fungos do Instituto de Botânica, São Paulo, SP, Brazil).
Biomass for DNA extraction was obtained by cultivating each isolate onto PYGs culture medium, prepared with a dilution of 50% sterile seawater, penicillin G 0.4 g L -1 and streptomycin sulphate 0.4 g L -1 , for seven days at 21 ºC.After incubation, the cells were harvested using a platinum loop.The DNA extraction followed the protocol described in the PureLink Genomic DNA kit (Invitrogen ® ).The ITS1-5.8S-ITS2 region was amplified using the primers UN-up18S42 and UN-lo28S22 (Robideau et al. 2011).PCR amplification was performed with the PCR SuperMix kit (Invitrogen ® ) for a final volume of 25 μl in a C1000 Touch™ Thermal Cycler Bio-Rad following the conditions described by Marano et al. (2014).Amplicons were purified with AxyPrep PCR Clean-up kit (Axygen ® ).Sequencing was performed using the same PCR primers in an ABI 3730 DNA Analyser (Life Technologies™).Assembly of contigs and correction of ambiguous bases were manually edited using Sequencher ™ version 4.1.4.
The ITS sequences obtained were compared against BLASTn (Basic Local Alignment Search Tool).For phylogenetic reconstruction, we used ITS rDNA sequences of our isolates and of the other isolates of Pseudozyma available at GenBank (table 1).We analysed 42 sequences from 23 species, using Cintractia axicola (Berk.)Cornu and Trichocintractia utriculicola (Henn.)M. Piepenbr.as outgroups.Sequences were aligned using MAFFT version 7 (Kazutaka & Daron 2013), the ambiguously aligned characters were removed manually using Geneious version 8 (Kearse et al. 2012) and the alignment was deposited in the Treebase (Boettinger & Lang 2011).The best fitting model of evolution was selected using the Akaike Information Criterion in jModeltest version 0.1.1 (Posada 2008).The Maximum Likelihood (ML) phylogenies were reconstructed with PhyML version 3.1 (Guindon & Gascuel 2003) using the TPm2uf + G substitution model, branch swapping by best of NNI and SPR, and support for nodes obtained by 1,000 bootstrap pseudo-replicates.Bayesian inferences (BI) were generated using MrBayes version 3.2.1 (Ronquist & Huelsenbeck 2003) with Markov Chain Monte Carlo (MCMC) methodology to calculate posterior probabilities of the phylogenetic trees.The program was run for 5 millions generations with the TPm2uf + G substitution model.The first 10% of the interactions were discarded as burn-in.

Results and Discussion
The specimens grew abundantly on Allium cepa L. used as bait and later onto PYGs (figure 1a), with a great number of cells produced in one week (figures 1b, 1c).
The BLASTn query showed that these specimens belong to Pseudozyma aphidis (alignment in the Treebase, http://purl.org/phylo/treebase/phylows/study/TB2:S21546).Our isolates of P. aphidis are well supported and clustered together with other isolates of the same species (90% of bootstrap support with high values of posterior probability (0.98), as shown in figure 2. This species has a close phylogenetic affinity with P. rugulosa (80% of bootstrap support and 0.98 of posterior probability), as previously demonstrated (Chang et al. 2008).
The culture collection numbers of the isolates obtained in this study and their respective sequences deposited in GenBank database are shown in table 1.
Although P. aphidis has been already reported in Brazil (Costa 2006, Tristão et al. 2012, Parahym et al. 2013) and worldwide, this is the first report from a mangrove area in São Paulo State.

Figure 2 .
Figure 2. Maximum likelihood tree of internal transcribed spacer (ITS) rDNA sequences of Pseudozyma spp.Maximum likelihood bootstrap support and posterior probability values large than 50% are indicated numerically, those under 50% are marked with (-).Those clades that were not present in the Bayesian trees are marked as (0).The scale bar represent the average number of substitutions per site