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, black spot (CBS) caused by alterations to the epicarp and mesocarp, but in our citricarpa is a major disease that has samples only hard spot lesions contained pycnidia. different symptoms on fruit. In this study, fruit of Both of these symptoms were accompanied by protein Citrus sinensis infected by G. citricarpa and showing inclusions. Epidermal and sub-epidermal cells located the symptoms false melanosis, freckle spot and hard in the oil-gland region were obliterated, causing alter- spot were cross-sectioned and analysed anatomically ations in these structures. All symptoms had regions and histochemically by light microscopy. Immuno- that stained strongly for lipids and phenols. histological assays were performed. All symptoms were accompanied by a thickening of the cuticle. False Keywords Citrus sinensis . Guignardia citricarpa . melanosis lesions did not contain pycnidia and Phyllosticta citricarpa . Necrotic symptoms 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 Introduction cells and their surrounding cells lysed, and a wound meristem began to form underneath them. Freckle spot Citrus black spot (CBS), caused by Guignardia citri- and hard spot lesions had very similar histological carpa 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. J. P. R. Marques : M. B. Spósito : A. F. S. Mello : L. Amorim : M. Mondin : B. Appezzato-da-Glória (*) 2004), which is controlled by four to five fungicide Escola Superior de Agricultura “Luiz de Queiroz”, sprays in the field (Timmer 1999). As the disease is Universidade de São Paulo, not found in Europe, fruit with black spot symptoms Cx. Postal 9, may not be imported into the European Union (Smith CEP 13418-900 Piracicaba, SP, Brazil e-mail: [email protected] and Charles 1998; Aguilar-Vildoso et al. 2002). The state of São Paulo produces 80% of the oranges con- Present Address: sumedinBrazilandistheworld’s largest citrus- A. F. S. Mello producing region but less than 1% of this total is Monsanto do Brasil Ltda, Rod. BR 452, Km 149,Cx. Postal 3077, exported, primarily due to the presence of CBS in CEP 38407-049 Uberlândia, MG, Brazil the groves (Neves et al. 2007). 440 Eur J Plant Pathol (2012) 133:439–448

Fig. 1 Healthy fruits of Citrus sinensis Osbeck ‘Valência’. a-g, ferric trichloride reaction (c). e. Epicarp stained with Xylidine Transversal sections of the epicarp and mesocarp. a, Epicarp Ponceau. f-g, Details of the mesocarp. g. Oil cavity epithelial with oil cavity. b-d, Details of the stomata after staining with cells. e: fruit epidermis, ep: epicarp, mesocarp (me), oc: oil toluidine blue (b) and fast green pH 2.5 (d) and after negative cavity, st: stomata, vb: vascular bundles

G. citricarpa is an ascomycete that produces asco- criterion of this disease is based on the presence of spores in pseudothecia on dead on the citrus pycnidia, which appear in the centres of hard spot and grove floor as well as pycnidia and conidia on fruits, sometimes on freckle spot lesions (Kotzé 2000). The dead leaves and dead twigs (Timmer 1999). The asco- cause of the diversity in citrus black spot symptoms is spores are predominantly transported by the wind, not known. It could be due to the size of the fruit when whereas the conidia are dispersed over short distances infection occurs (Kotzé 2000; Aguilar-Vildoso et al. by rain splashes (Kotzé 1963). After the spores have 2002), the weather (Brodrick and Rabie 1970), the been deposited, the infection process starts, and symp- concentration or type of the inoculum (Almeida toms begin to appear in 4 months (Baldassari et al. 2009). As no reliable, practical and effective inocula- 2008). Usually symptoms appear on ripe fruit, some- tion method has been reported in the literature, the times even after harvest. Three symptoms are usually symptoms development is only observed under natural associated with this disease (Brodrick and Rabie 1970; Kotzé 2000): hard spot, false melanosis and freckle Fig. 2 Fruits of Citrus sinensis (L.) Osbeck ‘Valência’ withb spot. Hard spot is the most common symptom and is symptoms of “False melanosis”. a-b, ‘False melanosis’ lesions. characterised by black, circular and depressed lesions, The arrow in b indicated the rupture in fruit surface. c-j, Trans- versal sections of the lesioned pericarp. c-e, Phenolic com- usually with pycnidia in the centre, but not always. It pounds in guard cells (arrows in c and d) and in the sub- appears usually after fruit colour changes from green stomatic chamber (arrows in e) after positive ferric trichloride to (Kotzé 2000). False melanosis is character- reaction. f, Presence of the between guard cells (arrow). ised by small black lesions with no depression, similar g, Installation of wound meristem (arrows) in the sub-stomatic chamber. h, Rupture of the epidermal and sub-epidermal layers to those seen in the melanosis symptom caused by (arrow) above the wound meristem. i, Detail of the wound tissue Diaporthe citri. It typically develops on green fruit composed of a layer of cells with phenolic compounds (arrow), and do not contain pycnidia (Kotzé 2000). Freckle one layer of cells with lipidic compounds (arrowhead) and some spot is characterised by red-to-brown depressed layers of meristematic cells. There are fungi hyphae on the surface. j, General view of the lesion indicated in b. The arrow lesions that develop pre-harvest. indicated the rupture in the fruit surface. e: fruit epidermis, ep: Since the symptoms are extremely variable and can epicarp, hy: hyphae, me: mesocarp, oc: oil cavities, st: stomata, be difficult to identify, the most reliable diagnostic wm: wound meristem Eur J Plant Pathol (2012) 133:439–448 441

infection. In the first report of a successful artificial lesions took 6 months to be produced on (Lemir inoculation with ascospores obtained in vitro, black spot et al. 2000), the most susceptible citrus species to G. 442 Eur J Plant Pathol (2012) 133:439–448 citricarpa (Kotzé 2000). It was not clear in that report under a stereomicroscope (Nikon, model SMZ-2T). Af- how the authors guarantee that the symptoms originated terwards, samples were fixed in Karnovsky solution from the artificial inoculation, because no control was (Karnovsky 1965), dehydrated in ethanol and embedded described. Even the latest reported method of artificial in plastic resin (Leica Historesin). Serial sections (5– inoculation of oranges does not allow a precise study for 7 μm thick) were cut on a rotary microtome and stained symptom development, because only false melanosis with toluidine blue O (Sakai 1973). The main classes of was consistently reproduced 150 days after inoculation metabolites in the lesions were investigated in sections on Pera sweet orange (Baldassari et al. 2009). from plastic resin-embedded samples using the follow- No hard spot symptoms developed on inoculated fruit of ing histochemical tests: Sudan black B (Jensen 1962)for Pera cv. Detached fruit cannot be inoculated because of total lipids, Ruthenium red (Johansen 1940) for pectins, the long incubation period of the disease. ferric trichloride (Johansen 1940) for phenolic com- Although the reproductive structures of the fungus pounds, Xylidine Ponceau for protein compounds and responsible for its spread are only present in fruit with fast green pH 2.5 for acid proteins (Cortelazzo 2007). hard spot or freckle spot symptoms, the European Union Images were captured digitally with a Leica DM LB does not allow the entry of fruit with any symptom. microscope with a video camera attached to a PC, using All symptoms are restricted to the orange peel, the IM50 image analysis software. reducing the price of diseased fruit on the fresh mar- For the immunohistochemical tests, the samples ket. Under high disease intensity, the fruit often drop were immersed in Bouin’s fixative solution (Sass prematurely, decreasing yield (Kotzé 2000). One of 1951), dehydrated in a graded ethanol series and em- the by-products of the industry that is bedded in Paraplast®. The paraffin blocks were sec- becoming increasingly valuable is the oil extracted tioned with a rotary microtome (5 μm thickness) and from the peels of citrus fruits. Fruits that show signs deposited on slides. Later, the paraffin was removed of hard spot surprisingly have higher oil content than using xylene, and the samples were rehydrated with a those that show false melanosis (Spósito et al. 2009). decreasing ethanol series. Toluidine blue (0.01%) in This difference could be explained by different phosphate-buffered saline solution (PBS) was used to changes in the pericarp associated with each symptom. inhibit the natural auto-fluorescence of the cuticle The aim of this study was to characterise anatomical- (Ruzin 1999). Subsequently, the samples were ly and histochemically the false melanosis, freckle spot blocked in a 1:1 mixture of PBS and Bovine serum and hard spot symptoms caused by Guignardia citri- albumin (BSA) for 5 min. The primary polyclonal carpa to better understand the colonisation process of antibody was obtained from fungal mycelia emulsified this pathogen and assess the damage caused by the fun- with incomplete Freund’s adjuvant (1:1) injected in- gus in naturally occurring symptoms on sweet oranges. tramuscularly in the thigh of a four-month-old New Zealand rabbit for antiserum production (Duarte et al. 2004). This primary polyclonal antibody was validat- Materials and methods ed on spore-traps of Guignardia spp. (Duarte et al. 2005). In this work the antibody diluted 1:100 in PBS, Fruit of Valencia sweet orange (Citrus sinensis was added to the samples, which were incubated for Osbeck) with symptoms of black spot as well as 1 h. The samples were washed again in PBS / BSA, healthy control fruits were collected in an orchard in and then the secondary antibody, fluorescein- the state of São Paulo. Six fruit with each symptom conjugated Goat Anti-Rabbit IgG (H+L) (KPL®), type were collected from different trees in the same was added. The samples were incubated for 2 h, orchard. Green fruit were collected with completely washed again in PBS / BSA, mounted in Vectashield developed false melanosis symptoms and mature fruit mounting medium and analysed with filters for detect- were collected with completely developed hard spot ing fluorescein isothiocyanate (FITC; 450 nm/ and freckle spot symptoms. Green and mature healthy 515 nm), rhodamine (546 nm/590 nm) and 4′,6-dia- fruit were used as controls. For each type of symptom midino-2-phenylindole (DAPI; 365 nm/397 nm) under and healthy tissues, ten 1-cm2 samples of peel, including a Zeiss Axiophot 2 fluorescence microscope coupled the epicarp and mesocarp, were collected with a scalpel. to a digital camera. Image processing was performed The surfaces of the diseased fruit were photographed with the Metasystems ISIS imaging software. Eur J Plant Pathol (2012) 133:439–448 443

Results outer epidermis with stomata (Fig. 1b-d) and a com- pact sub-epidermal parenchyma (Fig. 1e) with oil Healthy control fruit has an epicarp or flavedo (yellow glands (Fig. 1a and g). The mesocarp, or albedo (white tissue, Fig. 1a), composed of a thin cuticle–covered tissue), consists of parenchyma with large intercellular

Fig. 3 ‘Freckle spot’ lesions caused by Guignardia citricarpa figure c. Notice the cell hyperplasia and protein inclusions in fruit of Citrus sinensis (L.) Osbeck ‘Valência’ (a-b). c-h, (arrow). f, Distribution of protein inclusion in epicarp (arrows) transversal sections of the infected area stained with toluidine and mesocarp cells (arrowheads). g, Spherical protein inclu- blue (c-e) and Xylidine Ponceau (f-h). c, general view of epicarp sions inside the mesocarp cells (arrows). h-i, Parenchyma cells and mesocarp. Cell lyses (arrow) and collapse (*) around oil lining the mesocarp vascular bundles in the infected area (h) and cavities. d, Detail of the epicarp cells shown in figure c exhibit- non-infected area (i). ep: epicarp, me: mesocarp, oc: oil cavities, ing fungi hyphae (arrows). e, Detail of the mesocarp shown in vb: vascular bundle 444 Eur J Plant Pathol (2012) 133:439–448 spaces (Fig. 1f). The vascular bundles occurred only in Fig. 4 ‘Hard spot’ lesion caused by Guignardia citricarpa inb fruit of Citrus sinensis (L.) Osbeck ‘Valência’ showing pycnidia the mesocarp and were classified as collateral. (arrow) on fruit surface (a); b-l. Transversal sections of the False melanosis symptoms appeared in the peri- infected area; b-c. Deep alterations caused by fungal infection carps of green fruits as small (<3 mm) lesions on epicarp and mesocarp. c, Section stained with Ruthenium red (Fig. 2a) surrounded by numerous black spots shows the lysed and collapsed cells walls around the oil gland (arrows). d, Layer of cells (arrows) that exhibit hyperplasia and (<0.5 mm) that sometimes break the epicarp as a small protein inclusions. e-f, detail of the cells surrounding the oil pustule (Fig. 2b). The stomatal guard cells and sub- gland whose content react positively to Xylidine Ponceau and stomatal chambers as well as the intercellular space Sudan black B, respectively. g, Detail of the cells arrowed in below them showed the presence of phenolic com- figure d exhibiting hyperplasia and protein inclusions (arrows). h, Lipid content of the ep cells stained by Sudan black B and no pounds (Fig. 2c-e) whereas in healthy fruits the reac- stained fungi structures (arrow). i-k, Layer of cells lining the tion was negative (Fig. 1c). Hyphal structures were pycnidial cavity exhibiting fungi hyphae (arrows) stained with observed in the sub-stomatal chambers (Fig. 2f). The toluidine blue (i), Xylidine Ponceau (j) and acid fast green (k). l, cuticle was thickened in areas adjacent to the lesions Pycnidia releasing conidiospores stained by acid fast green. Notice the hyphae into the epicarp cells (arrows). co: conidio- (data not shown). The epidermal and sub-epidermal spores, e: epidermis, ep: epicarp, me: mesocarp, oc: oil cavities, cell layers had undergone necrosis, disruption and pc: pycnidia, vb: vascular bundle lysis, and a wound meristem was present (Fig. 2g). When the symptoms were severe (Fig. 2h), wounded close to the pycnidia (Fig. 4j). Deep alterations of the tissue were composed of an outer layer of cells with epicarp and mesocarp resulted in lysed and collapsed phenolic compounds, an inner layer of cells that accu- cells around the oil glands (Fig. 4b-d). These alter- mulated lipid compounds and a few layers of meriste- ations were not observed in healthy fruit (Fig. 1a). The matic cells, was visible on the surface (Fig. 2i). cells surrounding the oil glands had protein inclusions Hyphae were observed on the surface of damaged fruit (Fig. 4e) and lipids (Fig. 4f) which did not appear in (Fig. 2i). The disintegration of the epicarp tissues was healthy fruits (Fig. 1g). A layer of cells exhibiting limited, and there was no collapse of the oil cavity hyperplasia and protein inclusions appeared close to (Fig. 2j). No pycnidia were observed in this symptom. the lysed areas of the mesocarp (Fig. 4g). Other me- The freckle spot symptoms appeared on the peri- socarp cells stained positively for lipid content carps of mature fruits and were slightly depressed and (Fig. 4h). G. citricarpa structures were abundant in reddish with small brown dots (Fig. 3a-b). The cuticle fruits with this symptom (Fig. 4i-l). was thickened (data not shown). The depression of Immuno-histochemical tests showed that G. citri- this region resulted from the obliteration of epidermal carpa hyphae were associated with stomata in the and sub-epidermal cells (Fig. 3c-d) located in the oil- lesions of false melanose (Fig. 5a-b). The specificity gland region that caused alterations in these structures of the antibodies to the fungus was tested using the (Fig. 3c and f) when compared to the healthy fruit pycnidia as a positive control (Fig. 5c). G. citricarpa (Fig. 1a). The symptomatic area also showed cell hyphae were observed adjacent to but not within the plasmolysis and lysis in the epicarp and mesocarp oil cavity (Fig. 5d). The hyphae were also observed (Fig. 3c). A layer of cells exhibiting hyperplasia both intra- and intercellularly as well as near the (Fig. 3e) and protein inclusions (Fig. 3f-g) adjoined vascular bundles in the mesocarps of fruits with hard the lysed areas of the mesocarp. Parenchyma cells spot symptoms (Fig. 5e). lining the mesocarp vascular bundles in the symptom- atic area accumulated proteins (Fig. 3h). Protein was not observed in asymptomatic areas (Fig. 3i) nor in Discussion healthy fruit (Fig. 1e). Hard spot lesions appeared on the pericarps of Different symptoms of CBS seem to be related to mature fruits. Fruits with this symptom showed a very the host reaction to infection. In fruits with the depressed region in the epicarp containing one or more false melanosis symptom, the presence of phenolic com- pycnidia in the centre of the lesion (Fig. 4a). The pounds around the stomatal guard cells suggests depression results from cell obliteration in the epider- that the host reacted against pathogen infection. The mal and sub-epidermal layers on the upper side of the presence of hyphae close to the stomata reinforces this oil glands (Fig. 4b-c). Cuticle thickness was observed hypothesis. Eur J Plant Pathol (2012) 133:439–448 445

With artificial inoculation, false melanosis appears reacts faster to infection than when a low amount of only when a high concentration of inoculum (108 inoculum is used, accumulating phenolic compounds. conidia/ml) is applied to the fruit attached to the trees The effect of inoculum concentration on the induction and the incubation period is around 150 days of resistance has previously been observed in other (Almeida 2009; Baldassari et al. 2009). When the pathosystems (Singh et al. 2002). In contrast, hard plant is exposed to a high inoculum concentration, it spot symptoms were formed when the concentration 446 Eur J Plant Pathol (2012) 133:439–448

Fig. 5 Immunohitoschemical assays of ‘false melanose’ and c,TestwithG. citricarpa pycnidia to verify the positive reactivity ‘hard spot’ lesions submitted to Guignardia citricarpa antibody of antibody to the fungi cell wall (c3-c4). d3-d4, The G. citricarpa conjugated with fluorescein treatment. The figures in a horizontal hyphae occur around oil cavity (arrows). e, analysis of mesorcarp line are the same area in section and were made with three different tissue. e2, Autofluorescence of the structure that seems to be dead filters (first three columns) and then merged (fourth column). The protoplast. e3-e4, Mesocarp that shows numerous hyphae that cuticle (aeb) and xylem in the vascular bundle (e)showsan react positively to antibody (arrows). e: epidermis; ep: epicarp, autofluorescence. a3-a4 and b3-b4 shows the weakly fluorescence fm: false melanosis, hs: hard spot, me: mesocarp, oc: oil cavity, pc: of fluorescein in fungi structure related with fruit stomata (arrows). pycnidia, st: stomata, vb: vascular bundle of inoculum was 104 conidia/ml (Almeida 2009). In analysis showed that the changes extended even to the this study, no reaction to phenols was observed in any mesocarp, where both intra- and inter-cellular hyphae tissues with hard spot or flecked spot symptoms. were observed. According to Spósito et al. (2009)more All morphological symptoms of this disease are oil can be extracted from fruit with CBS symptoms than considered superficial and limited to the exocarp healthy fruit. The damage to the oil glands in hard spot (Cardoso-Filho 2003). However, the histological and freckle spot lesions due to pathogen colonisation Eur J Plant Pathol (2012) 133:439–448 447 observed in this study could favour oil overflow and Almeida, T. F. (2009). Expressão de sintomas, período de sus- promote the increase in extraction. cetibilidade e controle de Guignardia citricarpa, agente causal da mancha preta dos citros. PhD Thesis, Brazil: Intense cell division is a common sign of the pro- Universidade Estadual Paulista Júlio de Mesquita Filho. cess of plant defence to injury or infection. The speed Baldassari, R. B., Reis, R. F., & Goes, A. (2009). A new method of this process associated with plant biochemical for inoculation of fruit with Guignardia citricarpa,the responses characterises the host as susceptible or re- causal agent of citrus black spot. European Journal of , 123,1–4. sistant (Achor et al. 1997). Through the anatomical Baldassari, R. B., Wickert, E., & Goes, A. (2008). Pathogenic- studies, we noticed that false melanosis lesions could ity, colony morphology and diversity of isolates of not become hard spot lesions. One of the main features Guignardia citricarpa and G. mangiferae isolated from of false melanosis lesions that distinguish them from Citrus spp. European Journal of Plant Pathology, 120, 103–110. hard spot lesions is the presence of a wound meristem Brodrick, H. T., & Rabie, C. J. (1970). Light and temperature on the surface of the pericarp. This wound meristem effects on symptom development and sporulation of originates a small pustule by periclinal divisions. The Guignardia citricarpa Kiely, on Citrus sinensis (Linn) – outer layers of this pustule are constituted by cells with Osbeck. Phytophylactica, 2, 157 164. Cardoso-Filho, J. A. (2003). Efeito de extratos de albedo de lipid content. The wound meristem and the pustule do laranja (Citrus sinensis) e dos indutores de resistência not appear in hard spot lesion and freckle spot symp- ácido salicílico, acilbenzolar-S-metil e Saccharomyces cer- tom where the epidermis is maintained as a coating evisiae no controle de Phyllosticta citricarpa (Teleomorfo: tissue. Guignardia citricarpa). PhD Thesis, Brazil: Universidade de São Paulo. McOnie (1964) verified the presence of a region of Cortelazzo, A. L. (2007). Caracterização celular e bioquímica dense staining that did not stain for lipids. In this de sementes. (Campinas: Universidade de Campinas – region, proteins accumulated, and protein aggregates UNICAMP) were observed. Hard spot lesions showed a pattern of Duarte, K. M. R., Figueiredo, L. A., Gomes, L. H., Spósito, M. B., & Amorim, L. (2004). 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