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Couto D.,Norwich Research Park | Stransfeld L.,Norwich Research Park | Arruabarrena A.,National Agricultural Research Institute INIA | Zipfel C.,Norwich Research Park | And 2 more authors.
BMC Research Notes | Year: 2015

Background: Standard molecular biological methods involve the analysis of gene expression in living organisms under diverse environmental and developmental conditions. One of the most direct approaches to quantify gene expression is the isolation of RNA. Most techniques used to quantify gene expression require the isolation of RNA, usually from a large number of samples. While most published protocols, including those for commercial reagents, are either labour intensive, use hazardous chemicals and/or are costly, a previously published protocol for RNA isolation in Arabidopsis thaliana yields high amounts of good quality RNA in a simple, safe and inexpensive manner. Findings: We have tested this protocol in tomato and wheat leaves, as well as in Arabidopsis leaves, and compared the resulting RNA to that obtained using a commercial phenol-based reagent. Our results demonstrate that this protocol is applicable to other plant species, including monocots, and offers yield and purity at least comparable to those provided by commercial phenol-based reagents. Conclusions: Here, we show that this previously published RNA isolation protocol can be easily extended to other plant species without further modification. Due to its simplicity and the use of inexpensive reagents, this protocol is accessible and affordable and can be easily implemented to work on different plant species in laboratories worldwide. © 2015 Couto et al.; licensee BioMed Central.

Stewart S.,National Agricultural Research Institute INIA | Abeysekara N.,Iowa State University | Robertson A.E.,Iowa State University
Plant Disease | Year: 2014

Changes in pathotype structure of Phytophthora sojae populations have been attributed to deployment of race-specific resistant Rps genes in soybean that have been incorporated into commercial cultivars to reduce losses due to Phytophthora root and stem rot. To test this hypothesis, a cultivar rotation study was established from 2007 through 2010 in microplots at a site in Iowa with no history of soybean cultivation. All microplots were inoculated with P. sojae isolate PR1, race 1 (vir 7) prior to planting in year 1. Six rotations were tested: (i) continuous planting of a P. sojae-susceptible cultivar, (ii) continuous planting of a cultivar with high partial resistance to the pathogen, (iii) continuous planting of a cultivar with the Rps 1k gene, (iv) annual rotation of a susceptible with a resistant cultivar, (v) annual rotation of a partially resistant cultivar with a cultivar with the Rps 1k gene, and (vi) 4-year rotation of cultivars with Rps 1k, 1c, 3a, and 1k genes in year one, two, three, and four, respectively. The diversity of 121 isolates of P. sojae that were recovered by baiting from soil samples collected from the experiment were assessed using pathotyping and eight microsatellite markers, and compared with PR1. Changes in pathotype and multilocus genotypes (MLGs) were recorded at the second sampling date, indicating that P. sojae has the ability to evolve quickly. In total, 14 pathotypes and 21 MLGs were recovered over the 4-year experiment, and only 49 and 22% of the isolates had the same pathotype and MLG, respectively, as PR1. The number of isolates of P. sojae recovered varied among rotations, with more isolates recovered from rotations that included a cultivar with partial resistance. Gain of virulence was detected on Rps 1a, 1b, 1c, 1d, and 3a and was not dependent on rotation. Using simple-sequence repeat analysis, 10 alleles that were different from those of PR1 were detected throughout the 4-year period. Cultivar rotation affected the genetic structure of the P. sojae population. Recovery of isolates with different MLGs, genotypic diversity (G = 4.7), and gene diversity (UHe = 0.45) were greater under continuous rotation with partial resistance. Phytophthora root and stem rot causes economic losses in the north-central region of the United States annually. An improved understanding of the effect of Rps gene deployment on P. sojae diversity would lead to improved management practices and reduced losses. © 2014 The American Phytopathological Society.

Rivas-Rivera N.,University of the Republic of Uruguay | Eguren G.,University of the Republic of Uruguay | Carrasco-Letelier L.,National Agricultural Research Institute INIA | Munkittrick K.R.,University of New Brunswick
Ecotoxicology | Year: 2014

Sediment constitutes an important sink of endocrine disruptor compounds; however, the potential of sediments to act as a source of endocrine disruptors should be more extensively investigated. The main objective of this study was to determine whether exposure of immature common carp to Uruguay River sediments undergo physiological and endocrine alterations. The lower Uruguay River watershed supports intensive agricultural and forest production, receives municipal sewage discharge and industrial effluent, and a new large pulp mill was constructed in 2006. A 30-day semi-static assay was performed using sediments from four sites along the Uruguay River and compared with an unexposed group in dechlorinated water as a negative control. We focused on two upstream and two downstream sites of a new elemental chlorine free pulp mill. The results showed that plasma vitellogenin levels increased in fish along the river and significant differences were found between the exposed and unexposed groups. Condition factor and gonadosomatic index were not different; however, a significant difference in hepatosomatic index was observed in fish exposed to sediment from an industrial site. A significant reduction in primary spermatocyte accumulation was observed in the exposed group compared with that in the control group, and some individuals exposed to sediments from industrial sites presented with testis-ova. Our results suggest that Uruguay River sediments act as an important source of estrogenic compounds that could be responsible for the alterations observed. Future studies are needed to identify the causal agents and determine exposure routes. © 2014 Springer Science+Business Media.

Carrasco-Letelier L.,National Agricultural Research Institute INIA | Mendoza-Spina Y.,National Agricultural Research Institute | Branchiccela M.B.,Institute Investigaciones Biologicas Clemente Estable
Chemosphere | Year: 2012

Glyphosate-resistant soybean cultivation is expanding rapidly in Uruguay, with its land area having increased by 95 times during the past 10years. Because of the region's Neotropical conditions, insecticide use is required to ensure adequate soybean productivity. However, in areas shared by soybean crops and beekeepers - such as the southwestern zone of Uruguay (SWZU) - the use of insecticides can increase the risks of honeybee death and honey contamination. Uruguayan commercial and legal guidelines set out practices and field doses designed to prevent acute intoxication with insecticides. However, honeybees in the SWZU are predominantly a polyhybrid subspecies different from that used to set international reference values, and hence they may have a different acute toxicity response, thus rendering such precautions ineffective. The aim of this work was to assess the acute toxicity response of polyhybrid honeybees in the SWZU to cypermethrin (commercial formulation: Cipermetrina 25 Agrin®), chlorpyrifos (commercial formulation: Lorsban 48E®), and endosulfan (commercial formulation: Thionex 35®). Acute toxicity bioassays were conducted to determine the median lethal dose (LD50) of each insecticide for the honeybees. The results indicate that, compared with EU reference values, SWZU honeybees have a higher toxicological sensitivity to chlorpyrifos and endosulfan, and a lower toxicological sensitivity to cypermethrin, based on the commercial formulations tested. However, when these results were adjusted according to their field dose equivalents, only chlorpyrifos emerged as a potential problem for beekeeping, as the maximum recommended field dose of Lorsban 48E® for soybean crops in Uruguay is 23 times the corresponding LD50 for honeybees in the SWZU. © 2012 Elsevier Ltd.

Gutierrez L.,University of the Republic of Uruguay | German S.,National Agricultural Research Institute INIA | Pereyra S.,National Agricultural Research Institute INIA | Hayes P.M.,Oregon State University | And 16 more authors.
Theoretical and Applied Genetics | Year: 2015

Abstract: Diseases represent a major constraint for barley (Hordeum vulgare L.) production in Latin America. Spot blotch (caused by Cochliobolus sativus), stripe rust (caused by Puccinia striiformis f.sp. hordei) and leaf rust (caused by Puccinia hordei) are three of the most important diseases that affect the crop in the region. Since fungicide application is not an economically or environmentally sound solution, the development of durably resistant varieties is a priority for breeding programs. Therefore, new resistance sources are needed. The objective of this work was to detect genomic regions associated with field level plant resistance to spot blotch, stripe rust, and leaf rust in Latin American germplasm. Disease severities measured in multi-environment trials across the Americas and 1,096 SNPs in a population of 360 genotypes were used to identify genomic regions associated with disease resistance. Optimized experimental design and spatial modeling were used in each trial to estimate genotypic means. Genome-Wide Association Mapping (GWAS) in each environment was used to detect Quantitative Trait Loci (QTL). All significant environment-specific QTL were subsequently included in a multi-environment-multi-QTL (MEMQ) model. Geographical origin and inflorescence type were the main determinants of population structure. Spot blotch severity was low to intermediate while leaf and stripe rust severity was high in all environments. Mega-environments were defined by locations for spot blotch and leaf rust. Significant marker-trait associations for spot blotch (9 QTL), leaf (6 QTL) and stripe rust (7 QTL) and both global and environment-specific QTL were detected that will be useful for future breeding efforts.Key message: Multi-environment multi-QTL mixed models were used in a GWAS context to identify QTL for disease resistance. The use of mega-environments aided the interpretation of environment-specific and general QTL. © 2014, Springer-Verlag Berlin Heidelberg.

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