Phenotypic Identification of Zygomycetes David Ellis School of Biological Sciences University of Adelaide, Australia. Mycology Online | www.mycology.adelaide.edu.au
Zygomycetes • Pin or bread moulds. • Fast growing, cosmopolitan (870 species). • Primitive coenocytic [infrequently septate] hyphae. • Asexual reproduction by sporangiospores formed in sporangia. • Key genera: Rhizopus, Rhizomucor, Lichtheimia, Cunninghamella, Saksenaea and Aphophyomyces.
Zygospores A thick-walled sexual spore formed by the fusion of two similar gametangia; characteristic of the Zygomycetes.
Coenocytic hyphae are infrequently septate, multi-nucleate hyphae as in the Zygomycetes.
Haematoxylin and eosin (H&E) stained section of lung tissue showing the broad, infrequently septate, thin-walled hyphae of Lichtheimia (Absidia) corymbifera.
Rhizopus oryzae – culture showing masses of sporangia (“pin heads”).
Sporangium of Rhizopus showing sporangiospores sitting on the columella awaiting dispersal, the sporangial wall has undergone autolysis which leaves a “head” of closely packed sporangiospores.
Zygomycota Sporangia Characters • Arrangement of sporangiospores [multispored, sporangiola, merosporangium] • Arrangement of sporangiophores [unbranched often in groups or frequently branched] • Sporangium shape [pyriform, spherical, flask-shaped etc] • Sporangium size [<100 μm diam. or >100 μm diam.] • Columella [Present or Absent] • Apophyses [Present or Absent] • Sporangiophore height [<0.8 mm or >1 mm] • Rhizoids [Present or Absent] (look in the agar) • Sporangiospore size [<6 μm or >6 μm]
Identification of Zygomycetes Growth temperature studies ie. 35, 40, 45C. Tease mounts are best, use a drop of 95% alcohol as a wetting agent to reduce air bubbles. May need to induce sporulation in isolates of Saksenaea and Apophysomyces.
Mucor Lichtheimia (Absidia) Mutispored sporangia with columella, + apophysis apophysis columella collarette
Rhizomucor pusillus Multispored sporangia, columella, no-apophysis. primitive rhizoids columellae
Rhizopus Multispored sporangia, columella, apophysis, sporangiophores in clusters with rhizoids. rhizoids collasped columella
Apophysomyces complex Multispored sporangia, funnel-shaped apophysis and conspicuous pigmented sub-apical thickening below the apophysis.
Saksenaea vasiformis Flask shaped multispored sporangium, columella, apophysis and rhizoids.
Cunninghamella bertholletiae Sporangiola “single celled conidia” borne on swollen denticles on a terminal vesicle.
Syncephalestrum racemosum Terminal vesicle and merosporangia.
Mortierella wolfii Sporangia with acrotonous (terminal) branches, no columella, no apophysis and rhizoids.
Unknown
Unbranched sporangiophores arising from rhizoids Multispored sporangium, large >100 um, spherical, columella and apophysis present. Cultures fast growing, white cottony at first, becoming grey-black with sporulation, No growth at 45C.
R. microsporus R. microsporus varieties not supported by molecular data
Multispored sporangium, spherical, <100 um diam. unbranched sporangiophore Columella present, apophysis absent, spores spherical
Its’ not always that easy! • Many uncommon moulds may present (especially from non-sterile sites) so remember growth at 37C is an important selector for medical fungi. • Often can not see the essential identification characters. • Can not find a suitable key or reference. • Non-sporulating moulds (now use ITS + sequencing).
Mould identification 1. Need to see the required characters • sporulating culture • slide culture + good microscope • careful observation 2. Learn mycology terminology and keys 3. Practice and reference books