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Naturalis Repositorio Institucional Universidad Nacional de La Plata http://naturalis.fcnym.unlp.edu.ar Facultad de Ciencias Naturales y Museo Caracterización de especies fitopatógenas de Pythium y Phytophthora (Peronosporomycetes) en cultivos ornamentales del Cinturón verde de La Plata-Buenos Aires y otras áreas y cultivos de interés Palmucci, Hemilse Elena Doctor en Ciencias Naturales Dirección: Wolcan, Silvia Co-dirección: Steciow, Mónica Facultad de Ciencias Naturales y Museo 2015 Acceso en: http://naturalis.fcnym.unlp.edu.ar/id/20171205001560 Esta obra está bajo una Licencia Creative Commons Atribución-NoComercial-CompartirIgual 4.0 Internacional Powered by TCPDF (www.tcpdf.org) CARACTERIZACIÓN DE ESPECIES FITOPATÓGENAS DE PYTHIUM Y PHYTOPHTHORA (PERONOSPOROMYCETES) EN CULTIVOS ORNAMENTALES DEL CINTURÓN VERDE LA PLATA-BUENOS AIRES Y OTRAS ÁREAS Y CULTIVOS DE INTERÉS TESIS PARA OPTAR AL TÍTULO DE DOCTOR EN CIENCIAS NATURALES FACULTAD DE CIENCIAS NATURALES Y MUSEO UNIVERSIDAD NACIONAL DE LA PLATA HEMILSE ELENA PALMUCCI DIRECTOR: ING. AGR. SILVIA WOLCAN CODIRECTOR: DRA MÓNICA STECIOW AÑO 2015 1 AGRADECIMIENTOS A la Facultad de Ciencias Naturales y Museo (FCNYM) por brindarme la posibilidad de realizar este trabajo A la Ing Agr Silvia Wolcan y a la Dra Mónica Steciow por sus sugerencias y comentarios en la ejecución y escritura de esta tesis. A la Dra Gloria Abad, investigadora líder en Oomycetes en el “USDA- Molecular Diagnostic Laboratory (MDL)”, por su invalorable y generosa colaboración en mi formación a través de sus conocimientos, por brindarme la posibilidad de llevar a cabo los trabajos moleculares en el MDL-Maryland-USA y apoyar mi participación en workshops internacionales y reuniones de la especialidad. A la Ing Wolcan por aportar su valiosa colección para realizar parte de las tareas de identificación y por los significativos aportes realizados desde su experiencia. A la Dra Gloria Abad, a la Ing. Agr. Silvia Wolcan, al Dr R. Dehley y al Ing. Agr. Carlos Carloni por la provisión de valiosas referencias bibliográficas. Al Ing Agr M Sc Pablo Grijalba por acompañarme en la ardua tarea de introducirnos al mundo de los Oomycetes a través de nuestras tesis doctorales y los proyectos de investigación que compartimos, y por sus consejos y colaboración en aspectos biomoleculares. A mis compañeros del Proyecto Ubacyt, Lic. Elisa Fantino, Cecilia Herrera y estudiantes tesistas quienes colaboraron en el mantenimiento de la colección, la ejecución de algunas pruebas de patogenicidad y apoyo técnico en tareas de laboratorio. A los productores que facilitaron mi ingreso a sus establecimientos o me enviaron muestras enfermas, a los Ings Agrs Daniel Morisigue y Oscar Herrera, a los viveros “Plugsystem” y “La Facultad”, por facilitarme plantas para la realización de las pruebas de patogenicidad. A la Dra Mary Palm, Directora del que fuera el “USDA- MDL-Maryland-USA”, por facilitar mi estadía de entrenamiento. Al Dr Seoghan Kant de la “Penn State University” por los insumos donados para parte de los estudios moleculares y al Dr Balci de la “University of Maryland” por facilitar instrumental óptico para toma de fotografías microscópicas en 2012. Muy especialmente a mi esposo y mi hija que me acompañaron estos años con su amor y paciencia. 2 INDICE GENERAL AGRADECIMIENTOS 2 RESUMEN 8 ABSTRACT 12 INTRODUCCIÓN Evolución del conocimiento de los géneros Pythium y Phytophthora 17 en Argentina 1. Hábito nutricional y sintomatología. 17 2. Ubicación taxonómica. 20 3. Caracterización morfológica. 24 4. Supervivencia y dispersión del inóculo. Relación con el manejo de la 27 enfermedad 5. Criterios de clasificación: morfológico, molecular, filogenético. 31 6. Antecedentes sobre trabajos de relevamiento. 36 7. Estudios efectuados en Argentina 37 HIPÓTESIS 40 OBJETIVOS GENERALES Y ESPECÍFICOS 40 CARACTERIZACIÓN DEL SECTOR FLORÍCOLA Y DE LA REGIÓN EN ESTUDIO. 1. Caracterización general 43 2. Condiciones ambientales: clima y suelo 46 MATERIALES Y MÉTODOS 1. Antecedentes sobre especies fitopatógenas de los géneros Pythium 49 y Phytophthora en Argentina. 2. Prospección de enfermedades en el Cinturón verde La Plata- 50 Buenos Aires y otras áreas de interés. 3. Técnicas de aislamiento, purificación y mantenimiento de la colección 53 3.1 Aislamientos a partir de material sintomático. 53 3.2. Aislamiento desde agua de riego. 54 3.3. Medios de cultivo. 55 3.4. Purificación de los aislamientos. 55 3.5. Mantenimiento de la colección. 56 3.6. Depósito de los aislamientos en Colecciones micológicas 56 y de secuencias en Bancos Genéticos 4. Identificación y descripción de los aislamientos por métodos 57 micológicos clásicos. 3 4.1. Caracterización de la colonia 57 4.2. Caracterización morfológica 57 4.2.1. Pythium spp. 57 4.2.2. Phytophthora spp. 58 59 4.2.3. Estimulación de estructuras reproductivas 4.2.4. Observación de estructuras con microscopio óptico 60 4.3. Determinación de temperaturas de crecimiento 61 4.4. Identificación taxonómica 61 5. Pruebas de patogenicidad 62 5.1. Depósito de bloques de agar con inóculo en heridas 62 5.2. Colocación de agar con inóculo en contacto con las raíces 63 5.3. Aporte de inóculo utilizando un soporte orgánico 64 5,4. Inoculación con agua de riego 65 5.5. Inoculaciones en otros hospedantes 65 6. Identificación y caracterización de los aislamientos por métodos 65 moleculares 6.1. Extracción del ADN 65 6.2. Amplificación del rADN 65 6.3. Purificación y secuenciación del rADN 66 6.4. Construcción de árboles filogenéticos 67 7. Aspectos referidos a la bibliografía utilizada en el presente estudio 67 RESULTADOS PARTE I. ANTECEDENTES SOBRE ESPECIES FITOPATÓGENAS DE OOMYCETES: STATUS DE LOS GÉNEROS PYTHIUM Y PHYTOPHTHORA EN ARGENTINA 1. Introducción 2. El género Pythium en Argentina 72 3. El género Phytophthora en Argentina 94 PARTE II. CARACTERIZACIÓN DE ESPECIES FITOPATÓGENAS DE PYTHIUM Y PHYTOPHTHORA INTRODUCCIÓN 119 GÉNERO PYTHIUM 122 4 123 ESPECIES CON ESPORANGIOS LOBULADOS O FILAMENTOSOS P. aphanidermatum 123 P. dissotocum 140 P. graminicola 150 ESPECIES CON ESPORANGIOS ESFÉRICOS, SUBESFÉRICOS O ELIPSOIDALES A. OOGONIOS LISOS P. cylindrosporum 160 P. sylvaticum 169 P. ultimum var. ultimum 179 P. ultimum var. sporangiiferum 187 B. OOGONIOS ORNAMENTADOS P. irregulare 192 P. spinosum 201 C .ÓRGANOS SEXUALES AUSENTES P. intermedium 208 P. splendens 217 NUEVAS ESPECIES DE PYTHIUM Pythium sp. nov. 1 224 Pythium sp. nov. 2 233 GÉNERO PHYTOPYTHIUM Phytopythium chamaehyphon 242 ESPECIES DE PHYTOPHTHORA IDENTIFICADAS ESPECIES HETEROTÁLICAS ESPORANGIOS PAPILADOS Ph. capsici 253 Ph. nicotianae= parasitica 267 ESPECIES HETEROTÁLICAS ESPORANGIOS NO PAPILADOS Ph. cinnamomi 293 Ph. cryptogea 310 Ph. taxon kelmania 322 FILOGENIA DE ESPECIES DE PHYTOPHTHORA PRESENTES 329 EN ARGENTINA TABLAS RESUMEN DE RESULTADOS OBTENIDOS 333 DISCUSIÓN GENERAL 344 CONCLUSIONES 371 BIBLIOGRAFÍA CITADA 378 ANEXO I (Antecedentes de Pythium spp. en Argentina) 424 ANEXO II (Antecedentes de Phytophthora spp. en Argentina) 431 5 ÍNDICE DE TABLAS 1: Especies patógenas de Pythium citadas en Argentina. relación hospedante- 73 patógeno, síntomas y localidades donde fueron halladas 2: Distribución de Pythium spp. en regiones geográficas de Argentina en los 87 años 2009 y 2014 3: Número de hospedantes afectados por especies de Pythium dentro 89 de cada tipo de cultivo en los recuentos de 2009 y de 2014 4: Especies de Pythium citadas para el Cinturón verde La Plata-Buenos Aires 93 hasta el año 2009. 5: Especies de Pythium identificadas durante el presente estudio (2009-2014). 93 6: Especies patógenas de Phytophthora citadas en Argentina: relación 95 hospedante-patógeno, síntomas y localización geográfica 7: Especies de Phytophthora aisladas de suelo o agua 108 8: Especies de Phytophthora citadas para el Cinturón verde La Plata-Buenos 108 Aires hasta el año 2009. 9: Especies de Phytophthora identificadas durante el presente estudio (2009- 109 2013). 10: Número de hospedantes afectados por especies de Phytophthora dentro de 111 cada tipo de cultivo en los recuentos de 2009 y de 2014. 11: Distribución de Phytophthora spp. en regiones geográficas de Argentina 113 (año 2014). 12: Crecimiento promedio de los aislamientos de Ph. nicotianae a 20 y 28 °C. 282 13: Características culturales, morfológicas y biométricas de los aislamientos 282 de Ph. nicotianae. 6 ÍNDICE DE FIGURAS Fig. 1: Cinturón verde La Plata-Buenos Aires 47 Fig. 2: Número de especies de Pythium/ provincia en los años 2009-2014. 88 Fig. 3: Número total de hospedantes/tipo de cultivo afectados por especies 90 de Pythium (evaluaciones en 2009 y en 2014). Fig. 4: Frecuencia relativa de especies de Pythium (2014). 91 Fig. 5: Frecuencia relativa de Pythium spp. que afectan cultivos ornamentales 92 (2014). Fig.6: Número de especies de Phytophthora/tipo de cultivo (año 2014). 112 Fig. 7: Número de de especies de Phytophthora/provincia (2014). 115 Fig. 8: Frecuencia relativa de especies de Phytophthora (2014) 116 Fig. 9: Frecuencia relativa de Phytophthora spp. que afectan cultivos 117 ornamentales (2014) Figs. 10-17: Pythium aphanidermatum 126 Figs.18-21: P. dissotocum 141 Figs. 22-26: P. graminicola 151 Figs. 27- 31: P. cylindrosporum 164 Figs. 32-36: P. sylvaticum 173 Figs. 37-39: P. ultimum var. ultimum 183 Fig. 40-41: P. ultimum var. sporangiiferum 190 Figs. 42-44: P. irregulare 198 Figs. 45-47: P. spinosum 202 Figs. 48- 51: P. intermedium 213 Figs. 52-53: P. splendens 222 Figs. 54-59: Pythium sp. nov.1 226 Figs. 60-65: Pythium sp. nov.2 235 Figs. 66-70: Phytopythium chamaehyphon 244 Figs. 71-76: Ph. capsici
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  • Microbial Suppression of Pythium Root Rot in Soilless Systems

    Microbial Suppression of Pythium Root Rot in Soilless Systems

    Microbial Suppression of Pythium Root Rot in Soilless Systems Cora McGehee B.S. Louisiana State University, 2015 A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science At the University of Connecticut 2018 Copyright by Cora Shields McGehee 2018 ii APPROVAL PAGE Master Thesis Microbial Suppression of Pythium Root Rot in Soilless Systems Presented by Cora Shields McGehee, B.S. Major Advisor_________________________________________________________________ Rosa E. Raudales Associate Advisor______________________________________________________________ Wade H. Elmer Associate Advisor_______________________________________________________________ Richard J. McAvoy University of Connecticut 2018 iii Acknowledgements I am beyond grateful to my major advisor Dr. Rosa Raudales for her hard work and dedication to the lab and innovative research projects. Her guidance and patience has made me into a stronger researcher and diligent worker. I also want to thank the other members of my committee Dr. Wade Elmer and Dr. Richard McAvoy for their time and generous advice. I want to thank Frederick Pettit, Shelley Durocher, and Ronald Brine for their assistance in the various greenhouse projects conducted. Thank you Margery Daughtrey for supplying isolates for experiments. Special thanks to Juan Cabrera, Sohan Aziz, Steve Olenski, Joy Tosakoon, and Carla Caballero for giving your time to help with experiments. Lastly a thank you to the office staff, Christine Strand and Nicole Gabelman for their assistance and kindness. Special thanks to the U.S. Department of Agriculture via the Connecticut Department of Agriculture Specialty Crop Block Grant # AG151260 for its support and funding of this work. Thank you to friends and family who supported me over the phone during this intense intellectual pursuit.
  • Phytophthora Species from Xinjiang Wild Apple Forests in China

    Phytophthora Species from Xinjiang Wild Apple Forests in China

    Article Phytophthora Species from Xinjiang Wild Apple Forests in China Xiaoxue Xu 1, Wenxia Huai 1, Hamiti 2, Xuechao Zhang 3 and Wenxia Zhao 1,* 1 The Key Laboratory of National Forestry and Grassland Administration on Forest Protection, Research Institute of Forest Ecology Environment and Protection, Chinese Academy of Forestry, Beijing 100091, China; [email protected] (X.X.); [email protected] (W.H.) 2 Forest Bureau of Emin County, Xinjiang Uygur Autonomous Region, Emin 834600, China; [email protected] 3 Institute of Agricultural Sciences of Ili Kazakh Autonomous Prefecture, Yining 835000, China; [email protected] * Correspondence: [email protected]; Tel.: +86-010-6288-8998 Received: 28 August 2019; Accepted: 17 October 2019; Published: 21 October 2019 Abstract: Phytophthora species are well-known destructive forest pathogens, especially in natural ecosystems. The wild apple (Malus sieversii (Ledeb.) Roem.) is the primary ancestor of M. domestica (Borkh.) and important germplasm resource for apple breeding and improvement. During the period from 2016 to 2018, a survey of Phytophthora diversity was performed at four wild apple forest plots (Xin Yuan (XY), Ba Lian (BL), Ku Erdening (KE), and Jin Qikesai (JQ)) on the northern slopes of Tianshan Mountain in Xinjiang, China. Phytophthora species were isolated from baiting leaves from stream, canopy drip, and soil samples and were identified based on morphological observations and the rDNA internal transcribed spacer (ITS) sequence analysis. This is the first comprehensive study from Xinjiang to examine the Phytophthora communities in wild apple forests The 621 resulting Phytophthora isolates were found to reside in 10 different Phytophthora species: eight known species (P.
  • Recovery of Phytophthora Ramorum and Other Phytophthora Spp

    Recovery of Phytophthora Ramorum and Other Phytophthora Spp

    Mississippi State University Scholars Junction Theses and Dissertations Theses and Dissertations 1-1-2015 Recovery of Phytophthora Ramorum and Other Phytophthora Spp. in a Forest Adjacent to a Mississippi Ornamental Plant Nursery Devin Sterling Bily Follow this and additional works at: https://scholarsjunction.msstate.edu/td Recommended Citation Bily, Devin Sterling, "Recovery of Phytophthora Ramorum and Other Phytophthora Spp. in a Forest Adjacent to a Mississippi Ornamental Plant Nursery" (2015). Theses and Dissertations. 3738. https://scholarsjunction.msstate.edu/td/3738 This Graduate Thesis - Open Access is brought to you for free and open access by the Theses and Dissertations at Scholars Junction. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of Scholars Junction. For more information, please contact [email protected]. Template B v3.0 (beta): Created by J. Nail 06/2015 Recovery of Phytophthora ramorum and other Phytophthora spp. in a forest adjacent to a Mississippi ornamental plant nursery By TITLE PAGE Devin Sterling Bily A Thesis Submitted to the Faculty of Mississippi State University in Partial Fulfillment of the Requirements for the Degree of Master of Science in Forest Products in the Department of Sustainable Bioproducts Mississippi State, Mississippi December 2015 Copyright by COPYRIGHT PAGE Devin Sterling Bily 2015 Recovery of Phytophthora ramorum and other Phytophthora spp. in a forest adjacent to a Mississippi ornamental plant nursery By APPROVAL PAGE Devin Sterling Bily Approved: ____________________________________ Susan V. Diehl (Major Professor) ____________________________________ Richard E. Baird (Minor Professor) ____________________________________ Abdolhamid Borazjani (Committee Member/Graduate Coordinator) ____________________________________ George M. Hopper Dean College of Forest Resources Name: Devin Sterling Bily ABSTRACT Date of Degree: December 11, 2015 Institution: Mississippi State University Major Field: Forest Products Major Professor: Dr.