Alteraciones histopatológicas causadas por la roya Puccinia nakanishikii (Pucciniales: Pucciniaceae) en plantas de Cymbopogon citratus (Poaceae)

Introducción: Los aspectos histopatológicos e histoquímicos relacionados con el ataque de royas en plantas, así como su relación con los diferentes estados espóricos, son escasos en la literatura científica. Objetivos: Describir y analizar los aspectos histopatológicos e histoquímicos en Cymbopogon citratus y su relación con los diferentes estados espóricos de la roya Puccinia nakanishikii. Métodos: Durante abril y agosto 2013 se recolectaron hojas sanas e infectadas con Puccinia nakanishikii en la escarpa noroccidental de la meseta de Bucaramanga-Colombia. Las muestras con lesiones en diferentes etapas del desarrollo se fijaron y procesaron de acuerdo a protocolos estándar para la inclusión y corte en parafina y resina. Las secciones obtenidas en parafina (5-7 µm) fueron teñidas con Safranina-azul de Alcian y azul de Alcian-Hematoxilina. En tanto que las secciones obtenidas en resina (0.5 µm) se tiñeron con azul de Toluidina. También se elaboraron secciones a mano alzada para análisis de autofluorescencia. Las observaciones y registro fotográfico se efectuaron con microscopio fotónico y microscopía de epifluorescencia. Para observaciones con microscopía electrónica de barrido (MEB), las muestras se fijaron en Glutaraldehído, se deshidrataron con 2,2 dimetoxipropano, se desecaron a punto crítico y se metalizaron con oro. Resultados: Las hojas son por lo general hipostomáticas, con células epidérmicas largas y cortas formando filas paralelas y con la presencia de tricomas unicelulares espinosos y microtricomas. La superficie abaxial está cubierta por una densa capa de ceras epicuticulares y la adaxial está formada por agrupaciones de células buliformes y células epidérmicas de contorno rectangular o cuadrado. En el mesófilo no hay diferenciación entre parénquima de empalizada y esponjoso y su anatomía refleja el metabolismo C4 presente en esta gramínea. Se observó la formación de urediosoros y teliosoros hipófilos. Las urediosporas son la fase de reinfección y estas tienen de 4-5 poros germinativos ecuatoriales y su pared es equinulada. Las teliosporas son de pared lisa y de pedicelo persistente. Las urediosporas forman tubos de geminación por lo general sobre la superficie abaxial de la hoja y se desarrollan en dirección de los estomas, por donde penetran al interior del mesófilo. No se observó la presencia de apresorios. La epidermis se desprende y levanta por del desarrollo de las urediosporas y las paráfisis capitadas, a medida que el urediosoro crece. Con el avance de la infección, los tejidos fotosintéticos se desorganizan, pierden la autofluorescencia de la clorofila y las células sufren necrosis. Posteriormente, los tejidos vasculares se fragmentan y colapsan. Para este momento, la infección se ha extendido sobre toda la lámina foliar llevando a la muerte de la hoja y defoliación de la planta. Durante etapas avanzadas de la infección en los urediosoros se observaron picnidios, probablemente del hiperparásito Sphaerellopsis, asociados estrechamente a los tejidos infectados por la roya. Conclusiones: Puccinia nakanishikii se desarrolla sobre las hojas de Cymbopogon citratus produciendo urediosoros y teliosoros. Las urediosporas son la fase de reinfección, y las teliosporas solo se observaron en etapas avanzadas de la infección. La epidermis y los tejidos fotosintéticos son severamente afectados por la necrosis celular. En etapas avanzadas de la infección los tejidos vasculares se ven afectados.

Introduction: Histopathological and histochemical aspects linked to the attack of fungal rusts to plants, as well as its relation with the different spore stages are topics rather scarce in the scientific literature. Objective: To describe and analyze the histopathological and histochemical aspects of Cymbopogon citratus and its relation with the different stages of the spores from the rust fungi Puccinia nakanishikii. Methods: During the months April and August 2013, leaves healthy and infected by Puccinia nakanishikii were collected in the Northwestern scarp of the Bucaramanga-Colombia plateau. The samples with injuries on diverse developmental stages were fixated and processed according to the standard protocols for embedding and sectioning in paraffin and resin. Sections obtained from paraffin (5-7 µm) were stained with Safranin-Alcian blue and Alcian blue-Hematoxylin. On the other hand, sections obtained from resin (0.5 µm) were stained with Toluidine blue. Further, freehand sections were obtained for an autofluorescence analysis. The observations and photographic record were done via photonic microscope and epifluorescence microscope. For the observations via scanning electron microscopy (SEM), the samples were fixated in Glutaraldehyde, dehydrated with 2,2 dimethoxypropane, then desiccated to critical point and finally coated with gold. Results: The leaves are generally hypostomatic, with long and short epidermic cell forming parallel rows and showing unicellular prickle trichomes and micro-trichomes. The abaxial surface is covered by epicuticular wax forming a dense layer. The adaxial epidermis is formed by groupings of bulliform cells and epidermal cells with rectangular or squared contour. In the mesophyll, there is no differentiation between palisade and spongy parenchyma, its anatomy reflects the C4 metabolism. The formation of uredosori and teliosori both hypophyllous was observed. Urediniospores are the reinfecting agents phase, they have 4-5 equatorial germ pores and echinulate wall. Teliospores have smooth wall and a persistent pedicel. The urediniospores form a germ tube, generally on the abaxial leaf surface, these tubes develop towards the stomata reaching the mesophyll interior. No appressorium were observed. The epidermis limiting the uredosorus detaches due the development and pressure that exert both the urediniospores and capitate paraphyses. As the infection progresses, autofluorescense of the chlorophyll is lost and the cells undergo necrotic processes. Afterwards, the phloem collapses and the xylem becomes slightly disorganized. At this moment, the infection is extended along the whole leaf blade, resulting in the leaf death and the plant defoliation. On advanced stages of the infection, the uredosori showed pycnidia, probably belonging to the hyperparasite Sphaerellopsis, these structures were closely associated to the rust infected tissues. Conclusions: Puccinia nakanishikii develops on the leaves of Cymbopogon citratus producing uredosori and teliosori. Urediniospores are the reinfective stage, teliospores were only observed at late stages of the infection. The epidermis and photosynthetic tissue are severely affected by cell necrosis. The vascular tissues are deeply affected on the advances stages of the infection.

Anatomia foliar de soja infectada por Phakopsora pachyrhizi H. Sydow & Sydow e tratadas com extratos vegetais / Leaf anatomy of soybean infected with Phakopsora pachyrhizi H. Sydow & Sydow and treated with plant extracts

Folhas de Glycine max (L.) Merril,infectadas pelo fungo Phakopsora pachyrhizi H. Sydow & Sydow e tratadas com extratos vegetais foram avaliadas, visando determinar in vivo as modificações anatômicas nas diferentes estruturas/tecidos foliares, além de reconhecer prováveis mecanismos de defesa. Folhas de soja cultivar 181 provenientes do quinto nó foram inoculadas com fungo e tratadas com diferentes extratos vegetais, água e álcool 70%. Para comparação foram analisadas a anatomia das folhas sadia e infectada e realizadas medidas nas estruturas/tecidos foliares. Na folha infectada, observou-se destruição da epiderme e parênquima lacunoso, visível proliferação de tricomas e cutícula espessada, principalmente na face abaxial. Observou-se a presença de compostos fenólicos nas células da epiderme quando rompida, em função do crescimento micelial. Nas folhas infectadas e tratadas com os extratos vegetais de Azadirachta indica, Maytenus ilicifolia e Allium sativum, as estruturas/tecidos vegetais apresentaram aumento de espessura por alongamento celular.

Glycine max (L.) Merril leaves, infected by the fungus Phakopsora pachyrhizi H. Sydow & Sydow and treated with plant extracts, were evaluated with the aim of determining in vivo the anatomical modifications in different leaf structures and of identifying probable defense mechanisms. Leaves from the 181 soybean cultivar originated from the fifth node were inoculated with the fungus and treated with different plant extracts, water and alcohol at 70%. For comparison, the anatomy of the healthy and infected leaves was analyzed and the leaf structures were measured. In the infected leaf, there was destruction of the epidermis and lacunar parenchyma, apparent trichome proliferation and denser cuticle, especially on the abaxial surface. There were also phenolic compounds in ruptured epidermis cells, due to mycelium growth. In the infected leaves treated with Azadirachta indica, Maytenus ilicifolia and Allium sativum extracts, the plant tissues had an increase in thickness due to cell elongation.

Métodos de inoculação de Phakopsora euvitis Ono em Vitis labrusca L / Inoculation methods of Phakopsora euvitis Ono in Vitis labrusca L

A ferrugem é uma das principais doenças da videira, com ocorrência registrada em diversas regiões produtoras. Neste trabalho, foram avaliados três métodos de inoculação de Phakopsora euvitis em mudas de videira 'Isabel' com o objetivo de selecionar um método para determinação da patogenicidade. Mudas oriundas de bacelos de videira foram inoculadas com esporos de P. euvitis e mantidas sob temperatura entre 25±4°C e UR 85±10 por cento durante 13 dias. O delineamento experimental foi inteiramente casualizado, com 10 repetições. Os tratamentos foram compostos por: T1 - pincelamento de esporos; T2 - pulverização com suspensão de esporos com 2,7x10³ esporos mL-1; T3 - discos de folhas de videira com sintomas afixados em cinco folhas por planta; e T4 - Testemunha. O método do pincelamento de esporos foi o mais eficiente, com início dos sintomas a partir do quinto dia e, após 13 dias, 100 por cento das folhas apresentavam sinais do patógeno.

Rust is one of the most important diseases in grapevine, with occurrence recorded in many production areas. In this research it was evaluated three methods of Phakopsora euvitis inoculation on grapevine seedlings cv. 'Isabel' with the objective of selecting an inoculation method for rapid pathogenicity determination. Grapevine seedlings were inoculated with P. euvitis and maintained under conditions of 25±4C and UR 85±10 percent for 13 days. The experimental design was completely randomized, with 10 replications. The treatments were: T1 - brush of pathogen spores; T2 - spores suspension spraying (2.7x10³ spores mL-1); T3 - disks of grapevine leaves with symptoms stuck in five leaves per plant and T4 - control. The spore brushing method was the most efficient, showing early symptoms at five days, and 100 percent of the leaves showing pathogen symptoms 13 days after treatment.