Identificación molecular y diversidad genética de las especies de nematodos Globodera rostochiensis y G. pallida en regiones productoras de papa de Guatemala / Molecular identification and genetic diversity of the species of nematodesGlobodera rostochiensisandG. pallidain potato growing regions of Guatemala

En Guatemala, la producción del cultivo de papa se ve afectada por los nematodos Globodera rostochiensis y Globo-dera pallida. La capacidad de ambas especies para formar quistes complica su control y provoca el aumento de sus poblaciones. En Guatemala se reporta la presencia de ambas especies de nematodos por identificación morfológica, sin embargo, no se ha realizado una confirmación molecular. Este es el primer estudio para validar la presencia de ambas especies de nematodos por PCR múltiple y la determinación de la diversidad y estructura genética de las poblaciones utilizando marcadores moleculares. Se realizaron muestreos en cuatro departamentos productores de papa del país. La identificación por PCR se realizó con el cebador común ITS5 y los cebadores PITSr3 específico para G. rostochiensisy PITSp4 para G. pallida. La caracterización molecular se realizó con el marcador AFLP. Se confirmó la presencia de las dos especies de nematodos en los cuatro departamentos. Los índices de diversidad Shannon y heterocigosidad esperada revelaron mayor diversidad genética en G. rostochiensis (H = 0.311, He = 0.301) que en G. pallida (H = 0.035, He = 0.223). Los métodos NJ, DAPC y PCA exhibieron una débil estructura entre las poblaciones de ambas especies de nematodos. Los resultados sugieren un patrón de dispersión desde Quetzaltenango hacia el resto del país, atribuido a la comercialización de semilla contaminada con nematodos. Se sugiere promover programas de socialización sobre los beneficios del uso de semilla certificada, además de constantes monitoreos moleculares para un diagnóstico certero de ambas especies de nematodos.

In Guatemala, potato crop production is affected by the nematodes Globodera rostochiensis and Globodera pallida. The ability of both species to form cysts complicates their control and causes an increase in their populations. In Guatemala, both species of nematodes have been reported by morphological identification; however, molecular confirmation has not been carried out. It is the first study to validate the presence of both nematode species by multiplex PCR and determine the diversity and genetic structure of the populations using molecular markers. Sampling was carried out in four pota-to-producing departments of the country. PCR identification was performed with the common primer ITS5 and the primers PITSr3 specific for G. rostochiensis and PITSp4 for G. pallida. We performed molecular characterization with the AFLP marker. We confirmed the presence of the two nematode species in the four departments. Shannon diversity and expected heterozygosity indices revealed higher genetic diversity in G. rostochiensis (H = 0.311, He = 0.301) than in G. pallida (H = 0.035, He = 0.223). The NJ, DAPC, and PCA methods exhibited weak structure among populations of both nematode species. The results suggest a dispersal pattern from Quetzaltenango to the rest of the country, attributed to the commer-cialization of seed contaminated with nematodes. We suggest promoting socialization programs on the benefits of using certified seeds and constant molecular monitoring for an accurate diagnosis of both species of nematodes.

Histopathology of Andean Potato (Solanum tuberosum Andigenum group) varieties parasitized by the false root-knot nematode, Nacobbus aberrans / Histopatologia de variedades de batata andina (Solanum tuberosum grupo Andigenum) parasitadas pelo falso nematóide das galhas, Nacobbus aberrans

Abstract Landraces of the Solanum tuberosum Andigenum group are abundant and diverse. They are a valuable genetic resource possessing resistance to pests, diseases, and environmental stresses. In the Andean region, populations of the false root-knot nematode Nacobbus aberrans became specialized to infect native potatoes, being one of the major limiting factors affecting this crop. A better understanding of the host plant-parasite interactions is important in order to select tolerant or resistant plants to be included in management programs. Despite the close of association of N. aberrans with potato, and the great diversity of the S. tuberosum Andigenum group, few histopathological studies have been conducted. The aim of this work was to analyze histological alterations induced by different Argentine populations of the nematode in naturally infested roots of four Andean potato varieties (Collareja, Negra Imilla, Ojo de Señorita and Colorada). All the varieties showed hyperplastic tissue in the central zone of galls, where syncytia developed in close association with the nematode female. Syncytia were composed of modified hyperplastic tissue and parenchyma xylem cells. The results showed differences among varieties in their response to nematode populations, with Ojo de Señorita and Negra Imilla being the most susceptible ones. This study is the first describing histopathological alterations induced by N. aberrans in susceptible Andean potato landraces.

Resumo As variedades de Solanum tuberosum grupo Andigenum são abundantes e diversas, além de serem uma fonte importante de recursos genéticos antea resistência de pragas, doenças e estresse ambiental. Na região andina, populações de Nacobbus aberrans se especializaram em parasitar batatas nativas e representam o principal fator limitante que afeta a produção desse cultivo. Um melhor entendimento da interação hospedeiro-parasita é importante para a seleção de plantas tolerantes ou resistentes a serem incluídas nos programas de manejo. Apesar da íntima associação de N. aberrans com a batata e a grande diversidade de S. tuberosum grupo Andigenum, os estudos histopatológicos desenvolvidos são limitados. O objetivo do presente trabalho foi analisar as alterações histológicas de raízes de quatro variedades de batata andina (Collareja, Negra Imilla, Ojo de Señorita e Colorada) parasitadas naturalmente por distintas populações argentinas do nematóide. Em todas as variedades se observou tecido hiperplásico na zona central das galhas onde, também, se desenvolveram os sincícios em íntima relação com as fêmeas do nematóide. Os sincícios estavam constituídos por tecido hiperplásico modificado e por células parenquimáticas do xilema. Os resultados mostraram diferenças entre as variedades de batata frente às populações do nematóide, sendo Ojo de Señorita e Negra Imilla as que evidenciaram maior susceptibilidade. Este estudo constitui a primeira avaliação da histopalogia induzida por N. aberrans em variedades suscetíveis de batata andina.

Resistência de cultivares de batata a Myzus persicae (Sulz.)(Hemiptera: Aphididae) / Resistance of potato cultivars to Myzus persicae (Sulz.) (Hemiptera: Aphididae)

Aphids are the most important vectors of viruses infecting potato (Solanum tuberosum). We focused on the response of the aphid vector Myzus persicae (Sulzer) to five commercial potatocultivars: Ágata, Jaette Bintje, Mondial, Monalisa and Santè, by traditional antibiosis and antixenosis tests and by the EPG (Electrical Penetration Graph) technique, as a step forward to the design of effective management practices. Our aim was to identify plant factors involved in resistance of these cultivars against M. persicae, both at the surface and in deeper plant tissues. Results from the antixenosis test confirmed a strong preference of M. persicae for the Mondial cultivar. The antibiosis study indicated a lower population development of the aphid in 'Monalisa' when compared to 'Ágata' and 'Jaette Bintje'. EPG assays indicated that 'Santè' inhibited the initial feeding process of M. persicae, whereas 'Monalisa' showed a physical-type of resistance as demonstrated by a very high number of short probes. The cultivar Mondial showed average values for all EPG variables analyzed. The behavior in 'Jaette Bintje' indicated this cultivar was an ideal host for aphid feeding and reproduction. Together, the EPG data revealed the existence of pre and post-phloematics factors in the cultivars under study, which have important implications on the efficiency of transmission and spread of virus in potato by M. persicae.

Population dynamics and damage caused by the leafminer Liriomyza huidobrensis blanchard (Diptera: Agromyzidae), on seven potato processing varieties grown in temperate environment

The leafminer Liriomyza huidobrensis Blanchard is considered a key pest for potatoes in Argentina. Population dynamics and leaf damage caused by the leafminer on seven selected potato processing varieties were assessed at Balcarce during the 2002 and 2003 growing seasons. Adult population dynamic was monitored using yellow sticky traps, while leaf damage (punctures and mines) was assessed using a damage index scale from low to severe. Liriomyza huidobrensis adults were present throughout the growing season and the population increased along crop development. The same was true for all varieties regarding larval damage, being low on early crop stages and severe late in the season. Varieties were grouped in two different categories according to damage scale index. Shepody, Kennebec, Frital and Innovator showed a higher damage index when compared with Santana, Ranger Russet and Russet Burbank, which exhibited a lower damage. Moreover, it could be assumed that damage was related to the foliage greenness, with light green colored varieties (Shepody, Kennebec, Frital and Innovator) being more attractive and affected by L. huidobrensis.

Presence and significance of Bacillus thuringiensis Cry proteins associated with the Andean weevil Premnotrypes vorax (Coleoptera: Curculionidae)

The Andean weevil Premnotrypes vorax represents an important cause of damage to Colombian potato crops. Due to the impact of this plague on the economy of the country, we searched for new alternatives for its biological control, based on the entomopathogenic bacteria Bacillus thuringiensis. A total of 300 B. thuringiensis strains obtained from potato plantations infested with P. vorax were analyzed through crystal morphology, SDS-PAGE, PCR and bioassays. We used site- directed mutagenesis to modify the Cry3Aa protein. Most of the B. thuringiensis isolates had a bipyramidal crystal morphology. SDS-PAGE analyses had seven strains groups with σ-endotoxins from 35 to 135 kDa. The genes cry 2 and cry 1 were significantly more frequent in the P. vorax habitat (PCR analyses). Three mutant toxins, 1 (D354E), 2 (R345A, ∆Y350, ∆Y351), and 3 (Q482A, S484A, R485A), were analyzed to assess their activity against P. vorax larvae. Toxicity was low, or absent, against P. vorax for isolates, wild type cry 3Aa and cry 3Aa mutants. The genetic characterization of the collection provides opportunities for the selection of strains to be tested in bioassays against other insect pests of agricultural importance, and for designing Cry proteins with improved insecticidal toxicity. Rev. Biol. Trop. 57 (4): 1235-1243. Epub 2009 December 01.

El gorgojo andino Premnotrypes vorax es una causa importante de daño en los cultivos colombianos de este tubérculo. Debido al impacto que esta plaga tiene sobre la economía del país, nos interesamos en buscar alternativas nuevas para el control biológico de P. vorax, basadas en la bacteria entomopatógena Bacillus thuringiensis. Se recolectaron un total de 300 cepas de B. thuringiensis a partir de plantaciones de papa infestadas con P. vorax, las cuales fueron analizadas por medio de la morfología del cristal, SDS-PAGE, PCR y ensayos biológicos. La mayoría de los aislamientos de B. thuringiensis presentaron cristales bipiramidales. Los análisis de SDS-PAGE indicaron la presencia de siete grupos de cepas con σ- endotoxinas que variaban entre 35 a 135 kDa. Las pruebas con PCR demostraron que los genes cry 2 y cry 1 fueron significativamente más frecuentes en el medioambiente de P. vorax. Además, se utilizó la mutagénesis sitio-dirigida para modificar la proteína Cry3Aa. Se analizaron tres toxinas mutantes, 1 (D354E), 2 (R345A, ∆Y350, ∆Y351), y 3 (Q482A, S484A, R485A), para determinar su actividad contra larvas de P. vorax. Los ensayos de toxicidad señalaron escasa, o nula, actividad hacia P. vorax tanto para las cepas, la toxina Cry3Aa de referencia y las proteínas Cry3Aa mutantes. La caracterización genética de la colección puede proveer oportunidades para la selección de cepas que pueden evaluarse por medio de bioensayos contra otros insectos-plaga de importancia agrícola, y para el diseño de proteínas Cry con actividad toxica mejorada.

Uso de silício como indutor de resistência em batata a Myzus persicae (Sulzer) (Hemiptera: Aphididae) / Use of silicon as inductor of the resistance in potato to Myzus persicae (Sulzer) (Hemiptera: Aphididae)

The aphid Myzus persicae (Sulzer) is an important pest of potato and causes direct harm, due to the quantity of sap extracted and for being vector of important phytovirus. This work was carried out to evaluate the action of silicon as a resistance inducer of potato to M. persicae. Four treatments were tried: foliar fertilization with silicon acid at 1 percent; soil fertilization with 250 ml silicic acid solution at 1 percent; foliar fertilization with silicon acid at 1 percent + soil fertilization with 250 ml silicic acid solution at 1 percent; and a control. The treatments were applied thirty days after the explants emergence. Fifteen days after the application of the treatments, feeding preference and some biological aspects of the aphids were evaluated. After, the content of tannins and lignin present in the leaves and the activity of the enzymes peroxidase and phenylalanine ammonia-lyase were also determined. The silicon fertilization did not affect the preference of the aphids; however it reduced fecundity and the rate of population growth of the insects. The lignin percentage increased in the leaves of plants fertilized with silicon via soil and/or foliar and the percentage of tannins increased only in the leaves fertilized via soil plus foliar. The silicon acted as a resistance inducer to M. persicae in potato.

O pulgão Myzus persicae (Sulzer) é uma importante praga da batateira, causando danos diretos pela quantidade de seiva extraída e por ser vetor de importantes fitovírus. Este trabalho foi realizado para verificar a atuação do silício como indutor de resistência em batata a M. persicae. Foram utilizados quatro tratamentos: adubação foliar com solução de ácido silícico a 1 por cento; adubação via solo com 250 ml de solução de ácido silícico a 1 por cento; adubação foliar com solução de ácido silício a 1 por cento + adubação via solo com 250 ml de solução de ácido silícico a 1 por cento1; e a testemunha. Os tratamentos foram aplicados trinta dias após a emergência das brotações. Após quinze dias da aplicação dos tratamentos avaliou-se a preferência e alguns aspectos biológicos do pulgão. Posteriormente foi feita a quantificação do teor de taninos e de lignina presentes nas folhas e a determinação das atividades das enzimas peroxidase e fenilalanina amônia-liase. A adubação silicatada não afetou a preferência dos pulgões, porém diminuiu a fecundidade e a taxa de crescimento populacional dos insetos. A percentagem de lignina aumentou nas folhas das plantas adubadas com silício via solo e/ou foliar e a porcentagem de taninos aumentou apenas nas folhas das plantas adubadas via solo + foliar. O silício atuou como indutor de resistência a M. persicae em batata.

Effect of two solanaceous plants on developmental and population parameters of the tomato leaf miner, Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae)

Tuta absoluta (Meyrick) is an important tomato pest that also feeds on other host-plants from the Solanceae family. We studied the effect of two cultivated plants, tomato (Lycopersicum esculentum Mill.) and potato Solanum tuberosum L. on the development and populational parameters of T. absoluta related with host-plant suitability. Larval developmental time, pupal weight, mean fecundity and an index of host-plant quality (IPQ = pupal weight / frass weight) were estimated. Age-specific survivorship and fecundity life tables were constructed in the laboratory to evaluate the following populational parameters: net reproductive rate (Ro), intrinsic rate of increase (r) and generation time (T). Larval developmental time was shorter and pupal weight was higher (P < 0.0001) for larvae reared on tomato (P < 0.0001). Mean fecundity was not significantly different on both plants (P = 0.07) and food quality of host-plant was higher for tomato (P = 0.02). Mean population parameters on tomato were: Ro = 48.92; T = 27.98, r = 0.14; and on potato: Ro = 14.43; T = 32.35, r = 0.08. Although results showed that tomato was a more suitable host-plant and had a better nutritional quality than potato, when T. absoluta fed on potato the potential population increase requires attention. Under appropriate climatic conditions, spatial and temporal coincidence between crop and pest, T. absoluta could become a pest for the potato crop.

Tuta absoluta (Meyrick) es una importante plaga del tomate que puede utilizar también otras solanáceas como plantas huéspedes. Este trabajo evaluó el efecto del tomate, Lycopersicum esculentum Mill. y la papa, Solanum tuberosum L., sobre algunos aspectos del desarrollo y parámetros poblacionales relacionados con la calidad del alimento. Se midieron: duración del estado larval, peso pupal, fecundidad y un índice de la calidad alimentaria de la planta huésped (peso pupal / peso de heces). Por medio de tablas de vida en el laboratorio se obtuvieron datos de supervivencia y fecundidad específicas por edad con los cuales se calcularon: tasa reproductiva neta (Ro), tasa intrínseca de crecimiento (r) y tiempo generacional (T). Los individuos alimentados con tomate tuvieron una menor duración del estado larval (P < 0,0001), y un mayor peso pupal (P < 0,0001) que con papa. La fecundidad promedio fue marginalmente más alta en tomate (P = 0,07) mientras que la calidad nutricional fue mayor para el tomate (P = 0,025). Los parámetros poblacionales promedio fueron: Ro = 48,92; T = 27,98, r = 0,14, en tomate; y Ro = 14,43; T = 32,35, r = 0,08 en papa. Si bien los resultados indicaron que el tomate exhibió una mayor calidad nutricional que la papa, las cohortes de T. absoluta alimentadas con este huésped secundario, mostraron un crecimiento relativamente importante. Bajo condiciones climáticas favorables y una coincidencia espacial y temporal entre la polilla y el cultivo de papa, sus poblaciones podrían convertirse en una plaga para el mismo.

Characterization of a newly established potato tuber moth (Phthorimaea operculella Zeller) cell line.

BACKGROUND & OBJECTIVE: Potato tuber moth (PTM), Phthorimaea operculella Zeller is a widely distributed, devastating pest of potatoes attacking the foliage and infest the tubers in both field and store causing serious economic damage. As application of PTM granulovirus (PTM-GV) has shown significant reduction in damage, attempts were made to develop a new cell line from this insect to grow PTM-GV for use as a biopesticide. METHODS: Approximately 100 mg of insect eggs were collected, surface sterilized and crushed gently in a boiling tube aseptically. The tissues were washed with physiological saline, suspended in growth medium and incubated stationary at 28 degrees C. Morphology of cells was studied after staining with Giemsa. Besides karyological and growth curve studies, PCR amplification was also done for rapid amplified polymorphic DNA pattern. RESULTS: A new cell line from the embryonic tissue of PTM was maintained in Mitsuhashi Maramorosch medium supplemented with 10 per cent foetal bovine serum. It is in the 78th passage level and designated as NIV-PTM-1095. Random amplified polymorphic DNA profile analysis indicated this as a new cell line from potato tuber moth and differed from the profiles of two other lepidopteran cell lines maintained in the laboratory. Three different cell types were observed at the 40th passage level and comprised of epithelial-like cells (77%), fibroblast-like cells (20%) and giant cells (3%). The chromosome number varied from 54-176. The cell line had a cell doubling time of approximately 42 h during the logarithmic phase of growth. The cell line did not support the multiplication of any of the baculoviruses used in the study. INTERPRETATION & CONCLUSION: Since the new cell line is found to replicate PTM-GV, it may be useful for the propagation of PTM-GV in large scale. Studies to scale up the production of the GV in the cell line and field trials may lead to its widespread use as an eco-friendly biopesticide.