Eur J Plant Pathol (2012) 133:439–448 DOI 10.1007/s10658-011-9917-9
Histopathology of black spot symptoms in sweet oranges João Paulo Rodrigues Marques & Marcel Bellato Spósito & Alexandre Furtado Silveira Mello & Lilian Amorim & Matheus Mondin & Beatriz Appezzato-da-Glória
Accepted: 1 December 2011 / Published online: 23 December 2011 # KNPV 2011
Abstract In Brazil, citrus black spot (CBS) caused by Guignardia citricarpa is a major disease that has different symptoms on fruit. In this study, fruit of Citrus sinensis infected by G. citricarpa and showing the symptoms false melanosis, freckle spot and hard spot were cross-sectioned and analysed anatomically and histochemically by light microscopy. Immunohistological assays were performed. All symptoms were accompanied by a thickening of the cuticle. False melanosis lesions did not contain pycnidia and remained restricted to the epicarp or to the first layers of the mesocarp. The stomata in this type of lesion showed phenolic compounds in the guard cells and in the sub-stomatal chamber. In some samples, the guard cells and their surrounding cells lysed, and a wound meristem began to form underneath them. Freckle spot and hard spot lesions had very similar histological J. P. R. Marques : M. B. Spósito : A. F. S. Mello : L. Amorim : M. Mondin : B. Appezzato-da-Glória (*) Escola Superior de Agricultura “Luiz de Queiroz”, Universidade de São Paulo, Cx. Postal 9, CEP 13418-900 Piracicaba, SP, Brazil e-mail:
[email protected] Present Address: A. F. S. Mello Monsanto do Brasil Ltda, Rod. BR 452, Km 149,Cx. Postal 3077, CEP 38407-049 Uberlândia, MG, Brazil
alterations to the epicarp and mesocarp, but in our samples only hard spot lesions contained pycnidia. Both of these symptoms were accompanied by protein inclusions. Epidermal and sub-epidermal cells located in the oil-gland region were obliterated, causing alterations in these structures. All symptoms had regions that stained strongly for lipids and phenols. Keywords Citrus sinensis . Guignardia citricarpa . Phyllosticta citricarpa . Necrotic symptoms
Introduction Citrus black spot (CBS), caused by Guignardia citricarpa Kiely (Phyllosticta citricarpa Van der Aa), is an important disease in Brazil. All commercial varieties of oranges are susceptible to this disease (Spósito et al. 2004), which is controlled by four to five fungicide sprays in the field (Timmer 1999). As the disease is not found in Europe, fruit with black spot symptoms may not be imported into the European Union (Smith and Charles 1998; Aguilar-Vildoso et al. 2002). The state of São Paulo produces 80% of the oranges consumed in Brazil and is the world’s largest citrusproducing region but less than 1% of this total is exported, primarily due to the presence of CBS in the groves (Neves et al. 2007).
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Fig. 1 Healthy fruits of Citrus sinensis Osbeck ‘Valência’. a-g, Transversal sections of the epicarp and mesocarp. a, Epicarp with oil cavity. b-d, Details of the stomata after staining with toluidine blue (b) and fast green pH 2.5 (d) and after negative
ferric trichloride reaction (c). e. Epicarp stained with Xylidine Ponceau. f-g, Details of the mesocarp. g. Oil cavity epithelial cells. e: fruit epidermis, ep: epicarp, mesocarp (me), oc: oil cavity, st: stomata, vb: vascular bundles
G. citricarpa is an ascomycete that produces ascospores in pseudothecia on dead leaves on the citrus grove floor as well as pycnidia and conidia on fruits, dead leaves and dead twigs (Timmer 1999). The ascospores are predominantly transported by the wind, whereas the conidia are dispersed over short distances by rain splashes (Kotzé 1963). After the spores have been deposited, the infection process starts, and symptoms begin to appear in 4 months (Baldassari et al. 2008). Usually symptoms appear on ripe fruit, sometimes even after harvest. Three symptoms are usually associated with this disease (Brodrick and Rabie 1970; Kotzé 2000): hard spot, false melanosis and freckle spot. Hard spot is the most common symptom and is characterised by black, circular and depressed lesions, usually with pycnidia in the centre, but not always. It appears usually after fruit colour changes from green to orange (Kotzé 2000). False melanosis is characterised by small black lesions with no depression, similar to those seen in the melanosis symptom caused by Diaporthe citri. It typically develops on green fruit and do not contain pycnidia (Kotzé 2000). Freckle spot is characterised by red-to-brown depressed lesions that develop pre-harvest. Since the symptoms are extremely variable and can be difficult to identify, the most reliable diagnostic
criterion of this disease is based on the presence of pycnidia, which appear in the centres of hard spot and sometimes on freckle spot lesions (Kotzé 2000). The cause of the diversity in citrus black spot symptoms is not known. It could be due to the size of the fruit when infection occurs (Kotzé 2000; Aguilar-Vildoso et al. 2002), the weather (Brodrick and Rabie 1970), the concentration or type of the inoculum (Almeida 2009). As no reliable, practical and effective inoculation method has been reported in the literature, the symptoms development is only observed under natural Fig. 2 Fruits of Citrus sinensis (L.) Osbeck ‘Valência’ withb symptoms of “False melanosis”. a-b, ‘False melanosis’ lesions. The arrow in b indicated the rupture in fruit surface. c-j, Transversal sections of the lesioned pericarp. c-e, Phenolic compounds in guard cells (arrows in c and d) and in the substomatic chamber (arrows in e) after positive ferric trichloride reaction. f, Presence of the fungus between guard cells (arrow). g, Installation of wound meristem (arrows) in the sub-stomatic chamber. h, Rupture of the epidermal and sub-epidermal layers (arrow) above the wound meristem. i, Detail of the wound tissue composed of a layer of cells with phenolic compounds (arrow), one layer of cells with lipidic compounds (arrowhead) and some layers of meristematic cells. There are fungi hyphae on the surface. j, General view of the lesion indicated in b. The arrow indicated the rupture in the fruit surface. e: fruit epidermis, ep: epicarp, hy: hyphae, me: mesocarp, oc: oil cavities, st: stomata, wm: wound meristem
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infection. In the first report of a successful artificial inoculation with ascospores obtained in vitro, black spot
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lesions took 6 months to be produced on lemons (Lemir et al. 2000), the most susceptible citrus species to G.
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citricarpa (Kotzé 2000). It was not clear in that report how the authors guarantee that the symptoms originated from the artificial inoculation, because no control was described. Even the latest reported method of artificial inoculation of oranges does not allow a precise study for symptom development, because only false melanosis was consistently reproduced 150 days after inoculation on Pera sweet orange cultivar (Baldassari et al. 2009). No hard spot symptoms developed on inoculated fruit of Pera cv. Detached fruit cannot be inoculated because of the long incubation period of the disease. Although the reproductive structures of the fungus responsible for its spread are only present in fruit with hard spot or freckle spot symptoms, the European Union does not allow the entry of fruit with any symptom. All symptoms are restricted to the orange peel, reducing the price of diseased fruit on the fresh market. Under high disease intensity, the fruit often drop prematurely, decreasing yield (Kotzé 2000). One of the by-products of the orange juice industry that is becoming increasingly valuable is the oil extracted from the peels of citrus fruits. Fruits that show signs of hard spot surprisingly have higher oil content than those that show false melanosis (Spósito et al. 2009). This difference could be explained by different changes in the pericarp associated with each symptom. The aim of this study was to characterise anatomically and histochemically the false melanosis, freckle spot and hard spot symptoms caused by Guignardia citricarpa to better understand the colonisation process of this pathogen and assess the damage caused by the fungus in naturally occurring symptoms on sweet oranges.
Materials and methods Fruit of Valencia sweet orange (Citrus sinensis Osbeck) with symptoms of black spot as well as healthy control fruits were collected in an orchard in the state of São Paulo. Six fruit with each symptom type were collected from different trees in the same orchard. Green fruit were collected with completely developed false melanosis symptoms and mature fruit were collected with completely developed hard spot and freckle spot symptoms. Green and mature healthy fruit were used as controls. For each type of symptom and healthy tissues, ten 1-cm2 samples of peel, including the epicarp and mesocarp, were collected with a scalpel. The surfaces of the diseased fruit were photographed
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under a stereomicroscope (Nikon, model SMZ-2T). Afterwards, samples were fixed in Karnovsky solution (Karnovsky 1965), dehydrated in ethanol and embedded in plastic resin (Leica Historesin). Serial sections (5– 7 μm thick) were cut on a rotary microtome and stained with toluidine blue O (Sakai 1973). The main classes of metabolites in the lesions were investigated in sections from plastic resin-embedded samples using the following histochemical tests: Sudan black B (Jensen 1962) for total lipids, Ruthenium red (Johansen 1940) for pectins, ferric trichloride (Johansen 1940) for phenolic compounds, Xylidine Ponceau for protein compounds and fast green pH 2.5 for acid proteins (Cortelazzo 2007). Images were captured digitally with a Leica DM LB microscope with a video camera attached to a PC, using the IM50 image analysis software. For the immunohistochemical tests, the samples were immersed in Bouin’s fixative solution (Sass 1951), dehydrated in a graded ethanol series and embedded in Paraplast®. The paraffin blocks were sectioned with a rotary microtome (5 μm thickness) and deposited on slides. Later, the paraffin was removed using xylene, and the samples were rehydrated with a decreasing ethanol series. Toluidine blue (0.01%) in phosphate-buffered saline solution (PBS) was used to inhibit the natural auto-fluorescence of the cuticle (Ruzin 1999). Subsequently, the samples were blocked in a 1:1 mixture of PBS and Bovine serum albumin (BSA) for 5 min. The primary polyclonal antibody was obtained from fungal mycelia emulsified with incomplete Freund’s adjuvant (1:1) injected intramuscularly in the thigh of a four-month-old New Zealand rabbit for antiserum production (Duarte et al. 2004). This primary polyclonal antibody was validated on spore-traps of Guignardia spp. (Duarte et al. 2005). In this work the antibody diluted 1:100 in PBS, was added to the samples, which were incubated for 1 h. The samples were washed again in PBS / BSA, and then the secondary antibody, fluoresceinconjugated Goat Anti-Rabbit IgG (H+L) (KPL®), was added. The samples were incubated for 2 h, washed again in PBS / BSA, mounted in Vectashield mounting medium and analysed with filters for detecting fluorescein isothiocyanate (FITC; 450 nm/ 515 nm), rhodamine (546 nm/590 nm) and 4′,6-diamidino-2-phenylindole (DAPI; 365 nm/397 nm) under a Zeiss Axiophot 2 fluorescence microscope coupled to a digital camera. Image processing was performed with the Metasystems ISIS imaging software.
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Healthy control fruit has an epicarp or flavedo (yellow tissue, Fig. 1a), composed of a thin cuticle–covered
outer epidermis with stomata (Fig. 1b-d) and a compact sub-epidermal parenchyma (Fig. 1e) with oil glands (Fig. 1a and g). The mesocarp, or albedo (white tissue), consists of parenchyma with large intercellular
Fig. 3 ‘Freckle spot’ lesions caused by Guignardia citricarpa in fruit of Citrus sinensis (L.) Osbeck ‘Valência’ (a-b). c-h, transversal sections of the infected area stained with toluidine blue (c-e) and Xylidine Ponceau (f-h). c, general view of epicarp and mesocarp. Cell lyses (arrow) and collapse (*) around oil cavities. d, Detail of the epicarp cells shown in figure c exhibiting fungi hyphae (arrows). e, Detail of the mesocarp shown in
figure c. Notice the cell hyperplasia and protein inclusions (arrow). f, Distribution of protein inclusion in epicarp (arrows) and mesocarp cells (arrowheads). g, Spherical protein inclusions inside the mesocarp cells (arrows). h-i, Parenchyma cells lining the mesocarp vascular bundles in the infected area (h) and non-infected area (i). ep: epicarp, me: mesocarp, oc: oil cavities, vb: vascular bundle
Results
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spaces (Fig. 1f). The vascular bundles occurred only in the mesocarp and were classified as collateral. False melanosis symptoms appeared in the pericarps of green fruits as small (
