Aspergillus fumigatus is known as a fungus of concern for human health particularly in immunocompromised patients. This fungus has a ubiquitous presence in our environment and healthy individuals exhibit high innate immunity to prevent Aspergillus diseases due to efficient clearing of the spores from the lungs (Hohl and Feldmesser, 2007). The most lethal manifestation of Aspergillus diseases includes invasive aspergillosis (IA), an infection that is characterized by tissue invasive growth of the fungus. IA is known to develop in patients with neutropenia, but in recent years cases are increasingly observed in nonneutropenic critically ill patients including those with severe influenza and coronavirus disease 2019 (COVID-19) (Schwartz et al., 2020; Chong and Neu, 2021). A major improvement in the management of Aspergillus diseases was made with the introduction of the triazole class, including itraconazole, voriconazole, posaconazole and isavuconazole. These triazoles have become evidence-based first choice treatment options for both treatment and prophylaxis of Aspergillus diseases. Management of IA has however remained difficult despite the availability of the triazoles. Diagnosis is often challenging because clinical symptoms and biomarkers are not always specific for IA, patient groups have become more heterogenous, and because of the emergence of acquired triazole resistance. Although triazole resistance was long neglected to be an uncommon phenomenon (Patterson and Strek, 2014), over the past decades triazole resistance has increased substantially in some geographic regions, mainly due to A. fumigatus isolates harboring TR₃₄/L98H and TR₄₆/Y121F/T298A mutations in the triazole target gene; the cyp51A gene. Triazole resistance was shown to be associated with treatment failure and 20% excess mortality in patients with IA (Lestrade et al., 2019). The most likely explanation for the spread of these tandem repeat(TR)-mediated resistance mechanisms in A. fumigatus is the use of triazole fungicides in agriculture (Verweij et al., 2009; Schoustra et al., 2019a). As the number of resistance mutations and variations increases over time, understanding the relationship between triazole fungicide exposure and the development of triazole resistance mutations is critical to contain this problem.

In order to optimally create genetic variation for adaptation to a triazole-containing environment, A. fumigatus needs to complete its reproduction cycle. A. fumigatus may benefit from three reproduction modes including asexual, parasexual, and sexual reproduction, each of which have specific benefits and limitations (Zhang et al., 2021a). For A. fumigatus the sexual cycle was detected in vitro in 2009 by O’Gorman et al. (2009), who showed that A. fumigatus possesses a fully functional sexual cycle and described the teleomorph Neosatorya fumigata. The sexual cycle involves the fusion of two haploid nuclei of opposing mating-types MAT1-1 and MAT1-2 to produce a diploid zygote from which haploid spores are produced by meiotic cell division. Sexual mating with meiotic recombination increases genetic variation among progeny enabling the fungus to adapt and survive in changing environments. It also enables the fungus to get rid of deleterious mutations in contrast to strictly asexual species that instead are expected to gradually accumulate deleterious mutations, also named the Muller’s ratchet (Muller, 1964). Although the 50:50 distribution, or balancing selection, of mating types in environmental A. fumigatus isolates suggests that niches are present in nature that support sexual reproduction, to date such niches have not been reported and mating has only been achieved under laboratory conditions (Poggeler, 2002; Dyer et al., 2003; Varga and Toth, 2003; O’Gorman et al., 2009). Moreover, mature cleistothecia (fruiting bodies) were obtained after six months of incubation on a parafilm sealed oatmeal agar plate at 30°C in the dark, recently the supermaters were discovered, which require much shorter time to produce cleistothecia around 4 weeks (Sugui et al., 2011) and the authors were still unable to induce a sexual cycle on any other media or conditions (O’Gorman et al., 2009). It remains unclear why so specifically oatmeal agar and incubation in the dark has been successful for sexual reproduction of A. fumigatus, especially considering it will be difficult to identify a natural environment resembling these conditions. Most fungal species reproduce asexually when conditions are favorable for growth and reproduce sexually in nutrient-limiting conditions, which is in contrast to what has been observed for A. fumigatus so far (Dyer et al., 1992). The thick ascospore wall of A. fumigatus has been suggested to protect sexual spores from different environmental stresses including high temperature and ultraviolet radiation. Given that A. fumigatus is a typical soil fungus it can explain the induction of sex in the absence of light (Sugui et al., 2011).

Sexual recombination has been suggested to play a role in triazole resistance development in A. fumigatus, as the environmental route involves resistance mutations characterized by complex mechanisms such as a TR in combination with one or more SNPs in the cyp51A-gene (i.e. TR₃₄/L98H (Zhang et al., 2017). As triazole resistant isolates that harbor either the TR or the cyp51A-SNP are extremely rare, selection of TR-mediated resistance mutations through asexual reproduction is unlikely and has only been reported once in a clinical case (Hare et al., 2019). As such complex changes may have developed through meiotic recombination, we hypothesized that sexual recombination can play a role in the development of TR-mediated resistance mutations in A. fumigatus. Over the past years we have investigated A. fumigatus population dynamics in plant-waste storage heaps, especially those produced by flower bulb growers, which may contain high levels of triazole fungicide residues (Zhang et al., 2017; Schoustra et al., 2019a). Although samples from these sites were shown to contain very high A. fumigatus colony forming unit (CFUs) per gram (Schoustra et al., 2019a), supporting active asexual reproduction, several observations indicated that a sexual cycle might also be present in these hotspots. A study performed by our group in 2017 revealed the presence of TR₄₆-variants with three repeats ( TR 46 3 ) instead of two copies in the promoter region of the cyp51A gene as a possible result of unequal crossing over during meiosis (Zhang et al., 2017). Further indications of sex in composting flower bulb waste material were not a clonal expansion but a high genetic diversity found among A. fumigatus isolates recovered from these heaps and the fact that significant growth of A. fumigatus CFUs was recorded after a 70°C heat shock, a temperature that is known to kill asexual conidia. The question now arises whether flower bulb waste stockpiles or compost heaps can provide the conditions required for sex in A. fumigatus. In the current study we set out to find evidence for sexual reproduction of A. fumigatus in flower bulb-waste and to determine if this material could provide conditions that support sexual recombination.

We provide evidence for an active sexual reproduction of A. fumigatus in natural substrate in the environment, i.e. flower-bulb waste. Our observations are supported by laboratory experiments that confirm that A. fumigatus can form cleistothecia with viable ascospores in flower bulb waste material. A first indication for sexual reproduction of A. fumigatus in the environment was an observation made during investigation of dynamics of A. fumigatus triazole-resistant phenotypes in flower bulb waste heaps with and without triazole fungicide residues. After heat shock of samples obtained from both heaps A. fumigatus colonies were recovered, while we assumed that all Aspergillus conidia would not survive such heat exposure. Furthermore, microsatellite typing of these colonies showed a high genetic diversity, that is not consistent with (clonal) asexual reproduction but could point towards sexual reproduction (Zhang et al., 2017). Although several attempts were made prior to this study to identify and make cleistothecia or ascospores visible in flower bulb waste material, we were unsuccessful due to technical limitations. In this study however, we successfully used an optically flat micro engineered membrane, developed by Innosieve Diagnostics BV^® to detect and selectively capture A. fumigatus ascospores in flower bulb waste material.

Although a 70°C heat shock has been shown to be highly effective to kill Aspergillus conidia, we were concerned that the matrix used, i.e. degrading flower bulb waste material, may reduce killing efficacy and post-heat shock regrowth could be due to surviving conidia rather than ascospores. This is confirmed by the saline only ascospore spiked samples that showed a lower germination rate after 24 hours of heat shock compared to flower bulb waste material spiked samples. The flower bulb waste material therefore does provide a buffering effect to the heat shock on conidia. As Figure 1 bottom graphs of conidia spiked saline or waste material shown, we confirm that a one-hour heat shock is effective to eliminate >99.9% of the conidia, but not all. This is something to consider when experimentally investigating survival of ascospores. Additional experiments showed that ascospore survival occurs even after a heat shock as long as 96 hours. It will be interesting to test whether these results are true for all A. fumigatus crosses or whether the genomic background of the parental isolates can affect these outcomes (Swilaiman et al., 2020). The isolates used in this study, AfIR974 and AfIR964, are natural isolates collected from the environment in Ireland and therefore could mimic a realistic, natural crossing. Another parameter to consider, that was not tested for in this study, is the maturity of the cleistothecia that is known to affect viability of ascospores (Sugui et al., 2011). Ascospore viability can increase with the age of the cleistothecia starting at 17% at four weeks up to 95% at 20 weeks.

Finally, proof of flower bulb waste stockpiles as natural niche for sexual reproduction of A. fumigatus is shown with the results from the Innosieve^® grid-based detection. Where SEM analysis cannot be performed on the dense waste material, a solid phase cytometry sample preparation combined with a fluorescent fungal staining did confirm the presence of ascospore structures in four of eight waste material samples. Even though it was technically not possible to culture and identify the observed ascospores as A. fumigatus, these findings together support the occurrence of sex in A. fumigatus in flower bulb waste. We then tested whether flower bulb waste material can provide the required nutrients and triggers to induce a sexual cycle in A. fumigatus. O’Gorman et al. showed that A. fumigatus is able to undergo sexual reproduction after six months of incubation on parafilm sealed oatmeal agar at 30°C in the dark, while crosses using other media including 2% MEA (Oxoid), Czapek Dox agar and Aspergillus complete medium failed. Since then, many studies replicated these results including our laboratory by specifically using A. fumigatus isolates AfIR974 and AfIR964 from Sugui et al. (2011). Cleistothecia can be visible within four weeks in all cases using oatmeal agar as nutrient source. In this study, we show that a different nutrient source than oatmeal can induce a sexual cycle, namely samples from agricultural flower bulb waste stockpiles. Cleistothecia were visible as quickly as three weeks after inoculation of the mating isolates, showed the typical ascospores upon crushing the fruiting bodies and also germinating after heat shock of 70˚C. In our study only one of seven samples of flower bulb waste material was successful in inducing a sexual crossing. Sexual crossing was most frequent when inoculated in a glass tube (50%), which could be due to three-dimensional access to nutrients, compared to a flat (waste material) agar plate (<5% cleistothecia formed). Additional experiments have been conducted with other flower bulb waste material samples that we collected at a later time point. Again, we were able to induce sexual crosses on some of the flower bulb waste material samples but not all, and therefore it seems that due to the heterogenic composition of flower bulb waste material samples batch-to-batch differences are present (data not shown).

So why are flower bulb waste stockpiles beneficial for A. fumigatus to undergo sexual reproduction? The possible explanations are that (1) flower bulb waste provides favorable conditions for sex; several key factors for sexual reproduction under laboratory conditions that have been identified seem to be present in flower bulb waste. Stockpiles of flower bulb waste represent a multi-organic resource with inside the heap a warm, dark, low oxygen/high CO₂ environment. A. fumigatus isolates show a 50/50 balancing selection of the mating type genes (Paoletti et al., 2005; Swilaiman et al., 2020), which makes the chance of finding an opposing mating type in flower bulb waste stockpiles very likely. In addition, it is believed that environmental changes affect the switching between asexual and sexual reproduction. It has been argued that in many fungi, sex occurs at the end of the growing season when the conditions for somatic growth become adverse and at this moment the cost of sex is low (Chamberlain et al., 1997; Aanen and Hoekstra, 2007). Hence, a dynamic composting process with temperature gradients (20-70°C) and gas changes might provide suitable environments to undergo sex. (2) Sex could provide benefits for the survival of A. fumigatus in the flower bulb waste stockpiles. During the sexual cycle heat-resistant thick walled ascospores are produced that can cope with unfavorable conditions. Many planktonic organisms produce ‘resting’ stages by sexual reproduction when the environmental conditions deteriorate, such as Daphnia (Alekseev and Lampert, 2001) and in plants, seeds can survive unfavorable conditions as well (Maynard-Smith, 1978). In fungi, the outcome of sexual reproduction is most often resting spores that are used to survive in extreme conditions and to spread. For example, the dormant ascospores of Neurospora are activated by fire, and then start to germinate (Henney and Storck, 1964; Macfarlane, 1970). All resting spores are thickly encysted in order to survive through stressful times. In A. fumigatus, the sexual ascospores have a thick cell wall and the capacity to survive adverse conditions, while asexual spores are destined to germinate quickly or to act as fertilizing agents (spermatia) (Chamberlain et al., 1997; Aanen and Hoekstra, 2007; Kwon-Chung and Sugui, 2013). Related to this ecological specialization is the tendency of sexual reproduction to be induced when the environments are harsh. In response to the dynamic but harsh composting environment, A. fumigatus could increase its survival rate via sexual reproduction. Sexual reproduction also generates genetically variable genotypes via recombination between parental alleles. Sex can speed up adaptation and eliminate deleterious mutations much more efficiently compared with asexual growth (Bell, 1982; Camps et al., 2012; Losada et al., 2015). Therefore, highly diverse ascospores enhance the survival rate of a fungus and can maximize its success in changing environments. A changing environment can for example be a triazole containing waste heap, where triazole containing flower bulb waste is regularly added. Via sexual recombination, the resistant allele can be re-shuffled and may generate new variable resistance genotypes that may be better adapted to the changing triazole environment. Our previous work showed experimental crosses of two resistant TR46 isolates producing progeny harboring the triple TR46, which conferred an increased triazole resistance phenotype compared with the ancestors (Zhang et al., 2017). Even though we did not show the azole resistant haplotypes arise as a consequence of sex in this flower bulb waste, in any environment with triazole pressure and an active sexual cycle favorable new offspring genotypes can be selected.

In this study, we provide evidence that flower bulb waste is a natural niche for A. fumigatus to undergo a sexual cycle, which to date had only been observed under laboratory conditions. Our observation was supported by successful crosses using flower bulb waste samples as a nutrient under laboratory conditions. Recombination of A. fumigatus as observed in the population structure can now be explained by active sexual recombination in nature. Furthermore, our observations may provide an explanation for the development of TR-based triazole resistance mutations in the environment.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding author.

PV, JZ, AD, and ES initiated the project. PV, AD, and ES designed the methodology; JZ, AR, and ES carried out experiments and formal analyses. JZ and ES prepared the manuscript. PV, JZ, AD, and ES contributed to the commenting and editing of subsequent versions of the manuscript. All authors contributed to the article and approved the submitted version.

This publication is partly financed by the project ‘One health consequences of circularity’ with project number GROEN.2019.002 of the research program Green III which is (partly) financed by the Dutch Research Council (NWO).

PV reports grants from Gilead Sciences, Thermofisher, Mundipharma, Pfizer and F2G, and nonfinancial support from OLM and IMMY, outside the submitted work.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The handling editor declared a past collaboration with one of the authors (PEV).

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