Identification of Mucorales isolates from soil using morphological and molecular methods

Abstract

Background and purpose: Soil is the main habitat of saprophytic and pathogenic fungi. Mucoromycotina constitutes a large group of soil fungi, with certain opportunistic members causing systemic infections in immunocompromised hosts. The majority of human and animal infections are caused by the members of the genera Rhizopus, Mucor, Rhizomucor, Lichtheimia (Absidia), Cunninghamella, and Mortierella. Accordingly, in the present study, we aimed to isolate and identify the main genera of the order Mucorales, using molecular assays and morphological features.

Materials and methods: In total, 340 soil samples were collected from seven public parks throughout the city and sidewalk gardens in 14 municipal districts in Isfahan, Iran. All the samples were cultured on the appropriate media, incubated at 27°C for 2- 4 days, and examined daily for visible fungal growth. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was applied and macroscopic, microscopic, and physiological characteristics were assessed to identify fungal colonies.

Results: 400 pure colonies, belonging to the orders Mucorales and Mortierellales, including the genera Lichtheimia, Rhizopus, Rhizomucor, Mucor, Cunninghamella, and Mortierella, were identified. The genus Rhizopus (35.5%) was the most frequent isolate, followed by Mucor (32.25%) and Rhizomucor (27.5%).

Conclusion: The results emphasize the importance of opportunistic fungi in public areas and indicate the risk of exposure for immunocompromised individuals.

Keywords: Lichtheimia; Mortierella; Mucor; Mucorales; PCR-RFLP; Rhizomucor; Rhizopus.

Figure 1

Agarose gel electrophoresis of 18S rRNA PCR products of different species of Mucorales after restriction digestion with XhoII and CspsI. Lanes 1, 2, & 3: digestion with XhoII (Rhizomucor pusillus); Lanes 4, 5, & 6: undigested PCR products; Lanes 7 & 8: digestion with AseI (Rhizopus microsporus or R. azygosporus); Lanes 9, 10, & 11: undigested PCR products; Lanes 12, 13, 14, 15, & 16: digestion with AcII (Lichtheimia corymbifera or L. blakesleeana); Lane M: 100 bp molecular-size marker

Figure 2

Agarose gel electrophoresis of 18S rRNA PCR products of different species of Mucorales after restriction digestion with BmgBI (Lanes 1, 2, & 3), AfIII (Lanes 4, 5, 8, & 9), CspsI (Lanes 6 & 7), XmnI (Lanes 10, 11, 12, & 13), and PpumI (Lanes 14 & 15); Lane 1: Rhizopus spp. (except R. stolonifer); Lanes 2 & 3: undigested PCR products; Lanes 4, 5, 8, & 9: Mucor spp.; Lanes 6 & 7: Rhizopus arrhizus; Lanes 10, 11, 12, & 13: M. circinelloides, or M. racemosus, or M. ramosissimus, or M. plumbeus; Lanes 14 & 15: Rhizomucor spp.; Lane N: negative control; Lane M: 100 bp molecular-size marker