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1 CBS Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, The
Netherlands
2 Department of Food Science and Technology, Agro-Industry Faculty,
Kasetsart University, 10900 Bangkok, Thailand
3 Faculty of Technology and Management, Prince of Songkla University,
Suratthani Campus, 84100 Suratthani, Thailand
4 Center for Microbial Biotechnology, BioCentrum-DTU, Building 221,
Technical University of Denmark, DK-2800 Kgs Lyngby, Denmark
5 Department of Microbiology, Faculty of Science and Informatics, University
of Szeged, H-6701 Szeged, P.O. Box 533, Hungary
*
Correspondence: Robert A. Samson,
r.samson{at}cbs.knaw.nl
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Abstract |
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 TOP Abstract INTRODUCTIONMATERIAL AND METHODSRESULTSCONCLUSIONS AND RECOMMENDATION...References |
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Keywords Boscalid medium / calmodin / colony morphology / CREA / Ehrlich reaction / extrolites / molecular tools / tubulin
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INTRODUCTION |
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TOPAbstractINTRODUCTIONMATERIAL AND METHODSRESULTSCONCLUSIONS AND RECOMMENDATION...References |
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Black aspergilli are one of the more difficult groups concerning classification and identification and several taxonomic schemes have been proposed. New molecular approaches have shown that there is a high biodiversity, but that taxa are difficult to be recognised based solely on their phenotypic characters (Mosseray 1934a;b; Murakami 1976a;b; Murakami 1979a-d; Murakami and Noro 1979; Murakami and Yoshida 1979a, b; Murakami et al. 1979; Al-Musallam, 1980). Murakami (1979d) only reluctantly recommended to use nitrite as sole nitrogen-source as a diagnostic medium in Aspergillus taxonomy. 20 % tannic acid agar seems to be less useful for diagnostic purposes as most black aspergilli can grow on it (van Diepeningen et al. 2004).
In this paper we have compiled the most relevant methods to be used in the diagnostics of the known and accepted species. Some additional methods have been listed by Frisvad et al. (2007).
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MATERIAL AND METHODS |
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TOPAbstractINTRODUCTIONMATERIAL AND METHODSRESULTSCONCLUSIONS AND RECOMMENDATION...References |
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Cultures were three-point inoculated on media in 9 cm plastic Petri dishes using a dense conidium suspension and incubated in the dark at 25 °C, except where otherwise noted. The fungi were also grown at 15, 30 and 37 °C on CYA. The cultures were examined after 7 d of growth and further examined after 14 d. Colony diam were measured using a ruler.
Growth response of the ex type cultures of section Nigri at 15, 18, 21, 24, 27, 30, 33, 36 and 40 °C after 10 d incubation on MEA has also been recorded.
All species were examined using oil immersion with a Zeiss microscope with Normarski contrast at up to 1000 x magnification. Digital micrographs of colonies were taken with a Nikon Coolpix 990 and 995 camera. Microscopic slides were prepared from MEA plates and 60 % lactic acid was used as a mounting medium.
Growth on specific media
Boscalid medium: In this study, we tested the growth abilities of
all type strains of Aspergillus section Nigri on MEA-B (MEA
according to Samson et al.
(2004) with 10 mg/L boscalid
(Sigma) added after autoclaving). Each strain was 3-point inoculated with a
dense spore suspension onto MEA-B and inspected for growth and sporulation
after 3 and 7 d of incubation at 25 °C.
Creatine Sucrose Agar (CREA): The growth of the type strains have also been tested on CREA, which is a semi-selective medium useful for classification of various fungal cultures especially Penicillium species (Samson et al. 2004; Frisvad & Samson 2004). Each strain was 3-point inoculated with a dense spore suspension onto CREA and inspected for growth and sporulation after 3 and 7 d of incubation at 25°C.
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Ehrlich test
The Ehrlich test was used by Frisvad & Samson
(2004) to distinguish taxa of
Penicillium subgenus Penicillium and is based on the
detection of alkaloids reacting with Ehrlich reagent
(Lund 1995) using a filter
paper method. The Ehrlich reagent consists of 2 g of
4-dimethylamino-benzaldehyde in 96 % ethanol (85 ml) added to 15 ml 10 N HCl.
An four mm agar plug is cut out from the centre of a colony grown on CYA
(incubated for 5-9 d at 25°C) and a round piece (1 cm diam.) of the wetted
filter paper (Whatman No. 1) is placed on the mycelial side of the plug. If a
violet ring appears after 2-6 min, the culture contains cyclopiazonic acid or
related alkaloids. If the reaction comes after 7-10 min, it is regarded as
weak. After 10 min the violet ring will fade away. Some fungi produce
alkaloids that will react with Ehrlich reagent to give pink to red or yellow
rings.
Molecular analysis
Phylogenetic analysis of sequences of the ITS region of the rRNA gene
cluster, and parts of the β-tubulin and calmodulin genes have been
carried out as described previously (Varga
et al. 2007; Noonim
et al. 2008).
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RESULTS |
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TOPAbstractINTRODUCTIONMATERIAL AND METHODSRESULTSCONCLUSIONS AND RECOMMENDATION...References |
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Conidia
Light microscopic photographs of the conidia of type strains and
representative isolates of Aspergillus section Nigri are
shown in Figs
3-4.
Several species produce large conidia up to 7-9 µm including A.
carbonarius, A. ibericus, A. homomorphus, A. sclerotiicarbonarius and
A. sclerotioniger. Most other species produce conidia in the size
range of (2.5-)3-4.5(-5) µm. The ornamentation of the conidia is also
characteristic for some species, e.g. A. homomorphus and A.
ibericus produce conidia with spiny appearance, while other species
produce conidia which are smooth or nearly so, including A. vadensis
and A. lacticoffeatus.
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With respect to the uniseriate species, CREA is helpful for distinguishing between the 4 species. A. uvarum had poor growth and limited acid production while A. aculeatus and A. japonicus grew quite well and had medium acid production. In contrast, A. aculeatinus had quite good growth and good acid production.
Growth on MEA-B
Pollastro et al.
(2006) developed a
semi-selective medium for A. carbonarius based on malt extract agar
(MEA) amended with some antibiotics and fungicides. Among these, MEA-B (MEA
with Boscalid) was found to be an efficient semi-selective medium to detect
the presence of A. carbonarius while A. niger could not
grow. In this study, we tested the growth abilities of all type strains of
Aspergillus section Nigri on MEA-B with 10 mg/L boscalid.
The results are shown in Table
3 and Fig. 7. After
3 d incubation, good growth could be detected only in A. carbonarius, A.
sclerotioniger, A. homomorphus and A. sclerotiicarbonarius. No
visual growth was detected in A. ellipticus, A. niger, A. brasiliensis, A.
vadensis, A. piperis and A. costaricaensis.
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After 7 d of incubation, many strains could recover and grow. However, only 3 strains were able to sporulate: A. carbonarius, A. sclerotioniger and A. sclerotiicarbonarius. So MEA-B is a helpful selective medium for differentiation of the ochratoxigenic A. carbonarius from many other species in section Nigri. Moreover, A. ibericus, a closely related non-OTA-producer species, could be differentiated from A. carbonarius as this species could not grow well on this medium.
Ehrlich reaction
Lund (1995) reported an
easy useful reaction to identify some closely related Penicillia. The so-call
Ehrlich reaction method detects some indole secondary metabolites produced by
fungi by direct reaction of the Ehrlich reagent wetted on filter paper with
mycelial side of an agar plug. Samson & Frisvad
(2004) also suggested that
this method is useful for classification of Penicillium subgenus
Penicillium. In this paper, we tried this method to classify some
Aspergillus spp. in section Nigri. All type strains were
examined and the results were shown in
Table 4 and
Fig. 8.
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In positive results, violet ring or blue colour compounds appeared. Based on their response, species of Aspergillus section Nigri can be classified into groups. A. heteromorphus and A. homomorphus had the same positive results with yellow reaction with purple ring occurring within 5 min. A. costaricaensis also gave a positive result with the test but it reacted and formed a strikingly blue colour. A. brasiliensis, A. foetidus and A. niger gave positive results in the form of a yellow reaction. A. tubingensis, and the other related species gave negative result so this method is useful to discriminate them from the others. Furthermore, A. sclerotioniger could also give purple colour positive result at the sclerotial area.
Extrolites found in the black aspergilli
The production of the secondary metabolites is usually consistent in a
species, however, ochratoxin A production in A. niger is only found
in ca. 6 % of the strains. Ochratoxin A producing species of section
Nigri occurring on grapes, raisins and in wine include A.
carbonarius which species produces this compound very consistently, and
to a lesser extent A. niger. Four species recovered from coffee,
A. carbonarius, A. niger, A. lacticoffeatus and A.
sclerotioniger, all produce ochratoxin A
(Table 5). None of the other
species in section Nigri have been found to be ochratoxin producers.
Very old culture collection strains may have lost the ability to produce some
of the secondary metabolites otherwise characteristic of the species. The
consistency in production of malformins in A. niger and A.
tubingensis is not yet explored. Many of the secondary metabolites found
are as yet of unknown structure, but are often diagnostic for one or more
species in section Nigri.
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Molecular tools to distinguish black aspergilli
Several molecular tools have been used to distinguish black
Aspergillus species. Among these, restriction fragment length
polymorphisms (RFLPs) of both nuclear and mitochondrial DNAs (mtDNAs) have
been used successfully to identify new species. Kusters-van Someren et
al. (1990) used Western
blotting and DNA hybridisation with a pectin lyase (pelD) gene to
ascertain whether these methods could be used for rapid strain identification.
The DNA hybridisation experiments showed that the pelD gene is
conserved in all isolates belonging to the A. niger aggregate.
Hybridisation was also observed in DNAs of all A. foetidus strains.
The authors established three groups within the A. niger aggregate on
the basis of presence or absence of three other bands which hybridised
strongly to the pelD gene. As a continuation of this work,
Kusters-van Someren et al.
(1991) carried out a more
extensive study on nuclear DNA RFLPs of several black Aspergillus
collection strains. Two groups of strains were distinguished according to
their SmaI-generated ribosomal DNA (rDNA) patterns. The two groups
were also clearly distinguishable by their hybridisation patterns when pectin
lyase genes (pelA, pelB) and the pyruvate kinase (pki) gene
were used as probes in DNA hybridisation experiments. The two groups found
were proposed to represent different species, namely A. niger and
A. tubingensis. Examination of other species not belonging to the
A. niger aggregate was also carried out. A. foetidus
strains, classified into a different species by Al-Musallam
(1980), showed the same nuclear
DNA RFLPs as A. niger. A. helicothrix was found to represent only a
morphological variant of A. ellipticus, and A. aculeatus
exhibited the same SmaI-digested rDNA pattern as the A.
japonicus strains examined. Jaap Visser"s group detected further
differences in the nuclear genes encoding polygalacturonase II,
arabinoxylan-arabinofuranohydrolase and xylanase enzymes of A. niger
and A. tubingensis strains
(Bussink et al. 1991;
Graaff et al. 1994;
Gielkens et al.
1997).
SmaI digested repetitive DNA profiles hybridised with the ribosomal repeat unit of A. nidulans were found to have distinctive value among black aspergilli (Varga et al. 1994, 2000). A. ellipticus, A. heteromorphus, A. japonicus and A. carbonarius exhibited species specific hybridisation patterns, with the exception of A. carbonarius strain IN7, which revealed a slightly different profile than the other A. carbonarius strains examined. Among the strains of the A. niger species complex, four profiles were observed, among which rDNA types I and III were shown by A. niger and A. brasiliensis strains, respectively, while rDNA types II and II were characteristic of the A. tubingensis strains (Varga et al. 1994).
Parenicova et al. (2001) used RFLP analysis to distinguish A. japonicus and A. aculeatus isolates. The hybridisation probes were the A. niger pyruvate kinase (pkiA) and pectin lyase A (pelA) and Agaricus bisporus 28S rRNA genes, which revealed clear polymorphism between these two taxa. The A. niger pkiA and pelA probes placed six strains in an A. japonicus group and 12 isolates in an A. aculeatus group, which exhibited intraspecific variation when they were probed with the pelA gene. The application of these probes could also be used to distinguish other species in the section (Parenicova et al. 2000), including the recently described species A. vadensis (de Vries et al. 2005).
Wide-ranging mtDNA variation has also been observed both among collection strains and in natural populations of the A. niger species complex (Varga et al. 1993, 1994). Within the A. niger species complex, most isolates were classifiable as A. niger or A. tubingensis according to their HaeIII-BglII digested mtDNA patterns. The A. niger and A. tubingensis species could be grouped into 5 and 6 mtDNA types, respectively. Six of the 13 Brazilian isolates examined exhibited mtDNA and rDNA types different from those of all the other strains. Later these strains have been assigned to the A. brasiliensis species (Varga et al. 2007). The sizes of the mtDNAs of the black Aspergillus strains examined were highly variable. The mtDNA of type 3 was the largest (35 kb) followed by those of types 2f and 2e (34 kb and 32.5 kb, respectively). The smallest mtDNA molecule (26 kb) was that of type 2c. All the other mtDNA types had sizes in the range 28-31 kb. For A. japonicus isolates, the strains could be classified into seven different mtDNA RFLP groups based on their HaeIII-digested mtDNA profiles. Hybridisation data suggest that six of these mtDNA types have certain common features in their organisation, while mtDNA type 7, which was exhibited by A. aculeatus strains, probably have quite different mtDNA structure (Hamari et al. 1997). The sizes of A. japonicus mtDNAs were in the range of 43-50 kb. Among the 16 collection strains and field isolates of Aspergillus carbonarius examined, the HaeIII-digested mtDNA profiles revealed only slight variations, except for one field isolate (IN7), which exhibited completely different mtDNA patterns (Kevei et al. 1996). The mtDNAs of these strains were found to be much larger (45 to 57 kb) than those found earlier in the A. niger aggregate. The physical maps of the mtDNAs of A. carbonarius strain IN7 (which later was found to belong to the A. ibericus species; Varga J., unpubl. data) and the other A. carbonarius strains are quite different from each other, however, the order of the genes on these molecules seems to be conserved (Hamari et al. 1999).
Among other approaches, Megnegneau et al. (1993) applied the random amplified polymorphic DNA (RAPD) technique for examining variability among black aspergilli. By applying six random primers, they could differentiate A. carbonarius, A. japonicus, A. aculeatus, A. heteromorphus and A. ellipticus from each other, and could divide the A. niger species complex into two groups corresponding to the A. niger and A. tubingensis species. The RAPD technique could also be used successfully for the examination of genetic variability within A. carbonarius and A. japonicus species. A. carbonarius strain IN7 could readily be distinguished from the other A. carbonarius strains examined (Kevei et al. 1996). The strains representing the A. aculeatus species could also be distinguished from the other A. japonicus strains by using 4 random primers (Hamari et al. 1997), and A. brasiliensis could also be distinguished from the other strains of the A. niger species complex by RAPD analysis (Varga et al. 2000). AFLP analysis could successfully be used to distinguish among black Aspergillus species by Perrone et al. (2006a, 2006b), Serra et al. (2006) and Varga et al. (2007). Analysis of electrophoretic karyotypes among black aspergilli revealed the presence of high levels of intraspecific variability of the banding patterns observed (Megnegneau et al. 1993; Swart et al. 1994). However, the estimated total genome sizes did not differ significantly, ranging from 35.9 Mb in an A. niger strain to 43.8 Mb in an A. ellipticus strain. The average genome size of strains belonging to the A. niger species complex was 38.3 Mb. In general, electrophoretic karyotyping seems to be of little taxonomic value in such a variable group as black aspergilli.
Among the PCR based approaches, Accensi et al. (1999) used a PCR-RFLP technique to distinguish A. niger and A. tubingensis isolates. The authors used the restriction enzyme RsaI to digest the amplified ITS region of the isolates, and observed that isolates of the A. niger species complex exhibit two different RFLP patterns, N and T corresponding to A. niger and A. tubingensis isolates, respectively. The ITS region of A. niger contains the recognition site of RsaI (5"-GT/AC-3") at position 75, while that of A. tubingensis does not. However, in silico examination of the ITS region of black aspergilli indicated that pattern T is also shared by A. foetidus, A. vadensis, A. piperis and A. costaricaensis isolates, while all other species exhibit pattern N (data not shown). However, this method has been used to distinguish A. niger from A. tubingensis (Medina et al. 2005, Accensi et al. 2001, Martinez-Culebras & Ramon 2007, Bau et al. 2006). PCR-RFLP analysis of the ITS region using other restriction enzymes has also been used for species identification recently (Martinez-Culebras & Ramon 2007). The authors used HhaI, NlaIII and RsaI to distinguish between A. niger, A. tubingensis, A. carbonarius and A. aculeatus isolates came from grapes. Some "A. tubingensis-like" isolates exhibited characteristic RFLP profiles when NlaIII was used to digest the amplified fragment, which was found to be caused by a single point mutation in the ITS region. Gonzales-Salgado et al. (2005) developed species-specific primer pairs designed based on sequences of the ITS region for the identification of A. niger, A. tubingensis, A. heteromorphus, A. ellipticus and A. japonicus. Zanzotto et al. (2006) used PCR-RFLP analysis of the ITS, IGS and β-tubulin genes to distinguish between OTA-producing and non-producing isolates of the A. niger aggregate. Schmidt et al. (2004) developed species specific PCR primers based on AFLP fragments for the identification of A. carbonarius on coffee beans, while Atoui et al. (2007) and Mule et al. (2006) developed real time PCR approaches to identify A. carbonarius on grapes. The latter two groups used species-specific primer pairs designed from the acyltransferase (AT) domain of the polyketide synthase sequence and the calmodulin gene, respectively. Susca et al. (2007a) also developed species-specific primers based on partial calmodulin gene sequences to identify A. carbonarius and A. niger by PCR. Recently, Susca et al. (2007b) developed a PCR-single-stranded conformational polymorphism (SSCP) screening method based on the detection of sequence variation in part of the calmodulin gene. Using this approach, 11 species including A. brasiliensis, A. niger, A. tubingensis, A. foetidus, A. aculeatus, A. uvarum, A. japonicus, A. ellipticus, A. heteromorphus, A. carbonarius and A. ibericus could be distinguished based on their different PCR-SSCP profiles. A low-complexity oligonucleotide microarray (OLISA) has also been developed based on oligonucleotide probes obtained from sequences of the calmodulin gene for the detection of black aspergilli (A. carbonarius, A. ibericus and A. aculeatus/A. japonicus) from grapes (Bufflier et al. 2007).
Nowadays, sequence-based identification methods are widely used for species identification. In Aspergillus section Nigri, all species can be distinguished from each other using calmodulin sequence data, and all except one could be distinguished using β-tubulin sequence data (A. lacticoffeatus had identical β-tubulin sequences to some A. niger isolates; Samson et al. 2004, Varga et al. 2007; Fig. 9). The ITS data set can be used to distinguish 4 groups within the A. niger species complex: 1. A. niger and A. lacticoffeatus isolates; 2. A. brasiliensis; 3. A. costaricaensis; 4. A. tubingensis, A. foetidus, A. vadensis and A. piperis (Varga et al. 2007). Among the other black aspergilli, A. carbonarius and A. sclerotioniger exhibit identical ITS sequences, while most uniseriate species also have identical ITS sequences (including A. japonicus, A. aculeatus and A. uvarum). Yokoyama et al. (2001) used sequences of the mitochondrial cytochrome b gene to infer phylogenetic relationships among black aspergilli. A. japonicus/A. aculeatus, A. niger, A. tubingensis, A. carbonarius and A. ellipticus could be distinguished from each other based on phylogenetic analysis of amino acid data. However, A. tubingensis and A. niger isolates could not be clearly distinguished when nucleotide sequences were subjected to phylogenetic analysis.
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, RNA polymerase
2 and actin gene sequences (S.W. Peterson, personal communication). According
to recent data, cox1 is not appropriate to be used for species
identification in black aspergilli (Geiser
et al. 2007). The phylogenetic tree constructed based on
the cox1 sequences shows an overlap between intra- and interspecific
variation possibly due to past mitochondrial DNA recombination events. The
different molecular techniques applied for species delimitation in
Aspergillus section Nigri are summarised in
Table 6.
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CONCLUSIONS AND RECOMMENDATION FOR THE IDENTIFICATION OF BLACK ASPERGILLI |
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TOPAbstractINTRODUCTIONMATERIAL AND METHODSRESULTSCONCLUSIONS AND RECOMMENDATION...References |
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When using extrolite patterns it is noteworthy that asperazine can be used to distinguish A. tubingensis, A. foetidus and A. vadensis from A. niger and A. brasiliensis, while pyranonigrin A is present in all species in the Aspergillus niger complex, except A. brasiliensis, A. costaricaensis and A. vadensis. Secalonic acid D is produced by the uniseriate species only, except A. japonicus. However the biseriate A. homomorphus also produces secalonic acid D. A. ellipticus is entirely unique and produces extrolites found in section Candidi (terphenyllin and candidusins) and section Usti (austdiol). A. heteromorphus also has a unique combination of extrolites not found in any other Aspergillus species. Thus it seems that all section Nigri members can be identified based solely on extrolites.
In Aspergillus section Nigri, all species can be distinguished from each other using calmodulin sequence data, and all except one could be distinguished using β-tubulin sequence data. As discussed ITS can only be used for a rough classification of the uni- and biseriate species while only four groups of related taxa of A. niger can be identified.
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References |
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TOPAbstractINTRODUCTIONMATERIAL AND METHODSRESULTSCONCLUSIONS AND RECOMMENDATION...References |
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