Aspergillus Section Flavi, Need for a Robust Taxonomy

In a recent letter to the editor, Houbraken et al. (23) provide a series of recommendations to the microbiological community to prevent the taxonomic misidentification of genome-sequenced fungal strains. In the era of genomics and bioinformatics, postulating that 1 nucleotide (nt) within a gene can “correctly” identify a species does not seem plausible. However, the authors of the letter call this the “calmodulin barcode,” meaning nucleotide substitutions within a 506-nt region of the calmodulin gene (1). After the evolutionarily conserved rRNA (18S rRNA, internal transcribed spacer [ITS], 28S rRNA) and RNA polymerase II (2–4) showed no differences between Aspergillus flavus S- and Lmorphotypes, attention shifted toward the calmodulin gene. Thus, without sequencing 18S rRNA, 28S rRNA, or the largest RNA polymerase II subunit, at least 34 new species of Aspergillus were named by Houbraken, Frisvad, Visagie, and coworkers (1, 5, 6). However, in a phylogenetic tree of 152 Aspergillus section Flavi isolates using the calmodulin 506- nt region, 40 Aspergillus minisclerotigenes isolates had only two nucleotide substitutions in common, namely, 100C.A and 269A.G, both of which are silent mutations (Fig. 1). However, only 269A.G discriminates A. minisclerotigenes from A. flavus, since 100C.A is present in three A. flavus isolates (GenBank accession numbers MK451387, MK451365, and MG517986) identified by the authors of the letter. We all agree that species identification is important; paradoxically, the calmodulin barcode assigns species based on a single-nucleotide polymorphism (SNP), while there are between 133,000 and 179,000 SNPs within A. flavus S- and L-morphotypes, respectively (7). Another limitation of Aspergillus taxonomy is the chemotypes resulting from 30 genes in the aflatoxin biosynthesis gene cluster (ABC) (8), e.g., A. flavus produces B-aflatoxins and Aspergillus parasiticus produces B and G types (9). Despite that a single nucleotide change in one ABC gene can prevent aflatoxin production (10), the inheritance of the ABC is favored by environmental pressure (11), and Aspergillus spp. are not physically or reproductively isolated; intraspecies and interspecies crosses can result in gain of function, e.g., G-type aflatoxin production (9, 12, 13). Hence, a new species named by one author of the letter was later reversed to its initial name by the same author because of the chemotype, i.e., A. flavus S-morphotype to Aspergillus parvisclerotigenus (14) and back to A. flavus (6). Other groups utilized the calmodulin gene and a single deletion in the ABC to name three new Aspergillus species (15, 16)

Analysis of small RNA populationsgenerated in peanut leaves after exogenous application of dsRNA and dsDNA targeting aflatoxin synthesis genes

Previously, we have shown that RNA interference (RNAi) can prevent aflatoxin accumulation in transformed peanuts. To explore aflatoxin control by exogenous delivery of double-strand RNA (dsRNA) it is necessary to understand the generation of small RNA (sRNA) populations. We sequenced 12 duplicate sRNA libraries of in-vitro-grown peanut plants, 24 and 48 h after exogenous application of five gene fragments (RNAi-5x) related to aflatoxin biosynthesis in Aspergillus flavus. RNAi-5x was applied either as double-stranded RNA (dsRNA) or RNAi plasmid DNA (dsDNA). Small interfering RNAs (siRNAs) derived from RNAi-5x were significantly more abundant at 48 h than at 24 h, and the majority mapped to the fragment of aflatoxin efflux-pump gene. RNAi-5x-specific siRNAs were significantly, three to fivefold, more abundant in dsDNA than dsRNA treatments. Further examination of known micro RNAs related to disease-resistance, showed significant down-regulation of miR399 and up-regulation of miR482 in leaves treated with dsDNA compared to the control. These results show that sRNA sequencing is useful to compare exogenous RNAi delivery methods on peanut plants, and to analyze the efficacy of molecular constructs to generate siRNAs against specific gene targets. This work lays the foundation for non-transgenic delivery of RNAi in controlling aflatoxins in peanut

Sixteen Draft Genome Sequences Representing the Genetic Diversity of Aspergillus flavus and Aspergillus parasiticus Colonizing Peanut Seeds in Ethiopia

Draft genomes of 16 isolates of Aspergillus flavus Link and Aspergillus parasiticus Speare, identified as the predominant genotypes colonizing peanuts in four farming regions in Ethiopia, are reported. These data will allow mining for se- quences that could be targeted by RNA interference to prevent aflatoxin accumula- tion in peanut seeds

Sixteen Draft Genome Sequences Representing the Genetic Diversity of Aspergillus flavus and Aspergillus parasiticus Colonizing Peanut Seeds in Ethiopia

Draft genomes of 16 isolates of Aspergillus flavus Link and Aspergillus parasiticus Speare, identified as the predominant genotypes colonizing peanuts in four farming regions in Ethiopia, are reported. These data will allow mining for sequences that could be targeted by RNA interference to prevent aflatoxin accumulation in peanut seeds

Suppression of Aflatoxin Production in \u3ci\u3eAspergillus\u3c/i\u3e Species by Selected Peanut (\u3ci\u3eArachis hypogaea\u3c/i\u3e) Stilbenoids

Aspergillus flavus is a soil fungus that commonly invades peanut seeds and often produces carcinogenic aflatoxins. Under favorable conditions, the fungus-challenged peanut plant produces and accumulates resveratrol and its prenylated derivatives in response to such an invasion. These prenylated stilbenoids are considered peanut antifungal phytoalexins. However, the mechanism of peanut−fungus interaction has not been sufficiently studied. We used pure peanut stilbenoids arachidin-1, arachidin-3, and chiricanine A to study their effects on the viability of and metabolite production by several important toxigenic Aspergillus species. Significant reduction or virtually complete suppression of aflatoxin production was revealed in feeding experiments in A. flavus, Aspergillus parasiticus, and Aspergillus nomius. Changes in morphology, spore germination, and growth rate were observed in A. flavus exposed to the selected peanut stilbenoids. Elucidation of the mechanism of aflatoxin suppression by peanut stilbenoids could provide strategies for preventing plant invasion by the fungi that produce aflatoxins

Transformation of Major Peanut (Arachis hypogaea) Stilbenoid Phytoalexins Caused by Selected Microorganisms

The peanut plant accumulates defensive stilbenoid phytoalexins in response to the presence of soil fungi, which in turn produce phytoalexin-detoxifying enzymes for successfully invading the plant host. Aspergillus spp. are opportunistic pathogens that invade peanut seeds; most common fungal species often produce highly carcinogenic aflatoxins. The purpose of the present research was to evaluate the in vitro dynamics of peanut phytoalexin transformation/detoxification by important fungal species. This work revealed that in feeding experiments, Aspergillus spp. from section Flavi were capable of degrading the major peanut phytoalexin, arachidin-3, into its hydroxylated homolog, arachidin-1, and a benzenoid, SB-1. However, Aspergillus niger from section Nigri as well as other fungal and bacterial species tested, which are not known to be involved in the infection of the peanut plant, were incapable of changing the structure of arachidin-3. The results of feeding experiments with arachidin-1 and resveratrol are also reported. The research provided new knowledge on the dynamics of peanut stilbenoid transformations by essential fungi. These findings may contribute to the elucidation of the phytoalexin detoxification mechanism involved in the infection of peanut by important toxigenic Aspergillus spp

Characterization of small RNA populations in non-transgenic andaflatoxin-reducing-transformed peanut

Aflatoxin contamination is a major constraint in food production worldwide. In peanut (Arachis hypogaea L.), these toxic and carcinogenic aflatoxins are mainly produced by Aspergillus flavus Link and A. parasiticus Speare. The use of RNA interference (RNAi) is a promising method to reduce or prevent the accumula-tion of aflatoxin in peanut seed. In this study, we performed high-throughput sequencing of small RNApopulations in a control line and in two transformed peanut lines that expressed an inverted repeattargeting five genes involved in the aflatoxin-biosynthesis pathway and that showed up to 100% less aflatoxin B1 than the controls. The objective was to determine the putative involvement of the smallRNA populations in aflatoxin reduction. In total, 41 known microRNA (miRNA) families and many novel miRNAs were identified. Among those, 89 known and 10 novel miRNAs were differentially expressed in the transformed lines. We furthermore found two small interfering RNAs derived from the inverted repeat, and 39 sRNAs that mapped without mismatches to the genome of A. flavus and were present only in the transformed lines. This information will increase our understanding of the effectiveness of RNAi and enable the possible improvement of the RNAi technology for the control of aflatoxins

First draft genome of \u3ci\u3eThecaphora frezii\u3c/i\u3e, causal agent of peanut smut disease

Objectives: The fungal pathogen Thecaphora frezii Carranza & Lindquist causes peanut smut, a severe disease currently endemic in Argentina. To study the ecology of T. frezii and to understand the mechanisms of smut resistance in peanut plants, it is crucial to know the genetics of this pathogen. The objective of this work was to isolate the pathogen and generate the first draft genome of T. frezii that will be the basis for analyzing its potential genetic diversity and its interaction with peanut cultivars. Our research group is working to identify peanut germplasm with smut resistance and to understand the genetics of the pathogen. Knowing the genome of T. frezii will help analyze potential variants of this pathogen and contribute to develop enhanced peanut germplasm with broader and long-lasting resistance. Data description: Thecaphora frezii isolate IPAVE 0401 (here referred as T.f.B7) was obtained from a single hyphal-tip culture, its DNA was sequenced using Pacific Biosciences Sequel II (PacBio) and Illumina NovaSeq6000 (Nova). Data from both sequencing platforms were combined and the de novo assembling estimated a 29.3 Mb genome size. Completeness of the genome examined using Benchmarking Universal Single-Copy Orthologs (BUSCO) showed the assembly had 84.6% of the 758 genes in fungi_odb10

First draft genome of Thecaphora frezii, causal agent of peanut smut disease

Objectives: The fungal pathogen Thecaphora frezii Carranza & Lindquist causes peanut smut, a severe disease currently endemic in Argentina. To study the ecology of T. frezii and to understand the mechanisms of smut resistance in peanut plants, it is crucial to know the genetics of this pathogen. The objective of this work was to isolate the pathogen and generate the first draft genome of T. frezii that will be the basis for analyzing its potential genetic diversity and its interaction with peanut cultivars. Our research group is working to identify peanut germplasm with smut resistance and to understand the genetics of the pathogen. Knowing the genome of T. frezii will help analyze potential variants of this pathogen and contribute to develop enhanced peanut germplasm with broader and long-lasting resistance. Data description: Thecaphora frezii isolate IPAVE 0401 (here referred as T.f.B7) was obtained from a single hyphal-tip culture, its DNA was sequenced using Pacific Biosciences Sequel II (PacBio) and Illumina NovaSeq6000 (Nova). Data from both sequencing platforms were combined and the de novo assembling estimated a 29.3 Mb genome size. Completeness of the genome examined using Benchmarking Universal Single-Copy Orthologs (BUSCO) showed the assembly had 84.6% of the 758 genes in fungi_odb10.Instituto de Patología VegetalFil: Arias, Renee S. USDA-ARS National Peanut Research Laboratory (NPRL); Estados UnidosFil: Conforto, Erica Cinthia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patología Vegetal; ArgentinaFil: Conforto, Erica Cinthia. Consejo Nacional de Investigaciones Científicas y Técnicas. Unidad de Fitopatología y Modelización Agrícola (UFyMA); ArgentinaFil: Orner, Valerie A. USDA-ARS National Peanut Research Laboratory (NPRL); Estados UnidosFil: Carloni, Edgardo José. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Fisiología y Recursos Genéticos Vegetales; ArgentinaFil: Soave, Juan H. El Carmen S.A.; ArgentinaFil: Massa, Alicia N. USDA-ARS National Peanut Research Laboratory (NPRL); Estados UnidosFil: Lamb, Marshall C. USDA-ARS National Peanut Research Laboratory (NPRL); Estados UnidosFil: Bernanrdi Lima, Nelson. Consejo Nacional de Investigaciones Científicas y Técnicas. Unidad de Fitopatología y Modelización Agrícola (UFyMA); ArgentinaFil: Bernanrdi Lima, Nelson. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patología Vegetal; ArgentinaFil: Rago, Alejandro Mario. Instituto Nacional de Tecnología Agropecuaria (INTA). Centro de Investigaciones Agropecuarias (CIAP); ArgentinaFil: Rago, Alejandro Mario. Consejo Nacional de Investigaciones Científicas y Técnicas. Unidad de Fitopatología y Modelización Agrícola (UFyMA); Argentin

Analysis of small RnA populations generated in peanut leaves after exogenous application of dsRnA and dsDnA targeting aflatoxin synthesis genes

Previously, we have shown that RNA interference (RNAi) can prevent aflatoxin accumulation in transformed peanuts. To explore aflatoxin control by exogenous delivery of double-strand RNA (dsRNA) it is necessary to understand the generation of small RNA (sRNA) populations. We sequenced 12 duplicate sRNA libraries of in-vitro-grown peanut plants, 24 and 48 h after exogenous application of five gene fragments (RNAi-5x) related to aflatoxin biosynthesis in Aspergillus flavus. RNAi-5x was applied either as double-stranded RNA (dsRNA) or RNAi plasmid DNA (dsDNA). Small interfering RNAs (siRNAs) derived from RNAi-5x were significantly more abundant at 48 h than at 24 h, and the majority mapped to the fragment of aflatoxin efflux-pump gene. RNAi-5x-specific siRNAs were significantly, three to fivefold, more abundant in dsDNA than dsRNA treatments. Further examination of known micro RNAs related to disease-resistance, showed significant down-regulation of miR399 and up-regulation of miR482 in leaves treated with dsDNA compared to the control. These results show that sRNA sequencing is useful to compare exogenous RNAi delivery methods on peanut plants, and to analyze the efficacy of molecular constructs to generate siRNAs against specific gene targets. This work lays the foundation for non-transgenic delivery of RNAi in controlling aflatoxins in peanut