Aneuploidy Formation in the Filamentous Fungus Aspergillus flavus in Response to Azole Stress.

Aspergillus flavus is a mycotoxigenic fungus that contaminates many important agricultural crops with aflatoxin B1, the most toxic and carcinogenic natural compound. This fungus is also the second leading cause of human invasive aspergillosis, after Aspergillus fumigatus, a disease that is particularly prevalent in immunocompromised individuals. Azole drugs are considered the most effective compounds in controlling Aspergillus infections both in clinical and agricultural settings. Emergence of azole resistance in Aspergillus spp. is typically associated with point mutations in cyp51 orthologs that encode lanosterol 14α-demethylase, a component of the ergosterol biosynthesis pathway that is also the target of azoles. We hypothesized that alternative molecular mechanisms are also responsible for acquisition of azole resistance in filamentous fungi. We found that an aflatoxin-producing A. flavus strain adapted to voriconazole exposure at levels above the MIC through whole or segmental aneuploidy of specific chromosomes. We confirm a complete duplication of chromosome 8 in two sequentially isolated clones and a segmental duplication of chromosome 3 in another clone, emphasizing the potential diversity of aneuploidy-mediated resistance mechanisms. The plasticity of aneuploidy-mediated resistance was evidenced by the ability of voriconazole-resistant clones to revert to their original level of azole susceptibility following repeated transfers on drug-free media. This study provides new insights into mechanisms of azole resistance in a filamentous fungus. IMPORTANCE Fungal pathogens cause human disease and threaten global food security by contaminating crops with toxins (mycotoxins). Aspergillus flavus is an opportunistic mycotoxigenic fungus that causes invasive and noninvasive aspergillosis, diseases with high rates of mortality in immunocompromised individuals. Additionally, this fungus contaminates most major crops with the notorious carcinogen, aflatoxin. Voriconazole is the drug of choice to treat infections caused by Aspergillus spp. Although azole resistance mechanisms have been well characterized in clinical isolates of Aspergillus fumigatus, the molecular basis of azole resistance in A. flavus remains unclear. Whole-genome sequencing of eight voriconazole-resistant isolates revealed that, among other factors, A. flavus adapts to high concentrations of voriconazole by duplication of specific chromosomes (i.e., aneuploidy). Our discovery of aneuploidy-mediated resistance in a filamentous fungus represents a paradigm shift, as this type of resistance was previously thought to occur only in yeasts. This observation provides the first experimental evidence of aneuploidy-mediated azole resistance in the filamentous fungus A. flavus.

Why Do Plant-Pathogenic Fungi Produce Mycotoxins? Potential Roles for Mycotoxins in the Plant Ecosystem.

For many plant-pathogenic or endophytic fungi, production of mycotoxins, which are toxic to humans, may present a fitness gain. However, associations between mycotoxin production and plant pathogenicity or virulence is inconsistent and difficult due to the complexity of these host-pathogen interactions and the influences of environmental and insect factors. Aflatoxin receives a lot of attention due to its potent toxicity and carcinogenicity but the connection between aflatoxin production and pathogenicity is complicated by the pathogenic ability and prevalence of nonaflatoxigenic isolates in crops. Other toxins directly aid fungi in planta, trichothecenes are important virulence factors, and ergot alkaloids limit herbivory and fungal consumption due to insect toxicity. We review a panel discussion at the American Phytopathological Society's Plant Health 2021 conference, which gathered diverse experts representing different research sectors, career stages, ethnicities, and genders to discuss the diverse roles of mycotoxins in the lifestyles of filamentous fungi of the families Clavicipitaceae, Trichocomaceae (Eurotiales), and Nectriaceae (Hypocreales).

The Frequency of Sex: Population Genomics Reveals Differences in Recombination and Population Structure of the Aflatoxin-Producing Fungus Aspergillus flavus.

The apparent rarity of sex in many fungal species has raised questions about how much sex is needed to purge deleterious mutations and how differences in frequency of sex impact fungal evolution. We sought to determine how differences in the extent of recombination between populations of Aspergillus flavus impact the evolution of genes associated with the synthesis of aflatoxin, a notoriously potent carcinogen. We sequenced the genomes of, and quantified aflatoxin production in, 94 isolates of A. flavus sampled from seven states in eastern and central latitudinal transects of the United States. The overall population is subdivided into three genetically differentiated populations (A, B, and C) that differ greatly in their extent of recombination, diversity, and aflatoxin-producing ability. Estimates of the number of recombination events and linkage disequilibrium decay suggest relatively frequent sex only in population A. Population B is sympatric with population A but produces significantly less aflatoxin and is the only population where the inability of nonaflatoxigenic isolates to produce aflatoxin was explained by multiple gene deletions. Population expansion evident in population B suggests a recent introduction or range expansion. Population C is largely nonaflatoxigenic and restricted mainly to northern sampling locations through restricted migration and/or selection. Despite differences in the number and type of mutations in the aflatoxin gene cluster, codon optimization and site frequency differences in synonymous and nonsynonymous mutations suggest that low levels of recombination in some A. flavus populations are sufficient to purge deleterious mutations.IMPORTANCE Differences in the relative frequencies of sexual and asexual reproduction have profound implications for the accumulation of deleterious mutations (Muller's ratchet), but little is known about how these differences impact the evolution of ecologically important phenotypes. Aspergillus flavus is the main producer of aflatoxin, a notoriously potent carcinogen that often contaminates food. We investigated if differences in the levels of production of aflatoxin by A. flavus could be explained by the accumulation of deleterious mutations due to a lack of recombination. Despite differences in the extent of recombination, variation in aflatoxin production is better explained by the demography and history of specific populations and may suggest important differences in the ecological roles of aflatoxin among populations. Furthermore, the association of aflatoxin production and populations provides a means of predicting the risk of aflatoxin contamination by determining the frequencies of isolates from low- and high-production populations.

Fitness Cost of Aflatoxin Production in Aspergillus flavus When Competing with Soil Microbes Could Maintain Balancing Selection.

Selective forces that maintain the polymorphism for aflatoxigenic and nonaflatoxigenic individuals of Aspergillus flavus are largely unknown. As soils are widely considered the natural habitat of A. flavus, we hypothesized that aflatoxin production would confer a fitness advantage in the soil environment. To test this hypothesis, we used A. flavus DNA quantified by quantitative PCR (qPCR) as a proxy for fitness of aflatoxigenic and nonaflatoxigenic field isolates grown in soil microcosms. Contrary to predictions, aflatoxigenic isolates had significantly lower fitness than did nonaflatoxigenic isolates in natural soils across three temperatures (25, 37, and 42°C). The addition of aflatoxin to soils (500 ng/g) had no effect on the growth of A. flavus Amplicon sequencing showed that neither the aflatoxin-producing ability of the fungus nor the addition of aflatoxin had a significant effect on the composition of fungal or bacterial communities in soil. We argue that the fitness disadvantage of aflatoxigenic isolates is most likely explained by the metabolic cost of producing aflatoxin. Coupled with a previous report of a selective advantage of aflatoxin production in the presence of some insects, our findings give an ecological explanation for balancing selection resulting in persistent polymorphisms in aflatoxin production.IMPORTANCE Aflatoxin, produced by the fungus Aspergillus flavus, is an extremely potent hepatotoxin that causes acute toxicosis and cancer, and it incurs hundreds of millions of dollars annually in agricultural losses. Despite the importance of this toxin to humans, it has remained unclear what the fungus gains by producing aflatoxin. In fact, not all strains of A. flavus produce aflatoxin. Previous work has shown an advantage to producing aflatoxin in the presence of some insects. Our current work demonstrates the first evidence of a disadvantage to A. flavus in producing aflatoxin when competing with soil microbes. Together, these opposing evolutionary forces could explain the persistence of both aflatoxigenic and nonaflatoxigenic strains through evolutionary time.

Population Subdivision and the Frequency of Aflatoxigenic Isolates in Aspergillus flavus in the United States.

Consumption of food contaminated with aflatoxin, from crops infected by Aspergillus flavus, is associated with acute toxicosis, cancer, and stunted growth. Although such contamination is more common in the lower latitudes of the United States, it is unclear whether this pattern is associated with differences in the relative frequencies of aflatoxigenic individuals of A. flavus. To determine whether the frequency of the aflatoxin-producing ability of A. flavus increases as latitude decreases, we sampled 281 isolates from field soils in two north-south transects in the United States and tested them for aflatoxin production. We also genotyped 161 isolates using 10 microsatellite markers to assess population structure. Although the population density of A. flavus was highest at lower latitudes, there was no difference in the frequency of aflatoxigenic A. flavus isolates in relation to latitude. We found that the U.S. population of A. flavus is subdivided into two genetically differentiated subpopulations that are not associated with the chemotype or geographic origin of the isolates. The two populations differ markedly in allelic and genotypic diversity. The less diverse population is more abundant and may represent a clonal lineage derived from the more diverse population. Overall, increased aflatoxin contamination in lower latitudes may be explained partially by differences in the population density of A. flavus, not genetic population structure.

Patterns, causes and consequences of defensive microbiome dynamics across multiple scales.

The microbiome can significantly impact host phenotypes and serve as an additional source of heritable genetic variation. While patterns across eukaryotes are consistent with a role for symbiotic microbes in host macroevolution, few studies have examined symbiont-driven host evolution or the ecological implications of a dynamic microbiome across temporal, spatial or ecological scales. The pea aphid, Acyrthosiphon pisum, and its eight heritable bacterial endosymbionts have served as a model for studies on symbiosis and its potential contributions to host ecology and evolution. But we know little about the natural dynamics or ecological impacts of the heritable microbiome of this cosmopolitan insect pest. Here we report seasonal shifts in the frequencies of heritable defensive bacteria from natural pea aphid populations across two host races and geographic regions. Microbiome dynamics were consistent with symbiont responses to host-level selection and findings from one population suggested symbiont-driven adaptation to seasonally changing parasitoid pressures. Conversely, symbiont levels were negatively correlated with enemy-driven mortality when measured across host races, suggesting important ecological impacts of host race microbiome divergence. Rapid drops in symbiont frequencies following seasonal peaks suggest microbiome instability in several populations, with potentially large costs of 'superinfection' under certain environmental conditions. In summary, the realization of several laboratory-derived, a priori expectations suggests important natural impacts of defensive symbionts in host-enemy eco-evolutionary feedbacks. Yet negative findings and unanticipated correlations suggest complexities within this system may limit or obscure symbiont-driven contemporary evolution, a finding of broad significance given the widespread nature of defensive microbes across plants and animals.