Aspergillus labruscus sp. nov., a new species of Aspergillus section Nigri discovered in Brazil

A novel fungal species, Aspergillus labruscus sp. nov., has been found in Brazil during an investigation of the fungal species present on the surface of grape berries (Vitis labrusca L.) for use in the production of concentrated grape juice. It seems to be associated to V. labrusca, and has never been recovered from Vitis vinifera. This new species belonging to Aspergillus subgenus Circumdati section Nigri is described here using morphological characters, extrolite profiling, partial sequence data from the BenA and CaM genes, and internal transcribed spacer sequences of ribosomal DNA. Phenotypic and molecular data enabled this novel species to be clearly distinguished from other black aspergilli. A. labruscus sp. nov. is uniseriate, has yellow mycelium, poor sporulation on CYA at 25 °C, abundant salmon to pink sclerotia and rough conidia. Neoxaline and secalonic acid D were consistently produced by isolates in this taxon. The type strain of A. labruscus sp. nov. is CCT 7800 (T) = ITAL 22.223 (T) = IBT 33586 (T).

in Aspergillus and recommended DNA markers as a reliable means of identification. Information on living ex-type culture collection numbers and GenBank accession numbers for available representative ITS, CaM, BenA and RPB2 sequences were listed, and CaM sequences were proposed as important identification markers for Aspergillus section Nigri.
During an investigation of the fungal species in grapes for juice production, a new Aspergillus taxon in Aspergillus section Nigri was found on the surface of grape berries. This species was found only in the region of Serra Gaúcha, Rio Grande do Sul, and on one grape variety, "Bordô". It is described here as Aspergillus labruscus sp. nov. and this species does not produce ochratoxins or fumonisins.

Results and Discussion
Molecular identification. It is very difficult to identify fungi belonging to Aspergillus section Nigri due to the subtle morphological differences between species, and therefore DNA sequence information is increasingly being used for species identification and diagnosis.
Fungi belonging to section Nigri have been identified mainly using CaM gene sequences, because the internal transcribed spacer (ITS) of the nuclear ribosomal (nrDNA), accepted as the official DNA barcode for fungi 12 , is insufficient for correctly identifying all Aspergillus section Nigri species 11 . The CaM gene sequence contains more variation than the ITS and the nucleotide sequence database is complete for all accepted species 11 .
Thus, as an initial step, part of the CaM gene sequence was determined for 275 isolates of Aspergillus section Nigri found on the surface of the V. labrusca grape berries for four different Brazilian regions, to identify the species. All CaM gene sequences were compared using the Basic Local Alignment Search Tool (BLAST) against the NCBI database to recognize fungal species that have similar DNA sequences. The majority of gene sequences were similar (sequence identity ≥99%) to one of the 27 species belonging to Aspergillus section Nigri, but the CaM gene sequences from 23 isolates were found to be significantly different from all other sequences of species described so far (NCBI accessed 20 Dez, 2016). Using the BLAST tool it was found that the CaM gene sequences from these isolates are most similar to those from Aspergillus homomorphus, but with only 85% of sequence identity.
Samson et al. 11 provided an updated accepted species list for the genus Aspergillus, now containing 339 species, and to enhance the scientific value of the list, it includes information on living ex-type culture collection numbers and GenBank accession numbers for available representative ITS, CaM, BenA and RPB2 sequences. We generated a CaM-based phylogram of the 23 sequences not assigned to any described species using the BLAST tool, and those retrieved from GenBank for each Aspergillus section Nigri on Samson's updated list. The CaM-based phylogram placed our isolates on a branch clearly separated from all other species of Aspergillus section Nigri (Fig. 1). The novel isolates were found to belong to a clade including A. homomorphus and Aspergillus saccharolyticus.
Six isolates of the putative novel species were randomly selected for further analysis. The nrDNA (ITS), region and partial BenA gene were sequenced. As discussed by Samson et al. 11 , a frequent difficulty experienced with sequence-based identifications is comparing newly obtained sequences with verified sequence databases. GenBank is a public, archival database, which means that it accepts all sequences submitted and cannot always verify the taxonomic names attributed to the sequences, and therefore BLAST search results may give hits to misidentified sequences in the database. In an attempt to clean up misidentified GenBank sequences, the RefSeq initiative was launched (http://www.ncbi.nlm.nih.gov/refseq/), and lists only verified sequences 13 . For Aspergillus, all ex-type sequences were included in the RefSeq database. Using the BLAST tool with the "sequences from type material" (RefSeq) option (accessed 20 Jan, 2017), the ITS sequence from our novel isolates was found to be most   11 . Interestingly, the ITS amplicon length from all isolates of the putative novel species (540 bp), obtained with the ITS1-ITS4 primer-pair 14 , was smaller than that of all other Aspergillus section Nigri. They exhibit a 38-39 bp deletion in the ITS1 region when compared to A. aculeatus, A. homomorphus, A. japonicus and A. saccharolyticus (Fig. 2).
Although the BenA gene may vary in the number of introns, and the PCR sometimes results in the amplification of paralogous genes 15 , this locus has been frequently used for molecular identification of Aspergillus. Thus, the DNA sequences from the six isolates of the putative novel species were determined for a portion of BenA. Comparative BLAST sequence analyses of the six isolates revealed that they are more similar to A. homomorphus ex-type strain CBS 101889, with 85% of sequence identity. A BenA-based phylogram placed the six isolates on a branch undoubtedly separated from all other species of Aspergillus section Nigri (Supplementary Figure 1).
In conclusion, the ITS, BenA and CaM sequences harmonized to show that our isolates belong to a novel phylogenetic species. We suggest the name Aspergillus labruscus sp. nov. for this new phylogenetic species and as its type, strain CCT 7800 (T) = ITAL 22.223 (T) = IBT 33586 (T). The GenBank accession numbers of the ITS barcodes and alternative identification markers BenA and CaM deposited for this paper are present in Table 1.
Metabolite Analysis. Six strains of A. labruscus were examined for extrolites and they all produced secalonic acid D and neoxaline ( Table 2). The phylogenetically closely related species A. saccharolyticus differed from A. labruscus by producing aculene A and B 16 and homomorphosins. A. homomorphus 17 shares production of secalonic acid D with A. labruscus, but differs by producing homomorphosins and 3-methoxy-5-hydroxy-9 -phenyl-2,4,6,8-nanotetranoic acid lactone (Hoeck, Petersen, Frisvad, Gotfredsen and Larsen, personal communication). The ex type strain produced two as yet not structure elucidated extrolites, that have only been observed in A. labruscus. Metabolite analysis indicated that A. labruscus does not produce ochratoxins and fumonisins.
Macro and Micromorphology Analysis. Colony diameters after 7 days at 25 °C were as follows: CYA: 70-77 mm, MEA: 56-60 mm, YESA: 69-79 mm, OAT: 42-45 mm, CREA: 3-4 mm and CYA + 5% NaCl: no growth. On CYA at 10 °C conidia germinate, at 15 °C, 20 °C and 30 °C growth is evident with colony diameters of 9-17 mm, 59-71 mm and 56-86 mm, respectively. The species does not grow on CYA at 37 °C and 42 °C while at 33 °C the colony diameters vary from 0 to 22 mm, indicating that at this temperature the growth response varies among the isolates. At 25 °C in CYA, the species showed yellow mycelium, poor sporulation, abundant salmon to pink sclerotia production, and reverse pale yellow in color. Figure 3 shows the colonies of A. labruscus (CCT 7800) on different media and temperatures. Micromorphology characters are as follows: uniseriate, spherical conidial heads, brown colored stipes smooth with thick wall (3.2-6.1 µm), vesicle 43 × 60 µm, phialides 7.2-7.8 × 3.8 µm, conidia not uniform in size and shape, spherical to ellipsoidal 6.5-8 × 6.1-6.9 µm, black and  conspicuously rough. Figure 4 shows the morphology on CYA and MEA at 25 °C and the micromorphological characteristics. Morphologically A. labruscus is similar to Aspergillus sclerotioniger because both have yellow mycelium, poor sporulation on CYA at 25 °C, abundant sclerotia and rough conidia. However, they differ because A. sclerotioniger has biseriate conidiophore, yellow sclerotia and produces ochratoxin A 11 while A. labruscus is uniseriate, has pink to salmon sclerotia and does not produce this mycotoxin.
Although molecular data revealed that A. labruscus is most similar to A. homomorphus, A. saccharolyticus and A. aculeatus, the yellow mycelium, salmon to pink sclerotia and large spherical to ellipsoidal conidia (6.5-8 µm) differentiate A. labruscus from these species. Furthermore, A. labruscus differed from A. homomorphus and A. saccharolyticus because these two species were able to grow on CYA at 37 °C with diameters 18 mm and 7-10 mm, and on CYA + 5% NaCl with diameters of 68 mm and 17 mm, respectively.
Incidence and Ecology. Among a total of 89 grape samples collected from different regions and grape cultivars analyzed for fungal contamination, 5 samples (5.6%) revealed the presence of A. labruscus. In total, 23 isolates of this new species were found on the surface of grape berries, V. labrusca, cv. Bordô grown in Rio Grande do Sul (Serra Gaúcha). The origins of representative A. labruscus isolates are shown in Table 3. This new species was not found in the other Brazilian regions. Table 4 shows the frequency of A. labruscus on the samples from different vineyards of Rio Grande do Sul and the range of contamination. Serra Gaúcha has a distinct climatic condition for vineyards, with an annual average temperature of 17 °C, rainfall of 1,700 mm and relative humidity of 76%. While most of the world's vineyards grow the European cultivars (V. vinifera) which produces fine wine, in Brazil due to the high humidity conditions, this species was affected by fungal disease and did not adapt to Brazilian conditions. Thus, the rustic North American cultivars of V. labrusca were introduced, which showed high disease resistance, and today 80% of Brazilian vineyards grow the cultivar V. labrusca, especially in the State of Rio Grande do Sul.
It is interesting to note that A. labruscus has never been found on V. vinifera grapes. A similar situation is known for peanuts where the species Aspergillus arachidicola, has only been found on the wild type species Arachis glabrata, but not on the domesticated peanut, Arachis hypogea 18 .
Data on growth temperatures under laboratory condition as presented above showed that A. labruscus was able to grow at temperatures of 15 °C to 33 °C, with no growth at 37 °C. Most members of Aspergillus section Nigri can grow at 37 °C and even higher 19,20 , including the closely species A. homomorphus and A. saccharolyticus. A. labruscus has never been found before in European vineyards and grape products 21 indicating that A. labruscus have an association with V. labrusca rather than V.vinifera. Data on the growth temperature of A. labruscus show that this species is adapted to colder temperatures, which may be due to its North American origin and therefore well-adapted to the Serra Gaúcha region which is colder than the other regions in Brazil. In addition, data to be published later will show that this species has not been found in V. labrusca cv. Bordô vineyards in the North Brazilian region.
Enzyme production. Black aspergilli are particularly effective enzyme producers 20 and the three closely related species A. labruscus, A. homomorphus and A. saccharolyticus were examined for the production of two extracellular enzymes: tannase and caseinase (Table 5). On TAN medium used for tannase production, A. labruscus, A. homomorphus and A. saccharolyticus grew and showed good sporulation, but the tannase production was most pronounced in A. homomorphus, followed by A. labruscus and was poor in A. saccharolyticus. Wine fruits have a high content of tannin, and it would be expected that A. labruscus can produce tannase. On Pro agar, the three species grew and sporulated well, but caseinase was not produced by A. homomorphus while A. labruscus and A. saccharolyticus showed very good production of this enzyme. These two culture media for extracellular enzyme production showed good performance to differentiate these three species, showing that they are distinct and giving a different response in enzyme production. Since A. saccharolyticus is one of the best β-glucosidase producers known 22,23 , it should be investigated whether A. labruscus is also an effective producer of such enzymes.

MYCOBANK: MB815746
Etymology: "labrusca" is Latin for wild grape vine, on which this fungus species was found.

Methods
Grape samples and mycological analysis. The 89 grape samples were collected from four Brazilian states: Pernambuco (n = 22), São Paulo (n = 21), Paraná (n = 16) and Rio Grande do Sul (n = 30). In 2014, twenty bunches were collected from each field, close to harvest time between late January and early February in Paraná and Rio Grande do Sul, in April in Pernambuco, and in December in São Paulo. The V. labrusca grape cultivars studied were "Bordô", "Cora", "Concord", "Isabel", "Isabel Precoce", "Violeta", "Coder", "Rudder", "Niagara" and "Muscadine". These are the grape cultivars most widely used for juice production in Brazil. The samples were collected as described in Serra et al. 25 , across two diagonal transects. A total of 100 berries of each sample was plated according to Pitt and Hocking 26 but without surface disinfection. All Aspergillus section Nigri fungi were transferred to CYA (Czapek Yeast Extract agar, formulated) and incubated at 25 °C for seven days.

Molecular analysis.
A total of 275 Aspergillus section Nigri isolates obtained from the grapes were randomly selected and subjected to DNA extraction using a commercial extraction kit, Biopur Mini Spin Planta (Biopur, Brazil), according to the manufacturer's instructions. As an initial step to identify the isolates, partial amplification and sequencing of the CaM gene were performed as described in detail by Taniwaki et al. 27 . The CaM sequences obtained were aligned with type or neotype strain sequences from all Aspergillus section Nigri species obtained from the database maintained by NCBI (http://www.ncbi.nlm.nih.gov/). Alignment was performed using ClustalW 28 . MEGA6.0 software 29 was used to construct a Maximum Likelihood (ML) tree, based the Tamura-Nei model 30 . Of the twenty three isolates none were phylogenetically closely related to any of the species described so far, six were further investigated using the ITS1-5.8S-ITS2 region of nrDNA (ITS) and partial BenA gene sequences. Partial amplification and sequencing of the BenA gene and ITS region were performed as described in Taniwaki et al. 27 . The ITS and BenA sequences obtained were aligned with the type or neotype strain sequences from all Aspergillus section Nigri species using the above-mentioned approaches.
Extrolite analysis. Six isolates of A. labruscus (Table 2), two isolates of A. homomorphus (IBT 21893 and IBT 21894) and two isolates of A. saccharolyticus (IBT 28231 and IBT 30881 = CBS 127449) were analysed for small molecule extrolites by extracting 3 agar plugs from 7 day incubated cultures at an incubation temperature of 25 °C in darkness 31 . The extracts were analyzed by ultra high performance liquid chromatography using diode array detection (UHPLC-DAD) 32 and compared to authentic small molecule extrolite standards 33 .

Morphological characterization. Morphological characterization was performed based on Samson et
al. 11 . Briefly, a spore suspension was prepared in 0.5% agar dissolved in water. Petri dishes were inoculated in a three-point pattern. The following culture media (formulated) and incubation temperature were used: Czapek yeast autolysate (CYA) agar at 10 °C, 15 °C, 20 °C, 25 °C, 30 °C, 33 °C, 37 °C, and 42 °C; malt extract agar (MEA), oatmeal agar (OAT), creatine sucrose agar (CREA) and CYAS (Czapek yeast autolysate agar with 5% NaCl), at 25 °C. For comparison, two isolates of A. homomorphus (IBT 21893 and IBT 21894) and two isolates of A. saccharolyticus (IBT 28231 and IBT 30881 = CBS 127449) were also grown on CYA at 37 °C and CYAS at 25 °C. After seven days, macro and micromorphological characters of A. labruscus were examined under light optical microscopy and scanning electron microscopy. The slide for light optical microscopic observations was prepared using lactic acid and lactophenol cotton blue. For each structure 3 replicate measurements were performed. For scanning electron microscopy, a 0.5 × 0.5 cm plug was fixed for 24 h at 4 °C in 2% glutaraldehyde in 0.1 M of NaPO 4 buffer. The plug was washed three times for 15 min (each) in 0.1 M phosphate buffer (NaPO 4 ) and post-fixed in osmium tetroxide 1% buffer for 2 h at 25 °C in the dark. The plug was then washed again three times in 0.1 M phosphate (NaPO 4 ) buffer for 15 min and dehydrated in ethanol series (70, 80, 90 and 100%) for 10 min. It was then placed in a critical point dryer (Bal-Tec, CSDC 030) and subsequently sputter-coated with gold (Bal-Tec, SDC 050). The observation was performed using a FEI Quanta 200 ® microscope.