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 sequences that could be targeted by RNA interference to prevent aflatoxin accumulation in peanut seeds

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

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

Draft genome assembly of \u3ci\u3ePassalora sequoiae\u3c/i\u3e a needle blight pathogen on Leyland cypress

Objective: Passalora sequoiae (family Mycosphaerellaceae) causes a twig blight on Leyland cypress that requires numerous fungicide applications annually to minimize economic losses for ornamental plant nursery and Christmas tree producers. The objective was to generate a high-quality draft assembly of the genome of P. sequoiae as a resource for primer development to investigate genotype diversity. Data description: We report here the genome sequence of P. sequoiae 9LC2 that was isolated from Leyland cypress ‘Leighton Green’ in 2017 in southern Mississippi, USA. The draft genome was obtained using Pacific Biosciences (PacBio) SMRT and Illumina HiSeq 2500 sequencing. Illumina reads were mapped to PacBio assembled contigs to determine base call consistency. Based on a total of 44 contigs with 722 kilobase (kb) average length (range 9.4 kb to 3.4 Mb), the whole genome size was estimated at 31,768,716 bp. Mapping of Illumina reads to PacBio contigs resulted in a 1000 × coverage and were used to confirm accuracy of the consensus sequences

Draft Genome Sequence of \u3ci\u3eCercospora arachidicola\u3c/i\u3e, Causal Agent of Early Leaf Spot in Peanuts

Early leaf spot caused by Cercospora arachidicola S. Hori (teleomorph Mycosphaerella arachidis Deighton) is one of two important leaf spot diseases in peanut (Arachis hypogaea L.) responsible for significant economic loss to the industry (1, 2). Infections by C. arachidicola appear as small necrotic lesions on the leaves, petioles, or stems, which may be followed by premature defoliation, and, if left unmanaged on susceptible cultivars, can severely decrease yield (1). An effective, yet expensive, disease management strategy consists of multiple fungicide applications throughout the growing season (3). Other strategies such as strip-tillage instead of conventional tillage (4) or weather forecast models that predict disease outbreaks (5) can help minimize the number of fungicide treatments. However, the development of leaf-spot-resistant cultivars that require no fungicide application would be the most desirable means of control (6)

Development of nuclear microsatellite markers to facilitate germplasm conservation and population genetics studies of five groups of tropical perennial plants with edible fruits and shoots: rambutan (Nephelium lappaceum L.), sapodilla (Manilkara zapota (L.) P. Royen), lychee (Litchi chinensis Sonn.), mangosteen (Garcinia mangostana Linn. and Garcinia cochinchinensis (Lour.) Choisy) and bamboo (Bambusa vulgaris Schrad. ex J.C. Wendl and Guadua angustifolia Kunth)

Simple sequence repeat (SSR) enriched libraries for five groups of tropical perennial plants with edible fruits and shoots were prepared and sequenced in a GS-FLX Roche 454: sapodilla (Manilkara zapota (L.) P. Royen), lychee (Litchi chinensis Sonn.), mangosteen (Garcinia mangostana Linn. and G. cochinchinensis (Lour.) Choisy), rambutan (Nephelium lappaceum L.), and bamboo (Bambusa vulgaris Schrad. ex J.C. Wendl and Guadua angustifolia Kunth). For SSR development, these species were organized by their common names in five groups. A total of 3870 SSR primer sets were designed, using capillary electrophoresis 1872 nuclear SSRs were tested on 4 to 10 DNA samples within each plant group, that is 384 loci for each of the four groups of fruit trees and 336 loci for the bamboo group. Only 7.9% of the primers tested did not result in amplification. All 1872 SSRs are provided, we highlight 178 SSRs (between 26 and 47 per group) considered topquality polymorphic SSRs that amplified all the samples, had strong fluorescence signal, presented no stutters and showed minimum non-specific amplification or background fluorescence. A total of 66,057 contig sequences were submitted to GenBank Database. Markers presented here will be useful not only for conservation efforts in banks of germplasm, but also for in-depth analysis of population genetics which usually requires evaluation of large number of loci

Development of nuclear microsatellite markers to facilitate germplasm conservation and population genetics studies of five groups of tropical perennial plants with edible fruits and shoots: rambutan (Nephelium lappaceum L.), sapodilla (Manilkara zapota (L.) P. Royen), lychee (Litchi chinensis Sonn.), mangosteen (Garcinia mangostana Linn. and Garcinia cochinchinensis (Lour.) Choisy) and bamboo (Bambusa vulgaris Schrad. ex J.C. Wendl and Guadua angustifolia Kunth)

Simple sequence repeat (SSR) enriched libraries for five groups of tropical perennial plants with edible fruits and shoots were prepared and sequenced in a GS-FLX Roche 454: sapodilla (Manilkara zapota (L.) P. Royen), lychee (Litchi chinensis Sonn.), mangosteen (Garcinia mangostana Linn. and G. cochinchinensis (Lour.) Choisy), rambutan (Nephelium lappaceum L.), and bamboo (Bambusa vulgaris Schrad. ex J.C. Wendl and Guadua angustifolia Kunth). For SSR development, these species were organized by their common names in five groups. A total of 3870 SSR primer sets were designed, using capillary electrophoresis 1872 nuclear SSRs were tested on 4 to 10 DNA samples within each plant group, that is 384 loci for each of the four groups of fruit trees and 336 loci for the bamboo group. Only 7.9% of the primers tested did not result in amplifi- cation. All 1872 SSRs are provided, we highlight 178 SSRs (between 26 and 47 per group) considered top-quality polymorphic SSRs that amplified all the samples, had strong fluorescence signal, presented no stutters and showed minimum non-specific amplification or background fluorescence. A total of 66,057 contig sequences were submitted to GenBank Database. Markers presented here will be useful not only for conservation efforts in banks of germplasm, but also for in-depth analysis of population genetics which usually requires evaluation of large number of loci