@article{molo_white_cornish_gell_baars_singh_carbone_isakeit_wise_woloshuk_et al._2022, title={Asymmetrical lineage introgression and recombination in populations of Aspergillus flavus: Implications for biological control}, volume={17}, ISSN={["1932-6203"]}, url={https://doi.org/10.1371/journal.pone.0276556}, DOI={10.1371/journal.pone.0276556}, abstractNote={Aspergillus flavus is an agriculturally important fungus that causes ear rot of maize and produces aflatoxins, of which B1 is the most carcinogenic naturally-produced compound. In the US, the management of aflatoxins includes the deployment of biological control agents that comprise two nonaflatoxigenic A. flavus strains, either Afla-Guard (member of lineage IB) or AF36 (lineage IC). We used genotyping-by-sequencing to examine the influence of both biocontrol agents on native populations of A. flavus in cornfields in Texas, North Carolina, Arkansas, and Indiana. This study examined up to 27,529 single-nucleotide polymorphisms (SNPs) in a total of 815 A. flavus isolates, and 353 genome-wide haplotypes sampled before biocontrol application, three months after biocontrol application, and up to three years after initial application. Here, we report that the two distinct A. flavus evolutionary lineages IB and IC differ significantly in their frequency distributions across states. We provide evidence of increased unidirectional gene flow from lineage IB into IC, inferred to be due to the applied Afla-Guard biocontrol strain. Genetic exchange and recombination of biocontrol strains with native strains was detected in as little as three months after biocontrol application and up to one and three years later. There was limited inter-lineage migration in the untreated fields. These findings suggest that biocontrol products that include strains from lineage IB offer the greatest potential for sustained reductions in aflatoxin levels over several years. This knowledge has important implications for developing new biocontrol strategies.}, number={10}, journal={PLOS ONE}, author={Molo, Megan S. and White, James B. and Cornish, Vicki and Gell, Richard M. and Baars, Oliver and Singh, Rakhi and Carbone, Mary Anna and Isakeit, Thomas and Wise, Kiersten A. and Woloshuk, Charles P. and et al.}, editor={Nierman, William C.Editor}, year={2022}, month={Oct} }
 @article{moore_olarte_horn_elliott_singh_o'neal_carbone_2017, title={Global population structure and adaptive evolution of aflatoxin-producing fungi}, volume={7}, ISSN={2045-7758}, url={http://dx.doi.org/10.1002/ece3.3464}, DOI={10.1002/ece3.3464}, abstractNote={Aflatoxins produced by several species in Aspergillus section Flavi are a significant problem in agriculture and a continuous threat to human health. To provide insights into the biology and global population structure of species in section Flavi, a total of 1,304 isolates were sampled across six species (A. flavus, A. parasiticus, A. nomius, A. caelatus, A. tamarii, and A. alliaceus) from single fields in major peanut-growing regions in Georgia (USA), Australia, Argentina, India, and Benin (Africa). We inferred maximum-likelihood phylogenies for six loci, both combined and separately, including two aflatoxin cluster regions (aflM/alfN and aflW/aflX) and four noncluster regions (amdS, trpC, mfs and MAT), to examine population structure and history. We also employed principal component and STRUCTURE analysis to identify genetic clusters and their associations with six different categories (geography, species, precipitation, temperature, aflatoxin chemotype profile, and mating type). Overall, seven distinct genetic clusters were inferred, some of which were more strongly structured by G chemotype diversity than geography. Populations of A. flavus S in Benin were genetically distinct from all other section Flavi species for the loci examined, which suggests genetic isolation. Evidence of trans-speciation within two noncluster regions, whereby A. flavus SBG strains from Australia share haplotypes with either A. flavus or A. parasiticus, was observed. Finally, while clay soil and precipitation may influence species richness in Aspergillus section Flavi, other region-specific environmental and genetic parameters must also be considered.}, number={21}, journal={Ecology and Evolution}, publisher={Wiley}, author={Moore, Geromy G. and Olarte, Rodrigo A. and Horn, Bruce W. and Elliott, Jacalyn L. and Singh, Rakhi and O'Neal, Carolyn J. and Carbone, Ignazio}, year={2017}, month={Sep}, pages={9179–9191} }
 @article{horn_gell_singh_sorensen_carbone_2016, title={Sexual Reproduction in Aspergillus flavus Sclerotia: Acquisition of Novel Alleles from Soil Populations and Uniparental Mitochondrial Inheritance}, volume={11}, ISSN={["1932-6203"]}, DOI={10.1371/journal.pone.0146169}, abstractNote={Aspergillus flavus colonizes agricultural commodities worldwide and contaminates them with carcinogenic aflatoxins. The high genetic diversity of A. flavus populations is largely due to sexual reproduction characterized by the formation of ascospore-bearing ascocarps embedded within sclerotia. A. flavus is heterothallic and laboratory crosses between strains of the opposite mating type produce progeny showing genetic recombination. Sclerotia formed in crops are dispersed onto the soil surface at harvest and are predominantly produced by single strains of one mating type. Less commonly, sclerotia may be fertilized during co-infection of crops with sexually compatible strains. In this study, laboratory and field experiments were performed to examine sexual reproduction in single-strain and fertilized sclerotia following exposure of sclerotia to natural fungal populations in soil. Female and male roles and mitochondrial inheritance in A. flavus were also examined through reciprocal crosses between sclerotia and conidia. Single-strain sclerotia produced ascospores on soil and progeny showed biparental inheritance that included novel alleles originating from fertilization by native soil strains. Sclerotia fertilized in the laboratory and applied to soil before ascocarp formation also produced ascospores with evidence of recombination in progeny, but only known parental alleles were detected. In reciprocal crosses, sclerotia and conidia from both strains functioned as female and male, respectively, indicating A. flavus is hermaphroditic, although the degree of fertility depended upon the parental sources of sclerotia and conidia. All progeny showed maternal inheritance of mitochondria from the sclerotia. Compared to A. flavus populations in crops, soil populations would provide a higher likelihood of exposure of sclerotia to sexually compatible strains and a more diverse source of genetic material for outcrossing.}, number={1}, journal={PLOS ONE}, author={Horn, Bruce W. and Gell, Richard M. and Singh, Rakhi and Sorensen, Ronald B. and Carbone, Ignazio}, year={2016}, month={Jan} }
 @article{olarte_horn_singh_carbone_2015, title={Sexual recombination in Aspergillus tubingensis}, volume={107}, ISSN={["1557-2536"]}, DOI={10.3852/14-233}, abstractNote={Aspergillus tubingensis from section Nigri (black Aspergilli) is closely related to A. niger and is used extensively in the industrial production of enzymes and organic acids. We recently discovered sexual reproduction in A. tubingensis, and in this study we demonstrate that the progeny are products of meiosis. Progeny were obtained from six crosses involving five MAT1-1 strains and two MAT1-2 strains. We examined three loci, including mating type (MAT), RNA polymerase II (RPB2) and β-tubulin (BT2), and found that 84% (58/69) of progeny were recombinants. Recombination associated with sexual reproduction in A. tubingensis provides a new option for the genetic improvement of industrial strains for enzyme and organic acid production.}, number={2}, journal={MYCOLOGIA}, author={Olarte, Rodrigo A. and Horn, Bruce W. and Singh, Rakhi and Carbone, Ignazio}, year={2015}, pages={307–312} }
 @article{moore_elliott_singh_horn_dorner_stone_chulze_barros_naik_wright_et al._2013, title={Sexuality Generates Diversity in the Aflatoxin Gene Cluster: Evidence on a Global Scale}, volume={9}, ISSN={1553-7374}, url={http://dx.doi.org/10.1371/journal.ppat.1003574}, DOI={10.1371/journal.ppat.1003574}, abstractNote={Aflatoxins are produced by Aspergillus flavus and A. parasiticus in oil-rich seed and grain crops and are a serious problem in agriculture, with aflatoxin B₁ being the most carcinogenic natural compound known. Sexual reproduction in these species occurs between individuals belonging to different vegetative compatibility groups (VCGs). We examined natural genetic variation in 758 isolates of A. flavus, A. parasiticus and A. minisclerotigenes sampled from single peanut fields in the United States (Georgia), Africa (Benin), Argentina (Córdoba), Australia (Queensland) and India (Karnataka). Analysis of DNA sequence variation across multiple intergenic regions in the aflatoxin gene clusters of A. flavus, A. parasiticus and A. minisclerotigenes revealed significant linkage disequilibrium (LD) organized into distinct blocks that are conserved across different localities, suggesting that genetic recombination is nonrandom and a global occurrence. To assess the contributions of asexual and sexual reproduction to fixation and maintenance of toxin chemotype diversity in populations from each locality/species, we tested the null hypothesis of an equal number of MAT1-1 and MAT1-2 mating-type individuals, which is indicative of a sexually recombining population. All samples were clone-corrected using multi-locus sequence typing which associates closely with VCG. For both A. flavus and A. parasiticus, when the proportions of MAT1-1 and MAT1-2 were significantly different, there was more extensive LD in the aflatoxin cluster and populations were fixed for specific toxin chemotype classes, either the non-aflatoxigenic class in A. flavus or the B₁-dominant and G₁-dominant classes in A. parasiticus. A mating type ratio close to 1∶1 in A. flavus, A. parasiticus and A. minisclerotigenes was associated with higher recombination rates in the aflatoxin cluster and less pronounced chemotype differences in populations. This work shows that the reproductive nature of the population (more sexual versus more asexual) is predictive of aflatoxin chemotype diversity in these agriculturally important fungi.}, number={8}, journal={PLoS Pathogens}, publisher={Public Library of Science (PLoS)}, author={Moore, Geromy G. and Elliott, Jacalyn L. and Singh, Rakhi and Horn, Bruce W. and Dorner, Joe W. and Stone, Eric A. and Chulze, Sofia N. and Barros, German G. and Naik, Manjunath K. and Wright, Graeme C. and et al.}, editor={McDonald, Bruce A.Editor}, year={2013}, month={Aug}, pages={e1003574} }
 @article{olarte_horn_dorner_monacell_singh_stone_carbone_2012, title={Effect of sexual recombination on population diversity in aflatoxin production by Aspergillus flavus and evidence for cryptic heterokaryosis}, volume={21}, ISSN={["1365-294X"]}, DOI={10.1111/j.1365-294x.2011.05398.x}, abstractNote={Aspergillus flavus is the major producer of carcinogenic aflatoxins (AFs) in crops worldwide. Natural populations of A. flavus show tremendous variation in AF production, some of which can be attributed to environmental conditions, differential regulation of the AF biosynthetic pathway and deletions or loss-of-function mutations in the AF gene cluster. Understanding the evolutionary processes that generate genetic diversity in A. flavus may also explain quantitative differences in aflatoxigenicity. Several population studies using multilocus genealogical approaches provide indirect evidence of recombination in the genome and specifically in the AF gene cluster. More recently, A. flavus has been shown to be functionally heterothallic and capable of sexual reproduction in laboratory crosses. In the present study, we characterize the progeny from nine A. flavus crosses using toxin phenotype assays, DNA sequence-based markers and array comparative genome hybridization. We show high AF heritability linked to genetic variation in the AF gene cluster, as well as recombination through the independent assortment of chromosomes and through crossing over within the AF cluster that coincides with inferred recombination blocks and hotspots in natural populations. Moreover, the vertical transmission of cryptic alleles indicates that while an A. flavus deletion strain is predominantly homokaryotic, it may harbour AF cluster genes at a low copy number. Results from experimental matings indicate that sexual recombination is driving genetic and functional hyperdiversity in A. flavus. The results of this study have significant implications for managing AF contamination of crops and for improving biocontrol strategies using nonaflatoxigenic strains of A. flavus.}, number={6}, journal={MOLECULAR ECOLOGY}, author={Olarte, Rodrigo A. and Horn, Bruce W. and Dorner, Joe W. and Monacell, James T. and Singh, Rakhi and Stone, Eric A. and Carbone, Ignazio}, year={2012}, month={Mar}, pages={1453–1476} }
 @article{moore_singh_horn_carbone_2009, title={Recombination and lineage-specific gene loss in the aflatoxin gene cluster of Aspergillus flavus}, volume={18}, ISSN={["1365-294X"]}, DOI={10.1111/j.1365-294X.2009.04414.x}, abstractNote={Aflatoxins produced by Aspergillus flavus are potent carcinogens that contaminate agricultural crops. Recent efforts to reduce aflatoxin concentrations in crops have focused on biological control using nonaflatoxigenic A. flavus strains AF36 (=NRRL 18543) and NRRL 21882 (the active component of afla-guard®). However, the evolutionary potential of these strains to remain nonaflatoxigenic in nature is unknown. To elucidate the underlying population processes that influence aflatoxigenicity, we examined patterns of linkage disequilibrium (LD) spanning 21 regions in the aflatoxin gene cluster of A. flavus. We show that recombination events are unevenly distributed across the cluster in A. flavus. Six distinct LD blocks separate late pathway genes aflE, aflM, aflN, aflG, aflL, aflI and aflO, and there is no discernable evidence of recombination among early pathway genes aflA, aflB, aflC, aflD, aflR and aflS. The discordance in phylogenies inferred for the aflW/aflX intergenic region and two noncluster regions, tryptophan synthase and acetamidase, is indicative of trans-species evolution in the cluster. Additionally, polymorphisms in aflW/aflX divide A. flavus strains into two distinct clades, each harbouring only one of the two approved biocontrol strains. The clade with AF36 includes both aflatoxigenic and nonaflatoxigenic strains, whereas the clade with NRRL 21882 comprises only nonaflatoxigenic strains and includes all strains of A. flavus missing the entire gene cluster or with partial gene clusters. Our detection of LD blocks in partial clusters indicates that recombination may have played an important role in cluster disassembly, and multilocus coalescent analyses of cluster and noncluster regions indicate lineage-specific gene loss in A. flavus. These results have important implications in assessing the stability of biocontrol strains in nature.}, number={23}, journal={MOLECULAR ECOLOGY}, author={Moore, Geromy G. and Singh, Rakhi and Horn, Bruce W. and Carbone, Ignazio}, year={2009}, month={Dec}, pages={4870–4887} }