Inhibition of Aflatoxin Formation in Aspergillus Species by Peanut (Arachis hypogaea) Seed Stilbenoids in the Course of Peanut− Fungus Interaction

Common soil fungi, Aspergillus flavus and Aspergillus parasiticus, are opportunistic pathogens that invade preharvest peanut seeds. These fungi often produce carcinogenic aflatoxins that pose a threat to human and animal health through food chains and cause significant economic losses worldwide. Detection of aflatoxins and further processing of crops are mandated to ensure that contaminated agricultural products do not enter food channels. Under favorable conditions, the fungus-challenged peanut seeds produce phytoalexins, structurally related stilbenoids, capable of retarding fungal development. The purpose of the present study was to evaluate the potential influence of peanut phytoalexins on fungal development and aflatoxin formation in the course of peanut−fungus interaction. The present research revealed that during such interaction, aflatoxin formation was completely suppressed in A. flavus and A. parasiticus strains tested, when low concentrations of spores were introduced to wounded preincubated peanuts. In most of the experiments, when fungal spore concentrations were 2 orders of magnitude higher, the spores germinated and produced aflatoxins. Of all experimental seeds that showed fungal growth, 57.7% were aflatoxin-free after 72 h of incubation. The research provided new knowledge on the aflatoxin/phytoalexin formation in the course of peanut−fungus interaction

UPIC: Perl scripts to determine the number of SSR markers to run

We introduce here the concept of Unique Pattern Informative Combinations (UPIC), a decision tool for the cost-effective design of DNA fingerprinting/genotyping experiments using simple-sequence/tandem repeat (SSR/STR) markers. After the first screening of SSR-markers tested on a subset of DNA samples, the user can apply UPIC to find marker combinations that maximize the genetic information obtained by a minimum or desirable number of markers. This allows a cost-effective planning of future experiments. We have developed Perl scripts to calculate all possible subset combinations of SSR markers, and determine based on unique patterns or alleles, which combinations can discriminate among all DNA samples included in a test. This makes UPIC an essential tool for optimizing resources when working with microsatellites. An example using real data from eight markers and 12 genotypes shows that UPIC detected groups of as few as three markers sufficient to discriminate all 12- DNA samples. Should markers for future experiments be chosen based only on polymorphism-information content (PIC), the necessary number of markers for discrimination of all samples cannot be determined. We also show that choosing markers using UPIC, an informative combination of four markers can provide similar information as using a combination of six markers (23 vs. 25 patterns, respectively), granting a more efficient planning of experiments. Perl scripts with documentation are also included to calculate the percentage of heterozygous loci on the DNA samples tested and to calculate three PIC values depending on the type of fertilization and allele frequency of the organism

Microsatellite markers in Spanish lime (Melicoccus bijugatus Jacq., Sapindaceae), a neglected Neotropical fruit crop

Spanish lime (Melicoccus bijugatus Jacq.) is aNeotropical fruit tree cultivated, mainly, in orchards for self-consumption or local sale. The genus Melicoccus includes other nine species with edible fruits, some of these species are at risk of extinction. Like for the vast majority of tropical fruit trees, there is no information on the genetic diversity of Spanish lime and its related species, and this is mostly due to the lack of molecular markers. The objectives of this study were to present the first microsatellite markers developed for Spanish lime, testing its usefulness on a sample of cultivated accessions, as well as its transferability to Huaya India (M. oliviformis). To do this, we performed high-throughput sequencing of microsatellite-enriched libraries of Spanish lime using Roche 454, assembled 9567 DNA contig sequences and identified 10,117 microsatellites. After screening 384 of those microsatellites on four DNA samples, 31 polymorphic markers were used to screen 25 accessions of Spanish lime and five of Huaya India collected in Yucatan, Mexico. Genetic diversity was low in Spanish lime (A = 20.61, HE = 0.38) and similar for both sexes of this species. Neighbor-Joining and PCoA analyses clearly discriminated between the two Melicoccus species studied. Nine of the markers showed unique alleles for Huaya India. The set of microsatellite markers developed has a great potential to generate information in relation to conservation genetics, improvement of elite cultivars and breeding programs for Spanish lime and related species

Structure and genetic diversity in wild and cultivated populations of Zapote mamey (Pouteria sapota, Sapotaceae) from southeastern Mexico: its putative domestication center

Tropical fruit trees are an important component of the human diet; however, little is known about their genetic diversity levels. Zapote mamey (Pouteria sapota) is a tree native to southeastern Mexico and Central America, and Mexico is the leading producer in the world. Studies of the genetic diversity of Zapote mamey have been based on cultivated materials using morphological and biochemical characterization or dominant molecular markers. To gain a deeper understanding about the conservation status of Zapote mamey in its center of origin and domestication, we collected 188 individuals from eight wild and five cultivated populations in southeastern Mexico and characterized them using eight microsatellite loci. STRUCTURE, 3D-PCoA, and neighbor-joining analyses showed three groups in the wild gene pool and one group in the cultivated gene pool. FST values were significant between wild and cultivated gene pools, among the four groups observed and among the 13 populations collected (0.13, 0.25, and 0.36, respectively). Overall, we found low levels of genetic diversity (A = 2.77, HO = 0.29, HE = 0.39), permutation tests did not show significant differences between wild and cultivated gene pools. The Garza–Williamson index showed low values in both gene pools (wild = 0.16, cultivated = 0.11) and the Bottleneck program indicated a decrease in genetic diversity in both gene pools (wild, P = 0.027; cultivated, P = 0.054); both analyses suggest a potential genetic bottleneck within this species. This study can help to generate adequate sampling techniques and to develop effective management strategies for Zapote mamey of southeastern Mexico

Genome Sequences of Eight \u3ci\u3eAspergillus flavus\u3c/i\u3e spp. and One \u3ci\u3eA. parasiticus\u3c/i\u3e sp., Isolated from Peanut Seeds in Georgia

Aspergillus flavus and A. parasiticus fungi produce carcinogenic mycotoxins in peanut seeds, causing considerable impact on both human health and the economy. Here, we report nine genome sequences of Aspergillus spp., isolated from Georgia peanut seeds in 2014. The information obtained will lead to further biodiversity studies that are essential for developing control strategies

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

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

Evaluation of Leaf Spot Resistance in Wild \u3ci\u3eArachis\u3c/i\u3e Species of Section \u3ci\u3eArachis\u3c/i\u3e

Wild diploid Arachis species are potential sources of resistance to early (ELS) and late (LLS) leaf spot diseases caused by Passalora arachidicola (syn. Cercospora arachidicola Hori), and Nothopassalora personata (syn. Cercosporidium personatum (Berk. & Curt.) Deighton), respectively. Within section Arachis, limited information is available on the extent of genetic variation for resistance to these fungal pathogens. A collection of 78 accessions representing 15 wild species of Arachis section Arachis from the U.S peanut germplasm collection was evaluated for resistance to leaf spots. Screening was conducted under field (natural inoculum) conditions in Dawson, Georgia, during 2017 and 2018. Accessions differed significantly (P , 0.01) for all three disease variables evaluated, which included final defoliation rating, ELS lesion counts, and LLS lesion counts. Relatively high levels of resistance were identified for both diseases, with LLS being the predominant pathogen during the two years of evaluation. This research documents new sources of resistance to leaf spot diseases selected from an environment with high inoculum pressure. The presence of ELS and LLS enabled the selection of resistant germplasm for further introgression and pre-breeding

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