Over the past few years, the use of RNA interference (RNAi) for insect pest management has attracted considerable interest in academia and industry as a pest-specific and environment-friendly strategy for pest control. For the success of this technique, the presence of core RNAi genes and a functional silencing machinery is essential. Therefore, the aim of this study was to test whether the Neotropical brown stinkbug Euschistus heros has the main RNAi core genes and whether the supply of dsRNA could generate an efficient gene silencing response. To do this, total mRNA of all developmental stages was sequenced on an Illumina platform, followed by a de novo assembly, gene annotation and RNAirelated gene identification. Once RNAi-related genes were identified, nuclease activities in hemolymph were investigated through an ex vivo assay. To test the functionality of the siRNA machinery, E. heros adults were microinjected with ~28 ng per mg of insect of a dsRNA targeting the V-ATPase-A gene. Mortality, relative transcript levels of V-ATPase-A, and the expression of the genes involved in the siRNA machinery, Dicer-2 (DCR-2) and Argonaute 2 (AGO-2), were analyzed. Transcriptome sequencing generated more than 126 million sequenced reads, and these were annotated in approximately 80,000 contigs. The search of RNAi-related genes resulted in 47 genes involved in the three major RNAi pathways, with the absence of sid-like homologous. Although ex vivo incubation of dsRNA in E. heros hemolymph showed rapid degradation, there was 35% mortality at 4 days after treatment and a significant reduction in V-ATPase-A gene expression. These results indicated that although sid-like genes are lacking, the dsRNA uptake mechanism was very efficient. Also, 2-fold and 4-fold overexpression of DCR-2 and AGO-2, respectively, after dsRNA supply indicated the activation of the siRNA machinery. Consequently, E. heros has proven to be sensitive to RNAi upon injection of dsRNA into its hemocoel. We believe that this finding together with a publically available transcriptome and the validation of a responsive RNAi machinery provide a starting point for future field applications against one of the most important soybean pests in South America. The Neotropical brown stink bug (BS), Euschistus heros (Hemiptera: Pentatomidae), is one of the most important Pentatomidae pests in South America 1 , especially in soybean (Glycine max) with a reduction in seed quality and yield 2. Stink bugs use their piercing/sucking mouthparts to inject enzymes into the plant tissues to digest plant components and remove pre-digested fluids 3. Although rarely reported before the 70s 2,4 , since then population outbreaks 2,5 and rapid population growth have allowed expansion of the range of E. heros to all the major South American soybean production regions, including Brazil 2 , Paraguay 2 , and Argentina 6 .

RNA interference (RNAi) technology has been used in the development of approaches for pest control. The presence of some essential genes, the so-called “core genes,” in the RNAi machinery is crucial for its efficiency and robust response in gene silencing. Thus, our study was designed to examine whether the RNAi machinery is functional in the South American (SA) fruit fly Anastrepha fraterculus (Diptera: Tephritidae) and whether the sensitivity to the uptake of double-stranded RNA (dsRNA) could generate an RNAi response in this fruit fly species. To prepare a transcriptome database of the SA fruit fly, total RNA was extracted from all the life stages for later cDNA synthesis and Illumina sequencing. After the de novo transcriptome assembly and gene annotation, the transcriptome was screened for RNAi pathway genes, as well as the duplication or loss of genes and novel target genes to dsRNA delivery bioassays. The dsRNA delivery assay by soaking was performed in larvae to evaluate the gene-silencing of V-ATPase , and the upregulation of Dicer-2 and Argonaute-2 after dsRNA delivery was analyzed to verify the activation of siRNAi machinery. We tested the stability of dsRNA using dsGFP with an in vitro incubation of larvae body fluid (hemolymph). We identified 55 genes related to the RNAi machinery with duplication and loss for some genes and selected 143 different target genes related to biological processes involved in post-embryonic growth/development and reproduction of A. fraterculus . Larvae soaked in dsRNA (dsV-ATPase) solution showed a strong knockdown of V-ATPase after 48 h, and the expression of Dicer-2 and Argonaute-2 responded with an increase upon the exposure to dsRNA. Our data demonstrated the existence of a functional RNAi machinery in the SA fruit fly, and we present an easy and robust physiological bioassay with the larval stages that can further be used for screening of target genes at in vivo organisms’ level for RNAi-based control of fruit fly pests. This is the first study that provides evidence of a functional siRNA machinery in the SA fruit fly.

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Water deficit is responsible for a negative impact on agricultural systems. Several physiological, biochemical, and molecular processes are initiated as a response to water-deficit stress.Quantifying the differential expression of the genes involved in the response to water-deficit stress plays a key role in the development of molecular strategies for engineering water-stress tolerance in plants.Therefore, the present study aims to evaluate the expression of key genes involved in the water-deficit response process in peach trees cv. Chimarrita grafted onto two different rootstocks. The experimental design was completely randomized, with four treatments that correspond to the evaluation periods: day zero (control), the fourth, seventh, and ninth days of water-deficit stress. Leaf samples of each Chimarrita/rootstock combination were analyzed separately. The PLANT BREEDING -ArticleDifferential expression of the genes involved in responses to water-deficit stress in peach trees cv. Chimarrita grafted onto two different rootstocks expression of the genes related to ethylene biosynthesis, osmotic adjustment, and carbohydrate metabolism, namely ACC oxidase, GTL, SDH, SIP1, SOT1, S6PDH, and P5CS was quantified by real-time quantitative polymerase chain reaction (RT-qPCR). A differential response was observed in the expression of the SDH, GTL, P5CS, and SIP1 genes between Chimarrita/rootstock (Aldrighi 1 and Tsukuba 2) combinations. In both combinations of grafting, the S6PDH gene presented the highest level of expression at the fourth day of stress.These results show that genes related to carbohydrate and proline metabolism are important molecular markers to identify variability to water-deficit tolerance in Prunus persica.

Strategies which allow the unequivocal identification of genes expressed in response to different treatments and/or stress conditions are important to obtain reliable results in plant physiology studies. However, in order to analyze the expression levels of target genes through RT-qPCR technique, it is essential to use model genes with uniform expression levels under specific experimental conditions. The present study aimed at investigating the stability of reference genes in peach leaves cultivar scion Chimarrita grafted onto 'Aldrighi 1' and 'Tsukuba 2' rootstock, subjected to water deficit for a 9-day period. The experiment was conducted in a completely randomized design with four treatments that correspond the evaluations period: zero (control), 4 th , 7 th , and 9 th stress day of water deficit. For each treatment three biological replicates was used. Eight reference genes were analyzed, among them: ACT, CYP2, Ef-1α, GAPDH, TUA, TUB, UBQ10 and 18SrRNA. For the normalization and validation of RT-qPCR data were used the four major software programs currently available: geNorm, NormFinder, BestKeeper, and Comparative ΔC T. The results showed that there was no influence of different plant grafting combinations in the expression of all reference genes evaluated, yet genes TUA and CYP2 had the most stable expression in the leaves of the peach plants under water deficit for 9-d, whereas genes Ef-1α and ACT were the least stable ones for the same stress conditions.

WATER DEFICIT AFFECTS GAS EXCHANGE IN PEACH TREES CULTIVAR CHIMARRITA GRAFTED ONTO DIFFERENT ROOTSTOCKS  LETICIA NEUTZLING RICKES, ELSA KUHN KLUMB, CARLOS DAVI SANTOS E SILVA, MARCOS ANTONIO BACARIN, VALMOR JOÃO BIANCHI 1 Dr.(a) em Fisiologia Vegetal pela Universidade Federal de Pelotas, Instituto de Biologia, Departamento de Botânica, Campus Universitário, Capão do Leão, RS – Brasil, E-mail: leticiarickes@hotmail.com, carlosdavi_santos@yahoo.com.br 2 Mestra em Fisiologia Vegetal pela Universidade Federal de Pelotas, Instituto de Biologia, Departamento de Botânica, Campus Universitário, Capão do Leão, RS – Brasil, E-mail: elsakk91@yahoo.com.br 3 Prof. Dr. Universidade Federal de Pelotas, Instituto de Biologia, Departamento de Botânica, Campus Universitário, Capão do Leão, RS – Brasil, E-mail: bacarin@ufpel.edu.br, valmorjb@yahoo.com  1 ABSTRACT The aim was to investigate the effect of the water deficit on the gas exchange of Chimarrita plants grafted onto five different rootstocks (Tsukuba 1, Tsukuba 2, Tsukuba 3, Aldrighi 1, and Selection UFPel 0402), i.e., five combinations ‘Chimarrita’/rootstocks. The factorial scheme consisted of two water conditions (control and water deficit) and eight days of evaluation (0, 1st, 2nd, 3rd, 4th, 6th, 7th, and 9th, the latter two were the recovery period). Gas exchange was assessed with the aid of the portable CO2 infrared analyzer. Results were subjected to the analysis of variance (p≤0.05) by using Tukey’s test in order to compare the means between treatments at each day of stress for each ‘Chimarrita’/rootstocks combination. It was found that the different ‘Chimarrita’/rootstocks combinations showed a distinct physiological behavior regarding the initial water-deficit tolerance. Plants consisting of the combination ‘Chimarrita/Aldrighi 1’ had the highest initial water-deficit tolerance. The reduction in the net assimilation rate in face of the water deficit is not only related to the stomatal limitation, suggesting that there may also be non-stomatal limitations. Seventy-two hours after providing irrigation back to plants, all combinations evaluated showed an overall resilience of the physiological performance. Keywords: Prunus persica, abiotic stress, stomatal conductance, net assimilation rate.  RICKES, L. N.; KLUMB, E. K.; SILVA, C. D. S. e; BACARIN, M. A.; BIANCHI, V. J.DÉFICIT HÍDRICO AFETA AS TROCAS GASOSAS EM PESSEGUEIRO CULTIVAR CHIMARRITA ENXERTADA SOBRE DIFERENTES PORTAENXERTOS   2 RESUMO O objetivo foi avaliar o efeito do déficit hídrico sobre as trocas gasosas em plantas da cultivar Chimarrita enxertada sobre cinco diferentes portaenxertos (Tsukuba 1, Tsukuba 2, Tsukuba 3, Aldrighi 1 e Seleção UFPel 0402), ou seja, cinco combinações ‘Chimarrita’/portaenxertos. O esquema fatorial foi composto de duas condições hídricas (controle e déficit hídrico) e oito dias de avaliação (0, 1º, 2º, 3º, 4º, 6º, 7º e 9º, os dois últimos sendo período de recuperação). As trocas gasosas foram avaliadas com um analisador portátil a infravermelho de CO2. Os resultados foram submetidos a análise de variância (p≤0,05), utilizando-se o teste de Tukey para comparação de médias entre os tratamentos em cada dia de estresse de cada combinação ‘Chimarrita’/portaenxertos. Verificou-se que as diferentes combinações de ‘Chimarrita’/portaenxertos apresentaram comportamento fisiológico diferencial para a tolerância inicial ao déficit hídrico. Plantas compostas pelas combinações de enxertia entre ‘Chimarrita’/Adrighi 1’ foram as que apresentaram a maior tolerância inicial ao déficit hídrico. A redução da taxa assimilatória líquida perante o déficit hídrico, não está relacionada principalmente à limitação estomática, sugerindo-se que ocorrem também limitações não estomáticas. Após 72h do retorno da irrigação das plantas, todas as combinações avaliadas demonstraram total capacidade de recuperação do desempenho fisiológico. Palavras-chave: Prunus persica, estresse abiótico, condutância estomática, taxa assimilatória líquida.

-The state of Rio Grande do Sul is the largest peach productor in Brazil; however, it still possesses poor yield values when compared with other states. One of the problems associated with this is the occurrence of soils with drainage problems, mainly in Pelotas region, which depending on the year period, may undergo water deficit or flooding situations in the great majority of the years, which harm the crop development and yield. Among the harmful effects caused by these stresses stand out, the decrease in the net assimilation rate, closure of stomata, reduction of the cell activities, production of reactive oxygen species, membrane and protein destabilization. Thus, the aim of this study was to investigate in what magnitude of the gaseous exchange parameters of Prunus spp. rootstocks are influenced under drought and flood stress. In the experiment, gas exchange parameters net photosynthetic rate (A) stomata conductance (gs), intercellular carbon (Ci) and transpiration (E)] were evaluated in three Prunus spp. rootstocks (peach tree 'Capdeboscq' and plum trees 'Julior' and 'Marianna 2624') under three water conditions (control, water deficit and soil flooding) for seven days. The three rootstocks proved more susceptible to flooding than to water deficit, only varying in response time, which is intrinsic to each genotype, and that there is a genetic variability for the tolerance to the studied stresses. The variation on physiological response to the water deficit stress was later in both evaluated genotypes. However, in general, 'Julior' presented greater tolerance to both stresses when compared to the other rootstocks evaluated. Such information is useful to help in the choice of rootstocks for plant production, in the orchard management and for plant breeding programs, aiming at the selection of new genotypes with increased tolerance to these water stresses. Index terms: water stress, photosynthesis, peach tree, plum tree. PARÂMETROS DE TROCAS GASOSAS EM DIFERENTES PORTA ENXERTOS DE Prunus SPP. SUBMETIDOS AO ESTRESSE POR SECA E ALAGAMENTORESUMO-O Estado do Rio Grande do Sul é o detentor da maior produção de pêssegos do Brasil; entretanto, ainda possui valores baixos de produtividade, quando comparado com outros estados. Um dos problemas associados a isto é a ocorrência de solos com problemas de drenagem, principalmente na região de Pelotas que, dependendo do período do ano, podem sofrer situações de déficit hídrico ou de alagamento, na grande maioria dos anos, que prejudicam o desenvolvimento e a produtividade da cultura. Dentre os efeitos prejudiciais causados por estes estresses, destacam-se a diminuição na taxa assimilatória líquida, fechamento de estômatos, a redução das atividades celulares, a produção de espécies reativas de oxigênio e a desestabilização de membranas e de proteínas. Desta forma, o objetivo deste trabalho foi investigar em que magnitude os parâmetros de trocas gasosas de porta-enxertos de Prunus spp. são influenciados sob estresse por seca e alagamento. No experimento, foram avali...

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