A TaqMan real-time reverse transcription-PCR (real-time RT-PCR) procedure was developed, optimized, and compared with other routine methods to detect Potato virus Y (PVY) in dormant potato tubers. Three pairs of primers and probes were designed and evaluated for their suitability to facilitate the real-time RT-PCR detection of PVY for all strain groups including PVY O , PVY N , PVY N: O (0 PVY N-Wi ) and PVY NTN . Among the primer and probe combinations tested, the combination PVY-1 produced the lowest threshold cycle (Ct) value of 25.75. The procedure was further optimized by adjusting various parameters including primer/probe concentration, reaction volume, amplification cycles, and master mixes from different sources. The real-time RT-PCR was then employed to detect PVY from dormant tubers of different cultivars and potato fields, and the results were compared with those obtained from conventional RT-PCR, enzyme-linked immunosorbent assay (ELISA) on sprouts and grow-out testing. Out of 1,069 single-virus infected (PVY only) tubers tested, both formats of RT-PCR detected PVY in 52 samples, ELISA on sprouts in 45, ELISA on leaves in 54 and visual observations in 53. However, in 61 multiple-virus infected tubers tested, both formats of RT-PCR, and ELISA on both sprouts and leaves detected a similar number of positives, thus, making all the methods equally sensitive. Considering that ELISA requires sprouting of dormant potato tubers for PVY testing, growout testing takes approximately 6-8 weeks to obtain results, and conventional RT-PCR needs post-PCR processing, realtime RT-PCR offers a speedy alternative for large scale detection of PVY from dormant tubers. The method is therefore recommended for testing of PVY in potato tubers on a commercial scale in a diagnostic laboratory.Resumen Se desarrolló y optimizó un procedimiento de transcripción reversa de tiempo real de TaqMan (RT-PCR de tiempo real), y se comparó con otros métodos rutinarios para detectar al virus Y de la papa (PVY) en tubérculos de papa en dormancia. Se designaron tres pares de iniciadores y sondas y se evaluaron para su conveniencia para facilitar la detección de PVY por RT-PCR de tiempo real para todos los grupos de variantes, incluyendo PVY O , PVY N , PVY N:O (0PVY N-Wi ) y PVY NTN . Entre las combinaciones probadas de iniciadores y sondas, la combinación de PVY-1 produjo el valor del ciclo del umbral más bajo (Ct) con 25.75. El procedimiento se optimizó mas adelante mediante el ajuste de varios parámetros, incluyendo la concentración iniciador/ sonda, volumen de reacción, ciclos de amplificación, y muestras maestras de diferentes fuentes. Se empleó posteriormente el RT-PCR de tiempo real para detectar PVY de tubérculos en dormancia de diferentes variedades y campos de papa, y se compararon los resultados con los obtenidos por la RT-PCR convencional, la serología con enzimas conjugadas (ELISA), en pruebas de brotes y en crecimiento. De 1069 tubérculos probados infectados con un solo virus (PVY solamente), ambos formatos de RT-PCR detectaron P...

Forest harvesting of increasing intensities is expected to have intensifying impacts on the genetic diversity and population structure of postharvest naturally regenerated stands by affecting the magnitude of evolutionary processes, such as genetic drift, gene flow, mating system, and selection. We have tested this hypothesis for the first time by employing widely distributed boreal white spruce (Picea glauca) as a model and controlled, replicated experimental harvesting and regeneration experiment at the EMEND project site (http://www.emendproject.org). We used two approaches. First, genetic diversity and population structure of postharvest natural regeneration after five harvesting treatments (green tree retention of 75%, 50%, 20%, and 10%, and clearcut) were assessed and compared with those of the unharvested control (pristine preharvest old‐growth) in two replicates each of conifer‐dominated (CD) and mixed‐wood (MW) forest, using 10 (six EST (expressed sequence tag) and four genomic) microsatellite markers. Second, genetic diversity and population structure of preharvest old‐growth were compared with those of postharvest natural regeneration after five harvesting treatments in the same treatment blocks in one replicate each of CD and MW forests. Contrary to our expectations, genetic diversity, inbreeding levels, and population genetic structure were similar between unharvested control or preharvest old‐growth and postharvest natural regeneration after five harvesting treatments, with clearcut showing no negative genetic impacts. The potential effects of genetic drift and inbreeding resulting from harvesting bottlenecks were counterbalanced by predominantly outcrossing mating system and high gene flow from the residual and/or surrounding white spruce. CD and MW forests responded similarly to harvesting of increasing intensities. Simulated data for 10, 50, and 100 microsatellite markers showed the same results as obtained empirically from 10 microsatellite markers. Similar patterns of genetic diversity and population structure were observed for EST and genomic microsatellites. In conclusion, harvesting of increasing intensities did not show any significant negative impact on genetic diversity, population structure, and evolutionary potential of white spruce in CD and MW forests. Our first of its kind of study addresses the broad central forest management question how forest harvesting and regeneration practices can best maintain genetic biodiversity and ecosystem integrity.

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Fageria, M. S., Singh, M., Nanayakkara, U., Pelletier, Y., Nie, X., and Wattie, D. 2013. Monitoring current-season spread of Potato virus Y in potato fields using ELISA and real-time RT-PCR. Plant Dis. 97:641-644.The current-season spread of Potato virus Y (PVY) was investigated in New Brunswick, Canada, in 11 potato fields planted with six different cultivars in 2009 and 2010. In all, 100 plants selected from each field were monitored for current-season PVY infections using enzymelinked immunosorbent assay (ELISA) and real-time reverse-transcription polymerase chain reaction (RT-PCR) assay. Average PVY incidence in fields increased from 0.6% in 2009 and 2% in 2010 in the leaves to 20.3% in 2009 and 21.9% in 2010 in the tubers at the time of harvest. In individual fields, PVY incidence in tubers reached as high as 37% in 2009 and 39% in 2010 at the time of harvest. Real-time RT-PCR assay detected more samples with PVY from leaves than did ELISA. A higher number of positive samples was also detected with real-time RT-PCR from growing tubers compared with the leaves collected from the same plant at the same sampling time. PVY incidence determined from the growing tubers showed a significant positive correlation with the PVY incidence of tubers after harvest. Preharvest testing provides another option to growers to either top-kill the crop immediately to secure the seed market when the PVY incidence is low or leave the tubers to develop further for table or processing purposes when incidence of PVY is high.In recent years, increases in Potato virus Y (PVY) incidence in potato fields across North America have been observed by potato growers and researchers (6,8,13). As a consequence, PVY has become a serious concern for the seed potato industry, and the rejection of seed lots submitted for certification is not uncommon (10). PVY O is the most prevalent strain in New Brunswick, Canada. Other emerging strains in the province include the tobacco veinal necrosis strain (PVY N ), potato tuber necrosis strain (PVY NTN ), and recombinant N:O strain (PVY N:O or PVY N-Wi ) (12). The practical way to manage PVY is by testing for the whole complex of PVY regardless of the strain group and using seed potato with minimum PVY content (8). One of the major challenges seed potato growers in New Brunswick and other jurisdictions facing is current-season spread of PVY within and between fields by aphids (8). This PVY spread can be effectively managed by planting virus-free seed potato and reducing current-season spread of PVY by using appropriate crop management practices, particularly applications of mineral oil (2,4) and combined applications of mineral oil and insecticides (9).Different methods are currently used to detect the presence of PVY. Testing of tubers is traditionally performed using grow-out tests where plants are observed for symptoms in symptom-producing cultivars (17). The grow-out observation in symptomless cultivars is usually supplemented with enzyme-linked immunosorbent assay (ELISA) testing of leaves. However, th...