Culturing and Mating of Aspergillus fumigatus

Abstract

Aspergillus fumigatus is an opportunistic human fungal pathogen, capable of causing invasive aspergillosis in patients with compromised immune systems. The fungus was long considered a purely asexual organism. However, a sexual cycle was reported in 2009, with methods described to induce mating under laboratory conditions. The presence of a sexual cycle now offers a valuable tool for classical genetic analysis of the fungus, such as allowing determination of whether traits of interest are mono‐ or poly‐genic in nature. For example, the sexual cycle is currently being exploited to determine the genetic basis of traits of medical importance such as resistance to azole antifungals and virulence, and to characterize the genes involved. The sexual cycle can also be used to assess the possibility of gene flow between isolates. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. This unit describes protocols for culturing of A. fumigatus and for inducing sexual reproduction between compatible MAT1‐1 and MAT1‐2 isolates of the species. The unit also provides working methods for harvesting sexual structures, isolating single‐spore progeny and confirming whether sexual recombination has occurred. © The Authors. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Global Sexual Fertility in the Opportunistic Pathogen Aspergillus fumigatus and Identification of New Supermater Strains

“…Conidia were collected from two-month old cultures grown at room temperature on slopes of 2% MEA, and crosses were then set up in 9 cm Petri dishes containing 25 mL oat meal agar (OMA) as described by Ashton and Dyer [ 39 ]. This included a preliminary screen of the effect of incubation temperature between 28–37 °C on crossing success.…”

“…Three replicate crosses were set up for each cross, with worldwide isolates of known MAT1-1 genotype being crossed with the MAT1-2 tester strains, and MAT1-2 genotypes being crossed with the MAT1-1 tester strains, in addition to control crosses being set up between the Irish MAT1-1 and MAT1-2 tester strains. Crosses were incubated for four months, and sexual fertility was assayed by using a dissecting microscope to carefully inspect plates and then count the number of cleistothecia produced per 9 cm Petri dish, using a hoovering technique to ensure that all cleistothecia could be detected [ 24 , 39 ]. Where crosses were found to be infertile, crosses were incubated for up to 12 months in total before a final inspection (plates were resealed as necessary to prevent drying out of media).…”

“…Growth conditions were as identified by Swilaiman [ 38 ] as follows. Ascospores were isolated and 50,000 spores per mL suspensions were prepared as previously described [ 19 , 39 ]. 50 µL aliquots were transferred to sterile 0.2 mL PCR tubes and heat shocked for 30 min at either 65 °C, 75 °C, 80 °C or 85 °C to kill any contaminating conidia.…”

“…It has been reported that pyrimidine auxotrophy can influence the sexual fertility of Aspergillus species [ 45 , 46 ]. To assess fertility of the resulting pyrimidine auxotrophic strains, the sexual cycle was, therefore, induced on OMA plates supplemented with four different levels of both uridine and uracil (0.3, 0.6, 1.2 and 1.8 g/L of each chemical), using conditions described above [ 39 ], and numbers of cleistothecia formed per cross were scored after 3–4 months incubation.…”

Antifungal Activity of Thyme and Carvacrol Nanoemulsions Against Aspergillus fumigatus Isolated from Different Types of Cheese

“…From the fast-growing and saprophytic fungus is A. fumigatus that sporulates abundantly, releasing thousands of airborne conidia which are of relatively small size (2-to 3-µm in diameter) (Croft et al, 2016). However, it can spread easily in the environments related to the pathway of cheese production either during manufacturing, ripening or storage and produce mycotoxins (Kwon-Chung and Sugui, 2013; Ashton and Dyer, 2019). (Banjara et al, 2015;Mohamed et al, 2017;El-Shinawy et al, 2018).…”