Penicillium excelsum sp. nov from the Brazil Nut Tree Ecosystem in the Amazon Basin’

A new Penicillium species, P. excelsum, is described here using morphological characters, extrolite and partial sequence data from the ITS, β-tubulin and calmodulin genes. It was isolated repeatedly using samples of nut shells and flowers from the brazil nut tree, Bertolletia excelsa, as well as bees and ants from the tree ecosystem in the Amazon rainforest. The species produces andrastin A, curvulic acid, penicillic acid and xanthoepocin, and has unique partial β-tubulin and calmodulin gene sequences. The holotype of P. excelsum is CCT 7772, while ITAL 7572 and IBT 31516 are cultures derived from the holotype.


Introduction
Penicillium species are very important agents in the natural processes of recycling biological matter. Some species cause deterioration of all sorts of man-made goods; some rot fruit or spoil foods; some species secrete secondary metabolites (extrolites) such as mycotoxins (e.g. ochratoxins, patulin, citrinin), while other extrolites are used as pharmaceuticals, including antibiotics such as penicillin and the cholesterol-lowering agent lovastatin [1,2,3,4]. Some species are known for their production of organic acids and diverse enzymes that degrade a wide variety of complex biomolecules [1,2,3]. A variety of species are capable of producing or modifying biological chemicals, and this field is set for great expansion. A few species are directly involved in food production: this field is not likely to expand, because many species produce mycotoxins. Penicillium is an ascomycete genus and belongs to the family Aspergillaceae [4]. More than 350 species are currently accepted in this genus [5].
The Amazon rainforest has multiple ecosystems with a huge fungal biodiversity. It has an important role in the global weather balance and is the location of many native people. The equatorial climate is hot and humid, with an average temperature of 26°C and relative humidity 80-95%.
Brazil nuts are one of the most important products taken from the Amazon rainforest region. Brazil nut trees, Bertholletia excelsa Humb. & Bonp., grow wild, take 12 years to bear fruit, may live up to 500 years and reach up to 60 m high. Pollination of the unusual flowers requires wild, large bodied bees, especially from the family Euglossinae [6]. The fungal species most commonly isolated from brazil nuts are Aspergillus flavus, A. nomius, A. pseudonomius, A. niger, A. tamarii, Penicillium glabrum, P. citrinum, Rhizopus spp., Fusarium oxysporum [7,8,9,10,11,12] and A. bertholletius, a species described recently [13].
During a study of the mycobiota of the brazil nut tree ecosystem, including flowers, brazil nuts, soil, bees and ants, an undescribed Penicillium species was found. This species is described here as Penicillium excelsum sp. nov.

Sample collection, isolation and morphological examination
Samples were collected from the ecosystem of the brazil nut tree, Bertholletia excelsa in the Amazon rainforest in Para and Amazon States, Brazil. Sample collection and methodology have been described previously [13]. Briefly, samples of brazil nut kernels and shells, flowers and leaves, soil from beneath the trees, plus bees and ants. Collecting was carried out in collaboration with the Brazilian Ministry of Agriculture.
For fungal isolation, nuts and shell samples were disinfected in sodium hypochlorite solution, then plated onto dichloran 18% glycerol agar (DG18), according to the methodology of Pitt and Hocking [2]. Soil samples were mixed with sterile water containing peptone (0.1%), then serially diluted and spread plated onto DG18. Flower and leaf samples were surface disinfected as above and plated onto DG18 while bee and ant samples were plated on DG18 without surface disinfection. All plates were incubated at 25°C for 7 days, then all colonies of Penicillium species were transferred onto Czapek yeast extract agar [2] and incubated at 25°C for 7 days for further identification.
The Penicillium isolates were examined on standard identification media for Penicillium species according to Pitt [14] namely: Czapek yeast extract agar (CYA), malt extract agar (MEA, Oxoid), and 25% glycerol nitrate agar (G25N) at 25°C and also on CYA at 37°C and 42°C, plus oatmeal agar (OAT), creatine sucrose agar (CREA) and yeast extract sucrose (YES) agar [3]. The incubation time for all media was 7 days and plates incubated in the dark.
The standard conditions used for the description of Penicillium excelsum are taken from Pitt [14] and Frisvad and Samson [15]. Capitalized colours are from the Methuen Handbook of Colour [16].
DNA extraction, amplification, sequencing and phylogenetic analysis A standard phenol:chloroform extraction protocol [17] was used for genomic DNA isolation from an extype culture (ITAL 7572). The primer-pairs ITS1-ITS4 [18], Bt2a-Bt2b [19] and cmd5-cmd6 [20] were used to amplify the ITS1-5,8S-ITS2 region (ITS), partial β-tubulin gene (BenA) and partial calmodulin gene (CaM) respectively, adopting a standard amplification cycle, which ran 35 cycles with an annealing temperature of 55°C [5]. Excess primers and dNTPs were removed from the PCR product using the Wizard1 SV Gel and PCR Clean-Up System (Promega, Wisconsin, USA). Purified PCR products were sequenced in both directions using a BigDye1 Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, California, USA) according to the manufacturer's instructions. A volume of HiDiformamide (10 μl) was added to the sequencing products, which were processed in an ABI 3500XL Genetic Analyzer (Applied Biosystems). Contigs were assembled using the forward and reverse sequences with the programme SeqMan from the Laser Gene package (DNAStar Inc., Wisconsin, USA). All sequences were subjected to Basic Local Alignment Search Tool (BLAST) against the NCBI database to identify Penicillium species with similar DNA sequences. The ITS and BenA sequences were aligned by ClustalW algorithm using Mega5.1 software (21) with those from Penicillium subgenus Aspergilloides section Lanata-Divaricata type or neotype strains, as recently suggested by Visagie et al [5]. Phylogenetic trees were constructed with Mega5.1 software [21], using the Neighbor-Joining (NJ) and Maximum Likelihood (ML) methods based on the Tamura-Nei model [22]. To determine the support for each clade, a nonparametric bootstrap analysis was performed with 1,000 resamplings.

Extrolite analysis
Cultures were analysed by High Performance Liquid Chromatography (HPLC) with a diode array detector (HPLC-DAD) as described by Frisvad and Thrane [23] and modified by Houbraken et al. [24], as previously described [13]. Three agar plugs each from CYA and YES medium were pooled and extracted with 0.75 mL of a mixture of ethyl acetate/ dichloromethane/methanol (3:2:1)  Results and Discussion

Sources of the isolates
In total, 116 isolates of -the new species described here as Penicillium excelsum were found in brazil nut shells and kernels, from soil close to Bertholletia excelsa trees, and from flowers, bees and ants associated with Bertholletia trees. The origins of representative P. excelsum isolates are shown in Table 1. Soil may be the primary habitat of this species, as many species of Penicillium are soil fungi [4,25]. However, this study shows that P. excelsum also occurs on bees and ants, which may carry spores to the flowers, and other locations by contact or excreta which will all play a role in dispersal of this species.

Extrolites
HPLC-DAD analysis of extracts showed that several strains of P. excelsum produce andrastin A, penicillic acid, while some also produce xanthoepocin. Strain ITAL 3000 also produced curvulic acid. Related species also produce penicillic acid, for example P. brasilianum, P. cremeogriseum, P. ochrochloron P. pulvillorum and P. vanderhammenii [24,26,27]. P. pulvillorum and P. simplicissimum have also been reported to produce andrastin A, and P. brasilianum, P. ochrochloron, P. pulvillorum, P. rolfsii, P. simplicissimum and P. svalbardense have been reported to produce xanthoepocin [24,28]. Even though andrastin A, penicilllic acid, and xanthoepocin have been found in species outside section Lanata-Divaricata [15] the particular combination of these extrolites is mostly found in this section. P. excelsum produces a profile of extrolites close to that of P. brasilianum, P. ochrochloron, P. pulvillorum and P. rolfsii and the close relationship is confirmed by sequence and morphological data as shown in Figs 1, 2 and 3.

Phylogenetic analyses
P. excelsum ITS, BenA and CaM sequences were found to be different from all other sequences in NCBI (accessed 30 May, 2015). When the BLAST searches were performed using the option "sequences from type material" [29] the sequences harmonized in showing that P. excelsum is   most similar to P. ochrochloron neotype strain CBS 357.48 and P. pulvillorum neotype CBS 280.39. Both, P. pulvillorum and P. ochrochloron belong to Penicillium subgenus Aspergilloides section Lanata-Divaricata in the recent phylogenetic reclassification of Penicillium [4]. A more recent study [5] provided GenBank accession numbers to reference sequences for all accepted Penicillium species. Using these reference sequences, ITS-based phylograms (data not shown) generated using Neighbor-Joining and Maximum Likelihood techniques confirmed the placement of P. excelsum in section Lanata-Divaricata. Although the ITS phylograms of P. excelsum clustered and were differentiated from other species of section Lanata-Divaricata, the majority of bootstrap values of branches were low, meaning that the ITS tree was poorly resolved. The ITS region is accepted as the primary fungal barcode [30]; however, it is well known that the ITS region provides only poor resolution of many Penicillium species [5,31]. In consequence, it has been proposed [5] that β-tubulin (BenA) is an optimal secondary identification marker for Penicillium species. BenA-based phylograms, generated using both Neighbor-Joining and Maximum Likelihood methods, placed P. excelsum on a branch separated from all other species on Penicillium section Lanata-Divaricata (Figs 1 and 2). Neighbor-Joining and Maximum Likelihood based phylograms were consistent and reveled that P. excelsum represent a separated lineage within a clade composed of P. pulvillorum, P. svalbardense, P. piscarium, P. ochrochloron, P. rolfsii, and P. subrubescens.
On G25N at 7 days, 25°C, colonies 10-14 mm in diameter, low and dense, coloured buff with light sporulation; reverse brown to deep brown.
On YES agar at 7 days, 25°C, colonies 34-42 mm in diameter, moderate sporulation and a brown reverse.
At 37°C on CYA, colonies 8-22 mm in diameter, coloured grey to brown; soluble pigment brown, reverse deep brown.
At 42°C on CYA, no growth.

Distinguishing features
This species is classified in Penicillium subgenus Furcatum section Furcatum in the classification of Pitt [14] and Penicillium subgenus Aspergilloides section Lanata-Divaricata according to Houbraken and Samson [4].
Morphologically, P. excelsum differs from the closely related P. subrubescens, P. pulvillorum, P. piscarium, P. rolfsii, P. ochrochloron and P. svalbardense by having a combination of smooth stipes, the frequent formation of rami, and the production of large, ellipsoidal, smooth walled conidia. P. ochrochloron and P. rolfsii are similar, but have finely roughened conidia. P. subrubescens, P. pulvillorum, P. piscarium and P. svalbardense produce globose to subglobose conidia, and in addition the conidia of P. piscarium are distinctly rough-walled. P. excelsum grows well at 37°C, though not as well as P. rolfsii. Most isolates of P. subrubescens and P. pulvillorum produce a red reverse colour on malt extract agar, whereas the reverse of P. excelsum is pale brown.
This species is also distinguished by a unique profile of extrolytes and by unique DNA sequences in the ITS, BenA and CaM genes. This species is also notable in that cultures on CYA, MEA and YES agar cause the polystyrene plastic in Petri dishes to become opaque over time (Fig  4). The opaqueness cannot be removed using a scapel, as the chemical reaction with the plastic lids was irreversible. A volatile compound produced by the fungus as it grows must be responsible. A preliminary examination of the volatiles from P. excelsum showed that it produced large amounts of acetic acid. An HPLD-DAD analysis of the opaque layer on the Petri dish lid revealed no detectable extrolites, indicating that the compound responsible for the opaqueness is without a chromophore. This effect has not been reported from any Penicillium or Aspergillus species. Further studies will be carried out in order to determine these compounds.

Conclusion
P. excelsum represents a new important phylogenetic species after applying a polyphasic approach using morphological characters, extrolite data, ITS, BenA and CaM partial sequences. P. excelsum is distinguished by a combination of a unique profile of extrolites, DNA sequence, micro-morphological features and the unique capacity to render Petri dish lids irreversible opaque.