Histopathology of Phakopsora euvitis on Vitis vinifera
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Rust (Phakopsora euvitis) is an important fungal disease in grapevines grown in tropical and subtropical regions. Epidemiological works have been conducted on plant disease resistance, however, little is known about defense mechanisms of resistance to the disease. Leaves of Vitis vinifera cv. Moscato Giallo were inoculated with P. euvitis and lesions were formed with pustules surrounded by water-soaked halo 17 days after inoculation. Foliar tissue of injured and sound material was fixed and submitted to histological techniques. Emergence of pustules from stomata was observed. In water-soaked halo, hyphae of fungus were not observed. In this region, foliar blade presented mesophyll modified by cell hypertrophy with reduction of intercellular spaces and accumulation of pectic compounds. Hypertrophied cells showed parietal thickenings in the cellulose and pectin layers. In the areas delimited by water-soaked halo in the pustule region, the fungus grew vigorously in intercellular spaces of chlorophyll parenchyma; however, vascular bundles also restricted the advance of fungus where sheath cells present parietal pectic thickenings. Therefore, although Vitis vinifera cv. Moscato showed rust symptoms on leaves, pathogen colonization was limited by the formation of water-soaked haloes and vascular bundles, which resulted in minor injuries along the foliar limbo.
KeywordsAnatomy Fungal infection Grapevine rust Hipertrophy Pectin Resistance mechanisms
Grapevine cultivation covers an area of 7.1 million ha worldwide with a production of 74.5 million tons in 2016 (Faostat 2017). With the expansion of viticulture to subtropical and tropical regions, climatic conditions have caused changes in culture management, as well as problems with fungal diseases, such as grapevine rust caused by Phakopsora euvitis (Amorim et al. 2016; Primiano et al. 2017). Grapevine rust is an end-of-cycle disease, with symptoms on leaves beginning to appear during fruit ripening (Amorim et al. 2016). Plants with high severity of the disease may show early defoliation and consequent fruit burn caused by overexposure to sunlight. Early leaf drop decreases production, translocation and photoassimilates storage in the root system, compromising bud sprouting and production of the following crop (Candolfi-Vasconcelos and Koblet 1990; Nogueira Júnior et al. 2017). Typical symptoms caused by P. euvitis are yellow-orange pustules on the underside of the leaf, while tissue necrosis is observed on the top of the leaf (Primiano et al. 2017). The disease occurs frequently in Vitis labrusca L. (Angelotti et al. 2008). In this species, young leaves generally have a higher density of trichomes that can serve as a physical barrier to pathogen penetration (Karabourniotis et al. 1999). As the leaf expands the number of trichomes per unit area decreases, favouring the infectious process (Angelotti et al. 2008). The first symptoms can be observed from 5 to 13 d after infection, under favorable conditions of temperature and leaf wetness (Angelotti et al. 2014). In more advanced stages of the disease, coalescence of pustules occurs due to the emergence of new uredines, which feature lesion growth (Primiano et al. 2017). Although grapevine rust is considered a disease of tropical and subtropical regions, it has already been reported in temperate regions of Asia, Oceania, North America, and other countries in Central America and South America (Primiano et al. 2017). There are no reports of the disease occurrence in Europe and Africa (Angelotti et al. 2008). In Brazil, grapevine rust was first reported in 2001 (Tessmann et al. 2004) and quickly spread to other producing regions in the country (Primiano et al. 2017). Hennessy et al. (2007) related the resistance degree to rust of species and grapevine cultivars by the number of pustules formed in the leaves, and cultivars of Vitis vinifera were classified as susceptible or highly susceptible, as well as American grapes. Although grapevine rust can be a threat to grape-producing regions of Europe (Chatasiri and Ono 2008; Primiano et al. 2017), there have been no studies on the colonization of and responses of host V. vinifera to infection by P. euvitis.
Host resistance can be characterized by different defense mechanisms which are classified as constituting and induced and may be biochemical or structural (Agrios 2005). Among the structural mechanisms, cell thickness and composition are highlighted and some substances like phenols that can be produced before infection (Agrios 2005) or accumulated later (Adaskaveg 1992) represent biochemical mechanisms. Regarding defense mechanisms induced by infection caused by the pathogen, accumulation of lignin (Hückelhoven 2007; Marques et al. 2018), pectin (Lionetti et al. 2012), cellulose and callose (Agrios 2005; Hückelhoven 2007; Marques et al. 2018) may occur in cell walls. Among the biochemical mechanisms induced, the cytoplasm may display accumulation of chemicals that are toxic to the pathogen, such as reactive oxygen species, phenolic compounds, pectic nature, among other substances (Agrios 2005; Hückelhoven 2007). In grapevines, sheaths of vascular bundles are an important preformed barrier that limits colonization of Plasmopara viticola, causal agent of downy mildew (Unger et al. 2007).
Despite epidemiological studies on grapevine cultivar resistance to rust based on disease severity and the number of pustules formed per unit of leaf area (Hennessy et al. 2007; Angelotti et al. 2008), it is not known which defense mechanisms in V. vinifera act in the colonization process by P. euvitis. Therefore, the objective of this study was to analyze lesions caused by P. euvitis on leaf of V. vinifera cv. Moscato Giallo.
Seedlings of V. vinifera cv. Moscato Giallo grafted onto SO4 (V. berlandieri x V. riparia) were grown in 7 l pots containing a sterilized substrate of sand, clay and manure (1:1:1). The plants were grown a temperature-controlled chamber at 20 °C and in relative humidity of 70%. The plants were considered ready for inoculation with the pathogen P. euvitis 60 d after sprouting of buds when the plants presented nine expanded leaves. The inoculum of P. euvitis used in the experiments was obtained from leaves of V. labrusca cv. Niagara Rosada with rust symptoms from the experimental area of the University of São Paulo, Piracicaba, São Paulo, Brazil. Leaves with disease symptoms were taken to the laboratory where urediniospores of pustules were collected with a brush, and a spore suspension was prepared at concentration of 1 × 104 urediniospores mL−1 with a Neubauer chamber. The spore suspension was sprinkled on the abaxial face of leaves of all plants of V. vinifera cv. Moscato, which were kept for 24 h in a humid chamber at 25 °C in the dark (Angelotti et al. 2014). The plants used as control had the leaves sprayed with water.
The material for the histopathological analysis was collected from intermediate leaves (5th leaf) of six plants, with three plants inoculated with P. euvitis and three plants that were not inoculated (control). The material was collected 17 d after inoculation when symptoms were well developed on the leaves. Samples of healthy and injured tissues were fixed in the Karnovsky solution for 24 h (Karnovsky 1965). During this period, the samples were placed in a vacuum pump for better penetration of the fastener. The plant material was subsequently dehydrated through a graded alcohol series (10, 20, 30, 50, 70, 80, 90 and 100%) and embedded in plastic resin (Leica Historesin®, Heraeus Kulzer, Hanau, Germany). The samples were sectioned in rotational microtome Leica RM 2045 at 5 μm thickness. For the histopathological analyses, part of the cross sections was mounted on glass slides and subsequently stained with toluidine blue (O’Brien et al. 1964). After staining, the slides were mounted with synthetic resin Entellan® (Merck, Darmstadt, Germany). Images were documented with a camera Leica DFC310FX under a Leica DMLB microscope. For the histochemical tests, the other part of the cuts was placed in an aqueous solution of ruthenium red to identify the presence of acidic polysaccharides (Chamberlain 1932) and ferric chloride to identify the presence of nature phenolic compounds (Johansen 1940). Three different dyes were used for the differentiation of P. euvitis hyphae and plant cell walls. The fungal cell wall structure was visualized by staining the slides with Wheat Germ Aglutin – Alexa Fluor 488 (WGA AF 488) for 30 min, which reacts specifically to N-acetylglucosamine residues of chitin from the wall of the fungus and fluoresces green when examined under a 5-L filter (460-500 nm excitation; 515-485 nm emission) (Marques et al. 2018). Cellulose visualization was done by 1% Calcofluor White stain for 1 min, which reacts with cellulose and fluoresces in blue when examined under an A4 filter (340–380 nm excitation; 450–490 nm emission) (Hughes and McCully 1975) and Nile Red for 7 min, which indicates the presence of substances of lipophilic nature in red, when analyzed under an N2.1 filter (515–560 nm excitation; 590 nm emission) (Greenspan et al. 1985). Images from slides were captured digitally through a Leica DM5500B microscope with a video camera attached to a PC, using Leica IM50 image analysis software. Samples of symptomatic and asymptomatic leaves with rust from the grapevine were fixed in a Karnovsky solution adjusted to pH 7.2, using phosphate buffer (Karnovsky 1965, modified) dehydrated using a graded series of ethyl alcohol (10, 30, 50, 70, 90, and 100%), critical point-dried using CO2 (Horridge and Tamm 1969), mounted on aluminum stubs using double-sided carbon tape and coated with a 30–40-nm gold film. The images were captured using an LEO VP 435 scanning electron microscope at an acceleration voltage of 20 kV.
Rust lesions on leaves of V. vinifera cv. Moscato caused by P. euvitis often presented a water-soaked halo. In the area of the water-soaked halo, hyphae of the pathogen were not observed. Restriction of fungal growth to the pustule area is probably due to the drastic reduction of the intercellular spaces and parietal thickening of cell cellulosic mesophyll in the area of the water-soaked halo. This growth restriction of the fungus associated with the cellulosic parietal thickening in cells of the host has been observed in other phytosystems such as corn roots infected by Phytophthora cinnamomi, in barley leaves infected by Blumeria graminis f. sp. hordei (Hinch and Clarke 1982; Chowdhury et al. 2014) and sugar cane showing a defense response to infection by Sponsorium scitamineum (Marques et al. 2018). The soaked halo aspect around the lesion is probably due to the accumulation of pectic substances that present a hydrophilic nature (Appezzato-da-Glória et al. 2004; Albersheim et al. 2011). Pectin accumulation can be related to strengthening parietal tissue when associated with the binding of Ca2+ ions to homogalacturans with a low degree of methyl esterification forming calcium pectate links, which gives greater rigidity to cell walls and consequent reduction of symptoms (Platero and Tejerina 1976; Peaucell et al. 2012). Further studies on immunostaining should be conducted to understand pectin composition and distribution and its association with the defense process in the pathosystem Vitis vinifera – Phakopsora euvitis. The increase in the volume of mesophyll cells in the water-soaked halo, as well as accumulation of phenols near the infection site, was also observed in soybean cultivars (Glycine max L.) resistant to rust caused by Phakopsora pachyrhizi (Keogh et al. 1980). The vascular bundle sheath in V. vinifera cv. Moscato Giallo restricted the access of P. euvitis to vascular tissues, which exhibited pectic parietal thickenings in cells. This limitation of mycelial growth in the vascular bundle sheath was also observed for the pathosystem Plasmopara viticola – Vitis vinifera cv. Müller-Thurgau (Unger et al. 2007). Restriction of biotrophic mutualist fungi by vascular bundles is often attributed to the restriction of production of enzymes that degrade lignin (Bellincampi et al. 2014).
In the pathosystem Phakopsora euvitis - Vitis vinifera cv. Moscato Giallo, the host presented induced defense mechanisms. The water-soaked halo, along with changes in the vascular bundle sheath cells, delineated injuries and acted as structural barrier on fungal colonization. The accumulation of pectic substances and the parietal thickenings in the cells of the cellulosic halo and vascular bundle sheath restricted fungal colonization, which resulted in the formation of small pustules over the foliar limbo.
The authors gratefully acknowledge the financial support of the São Paulo Research Foundation (FAPESP Proc: 2013/24003-9). We thank the Center of Support in Electron Microscopy Applied to Agriculture, ESALQ, USP, at the laboratory of Plant Anatomy of ESALQ (LanVeg) for the use of equipment. We also thank Msc. Marli Kasue Missake Soares for helping in material preparation for the anatomical analysis. BLN thanks Dr. Antonio F. Nogueira Jr. for his comments to the manuscript.
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