Desarrollo y aplicacion de PCR multiple para la deteccion simultanea de tres virus de ADN en camote (Ipomoea batatas L.)

El virus colusivo del camote (SPCV, genero Cavemovirus), el virus del aclaramiento de venas del camote (SPVC, genero Solendovirus) y el virus del enrollamiento de hojas del camote (SPLCV, genero Begomovirus) son virus con genoma de ADN, presentes en el camote en infecciones virales simples o multliples. La identificacion y deteccion de estos virus es complicada, ya que con frecuencia son asintomaticos y estan en concentraciones bajas en las plantas de camote. Se desarrollo una PCR multiple (mPCR) con el objetivo de lograr la deteccion simultanea de SPVC, SPVCV y SPLCV (y Begomovirus relacionados); para ellos se seleccionaron cebadores especificos para SPCV y SPVCV, y se utilizaron cebadores degenerados para Begomovirus (desarrollados por Li et al. 2004). Para la optimizacion de parametros se usaron plantas de camote con infecciones simples y mixtas. Se optimizo la concentracion de cebadores (0,1-0, 3uM), MgCl2 (2,5-8,0mM), dNTPs (0.2-0.8 mM), Taq-polimerasa (2-4U), parametros en el termociclador (temperatura de hibridacion de 48-62 oC y el numero de ciclos de 29-35), y la cantidad de acidos nucleidos (50-300 ng). Para validar le mPCR se uso plantas de camote de una coleccion de germplasma in vitro que estaban infectados con los virus en estudio y fueron confirmados por clonacion y secuenciamiento de las amplificaciones obtenidas. Los pares de cebadores especificos seleccionados para cada virus pudieron amplificar fragmentos de ADN en tamanos esperados, a una concentracion final de 0.16 uM para cebadores de SPCV y SPVCV, y 0.2uM para SPLCV. Ademas la concentracion de ADN adecuada esta entre 50-100 ng, con 30 ciclos termicos y 53 oC de temperatura de hibridacion. Este ensayo demostro ser simple, sensible y confiable para el diagnostico de rutina de SPCV, SPVCV y SPLCV (y Begomovirus relacionados). El ensayo de mPCR sera util para programas de cuarentena, como un metodos rapido y rentable para un gran numero de muestras

Cognitive-behavior therapy for children and adolescents with anxiety disorders:A meta-analysis of secondary outcomes

Anxiety-focused cognitive-behavioral therapy (CBT) effectively reduces anxiety in children and adolescents. An important remaining question is to what extent anxiety-focused CBT also affects broader outcome domains. Additionally, it remains unclear whether parental involvement in treatment may have impact on domains other than anxiety. A meta-analysis (nstudies = 42, nparticipants = 3239) of the effects of CBT and the moderating role of parental involvement was conducted on the following major secondary outcomes: depressive symptoms, externalizing behaviors, general functioning, and social competence. Randomized controlled trials were included when having a waitlist or active control condition, a youth sample (aged<19) with a primary anxiety disorder diagnosis receiving anxiety-focused CBT and reported secondary outcomes. Controlled effect sizes (Cohen's d) were calculated employing random effect models. CBT had a large effect on general functioning (-1.25[-1.59;0.90], nstudies = 17), a small to moderate effect on depressive symptoms (-0.31[-0.41;-0.22], nstudies = 31) and a small effect on externalizing behaviors (-0.23[-0.38;-0.09], nstudies = 12) from pre-to post-treatment. Effects remained or even further improved at follow-up. Social competence only improved at follow-up (nstudies = 6). Concluding, anxiety-focused CBT has a positive effect on broader outcome domains than just anxiety. Higher parental involvement seemed to have beneficial effects at follow-up, with improvements in general functioning and comorbid symptoms

Induction of somatic embryogenesis in recalcitrant sweetpotato (Ipomoea batatas L.) cultivars

Genetic transformation is considered as one of the most promising options for improvement of crop traits. Current transformation methods for sweetpotato depend on plant regeneration through organogenesis or somatic embryogenesis. Somatic embryogenesis and plant regeneration at a high frequency has been  restricted to a few sweetpotato varieties. Three auxins namely: 2,4-dichlorophenoxyacetic acid (2,4-D),  4-fluoroamphetamine (4-FA) and 4,5-trichlorophenoxyacetic acid (2,4,5-T) were investigated in this study for enhancing somatic embryogenesis from various plant organs of recalcitrant African sweetpotato cultivars.  2,4-D was found to be the best (p . 0.05) for induction of embryogenic callus. Cultivar Bwanjule had the highest  (20.2%) embryogenic callus frequency among the five African cultivars tested. The highest number of plants in this study was regenerated from the non-African cultivar variety Jonathan on media supplemented with 0.2 mg Zeatin. The emergence of roots from callus of recalcitrant Ugandan cultivars and the comparable high embryogenic responses in this work demonstrate the potential for regenerating plants from African  cultivars that have not been regenerated before. The regeneration of roots in this work could be useful for the initiation of root cultures. The most important application of this work is in genetic transformation of sweet potato, particularly for improvement of resistance to weevils.Key words: Embryogenesis, plant growth regulators, plant regeneration, Ipomoea batatas

EVALUATION OF BIOSSAYS FOR TESTING Bt SWEETPOTATO EVENTS AGAINST SWEETPOTATO WEEVILS

Sweetpotato weevil ( Cylas puncticollis ) Boheman is a serious pest throughout Sub-Saharan Africa region and is a big threat to sweetpotato cultivation. Ten transgenic sweetpotato events expressing Cry7Aa1, Cry3Ca1, and ET33-34 proteins from Bacillus thuringiensis (Bt) were evaluated for resistance against C. puncticollis. Four bioassays used were: (i) 1st instar larva in an artificial diet using root powder of transgenic events, (ii) whole root of transgenic events infested with female adults, (iii) root chip, and (iv) small root of transgenic events both infested with egg-plugs. DAS-ELISA analysis showed variation in Cry protein concentration among the events that ranged between 0.1 and 0.394 \ub5g g-1 of root fresh weight. The highest protein quantity was observed in the event carrying the ET33-34 transcript. In general, transgenic events had no significant effect on larval survival (P = 0.28) and pupal (P = 0.86) development, though maximum pupation of 96% was observed on event CIP410009.12 that had very low expression levels of cry3Ca1 gene. Root chips were prone to damage by fungi and desiccation especially during the first larval instar. The whole root bioassay had less handling injuries to weevils compared with other methods. However, it requires a large number of adult females for oviposition and roots per event to be tested to obtain efficacy results with statistical rigour. The small root egg plug bioassay required much fewer roots and experimental insects to assess larval mortality and development. The root chip method was the least desirable because of susceptibility to fungal and bacterial contamination. In addition, this method was the most labour intensive in terms of frequent replacement of root chips for weevil development. Hence, the most appropriate method for testing Bt efficacy in sweetpotato is the small root egg-plug bioassay. Nonetheless, none of the transgenic events tested provided weevil control probably because of low Cry protein expression in storage roots.La charan\ue7on de la patate douce ( Cylas puncticollis ) Boheman constitue une importante peste \ue0 travers la r\ue9gion d\u2019Afrique sub-saharienne et une grande contrainte \ue0 la production de la culture. Dix patate douce transg\ue9niques d\u2019\ue9v\ue9n\ue9ments exprimant les prot\ue9ines Cry7Aa1, Cry3Ca1, et ET33-34 des Bacillus thuringiensis (Bt) \ue9taient \ue9valu\ue9es pour r\ue9sistance contre C. puncticollis. Quatre bioassais utilis\ue9s \ue9taient: (i) une larve de premier instar dans une alimentation artificielle utilisant une poudre racinaire d\u2019\ue9v\ue9nement transg\ue9nique, (ii) racine entire d\u2019\ue9v\ue9nement transg\ue9niques infest\ue9s avec adultes femelles, (iii) un morceau racinaire, et (iv) petite racine d\u2019\ue9v\ue9nements transg\ue9niques tous infest\ue9s avec des oeufs. L\u2019analyse DAS-ELISA a montr\ue9 une variation dans la concentration en protein Cry parmi les \ue9v\ue9nements variant de 0.1 \ue0 0.394 \ub5g g-1 de poids de raciness fra\ueeches. La quqntit\ue9 la plus \ue9lev\ue9e de prot\ue9ines \ue9tait observ\ue9e dans l\u2019\ue9v\ue9nement portant le relev\ue9 ET33-34. En g\ue9n\ue9ral, les \ue9v\ue9nements transg\ue9niques n\u2019avaient aucun effet significatif sur la survie de larves (P = 0.28) et le d\ue9veloppement de la nimphe (P = 0.86), bien que la pupation maximale de 96% \ue9tait observ\ue9e sur l\u2019\ue9v\ue9nement CIP410009.12 qui avait un tr\ue8s bas niveau d\u2019expression du g\ue8ne cry3Ca1. Les morceaux de raciness \ue9taient susceptibles d\u2019\ueatre endommag\ue9s par les champignons et la dessication sp\ue9cialement durant le premier instar larvaire. La racine enti\ue8re bioassay \ue9tait moins affct\ue9e par la charan\ue7on et avait moins de blessures en comparaison \ue0 d\u2019autres m\ue9thodes. Par ailleurs, il n\ue9cessite un grand nombre d\u2019adultes femelles pour l\u2019oviposition et raciness par \ue9v\ue9nement devrant \ueatre test\ue9s pour obtenir des r\ue9sultats efficaces avec rigueur statistique. Le bioassai de petits oeufs racinaires ongt n\ue9cessit\ue9 un peu moins de raciness et insectes exp\ue9rimentaux pour \ue9valuer la mortalit\ue9 larvaire et leur d\ue9veloppement. La m\ue9thode de morceaux racinaires \ue9tait la moins desirable \ue0 cause de la contamination fongique et bact\ue9rienne. En plus, cette m\ue9thode exigeait plus de travail intensif en terme remplacement fr\ue9quent de morceaux de racines pour le d\ue9veloppement de charan\ue7on. Ainsi, la m\ue9thode la plus appropri\ue9e pour tester l\u2019efficacit\ue9 du Bt dans la patate douce est le bioassai de petis oeufs racinaires. N\ue9amoins, aucun des \ue9v\ue9nements transg\ue9niques test\ue9s n\u2019a fourni un control probable de la charan\ue7on suite \ue0 une basse expression de la protein Cry dans les raciness de stockage

Phytosanitary Interventions for Safe Global Germplasm Exchange and the Prevention of Transboundary Pest Spread: The Role of CGIAR Germplasm Health Units

The inherent ability of seeds (orthodox, intermediate, and recalcitrant seeds and vegetative propagules) to serve as carriers of pests and pathogens (hereafter referred to as pests) and the risk of transboundary spread along with the seed movement present a high-risk factor for international germplasm distribution activities. Quarantine and phytosanitary procedures have been established by many countries around the world to minimize seed-borne pest spread by screening export and import consignments of germplasm. The effectiveness of these time-consuming and cost-intensive procedures depends on the knowledge of pest distribution, availability of diagnostic tools for seed health testing, qualified operators, procedures for inspection, and seed phytosanitation. This review describes a unique multidisciplinary approach used by the CGIAR Germplasm Health Units (GHUs) in ensuring phytosanitary protection for the safe conservation and global movement of germplasm from the 11 CGIAR genebanks and breeding programs that acquire and distribute germplasm to and from all parts of the world for agricultural research and food security. We also present the challenges, lessons learned, and recommendations stemming from the experience of GHUs, which collaborate with the national quarantine systems to export and distribute about 100,000 germplasm samples annually to partners located in about 90 to 100 countries. Furthermore, we describe how GHUs adjust their procedures to stay in alignment with evolving phytosanitary regulations and pest risk scenarios. In conclusion, we state the benefits of globally coordinated phytosanitary networks for the prevention of the intercontinental spread of pests that are transmissible through plant propagation materials

Gene expression polymorphism underpins evasion of host immunity in an asexual lineage of the Irish potato famine pathogen

BACKGROUND: Outbreaks caused by asexual lineages of fungal and oomycete pathogens are a continuing threat to crops, wild animals and natural ecosystems (Fisher MC, Henk DA, Briggs CJ, Brownstein JS, Madoff LC, McCraw SL, Gurr SJ, Nature 484:186-194, 2012; Kupferschmidt K, Science 337:636-638, 2012). However, the mechanisms underlying genome evolution and phenotypic plasticity in asexual eukaryotic microbes remain poorly understood (Seidl MF, Thomma BP, BioEssays 36:335-345, 2014). Ever since the 19th century Irish famine, the oomycete Phytophthora infestans has caused recurrent outbreaks on potato and tomato crops that have been primarily caused by the successive rise and migration of pandemic asexual lineages (Goodwin SB, Cohen BA, Fry WE, Proc Natl Acad Sci USA 91:11591-11595, 1994; Yoshida K, Burbano HA, Krause J, Thines M, Weigel D, Kamoun S, PLoS Pathog 10:e1004028, 2014; Yoshida K, Schuenemann VJ, Cano LM, Pais M, Mishra B, Sharma R, Lanz C, Martin FN, Kamoun S, Krause J, et al. eLife 2:e00731, 2013; Cooke DEL, Cano LM, Raffaele S, Bain RA, Cooke LR, Etherington GJ, Deahl KL, Farrer RA, Gilroy EM, Goss EM, et al. PLoS Pathog 8:e1002940, 2012). However, the dynamics of genome evolution within these clonal lineages have not been determined. The objective of this study was to use a comparative genomics and transcriptomics approach to determine the molecular mechanisms that underpin phenotypic variation within a clonal lineage of P. infestans. RESULTS: Here, we reveal patterns of genomic and gene expression variation within a P. infestans asexual lineage by comparing strains belonging to the South American EC-1 clone that has dominated Andean populations since the 1990s (Yoshida K, Burbano HA, Krause J, Thines M, Weigel D, Kamoun S, PLoS Pathog 10e1004028, 2014; Yoshida K, Schuenemann VJ, Cano LM, Pais M, Mishra B, Sharma R, Lanz C, Martin FN, Kamoun S, Krause J, et al. eLife 2:e00731, 2013; Delgado RA, Monteros-Altamirano AR, Li Y, Visser RGF, van der Lee TAJ, Vosman B, Plant Pathol 62:1081-1088, 2013; Forbes GA, Escobar XC, Ayala CC, Revelo J, Ordonez ME, Fry BA, Doucett K, Fry WE, Phytopathology 87:375-380, 1997; Oyarzun PJ, Pozo A, Ordonez ME, Doucett K, Forbes GA, Phytopathology 88:265-271, 1998). We detected numerous examples of structural variation, nucleotide polymorphisms and loss of heterozygosity within the EC-1 clone. Remarkably, 17 genes are not expressed in one of the two EC-1 isolates despite apparent absence of sequence polymorphisms. Among these, silencing of an effector gene was associated with evasion of disease resistance conferred by a potato immune receptor. CONCLUSIONS: Our findings highlight the molecular changes underpinning the exceptional genetic and phenotypic plasticity associated with host adaptation in a pandemic clonal lineage of a eukaryotic plant pathogen. We observed that the asexual P. infestans lineage EC-1 can exhibit phenotypic plasticity in the absence of apparent genetic mutations resulting in virulence on a potato carrying the Rpi-vnt1.1 gene. Such variant alleles may be epialleles that arose through epigenetic changes in the underlying genes