Caracterización morfológica e histopatología de Peronospora ciceris en hojas y semillas de garbanzo (Cicer arietinum L.)
Dagoberto Fierro-Corrales1; Miguel Ángel Apodaca-Sánchez1; José Alberto Quintero-Benítez1; Santos Gerardo Leyva-Mir2; Jorge Luis Flores-Sánchez3; Juan Manuel Tovar-Pedraza3*
1 Universidad Autónoma de Sinaloa, Escuela Superior de Agricultura del Valle del Fuerte, Protección Vegetal. Calle 16 Av. Japaraqui S/N, Juan José Ríos, Ahome, Sinaloa, C.P. 81110, MÉXICO.
2 Universidad Autónoma Chapingo, Departamento de Parasitología Agrícola. Carretera México-Texcoco km. 38.5, Chapingo, Estado de México, C.P. 56230, MÉXICO.
3 Colegio de Postgraduados, Campus Montecillo, Instituto de Fitosanidad, Fitopatología. Carretera México-Texcoco km. 36.5, Montecillo, Estado de México, C.P. 56230, MÉXICO. Correo-e: jmtovar@colpos.mx (*Autor para correspondencia).
]]>Received: February 23, 2014.
Accepted: April 12, 2014.
Abstract
Chickpea downy mildew is one of the most destructive diseases found sporadically in chickpea fields in the counties of Salvador Alvarado and Guasave, Sinaloa, Mexico. The aims of this study were to morphologically characterize the species of Peronospora causing downy mildew in chickpea, and to describe the histological changes induced by the pathogen in chickpea seeds and leaflets at different stages of infection (initial, intermediate and advanced). Samples of healthy and naturally-infected chickpea cv. "Blanco Sinaloa-92" leaflets and seeds were collected for morphological characterization of the pathogen and histological analysis. Cross-sections of leaflets and seeds were obtained with a manual rotary microtome and processed using the safranin-fast green differential staining technique. Morphological characterization indicated that Peronospora ciceris is the causative species of downy mildew symptoms in Sinaloa, Mexico. Histological evidence in leaflets with initial stages of infection showed palisade parenchyma with cell disruption, followed by hypertrophy and rupturing of cells. In addition, abundant coenocytic mycelial growth colonizing the vascular system and causing rupturing of the xylem vessels and phloem sieve tubes was observed. In leaflets with intermediate symptoms, the presence of intercellular mycelium, chloroplast degradation and loss of cell nuclei was detected. Leaflets with advanced symptoms exhibited damage in all tissues, showing massive cell destruction caused by extensive mycelial colonization in the epidermis, mesophyll, phloem and xylem. In seeds, intercellular mycelium was only observed in the embryonic cells.
Keywords: Downy mildew, oomycete, severity.
Resumen
]]> El mildiu del garbanzo, es una de las enfermedades más destructivas que se ha encontrado esporádicamente en campos de garbanzo, localizados en los municipios de Salvador Alvarado y Guasave, Sinaloa, México. Los objetivos de este estudio fueron caracterizar morfológicamente a la especie de Peronospora, causante del mildiu del garbanzo; así como, describir los cambios histológicos que induce el patógeno en semillas y foliolos de garbanzo, con diferentes estados de infección (inicial, intermedia y avanzada). Muestras de foliolos y semillas de garbanzo cv. "Blanco Sinaloa-92", sanos e infectados naturalmente, se recolectaron para la caracterización morfológica del patógeno y su análisis histológico. Secciones transversales de foliolos y semillas se obtuvieron con un micrótomo de rotación manual y se procesaron mediante la técnica de tinción diferencial safranina verde-rápido. La caracterización morfológica indicó que Peronospora ciceris, es la especie causante de los síntomas de mildiu en Sinaloa, México. Los daños histológicos en foliolos con etapas iniciales de infección, mostraron parénquima en empalizada con desorganización celular, seguida por hipertrofia y rompimiento de células; además, se observó abundante crecimiento micelial cenocítico colonizando el sistema vascular y provocando rompimiento de los vasos del xilema y tubos cribosos del floema. En foliolos con síntomas intermedios, se detectó la presencia de micelio intercelular, degradación de cloroplastos y pérdida de núcleos celulares. Foliolos con síntomas avanzados exhibieron daño en todos los tejidos, observándose destrucción celular masiva, provocada por extensiva colonización micelial en epidermis, mesófilo, floema y xilema. En semillas, únicamente se observó micelio intercelular distribuido en las células embrionarias.Palabras clave: Mildiu, oomicete, severidad.
Introduction
The chickpea (Cicer arietinum L.) is cultivated in about 50 countries and is the third most important legume in the world, after the bean (Phaseolus vulgaris L.) and pea (Pisum sativum L.). This is due to its high nutritional value, which has made it an integral part of the daily diet of millions of people (Knights et al., 2007).
During the 2012 cycle in Mexico, 137,861 and 17,476 ha were seeded with chickpea for grain and fodder respectively. The states of Sinaloa, Sonora, Guanajuato, Baja California Sur and Michoacán were the main producers of chickpea for grain. In this regard, the state of Sinaloa stands out because it accounted for approximately 70 % of national production in the autumn-winter cycle, with an average yield of 2.01 t∙ha-1 (Servicio de Información Agroalimentaria y Pesquera [SIAP], 2013).
The gap between average and potential chickpea production is mainly due to the high incidence of disease and poor agricultural management. The main diseases affecting chickpea worldwide are: blight (Ascochyta rabiei), Fusarium wilt (Fusarium oxysporum f. sp. ciceris), gray mold (Botrytis cinerea), dry root rot (Rhizoctonia solani), rust (Uromyces ciceris arietini), neck rot (Sclerotium rolfsii) and stem rot (Sclerotinia sclerotiorum) (Chen, Sharma, & Muehlbauer, 2011; Ghosh, Sharma, Telangre, & Pande, 2013; Nene et al., 2012).
Chickpea production in Sinaloa is based on the production of quality grain, which has given it worldwide recognition. Much of this success is due to the production of varieties with features demanded by the international market, but also for their resistance to diseases originating in the soil. An example of these tolerant varieties is called Blanco Sinaloa-92, which has allowed the Mexican chickpea to be exported to more than 40 countries (Valenzuela-Herrera et al., 2012). However, this variety is highly susceptible to downy mildew, especially when there is high humidity and moderate temperatures (Salinas-Pérez, Cortez-Moncada, & Macías-Cervantes, 2008).
Downy mildews (order Peronosporales) are obligated biotrophic pathogens that cannot complete their life cycle without the presence of the host's living tissue, and they cannot be cultivated in axenic medium. Also, this group of pathogenic oomycetes is responsible for causing severe diseases in economically important crops (Webster & Weber, 2007).
In India, Agarwal, Kulshreshtha, Bhalla, and Sarbhoy (2003) described for the first time Peronospora ciceris as the causal agent of chickpea downy mildew. However, the disease had already appeared in Israel and Mexico in the late 70's (Nene, 1980). In Mexico, chickpea downy mildew has been detected sporadically ever since the first outbreak occurred in Sinaloa and Baja California during the 1996-1997 cycle, with Peronospora sp. reported as the causal agent (Carrillo-Fasio, AllendeMolar, & García-Estrada, 2012).
]]> Symptoms of the disease, called downy mildew, occur in chickpea leaflets in the form of light green spots, which are diffuse at the beginning. Subsequently, the infection progresses on the leaflet irregularly and acquires a dull yellow color. If the conditions of temperature (20 - 24 °C) and humidity (> 85 %) are favorable for its development, fungal growth with a hairy and dark appearance can be seen on the underside of the leaf (Carrillo-Fasio et al., 2012).Globally, no histopathological studies describing the damage induced by the causal agent of chickpea downy mildew have been published. Moreover, in Mexico there is no taxonomic confirmation of which species of Peronospora causes this disease. Therefore, this study was conducted in order to morphologically characterize the causal agent of chickpea downy mildew and determine the histological and cellular changes induced by this oomycete in both chickpea seeds and leaflets with different levels of infection.
MATERIALS AND METHODS
Plant material
Samples of leaflets and seeds (500 g) of chickpea cv. "Blanco Sinaloa-92," both healthy and with presence of downy mildew symptoms, were collected in several lots in Valle del Fuerte and Évora, Sinaloa (Figure 1).
Leaflet samples were grouped according to the severity level of symptoms corresponding to different stages of downy mildew infection on each leaflet. The severity scale was established as follows: a) healthy leaflet: no symptoms of the disease, b) leaflet with initial symptoms: presents ovoid or round pale-green spots (1 - 10 % severity), c) leaflet with intermediate symptoms' chlorotic spots (11 - 40 % severity), d) leaflet with advanced symptoms: in this stage of disease development, the leaflets have brown to black spots, and their tissue is necrotic (> 41%). In the initial and intermediate symptoms, the presence of the fungus sporulating on the surface of the infected leaflets was observed (Figure 2).
Morphological characterization
To carry out the quantitative and qualitative characterization of the reproductive structures of the fungus (sporangiophores and sporangia), 10 chickpea leaflets with abundant Peronospora sp. sporulation were fixed in Carnoy's solution (96 % ethyl alcohol and glacial acetic acid, 1:1) in 50-mL amber vials; they were placed uncovered in a Kitasato flask and the air inside the leaflets was displaced with a vacuum pump; the Carnoy's solution was infiltrated properly. The vials were kept capped at room temperature (2025 °C) for six months until the Carnoy's solution had extracted the chlorophyll from the tissues; this process enabled adequate observation of the samples under a compound microscope (34936 Ultraphot II, Carl Zeiss®, Germany). Of the leaflets fixed in Carnoy's solution, nine were randomly selected, each arranged on a slide with lactofenol and a coverslip placed on top of it. The counting of sporangiophores emerging from a stoma and the morphometric characterization of 100 sporangia were performed using the compound microscope at a magnification of 40 X, thoroughly examining each leaflet. At the same time as the sporangiophore count, the search for oospores embedded among the intercellular spaces of the chickpea leaflet tissue was also conducted.
]]>Leaflet preparation for histological studies
Samples of leaflets, at each stage of infection, were fixed in FAA solution (formaldehyde [100 mL], glacial acetic acid [50 mL], absolute ethanol [50 mL] and distilled water [350 mL]), where they remained for at least 24 h. After the fixation time, the samples were washed with tap water for 30 to 60 min. The samples were dehydrated using an automatic tissue processor (Tissue-Tek II, model 4640-B, Sakura Finetechnical®, Japan), where they passed through ethyl alcohol solutions (50, 70, 96 and 100 %), followed by a change to absolute ethanol-xylene (1:1), two changes of pure xylene and one in paraplast (SIGMA Chemical®, USA); tissues were kept for 3 h in each solution.
Inclusion of the tissues consisted of embedding the samples in cubic bond paper molds, pre-filled with molten paraplast; the tissues were oriented in both a cross and paradermal plane. The paraplast cubes were fixed in wooden blocks (2 x 1 x 3 cm) and mounted in a manual rotary microtome (Spencer 820, American Optical®, USA), where paradermal and cross cuts of 8 μm in thickness were made; subsequently, the cut strips were placed in a water bath (60 °C water plus 3 % gelatin) and mounted on slides.
The sections were stained using the safranin-fast green differential staining technique described by Leyva-Mir, Cárdenas-Soriano, Tovar-Pedraza, Huerta-Espino, and Villaseñor-Mir (2012) with some modifications, which began with dewaxing the cuts in three changes of xylene (3 min in each one), after which they were hydrated with a graded ethyl alcohol series (100, 96, 70 and 50 %) for 3 min each. Staining in 1 % safranin (prepared in 50 % ethyl alcohol) was performed for 3 h, and then the sections were dehydrated in an ethyl alcohol series of 50, 70 and 96 % (3 min in each one) and stained with fast green (Técnica Química®, Mexico) at 1 % (prepared in 96 % ethyl alcohol) for 30 s; then they were washed and dehydrated by being passing through ethyl alcohol at 96 and 100 % for 3 min in each one. Finally, the stained sections were passed through three changes of xylene (3 min in each one) and covered with synthetic resin and coverslips.
Out of a total of 30 randomly-selected leaflets fixed in FAA solution, 80 permanent preparations stained with safranin-fast green were obtained. The preparations were analyzed in detail under a compound microscope (34936 Ultraphot II, Carl Zeiss®, Germany).
Seed preparation for histological studies
The seeds were selected by color: normal and dark brown. Of the dark brown seeds, 10 were taken randomly and included in paraffin, following the same procedure used to include the chickpea leaflets in paraffin, but in this case the grains remained for 5 h in each change and were cut at 12-μm thickness. The histological seed sections were stained with safranin-fast green, according to the methodology described for leaflets. Finally, the preparations were examined systematically using a compound microscope.
]]> RESULTS AND DISCUSSION
Morphological characterization
The preparations examined under light microscopy showed that the sporangiophores emerging from each stoma were arranged in fascicles (1 - 7), measured from 149.9 - 471.7 x 60 - 276.7 μm (most often occurring in the range from 210 - 330 x 100 - 200 μm) and branched dichotomically from two to ten times (most often appearing with five to six branches) (Figure 3 A and B). The terminal branches were acute to sub-acute, ending in rounded tips (Figure 3 C). The sporangia were ovoid to sub-elliptic, hyaline, and 21.87 - 34.99 x 13.8 - 24.54 μm, with a granular, thin-walled and smooth cytoplasm (Figure 3 D), whereas the mycelia were coenocytic, filiform and distributed intercellularly within the mesophyll. All morphological characters and measurement ranges agreed with those reported by Agarwal et al. (2003) for Peronospora ciceris.
It is important to note that morphological characterization is a reliable tool for identifying Peronospora ciceris, as demonstrated by Agarwal et al. (2003), who made a comparison between the morphological characters presented by different Peronospora spp. related to Peronospora ciceris, which can be differentiated by more than one morphological character (length, number of conidiophore branches and the shape and size of sporangia and oospores).
Anatomical description of healthy tissues
Healthy leaflets had an adaxial and abaxial epidermis formed by a layer of cells, usually tubular in a horizontal direction, apparently without chloroplasts (Figure 4 A). A large number of stomata were observed, but it was not possible to see the cuticle. The palisade parenchyma is constituted by four to five layers of vertically-arranged cells; these cells have a central nucleus and a large number of chloroplasts. The spongy parenchyma is composed of two to three layers of irregularly-shaped or almost round cells. Similarly, these cells showed a nucleus and abundant chloroplasts; the xylem was made up of large conducting vessels oriented towards the adaxial epidermis, and the phloem by sieve tubes oriented towards the abaxial epidermis.
Histological alterations in naturally-infected leaflets
Infected leaflet sections with initial symptoms showed cell disruption of the mesophyll (Figure 4 B) due to the presence of mycelia in this plant organ. In chickpea leaflet sections with intermediate symptoms, the presence of intercellular mycelium (Figure 4 C), collapse of the epidermis (adaxial and abaxial), degradation of a large number of chloroplasts, loss of cell nuclei, phenol accumulation, cell necrosis and hyperplasia in some cells were detected (Figure 4 D). Some of these changes and accumulation of phenolic compounds were recorded by Reuveni and Cohen (1978) in tobacco plants infected by P. tabacina. Also, accumulation of phenolic compounds, lignification and callus deposition have been described for many systemic mildews (Mauch-Mani, 2002).
]]> In leaflet sections with advanced symptoms, damage caused by P. ciceris mycelium was easily found throughout the leaf, showing massive destruction of host cells, both in the epidermis (abaxial and adaxial) and in the mesophyll and vascular bundles (Figure 4 E), causing a rupture in the xylem vessels and phloem sieve tubes, which probably caused the blockage of liquids, as well as of nutrients, and therefore rapid necrosis of the leaflets. This coincides with the findings of Milholland, Papadopoulou, and Daykin (1981), who found P. tabacina intercellular mycelium growing through the cambium, xylem and phloem of systemically-infected tobacco plants. For their part, Wehtje and Zimmer, in 1978, found Plasmopara halstedii mycelium colonizing the vascular bundles of experimentally-infected sunflower seedlings.No oospores were observed in chickpea leaflets under either of the two techniques developed for this purpose: leaflets fixed in Carnoy's solution and in FAA solution included in paraffin; this contrasts with the results of Agarwal et al. (2003), who reported the presence of P. ciceris oospores in chickpea leaflets. This suggests that P. ciceris is possibly heterothalic, and that in the region under study it failed to form oospores for its survival as indicated by Smith and Price in 1997 for Hyaloperonospora parasitica.
Histological alterations in naturally-infected seeds
Our histological evidence showed mycelium distributed intercellularly in the seed embryo (Figure 4 F), coinciding with the findings of Adenle and Cardwell (2000), who reported the presence of mycelium in the embryo of maize seeds infected by Peronosclerospora sorghi, whereas Shetty, Mathur, and Neergaard (1980) recorded latent mycelium of Sclerospora graminicola in embryos of pearl millet (Pennisetum glaucum) seeds.
Several histological studies have reported the presence of Peronospora spp. oospores embedded in the cover of beet, pea and soybean seeds (Agarwal et al., 2006; Roongruangsree, Olson, & Lange, 1988), which act as survival structures during adverse conditions; they also represent the source of primary inoculum, which causes the systemic invasion in seedlings at the beginning of the next cycle, which begins with the epidemic's development in the field (Adenle & Cardwell, 2000; Agarwal et al., 2006; Singh & Mathur, 2004). However, in this study the presence of P. ciceris oospores in seeds was not detected, confirming the observations of Agarwal et al. (2003), who only noted the formation of oospores in chickpea leaflets. Also, studies conducted by Van der Gaag, Frinking, and Geerds (1993) showed oospores of P. vicia f. sp. fabae in all the aerial parts of bean plants, except seeds. This reinforces our belief that P. ciceris is heterothallic, and that in the region under study it was unable to form oospores for its survival, and thus the only way to do so would be as mycelium in the seed as reported by Sugha, Develash, Singh, Thakur, and Tyagi (1996), for the case of Peronospora destructor.
CONCLUSIONS
The morphological characters of the oomycete causing downy mildew symptoms in Sinaloa, Mexico, corresponded to those described for Peronospora ciceris. To the best of our knowledge, this is the first histopathological study showing a large part of the process of mycelial colonization and histological alterations induced by P. ciceris in the epidermis, mesophyll, phloem and xylem of leaflets, as well as in embryonic cells of chickpea seeds. These histological data can be used as a component in future studies aimed at evaluating various strategies for managing the disease.
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