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

Examination of Selenium Incorporation and Product Formation in the Nitrogenase FeMo-Cofactor

Nitrogenase is the only known enzyme to convert the triply bonded atmospheric dinitrogen (N2) to bioavailable ammonia (NH3) in an ambient environment, breaking one of the strongest chemical bond in nature in the process. Industrially, the Haber-Bosch process is also capable of reducing dinitrogen to ammonia, and is essential for worldwide food production 1,2. Due to the high temperatures and pressures required for the Haber-Bosch process (between 300-550ºC and 15-25 MPa) and its requirement for molecular hydrogen, it has become paramount to scientifically investigate the biological processes of nitrogen fixation to ultimately develop more efficient methods to produce bioavailable ammonia. Nitrogenase utilizes two component proteins, the Fe-protein and the MoFe-protein, to reduce ammonia in an ATP-hydrolysis dependent and electron-intensive reaction. Besides the canonical dinitrogen reduction reaction, nitrogenase can reduce a variety of other substrates including: acetylene (C2H2), carbon dioxide (CO2), carbon monoxide (CO), carbonyl sulfide (COS), nitrous oxide (N2O), diazene (N2H2), and more 3-11. CO has long been of interest to the study of the mechanism of nitrogenase, owing to its isoelectronic identity to N2, and its potent inhibitor properties at well as its ability to serve as a weak substrate 12,13. Like CO, cyanide compounds (X-CN) are also of interest to the study of nitrogenase due to the isoelectronic nature of CN- to N2. However, cyanide compounds serve as particularly interesting spectroscopic and crystallographic tools, because X in X-CN can be substituted for more significant sulfur or selenium (Se). In this study, we investigate the substrate properties of SeCN-, with Se-incorporation into the active site FeMo-cofactor and concurrent reduction of SeCN- to methane (CH4). This study serves as yet another link between substrate reduction in nitrogenase. Part of this work describes the incorporation of Se into the cofactor as a vehicle for high-resolution study of nitrogenase under turnover using spectroscopy and crystallography, while another part describes a proposal for future work on the trapping of enzyme intermediates by fast-growing crystallography.</p

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

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

The “speed limit” for macromolecular crystal growth

A simple “diffusion‐to‐capture” model is used to estimate the upper limit to the growth rate of macromolecular crystals under conditions when the rate limiting process is the mass transfer of sample from solution to the crystal. Under diffusion‐limited crystal growth conditions, this model predicts that the cross‐sectional area of a crystal will increase linearly with time; this prediction is validated by monitoring the growth rate of lysozyme crystals. A consequence of this analysis is that when crystal growth is diffusion‐limited, micron‐sized crystals can be produced in ~1 s, which would be compatible with the turnover time of many enzymes. Consequently, the ability to record diffraction patterns from sub‐micron sized crystals by X‐ray Free Electron Lasers and micro‐electron diffraction technologies opens the possibility of trapping intermediate enzyme states by crystallization