Three New Species of Aspergillus from Amazonian Forest Soil(Ecuador)

Donatella Mares Æ Elisa Andreotti Æ Maria Elena Maldonado ÆPaola Pedrini Æ Chiara Colalongo Æ Carlo Romagnoli

Received: 13 March 2008 / Accepted: 20 April 2008 / Published online: 2 July 2008

� Springer Science+Business Media, LLC 2008

Abstract From an undisturbed natural forest soil in

Ecuador, three fungal strains of the genus Aspergillus were

isolated. Based on molecular and morphological features

they are described as three new species, named A. quiten-

sis, A. amazonicus, and A. ecuadorensis.

Introduction

As part of a collaborative project with the University of Quito

(Ecuador) to find new microorganisms from undisturbed

natural places as possible sources of pharmacologically

active compounds, we isolated a few fungal strains from soil

samples from the Amazonian forest (Ecuador). The Ama-

zonian forest was chosen because it is a place with little

anthropogenic influence, where, to date, there is a consid-

erable biodiversity of animals, plants, and microorganisms.

The first step in this work was to study whether the

microorganisms were able to perform Baeyer-Villinger

(BV) oxidations. Three fungal isolates were able to induce

the oxidation of cyclic ketones to corresponding lactones

[2]. These compounds are interesting, since they are

starting products for the synthesis of several pharmaceuti-

cally active compounds, such as prostaglandins [7]. In this

work we studied in detail these three fungal isolates and

performed morphological observations and molecular

investigations to determine their systematic position.

Materials and Methods

Microorganisms

The three fungi most interesting for their enzymatic prop-

erties were isolated in the year 2004 from a soil collected in

the Amazonian forest, near the town of Makas (Ecuador).

The fungi were arbitrarily assigned the following alpha-

numerical codes: E 19 C, E 19 D, and E 19 F. The isolated

microorganisms were maintained in vials containing Sab-

ouraud dextrose broth (SD; Difco, Detroit, MI) and 20%

glycerol in liquid nitrogen at -190�C. Morphological

observations were carried out on fungi grown for 20 days

on the same solidified culture medium at 26� ± 2�C.

Morphological Analyses

Macroscopic colony characters (color of the aerial part and

of the reverse mycelia, texture, topography, and edges of

colonies) and microscopic morphological characteristics

were recorded and illustrated using a Leika stereomicro-

scope and a Cambridge Stereoscan 360 scanning electron

D. Mares � P. Pedrini

Dipartimento di Biologia ed Evoluzione, Universita di Ferrara,

C. so Ercole I d’Este, 32, I-44100 Ferrara, Italy

E. Andreotti � C. Romagnoli (&)

Dipartimento del Museo di Paleobiologia e dell’Orto Botanico,

Universita di Modena e Reggio E, v. le Caduti in Guerra 127,

I-41100 Modena, Italy

e-mail: carlo.romagnoli@unimore.it

M. E. Maldonado

Centro de Investigacion y Valoracion de la Biodiversidad,

Universidad Politecnica Salesiana (CIVABI-UPS), Avenida 12

de Octubre N 24-22 y Wilson, Casilla Postal 17 12 536,

Quito, Ecuador

C. Colalongo

Dipartimento di Scienze e Tecnologie Agroambientali,

University of Bologna, Via Fanin 44, 40127 Bologna, Italy

123

Curr Microbiol (2008) 57:222–229

DOI 10.1007/s00284-008-9178-9

microscope (Cambridge, UK), owned by the Electron

Microscopic Centre of the University of Ferrara, Italy.

Munsell color charts were used for description of the

colors [6]. Living cultures are deposited at the culture

collection of the Department of Biology and Evolution

(Ferrara University, Italy). Ultrastructural observations

were made by harvesting small samples from a 20-day

cultures of fungi grown on SDA. The samples were pro-

cessed using methods described previously [5].

Isolation and Analysis of Nucleic Acids

Nucleic acids were extracted as described by Griffin et al.

[3] with slight modifications. Briefly, about 50 mg of

fungal mycelia from each isolate was placed in a sterile

1.5-ml microcentrifuge tube. To each tube 400 ll of AP1

buffer (DNeasy Plant Mini Kit; Qiagen) was added and

freeze/thaw was used to lyse fungal cells. Freeze/thaw was

repeated five times using liquid nitrogen and a boiling-

water bath. After the last freezing step samples were boiled

for 30 min with DNA from plant tissue using the DNeasy

Plant Mini Kit. Fungal DNA was eluted in 50 ll AE buffer

and stored at 4�C until use. PCR amplification of the ITS1

and ITS2 regions was performed with universal fungal

primers ITS1 (50-TCCGTAGGTGAACCTGCGG-30) and

ITS4 (50-TCCTCCGCTTATTGATATG-30), as described

by White et al. [12]. The full ITS region was amplified by

PCR in a final volume of 50 ll of the following reaction

mixture: *100 ng template DNA; 0.3 lM (each) forward

(ITS1) and reverse (ITS4) primers; 100 lM (each) dATP,

dCTP, dGTP, and dTTP; 1 9 recombinant Taq DNA

polymerase reaction buffer; 2 mM MgCl2 and 2 U of

recombinant Taq DNA polymerase (Invitrogen Life

Technologies, Carlsbad, CA, USA). Samples were over-

layed with a drop of mineral oil and amplified in a Cetus

Perkin Elmer PCR machine. An initial denaturation step

(94�C for 4 min) was followed by 40 cycles, each con-

sisting of DNA denaturation at 94�C for 30 s, primer

annealing at 50�C for 30 s, and elongation at 72�C for

1 min. The last step was a final extension at 72�C for

10 min. PCR products of *600 bp were visualized by

1.2% agarose gel electrophoresis and ethidium bromide

staining. The amplified ITS regions were then purified from

primers and salts using Nucleospin Extract II spin columns

(Machery-Naghel). Direct sequencing of the fragments was

performed by MWG Biotech using Big Dye terminator

reaction chemistry. Sequences were determined from both

strands using ITS1 and ITS4 as primers.

Data Analysis

Alignment of the three sequences obtained was performed

by ClustalW software (http://www.ebi.ac.uk/clustalw/).

Search comparisons were also made between the three

fungal sequences and other ITS regions available in the

GenBank database using MEGABLAST for sequence

identification.

Results

Our research focused on the three samples of isolated fungi

showing the most interesting enzymatic properties, as

detailed in previously published work [2]. The three iso-

lated fungi were macroscopically very similar. However,

they appeared to be different enzymatically. To verify the

effective diversity among the three isolates, macroscopic

and microscopic analyses were carried out.

On SDA medium all colonies grew slowly, reaching

diameters of from 1.5 to 3.0 cm in 2 weeks at 26� ± 2�C.

SEM observations were made on the 20th day from the

inoculum, when the colonies reached a diameter of 4 cm.

All three fungi showed mycelia flat in topography, granular

in texture, and brown in color, with small floccose and

white areas (Fig. 1). The brown pigmentation of the

mycelia varied lightly among the three strains: in E19C the

surface was blackish-red, in E19D the same color was more

dusky-brown, whereas in E19F the colony showed a

moderate reddish-brown color. Reverse of the cultures

showed a reddish-brown color. Vegetative mycelia were

largely submerged, and surface growth consisted of bran-

ched hyphae with many conidiophores born from an aerial

mycelium (Fig. 2).

All conidiophores had a swollen tip (vesicle) located at

the apex of the stalk, on which conidiogenous cells

Fig. 1 Brown pigmentation of the three Aspergillus mycelia, with

small floccose and white areas

D. Mares et al.: Three New Species of Aspergillus from Amazonian Forest Soil (Ecuador) 223

123

(phialides) and conidia were mounted; this type of conid-

iophore is characteristic of Aspergillales. In all three

isolated strains the conidial heads were globular, radiate,

and typically brown in color. They formed at the end of a

long, straight, colorless, and lightly wrinkled stalk (Fig. 3).

In fact the conidiophore hypha enlarged at its apex to form

a globose structure, known as a vesicle; in the three iso-

lated fungi the vesicles were fertile over the whole surface

(Fig. 4) and were characterized by uni- or biseriate phia-

lides bearing yellow-brown or brown conidia. The

differences among the three fungi in relation to these

characters are reported in Table 1.

In E19C the number of conidiophores, seen at various

development stages, was quite high (Fig. 5). They were

hyaline and 250 ± 5 lm long, and in the upper part they

widened to a cinnamon-brown vesicles, close-woven,

measuring about 50 lm in diameter, with uniseriate phia-

lides over the whole surface (Fig. 6). From phialides, small

dusky-brown conidia were born; they were round, mea-

sured 2.0 ± 0.4 lm in diameter, and were arranged in long

chains (Figs. 7 and 8).

In E19D fungus, the conidiophores were less abundant

in comparison to E19C, but they were longer (about

320 ± 10 lm) and came off with dusky-brown, almost-

black vesicles with tightly adherent conidia, that were tied

to truncated phialides (Fig. 9). The head averaged

48 ± 2 lm and showed only one or two layers of adherent

conidia. These were slightly lemon-shaped and measured

1.9 ± 0.2 9 3.0 ± 0.4 lm, with a kind of spur on both

sides (Fig. 10).

Even E19F had conidiophores with a brown head, from

which many conidia departed, forming short chains

adherent to the head (Fig. 11). Conidiophore stalk length

and vesicle diameter were even smaller: about 200 ± 4 and

36 ± 6 lm, respectively. Conidia were globose in shape

and had an average diameter of 1.65 ± 0.5 lm (Fig. 12).

For molecular identification of our three isolates (E19C,

E19D, E19F) the ITS 1-5.8S-ITS 2 regions were sequenced

using primers ITS 1 and ITS 4. This yielded an identical

sequence of 538 bp for all three strains. The best BLAST

hits of the three identical sequences, obtained after a search

in the GenBank database, were ITS sequences from a few

species of Emericella (E. falconensis, AB249004; E. sim-

ilis, AB248996; E. violacea, AB248985; E. fruticulosa,Fig. 2 Overview of vegetative mycelia showing many conidio-

phores. SEM; bar, 50 lm

Fig. 3 Detail of a conidiophore with globular and radiate conidial

heads, formed at the end of a long, straight, and wrinkled stalk. SEM;

bar, 20 lm

224 D. Mares et al.: Three New Species of Aspergillus from Amazonian Forest Soil (Ecuador)

123

AB248969) and Aspergillus versicolor (AY373882) with

88% sequence identity.

Nucleotide Sequence Accession Numbers

The sequences of the ITS regions of the three fungi

described in this study were deposited in the GenBank

database and assigned accession numbers EF 608616 (A.

quitensis; E19C), EF 608614 (A. amazonicus; E19D), and

EF 608615 (A. ecuadorensis; E19F).

Discussion

The three fungi selected from the Amazonian forest soil

and tested for their BV activity showed different enzymatic

activity [2], although their macroscopical morphology was

very similar. Our morphological observations indicate that

they belong to the Aspergillus group for the presence of

characteristic conidiophores. Since relatively few aspergilli

produce cleistothecia and ascospores [8], we chose to study

other characteristics of the colonies as a basis for their

identification: we used scanning electron microscopy to

characterize morphologically spore-bearing structures and

employed molecular methods by sequencing the ITS1-

5.8S-ITS2 region of rDNA [9].

Using the identification keys of Rapel and Fennell [8],

the distinctive morphological characters indicate that all

three isolated samples are members of the Aspergillus

wentii group. A. wentii Wehmer is a cosmopolitan fungus

that lives all over the world in the soil and on a great

variety of organic substrates. Nevertheless, in A. wentii the

Fig. 4 Detail of a vesicle fertile over its whole surface. SEM; bar,

5 lm

Table 1 Characteristics of

conidiophore branches, vesicles,

phialides, and conidia of the

three isolated fungi

Note: Measurements are the

average of 50 SEM

determinations ± standard error

E19C E19D E19F

Conidiophores

Stalks

Length (lm) 250 ± 5 320 ± 10 200 ± 4

Color Colorless Colorless Colorless

Vesicles

Diameter (lm) 50 ± 3 48 ± 2 36 ± 6

Color Cinnamon-brown Dusky-brown/black Brown

Conidia

Arrangement Long chains 1 or 2 layers Short chains

Shape Round Oval Globular

Color Dusky-brown Dusky-brown/black Brown

Measurements (lm) 2 ± 0.4 1.9 ± 0.2 9 3.0 ± 0.4 1.65 ± 0.5

Phialides Uniseriate Biseriate Uniseriate

Fig. 5 Several conidiophores in the E19C strain. SEM; bar, 100 lm

D. Mares et al.: Three New Species of Aspergillus from Amazonian Forest Soil (Ecuador) 225

123

conidiophore stalks (that may reach several millimeters in

length) and the heads (diameter of C500 lm [8] are much

bigger than those observed in our samples. Thus, we think

that our isolated fungi are not A. wentii strain, but they

could be another species belonging to the same group.

These morphological data were confirmed by BLAST

analyses. We found that the ITS regions of our three fungi

were at least 12% different from other fungal ITS

sequences present in the GeneBank database. Samson et al.

[11] suggest ITS sequence comparison as the molecular

marker of choice for species recognition. Indeed, ITS

sequence comparison has been widely used and many

sequences are now available in public databases. Balajee

et al. [1] indicate that the ITS region is the best tool for

intersection-level classification of Aspergillus. Because of

the great sequence diversity we observed with other spe-

cies, we can confidently conclude that our three fungi have

not been described previously by molecular data. The

closest sequence similarity we found was with a limited

number of Emericella sp. and Aspergillus sp., as shown

under Results.

As ITS regions 1 and 2 yielded an identical sequence for

all three strains, we were not able to distinguish the three

fungal strains by the ITS method, and further molecular

Fig. 6 Uniseriate phialides adherent to the surface of E19C conid-

iophores. SEM; bar, 10 lm

Fig. 7 Conidiophore head of E19C with many round conidia. SEM;

bar, 20 lm

Fig. 8 Detail of an E19C conidial head showing conidia arranged in

long chains. SEM; bar, 10 lm

Fig. 9 E19D conidiophore showing tightly adherent conidia, tied to

truncated phialides. SEM; bar, 10 lm

226 D. Mares et al.: Three New Species of Aspergillus from Amazonian Forest Soil (Ecuador)

123

investigations will be performed to clarify this. However,

unambiguous species distinction based on internally tran-

scribed spacers is controversial in the literature [1]. In

recent papers some authors have underscored the impor-

tance of morphological characterization to identify fungi at

the species level [4, 9, 10, 11].

SEM observations of our three isolates seem to indicate

that they are three different species; this is suggested by the

dimensions and shapes of conidiophore stalks, vesicles, and

conidia. Further proof that our isolates represent three new

species comes from enzymatic data obtained in previously

published work [2]. Indeed, E19C supplied a low amount

of oxidation products, whereas E19D and E19F oxidized

the substrate to correspondent lactone with a high enan-

tiomeric excess.

In conclusion, we suggest that E19C, E19D, and E19F

are classified as three different, new, separate species,

never previously described, which can be important as

possible sources of pure enantiomers [2]. So we have

classified and deposited the sequences from E19C, E19D,

and E19F in the GenBank database (accession numbers EF

608616 [A. quitensis], EF 608614 [A. amazonicus], EF

608615 [A. ecuadorensis]). We have assigned them the

following names: E19C, A. quitensis; E19D, A. amazoni-

cus; and E19F, A. ecuadorensis.

To better clarify the taxonomic position of the three

fungi, our purpose is to conduct further molecular studies

with the aim of confirming the morphological and enzy-

matic data. In particular, we are planning to sequence genes

informative at the interspecific and intraspecific level.

Taxonomy

E19C, Aspergillus quitensis D. Mares sp. nov. (Figs. 5–8)

Teleomorph: Not Known

In agaro SDA, 26 ± 2�C, / coloniae/ crescentes lente,

ustae-brunneae, granulosae vel floccosae cum aliquis cin-

erariis areis. /Mycelium/ partim superficiale et partim in

substrato immersum. /Conidiophorae/ numerosae, singulae,

simplices, rectae, stipites sunt erecti, hyalini, 250 ± 50 mm

Fig. 10 Same as Fig. 9; magnification of slightly lemon-shaped

conidia, with a sort of spur on both sides. SEM; bar, 5 lm

Fig. 11 E19F Aspergillus conidiophore head, with short chains of

conidia. SEM; bar, 10 lm

Fig. 12 Same as Fig. 11; magnification of phialides and conidia

globose in shape. SEM; bar, 5 lm

D. Mares et al.: Three New Species of Aspergillus from Amazonian Forest Soil (Ecuador) 227

123

longi et vesicae sunt globosae, 50 ± 2 mm latae, fertiles in

tota esteriore parte, cinnamum-brunneae. /Phialides/ uni-

verticillati. /Conidia/ globosa 2.0 ± 0,4 mm, atro-brunnea,

sicut longae catenae vinculate.

Anamorph: In SDA, at 26� ± 2�C, slow-growing colo-

nies, flat, blackish-red, granular in texture except in

somewhat floccose gray areas; exudate lacking; reverse

dusky-brown. Mycelium superficial and immersed in the

substratum, composed of septate, branched, and smooth

hyphae. Conidiophores abundant, single, unbranched,

straight, with erect and hyaline stalks, 250 ± 5 lm long.

Vesicles globose, cinnamon-brown, 50 ± 2 lm in diame-

ter. Phialides uniseriate over the whole surface. Conidia

small, round, 2.0 ± 0.4 lm in diameter, dusky-brown, and

arranged in long chains.

Specimen examined: Ecuador, Makas, from soil, 25

April 2004.

Etymology: The specific epithet quitensis refers to

Quito, the capital of Ecuador.

E19D, Aspergillus amazonicus D. Mares sp. nov.

(Figs. 9 and 10)

Teleomorph: Not Known

In agaro SDA, 26 ± 2�C, / coloniae/ crescentes lente,

floccosae atro-brunneae. /Mycelium/ partim superficiale et

partim in substrato immersum. /Conidiophorae/ singulae,

simplices, rectae, stipites sunt erecti, hyalini, 320 ± 10 mm

longi et vesicae sunt globosae, 48 ± 2 mm latae, fertiles in

tota esteriore parte, atro-brunneae vel atrae. /Phialides/

biverticillati et trunci. /Conidia/ ovoidea vel ellipsoidea 1.9

± 0, 2 x 3, 0 ± 0, 4 mm, atro-brunnea vel atra, portata in 1

vel 2 strata adhaerentia exteriori parti vesicae.

Anamorph: In SDA, at 26� ± 2�C, slow-growing colo-

nies, flat, floccose, dusky-brown; exudate clear; reverse

dusky-brown. Mycelium superficial and immersed in the

substratum, composed of septate, branched, and smooth

hyphae. Conidiophores single, unbranched, straight, with

erect and colorless stalks, 320 ± 10 lm long. Vesicles

globose and fertile over the entire surface, almost black,

48 ± 2 lm in diameter. Phialides biseriate and truncated.

Conidia oval or ellipsoidal, 1.9 ± 0.2 9 3.0 ± 0.4 lm,

almost black, tightly adherent to vesicle surface, forming

one or two closely packed layers.

Specimen examined: Ecuador, Makas, from soil, 25

April 2004.

Etymology: The specific epithet amazonicus refers to the

place of origin.

E19F, Aspergillus ecuadorensis D. Mares sp. nov.

(Figs. 11 and 12)

Teleomorph: Not Known

In agaro SDA, 26 ± 2�C, / coloniae/ crescentes lente, floc-

cosae, brunneae. Albi umores exudant. /Mycelium/ partim

superficiale et partim in substrato immersum. /Conidiopho-

rae/ singulae, simplices, rectae, stipites sunt erecti, hyalini,

200 ± 4 mm longi et vesicae sunt globosae, 36 ± 6 mm

latae, fertiles in tota exteriore parte, atro-brunneae vel atrae.

/Phialides/ univerticillati, et ampulliformes. /Conidia/ glob-

osa 1.65 ± 0.5 mm, atro-brunnea vel atra, sicut catenae

vinculata et adhaerentia exteriori parti vesicae.

Anamorph: In SDA, at 26o ± 2�C, slow-growing colo-

nies, flat, floccose, moderate reddish-brown; exudate clear;

reverse dusky-brown. Mycelium superficial and immersed in

the substratum, composed of septate, branched, and smooth

hyphae. Conidiophores single, unbranched, straight, with

erect and colorless stalks, 200 ± 4 lm long. Vesicles glo-

bose and fertile over the entire surface, brown, 36 ± 6 lm in

diameter. Phialides uniseriate. Conidia globular, 1.65 ±

0.5 lm, brown, arranged in short chains and adherent to the

vesicle surface.

Specimen examined: Ecuador, Makas, from soil, 25

April 2004.

Etymology: The specific epithet ecuadorensis refers to

the nation of origin.

Acknowledgments The authors wish to thank Prof. Marisa Ferra-

resi, Dr. Mirco Iotti, and the staff of the Electron Microscopy Center

at Ferrara University for their skillful assistance. This work was

supported by research grants from MIUR-URST (Ministero dell’Ist-

ruzione, dell’Universita e della Ricerca-Area Universita e Ricerca

Scientifica e Tecnologica) of Italy.

References

1. Balajee SA, Houbraken J, Verweij PE, Hong S-B, Yaghuchi T,

Varga J, Samson RA (2007). Aspergillus species identification in

the clinical setting. Stud Mycol 59:39–46

2. Fantin F, Giovannini PP, Guerrini A, Maietti S, Medici A, Pedrini

P (2006) Enantioselective Baeyer-Villiger oxidation of bicy-

cle[3.2.0]hept-2-en-6-one with fungi: optimization of biotransfor-

mation and use of TiO2 as support of cell growth. Biotechnol Lett

28:805–810

3. Griffin DW, Kellogg CA, Peak KK, Shinn EA (2002) A rapid and

efficient assay for extracting DNA from fungi. Lett Appl

Microbiol 34:210–214

4. Hawksworth D, Kirk P, Sutton B, Pegler D (1996) Ainsworth’s and

Bisby’s dictionary of the fungi, 8th edn. CABI, Wallingford, UK

5. Mares D, Romagnoli C, Benvegnu R et al (2002). Mannan-

mediated antifungal activity on Epidermophyton floccosum by 3-

methyl-5-aminoisoxazole-4-thiocyanate, a new azole derivative.

Fung Gen Biol 36:47–57

228 D. Mares et al.: Three New Species of Aspergillus from Amazonian Forest Soil (Ecuador)

123

6. Munsell (1990) Munsell soil color charts. MacBeth Division,

Kollmorgen Instruments Corp. Baltimore, MD

7. Newton RF (1982) In: Roberts SM, Scheimann F (eds) New

synthetic routes to prostaglandins and thromboxanes. Academic

Press, New York, p 61

8. Raper KB, Fennell DI (1965). The genus Aspergillus. Williams

and Wilkins, Baltimore, MD

9. Samson RA, Hong SB, Frisvad JC (2006) Old and new concepts

of species differentiation in Aspergillus. Med Mycol 44:S133–

S148

10. Samson RA, Varga J, Witiak SM, Geiser DM (2007) The species

concept in Aspergillus: recommendations of an international

panel. Stud Mycol 59:71–73

11. Taylor JW, Jacobson DJ, Kroken S Kasuga et al (2000) Phylo-

genetic species recognition and species concepts in fungi. Fung

Gen Biol 31:21–32

12. White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and

direct sequencing of fungal ribosomal RNA genes for phyloge-

netics. In: Innis M, Gelfand D, Sninsky J, White T (eds) PCR

protocols: a guide to methods and application. Academic Press,

San Diego, CA, pp 315–322

D. Mares et al.: Three New Species of Aspergillus from Amazonian Forest Soil (Ecuador) 229

123