Investigating the inoculum dynamics of Cladosporium on the surface of raspberry fruits and in the air

Raspberry production is under threat from the emerging fungal pathogenic genus Cladosporium. We used amplicon-sequencing, coupled with qPCR, to investigate how fruit age, fruit location within a polytunnel, polytunnel location and sampling date affected the fruit epiphytic microbiome. Fruit age was the most important factor impacting the fungal microbiome, followed by sampling date and polytunnel location. In contrast, polytunnel location and fruit age were important factors impacting the bacterial microbiome composition, followed by the sampling date. The within-tunnel location had a small significant effect on the fungal microbiome and no effect on the bacterial microbiome. As fruit ripened, fungal diversity increased and the bacterial diversity decreased. Cladosporium was the most abundant fungus of the fruit epiphytic microbiome, accounting for nearly 44% of all fungal sequences. Rotorod air samplers were used to study how the concentration of airborne Cladosporium inoculum (quantified by qPCR) varied between location (inside and outside the polytunnel) and time (daytime vs. nighttime). Quantified Cladosporium DNA was significantly higher during the day than the night and inside the polytunnel than the outside. This study demonstrated the dynamic nature of epiphytic raspberry fruit microbiomes and airborne Cladosporium inoculum within polytunnels, which will impact disease risks on raspberry fruit.Biotechnology and Biological Sciences Research Council, Grant/Award Number: BB/T509073/

Fast purification of the filamentous Potato virus Y using monolithic chromatographic supports

International audienceObtaining pure virus suspensions is an essential step in many applications, such as vaccine production, antibody production, sample preparation for procedures requiring enrichment in viruses and other in vitro characterizations. Purification procedures usually consist of complex, long lasting and tedious protocols involving several ultracentrifugation steps. Such complexity is particularly evident in the case of plant viruses, where the virus needs to be isolated from the complex plant tissue matrix. Convective Interaction Media (CIM) monoliths are chromatographic supports that have been successfully utilized for the purification of large bio-molecules such as viruses, virus like particles and plasmids from various matrixes. In this study a CIM monolith based procedure was developed for the fast purification from plant tissue of the filamentous Potato virus Y(PVY) (virion size, 740 nm x 11 nm), which is one of the most important plant viruses causing great economical losses in potato production. Different mobile phases, chemistries and sample preparation strategies were tested. The presence of the virus in the chromatographic fraction was monitored with viral RNA quantification (RT-qPCR), viral protein purity estimation (SDS-PAGE) and viral particle integrity observation (transmission electron microscopy). The optimized procedure involves initial clarification steps, followed by chromatography using CIM quaternary amine (QA) monolithic disk column. In comparison to classical purification procedure involving ultracentrifugation through sucrose and caesium chloride, the developed CIM-QA purification achieved comparable yield, concentration and purity. Plant nucleic acids were successfully removed. Purification showed good reproducibility and moreover it reduced the purification time from four working days required for classic purification to a day and a half. This is the first study where a filamentous virus was purified using CIM monolithic supports. The advantages of this new purification procedure make it an attractive method in serological diagnostic tool production, which requires purified viruses for the immunization step. Moreover, the outcome of this study could serve as starting point for the improvement of the purification methods of other important filamentous viruses. (C) 2012 Elsevier B.V. All rights reserved

From Endophyte Community Analysis to Field Application: Control of Apple Canker (<i>Neonectria ditissima</i>) with <i>Epicoccum nigrum</i> B14-1

Apple canker, caused by Neonectria ditissima (Tul. and C. Tul.) Samuels and Rossman, is a major disease of apples (Malus domestica) worldwide. N. ditissima infects through natural and artificial wounds. Infected wood develops canker lesions which girdle branches and main stems causing reduced yield and tree death. N. ditissima is difficult to control; removal of inoculum (cankers) is expensive and therefore seldom practiced, whilst effective chemical products are being banned and no biocontrol products have been found to be effective against N. ditissima. This study used cues from a previous apple endophyte community analysis to isolate and test fungal endophytes belonging to the genus Epicoccum as potential endophytic biocontrol agents. Epicoccum nigrum B14-1, isolated from healthy apple trees, antagonised N. ditissima in vitro and reduced the incidence of N. ditissima infections of leaf scars by 46.6% and pruning wounds by 5.3% in field conditions at leaf fall. Autumn application of B14-1 conidia increased E. nigrum abundance in apple tissues at 10–20 days post-inoculation by ca. 1.5×, but this returned to control levels after one year. E. nigrum B14-1 did not cause detrimental effects on apple foliage, buds, fruit, or growth and could therefore present a new biocontrol agent to manage N. ditissima in commercial apple production

Molecular evolution and phylogeography of Potato virus Y based on the CP gene

Potato virus Y (PVY) is an important plant pathogen with a wide host range that includes, among others, potato, tobacco, tomato and pepper. The coat protein (CP) of PVY has been commonly used in phylogenetic studies for strain classification. In this study, we used a pool of 292 CP sequences from isolates collected worldwide. After detecting and removing recombinant sequences, we applied Bayesian techniques to study the influence of geography and host species in CP population structure and dynamics. Finally, we performed selection and covariation analyses to identify specific amino acids involved in adaptation. Our results show that PVY CP diversification is significantly accounted for by both geographical and host-driven adaptations. Amino acid positions detected as positively selected concentrate in the N-terminal region of the protein. Some of these selected positions may discriminate among strains, and to a much lesser extent, between potato and non-potato isolates.This research was supported by the Spanish Ministry of Science and Innovation grant BFU2009-06993 to S. F. E. and J. M. C was supported by the JAE-Doc program from CSIC. We thank Mario A. Fares for fruitful bioinformatics guidance. PVY isolates used in this work were available thanks to PVYwide Organization members (Carole Balmelli, Peter Dedic, Stewart Gray, Christophe Lacomme, Kestin Lindner, Benoit Moury, Maja Ravnikar, Laura Tomassoli, Rene Van der Vlugt, and Christina Varaveri). The authors do not have a commercial or any other association that might pose a conflict of interest.Cuevas Torrijos, JM.; Delaunay, A.; Rupar, M.; Jacquot, E.; Elena Fito, SF. (2012). Molecular evolution and phylogeography of Potato virus Y based on the CP gene. Journal of General Virology. 93(11):2496-2501. https://doi.org/10.1099/vir.0.044347-0S24962501931

Fluorescently tagged[i] potato virus Y[/i]: a versatile tool for functional analysis of plant-virus interactions

BGPI : équipe 6International audiencePotato virus Y (PVY) is an economically important plant virus that infects Solanaceous crops such as tobacco and potato. To date, studies into the localization and movement of PVY in plants have been limited to detection of viral RNA or proteins ex vivo. Here, a PVY N605 isolate was tagged with green fluorescent protein (GFP), characterized and used for in vivo tracking. In Nicotiana tabacum cv. Xanthi, PVY N605-GFP was biologically comparable to nontagged PVY N605, stable through three plant-to-plant passages and persisted for four months in infected plants. GFP was detected before symptoms and fluorescence intensity correlated with PVY RNA concentrations. PVY N605-GFP provided in vivo tracking of long-distance movement, allowing estimation of the cell-to-cell movement rate of PVY in N. tabacum cv. Xanthi (7.1 +/- 1.5 cells per hour). PVY N605-GFP was adequately stable in Solanum tuberosum cvs. Desiree and NahG-Desiree and able to infect S. tuberosum cvs. Bintje and Bea, Nicotiana benthamiana, and wild potato relatives. PVY N605-GFP is therefore a powerful tool for future studies of PVY-host interactions, such as functional analysis of viral and plant genes involved in viral movement