Evaluación en campo de la resistencia de líneas transgénicas de ciruelo europeo (Prunus domestica L.) a Plum pox virus

La sharka es la enfermedad viral más grave de frutales de hueso. Afecta especialmente a albaricoquero, melocotonero y ciruelo produciendo deformación, pérdida de calidad y caída prematura de los frutos. Esto causa importantes pérdidas económicas en la industria de frutales de hueso. El agente causante de la sharka es Plum pox virus (PPV). PPV se transmite a través de pulgones y mediante injerto, siendo el transporte y uso ilegal de material infectado la principal causa de la dispersión de la enfermedad a larga distancia. Existen programas de mejora encaminados a la obtención de variedades de prunus resistentes a PPV. Sin embargo, el largo tiempo que requiere esta técnica unido a la naturaleza poligénica de la resistencia y a la falta de fuentes de resistencia compatibles han hecho que se obtengan escasos éxitos y únicamente en albaricoquero. Una técnica alternativa o complementaria es el uso de la resistencia derivada del patógeno (PDR) mediante la obtención de plantas transgénicas que expresan un gen viral. Esto confiere a la planta resistencia frente al virus del cual procede el gen y frente a virus relacionados. En este sentido, Scorza et al. (1994) obtuvieron líneas de ciruelo europeo (Prunus domestica L.) que expresan el gen de la proteína de la cápsida de PPV. En condiciones de invernadero estas líneas transgénicas mostraron distintos grados de sensibilidad a la infección por PPV, con excepción de la línea C5 que demostró ser altamente resistente a la infección natural (a través de pulgones) de PPV. Cuando las plantas CS se inocularon mediante injerto, se desarrolló una infección muy leve y sólo en zonas localizadas de las plantas

La Sharka tipo Marcus (M), una grave enfermedad del melocotonero

Plum pox virus (PPV) causa la enfermedad de la sharka en frutales de hueso. Se trata de la enfermedad viral más grave del albaricoquero y ciruelo y también del melocotonero, cuando están presentes ciertos aislados del virus denominados Marcus (M) o tipos recombinantes con M (Rec). En España sólo se han dispersado aislados comunes o D del virus, pero existe riesgo real de introducción de aislados M agresivos en melocotonero, que podrían incidir gravemente en la producción temprana española. De hecho, un foco de PPV-M fue detectado y erradicado en Aragón en 2002. Los aislados Marcus se están dispersando por la cuenca del Mediterráneo y tienen una incidencia importante en ltalia y Francia, países con los que se mantiene un frecuente tráfico de material vegetal. La precaución y la vigilancia de fruticultores, viveristas y técnicos, debiera poderevitarla introducción y dispersión de aislados agresivos PPV-M en España. Se dispone técnicamente de sistemas de diagnóstico específicos que permiten la detección específica de cepas Marcus de forma fiable. Estos métodos, disponibles comercialmente, han sido transferidos a los Servicios de Sanidad Vegetal de las distintas Comunidades Autónomas

Biological control of strawberry soil-borne pathogens Macrophomina phaseolina and Fusarium solani, using Trichoderma asperellum and Bacillus spp.

In south-western Spain, Macrophomina phaseolina and Fusarium solani were found to be associated in strawberry plants with, respectively, charcoal rot, and crown and root rot symptoms. For management of both fungal diseases, the antagonistic effects of two commercial formulations, one based on Trichoderma asperellum T18 strain (Prodigy®) and the other on Bacillus megaterium and B. laterosporus (Fusbact®), were evaluated in vitro and under controlled environment and field conditions. Two inoculation methods (root-dipping and soil application) and two application times (pre- and post-pathogen inoculation, as preventive and curative treatments, respectively) were assessed. Dual plate confrontation experiments demonstrated the antagonistic effects of T. asperellum and Bacillus spp. by inhibiting radial growth of M. phaseolina and F. solani by more than 36%. Preventive application of T. asperellum by root-dipping reduced the incidence of charcoal rot (up to 44% in a growth chamber and up to 65% under field conditions) and also reduced disease progression, the percentage of crown necrosis, as well as the level of infection measured as ng of pathogen DNA g-1 plant by quantitative real-time PCR. This treatment was also the most effective for reduction of crown and root rot caused by F. solani (up to 100% in a greenhouse and up to 81% under field conditions). These results were nearly comparable with the control achieved using chemical fungicides. The Bacillus spp.-based formulation was also effective for control of charcoal rot and showed variable results for control of F. solani, depending on the growth conditions

Direct sample preparation methods for the detection of Plum pox virus by real-time RT-PCR

Direct systems to process plant materials allowed high-throughput testing of Plum pox virus (PPV) by real-time reverse transcription (RT)-PCR without nucleic acids purification. Crude plant extracts were diluted in buffer or spotted on membranes to be used as templates. Alternatively, immobilized PPV targets were amplified from fresh sections of plant tissues printed or squashed onto the same supports, without extract preparation. Spot real-time RT-PCR was validated as a PPV diagnostic method in samples collected during the dormancy period and showed high sensitivity (93.6%), specificity (98.0%), and post-test probability (97.9%) towards sharka disease. In an analysis of 2919 Prunus samples by spot real-time RT-PCR and DASI-ELISA 90.8% of the results coincided, demonstrating high agreement (k = 0.77 +/- 0.01) between the two techniques. These results validate the use of immobilized PPV targets and spot real-time RT-PCR as screening method for large-scale analyses. [Int Microbiol 2009; 12(1): 1-6

Field trials of plum clones transformed with the Plum pox virus coat protein (PPV-CP) gene

Transgenic clones C2, C3, C4, C5, C6, and PT-6, of plum (Prunus domestica L.) transformed with the coat protein (CP) gene of Plum pox virus (PPV), PT-23 transformed with marker genes only, and nontransgenic B70146 were evaluated for sharka resistance under high infection pressure in field trials in Poland and Spain. These sites differed in climatic conditions and virus isolates. Transgenic clone C5 showed high resistance to PPV at both sites. None of the C5 trees became naturally infected by aphids during seven (Spain) or eight (Poland) years of the test, although up to 100% of other plum trees (transgenic clones and nontransgenic control plants) grown in the same conditions showed disease symptoms and tested positively for PPV. Although highly resistant, C5 trees could be infected artificially by chip budding or via susceptible rootstock. Infected C5 trees showed only a few mild symptoms on single, isolated shoots, even up to 8 years post inoculation. These results clearly indicate the long-term nature and high level of resistance to PPV obtained through genetically engineered resistance

Differences in coping strategies among young adults and the elderly with cancer

Background: Coping with cancer and the oncologist-patient relationship can vary depending on the patient's age. Our aim is to examine and compare young and elderly adults with non‐metastatic, resected cancer. Methods: Two groups of patients were selected, young ( 70) with a diagnosis of non‐metastatic, resected cancer requiring adjuvant chemotherapy from a pre‐exiting, national database (NEOCOPING Study). Epidemiological variables were collected and subjects' emotional responses, perceptions of the physician-patient relationship, support network, fears, and regret about the decision to receive chemotherapy were assessed with questionnaires validated in previous studies: Mini‐Mental Adjustment to Cancer, Brief Summary Inventory (18 items), European Organization for the Research and Treatment of Cancer Quality of Life Questionnaire‐C30, Shared Decision‐Making Questionnaire‐Physician's version, Shared Decision‐Making Questionnaire‐Patient's version, and Informed Risk (physician and patient versions). Results: Data from 46 young and 46 elderly participants were collected. The most common neoplasms in both groups were breast (50%) and colorectal (22%). The younger adults had a higher level of education and were actively employed (72% vs. 7%). The leading coping strategy in the younger cohort was hope, and resignation among the elderly. Young adults sought more social support and the impact of diagnosis was more negative for them than for older individuals. No significant differences were detected in quality of life; both age groups demanded more time at their first visit with the doctor, while the older group exhibited greater satisfaction with shared decision‐making. At the end of adjuvant chemotherapy, neither age group regretted their decision to receive said treatment. Conclusion: Higher levels of education, greater demands of the labour market, and the advent of the age of information have entailed drastic changes in the physician-patient relationship paradigm. This is especially true in the younger cancer patient population, who require more information and active participation in decision‐making, can display more anxiety about their diagnosis, but also greater capacity to fight

Identification and characterization of Diaporthe spp. associated with twig cankers and shoot blight of almonds in Spain

[EN] Two hundred and twenty-fiveDiaportheisolates were collected from 2005 to 2019 in almond orchards showing twig cankers and shoot blight symptoms in five different regions across Spain. Multilocus DNA sequence analysis with five loci (ITS,tub,tef-1 alpha,calandhis), allowed the identification of four knownDiaporthespecies, namely:D. amygdali,D. eres,D. foeniculinaandD. phaseolorum. Moreover, a novel phylogenetic species,D. mediterranea, was described.Diaportheamygdaliwas the most prevalent species, due to the largest number of isolates (85.3%) obtained from all sampled regions. The second most frequent species wasD. foeniculina(10.2%), followed byD. mediterranea(3.6%),D.eresandD. phaseolorum, each with only one isolate. Pathogenicity tests were performed using one-year-old almond twigs cv. Vayro and representative isolates of the different species. Except forD. foeniculinaandD. phaseolorum, allDiaporthespecies were able to cause lesions significantly different from those developed on the uninoculated controls.Diaporthe mediterraneacaused the most severe symptoms. These results confirmD. amygdalias a key pathogen of almonds in Spain. Moreover, the new species,D. mediterranea, should also be considered as a potential important causal agent of twig cankers and shoot blight on this crop.Research funded by the Instituto Nacional de Investigacion y Tecnologia Agraria y Alimentaria (INIA), grants RTA2017-00009-C04-01, -02, -03 and -04 and with matching funds from the European Regional Development Fund (ERDF). G. Elena and C. Agusti-Brisach were supported by the Spanish post-doctoral grants "Juan de la Cierva-Formacion" and "Juan de la Cierva-Incorporacion", respectively. J. Luque and X. Miarnau were partially supported by the CERCA program, Generalitat de Catalunya. D. Gramaje was supported by the Ramon y Cajal program, Spanish Government (RYC-2017-23098).León Santana, M.; Berbegal Martinez, M.; Rodríguez-Reina, JM.; Elena, G.; Abad Campos, P.; Ramón-Albalat, A.; Olmo, D.... (2020). Identification and characterization of Diaporthe spp. associated with twig cankers and shoot blight of almonds in Spain. Agronomy. 10(8):1-23. https://doi.org/10.3390/agronomy10081062S123108Food and Agriculture Organization of the United Nationshttp://www.fao.org/faostat/es/#datDiogo, E. L. F., Santos, J. M., & Phillips, A. J. L. (2010). Phylogeny, morphology and pathogenicity of Diaporthe and Phomopsis species on almond in Portugal. Fungal Diversity, 44(1), 107-115. doi:10.1007/s13225-010-0057-xTuset, J. J., & Portilla, M. A. T. (1989). Taxonomic status of Fusicoccum amygdali and Phomopsis amygdalina. Canadian Journal of Botany, 67(5), 1275-1280. doi:10.1139/b89-168TUSET, J. J., HINAREJOS, C., & PORTILLA, M. T. (1997). Incidence of Phomopsis amygdali, Botryosphaeria berengeriana and Valsa cincta diseases in almond under different control strategies. EPPO Bulletin, 27(4), 449-454. doi:10.1111/j.1365-2338.1997.tb00664.xUdayanga, D., Liu, X., Crous, P. W., McKenzie, E. H. C., Chukeatirote, E., & Hyde, K. D. (2012). A multi-locus phylogenetic evaluation of Diaporthe (Phomopsis). Fungal Diversity, 56(1), 157-171. doi:10.1007/s13225-012-0190-9Rossman, A. Y., Adams, G. C., Cannon, P. F., Castlebury, L. A., Crous, P. W., Gryzenhout, M., … Walker, D. M. (2015). Recommendations of generic names in Diaporthales competing for protection or use. IMA Fungus, 6(1), 145-154. doi:10.5598/imafungus.2015.06.01.09Gomes, R. R., Glienke, C., Videira, S. I. R., Lombard, L., Groenewald, J. Z., & Crous, P. W. (2013). Diaporthe: a genus of endophytic, saprobic and plant pathogenic fungi. Persoonia - Molecular Phylogeny and Evolution of Fungi, 31(1), 1-41. doi:10.3767/003158513x666844Gao, Y., Liu, F., Duan, W., Crous, P. W., & Cai, L. (2017). Diaporthe is paraphyletic. IMA Fungus, 8(1), 153-187. doi:10.5598/imafungus.2017.08.01.11Dissanayake, A. (2017). The current status of species in Diaporthe. Mycosphere, 8(5), 1106-1156. doi:10.5943/mycosphere/8/5/5Santos, L., Alves, A., & Alves, R. (2017). Evaluating multi-locus phylogenies for species boundaries determination in the genusDiaporthe. PeerJ, 5, e3120. doi:10.7717/peerj.3120Lawrence, D. P., Travadon, R., & Baumgartner, K. (2015). Diversity of Diaporthe species associated with wood cankers of fruit and nut crops in northern California. Mycologia, 107(5), 926-940. doi:10.3852/14-353Gramaje, D., Agustí-Brisach, C., Pérez-Sierra, A., Moralejo, E., Olmo, D., Mostert, L., … Armengol, J. (2012). Fungal trunk pathogens associated with wood decay of almond trees on Mallorca (Spain). Persoonia - Molecular Phylogeny and Evolution of Fungi, 28(1), 1-13. doi:10.3767/003158512x626155GARDES, M., & BRUNS, T. D. (1993). ITS primers with enhanced specificity for basidiomycetes - application to the identification of mycorrhizae and rusts. Molecular Ecology, 2(2), 113-118. doi:10.1111/j.1365-294x.1993.tb00005.xTravadon, R., Lawrence, D. P., Rooney-Latham, S., Gubler, W. D., Wilcox, W. F., Rolshausen, P. E., & Baumgartner, K. (2015). Cadophora species associated with wood-decay of grapevine in North America. Fungal Biology, 119(1), 53-66. doi:10.1016/j.funbio.2014.11.002O’Donnell, K., & Cigelnik, E. (1997). Two Divergent Intragenomic rDNA ITS2 Types within a Monophyletic Lineage of the FungusFusariumAre Nonorthologous. Molecular Phylogenetics and Evolution, 7(1), 103-116. doi:10.1006/mpev.1996.0376Glass, N. L., & Donaldson, G. C. (1995). Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes. Applied and Environmental Microbiology, 61(4), 1323-1330. doi:10.1128/aem.61.4.1323-1330.1995Weir, B. S., Johnston, P. R., & Damm, U. (2012). The Colletotrichum gloeosporioides species complex. Studies in Mycology, 73, 115-180. doi:10.3114/sim0011Udayanga, D., Castlebury, L. A., Rossman, A. Y., & Hyde, K. D. (2014). Species limits in Diaporthe: molecular re-assessment of D. citri, D. cytosporella, D. foeniculina and D. rudis. Persoonia - Molecular Phylogeny and Evolution of Fungi, 32(1), 83-101. doi:10.3767/003158514x679984Thompson, J. D., Higgins, D. G., & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research, 22(22), 4673-4680. doi:10.1093/nar/22.22.4673Kumar, S., Stecher, G., Li, M., Knyaz, C., & Tamura, K. (2018). MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms. Molecular Biology and Evolution, 35(6), 1547-1549. doi:10.1093/molbev/msy096Vaidya, G., Lohman, D. J., & Meier, R. (2011). SequenceMatrix: concatenation software for the fast assembly of multi-gene datasets with character set and codon information. Cladistics, 27(2), 171-180. doi:10.1111/j.1096-0031.2010.00329.xRonquist, F., Teslenko, M., van der Mark, P., Ayres, D. L., Darling, A., Höhna, S., … Huelsenbeck, J. P. (2012). MrBayes 3.2: Efficient Bayesian Phylogenetic Inference and Model Choice Across a Large Model Space. Systematic Biology, 61(3), 539-542. doi:10.1093/sysbio/sys029Stamatakis, A. (2014). RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics, 30(9), 1312-1313. doi:10.1093/bioinformatics/btu033Felsenstein, J. (1985). CONFIDENCE LIMITS ON PHYLOGENIES: AN APPROACH USING THE BOOTSTRAP. Evolution, 39(4), 783-791. doi:10.1111/j.1558-5646.1985.tb00420.xDuthie, J. A. (1997). Models of the Response of Foliar Parasites to the Combined Effects of Temperature and Duration of Wetness. Phytopathology®, 87(11), 1088-1095. doi:10.1094/phyto.1997.87.11.1088Agricolae: Statistical Procedures for Agricultural Research. R Package Version 1.2-3http://CRAN.R-project.org/package=agricolaeVan Niekerk, J. M., Groenewald, J. Z., Farr, D. F., Fourie, P. H., Halleen, F., & Crous, P. W. (2005). Reassessment ofPhomopsisspecies on grapevines. Australasian Plant Pathology, 34(1), 27. doi:10.1071/ap04072Lesuthu, P., Mostert, L., Spies, C. F. J., Moyo, P., Regnier, T., & Halleen, F. (2019). Diaporthe nebulae sp. nov. and First Report of D. cynaroidis, D. novem, and D. serafiniae on Grapevines in South Africa. Plant Disease, 103(5), 808-817. doi:10.1094/pdis-03-18-0433-reGuarnaccia, V., Groenewald, J. Z., Woodhall, J., Armengol, J., Cinelli, T., Eichmeier, A., … Crous, P. W. (2018). Diaporthe diversity and pathogenicity revealed from a broad survey of grapevine diseases in Europe. Persoonia - Molecular Phylogeny and Evolution of Fungi, 40(1), 135-153. doi:10.3767/persoonia.2018.40.06Varjas, V., Vajna, L., Izsépi, F., Nagy, G., & Pájtli, É. (2017). First Report of Phomopsis amygdali Causing Twig Canker on Almond in Hungary. Plant Disease, 101(9), 1674. doi:10.1094/pdis-03-17-0365-pdnMichailides, T. J., & Thomidis, T. (2006). First Report of Phomopsis amygdali Causing Fruit Rot on Peaches in Greece. Plant Disease, 90(12), 1551-1551. doi:10.1094/pd-90-1551cLópez-Moral, A., Lovera, M., Raya, M. del C., Cortés-Cosano, N., Arquero, O., Trapero, A., & Agustí-Brisach, C. (2020). Etiology of Branch Dieback and Shoot Blight of English Walnut Caused by Botryosphaeriaceae and Diaporthe Species in Southern Spain. Plant Disease, 104(2), 533-550. doi:10.1094/pdis-03-19-0545-reAdaskaveg, J. E., Förster, H., & Connell, J. H. (1999). First Report of Fruit Rot and Associated Branch Dieback of Almond in California Caused by a Phomopsis Species Tentatively Identified as P. amygdali. Plant Disease, 83(11), 1073-1073. doi:10.1094/pdis.1999.83.11.1073cFarr, D. F., Castlebury, L. A., & Pardo-Schultheiss, R. A. (1999). Phomopsis amygdali causes peach shoot blight of cultivated peach trees in the southeastern United States. Mycologia, 91(6), 1008-1015. doi:10.1080/00275514.1999.12061111Mostert, L., Crous, P. W., Kang, J.-C., & Phillips, A. J. L. (2001). Species of Phomopsis and a Libertella sp. occurring on grapevines with specific reference to South Africa: morphological, cultural, molecular and pathological characterization. Mycologia, 93(1), 146-167. doi:10.1080/00275514.2001.12061286KANEMATSU, S., YOKOYAMA, Y., KOBAYASHI, T., KUDO, A., & OHTSU, Y. (1999). Taxonomic Reassessment of the Causal Fungus of Peach Fusicoccum Canker in Japan. Japanese Journal of Phytopathology, 65(5), 531-536. doi:10.3186/jjphytopath.65.531Dai, F. M., Zeng, R., & Lu, J. P. (2012). First Report of Twig Canker on Peach Caused by Phomopsis amygdali in China. Plant Disease, 96(2), 288-288. doi:10.1094/pdis-04-11-0321Bai, Q., Zhai, L., Chen, X., Hong, N., Xu, W., & Wang, G. (2015). Biological and Molecular Characterization of Five Phomopsis Species Associated with Pear Shoot Canker in China. Plant Disease, 99(12), 1704-1712. doi:10.1094/pdis-03-15-0259-reMeng, L., Yu, C., Wang, C., & Li, G. (2018). First Report of Diaporthe amygdali Causing Walnut Twig Canker in Shandong Province of China. Plant Disease, 102(9), 1859-1859. doi:10.1094/pdis-01-18-0192-pdnSantos, L. (2017). Diaporthe species on Rosaceae with descriptions of D. pyracanthae sp. nov. and D. malorum sp. nov. Mycosphere, 8(5), 485-511. doi:10.5943/mycosphere/8/5/