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1 Identification of Aspergillus Aspergillus a common genus with approx. 300 species several ascomycetous teleomorphs has preference for warmer climates important for biotechnology, producers of mycotoxins and opportunistic human pathogens Nomenclature of infrageneric taxa of the genus Aspergillus (group- names following the classification of Raper and Fennell,1965) Subgenus Section Aspergillus Aspergillus (A. glaucus-group) Restricti (A. restrictus-group) Fumigati Fumigati (A. fumigatus-group) Cervini (A. cervinus-group) Ornati Ornati (A. ornatus-group) Clavati Clavati (A. clavatus-group) Nidulantes Nidulantes (A. nidulans-group) Versicolor (A. versicolor-group) Usti (A. ustus-group) Terrei (A. terreus-group) Flavipedes (A. flavipes-group) Circumdati Wentii (A. wentii-group) Flavi (A. flavus-group) Nigri (A. niger-group) Circumdati (A. ochraceus-group) Candidi (A. candidus-group) Cremei (A. cremeus-group) Sparsi (A. sparsus-group)

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Page 1: Identification of Aspergillus - KNAW · Aspergillus niger CBS 101886 Aspergillus niger CBS 618.78 Aspergillus niger CBS 554.65 T Aspergillus niger CBS 120.49 Aspergillus brasiliensis

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Identification of Aspergillus

Aspergillus a common genus with

approx. 300 species several ascomycetous

teleomorphs has preference for

warmer climates important for

biotechnology, producers of mycotoxins and opportunistic human pathogens

Nomenclature of infrageneric taxa of the genus Aspergillus (group-names following the classification of Raper and Fennell,1965)

Subgenus SectionAspergillus Aspergillus (A. glaucus-group)

Restricti (A. restrictus-group)Fumigati Fumigati (A. fumigatus-group)

Cervini (A. cervinus-group)Ornati Ornati (A. ornatus-group)Clavati Clavati (A. clavatus-group)Nidulantes Nidulantes (A. nidulans-group)

Versicolor (A. versicolor-group)Usti (A. ustus-group)Terrei (A. terreus-group)Flavipedes (A. flavipes-group)

Circumdati Wentii (A. wentii-group)Flavi (A. flavus-group)Nigri (A. niger-group)Circumdati (A. ochraceus-group)Candidi (A. candidus-group)Cremei (A. cremeus-group)Sparsi (A. sparsus-group)

Page 2: Identification of Aspergillus - KNAW · Aspergillus niger CBS 101886 Aspergillus niger CBS 618.78 Aspergillus niger CBS 554.65 T Aspergillus niger CBS 120.49 Aspergillus brasiliensis

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Monascus purpureusSclerocleista ornataSclerocleista thaxteri

A. raperiA. ustusA. insuetusA. pseudodeflectus

Emericella rugulosaEmericella nidulans

A. versicolorA. sydowiiA. protuberus

A. rambelliiA. ochraceoroseusA. sparsusA. biplanus

Eurotium echinulatumEurotium rubrum

Eurotium chevalieriA. clavatoflavus

Warcupiella spinosaChaetosartorya chrysellaA. wentii

A. restrictusA. penicillioides

A. auricomusNeopetromyces muricatusA. ochraceus

A. flavusPetromyces alliaceus

A. pseudotamarii

A. cervinusA. kanagawaensis

Neosartorya pseudofischeriA. fumigatus

Neosartorya fennelliae

A. campestrisA. candidus

Neocarpenteles acanthosporusA. clavatus

Dichotomomyces cejpii

A. carbonariusA. nigerA. tubingensis

A. carneusA. terreus var. africanusA. terreus

A. allahabadiiA. niveus

Fennellia flavipesA. iizukae

Nidulantes

Ornati

Usti

Raperi

Ochraceorosei

Sparsi

CremeiWarcupi

Zonati

Aspergillus

Restricti

Circumdati

Flavi

Fumigati

Cervini

Clavati

Flavipedes

Nigri

Candidi

TerreiNeighbor-joining tree of ITS sequences of selected Aspergilli

• 8 subgenera, 22 sections

CircumdatiRestricti

Candidi

Ascomycetous genera with an Aspergillus anamorph

Aspergillus anamorph Teleomorph

Section Aspergillus: Eurotium Link:Fr. Section Fumigati: Neosartorya Malloch & Cain. Section Ornati: Warcupiella Subram., Sclerocleista Subram.,

Hemicarpenteles Sarbhoy & ElphickSection Nidulantes: Emericella Berk. & Br. Section Flavipedes: Fennellia Wiley & SimmonsSection Circumdati: Neopetromyces Frisvad & SamsonSection Flavi: Petromyces Malloch & CainSection Cremei: Chaetosartorya Subram.

Overview identification criteria

Macro-morphology:Colony color

Colony size

Micro-morphology

Molecular based identification(sequencing)

Page 3: Identification of Aspergillus - KNAW · Aspergillus niger CBS 101886 Aspergillus niger CBS 618.78 Aspergillus niger CBS 554.65 T Aspergillus niger CBS 120.49 Aspergillus brasiliensis

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Colony colors and size in Aspergillus

Colony colors and size in Aspergillus

Micro-morphological criteria

conidiophores and conidia

columnar or radiate conidial heads

Hülle cells

ascomata

ascospores

Page 4: Identification of Aspergillus - KNAW · Aspergillus niger CBS 101886 Aspergillus niger CBS 618.78 Aspergillus niger CBS 554.65 T Aspergillus niger CBS 120.49 Aspergillus brasiliensis

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Conidiophores and conidia

Footcell

stipe

Conidial head

Conidiogenous cells of Aspergillus are phialides

Page 5: Identification of Aspergillus - KNAW · Aspergillus niger CBS 101886 Aspergillus niger CBS 618.78 Aspergillus niger CBS 554.65 T Aspergillus niger CBS 120.49 Aspergillus brasiliensis

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UNISERIATE CONIDIAL HEADS

BISERIATE CONIDIAL HEADS

SHAPES OF VESICLES IN ASPERGILLUS

clavateglobose subclavate

Page 6: Identification of Aspergillus - KNAW · Aspergillus niger CBS 101886 Aspergillus niger CBS 618.78 Aspergillus niger CBS 554.65 T Aspergillus niger CBS 120.49 Aspergillus brasiliensis

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Micro-morphological criteria

conidiophores and conidia

columnar or radiate conidial heads

Hülle cells

ascomata

ascospores

RADIATE CONIDIAL HEADS

Page 7: Identification of Aspergillus - KNAW · Aspergillus niger CBS 101886 Aspergillus niger CBS 618.78 Aspergillus niger CBS 554.65 T Aspergillus niger CBS 120.49 Aspergillus brasiliensis

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COLUMNAR CONIDIAL HEADS

COLUMNAR CONIDIAL HEADS

RADIATE CONIDIAL HEADS SPLITTING INTO COLUMNS

Page 8: Identification of Aspergillus - KNAW · Aspergillus niger CBS 101886 Aspergillus niger CBS 618.78 Aspergillus niger CBS 554.65 T Aspergillus niger CBS 120.49 Aspergillus brasiliensis

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Micro-morphological criteria

columnar or radiate conidial heads

conidiophores and conidia

Hülle cells

ascomata

ascospores

Emericella nidulans Anamorph: Aspergillus nidulans

Hülle cells

Page 9: Identification of Aspergillus - KNAW · Aspergillus niger CBS 101886 Aspergillus niger CBS 618.78 Aspergillus niger CBS 554.65 T Aspergillus niger CBS 120.49 Aspergillus brasiliensis

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Hülle cells

conidia

Irregularly shaped Hüllecells of Aspergilluscalidoustus embedded in conidia

Micro-morphological criteria

columnar or radiate conidial heads

conidiophores and conidia

Hülle cells

ascomata

ascospores

Page 10: Identification of Aspergillus - KNAW · Aspergillus niger CBS 101886 Aspergillus niger CBS 618.78 Aspergillus niger CBS 554.65 T Aspergillus niger CBS 120.49 Aspergillus brasiliensis

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Example:Aspergillus fumigatus

mostly not common in indoor environments

important in clinical environments

incubation temperature important

Blue green colonyGrowing fastCovering the diskIn 5-7 days at 37°C

Typical columnar heads

Page 11: Identification of Aspergillus - KNAW · Aspergillus niger CBS 101886 Aspergillus niger CBS 618.78 Aspergillus niger CBS 554.65 T Aspergillus niger CBS 120.49 Aspergillus brasiliensis

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Typical isolates Clinical isolates

Aspergillus penicillioides(xerophilic, no growth on MEA and CYA)

Aspergillus versicolor (strong odour; moderate xerophilic)

Page 12: Identification of Aspergillus - KNAW · Aspergillus niger CBS 101886 Aspergillus niger CBS 618.78 Aspergillus niger CBS 554.65 T Aspergillus niger CBS 120.49 Aspergillus brasiliensis

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MEDIA FOR IDENTIFICATION

Malt Extract Agar (MEA) for macro- and micro morphology

Czapek Yeast Agar (CYA) for colony color and size

For xerophilic Aspergilli: MEA and CZ with 20% sucrose

Incubation temperature: 25°C Incubation time: 7 days (or longer) 3 point position

Microscopic preparation of Aspergillus

Lactic acid with analine blue/cotton blueDrop of alcoholPreparations should be made from Malt Extract Agar

IDENTIFICATION CRITERIA

Macro-morphologyColony color

Colony size

Micro-morphologyColumnar or radiate conidial heads

Conidial heads: uni- or biseriate

Hülle cells

(Ascomata, ascospores)

Molecular based identification (sequencing)

Page 13: Identification of Aspergillus - KNAW · Aspergillus niger CBS 101886 Aspergillus niger CBS 618.78 Aspergillus niger CBS 554.65 T Aspergillus niger CBS 120.49 Aspergillus brasiliensis

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2gbII27E7IA.ustusIbT 2gbII27D6IA.ustusIbT

2gbII27F3IA.ustusIbT 2gbII27E3IA.ustusIbT 2gbII27E2IA.ustusIbT 2gbII27E9IA.ustusIbT

gbII27F5IA.ustusIBt gbII27E8IA.ustusIBt 2gbII27F6IA.ustusIbT 2gbII27D3IA.ustusIbT 2gbII27F2IA.ustusIbT gbII27D8IA.ustusIBt

gbII27E6IA.ustusIBt gb||CBS112452|N2|AinsuetusCBSB 2gbII27D2IA.ustusIbT gb||20D2|N2|AustusCBSbt2200607 2gbII27F1IA.ustusIbT 2gbII27F5IA.ustusIbT

gbII21A2bIA.pseudodeflectusIBt gbII27E4IA.ustusIBt gb||CBS596.65|N2|AinsuetusCBSB

gb||2CBS107.25|N2|T AinsuetusC gb||CBS119.27 - |N2|AinsuetusC

gbII27E5bIA.ustusIBt gbII27E1IA.ustusIBt

gbII27D9IA.ustusIBt gb||CBS561.65|N2|AinsuetusCBSB gb||IBT16751|N2|AgranulosusCBS

gb||CBS113227|N2|AinsuetusCBSB gb||CBS209.92|N2|AinsuetusCBSB

gb||CBS119.58|N2|AgranulosusCB gb||2CBS261.67|N2|T AustusCBSB

gb||20D5 - |N2|AustusCBSbt2200 gb||27E6|AustusIBT

gb||CBS495.65|N2|T ApuniceusCB gb||2CBS495.65|N2|T ApuniceusC 2gbII27D5IA.ustusIbT 2gbII27D4IA.ustusIbT

gb||21A4|N2|AustusCBSBT2200706 gb||27D7I|AustusIbt gb||27E8|AustusIbt

gb||CBS583.65|N2|AversicolorCB

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A. ustus

A. puniceus

A. granulosus

A. keveii

A. versicolor

A. calidoustus

A. pseudodeflectus

A. insuetusAspergillus sp.

A.calidoustus is also frequently isolated from indoor environments

(partial β-tubulin data)

Aspergillus heteromorphus CBS 117.55 TAspergillus ellipticus CBS 707.79 TAspergillus ellipticus CBS 677.79

Aspergillus carbonarius CBS 101697Aspergillus carbonarius CBS111.26 NTAspergillus carbonarius CBS 116.49

Aspergillus carbonarius CBS 126.49Aspergillus sclerotioniger CBS 115572 TAspergillus niger CBS 101883 TAspergillus niger CBS 101884Aspergillus niger CBS 101698Aspergillus niger CBS 557.65Aspergillus niger CBS 420.64Aspergillus niger CBS 101705Aspergillus niger CBS 101699Aspergillus niger CBS 101886Aspergillus niger CBS 618.78Aspergillus niger CBS 554.65 TAspergillus niger CBS 120.49Aspergillus brasiliensis CBS 101740

Aspergillus vadensis CBS 113365 TAspergillus tubingensis CBS 134.48 TAspergillus tubingensis CBS136.52Aspergillus tubingensis CBS 425.65Aspergillus tubingensis CBS 126.52Aspergillus tubingensis CBS 161.79Aspergillus tubingensis CBS 115657Aspergillus tubingensis CBS 117.32Aspergillus acidus CBS 564.65 TAspergillus acidus CBS 565.65Aspergillus piperis CBS 112811 TAspergillus costaricaensis CBS 115574 T

Aspergillus homomorphus CBS 101889Aspergillus homomorphus CBS 101888

Aspergillus "aculeatus” CBS 620.78Aspergillus "aculeatus” CBS 114.80

Aspergillus aculeatus CBS 119.49Aspergillus aculeatus CBS 172.66 TAspergillus japonicus CBS 115.80Aspergillus japonicus CBS 113.48Aspergillus japonicus CBS 568.65Aspergillus japonicus CBS 611.78Aspergillus japonicus CBS 101.14 Aspergillus japonicus CBS 522.89Aspergillus japonicus CBS 114.51 T

Aspergillus flavus CBS 100927 NT10 changes

10088

58

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9354

538593

92100

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Section Nigri‘black Aspergilli’

Morphological similar species;molecular tools and/or extrolite patterns recommended

www.studiesinmycology.org; www.cbs.knaw.nl; www.aspergilluspenicillium.org