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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.

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.

The use of resistant genotypes is the preferred method to control orange rust of sugarcane (Saccharum spp) caused by Puccinia kuehnii. This approach has been adopted in Brazil but outbreaks of the disease on previously resistant varieties showed that the efficacy of this method is limited and requires a better understanding of pathogen diversity. Nevertheless, adequate molecular markers for examining pathogen diversity at population level are not available, which limits the success of orange rust control by genetic resistance. Therefore, two independent investigations were conducted to examine genetic diversity of P. kuehnii from Sao Paulo state, the most important sugarcane growing state of Brazil. First, simple‐sequence repeat (SSR) markers were developed in the present work and genotypic diversity of orange rust isolates from different locations investigated. Second, phenotypic diversity was examined by the single‐pustule inoculation technique on P. kuehnii isolates retrieved from three susceptible commercial sugarcane cultivars. A total of 96 SSR markers were generated and tested for this species. Subsequently, 29 isolates of P. kuehnii were fingerprinted with nine SSR markers to estimate the genotypic diversity by neighbour‐joining and 3D principal coordinates. The 29 isolates of the pathogen clustered into four main groups, which were identified by three SSR markers (NPRL_PK_108a, NPRL_PK_162_spka and NPRL_PK_221_spka). Phenotypic data at 21 days after the single‐pustule inoculation showed that P. kuehnii from highly susceptible commercial cultivars harboured a small proportion of variants capable of causing disease on resistant cultivars. A differential reaction was demonstrated for the most virulent variant in a repeated experiment confirming the existence of races within P. kuehnii in Brazil.

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 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.

Spanish lime (Melicoccus bijugatus Jacq.) is a Neotropical 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.

Background Peanut smut is a disease caused by the fungus Thecaphora frezii Carranza & Lindquist to which most commercial cultivars in South America are highly susceptible. It is responsible for severely decreased yield and no effective chemical treatment is available to date. However, smut resistance has been identified in wild Arachis species and further transferred to peanut elite cultivars. To identify the genome regions conferring smut resistance within a tetraploid genetic background, this study evaluated a RIL population {susceptible Arachis hypogaea subsp. hypogaea (JS17304-7-B) × resistant synthetic amphidiploid (JS1806) [A. correntina (K 11905) × A. cardenasii (KSSc 36015)] × A. batizocoi (K 9484)4×} segregating for the trait. Results A SNP based genetic map arranged into 21 linkage groups belonging to the 20 peanut chromosomes was constructed with 1819 markers, spanning a genetic distance of 2531.81 cM. Two consistent quantitative trait loci (QTLs) were identified qSmIA08 and qSmIA02/B02, located on chromosome A08 and A02/B02, respectively. The QTL qSmIA08 at 15.20 cM/5.03 Mbp explained 17.53% of the phenotypic variance, while qSmIA02/B02 at 4.0 cM/3.56 Mbp explained 9.06% of the phenotypic variance. The combined genotypic effects of both QTLs reduced smut incidence by 57% and were stable over the 3 years of evaluation. The genome regions containing the QTLs are rich in genes encoding proteins involved in plant defense, providing new insights into the genetic architecture of peanut smut resistance. Conclusions A major QTL and a minor QTL identified in this study provide new insights into the genetic architecture of peanut smut resistance that may aid in breeding new varieties resistant to peanut smut.

Peanut smut caused by Thecaphora frezii is a severe fungal disease currently endemic to Argentina and Brazil. The identification of smut resistant germplasm is crucial in view of the potential risk of a global spread. In a recent study, we reported new sources of smut resistance and demonstrated its introgression into elite peanut cultivars. Here, we revisited one of these sources (line I0322) to verify its presence in the U.S. peanut germplasm collection and to identify single nucleotide polymorphisms (SNPs) potentially associated with resistance. Five accessions of Arachis hypogaea subsp. fastigiata from the U.S. peanut collection, along with the resistant source and derived inbred lines were genotyped with a 48K SNP peanut array. A recently developed SNP genotyping platform called RNase H2 enzyme-based amplification (rhAmp) was further applied to validate selected SNPs in a larger number of individuals per accession. More than 14,000 SNPs and nine rhAmp assays confirmed the presence of a germplasm in the U.S. peanut collection that is 98.6% identical (P < 0.01, bootstrap t-test) to the resistant line I0322. We report this germplasm with accompanying genetic information, genotyping data, and diagnostic SNP markers. Fil: Massa, Alicia N.. National Peanut Research Laboratory; Estados Unidos Fil: Bressano, Marina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Fitopatología y Fisiología Vegetal; Argentina Fil: Soave, Juan H.. Criadero El Carmen; Argentina Fil: Buteler, Mario I.. Criadero El Carmen; Argentina Fil: Seijo, José Guillermo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Botánica del Nordeste. Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias. Instituto de Botánica del Nordeste; Argentina Fil: Sobolev, Victor S.. National Peanut Research Laboratory; Estados Unidos Fil: Orner, Valerie A.. National Peanut Research Laboratory; Estados Unidos Fil: Oddino, Claudio Marcelo. Criadero El Carmen; Argentina Fil: Soave, Sara J.. Criadero El Carmen; Argentina Fil: Faustinelli, Paola Carmen. National Peanut Research Laboratory; Estados Unidos Fil: de Blas, Francisco Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto Multidisciplinario de Biología Vegetal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto Multidisciplinario de Biología Vegetal; Argentina Fil: Lamb, Marshall C.. National Peanut Research Laboratory; Estados Unidos Fil: Arias, Renee S.. National Peanut Research Laboratory; Estados Unidos

BackgroundGroundnut pre- and post-harvest contamination is commonly caused by fungi from the GenusAspergillus.Aspergillus flavusis the most important of these fungi. It belongs to sectionFlavi; a group consisting of aflatoxigenic (A. flavus,A. parasiticusandA. nomius) and non-aflatoxigenic (A. oryzae,A. sojaeandA. tamarii) fungi. Aflatoxins are food-borne toxic secondary metabolites ofAspergillusspecies associated with severe hepatic carcinoma and children stuntedness. Despite the well-known public health significance of aflatoxicosis, there is a paucity of information about the prevalence, genetic diversity and population structure ofA. flavusin different groundnut growing agro-ecological zones of Uganda. This cross-sectional study was therefore conducted to fill this knowledge gap.ResultsThe overall pre- and post-harvest groundnut contamination rates withA. flavuswere 30.0 and 39.2% respectively. Pre- and post-harvest groundnut contamination rates withA. flavusacross AEZs were; 2.5 and 50.0%; (West Nile), 55.0 and 35.0% (Lake Kyoga Basin) and 32.5 and 32.5% (Lake Victoria Basin) respectively. There was no significant difference (χ2 = 2,p = 0.157) in overall pre- and post-harvest groundnut contamination rates withA. flavusand similarly no significant difference (χ2 = 6,p = 0.199) was observed in the pre- and post-harvest contamination of groundnut withA. flavusacross the three AEZs. The LKB had the highest incidence of aflatoxin-producingAspergillusisolates while WN had no singleAspergillusisolate with aflatoxin-producing potential.Aspergillusisolates from the pre-harvest groundnut samples had insignificantly higher incidence of aflatoxin production (χ2 = 2.667,p = 0.264) than those from the post-harvest groundnut samples. Overall,A. flavusisolates exhibited moderate level (92%,p = 0.02) of genetic diversity across the three AEZs and low level (8%,p = 0.05) of genetic diversity within the individual AEZs. There was a weak positive correlation (r = 0.1241,p = 0.045) between genetic distance and geographic distance amongA. flavuspopulations in the LKB, suggesting that genetic differentiation in the LKB population might be associated to geographic distance. A very weak positive correlation existed between genetic variation and geographic location in the entire study area (r = 0.01,p = 0.471), LVB farming system (r = 0.0141,p = 0.412) and WN farming system (r = 0.02,p = 0.478). Hierarchical clustering using the unweighted pair group method with arithmetic means (UPGMA) revealed two main clusters of genetically similarA. flavusisolates.ConclusionsThese findings provide evidence that genetic differentiation inA. flavuspopulations is independent of geographic distance. This information can be valuable in the development of a suitable biocontrol management strategy of aflatoxin-producingA. flavus.

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.

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.

Drought is one of the main constraints in peanut production in West Texas and eastern New Mexico regions due to the depletion of groundwater. A multi-seasonal phenotypic analysis of 10 peanut genotypes revealed C76-16 (C-76) and Valencia-C (Val-C) as the best and poor performers under deficit irrigation (DI) in West Texas, respectively. In order to decipher transcriptome changes under DI, RNA-seq was performed in C-76 and Val-C. Approximately 369 million raw reads were generated from 12 different libraries of two genotypes subjected to fully irrigated (FI) and DI conditions, of which ~329 million (90.2%) filtered reads were mapped to the diploid ancestors of peanut. The transcriptome analysis detected 4,508 differentially expressed genes (DEGs), 1554 genes encoding transcription factors (TFs) and a total of 514 single nucleotide polymorphisms (SNPs) among the identified DEGs. The comparative analysis between the two genotypes revealed higher and integral tolerance in C-76 through activation of key genes involved in ABA and sucrose metabolic pathways. Interestingly, one SNP from the gene coding F-box protein (Araip.3WN1Q) and another SNP from gene coding for the lipid transfer protein (Aradu.03ENG) showed polymorphism in selected contrasting genotypes. These SNPs after further validation may be useful for performing early generation selection for selecting drought-responsive genotypes.

Groundnut (Arachis hypogaea L.) is an important cash crop in the lowland areas of Ethiopia. However, prevalence of Aspergillus invasions and subsequent aflatoxin contamination compromises the quality of groundnut kernels. This study was conducted to evaluate the effect of farm yard manure (FYM) and seed treatments against Aspergillus species pod colonization and aflatoxin accumulation under field conditions. The inhibitory efficacy of Trichoderma species as biocontrol agents was also assessed. A total of 20 treatment combinations including pre-planting applications of FYM at 0, 2.5, 5, and 7.5 tons/ha and seed treatments with carbendazim at 2 g/kg and mancozeb at 3 g kg-1, and Trichoderma harzianum and Trichoderma viride each at 5 g/kg as well as untreated seed as control were used. Treatments were laid out in a randomized complete block design (RCBD) in three replications. The experiment was conducted in two consecutive seasons (2014 and 2015) at Babile Haramaya University sub-Research Station. The highest pod and seed yields (1901.5 and 1281.5 kg/ha, respectively) were recovered from plots treated with T. harzianum at 5 g/kg seed. A. flavus was abundantly recorded in control plots, which could be responsible for the high aflatoxin B1 (5704.4 µg/kg) and B2 (2219.0 µg/kg) contamination. However, plots treated with T. harzianum at 5 g/kg seed and FYM at 5 tons/ha + T. harzianum at 5 g/kg were free from aflatoxin. Integrations of T. harzianum as biocontrol seed treatment and soil amendment with FYM were effective in the pre-harvest management of Aspergillus spp. and aflatoxins contamination. Key words: Aflatoxin, Aspergillus, carbendazim, farm yard manure, groundnut, mancozeb, Trichoderma species.

New technologies are needed to eliminate mycotoxins and/or fungal pathogens from agricultural products. RNA interference (RNAi) has shown potential to control fungi associated with crops. In RNAi, double-stranded RNA (dsRNA) targets homologous mRNA for cleavage, and can reach the mRNA of pathogens in contact with the plant. The key element in this process is the movement of RNA signals cell-to-cell and over long distances within the plant, and between host plants and parasites. In this study, we selected a regulatory gene in the aflatoxin biosynthesis pathway, aflS/aflR, necessary for the production of aflatoxins in Aspergillus spp. We designed a Dicer-substrate RNA (DsiRNA) to study the movement and stability of the duplex over time in in vitro peanut plants using stem-loop primers and RT-PCR for DsiRNA detection. The preliminary results demonstrated that DsiRNA was absorbed and moved away from the point of application, spread systemically and was transported rapidly, most likely through the phloem of the shoot, to the sink tissues, such as new auxiliary shoots, flowers and newly formed pegs. The DsiRNA remained detectable for at least 30 days after treatment. This is the first time that movement of exogenous DsiRNA in in vitro peanut plants has been described. Since DsiRNA was detectable in the pegs 15 days after treatment, aflatoxin reduction may be possible if the duplexes containing part of the aflatoxin biosynthesis pathogen gene induce silencing in the peanut seeds colonised by Aspergillus spp. The application of small RNAs could be a non-transformative option for mycotoxin contamination control.

Currently, some species of Sapindaceae are important fruit crops worldwide. The Huaya India (Meliccocus oliviformis, Sapindaceae) is a neglected Neotropical fruit tree consumed locally in the Maya Lowlands of Mexico, where it exists in both wild and domesticated forms. Our objective was to evaluate the genetic diversity of the Huaya India in its possible domestication area and thus generate knowledge that serves as the basis for a commercial management. A total of 450 individuals collected from 15 natural vegetation sites and 15 Maya villages, were characterized using nine microsatellite loci and population genetics approaches were applied. STRUCTURE, Neighbor-Joining and PCoA analyses suggested the existence of three main groups: 1) one composed by 14 natural vegetation sites, 2) one integrated by 10 Maya villages plus one natural vegetation site, 3) one composed by five Maya villages. At the species level, genetic differentiation was high (FST = 0.562) and gene flow was low (Nm = 0.395); between genetic groups, differentiation was low and gene flow was high. Genetic diversity was low at the level species (HE = 0.19) and higher in the group composed for only natural vegetation sites. When we considered only two groups (natural vegetation sites vs Maya villages) to explore a possible bottleneck as a consequence of human management, the natural vegetation sites showed higher, and significant, genetic diversity (HE = 0.231) than the Maya villages (HE = 0.152). This study can serve as a basis to develop management strategies for Huaya India in the Maya Lowlands of Mexico, but without compromising its conservation.

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 accumulation of aflatoxin in peanut seed. In this study, we performed high-throughput sequencing of small RNA populations in a control line and in two transformed peanut lines that expressed an inverted repeat targeting 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 small RNA 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.

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.

Peanut smut caused by Thecaphora frezii Carranza & Lindquist has been an issue for farmers and the peanut industry (Arachis hypogaea L.) in Argentina since the mid-1990s. This disease causes pod malformation due to hypertrophy of seed tissues; in addition, colonized cells filled with teliospores give seeds a smutted mass appearance. Incidence may reach up to 52% in commercial plots, with up to 35% yield losses. Cultural management strategies and chemical treatment have not been effective; therefore, growing resistant varieties is likely to be the most effective control method for this disease. This study is aimed to identify sources of resistance in wild Arachis and to develop pre-breeding materials for transferring the trait to cultivated peanut. After 3 yr of field trials using a randomized complete block design, the seven accessions of wild species assayed were resistant to smut. An amphidiploid [A. correntina(Burkart) Krapov. & W.C. Greg. × A. cardenasii Krapov. & W.C. Greg.] × A. batizocoi Krapov. & W.C. Greg.)4× was obtained and subsequently crossed with and experimental line of A. hypogaea for the development of a recombinant inbred line (RIL) population (89 lines). The RIL population showed a high phenotypic variability for resistance to peanut smut. The amphidiploid and 22 RILs were highly resistant, illustrating the effective transmission of resistance to peanut smut from the wild diploids into A. hypogaea. The development of RILs with resistance derived from wild species is a significant step towards the development of new peanut cultivars with different sources of resistance to peanut smut. Fil: de Blas, Francisco Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto Multidisciplinario de Biología Vegetal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto Multidisciplinario de Biología Vegetal; Argentina Fil: Bressano, Marina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Córdoba; Argentina Fil: Teich, Ingrid. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Fisiología y Recursos Genéticos Vegetales; Argentina Fil: Balzarini, Monica Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Córdoba; Argentina Fil: Arias, Renee S.. United States Department of Agriculture; Estados Unidos Fil: Manifesto, María. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación de Recursos Naturales. Instituto de Recursos Biológicos; Argentina Fil: Costero, Beatriz. Universidad Nacional de Córdoba; Argentina Fil: Oddino, Claudio Marcelo. Universidad Nacional de Río Cuarto; Argentina Fil: Soave, Sara Josefina. Universidad Nacional de Río Cuarto; Argentina Fil: Soave, Juan A.. Universidad Nacional de Río Cuarto; Argentina Fil: Buteler, Mario I.. Universidad Nacional de Río Cuarto; Argentina Fil: Massa, Alicia N.. No especifíca; Fil: Seijo, José Guillermo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Botánica del Nordeste. Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias. Instituto de Botánica del Nordeste; Argentina

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.

Peanut (Arachis hypogaea) is a botanically indeterminate plant where flowering, fruit initiation, and pod maturity occurs over an extended time period during the growing season. As a result, the maturity and size of individual peanut pods vary considerably at harvest. Immature kernels that meet commercial edible size specifications negatively affect quality during processing due to their increased propensity for off flavors, higher moisture and water activity, and variable roasting properties. As peanuts progress toward maturation, late season flowers set within 40 days till harvest will not have sufficient time to develop into mature, marketable pods prior to harvest. Research was conducted to determine the effect of late season flower termination on peanut yield, grade, and seed germination. Diflufenzopyr-Na (Diflufenzopyr) (BASF Biosciences), a synthetic auxin transport inhibitor, and the herbicide glyphosate were applied at three sub-lethal rates along with a “hand flower removal” and a non-treated control in both irrigated and non-irrigated plots. No differences in non-irrigated pod yield across all treatments were detected. Glyphosate at 56 and 112 g/ha increased non-irrigated sound mature kernels plus sound splits (SMK+SS) and decreased other kernels (OK). Non-irrigated seed germination was negatively affected by glyphosate. Diflufenzopyr at 17 and 25 g/ha increased irrigated peanut yield. Glyphosate at 112 and 168 g/ha increased irrigated SMK+SS and decreased OK and germination.

This study was conducted to assess major Aspergillus species and aflatoxins associated with groundnut seeds and cake in Eastern Ethiopia and evaluate growers’ management practices. A total of 160 groundnut seed samples from farmers’ stores and 50 groundnut cake samples from cafe and restaurants were collected. Fungal isolation was done from groundnut seed samples. Aspergillus flavus was the dominant species followed by Aspergillus parasiticus. Aflatoxin analyses of groundnut seed samples were performed using ultra performance liquid chromatography; 22.5% and 41.3% of samples were positive, with total aflatoxin concentrations of 786 and 3135 ng g−1 from 2013/2014 and 2014/2015 samples, respectively. The level of specific aflatoxin concentration varied between 0.1 and 2526 ng g−1 for B2 and B1, respectively. Among contaminated samples of groundnut cake, 68% exhibited aflatoxin concentration below 20 ng g−1, while as high as 158 ng g−1 aflatoxin B1 was recorded. The study confirms high contamination ...

Macrophomina crown and root rot has become a significant soil-borne disease issue in California. For many locations in the state, the disease is associated with fields that are no longer pre-plant,...

Field-evolved resistance of fall armyworm, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae), to the bacterium Bacillus thuringiensis (Bt) has been reported worldwide as one of the most serious threats to the sustainability of Bt maize crop. Therefore, it is important to assess the magnitude of adult survival and the possibility of cross-resistance of fall armyworm neonates exposed to Bt proteins. In this study, bioassays were used to examine susceptibility of two field-collected Cry1Fa-resistant strains of fall armyworm from Puerto Rico (456RR, 512RR) and their crosses with a susceptible strain (Monsanto SS) (456SR-RS, 512SR-RS). LC50 values varied in both Cry1Fa-resistant strains and in their backcrosses with the susceptible strain. The two RR strains were more tolerant to Cry1Fa and Cry1Ac proteins in earlier instars of development than were their crosses. Greater survival to the adult stage was obtained in the 512 RR strain and their RS-SR crosses when exposed to all concentrations of Cry1Ac and 1Fa. Survival to adult in the 456 RR was much greater when exposed to Cry1Fa than to Cry1Ac. Adults of 456 RR and their crosses survived only when exposed to the lowest concentrations of Cry1Ac. Our data confirmed great resistance to Cry1Fa and Cry1Ac in S. frugiperda larvae from Puerto Rico. However, based on the larvae that survived the 7-day diet bioassay and developed to pupae and adult maturity on regular diet, their LC50 values were less for both resistant strains and their crosses.

Aflatoxin contamination in peanut seeds is still a serious problem for the industry and human health. No stable aflatoxin resistant cultivars have yet been produced, and given the narrow genetic background of cultivated peanuts, wild species became an important source of genetic diversity. Wild peanut seeds, however, are not abundant, thus, an effective method of screening for aflatoxin accumulation using minimal seeds is highly desirable. In addition, keeping record of genetic fingerprinting of each accession would be very useful for breeding programs and for the identification of accessions within germplasm collections. Results: In this study, we report a method of screening for aflatoxin accumulation that is applicable to the small-size seeds of wild peanuts, increases the reliability by testing seed viability, and records the genetic fingerprinting of the samples. Aflatoxin levels observed among 20 wild peanut species varied from zero to 19000 ng.g-1 and 155 ng.g-1 of aflatoxin B1 and B2, respectively. We report the screening of 373 molecular markers, including 288 novel SSRs, tested on 20 wild peanut species. Multivariate analysis by Neighbor-Joining, Principal Component Analysis and 3D-Principal Coordinate Analysis using 134 (36 %) transferable markers, in general grouped the samples according to their reported genomes. The best 88 markers, those with high fluorescence, good scorability and transferability, are reported with BLAST results. High quality markers (total 98) that discriminated genomes are reported. A high quality marker with UPIC score 16 (16 out of 20 species discriminated) had significant hits on BLAST2GO to a pentatricopeptide-repeat protein, another marker with score 5 had hits on UDP-D-apiose synthase, and a third one with score 12 had BLASTn hits on La-RP 1B protein. Together, these three markers discriminated all 20 species tested. Conclusions: This study provides a reliable method to screen wild species of peanut for aflatoxin resistance using minimal seeds. In addition we report 288 new SSRs for peanut, and a cost-effective combination of markers sufficient to discriminate all 20 species tested. These tools can be used for the systematic search of aflatoxin resistant germplasm keeping record of the genetic fingerprinting of the accessions tested for breeding purpose. Fil: Arias, Renee S.. National Peanut Research Laboratory; Estados Unidos Fil: Sobolev, Victor S.. National Peanut Research Laboratory; Estados Unidos Fil: Massa, Alicia N.. National Peanut Research Laboratory; Estados Unidos Fil: Orner, Valerie A.. National Peanut Research Laboratory; Estados Unidos Fil: Walk, Travis E.. National Peanut Research Laboratory; Estados Unidos Fil: Ballard, Linda L.. Usda Agricultural Research Service; Estados Unidos Fil: Simpson, Sheron A.. Usda Agricultural Research Service; Estados Unidos Fil: Puppala, Naveen. New Mexico State University Las Cruces; México Fil: Scheffler, Brian E.. Usda Agricultural Research Service; Estados Unidos Fil: de Blas, Francisco Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto Multidisciplinario de Biología Vegetal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto Multidisciplinario de Biología Vegetal; Argentina Fil: Seijo, José Guillermo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Botánica del Nordeste. Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias. Instituto de Botánica del Nordeste; Argentina

Charcoal rot of soybean (Glycine max (L.) Merr.), caused by Macrophomina phaseolina (Tassi) Goid., is an economically important disease worldwide. In recent years, charcoal rot has become a concern for farmers in north-western Argentina. The present work aimed to (1) evaluate disease incidence of charcoal rot; (2) assess morphological and genetic characteristics of M. phaseolina isolates from soybean and other hosts collected in north-western Argentina; and (3) compare genetic diversity between soybean isolates of M. phaseolina from Argentina and the USA. Incidence of charcoal rot evaluated in 11 locations during five growing seasons (2008–2012) was 1%, 1%, 1%, 5–10% and 90%, respectively, indicating a gradual increase over the cropping seasons. Cluster analysis by AFLP of Argentinean isolates exhibited one main group, with isolate Mp56 separated at a genetic distance of 0.70. Four main groups were identified using SSR markers at the same genetic distance, with Mp56 and Mp53 separated from them. There was no clear association between AFLP, SSR profiles, morphological characteristics or host of origin within isolates from Argentina. However, using principal coordinate analysis of SSRs, 22 isolates of M. phaseolina from soybean from north-western Argentina were clearly distinguished from 11 isolates previously collected from two states in the USA. Our results confirmed the increasing importance of charcoal rot in north-western Argentina and identified characteristics of isolates that may be useful for breeding for disease resistance and developing integrated management programmes for charcoal rot in Argentina in the future. Fil: Reznikov, Sebastian. Estación Experimental Agroindustrial Obispo Colombres (EEAOC) - Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Instituto de Tecnología Agroindustrial del Noroeste Argentino (ITANOA; Argentina Fil: Vellice, Gabriel. Estación Experimental Agroindustrial Obispo Colombres (EEAOC) - Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Instituto de Tecnología Agroindustrial del Noroeste Argentino (ITANOA); Argentina Fil: Mengistu, Alemu. USDA-ARS Crop Genetics Research Unit; Estados Unidos Fil: Arias, Renne Silva. USDA-ARS-National Peanut Research Laboratory; Estados Unidos Fil: Gonzalez, Victoria. Estación Experimental Agroindustrial Obispo Colombres (EEAOC) - Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Instituto de Tecnología Agroindustrial del Noroeste Argentino (ITANOA); Argentina Fil: de Lisi, Vicente. Estación Experimental Agroindustrial Obispo Colombres (EEAOC) - Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Instituto de Tecnología Agroindustrial del Noroeste Argentino (ITANOA); Argentina Fil: Garcia, Maria Gabriela. Estación Experimental Agroindustrial Obispo Colombres (EEAOC) - Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Instituto de Tecnología Agroindustrial del Noroeste Argentino (ITANOA); Argentina Fil: Rocha, Carla Maria Lourdes. Estación Experimental Agroindustrial Obispo Colombres (EEAOC) - Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Instituto de Tecnología Agroindustrial del Noroeste Argentino (ITANOA); Argentina Fil: Pardo, Esteban Mariano. Estación Experimental Agroindustrial Obispo Colombres (EEAOC) - Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Instituto de Tecnología Agroindustrial del Noroeste Argentino (ITANOA); Argentina Fil: Castagnaro, Atilio Pedro. Estación Experimental Agroindustrial Obispo Colombres (EEAOC) - Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Instituto de Tecnología Agroindustrial del Noroeste Argentino (ITANOA); Argentina Fil: Ploper, Leonardo Daniel. Estación Experimental Agroindustrial Obispo Colombres (EEAOC) - Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Instituto de Tecnología Agroindustrial del Noroeste Argentino (ITANOA); Argentina

Pouteria sapota is known for its edible fruits that contain unique carotenoids, as well as for its fungitoxic, anti-inflammatory and anti-oxidant activity. However, its genetics is mostly unknown, including aspects about its genetic diversity and domestication process. We did high-throughput sequencing of microsatellite-enriched libraries of P. sapota, generated 5223 contig DNA sequences, 1.8 Mbp, developed 368 microsatellites markers and tested them on 29 individuals from 10 populations (seven wild, three cultivated) from Mexico, its putative domestication center. Gene ontology BLAST analysis of the DNA sequences containing microsatellites showed potential association to physiological functions. Genetic diversity was slightly higher in cultivated than in the wild gene pool (HE = 0.41 and HE = 0.35, respectively), although modified Garza–Williamson Index and Bottleneck software showed evidence for a reduction in genetic diversity for the cultivated one. Neighbor Joining, 3D Principal Coordinates Analysis and assignment tests grouped most individuals according to their geographic origin but no clear separation was observed between wild or cultivated gene pools due to, perhaps, the existence of several admixed populations. The developed microsatellites have a great potential in genetic population and domestication studies of P. sapota but additional sampling will be necessary to better understand how the domestication process has impacted the genetic diversity of this fruit crop.

Aflatoxins are among the most powerful carcinogens in nature. The major aflatoxin-producing fungi are Aspergillus flavus and A. parasiticus. Numerous crops, including peanut, are susceptible to aflatoxin contamination by these fungi. There has been an increased use of RNA interference (RNAi) technology to control phytopathogenic fungi in recent years. In order to develop molecular tools targeting specific genes of these fungi for the control of aflatoxins, it is necessary to obtain their genome sequences. Although high-throughput sequencing is readily available, it is still impractical to sequence the genome of every isolate. Thus, in this work, the authors proposed a workflow that allowed prescreening of 238 Aspergillus section Flavi isolates from peanut seeds from Georgia, USA. The aflatoxin biosynthesis cluster (ABC) of the isolates was fingerprinted at 25 InDel (insertion/deletion) loci using capillary electrophoresis. All isolates were tested for aflatoxins using ultra-high-performance liquid chromatography. The neighbor-joining, three-dimension (3D) principal coordinate, and Structure analyses revealed that the Aspergillus isolates sampled consisted of three main groups determined by their capability to produce aflatoxins. Group I comprised 10 non-aflatoxigenic A. flavus; Group II included A. parasiticus; and Group III included mostly aflatoxigenic A. flavus and the three non-aflatoxigenic A. caelatus. Whole genomes of 10 representative isolates from different groups were sequenced. Although InDels in Aspergillus have been used by other research groups, this is the first time that the cluster analysis resulting from fingerprinting was followed by whole-genome sequencing of representative isolates. In our study, cluster analysis of ABC sequences validated the results obtained with fingerprinting. This shows that InDels used here can predict similarities at the genome level. Our results also revealed a relationship between groups and their capability to produce aflatoxins. The database generated of Aspergillus spp. can be used to select target genes and assess the effectiveness of RNAi technology to reduce aflatoxin contamination in peanut.

Smut disease caused by the fungal pathogen Thecaphora frezii Carranza & Lindquist is threatening the peanut production in Argentina. Fungicides commonly used in the peanut crop have shown little or no effect controlling the disease, making it a priority to obtain peanut varieties resistant to smut. In this study, recombinant inbred lines (RILs) were developed from three crosses between three susceptible peanut elite cultivars (Arachis hypogaea L. subsp. hypogaea) and two resistant landraces (Arachis hypogaea L. subsp. fastigiata Waldron). Parents and RILs were evaluated under high inoculum pressure (12000 teliospores g -1 of soil) over three years. Disease resistance parameters showed a broad range of variation with incidence mean values ranging from 1.0 to 35.0% and disease severity index ranging from 0.01 to 0.30. Average heritability (h 2 ) estimates of 0.61 to 0.73 indicated that resistance in the RILs was heritable, with several lines (4 to 7 from each cross) showing a high degree of resistance and stability over three years. Evidence of genetic transfer between genetically distinguishable germplasm (introgression in a broad sense) was further supported by simple-sequence repeats (SSRs) and Insertion/Deletion (InDel) marker genotyping. This is the first report of smut genetic resistance identified in peanut landraces and its introgression into elite peanut cultivars. This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Fil: Bressano, Marina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Fitopatología y Fisiología Vegetal; Argentina. Universidad Nacional de Córdoba; Argentina Fil: Massarini, Alicia. Usda Ars National Peanut Research Laboratory; Estados Unidos. Universidad de Buenos Aires. Facultad de Ingeniería. Postgrado-Maestría en Política y Gestión de la Ciencia y Tecnología; Argentina Fil: Arias, Renee S.. Usda Ars National Peanut Research Laboratory; Estados Unidos Fil: de Blas, Francisco Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto Multidisciplinario de Biología Vegetal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto Multidisciplinario de Biología Vegetal; Argentina Fil: Oddino, Claudio Marcelo. Criadero El Carmen; Argentina Fil: Faustinelli, Paola Carmen. Usda Ars National Peanut Research Laboratory; Estados Unidos Fil: Soave, Sara Josefina. Criadero El Carmen; Argentina Fil: Soave, Juan H.. Criadero El Carmen; Argentina Fil: Pérez, Maria A.. Universidad Católica de Córdoba. Facultad de Ciencias Agropecuarias; Argentina Fil: Sobolev, Victor S.. Usda Ars National Peanut Research Laboratory; Estados Unidos Fil: Lamb, Marshall C.. Usda Ars National Peanut Research Laboratory; Estados Unidos Fil: Balzarini, Monica Graciela. Universidad Nacional de Córdoba; Argentina. Instituto Nacional de Tecnologia Agropecuaria. Centro de Investigaciones Agropecuarias. Unidad de Fitopatologia y Modelizacion Agricola. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Unidad de Fitopatologia y Modelizacion Agricola.; Argentina Fil: Buteler, Mario I.. Criadero El Carmen; Argentina Fil: Seijo, José Guillermo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Botánica del Nordeste. Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias. Instituto de Botánica del Nordeste; Argentina

Aspergillus flavus accumulates carcinogenic aflatoxins in peanuts, mainly in immature kernels during drought. Aspergillus flavus invasion induces accumulation of phytoalexins, mostly stilbenoids in peanut, as a plant defence mechanism. Because fungal laccases are often related to pathogenicity and can degrade stilbenoids, this study reports for the first time the expression of A. flavus laccases in the presence of kernels, hulls and low water potential in relation to the accumulation of phytoalexins in peanut kernels. Packed-cell volume (PCV) of A. flavus biomass was significantly higher (P ≤ 0·01) in the presence of mature kernels, dead kernels, and mature and immature peanut hulls than the control. The presence of kernels and hulls lowered the level of expression of three A. flavus laccases by 4–6-fold (P < 0·01), whereas 3% sucrose up-regulated them by 35–304-fold, and low water potential (−1·1 MPa) up-regulated them by 85–248-fold (P < 0·01). Phytoalexins that accumulated in peanut kernels in the presence of A. flavus and were quantified by HPLC-DAD-MS were primarily the stilbenoids: 3′-isopentadienyl-3,5,4′-trihydroxystilbene (IPD), chiricanine-A, arachidin-2, arachidin-3 and arahypin-1. Apparent degradation of phytoalexins was observed when using a priori induction of phytoalexins in seeds in combination with a priori induction of laccases in A. flavus. The up-regulation of laccase expression observed at −1·1 MPa and at high sucrose concentration could be contributing to peanut invasion in immature kernels under drought conditions.

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.

This is the first report of sequence-specific microsatellite markers (simple sequence repeats [SSRs]) of fall armyworm, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae), an economically important pest of crops on the Americas. We isolated 192 microsatellite markers by using pyrosequencing and screened 15 individuals from eight isofamilies collected from three geographical areas: Puerto Rico (PR), Texas (TX), and Mississippi (MS). Isofamilies resistant to Cry toxins from Bacillus thuringiensis (Berliner) (Bacillales: Bacillaceae) also were included. Cluster analysis was performed to determine the potential use of these SSRs in discriminating populations, and colonies were grouped with a reliability of 100% estimated by bootstrap. In this analysis, colonies from TX grouped away from those from PR, but the two MS isofamilies grouped with TX and PR separately. Genetic distance within isofamilies ranged between 0.22 and 0.56, and the minimum distance between isofamilies was 0.83. Unique pattern informative combination (UPIC) scores were calculated, and the 80 SSR markers that had UPIC scores of ≥1 are listed according to their discriminating potential. UPIC scores allow reducing costs by choosing fewer and highly informative markers for future studies. From the best 125 markers, 103 had a maximum of two alleles per sample, making them ideal candidates for population genetic studies. BLAST screening of the sequences points to potential biological meaning of marker polymorphisms. The percentage of alleles shared by the three geographic areas was 14%. The markers reported will significantly enrich the pool of molecular markers available for S. frugiperda. In addition, they could be used for monitoring migration of populations, in the development of biocontrol agents and for management practices in general.

One hundred and eighty-two microsatellites or simple-sequence-repeat (SSR) markers for Macrophomina phaseolina were developed. These were tested on 24 isolates of M. phaseolina obtained from seven plant species, and the genetic variation of isolates was studied in relation to potential biological processes that could be affected in this fungus. A total of 120 SSR markers were polymorphic, amplifying >90% of the 24 isolates tested. Thirty percent of the markers showed multiple alleles on individual samples. A large number of markers showed unique alleles in isolates collected from pumpkin and snap bean. DNA sequences corresponding to 43 markers had significant hits on BLAST xa nd⁄or BLAST2GO, and the polymorphism of 36 of those markers showed specific allele patterns for one or more plant host origin of the isolates. Additional tests on growth rate and copper resistance of the isolates identified markers that could be related to those traits. In addition, 27 markers were monomorphic and amplified all 24 isolates. Whereas polymorphic markers can be used for population genetics studies of M. phaseolina, the group of 27 monomorphic markers could help in the fast identification of this species in clinical specimens. The SSR markers developed here will enrich the limited molecular marker resource in M. phaseolina and could be used as the basis for more in-depth studies of the host-pathogen interactions of M. phaseolina.

Summary We studied the effects on plant growth and from insertion of five cisgenes that encode proteins involved in gibberellin metabolism or signalling. Intact genomic cop- ies of PtGA20ox7, PtGA2ox2,Pt RGL1_1, PtRGL1_2 and PtGAI1 genes from the gen- ome-sequenced Populus trichocarpa clone Nisqually-1 were transformed into Populus tremula · alba (clone INRA 717-1B4), and growth, morphology and xylem cell size characterized in the greenhouse. Each cisgene encompassed 1-2 kb of 5¢ and 1 kb of 3¢ flanking DNA, as well as all native exons and introns. Large numbers of inde- pendent insertion events per cisgene (19-38), including empty vector controls, were studied. Three of the cisgenic modifications had significant effects on plant growth rate, morphology or wood properties. The PtGA20ox7 cisgene increased rate of shoot regeneration in vitro, accelerated early growth, and variation in growth rate was correlated with PtGA20ox7 gene expression. PtRGL1_1 and PtGA2ox2 caused reduced growth, while PtRGL1_2 gave rise to plants that grew normally but had sig- nificantly longer xylem fibres. RT-PCR studies suggested that the lack of growth inhi- bition observed in PtRGL1_2 cisgenic plants was a result of co-suppression. PtGAI1 slowed regeneration rate and both PtGAI1 and PtGA20ox7 gave rise to increased variance among events for early diameter and volume index, respectively. Our work suggests that cisgenic insertion of additional copies of native genes involved in growth regulation may provide tools to help modify plant architecture, expand the genetic variance in plant architecture available to breeders and accelerate transfer of alleles between difficult-to-cross species.

The genus Chionanthus (Oleaceae Hoffmans. & Link) includes deciduous or evergreen trees and shrubs distributed widely in tropical and sub-tropical areas, including a few temperate species. Although Chionanthus species are planted as ornamental garden plants and commercialized for natural products, genetic information for Chionanthus spp. is lacking. We created microsatellite-enriched libraries of Chionanthus retusus Lindl. & Paxton, assembled 1072 contigs, and detected 1010 repeats. The frequency of the repeats decreased with the increase in repeat length, and the most abundant motifs were: AG, AC, AAG, ACC, AT, and ACTC. We screened 384 markers on 12 accessions of four related taxa that included C. retusus, Chionanthus virginicus L., Chionanthus pygmaeus Small, and Osmanthus americanus (L.) Benth. & Hook. A total of 195 simple sequence repeat (SSR) markers amplified and discriminated six accessions of C. retusus and 57 SSR markers amplified and discriminated across the four Oleaceae species screened. To identify the best markers to use in future experiments, the “Unique Pattern Informative Combination” (UPIC) values were calculated for all the markers and the 100 markers that were most effective are reported here. The percentage of heterozygous loci across the 384 markers was lowest for C. retusus (29.3%) and highest for O. americanus (68.9%). The SSR markers developed here could assist in taxonomy and hybridization investigations for breeding programs and authentication of varieties used as medicinal plants.

The fall armyworm, Spodoptera frugiperda (J. E. Smith), is one of the most important insect pests on the American continent. Its control has relied primarily on multiple applications of insecticides that can amount to 1,000 g of active ingredient per hectare on some of approximately 30 crops the insect damages. The use of genetically engineered crops that express Bacillus thuringiensis (Bt) Berliner toxins, Bt-corn, Zee meys L.; and Bt-cotton, Gossypium hirsutum L.; are other ways to control this insect. However, fall armyworm is one of the Lepidoptera species least susceptible to Bt proteins, and a case of high tolerance to Bt-corn has already being reported. We found the susceptibility to Cry1Ac and Cry1Fa proteins of Bt in 133 isofamilies from five regions of three countries was similar to the susceptibility of two Bt-susceptible laboratory colonies to these proteins. Four isofamilies from Puerto Rico were very tolerant to Cry1Fa and not so tolerant to Cry1Ac. Two of the four isofamilies were backcrossed with a Bt-susceptible laboratory colony and their progeny was as susceptible to both Bt proteins as was the Bt-susceptible colony, indicating that resistance to Bt is a recessive trait.

We investigated the efficiency of RNA interference (RNAi) in Arabidopsis using transitive and homologous inverted repeat (hIR) vectors. hIR constructs carry self-complementary intron-spliced fragments of the target gene whereas transitive vectors have the target sequence fragment adjacent to an intron-spliced, inverted repeat of heterologous origin. Both transitive and hIR constructs facilitated specific and heritable silencing in the three genes studied (AP1, ETTIN and TTG1). Both types of vectors produced a phenotypic series that phenocopied reduction of function mutants for the respective target gene. The hIR yielded up to fourfold higher proportions of events with strongly manifested reduction of function phenotypes compared to transitive RNAi. We further investigated the efficiency and potential off-target effects of AP1 silencing by both types of vectors using genome-scale microarrays and quantitative RT-PCR. The depletion of AP1 transcripts coincided with reduction of function phenotypic changes among both hIR and transitive lines and also showed similar expression patterns among differentially regulated genes. We did not detect significant silencing directed against homologous potential off-target genes when constructs were designed with minimal sequence similarity. Both hIR and transitive methods are useful tools in plant biotechnology and genomics. The choice of vector will depend on specific objectives such as cloningmore » throughput, number of events and degree of suppression required.« less