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Species in the genus Aspergillus have been classified primarilybased on morphological features. Sequencing of house-hold genes has also beenused in Aspergillus taxonomy and phylogeny, while extrolites andphysiological features have been used less frequently. Three independent waysof classifying and identifying aspergilli appear to be applicable: Morphologycombined with physiology and nutritional features, secondary metaboliteprofiling and DNA sequencing. These three ways of identifyingAspergillus species often point to the same species.
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This consensusapproach can be used initially, but if consensus is achieved it is recommendedto combine at least two of these independent ways of characterising aspergilliin a polyphasic taxonomy. The chemical combination of secondary metabolitesand DNA sequence features has not been explored in taxonomy yet, however.Examples of these different taxonomic approaches will be given forAspergillus section Nigri. INTRODUCTIONThe genus Aspergillus and its teleomorphs contain a large numberof species some of which have been exploited for biotechnologicallyinteresting products for centuries. In particular Aspergillus niger hasbeen used for fermentation of Puer tea and Awamori, citric acid production(;),extracellular enzyme production, for biotransformations of chemicals,and as a producer of antioxidants.
Niger strains appear to beable to (over)produce citric acid , suggesting that this ability is probably an essentialfeature of the species. It is therefore tempting to turn this phenomenonaround and use such a chemical feature as a taxonomic diagnostic tool. Otherspecies in the section Nigri such as A.
Carbonarius andA. Aculeatus are able to produce citric acid, soit is necessary to use a whole profile of such chemical features tocircumscribe a species. Several types of tests and measurements can be used inAspergillus taxonomy , but some of these require special equipment and may not all bediagnostic. In some cases it is only the combination of some of those featuresthat may work in classification and identification. Some features arespecially suited for cladisitic studies, especially DNA sequence data. Bothcolour and physiological tests were used in early taxonomic research byMurakami and Murakamiet al.
,including pigment production in Czapek agar, growth on nitrite as solenitrogen source, acid production, extracellular enzyme production and reactionof broth with FeCl 3. However, these detailed studies were mostlyignored by the Aspergillus community. Raper & Fennell (1965) didnot use any chemical, biochemical or physiological characters, but in latertaxomomic studies of Aspergillus physiological tests andsecondary metabolites (for example; Frisvad et al.,;;)have been introduced. In addition to their use in chemotaxonomy, manysecondary metabolites have bioactive properties. Mycotoxins are of particularinterest, because Aspergillus species produce some of the mostimportant mycotoxins.
In this review we focus mainly on the use ofsecondary metabolites and nutritional tests in Aspergillus taxonomyand the reasons why they may work very efficiently in some cases, and lesssatisfactory in other cases. Aflatoxin production is used as an example caseto the genetical background on why certain strains in a species do not producemycotoxins and others do.
Type of featureSpecialized equipment needed?Specialized equipment present in mycological labs?Level of diagnostic powerIn useMicromorphologyMicroscopeYesMacromorphology(Camera, colourimeter)YesPhysiology(Incubators etc.)YesNutritional testsNo(Yes)+RareSecondary metabolites, volatilesGCRarely+RareSecondary metabolites, non-volatileTLCOccasionallyRareHPLC-DADRarelyRareHPLC-MSRarelyRarediMSRarelyRareExtracellular enzymesGE, CERarely+RareDNA sequencingPCR, sequencingOccasionally. Extrolites in AspergillusThe fungal exo-metabolome , cell-wall metabolome and certain parts ofthe endo-metabolome are produced as a reaction to the biotic and abioticenvironment, and consists of secondary metabolites, overproduced organicacids, accumulated carbohydrates (e.g. Trehalose and polyols), extracellularenzymes, hydrophobins, adhesins, expansins, chaperones and other molecules.Those metabolites that are secreted or are accumulated in the cell wall arepart of the exo-interactome. Exo-metabolites are secreted and consist mainlyof secondary metabolites, overproduced organic acids, extracellular enzymesand other bioactive secreted proteins. The cell wall metabolome consists ofstructural components (melanin, glucan etc.), epitopes, and certainpolyketides and alkaloids that probably protect fungal propagules in beingeaten by insects, mites and other animals(;).The endo-metabolome consists of primary metabolites in constant change andinternal interaction (the interactome and fluxome). These primary metabolitesare of no interest for taxonomy.
However, the profile of accumulatedcarbohydrates, such as trehalose and mannitol, may change as a reaction to theenvironment in a more species-specific manner (Henriksen et al.1988). The same may be the case for certain chaperones, i.e. Those thatparticipate in the reaction to changes in the environment or stress based onextreme environments. Only a fraction of all these molecules have been used intaxonomy. In general those metabolites that are of ecologicalinterest can be called extrolites, because they are outwards directed.
Themolecules used most in species recognition have been secondary metabolites,because the profiles of these are highly species specific(; ). In some cases several isolates in a species do not producethe secondary metabolite expected and this is especially common concerningaflatoxin and ochratoxin production (see below). However the“chemoconsistency” is usually much more pronounced for othersecondary metabolites. For example in the case of Aspergillus sectionNigri, each species is characterised by a specific profile (see for acomplete Table in ) which also shows relationships among the taxa. Based on suchprofiles a “chemophylogeny” can be seen in section Nigri or at least anagreement in taxonomic and phylogenetic grouping. Classification of the blackaspergilli using morphological, physiological, and chemical features resultsin a grouping of the black aspergilli that is in very good agreement with acladification of the same aspergilli using β-tubulin sequencing(;).For example A. Carbonarius, A.
Sclerotioniger, A. Ibericus and A.sclerotiicarbonarius in the suggested series“ Carbonaria” have relatively large rough-walled conidia,a relatively low growth rate at 37° C, moderate citric acid production andother characters in common and at the same time they belong to the same cladeaccording to β-tubulin sequencing. Series Nigri:Subseries Nigri:Aspergillus nigerAspergillus lacticoffeatusAspergillus brasiliensisSubseries Tubingensis:Aspergillus tubingensisAspergillus vadensisAspergillus foetidusAspergillus piperisAspergillus costaricaensisSeries Carbonaria:Aspergillus carbonariusAspergillus sclerotionigerAspergillus ibericusAspergillus sclerotiicarbonariusSeries Heteromorpha:Aspergillus heteromorphusAspergillus ellipticusSeries Homomorpha:Aspergillus homomorphusSeries Aculeata:Aspergillus aculeatusAspergillus aculeatinusAspergillus uvarumAspergillus japonicus.
Some of the secondary metabolites are secreted as volatiles, especiallyterpenes and certain small alcohols. Other secondary metabolites stay in theconidia, sclerotia or other propagules or are secreted in to the growthmedium. Volatile metabolites can be separated and detected by GC-MS, whereasmost other secondary metabolites are extracted by organic solvents andseparated and detected by HPLC-DAD-MS.
Proteins of interest may be separatedby 2D-gel electrophoreses or capillary electrophoresis and detected (andidentified) by MS. A more indirect detection, followed by chemometrictreatment of the data may also be used. For example, extracts of fungi may beanalysed by direct inlet electrospray mass spectrometry.Filamentous fungi can also be characterised by quantitative profiles offatty acids (Blomquist et al. 1998), their pattern of utilisation ofC- and N-sources, their temperature, water activity, pH, atmosphere, redoxrelationships (; ) etc.Isolates of Aspergillus have mostly been characterised by theirprofiles of secondary metabolites, by their growth rate at certaintemperatures and water activities, their growth on creatine-sucrose agar andthe color of the conidia, in addition to morphology. As can be seen from thediscussion above, many other potential means of characterising the phenome ofaspergilli exist.
Of all the phenotypic features it is strongly recommended touse secondary metabolites in species descriptions, in addition tomorphological and DNA sequence features. However, water and temperaturerelationships should also be used, at least for culturable fungi such as theaspergilli.
A minimum standard for the features that need to be characterisedfor a species description should be made as an international collaborativeeffort. Chemotaxonomy and secondary metabolite profilingAs mentioned in the previous section, the molecules used most often inspecies recognition have been secondary metabolites, due to their high speciesspecificity (;).In other words practically all species produce a unique combination ofdifferent types of small organic compounds such as polyketides, non-ribosomalpeptides, terpenoids as well as many other compounds of mixed biosyntheticorigin. Some of these compounds are even unique to a single species. The factthat secondary metabolites are indeed excellent phenotypic characters forspecies recognition is backed up by the recent studies on full genomesequencing of important aspergilli concluding that major genomic differencesbetween species are often related to the number and similarity of polyketideand non-ribosomal peptide synthase genes(;;).Thus in various scenarios detection of a unique mixture or in some casesone or a few biomarkers can be used for species recognition. The use of growth and enzyme profiles for species recognition in theblack aspergilliBlack aspergilli are found throughout the world except for the arcticregions.
This means that these fungi encounter highly different biotopes withstrong variations in the crude carbon sources they utilise for growth. Thisraises the question whether strains that were isolated from different biotopeshave adapted to the carbon sources in their environment and are thereforedifferent in their enzyme and growth profile with respect to a range ofdifferent carbon sources (nutritional tests). Also, one might expect thatdifferent black aspergilli occupy different ecological niches and thereforehave different growth and enzyme profiles. Murakami et al. have studied this onsome black aspergilli, but many new species have been described since. Acomparison of A.
Tubingensis, A. Foetidus andA. Japonicus on 7 carbon sources revealed clearly different growthprofiles for each species, and demonstrated that A. Niger and A.tubingensis were most similar. The growth profile of A.vadensis was remarkable in that growth on glycerol, D-galacturonate andacetate was poor compared to the other species. Foetidus andA.
Japonicus grew poorly on xylitol, while A. Tubingensisgrew poorly on citrate. Recently, a more elaborate study was performed inwhich differences between A. Niger isolates were compared todifferences between the black Aspergillus species (Meijer, Houbraken,Samson & de Vries, unpubl.
For this study 17 true A. Nigerisolates (verified by ITS and β-tubulin sequencing) from differentlocations throughout the world were compared to type strains of the differentblack Aspergillus species and grown on different monosaccharides. Nodifferences in growth on specific carbon sources was observed between theA. Niger isolates, while significant differences were observedcompared to the different species, demonstrating that adaptation of strains totheir environment with respect to carbon source utilisation does not occur inA. Most remarkable was the finding that of all the blackaspergilli, only A. Brasiliensis was able to grow significantly onD-galactose, but growth differences between the species were also observed onD-fructose, D-xylose, L-arabinose and galacturonic acid (Meijer, Houbraken,Samson & de Vries, unpubl.
Niger isolates and thedifferent type strains were also grown in liquid medium with wheat bran orsugar beet pulp as a carbon source. Culture filtrate samples were taken after1 and 2 d and analysed on SDS-PAGE. The SDS-PAGE profiles were found to behighly similar between the different A.
Niger isolates, whilesignificant differences were observed between the different species. Thisindicates that protein profiles could be used as a fast screen for speciesidentification (Meijer, Houbraken, Samson & de Vries, unpubl.results).As growth and protein profiles require only relatively low-techinfrastructure these characteristics could be extremely helpful in initialscreens to determine the identity of an isolate. However, for conclusiveidentification, these tests should be followed by sequencing the ITS and theβ-tubulin region and would be significantly strengthened by metaboliteanalysis as described in this paper. So far, using growth characteristics ondefined media and specific carbon sources has received little attention intaxonomy where traditionally undefined media like malt extract agar, potatodextrose agar and mout extract agar are used for morphological analysis. Theexample of growth on minimal medium with D-galactose as sole carbon source forA. Brasiliensis as the only species from the black aspergilli(Meijer, Houbraken, Samson & de Vries, unpubl.
Data), demonstrates thatthis is an unexplored area that might be a significant asset in multifactorspecies identification. Use of Ochratoxin A in identification of aspergilliThere are more than 20 species cited as ochratoxin A-producing fungi in thegenus Aspergillus (;; ) However, few of them are known to be regularly thesource of ochratoxin A (OTA) contamination of foods. OTA contamination offoods was until recently believed to be caused only by Aspergillusochraceus and by Penicillium verrucosum, which affect mainlydried stored foods and cereals respectively, in different regions of theworld.
However, recent surveys have clearly shown that someAspergillus species belonging to the section Nigri (e.g.A niger and A. Carbonarius), are sources of OTA in foodcommodities such as wine, grapes and dried vine fruits.
Petromycesalliaceus has been cited as a possible source for the OTA contamination,occasionally observed in figs. Recently, new OTA-producing species havebeen described from coffee (e.g. Lacticoffeatus, A.
Sclerotioniger, A.westerdijkiae and A. Steynii)(;),and recent results indicated that A.
Westerdijkiae, A. Steynii, A.ochraceus, A. Carbonarius are responsible for theformation of OTA in this product (; ).On the other hand, not all the strains belonging to an ochratoxigenicspecies are necessarily producers. Several methods have been developed todetect OTA producing fungi. Traditional mycological methods are time consumingand require taxonomical and chromatography expertise, however the agar plugmethod is quite simple (Filtenborg & Frisvad 1981;).
Different molecular diagnostic methods for an earlydetection of ochratoxigenic fungi, using mainly PCR techniques, have been alsoproposed. One of the goals of these techniques is to differentiate betweentoxigenic and non-toxigenic strains belonging to species known to produce OTA.To date, one of the problems is that little is known about the genes involvedin the OTA biosynthesis (;; ).
A full characterisation of the gene clustersresponsible for ochratoxin A production in the different species will showwhether all isolates in any of the species reported to produce OTAactually have the gene cluster required. The inability to produce OTA may becaused by silent genes or by mutations in functional or regulatory genes.OTA production is included as a character for taxonomical purposes inclassification (e.g. Extrolite profiles for describing species) and also foridentification (e.g. Synoptic key to species).
As is well known in taxonomy,one difficulty in devising identification schemes is that the results ofcharacterisation tests may vary depending on different conditions such as theincubation temperature, the length of incubation period, the composition ofthe medium, and the criteria used to define a positive or negative mycotoxinor extrolite production. In general the presence of a secondary metabolite isa strong taxonomic character, while the absence of a secondary metabolite issimply no information. Ochratoxin A production is a very consistent propertywhen monitored on YES agar for most species known to produce it, whereas otherspecies, such as A. Niger, have few strains producing it. Perhaps,for these reasons we can find some confusing or controversial data about theability to produce OTA by some species in the literature. Very often a way to solve such a problem is to record thewhole profile of secondary metabolites, because several other secondarymetabolites than ochratoxin are consistently produced, in this example, byAspergillus niger. Aflatoxin biosynthesis and regulationAflatoxin is the best studied fungal polyketide-derived metabolite.Aflatoxins are produced by an array of different Aspergillus species,but have not yet been found outside Aspergillus.
Aflatoxins have beenfound in three phylogenetically different groups of aspergilli: A. Parasiticus, A.
Parvisclerotigenus, A. Bombycis, and A.pseudotamarii in section Flavi, A. Shoe and boot designing manual pdf.
Ochraceoroseus and A.rambellii in section Ochraceorosei and Emericellaastellata and E. Venezuelensis in section Nidulantes. However, sterigmatocystin is also produced byphylogenetically widely different fungi such as Chaetomium species(;),Monocillium nordinii and Humicola fuscoatra.The genes for production of sterigmatocystin in E.
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Nidulans ( A.nidulans) and aflatoxin in A. Parasiticus, and A.nomius are clustered. At least some of the genes required forproduction of aflatoxins are present in species of Aspergillus notknown to be able to make aflatoxins or its precursors, such as A. Niger, and A. TheST gene cluster from A. Nidulans contains most of the genes found inthe A.
Flavus-type aflatoxin cluster, except that gene order andregulation of gene expression are different.In the aflatoxin biosynthesis gene cluster from A. Ochraceoroseus, aspecies more related to A. Nidulans than to A. Flavus, thegenes are similar to those in the biosynthesis cluster of A.nidulans, but are separated into at least two clusters.Dothistromin, produced by D. Septosporum, is an oxidation product ofthe aflatoxin biosynthesis intermediate versicolorin A.
The genes involved indothistromin biosynthesis are organised into at least 3 different clusters. These differences in cluster organisation could reflect theevolutionary processes involved in the formation of the AF biosynthesiscluster in section Flavi aspergilli.The genes in the ST and AF cluster are presumably co-ordinately regulatedby the Gal4-type (Cys 6Zn 2) DNA-binding protein, AflR.Most of the AF biosynthetic genes in section Flaviaflatoxin-producing species have AflR-binding sites in their promoter regionsand not in the promoter regions of genes neighbouring the cluster. In the STcluster of A. Nidulans, only a few genes have recognisableAflR-binding sites in their promoters. This difference and the fact thatglobally acting transcription factors putatively affect gene expression couldaccount for the differences in regulation of cluster gene transcription inresponse to environmental and nutritive signals of the differentaflatoxin-producing species.In addition to AflR, upstream regulatory proteins such as LaeA, a putativeRNA methyltransferase, (;;; ) control secondary metabolism possibly by affecting chromatinorganisation in subtelomeric regions, where most of these polyketidebiosynthesis clusters are located. Location of the genes in the cluster isimportant to their abilities to be transcribed.Protein factors that affect developmental processes such as formation ofsclerotia and conidia also affect aflatoxin formation(;)(Lee & Adams,;).Aflatoxin/ST/dothistromin biosynthesis begins with a hexanoylCoA starterunit synthesised by two non-primary metabolism FASs, encoded by genes in thecluster. These FASs form a complex with the PKS.
This complex allowsa unique domain in the PKS to receive hexanoylCoA prior to iterative additionof malonylCoA units. It was hypothesised that addition of malonylCoA continuesuntil the polyketide chain fills the cavity of the PKS and is excised by athioesterase that also acts as a Claisen-like-cyclase.The starter unit ACP transacylase domain (SAT) is found near the N-terminus ofthe AF/ST/DT PKSs. SAT domains have now been implicated in the formation ofmany fungal polyketides.Although the functions of most of the oxidative enzymes encoded by AF/STcluster genes are now well understood, there are still some enzymes whose rolehas not been established. The highly similar short chain alcoholdehydrogenases, NorB and NorA, may be necessary for the oxidativedecarboxylation required to convert open chain AFB1 and AFG1 precursors toAFB1 and AFG1.
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Mutation of a gene, nadA, previously predicted to bepart of a sugar cluster adjoining the AF cluster, prevents formation of AFG1,but not AFB1. NadA may be involved in ring opening of a putative epoxideintermediate formed in the conv. The genes avfA andordB ( aflX) also encode proteins predicted to have acatalytic motif for a flavin-dependent monooxygenase.Insertional inactivation of ordB led to a leaky mutant thataccumulated versicolorin A at the expense of AF. Although avfAmutants accumulate averufin , the role of AvfA in the averufin oxidation tohydroxyversicolorone has not been established. Another enzyme, CypX, wasproven to be required for the first step of the conv. Process.AvfA may catalyze opening of an epoxide intermediate to an unstable aldehyde,which would be expected to immediately condense to hydroxversicolorone.
Asimilar step can be imagined for the conv. Of VerA to ST in which anotherpredicted intermediate epoxide might require an enzyme to catalyze the openingof its ring to a form an unstable intermediate that would be subsequently bythe enzymes Ver-1 and AflY to generate the expected precursor(; ). The genes in the AF cluster, hypB1 andhypB2, are predicted to encode hypothetical oxidases. Similar genesare found in other clusters, for example, in the A. Terreus emodinbiosynthesis cluster.
Deletion of the gene for HypB2 gave leaky mutants thataccumulate OMST and norsolorinic acid, while deletion of hypB1 gavemutants with reduced ability to produce AF. From the chemical structures ofHypB1 and HypB2 we predict they are dioxygenases that catalyze the oxidations,respectively, of the anthrone initially produced by PksA and the OMST epoxideintermediate resulting from oxidation of OMST by OrdA during the conv. Of OMSTto AFB1 and G1. CONCLUSIONSSeveral chemical features may be used for classifying, identifying andcladifying Aspergillus species, but only a fraction of these havebeen used to any great extent. However as shown here many of these featureshave shown to be promising in Aspergillus section Nigri.Nutritional tests, fatty acid profiling, extracellular enzyme production,volatile secondary metabolites have been used sparingly, while secondarymetabolite profiling has been used quite extensively for taxonomic purposes inseveral Aspergillus sections. Together with morphology, physiology,nutritional tests and DNA sequence features, a stable polyphasicclassification can be suggested for Aspergillus species.
Any of thosekinds of characterisation methods alone may give occasional unambiguousresults, but together they are very effective in discovering species andidentifying isolates of Aspergillus. A minimum standard fordescribing new species and for an unequivocal classification andidentification of Aspergillus species should be developed.