Diagnosis of grapevine esca disease by immunological detection of Phaeomoniella chlamydospora

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Fleurat-Lessard et al.

Grapevine esca disease diagnosis

455

Diagnosis of grapevine esca disease by immunological detection of Phaeomoniella chlamydospora _106 455..463

P. FLEURAT-LESSARD1, E. LUINI1, J.-M. BERJEAUD2 and G. ROBLIN1 1

Laboratoire de Biochimie, Physiologie et Biologie Moléculaire Végétales, FRE CNRS 3091, University of Poitiers, 40 Avenue du Recteur Pineau, F-86022 Poitiers cedex, France 2 Laboratoire de Chimie et Microbiologie de l’eau, University of Poitiers, UMR CNRS 6008, 40 Avenue du Recteur Pineau, F-86022 Poitiers cedex, France Corresponding author: Dr Gabriel Roblin, fax +33 5 49 45 46 81, email [email protected] Abstract Background and Aims: Esca is a devastating disease affecting grapevines all around the world induced by a complex of xylem-inhabiting fungi. Among them, Phaeomoniella chlamydospora has been considered as an early causal agent of the disease facilitating the access of opportunistic saprophytes whose mode of action should be further investigated. P. chlamydospora secreted into its culture medium a variety of polypeptides, the biochemical nature of which permitted us to develop a method of detection based on a serological test. Methods and Results: Polyclonal antibodies raised in rabbit against the polypeptide fraction recognised secreted fungal proteins in low amounts (commonly 1 ng). These antibodies showed a valuable specificity because they cross-reacted with polypeptides excreted by various strains of P. chlamydospora but not with those secreted by many other fungal pathogens commonly found in other grapevine infections. Importantly, as shown by the enzyme-linked immunosorbent assay test and immunolocalisation on ultrathin sections, they did not cross-react with the secreted polypeptides of various fungi intervening in other wood decay diseases, namely Eutypa dieback (Eutypa lata) and Black Dead Arm (Diplodia seriata and Neofusicoccum parvum). Using a serological approach, the presence of P. chlamydospora was detected in canes of selectively infected cuttings. Conclusions: The antibodies raised in rabbit against polypeptides secreted by P. chlamydospora are useful tools to specifically detect the presence of the pathogen. Significance of the Study: These results allow us to propose a reliable dot blot method to detect grapevine infection by P. chlamydospora. This method is non-destructive for grapevines, simple and rapid. Keywords: diagnosis, esca, Phaeomoniella chlamydospora, Vitis vinifera, wood disease

Introduction Esca is a devastating disease of grapevines that affects vineyards in major grape-producing areas around the world (Chiarappa 1959). Two features allow for the diagnosis of this disease, namely, external symptoms on leaves, grape bunches and berries, and internal features characterised by degradation of woody tissues. Esca is insidious because the first visible foliar symptoms may develop 3–5 years after planting of grapevines (Whiting et al. 2001). Symptoms can be graduated, occurring in mild or severe forms (Chiarappa 1959, Larignon and Dubos 1997). Generally, in the mild form, the leaves at the beginning of summer present chlorotic interveinal areas that later become necrotic. The severe form of the disease, called apoplexy, is characterised by the sudden and rapid wilting of the foliage leading to the death of the vines which occurs particularly during hot summers (Mugnai et al. 1999). The monitoring of the spread of this disease is complicated by the difference in the extent of external symptoms which varies within a vineyard and doi: 10.1111/j.1755-0238.2010.00106.x © 2010 Australian Society of Viticulture and Oenology Inc.

on the same plant from 1 year to another, depending on various factors. Climatic variations may intervene because symptoms of the mild form appear more frequently during rainy and mild summers (Surico et al. 2000). Furthermore, the soil type also influences disease development as observed in vineyards established in deep and rich soils which seem more sensitive to esca (Geoffrion 1971, Mugnai et al. 1999). Additionally, the type of vine training is also a factor of importance and, in particular, a pruning system requiring more cuttings creates favourable conditions for the development of the disease (Mugnai et al. 1999, Borgo et al. 2008). Characteristic symptoms inside the trunk and arms are indicative of chronic esca. In early stages, wood deterioration is indicated in longitudinal sections by black streaks. On adult vines (about 10 years), the most common symptom is white rot invading the centre of the trunk which changes the hard wood to a soft and friable mass. This rotted area is lined at its border with non-decayed wood by a thick brown-red area (Mugnai et al. 1999).

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The causal agents of this disease have been a source of controversy for many years. According to Larignon and Dubos (1997), esca is a complex disease involving successive fungal infections. Based on isolation and identification of the fungal pathogens associated with this disease, it has now been accepted that Phaeomoniella chlamydospora and Phaeoacremonium aleophilum are associated with the early stages of the infection (Péros et al. 2008) and were, respectively, isolated from the black streaks and from the brown-red areas observed in the xylem tissues (Mugnai et al. 1999). Infection of vines by these agents causes darkening of xylem vessels, with production of tyloses and gummy deposits resulting in occlusion of the vessels (Scheck et al. 1998, Pascoe and Cottral 2000, Trocoli et al. 2001). Moreover, vascular alteration was observed in spurs 4 months after inoculation with spore suspensions deposited on pruning wounds (Feliciano et al. 2004). These data stress the importance of P. chlamydospora in the spreading of the disease, allowing infection by other pathogens, namely, Phaeoacremonium aleophilum and, in the last stage of wood degradation, Fomitiporia mediterranea isolated from the white-rotted wood (Mugnai et al. 1999). Moreover, P. chlamydospora is also associated with Petri disease observed in young grapevines (Graniti et al. 2000, Retief et al. 2006). To date, the routine detection of the disease is firstly performed by visual observation of the foliar symptoms. This observation is completed by harvesting a piece of woody tissue in stocks and by identifying the growing fungus through microscope observations of the mycelium and the conidia. A second method may be based on the identification of extra-cellular compounds secreted by the pathogen as advocated by Mahoney et al. (2005) in the case of Eutypa dieback. This method can also be considered in the case of esca because particular biomolecules were isolated in xylem sap and leaves of diseased vines (Tabacchi et al. 2000, Bruno and Sparapano 2006b). A third type of approach could include molecular biology methods. Thus, Random Amplification Polymorphic DNA (RAPD) analysis, employing seven markers, was used to study the genetic diversity in populations of P. chlamydospora sampled in South-western France (Borie et al. 2002). In the same direction, a polymerase chain reaction (PCR) assay using species-specific primers also provided a highly sensitive diagnostic tool of P. chlamydospora in soils (Whiteman et al. 2002, Damm and Fourie 2005, Ridgway et al. 2005) and in the woody tissues of grapevine rootstock (Fourie and Halleen 2004, Overton et al. 2004, Retief et al. 2006). A fourth type of assay may implicate serological techniques. Thus, recently, Andolfi et al. (2009) have raised antibodies in rats directed against exopolysaccharides produced by P. chlamydospora, allowing them to detect signals in the symptomatic leaves of esca-affected grapevines. The method proposed in the present work is based on an immunological approach which has been successfully used in other wood decay diseases (Clausen 1997). It has been previously reported that P. chlamydospora secreted in its culture medium polypeptide metabolites showing various biological effects, in particular enzyme activities (Santos et al. 2006, Bruno and Sparapano 2006a). The

Australian Journal of Grape and Wine Research 16, 455–463, 2010

proteinic nature of these compounds therefore offers the opportunity for development of a method based on serological tests. The aims of this study were: (i) to describe the various steps in the development of the serological procedure; (ii) to assess the specificity and sensitivity of the used antibodies; and (iii) to propose a protocol using dot blots for the detection of P. chlamydospora in infected grapevines. Materials and methods Fungal growth conditions The strains of P. chlamydospora P.W. Crous & W. Gams (PC-PC 37, PC-PC 3, PC-PC 21 and PC-PC-32) were isolated from necrotic wood in vineyards from the Cognac area. The strains of Phaeoacremonium aleophilum (PA-PC 24), Diplodia seriata (Bo F99.6) and Neofusicoccum parvum (Bd F00.21) as well as the many other species used were isolated in the Cognac area. All strains were generously provided by Dr P. Larignon (IFV Nîmes). Eutypa lata BI 1 was isolated near Poitiers and Botrytis cinerea (916 T) was isolated in the Bordeaux area. The mycelia were grown on yeast nitrogen base minimal medium (YNBm, DIFCO 233520, Detroit, MI, USA) at 1.7 g/L supplemented with 50 mmol/L glucose (10 g/L) and 40 mmol/L glutamine (5.84 g/L) as carbon and nitrogen sources, respectively. These nutrient sources were chosen because they represent the compounds most frequently transported in the xylem vessels in vines (Glad et al. 1992). The cultures were performed in the dark at 21 ⫾ 1°C on a rotary shaker (125 rpm). For solid media, agar was added in the above medium at 2 g/L (Amborabé et al. 2005). Plant material Vitis vinifera L.var. Ugni Blanc, the most common variety used in production in Cognac, is particularly sensitive to esca. Cuttings were prepared from canes harvested in vineyards (clone 479) and formed by two nodes separated by an internode. The cuttings were maintained in a greenhouse at 70 ⫾ 10% relative humidity and 22°C. They were watered daily with Snyder solution and illuminated 16 h per day with natural light or, when photon flow rates decreased below 357 mmol/m2/s, sodium vapor lamps (Philips SON-T, 423 W). Inoculation was performed by inserting a plug of mycelium (5-mm diameter) harvested on a culture of P. chlamydospora in a hole (5-mm diameter, 20-mm deep) drilled at the top of the cuttings. Observations were made on ten 1-year-old inoculated cuttings and controls were non-inoculated vines cultured in the same conditions. The assays were carried out when cuttings bore 7–9 mature leaves. Grapevine 41BT cells were obtained from a Vitis vinifera ¥ Vitis berlanderi hybrid grown in Murashige and Skoog medium whose composition has been previously reported (Octave et al. 2008). Cells were grown at 26°C in the dark under constant agitation (170 rpm). Isolation of polypeptides and electrophoresis Isolation of the fungal polypeptides was made as previously described (Octave et al. 2006a). The secretion has been correlated with the mycelial development which © 2010 Australian Society of Viticulture and Oenology Inc.

Fleurat-Lessard et al.

reached a maximum after 30 days in our experimental conditions (data not shown). In a first step, mycelia were removed from culture medium by filtration (Whatman 1). On the one hand, the filtrate containing the secreted polypeptides was centrifuged and purified by elution through a Sephadex G25 column (PD 10, GE Healthcare Europe GmbH, Orsay, France). The first 3 mL of eluate (fraction ‘PF’) containing polypeptides with a molecular mass higher than 5 kDa was used in the experiments. On the other hand, the pellet was ground in a mini beadbeater (0.6-mm diameter, Biospec Products, Bartlesville, OK, USA) at 5000 rpm. After centrifugation for 5 min at 5000 ¥ g, the supernatant containing the structural polypeptides was retrieved and treated as the secreted polypeptides. Proteins of leaf and suspension culture cells were extracted from 1 g of material ground in a mortar and pestle in liquid nitrogen and resuspended in 20 mmol/L MES/NaOH buffer (pH 5.5) supplemented with 20 mmol/L dithiothreitol and 100 mg/mL polyvinylpolypyrrolidone. After filtration of the debris, the filtrate was centrifuged at 4°C for 15 min at 15 000 ¥ g. The supernatant was resuspended and purified as previously on Sephadex G25 columns. Protein concentration was measured according to Bearden (1978) with bovine serum albumin (BSA) as the standard. Electrophoresis of protein extracts was performed in denaturating and non-denaturating conditions generally on 10% polyacrylamide gels. SDS-denaturated proteins were analysed according to Laemmli (1970) and nondenaturated proteins according to Hames (1996). Migration was performed under a constant current of 18 mA. The plusOne silver staining kit (GE Healthcare Europe GmbH) was used to reveal proteins and molecular markers (SeeBlueR Plus2 Pre-stained standard) were purchased from Invitrogen (Cergy-Pontoise, France). Antiserum production, Western blot analysis and ELISA The PF fraction obtained previously was lyophilised and resuspended in phosphate buffered saline (PBS) at pH 7.4. Two rabbits were immunised by an initial injection containing 1 mg of the protein antigen suspension followed by three booster injections on 14, 28 and 42 days, respectively, after the initial injection. Bleeding was performed 26 days after the last injection. The polyclonal antibody solution was obtained by purification of IgGs from the collected serum through a protein A column (GE Healthcare Europe GmbH). Immunisation was performed from the total proteinic fraction possibly synthesised by P. chlamydospora in vivo and found as a whole in the host plant, with the aim of obtaining high sensitivity in the diagnostic method. For Western blot analysis, the PF proteins separated by electrophoresis were tranferred from polyacrylamide gels onto a nitrocellulose membrane (HybondTM–ECLTM, GE Healthcare Europe GmbH) under semi-dry conditions (FastBlot B31, Biometra biomedizinische Analytik GmbH, Göttingen, Germany). Transfer was carried out in a medium composed of 25 mmol/L Tris, 192 mmol/L glycine and 20% methanol, for 35 min under a constant © 2010 Australian Society of Viticulture and Oenology Inc.

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current of 5 mA per cm2 of gel. The efficiency of the transfer was verified by using 0.2% (w/v) Ponceau red, 3% (v/v) trichloracetic acid for 10 min. The membrane was then thoroughly rinsed in distilled water. Nonspecific sites were saturated by incubation of the membrane overnight at 4°C in the saturation buffer composed of 20 mmol/L PBS pH 7.4, 3% (w/v) powdered milk and 0.2% (w/v) Tween 20. The polyclonal antibody raised against the PF fraction, diluted 1/1000 in the saturation medium, was then applied for 2 h at room temperature. The membrane was washed 3 times in the saturation medium and treated with the anti-rabbit-peroxidase (HRP) coupled secondary antibody (diluted at 1/2000 in the saturation medium) for 90 min. Successive washings in the saturation medium, PBS-powdered milk and PBS were then made. The antigen-antibody complexes were revealed by the kit ECL RPN 2106 (GE Healthcare Europe GmbH) containing the substrate of the conjugated peroxidase. The subsequent light emission was monitored on film (Hyperfilm, GE Healthcare Europe GmbH) treated with 1/10 Ilford Ilfotec LC 29 and 1/10 Ilford Rapid Fixer. For the enzyme-linked immunosorbent assay (ELISA) test, samples were diluted in PBS to obtain each desired protein concentration. Samples (100 mL) were transferred into individual wells (96 well-plates, Costar 3369) for 120 min at room temperature. The unspecific sites were saturated by four incubations in the saturation buffer for 15 min each. Primary antibody (100 mL) diluted in the saturation buffer was added to each well at room temperature for 120 min. After washing three times for 5 min each with the saturation buffer, treatment with 1/2000 diluted secondary antibody coupled to peroxidase was performed for 90 min at room temperature. A series of washings were subsequently carried out in order to eliminate traces of milk proteins. The revelation was obtained by the addition of 100 mL of 0.05 mol/L citrate/0.15 mol/L phosphate buffer containing 0.5 g/L O-phenylene diamine and 0.25% H2O2. The yellow staining was blocked after 5 min by addition of 50 mL 2 N sulfuric acid. The staining intensity was measured by the absorbance at 492 nm in a microplate reader (Sunrise reader, Tecan, Lyon, France). Dot blots were performed following application of small quantities of PF fraction or after application of sections of canes extruding sap onto the nitrocellulose membrane for 10 s. The proteins were allowed to dry on the membrane for 30 min. The method leading to detection of antigens was similar to that described previously for Western blotting: blocking of non-specific sites, incubation for 120 min in primary antibody (1/250), incubation in secondary antibody (1/2 000) coupled to peroxidase for 90 min, revelation using the ECL kit. Microscopy and in situ immunodetection The samples of mycelia and plant tissues were treated for microscopic analyses as previously described (Octave et al. 2006b). Thin sections (70 nm) of the prepared and embedded materials were collected on gold grids and immunotreated (Octave et al. 2006b). Briefly, the sections on the grids were rehydrated and treated with

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0.5 mol/L NaIO4 for 25 min and 0.1 mol/L HCl for 8 min to liberate the antigen sites. The non-specific sites were saturated by addition of 10 mM PBS (pH 7.2), 0.1% Triton X-100, 0.2% Tween 20, 0.1% BSA and 1/50 normal goat serum (NGS, Nordic, Tebu, Le Perray en Yvelines, France) for 3 ¥ 5 min. The primary antibody was diluted (final titre 100 mg/mL) in the saturation medium and applied to the samples overnight at 4°C. The grids were then saturated in Tris buffered saline (TBS) (pH 8.2), 0.1% Triton X-100, 0.2% Tween 20, 0.1% BSA, 1/20 NGS for 1 h. The secondary antibody coupled to gold particles (15 nm, EMGAR) was applied in the TBS solution for 3 h. After washing in TBS (3 ¥ 15 min) and distilled water for 15 min, the samples on the grids were observed by transmission electron microscopy (JEOL JEM 1010 80 kV). Results Excretion of polypeptides by P. chlamydospora in its culture medium As observed in Figure 1, the electrophoretic pattern of the PF fraction isolated from a liquid culture medium and eluted from the Sephadex G25 column showed around ten bands under non-denaturating conditions. Polypeptides covered a large range of molecular masses ranging from 20 to 200 kDa. By comparison, the electrophoretic pattern under denaturating conditions showed a large increase in number of visible bands (around 20) particularly in the range of low molecular mass (from 6 to 36 kDa), indicating the presence of protein complexes in the PF fraction. Production and properties of antibodies raised against the PF fraction Before immunisation of the rabbits, control assays with the pre-immune sera from four rabbits showed that no non-specific reaction occurred in Western blots obtained from the nutritive YNBm medium, the culture medium in which P. chlamydospora was grown for 40 days and the polypeptide fraction PF, as well as the protein extracts

Figure 1. Typical electrophoretic pattern in (a) non-denaturating and (b) denaturating conditions of proteins excreted by Phaeomoniella chlamydospora (PC-PC 37) after 40 days in culture. kDa, kilodaltons; M, molecular mass markers; acrylamide, 10% in (a), 13% in (b); 10 mg per lane.

Australian Journal of Grape and Wine Research 16, 455–463, 2010

from grapevine leaves harvested in a greenhouse. However, at the high serum concentration used, a faint labelling of the proteins extracted from the mycelium and from the leaf extracts (one band at 150 kDa) can be observed (e.g. see Figure 2a). Two rabbits, R1 and R2, were selected and immunised with the PF fractions. As shown in Figure 2b, production of antibodies was successful in both rabbits. Both labelling patterns obtained were similar, showing recognition of polypeptides with an apparent molecular mass ranging from 40 to 180 kDa. Note that the intensity of the labelling of the antibodies raised in R2 was higher than those raised in R1. Consequently, anti-R2 was chosen in the following experiments. It should be noted that there is little correlation between the immunoblots and the secreted proteins separated in electrophoresis gels, suggesting that the most abundant proteins are not the most antigenic ones. In addition, the fact that the immunoblots were smeared is presumably because of reaction with glycoproteins. With the goal of the development of a reliable diagnostic, a criterion to be considered was the level of sensitivity, which should be high, because the polypeptide concentration in plants were expected to be low. Data on ELISA tests (Figure 3a) showed the amplitude of the signal as a function of the protein amount at various antibody concentrations. A visible response was obtained using 0.1 ng protein with an antibody dilution of 1/5000 and lower. Saturation was obtained at 10 ng protein regardless of antibody dilution, including as low as 1/50 000. From these results (Figure 3a,b), standardised conditions were selected for further experiments: 1/10 000 antibody dilution and 10 ng protein per well with the 0.4 absorbance unit was chosen as the reference value. Using the said conditions, the antibodies, directed towards the PF fraction, did not recognize the polypeptides extracted from grapevine cells in neither culture and leaves nor fungal hyphae (Figure 3b).

Figure 2. Western blot analysis after non-denaturating electrophoresis of (a) protein fraction PF, proteins extracted from mycelium (Myc), grapevine cells in culture (C), grapevine leaf (L) and nutritive medium (Y) after reaction with a rabbit pre-imminune serum diluted at 1/50 and (b) proteins excreted by Phaeomoniella chlamydospora (PC-PC 37) using antibodies raised in two rabbits (R1, R2) at various dilutions as indicated. kDa, kilodaltons; acrylamide, 10%. Antibody titre, R1: 2.10 mg/mL, R2: 5.20 mg/mL; 1/2000 diluted goatantirabbit-HRP coupled antibody. ECL kit. © 2010 Australian Society of Viticulture and Oenology Inc.

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Figure 4. Western blot analysis after non-denaturating electrophoresis of proteins excreted by various strains of Phaeomoniella chlamydospora as indicated using antibodies raised in R2. kDa, kilodaltons; acrylamide, 10%; 10 mg protein per lane. Titre of antibody raised in R2, 5.20 mg/mL, antibody dilution, 1/1000; 1/2000 diluted goat-antirabbit-HRP coupled antibody. ECL Kit.

Table 1. Result of enzyme-linked immunosorbent assay showing the variation in absorbance at 492 nm monitored in the presence of the polypeptide fraction (10 ng protein) secreted by various fungal species found on grapevines. Fungus Figure 3. Enzyme-linked immunosorbent assay showing (a) the variation in absorbance at 492 nm as a function of protein amount, using various dilutions of antibodies raised in R2 as indicated (Primary antibody titre, 5.20 mg/mL) and (b) the labelling induced by PF and by proteins extracted from 41BT cells in culture, from vine leaves and from hyphae (Pch) (1 and 10 ng). (mean ⫾ SD; n = 5). Primary antibody titre, 5.20 mg/mL; dilution 1/10 000; 1/2000 diluted goat-antirabbit-HRP coupled antibody.

The specificity of the antibodies has been assayed against PF from four strains of P. chlamydospora because the genetic diversity of P. chlamydospora may constitute a source of variability (Borie et al. 2002). It is thus crucial that the antibodies raised towards a single strain were capable of recognising some of the proteins secreted from other strains. Western blot analysis (Figure 4) confirmed that the proteins secreted from various strains of P. chlamydospora were recognised by the antibodies directed towards the secreted proteins of strain PC-PC37, although differences in response intensity were observed in some areas of the transferred bands from a strain to another (e.g. see 98 kDa bands). The data obtained with the global response measured by the ELISA showed that the intensity of the reaction presented variations, although low, according to the origin of the excreted antigen (data not shown). However, the possibility existed that antibodies raised against the P. chlamydospora secreted polypeptides may cross-react with polypeptides secreted by other pathogenic fungi colonising grapevines, particularly also found in esca (e.g. Phaeoacremonium aleophilum) or causing other © 2010 Australian Society of Viticulture and Oenology Inc.

Phaeomoniella chlamydospora Alternaria sp. Botrytis cinerea Cladosporium sp. Cylindrocarpon destructans Diplodia seriata Epicoccum sp. Eutypa lata Fusarium sp. Neofusicoccum parvum Paecilomyces sp. Penicillium sp. Pestalotia sp. Phaeoacremonium aleophilum Phoma sp. Phomopsis viticola Pullularia sp. Trichoderma atroviride Trichoderma harzanium Verticillium cephalosporium

OD 492 nm

SE

0.3262 0.0024 0.0002 0.0036 0.0018 0.0016 0.0030 0.0023 0.0069 0.0108 0.0126 0.0066 0.0064 0.0058 0.0104 0.0110 0.0070 0.0080 0.0054 0.0064

0.0102 0.0058 0.0001 0.0027 0.0017 0.0016 0.0019 0.0020 0.0004 0.0018 0.0040 0.0006 0.0034 0.0035 0.0056 0.0035 0.0032 0.0062 0.0016 0.0025

Value ⫾ SD; n = 5. Antibody titre: 5.2 mg/mL; dilution 1/10000; 1/2000 diluted goat-antirabbit-HRP coupled antibody.

wood decay diseases such as Eutypa dieback (E. lata) and Black Dead Arm (D. seriata and N. parvum). The absence of significant labelling obtained by ELISA carried out on the excreted proteins by the species listed in Table 1 indicated that P. chlamydospora antibodies possessed high species specificity. The very low signal against extracts obtained from some fungi following the application of 10 ng protein was not amplified even with 100 times the

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Figure 5. In situ detection in various fungal species of antigenic sites by antibodies raised in R2 against PF fraction of Phaeomoniella chlamydospora. (a) Observation of labelled P. chlamydospora hyphae in confocal microscopy. (b) Gold immunolabelling (black arrows) in external parietal area (W) of hyphae and mucus (M) surrounding hyphae. As controls: no labelling using pre-immune serum (c), omission of primary antibodies (d), omission of secondary antibody (e) and treatment with primary antibody saturated with PF proteins (f). Absence of cross reaction with Phaeoacremonium aleophilum (g) and Eutypa lata (h). Titre of antibody raised in R2, 5.20 mg/mL, antibody dilution 1/50; 1/50 goat anti rabbit antibody coupled with 15 nm gold grains. PFA/Gluta/OsO4, thin sections, uranyl/lead. Scale bars indicate 1 mm. N, nucleus, V, vacuole.

amount of protein (data not shown), indicating that the monitored response resulted from non-specific labelling. The absence of recognition has been verified by Western blot performed in conditions described previously (i.e. 10 ng protein per well, 1/1000 antibody dilution) and which gave no detectable signal (data not shown). Additionally, an in situ analysis was carried out using an immunocytochemical approach. Using confocal microscopy, the hyphae appeared strongly labelled in the wall area (Figure 5a). Observations on ultrathin sections of hyphae confirmed that the labelling occurred mainly on the external border of the hyphal wall and in the surrounding mucus area (Figure 5b). Control experiments made with preimmune serum (Figure 5c), omission of the anti-PF antibody (Figure 5d), omission of the secondary antibody (Figure 5e) and using saturated antibody (Figure 5f) showed the specificity of the labelling. In addition, no specific labelling was found on Phaeoacremonium aleophilum (Figure 5g) and E. lata (Figure 5h), D. seriata and N. parvum (data not shown).

Australian Journal of Grape and Wine Research 16, 455–463, 2010

Figure 6. Recognition of antigenic sites corresponding to Phaeomoniella chlamydospora secreted proteins. (a) No labelling in vessels (V) fibres (F) and vessel-associated cells (white arrows) of non-infected cuttings whereas (b) green FITC-labelling (white triangle) in hyphae occurring in cuttings selectively infected by P. chlamydospora. (c) Detail of immunolabelling at the subcellular scale: very few grains of non-specific labelling in a hypha (H) contained in a vessel of control cuttings whereas (d) high labelling with many 15-nm gold grains (dark arrows) around a hypha (H) and in the middle lamella of infected cuttings (LW, lignified wall). (e). Crosssection in a cane at 80 cm from the trunk in an apparently healthy vine (control) and (f) in an esca-infected vine showing a high green FITC-labelling (triangle) in vessel-associated cells (arrow). Scale bars indicate 10 mm in a, b, e, f and 1 mm in c and d.

Taken together, these results allow us to conclude that antibodies raised in rabbits against proteins secreted by P. chlamydospora in vitro could be useful tools to specifically detect proteins possibly secreted by P. chlamydospora in infested grapevines. Diagnosis of grapevine infection by P. chlamydospora. Assays were carried out to detect the presence of the pathogen in tissues of cuttings which have been selectively infected. Compared with uninfected cuttings (Figure 6a), a labelling was observed around the hyphae which had invaded the vessels and the fibres of xylem (Figure 6b). A detailed view of hyphae in non-infected cuttings showed scarce non-specific labelling (Figure 6c). In the hyphae observed in P. chlamydospora infected cuttings, a high labelling occurred at the border of the cytoplasm near plasmalemma, in the fungal wall and in its external border close to the lignified wall of the vessels. Grains of labelling were also abundant in the middle lamella that joined vessels and fibres (Figure 6d). A second area of detection was then assayed in canes in order to observe whether the secreted molecules were able to be transported at distance. In apparently healthy © 2010 Australian Society of Viticulture and Oenology Inc.

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Figure 7. Dot blots on nitrocellulose membrane performed from upper parts (U) and parts near the stock (L) of canes sampled on cuttings uninfected and selectively infected by Phaeomoniella chlamydospora. PF: control of immunoreaction using 0.1 mg protein. S1: control section and S2: section of the trunk showing the dark necrosis (N). Titre of antibody raised in R2, 5.20 mg/mL, antibody dilution 1/250, secondary antibody 1/2000 GAR-HRP, Kit ECL.

vines, no labelling was detected in canes sampled at 80 cm from the trunk (Figure 6e), whereas in those from esca-infected vines high immunolabelling was observed around the vessels in the small vessels associated cells (Figure 6f). The said different methods used require timeconsuming sample preparation: protein extraction for the ELISA and Western blot analysis, and chemical fixation and preparation of the ultrathin sections for microscopy. Therefore, it was not possible to use these methods for routine diagnosis of esca disease. Because the molecules secreted by the pathogen were found in the aerial parts of the grapevine (Figure 6f), we developed a protocol based on dot-blots on nitrocellulose membrane. In this assay, canes were sectioned and the cut section was immediately applied on the nitrocellulose membrane. As seen in Figure 7, an intense signal was obtained from the sap extruding from canes sampled on the infected vines compared with canes sampled from uninfected vines. Note however that a faint signal was observed in an artificially non-inoculated cutting, addressing whether infection would happen before harvesting of the cutting as reported in previous observations (see Discussion). It should be stressed that the signal obtained in the upper parts of the canes, far from the infection area, was inferior to that of the lower parts of the canes near the stock. This can be linked to the reduced amount of sap extruded at the upper level. This serological test was correlated with the presence of the fungus in the stock (S2; Figure 7). Compared with uninfected controls (section S1), it was verified that the trunk presented the brown wood area (section S2) characteristic of the tissue infection by P. chlamydospora. Discussion Grapevines are affected around the world by devastating diseases resulting from the colonisation of the vascular tissues by fungal pathogens. In particular, esca severely reduces yields and shortens vineyard production life, therefore causing important economic losses (Mugnai et al. 1999). The entire mode of implication of the fungal pathogens in this type of disease is far from accurately known (Graniti et al. 2000). In particular, the internal localisa© 2010 Australian Society of Viticulture and Oenology Inc.

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tion of the causal agents in the woody tissues of plants slows down the attainment of knowledge of the behaviour of the fungi in relation to their biotic environment. Indeed, researchers lack methods of investigations which are relatively simple and yield results quickly. In this scope, a diagnostic method allowing the early assessment of plant infection would be of particular interest. To date, the diagnosis of the disease is mainly performed by foliar observations, but this method proves to be imprecise because symptoms vary from 1 year to another on the same stock. The contamination may be demonstrated by sectioning of arms or trunks in order to observe the characteristic black necrosis that develops in the wood tissues because of the pathogen (Figure 7). This procedure on relatively old vines results in significant damage for the sectioned stock. The subsequent characterisation of the pathogen involves culturing and isolation of the fungus from the infected plant material, processes that are prolonged because of the slow growth of the pathogen (Valtaud et al. 2009). Molecular biology assays (PCR) may be used for the detection of the pathogen (Borie et al. 2002), but they require appropriate choice of primers to ensure a good specificity. Traditional analyses carried out on isolated and cultured fungal samples from infected plant material, although precise, are time consuming, needing about 4 weeks (Retief et al. 2005). DNA extraction from plant tissues and the following amplifications with specific primers allowed the intermediate steps to be bypassed, allowing results in about 1 day (Ridgway et al. 2002, Retief et al. 2005). Thus, nested-PCR, one-tube nested-PCR and quantitative PCR (SYBR-green) methods have been successfully developed for the direct detection of P. chlamydospora in wood samples from nurseries and fields (Tegli et al. 2000, Overton et al. 2004, Abbatecola et al. 2006, Retief et al. 2006, Edwards et al. 2007, Romanazzi et al. 2009). Nethertheless, Retief et al. (2005) have emphasised that molecular techniques are unable to distinguish between dead and viable pathogen tissue. A drawback of this method is that it needs a piece of woody tissue to be harvested in situ, with the risk that the harvesting may be performed out of the contaminated area, thus giving a false negative (Ridgway et al. 2005). A second approach has been proposed by Mahoney et al. (2005) in the case of Eutypa dieback based on detection of particular metabolites secreted by fungi in infected plant tissues. This method also requires a time-consuming isolation and analysis of the extracted products. In the case of esca, this method may be used in restricted conditions because the same compounds (scytalone, pullulans) are secreted by many grapevine pathogens (Evidente et al. 2000, Sparapano et al. 2000, Tabacchi et al. 2000), thereby reducing method specificity. Recent assays have implicated the production of antibodies raised against exopolysaccharides produced by P. chlamydospora. (Andolfi et al. 2009). These metabolites presenting toxic properties were detected in the symptomatic leaves of esca-affected grapevines, while healthy and asymptomatic leaves did not give a serological signal. Therefore, this method will be very useful to study the mechanisms underlying the development of external

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symptoms but will only assess the presence of the pathogen in a particular physiological state of the plants linked to the expression of disease symptoms on leaves. The serological assay that we have developed here, based on the secretion of a number of polypeptides by P. chlamydospora (Figure 1) appears to be a reliable method for identification of this fungal pathogen. Indeed, the obtained antibodies were highly sensitive and more importantly specific. These antibodies recognised the presence of the fungal antigens in expected localised areas (i.e. walls and mucilage surrounding the hyphae) in accordance with the extracellular origin of the polypeptides used to raise the antibodies. By contrast, they did not recognise the antigens displayed either by the mycelial components or by the mucilage surrounding other pathogens involved either in esca (Phaeoacremonium aleophilum) or in other wood diseases frequently encountered in grapevines, such as Eutypa dieback (E. lata) and Black Dead Arm (D. seriata and N. parvum). The test proposed based on dot blots run from the flowing sap in grapevines is somewhat simple and rapid because results may be simultaneously obtained on numerous vine samples, only 4 h after harvesting. Furthermore, it does not need an extensive laboratory material. More importantly, it is not destructive for the vine stock because it can be made from a piece of cane because the polypeptide metabolites travel at distance from the infected area (Figure 6). However, the reduction in the amount of the exuded sap on standing vines constitutes a selective difficulty. Consequently, the test should be carried out preferentially in the humid season, possibly at the end of spring. The major drawback of this method is linked to the limited antibody pool, even if a large number of assays (more than 1000) can be made following each host immunisation as described in this work. Nevertheless, for each new pool of antibodies, the host selection and verification of the sensitivity and specificity of the immunosera would be required. A considerable improvement in this approach would be the production of a monoclonal antibody. This step, however, needs extended research in order to isolate the convenient polypeptide associated with the antibody offering the highest sensitivity and, more importantly, the best specificity. Once realised, the same antibody could be used in a repetitive manner, thereby allowing comparison in the successive sets of data. Nevertheless, this tool in the present state may be useful in two applications. Firstly, it may contribute to gaining new knowledge about the disease. Thus, it might help to reveal the link between the seasonal fungal metabolism and the variable expression of the foliar symptoms, and to investigate the influence of environment on fungal biology in vivo, and thereby perhaps help predict the spread of the disease. Secondly, this test may be used in practical procedures. In particular, it may allow to diagnose early in the infection of vines planted for some years and to direct towards a curative method consisting of an early surgical excision of the stocks. In this way, damage could be reduced on a single stock and, on average, would not greatly modify the mean age of a

Australian Journal of Grape and Wine Research 16, 455–463, 2010

grapevine area, resulting in the maintenance of the typicity of the produced wine. A second application may be proposed to the nurseries to allow checking whether cuttings are infected or not and therefore to ensure the production of high-quality grapevines. Indeed, it has been shown that infected rootstock material is the primary means of disease spread (Ridgway et al. 2002, Fourie and Halleen 2004, Retief et al. 2005, 2006), which seems to be observed in the course of our experiments as illustrated by the small positive signal in the first position in the uninoculated cuttings obtained in Figure 7. However, several further tests should be carried out in standing vines before an extended practical use in order to establish the relationship between the extent of the rootstock colonisation and the degree of detection of the pathogen.

Acknowledgements We are grateful to the service Interdisciplinaire de Microscopie et d’Imagerie Scientifiques, UFR SFA of Poitiers University and to JM Pérault for technical assistance. We thank Dr J-P. Péros (INRA Montpellier) and Dr P. Larignon (IFVV Rodilhan) for providing selected fungal isolates. This work was supported by the Bureau National Interprofessionnel du Cognac. We are indebted to Mrs Tracey Barnes and Dr Manilduth Ramnath for the improvement of the English manuscript.

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Manuscript received: 3 December 2009 Revised manuscript received: 16 March 2010 Accepted: 18 May 2010

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