About the Author
Peter Weingartner took his Ph.D. from Michigan State University in 1969 and has been a plant pathologist at the University of Florida Agricultural Research and Education Center in Hastings, Florida since 1969. He presently covers research and extension assignments in plant pathology and nematology for vegetables in northeast Florida. His research has concentrated on nematode and disease management in potatoes and downy mildew disease of cabbage. Weingartner has published numerous articles, reports, and other publications in plant pathology, nematology, and integrated pest management (IPM). He served as editor of the nematology section of Fungicide and Nematicide Tests and is currently co-editing the second edition of the Potato Disease Compendium, published by APS Press.
Identification of
Late Blight
and Phytophthora infestans
Pete Weingartner
University of Florida, IFAS, Hastings REC
The first step in identifying late blight is to find the disease in the field. Since the pathogen requires moisture the disease is often first detected in field locations subject to long periods of wetness. Examples of these include rows along irrigation furrows, within drip and wheel zones of center pivots, and in protected or shaded areas near woods or wind breaks.
Symptoms of late blight vary widely depending upon such factors as moisture, temperature, light intensity, cultivar, and age of plants. Foliar lesions are usually first visible as small light to dark green irregularly shaped spots which rapidly expand. As lesions age, the centers become necrotic, turning brown to black. Expanding lesions on some potato cultivars are often bordered by a light green halo. Under moist conditions profuse sporulation occurs, especially on the underside of the leaf. Presence of white "fuzz" on a lesion is often a useful distinguishing characteristic to help differentiate late blight infections from other diseases.
Lesions can also occur on petioles and stems, often killing entire leaves or branches of affected plants. Stem lesions and infection of plant terminals have been especially prevalent with new strains of P. infestans. Terminal leaves on newly lesioned stems often roll upward and margins turn red to purple mimicking symptoms of leaf roll virus, purple top, or plants with rhizoctonia stem canker. Stem lesions are especially dangerous aspects of new strains of P. infestans because stems are difficult to cover and protect with protectant fungicides. In addition, stem lesions often are undetected, are long lasting, and can produce copious amounts of inoculum during an epidemic.
Old stem lesions on dead plants can easily be mistaken for advanced symptoms of black leg caused by Erwinia spp. Other diseases having foliar symptoms which can be confused with late blight include early blight (Alternaria solani), gray mold (Botrytis cinerea), and white mold (Sclerotinia sclerotiorum). Leaf scald caused by moisture stress and dried freeze injury can also mimic late blight symptoms.
Late blight also affects potato tubers. Relative susceptibility of tubers can differ from that of foliage. Tuber lesions are irregular in shape, brown to purplish in color, and are slightly depressed. Internally, lesions are dry, tan in color, without distinct margins, and are usually confined to the outer 1-2 cm of the tuber flesh. Sporulation can be induced, especially on freshly cut surfaces of tubers held in moisture chambers. Tuber lesions are readily invaded by secondary pathogens which results in decay and often makes tuber diagnosis difficult. Tuber symptoms caused by A. solani, Sclerotium rolfsii, P. erythroseptica, Pythium spp., and Erwinia spp. can be confused with tuber symptoms of late blight. Commercial Elisa kits are available for identifying Phytophthora spp. in tubers, and recently developed PCR based techniques (polymerase chain reaction) show promise for accurately diagnosing late blight in diseased tubers. Diagnosis in tubers is especially important in seed certification.
Appropriate collection and handling of field samples is a prerequisite to having suitable tissue for laboratory analyses. Ideally, the pattern of sampling and number of samples taken should be determined by the precision desired and the distribution (e.g., random or aggregated) of late blight in the field. Lacking information on distribution, multiple samples should be taken. Multiple samples are especially important if reliable identification of fungicide resistance, mating and genotype are desired. A small number of samples taken in multiple fields or multiple locations within large fields are preferred to a large number of samples taken within a limited area in a single field. Samples should be taken before application of fungicides, especially phenylamide fungicides, and consist of active lesions on leaflets or stems having a majority of healthy tissue. Samples should be carefully placed in a plastic bag containing a damp (but not saturated) paper towel. When closing the bag, care should be taken to maintain air space around the sample. Samples ship better in boxes than in envelopes. Tissue collected when temperatures exceed 70-75 F should be cooled before shipping and all samples should be shipped using overnight delivery whenever possible.
In the laboratory P. infestans can be identified microscopically. The fungus belongs to Phytophthora morphological Group IV (see pps 138-139 in Erwin and Riberio) and can be identified by the following morphological characteristics. Sporangia of P. infestans are compoundly branched and develop sympodially which results in swellings along the sporangiophore as it produces successive crops of sporangia. The sporangia are caducous (i.e., deciduous) which results in the fungus having a short (< 3 m m) pedicel or attachment scar at the base. The sporangia are semipapillate, having a small plug or pimple at the tip where zoospores will be released. The spore shape is ovoid—limoniform and they are 21-38 long X 12-23 m m wide (average-29 X 19 m m), and have a length to breadth ratio of 1.6. The fungus is heterothallic (i.e., self infertile), the antheridium is amphigynous and wraps around the oogonium and is 17 long X 16m m wide. The oogonia are 31-50 m m in diameter (mean= 30 m m) and the wall is 3.5 m m thick. Oospores are plerotic and are not readily formed in host tissue.
Isolation of P. infestans in pure culture is difficult. Use of selective media amended with fungicides and antibiotics, and isolation and transfer of single sporangia growing from thin slices of water agar or potato tuber slices placed on active lesions are widely used methods for isolating P. infestans. (See Erwin and Riberio for several recipes). Once in pure culture mating type is determined by crossing known A1 and A2 mating types with unknown isolates. Sensitivity to phenylamide fungicides can be assayed by floating infected leaf disks on dilutions of the fungicide; however, the preferred method is to grow the pathogen on agar plates amended with different concentrations of the chemical. Pathogenic races of P. infestans are designated according to their virulence/avirulence on differential potato clones having different R genes for resistance. Eleven different pathogenic races are known. Recently DNA methods have been devised for identifying some pathogenic races. Clones for differentiating among P. infestans races are available from USDA/ARS Inter-regional Potato Introduction Station at Sturgeon Bay, Wisconsin.
Molecular assays are used to determine genotypes. Allozyme electrophoresis is widely used, especially since Goodwin’s description of the easy to use cellulose acetate electrophoresis (CAE) method for the Glucose-6-phosphate isomerase and Peptidase loci for identifying the most common US genotypes (US1, 6, 7, and 8). The number of differentiating allozymes in P. infestans is limited and more precise identification of genotypes requires DNA analysis. Methods employed include PCR and use of RAPD’s (randomized amplified polymorphic DNA) and RFLP’s (restriction fragment length polymorphisms). The CAE assay can be performed using field samples. Although RAPD and RFLP analyses are done with pure cultures several laboratories are developing PCR techniques which can be used with diseased tissue. (e.g. J. B. Ristaino, NC State and P. W. Tooley, USDA/ARS, Ft. Detrick.)
Selected References
Carter, D. A., S. A. Archer, K. W. Buck, D. S. Shaw, and R. C. Shattock. 1991. DNA Polymorphisms in Phytophthora infestans: The UK experience. pp. 272-294. In Phytophthora. J. A. Lucas, R. C. Shattock, D. S. Shaw, and L. R. Cooke, eds. University Press, Cambridge, UK. 447 pp.
Carter, D. A., S. A. Archer, K. W. Buck, D. S. Shaw, and R. C. Shattock. 1990. Restriction fragment length polymorphisms of mitochondrial DNA of Phytophthora infestans. Mycological Research 94:1123-1128.
Erwin, D. C., and O. K. Ribeiro. 1996. Phytophthora diseases Worldwide. APS Press, St. Paul, MN 562 pp.
Erwin D. C., S. Barnicki-Garcia, and P. H. Tsao eds. 1983. Phytophthora: Its Biology, Taxonomy, Ecology, and Pathology. APS, St. Paul, MN 392 pp.
Fry, W. E., S. B. Goodwin, J. M. Matuszak, L. J. Spielman, and M. G. Milgroom. 1992. Population genetics and intercontinental migrations of Phytophthora infestans. Annual Review Phytopathology 30:107-129.
Goodwin, S. B. 1991. DNA polymorphism in Phytophthora infestans: The Cornell Experience. Pp. 256-271. In Phytophthora. J. A. Lucas, R. C. Shattock, D. S. Shaw, and L. R. Cooke, eds. University Press, Cambridge, UK. 447 pp.
Goodwin, S. B., A. Drenth, and W. E. Fry. 1992. Cloning and genetic analyses of two highly polymorphic, moderately repetitive nuclear DNA’s from Phytophthora infestans. Current Genetics 22:107-115.
Goodwin, S. B., R. E. Schneider, and W. E Fry. 1995. Use of Cellulose Acetate Electrophoresis for Rapid Identification of Allozyme Genotypes of Phytophthora infestans. Plant Disease 79:181-185.
Henson, J. M., and R. French. 1993. The Polymerase Chain Reaction and Plant Disease Diagnosis. Annual Review of Plant Pathology 31:81-109.
Hooker, W. J. ed. 1981. Compendium of Potato Diseases 125 pp. APS Press, St. Paul, MN.
Maufrand, R., S. A. Archer, K. W. Buck, D. S. Shaw, and R. C. Shattock. 1995. The use of RAPD markers in genetic studies of Phytophthora infestans. pp. 55-63. In Phytophthora infestans 150. L. J. Donley, E. Brannon, L. R. Cooke, T. Keane, and E. O’Sullivan, eds. Boole Press, Dublin Ireland.
Michelmore, R. W. and S. H. Hulbert. 1987. Molecular markers for genetic analyses of Phytopathogenic fungi. Annual Review Phytopathology 25:383-404.
Miller, Sally A., and R. R. Martin. 1988. Molecular Diagnosis of Plant Disease. Annual Review Phytopathology 26:409-432.
Spielman, L. J. 1991. Isozymes and the population genetics of Phytophthora infestans. pp. 231-241. In Phytophthora. J. A. Lucas, R. C. Shattock, D. S. Shaw, and L. R. Cooke, eds. University Press, Cambridge, UK. 447 pp.
Stamps, D. J., G. M. Waterhouse, F. J. Newhook, and G. S. Hall. 1990. Revised Tabular Key to the Species of Phytophthora. 28pps. Mycological Paper No. 162. CAB International Mycological Institute, Kew Surry, UK.
Tooley, P. W., C. D. Therrien, D. L. Ritch. 1989. Mating type, race composition, and isozyme analysis of Peruvian isolates of Phytophthora infestans. Phytopathology 79:478-481.
Tooley, P. W., W. E. Fry, and M. J. Villarreal Gonzalez. 1985. Isozyme characterization of sexual and asexual Phytophthora infestans populations. The Journal of Heredity 76:431-435.
IDENTIFICACION DE TIZON TARDIO Y PHYTOPHTHORA INFESTANS
PETE WEINGARTNER
Una identificacion precisa de tizon tardio y su patogeno Phytophthora infestans, es esencial para un manejo efectivo de la enfermedad. Simtomas de la hoja o del tuberculo deberan ser examinados con un microscopio los esporangiosporas y esporangios son usualmente suficientes para la mayoria de las lesiones de campo; sin embargo, para muchas de los laboratorios pruebas sofisticadas son requesitos para una confiable determinacion de sensibilidad a phenylamide fungicida, tipo de apareamiento, y genotipos del patogeno.
Los simtomas de tizon tardio varian dependiendo de la humedad, temperatura, intensidad de luz, variedad, y edad de la planta. Lesiones foliares son usualmente primeramente visibles como manchas irregulares de color verde claro abscuro que se expanden rapidamente. El centro de las lesiones se hace necrotico, volviendose de color cafe a negro. Las lesiones expandidas son frecuentemente bordeados por un halo verde claro. Bajo condiciones humedas una profusa esporulacion ocurre, especialmente en la parte inferior de las hojas sueltas. Tambien ocurren lesiones en los petioles, y tallos que frecuentemente matan todas las hojas de la rama de la planta afectada. Lesiones del tallo e infecciones de la parte terminal de la planta han estado teniendo especial prevalencia con los nuevos biotipos de P. infestans. Hojas terminales en tallos con recientes lesiones frecuentemente se enrrollan hacia arriba y los margenes se tornan de color rojo purpura parecidos a los simtomas del virus leaf roll o plantas con llagas de rhizoctonia. Lesiones viejas en los tallos o plantas muertas pueden ser facilmente confundidos con sintomas avanzados de cuello negro causado por Erwinia spp. Otras enfermedades que tienen simtomas en el foliaje pueden ser confundidos con tizon tardio incluyendo a tizon temprano (Alternaria solani), moho gris (Botrytis cinera), y moho blanco (Sclerotina sclerotiorum). Tizon tardio tambien puede afectar los tuberculos de la papa. Una suceptibilidad relativa de los tuberculos puede diferir del foliaje. las lesiones de los tuberculos son de forma irregular, de color cafe a purpura, y son ligeramente deprimidos. Interiormente, las lesiones son secas, de color cafe canela a cafe, sin margenes distintivos, y usualmente estan confinados de 1 a 2 cm fuera de la pulpa de los tuberculos.La esporulacion puede ser inducido, especialmente en en la superficie de los cortes frescos o en tuberculos colocados en camaras humedas. Tuberculos con lesiones son facilmente invadidos por patogenos secundarios que provoca un deterioramiento y frecuentemente dificulta la diagnosisde de los tuberculos. Juegos comerciales de Elisa estan disponibles para indentificacion de Phytophthora spp. en tuberculos y recientemente han sido desarrolados tecnicas basadas en PCR (Reacciones en cadena de Polimerasas) que muestra ser muy promisorio para la diagnosis de tuberculos enfermos con tizon tardio.
Identificacion a resistencia a fungicides, apareamientos y genotipos en poblaciones de P. infestans requiere adicionales pruebas. Muestras de campo en condiciones adecuadas son esenciales para una identificacion confiable de las poblaciones de P. infestans. Idealmente, el modelo y numero de muestras deberan ser determinados con la presicion requerida y la distribucion (e.g. al hazar o colectivamente) de tizon tardio en el campo. Cuando no existe buena informacion en cuanto a la distribucion, muchas muestras deberan ser tomadas. La muestras deberan ser sacadas en diferentes secciones del campo y en forma separada. Muestras sacadas de muchos campos son preferibles enves de muchas muestras sacadas de un solo campo. La muestras deberan ser sacadas antes de la aplicacion de fungicidas y con lesiones consistentes y activos de hojas sueltas o tallos mostrando una mayor proporcion de tejido sano.
Aislamientos de P. infestans en cultivos puros es dificil. Usando medios selectivos agregando fungicidas y antibioticos, aislar y transferir un esporangio solo a un medio de cultivo delgado de agar agua o rodajas de tuberculo de papa colocados sobre las lesiones son metodos ampliamente usados para aislar P. infestans. Una vez que estan en cultivo puro los typos de apareamiento son determinados por cruzamientos de los tipos de apareamiento A1 y A2 conocidos con los aislamientos de los tipos de apareamiento desconocidos. Sensitividad al fungicida phenylamide puede ser examinado flotando pedasos de hojas infectadas en una dilucion de fungicida; sin embargo, el metodo preferido es hacer crecer al patogeno en platos de agar agregando diferentes consentaciones del quimico. Razas patogenicas de P. infestans son designados de acuerdo a su virulencia/avirulencia en clones de papa que tienen diferentes R resistentes genes. Once diferentes razas patogenicas son conocidas. Reciemtemente metodos de DNA han sido creados para identificar algunas de las razas patogenicas.
Pruebas moleculares son usados para determinar genotipos. Allosyme electroforesis es ampliamente usado, especialmente desde la descripcion de Goodwin en la prueba de usar elestroforesis de acetato de celulosa (CAE) metodo para isomeros de Glucosa-6-fosfato y Pestidasa loci par identificar los genotipos mas comunes de US (US1,6,7, y 8). El numero de los diferentes allozymes en P. infestans es limitado y requiere de un analices de DNA par una identificacion mas precisa de los genotipos. Los metodos empleados incluye PCR y uso de RAPD's ( Polimeros de DNA al hazar y amplificados) y RFLP's. Las pruebas de CAE pueden ser ejecutados usando muestras de campo. Aunque muestras de RAPD y RFLP son hechos con cultivos puros muchos laboratorios estan desarrollando tecnicas de PCR los cuales pueden ser usados con tejidos enfermos.