How to differentiate Aspergillus species?

Differentiating Aspergillus species primarily involves a combination of macroscopic and microscopic morphological characteristics, supported by specific culture conditions and, increasingly, molecular techniques.

How to Differentiate Aspergillus Species?

The differentiation of Aspergillus species relies on a systematic approach that combines traditional mycological methods with modern molecular techniques, providing both phenotypic and genotypic insights.

1. Morphological and Cultural Identification

Morphological identification is the cornerstone of Aspergillus differentiation, providing readily observable characteristics. This process begins with specific culture conditions to ensure optimal growth and sporulation.

a. Culture Conditions and Macroscopic Features

For reliable morphological identification, isolates are usually inoculated at three points on Czapek Dox agar (CZA) and 2% malt extract agar (MEA) and incubated at 25°C. Most species sporulate within 7 days. These media provide essential nutrients and conditions for characteristic colony development.

Descriptions are primarily based on:

• Colony Pigmentation: The color of the conidial heads (e.g., black for A. niger, yellow-green for A. flavus, blue-green for A. fumigatus, white/cream for A. candidus) is a key differentiator. The color of the colony reverse (underside) can also be important.
• Colony Morphology:
  • Texture: Colonies can appear velvety, floccose (cottony), granular, or powdery.
  • Growth Rate: Fast-growing species like A. fumigatus contrast with slower-growing ones.
  • Presence of Exudates: Some species produce droplets of pigment on the colony surface.
  • Presence of Sclerotia: Hard, survival structures found in some species (e.g., A. flavus, A. parasiticus).

b. Microscopic Features

Once sufficient growth is achieved, microscopic examination of the conidial head is crucial. Key features include:

• Conidiophore: The stalk supporting the conidial head. Its length, width, and wall texture (smooth, rough) are important.
• Vesicle: The swollen apex of the conidiophore from which phialides arise. Vesicle shape (globose, clavate, pyriform), size, and whether it is fertile over the entire surface or only part of it (hemispherical) are diagnostic.
• Phialides: Flask-shaped cells that produce conidia.
  • Uniseriate: Phialides arise directly from the vesicle.
  • Biseriate: Metulae (intermediate cells) arise from the vesicle, and phialides arise from the metulae. The arrangement (uniseriate vs. biseriate) is a primary taxonomic criterion.
• Conidia: Asexual spores. Their shape (globose, elliptical), size, color, and surface texture (smooth, echinulate, rough) are vital characteristics.

2. Molecular Identification

While morphological methods are foundational, molecular techniques offer precise and rapid identification, especially for closely related or atypical isolates.

• DNA Sequencing:
  • ITS Region (Internal Transcribed Spacer): Often used as a primary barcode for fungal identification.
  • Beta-Tubulin (BenA) and Calmodulin (CaM) Genes: These genes are commonly used as secondary barcodes, providing higher resolution for species within Aspergillus sections where ITS may not be discriminative enough (e.g., Aspergillus section Fumigati, Flavi).
• PCR-based methods: Real-time PCR assays can detect specific Aspergillus species rapidly from clinical samples.

3. Physiological and Biochemical Characteristics

Some physiological or biochemical tests can provide supplementary information for differentiation, although they are less commonly used as primary identification methods.

• Growth at Different Temperatures: Some species exhibit distinct growth patterns at varying temperatures (e.g., A. fumigatus is thermotolerant, growing well at 37°C or higher).
• Production of Secondary Metabolites: Certain species produce specific mycotoxins or other metabolites, which can be detected through analytical methods, though this is usually for specific research or food safety applications rather than routine identification.

Key Differentiation Features at a Glance

By combining these methods, mycologists and diagnosticians can accurately differentiate Aspergillus species, which is critical for clinical diagnosis, agricultural management, and industrial applications.