Documentos de Académico
Documentos de Profesional
Documentos de Cultura
Robert A. Samson1*, Jos A.M.P. Houbraken1, Angelina F.A. Kuijpers1, J. Mick Frank2 and Jens C.
Frisvad3
1
Centraalbureau voor Schimmelcultures, P.O. Box 85167, 3508 AD Utrecht, the Netherlands; 233 Tor Road, Farnham,
Surrey, GU9 7BY, U.K.; 3Center for Microbial Biotechnology, BioCentrum-DTU, Building 221, Technical University of
Denmark, DK-2800 Kgs. Lyngby, Denmark
Abstract: Aspergillus section Nigri includes some of the most important species for biotechnology and its species are of
widespread occurrence. During our surveys of various food products and tropical soil we isolated several aspergilli belonging
to section Nigri. In this paper, four new sclerotium and/or ochratoxin A producing species belonging to this section are
proposed. In addition, based on a polyphasic approach using traditional characters, extrolites and -tubulin sequencing, a
provisional revision and synoptic key of section Nigri is proposed. Aspergillus costaricaensis was isolated from soil in Costa
Rica and produces large pink to greyish brown sclerotia. Aspergillus lacticoffeatus was found on coffee beans in Venezuela
and Indonesia, and is an effective producer of ochratoxin A. Aspergillus piperis was isolated from black ground pepper and
produces large yellow to pink brown sclerotia. Aspergillus sclerotioniger was isolated from a green coffee bean and produces
large yellow to red brown sclerotia and abundant ochratoxin A. The species A. homomorphus is validated. The ochratoxin A
producing black aspergilli are revised. Fifteen species are provisionally accepted in Aspergillus section Nigri, four of these
produce ochratoxin A. Ochratoxin A producing species of section Nigri occurring on grapes, raisins and in wine include A.
carbonarius and to a lesser extent A. niger. Four species recovered from coffee, viz. A. carbonarius, A. niger, A. lacticoffea-
tus and A. sclerotioniger, all produce ochratoxin A, but other species of Nigri also occur on this substrate, including A.
japonicus and A. tubingensis. The 10 species not producing ochratoxin A are especially interesting for biotechnological
exploration, as many other extrolites are produced by these species.
Taxonomic novelties: Aspergillus costaricaensis Samson & Frisvad sp. nov., Aspergillus homomorphus Frisvad & Samson
sp. nov., Aspergillus lacticoffeatus Samson & Frisvad sp. nov., Aspergillus piperis Samson & Frisvad sp. nov., Aspergillus
sclerotioniger Steiman, Guiraud, Sage & Seigle-Mur. ex Samson & Frisvad sp. nov.
Key words: Aspergillus niger, black aspergilli, ochratoxin A, pyranonigrin, sclerotia.
45
SAMSON ET AL.
eromorphus were invalidly described (no designated The names of colours are based on Kornerup & Wan-
type, International Code of Botanical Nomenclature scher (1978). The cultures used for the molecular
Art. 37) (Steiman et al. 1994; see Mouchacca 1999). study were grown in 2 mL malt peptone (MP) broth
Recently, a new species A. vadensis, with a different using 10 % (v/v) of malt extract (Brix 10) and 0.1 %
extrolite profile, colony characters and unusually low (w/v) bacto peptone (Difco) in 15 mL polystyrene
citric acid production, was proposed (de Vries et al. centrifuge tubes. The cultures were incubated at 25 C
2004a, b). without agitation for 7 d in light/darkness. The strains
Ueno et al. (1991) were the first to report on ochra- selected included 1 to 8 representatives of the major
toxin A (OA) production by a black Aspergillus taxa accepted by Al-Musallam (1980), Kozakiewicz
species, A. foetidus. This was later confirmed by (1989) and Abarca et al. (2004) (see Table 1) in
Tren et al. (1996) and Magnoli et al. (2003). Abarca addition to the new taxa described here and in de
et al. (1994) reported that two strains of A. niger Vries et al. (2004b).
produced OA, which was confirmed in numerous
studies (Ono et al. 1995, Tren et al. 1996, 1997, Extrolite analysis
Nakajima et al, 1997, Heenan et al. 1998, Accensi et Extrolites (includes secondary metabolites; for defini-
al. 2001, Urbano et al. 2001, Dalcero et al. 2002, Da tion see Samson & Frisvad 2004) were analysed by
Rocha et al. 2002, Abarca et al. 2003, Magnoli et al. HPLC using alkylphenone retention indices and diode
2003, Taniwaki et al. 2003, Surez-Quiroz et al. array UV-VIS detection as described by Frisvad &
2004). Horie (1995) reported OA in A. carbonarius, Thrane (1987), with minor modifications as described
which was confirmed by Wicklow et al. (1996), Tren by Smedsgaard (1997). Standards of ochratoxin A and
et al. (1996), Heenan et al. (1998), Varga et al. B, aflavinine, asperazine, austdiol, kotanin and other
(2000), Joosten et al. (2001), Da Rocha et al. (2002), extrolites from the collection at Biocentrum-DTU
Cabanes et al. (2002), Sage et al. (2002), Abarca et al. were used to compare with the extrolites from the
(2003), Battilani et al. (2003), Taniwaki et al. (2003), species under study. Pyranonigrin A was identified by
Bell et al. (2004) and Sage et al. (2004). Varga et al. comparison with literature UV and MS data (Hiort
(2000) tested about 160 black Aspergillus strains from 2003, Hiort et al. 2004)
collections and from field isolates for OA production
using an immunochemical method and thin layer DNA Extraction, sequencing and analysis
chromatography. The strains examined included 12 A. The total fungal genomic DNA was isolated using
carbonarius and 45 A. japonicus strains from culture UltracleanTM Microbial DNA Isolation Kit (MoBio,
collections and field isolates from all over the world, Solana Beach, U.S.A.) according to the manufac-
including about 100 strains belonging to the A. niger turers instructions. Amplification of -tubulin gene
species complex. was mostly performed using the primers Bt2a and
Ochratoxin A production was detected in about 6 Bt2b. Some strains in this study Bt-T10 (5'ACG ATA
% of the strains from the A. niger species complex GGT TCA CCT CCA GAC 3') an Bt2b (Glass 1995).
(Abarca et al. 1994, Tren et al. 1996). Of the 13 A. PCR was performed in a 25 L reaction mixture
carbonarius strains tested, six produced both OA and containing 1 L of genomic DNA (10 ng/L), 0.75 L
ochratoxins B (Fig. 8, Tren et al. 1996, Wicklow et of MgCl2 (50mM provided with BioTaq), 2.5 L
al. 1996). Aspergillus ellipticus, A. heteromorphus, A. Buffer with 10 NH4 (provided with BioTaq), 17.8 L
japonicus and A. tubingensis strains did not produce of ultra pure sterile water, 1.85 L dNTP (1 mM),
detectable amounts of ochratoxins. However, A. 0.50 L of each primer (10 pmol/L) and 0.1 L
japonicus was later claimed to produce OA (Dalcero BioTaq polymerase (5 U/L, BiotaqTM DNA Poly-
et al. 2002, Battilani et al. 2003). merase, Bioline Randolph, U.S.A.). Amplification was
During our surveys of coffee, black pepper and performed in a GeneAmp PCR system 9700 (AB
soil, several isolates of black aspergilli were recov- Applied Biosystems, Nieuwerkerk a/d IJssel, The
ered. The purpose of this paper is to describe four new Netherlands); programmed for 5 min 94 C followed
species from section Nigri, distinguished from previ- by 35 cycles of 1 min denaturation at 94 C followed
ously known species by large sclerotia or unusual by primer annealing 1 at 55 C and primer extension 1
conidial colours. Furthermore we wanted to suggest a min. at 72 C and a final 7 min elongation step at 72
provisional revision of this industrially important
C. After amplification of the -tubulin gene, excess
section of Aspergillus based on a relatively small
primers and dNTPs were removed from the reaction
number of typical strains of each taxon.
mixture using a commercial GFX column, PCR DNA
Purification kit (Amersham Bioscience, Roosendaal,
The Netherlands). The purified PCR fragments were
MATERIALS AND METHODS resuspended in 50 L of TE buffer. The PCR frag-
ments were directly sequenced in both directions with
The methods and media for isolation and identifica- the primers Bt2a or BtT10 and Bt2b using a
tion followed the procedures of Samson et al. (2004).
46
NEW SPECIES OF ASPERGILLUS SECTION NIGRI
DYEnamic ET Terminator Cycle Sequencing Kit 3700 Genetic Analyzer (AB Applied Biosystems,
(Amersham Bioscience, Roosendaal, The Nether- Nieuwerkerk a/d IJssel, The Netherlands). A concen-
lands). The sequence PCR reaction mixture, total sus was computed from the forward and reverse
reaction mix is 10 L, contained 1 L of template sequences with software package Seqman and Editseq
DNA (1015 ng/L), 4 L Dye terminator RR mix, 4 from the lasergene package (DNAStar Inc., Madison,
L ultra pure sterile water and 1 L primer Bt2a or WI). The alignments of the partial -tubulin gene
Bt2b (4 pmol/L). The reaction was performed in a sequence data were performed using the software
GeneAmp PCR system 9700 run in 9600 mode (AB package BioNumerics from Applied Maths and man-
Applied Biosystems, Nieuwerkerk a/d Yssel, The ual adjustments for improvement were made by eye
Netherlands); programmed for 25 cycles of 10 s where necessary. The phylogenetic analyses of se-
denaturation at 96 C followed by primer annealing 5 quence data were done using PAUP (Phylogenetic
s at 50 C and primer extension 4 min at 60 C. Se- Analysis Using Parsimony) v. 4.0b10 (Swofford
quencing products were purified according to the 2000). Alignment gaps were treated as fifth character
manufacturers recommendations with Sephadex G-50 state, missing data were identified by ?, uninforma-
superfine column (Amersham Bioscience, Roosen- tive characters were excluded and all characters were
daal, The Netherlands) in a multiscreen HV plate unordered and equal weight. Maximum parsimony
(Millipore, Amsterdam, The Netherlands) and with analysis was performed for all data sets using the
MicroAmp Optical 96-well reaction plate (AB Ap- heuristic search option. The robustness of the most
plied Biosystems, Nieuwerkerk a/d IJssel, The Nether- parsimonious trees was evaluated by 1000 bootstrap
lands). The samples were analyzed on an ABI PRISM replications (Hillis & Bull 1993). Other measures
48
NEW SPECIES OF ASPERGILLUS SECTION NIGRI
including tree length, consistency index, retention toxin A, pyranonigrin A, orlandin (see Cutler et al.
index and rescaled consistency index (CI, RI and RC) 1979), kotanin and desmethylkotanin. All eight strains
were also calculated. of A. niger investigated produced pyranonigrin A and
naphtho--pyrones. CBS 101705, CBS 101698 and
CBS 554.65 produced orlandin, kotanin and des-
RESULTS methylkotanin and CBS 618.78, CBS 420.64, CBS
101705, and CBS 101698 produced ochratoxin A and
All strains of the black aspergilli produced a large B. A. piperis CBS 112811 produced aurasperone B,
number of known and as yet unknown extrolites. 14-epi-14-hydroxy-10,23-dihydro-24,25-
Some of the most important extrolites are listed in dehydroaflavinine, and 10,23-dihydro-24,25-
Table 1. Two strains of Aspergillus aculeatus (CBS dehydroaflavinine. Aspergillus sclerotioniger CBS
172.66 and CBS 119.49) produced secalonic acid D as 115572 produced pyranonigrin A, naphtho--pyrones,
earlier reported for this taxon (Andersen et al. 1977) ochratoxin A and B, and compounds related to fu-
and in addition they both produced neoxaline. The nalenone or corymbiferan-lactones. All eight strains of
latter metabolite was first reported from A. japonicus A. tubingensis produced asperazine, except CBS
(Hirano et al. 1979, Konda et al. 1980), but we only 161.79. The latter strain produced tubingensin A and
found neoxaline in A. aculeatus in this study. Two B (TePaske et al. 1989b, c), dihydrotubingensin A and
other strains identified as A. aculeatus were quite B (Sings et al. 2001) and 14-epi-14-hydroxy-10,23-
dissimilar to the two typical strains above: CBS dihydro-24,25-dehydroaflavinine, 10,23-dihy-dro-
620.78 produced secalonic acid D and some indole 24,25-dehydroaflavinine and 10,23-dihydro-24,25-
compounds, while CBS 114.80 produced the same dehydro-21-oxo-aflavinine (TePaske et al. 1989a)
indole compounds and okaramin H and I. Those indicating a difference between CBS 161.79 and other
okaramins have earlier been reported from A. aculea- strains of A. tubingensis. All eight strains of A. tubin-
tus (Hayashi et al. 1999). A. brasiliensis CBS 101740 gensis (Table 1) also produced pyranonigrin A and
produced some naphtho--pyrones including naphtho--pyrones. Aspergillus vadensis CBS 113365
aurasperone B (Tanaka et al. 1972) and a series of produced nigragillin, asperazine, aurasperone B (a
compounds that have not been structure elucidated naphtho--pyrone) and a polar orlandin-like com-
yet. pound.
All four strains of A. carbonarius produced Among the isolates listed in Table 1, four species
pyranonigrin A (earlier reported from A. niger, Hiort were able to produce OA. Ochratoxin A was consis-
et al. 2004), ochratoxin A and naphtho--pyrones. tently produced by A. carbonarius strains, in agree-
Aspergillus costaricaensis produced trace amounts of ment with most other studies on this species (Abarca
aurasperone B, pyranonigrin A, 14-epi-14-hydroxy- et al. 2004). Ochratoxin A was only produced by
10,23-dihydro-24,25-dehydroaflavinine, 10,23-dihy- some strains of A. niger sensu stricto, also in agree-
dro-24,25-dehydroflavinine (those aflavinines were ment with numerous studies (Abarca et al. 2004). The
found earlier in A. tubingensis CBS 161.79 = NRRL other producers of OA were the new species that are
4700, TePaske et al. 1989a), a funalenone-like com- described below, namely A. lacticoffeatus and A.
pound (see Inokoshi et al. 1999) or a corymbiferan sclerotioniger. Both of these new species were iso-
lactone-like compound (see Overy & Blunt 2004). A. lated from coffee. On the other hand OA production
ellipticus CBS 677.79 and CBS 707.79 both produced by A. japonicus (Dalcero et al. 2002, Battilani et al.
austdiol earlier reported from Aspergillus ustus (Vleg- 2003) was not confirmed. Similarly, no strains of A.
gaar et al. 1974). Aspergillus foetidus CBS 564.65 and foetidus sensu stricto produced OA. The strain CBS
CBS 565.65 both produced asperazine, earlier errone- 618.78 has been identified by different authors as A.
ously reported from A. niger (Varoglou et al. 1997). foetidus, A. foetidus var. citricus or A. citricus and
The strain examined by Varoglou et al. was actually produced OA (Tren et al. 1996). It was listed among
an A. tubingensis (unpublished results, J.C. Frisvad). isolates of A. foetidus by Raper & Fennell (1965).
Furthermore CBS 564.65 and CBS 565.65 produced Ochratoxin A production by this strain was confirmed
antafumicin (Fujimoto et al. 1993). Both strains also here, but this strain has been shown to be A. niger and
produced naphtho--pyrones and pyranonigrin A. not A. foetidus (Kusters-van Someren et al. 1991,
Aspergillus heteromorphus CBS 117.55 produced Parenicov et al. 1997, Accensi et al. 1999).
several as yet unknown extrolites, including some Sclerotium production was not necessarily corre-
indol-alkaloids. Aspergillus homomorphus CBS lated with OA production. It was suggested by Wick-
101889 and A. pseudoheteromorphus had identical low et al. (1996), that ochratoxin A was associated
profiles of extrolites, including secalonic acid D. with sclerotium production of A. carbonarius. Asper-
Aspergillus japonicus CBS 101.14, CBS 114.51 and gillus carbonarius occasionally produced sclerotia and
CBS 522.89 did not produce any known extrolites. OA, but non-sclerotial strains of A. carbonarius could
Aspergillus lacticoffeatus CBS 101886, CBS 101883, also produce ochratoxin A. A. tubingensis occasion-
CBS 101884 and CBS 101885 all produced ochra- ally produces sclerotia but never produces ochratoxin
49
SAMSON ET AL.
50
NEW SPECIES OF ASPERGILLUS SECTION NIGRI
Table 2. Production of sclerotia, ochratoxin A and other extrolites by species in Aspergillus section Nigri.
Species Ochratoxin A Sclerotia Pyranonigrin N--P1 Asp2 SeD3 Ant4 Afl5 Cor6 Kot7
A. aculeatus +/ +
A. brasiliensis +
A. carbonarius + +/ + +
A. costaricaensis + + + +
A. ellipticus +
A. foetidus + + + +
A. heteromorphus
A. homomorphus8 +
A. japonicus
A. lacticoffeatus + + +
A. niger +/ + + +/
A. piperis + + + +
A. sclerotioniger + + + + +
A. tubingensis +/ + + +
A. vadensis - + +
1
N--P: Naphtho--pyrones; 2Asp = asperazine; 3SeD = secalonic acid D; 4Ant = antafumicin; 5Afl = aflavinines; 6Cor =
Corymbiferan lactones; 7Kot = Kotanins (kotanin, desmethylkotanin, orlandin); 8A. pseudoheteromorphus was not different
from A. homomorphus, but none of the species have been validly described.
51
SAMSON ET AL.
52
NEW SPECIES OF ASPERGILLUS SECTION NIGRI
Fig. 2. Aspergillus costaricaensis. Seven-day-old cultures on A. CYA and B. MEA. C. Conidiophore. D. Detail of a conidio-
phore showing large metulae. E. Detail of a 7-day-old colony showing sclerotia. F. Conidia. G. Scanning electron micrograph
photo of conidia. Scale bars: C, D, F = 10 m, E = 1 mm, G = 1 m.
53
SAMSON ET AL.
Fig. 3. Aspergillus lacticoffeatus. Seven-day-old cultures on A. CYA and B. MEA. C, D. Conidiophores. E. Conidia. F. Scan-
ning electron micrograph photos of conidia. Scale bars:: CE = 10 m, F = 1 m.
54
NEW SPECIES OF ASPERGILLUS SECTION NIGRI
Fig 4. Aspergillus piperis. Seven-day-old cultures on A. CYA and B. MEA. C, D. Conidiophores. E. Conidia. F. Scanning
electron micrograph photo of conidia. Scale bars: CE = 10 m, F = 1 m.
55
SAMSON ET AL.
Fig. 5. Aspergillus sclerotioniger. Seven-day-old cultures on A. CYA and B. MEA. C. Conidiophore. D. Conidial head. E.
detail of a 7-day-old colony showing sclerotia. F. Conidia. G. Scanning electron micrograph photo of conidia. Scale bars: CD,
FG = 10 m, E = 2 mm.
56
NEW SPECIES OF ASPERGILLUS SECTION NIGRI
Other strains: CBS 101885 = IBT 22029, ex surface disin- many aspergilla are produced on all media; hyphae
fected ripe green arabica coffee bean, farm Agua Blanco, inconspicuous, white; large sclerotia (1-17 mm)
Rubio district, Venezuela, J.M. Frank; CBS 101884 = IBT abundantly produced on all media, white when young
22030, ex surface disinfected ripe green arabica coffee becoming yellow to pink brown at age; exudate pre-
bean, farm Agua Blanco, Rubio district, Venezuela, J.M.
sent like small hyaline droplets; reverse uncoloured,
Frank; CBS 101886 = IBT 22032, ex soil under robusta
cherry coffee of a compacted soil drying yard, Karangsari, pale to creamy. Conidial heads radiate; stipes (300)
Pulo Pannggung subdistrict, Sumatra, Indonesia, J.M. 4003000 (7)1215(20) m, walls thick, smooth,
Frank hyaline; vesicles (40)4550(55) m wide, nearly
spherical; biseriate; metulae covering virtually the
Colony diameters at 7 d 25 C, in mm: CYA: 7176 entire surface of the vesicle, measuring (20)2530(
mm, MEA 5270 mm, YES: 7580 mm, OAT: 3236 35) 36 m; phialides (5.5)67.5(8) 34 m;
mm, CREA: 3244 mm, thin colonies with poor conidia subglobose to broadly ellipsoidal, 2.83.6
sporulation, strong acid production, CYA at 37 C: 2.83.4 m, smooth when young to very rough with
5975 mm. Colony colours and texture. Conidial irregular bars/striations.
areas first white then becoming hair brown (5E4) to
dark blonde (5D4) and densely packed on CYA25, Extrolites: Aurasperone B, 14-epi-14-hydroxy-10,23-
hyphae usually inconspicuous, no sclerotia on any dihydro-24,25-dehydroaflavinine, and 10,23-dihydro-
medium, no exudates present, reverse cream to light 24,25-dehydroaflavinine.
brown on CYA, colony granular, sometimes sulcate.
The conidial heads are globose at first and later occa- Distinguishing features: This species is characterized
sionally developing into several conidial columns on by its yellow to pink brown sclerotia, subglobose to
each head. Colonies on CZ similar as on CYA, only broadly ellipsoidal and distinctly roughened conidia.
reverse is uncoloured on CZ. Growth on YES is
characterized by sulfur yellow mycelium formation. Aspergillus sclerotioniger Samson & Frisvad sp.
Conidial heads radiate; stipes short (200)3001200 nov. MycoBank MB500010.
(7)1015(18) m, walls thick, smooth, orange
brown; vesicles (40)4560(65) m wide, nearly Aspergillo carbonario similis, capitulis biseriatis, sed
spherical; biseriate; metulae covering virtually the mycelio luteo, sclerotiis luteis vel aurantiacis vel rubro-
entire surface of the vesicle, measuring 1225 36 brunneis, hyphis spicularibus luteis in agaro YES formatis
m; phialides 710 34 m; conidia subglobose, et conidiis majoribus differens. Typus CBS H-13433.
3.54.1 3.43.9 m, usually smooth to very finely
roughened. No sclerotia observed Type: CBS 115572 = IBT 22905 ex surface disinfected
green Arabica coffee bean, Karnataka, India, J.M. Frank.
Extrolites: Ochratoxin A, ochratoxin B, pyranonigrin
A, orlandin, kotanin. Colony diameters at 7 d 25 C, in mm: CYA: 7178
mm, MEA 6072 mm, YES: 7280 mm, OAT: 4256
Distinguishing features: This species is characterized mm, CREA: 1925 mm, thin colonies with poor
by its hair brown to dark blonde colonies, biseriate sporulation, strong acid production, CYA at 37 C: 7
conidial heads with large vesicles and smooth to very 16 mm. Colony colours and texture. On CYA25 and
finely roughened conidia. MEA only a few conidiophores are produced, conidial
areas are black; mycelium yellow, conspicuous;
Aspergillus piperis Samson & Frisvad sp. nov. sclerotia abundantly present, large (11.6 mm),
(sub)globose, yellow to orange to red brown covered
MycoBank MB500009.
by yellow mycelium. Reverse on CYA pale, on MEA
mediumyellow. Conidial heads radiate; stipes short
Aspergillo nigro similis, capitulis biseriatis, sed sclerotiis
luteis vel roseo-brunneis et conidiis subglobosis vel late (400)500800(1200) (12)1416(18) m, walls
ellipsoideis distincte asperatis differens. Typus CBS H- thick, smooth, hyaline; vesicles (30)3545(50) m
13434. wide, pyriform; biseriate; metulae covering three
quarters of the vesicle, measuring 814 46 m;
Type: CBS 112811 = IBT 26239, ex grounded black pepper phialides 6.59.5 35 m; conidia subglobose,
of tropical origin, Kgs. Lyngby, Denmark, K.F. Nielsen. (4.7)56(6.4) (4.5)4.95.6(6.1) m, smooth
when young, becoming verruculose, dark brown.
Colony diameters at 7 d 25 C, in mm: CYA: 6075
mm, MEA 5978 mm, YES: 7983 mm, OAT: 4554 Extrolites: Ochratoxin A, ochratoxin B, traces of
mm, CREA: 4348 mm, thin colonies with poor aurasperone B, and pyranonigrin A. The isolates
sporulation, strong acid production, CYA at 37 C: produce a compound with a chromophore like that of
6482 mm. Colony colours and texture. Conidial the corymbiferans produced by Penicillium hordei
areas black and sparsely produced, after sub-culturing
57
SAMSON ET AL.
58
NEW SPECIES OF ASPERGILLUS SECTION NIGRI
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