PROHEXADIONE-CA INDUCES REDUCTION IN BACTERIAL BLIGHT SEVERITY AND ALTERATION IN PHENOLIC CONTENT IN WALNUTS

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1 008_COST(Solar)_S :30 Pagina 47 Journal of Plant Pathology (2012), 94 (1, Supplement), S1.47-S1.52 Edizioni ETS Pisa, 2012 S1.47 PROHEXADIONE-CA INDUCES REDUCTION IN BACTERIAL BLIGHT SEVERITY AND ALTERATION IN PHENOLIC CONTENT IN WALNUTS A. Solar 1, J. Jakopic 2, V. Nour 3, M. Mikulic-Petkovsek 2, R. Veberic 2, M. Botu 3 and F. Stampar 2 1 Department of Agronomy, Experimental Field for Nut Crops, Biotechnical Faculty, University of Ljubljana, Vinarska 14, 2000 Maribor, Slovenia 2 Department of Agronomy, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000 Ljubljana, Slovenia 3 University of Craiova, Faculty of Agriculture and Horticulture, Department of Horticulture and Food Science, 13, Al. I. Cuza Str., Craiova , Romania SUMMARY Prohexadione-Ca (ProCa) was applied in an adult walnut orchard of cv. Franquette to test its ability to reduce severity of Xanthomonas arboricola pv. juglandis (Xaj) infection, and the effect on the contents of phenolic compounds in walnut fruits. ProCa was used three times over at 8-10 day interval, starting before the stigmas emergence at mg l -1 concentration (treatment R1), and at the stage of brown stigmas at mg l -1 concentration (treatment R2). Blight severity was assessed within the orchard at the same time as the husk tissues from symptomatic fruits were sampled for phenolic analyses, which were conducted using HPLC with PDA and MS detection. ProCa caused a significant reduction in Xaj severity regardless of the concentration and the first application date. The induced defence reaction was maintained throughout the season. Within a 15-day interval after the ProCa treatments, the walnut husks showed up to a 14 fold higher content of different phenolics than before fruit maturation. The treated fruits had a higher total phenolic content (TPC), more hydroxycinnamic acids (HC), and flavanols, whilst the contents of flavonols and gallic acid had decreased due to ProCa. The expressed potential of ProCa for triggering the defense mechanisms of walnut through the regulation of phenolic synthesis should be confirmed by additional experiments with the final aim of including this product in a sustainable control against Xaj. Key words: Juglans regia L., Xanthomonas arboricola pv. juglandis, Regalis, disease severity, phenolic content, defence mechanism. The good health status of plant material has a crucial impact on the quantity and quality of the yield, fruiting regularity and, consequently, the economic efficiency of walnut production. Bacterial blight induced by Xanthomonas arboricola pv. juglandis (Xaj) known to be the most severe disease of common walnut (Juglans regia) in Corresponding author: A. Solar Fax: anita.solar@bf.uni-lj.si all production areas, may cause up to 100% crop losses and a significant reduction in tree vigour (Mulrean and Schroth, 1982). Normally, the disease is chemically controlled using preventive spraying with copper bactericides. Up to five treatments, applied within the period between bud-breaking and the lignified shell stage, is recommended in adult orchards (Ninot et al., 2002). In practice, more treatments per year are frequently reported (Gonçalves et al., 2001; Buchner et al., 2010; Chevallier et al., 2010). Due to the limited choice of officially registered products, the difficult coverage of large trees with a spraying mixture, the development of bactericide-resistant populations of the pathogen, the significant impact of weather conditions on disease incidence and severity, it is very difficult to control the disease by usual chemical means (Gardan et al., 1993; Olson et al., 1997; Radix et al., 1998; Garcin and Duchesne, 2001). Planting genetically resistant or tolerant cultivars, and the use of alternative products for spraying seem to be the most promising strategy for overcoming the economically important reductions of walnut yield caused by bacterial blight. Prohexadione-calcium (calcium 3-oxido-4-propionyl- 5-oxo-cyclohexene carbohydrate) could act as an alternative product in a new strategy for walnut-blight control. It was originally developed as a plant growth regulator that reduces shoot growth by inhibiting late stage of giberellin biosynthesis (Evans et al., 1999; Rademacher, 2000). Disease incidence and severity have also been reported as being reduced by the multiple effects of ProCa. Apart from the morphological, histological and physiological effects, such as the inhibition of internode elongation, lack of the formation of new susceptible leaves, lignification, modification of canopy density, and promotion of the early physiological maturation of leaves (Biehn et al., 1966; Winkler, 1997; Spinelli et al., 2010), alterations in flavonoid metabolism caused by ProCa contribute to increased resistance against some fruit tree diseases (Roemmelt et al., 2003; Bazzi et al., 2003a; Gosch et al., 2003; Halbwirth et al., 2003; Rademacher, 2004; Spinelli et al., 2005). The purpose of this contribution was to investigate the potential of ProCa for reducing the severity of walnut blight and its effect on the contents of phenolic

2 008_COST(Solar)_S :30 Pagina 48 S1.48 Prohexadione-Ca impact on walnut blight Journal of Plant Pathology (2012), 94 (1, Supplement), S1.47-S1.52 compounds in walnut fruits, as previously shown for pome fruits infected by fire-blight (Erwinia amylovora). This study was conducted on thirteen-year-old trees of cv. Franquette, growing on slope at the Gacnik Experimental Station for Fruitgrowing at Maribor (northeast Slovenia). The trees were planted at a distance 8 x 6 m, and trained as a goblet with three to four scaffold branches on a trunk of cm. Single tree plots within a completely randomized block design with four replications were included in the trial. The trees were sprayed with Prohexadione-calcium, formulated as Regalis (wettable granular with 10% ProCa). The spray solution was applied using handgun equipment to run-off. Three ProCa treatments were applied during spring With treatment Regalis 1 (R1), the selected trees received an application of ProCa at 125 mg l -1 at phenological stages Ef (just before stigma emergence), Gf (brown stigmas), and 10 days later. With treatment Regalis 2 (R2), ProCa was used at a concentration 250 mg l -1 during the Gf stage (brown stigmas), 10 and 20 days later. The treated trees were compared with the untreated controls to see whether ProCa had affected bacterial blight severity and the contents of phenolic compounds. Twenty fruits per tree were observed at two sampling dates, SD1 and SD2, to assess disease severity. SD1 was 15 days after the third ProCa application (R1), and 5 days after the third ProCa application (R2), respectively. SD2 was before fruit maturation (R1, R2). Blight severity was quantified according to necrotic fruit tissue expressed as % of the fruit surface and arranging symptomatic fruits into six severity classes (Solar et al., 2012). Simultaneously, fruits were harvested for phenolic content determination. At each sampling date, ten representative infected fruits were collected from each tree. Twenty fruits were then randomly combined into four repetitions, each containing five fruits. Immediately after sampling, fruits were immersed in liquid nitrogen and stored at -20ºC. For the extraction of individual phenolic compounds, infected husk tissue (exocarp + mesocarp, 1 mm thick) with a surrounding narrow zone of healthy tissue (1-2 mm) was excised from diseased fruits. The husk of each sample was ground to a fine powder using liquid nitrogen, aliquots (0.30 g) were placed into test tubes that, after the addition of 3 ml methanol containing 1% of 2,6-di-tetr-butyl-4- metylphenol (BHT), were placed in an ultrasonic bath for 1 h to extract phenolic compounds. After extraction the samples were centrifuged for 7 min at 10,000 rpm. The supernatant was filtered through a 0.45 µm polyamide filter and transferred into a vial prior to injection in a Thermo Finningan Surveyor HPLC system (Torrance, USA) with a diode array detector. A Phenomenex HPLC column C18 (150 x 4.6 mm, Gemini 3µm) protected with a phenomenex security guard column, was used. The elution solvents were 1% aqueous formic acid with 5% acetonitrile (A) and pure acetonitrile (B). Samples were eluted according to the linear gradient: 0-5 min, 3% to 9% B; 5-15 min, 9% to 16% B; min, 16% to 50% B; min, 50% isocratic. Compounds were identified by comparing retention times and their UV-VIS spectra from 200 to 600 nm, as well as by the addition of an external standard using a mass spectrometer (Thermo Scientific, LCQ Deca XP MAX, Torrance, USA) with an electrospray interface (ESI) operating in negative ion mode. The analyses were carried out using full-scan data dependent MS n scanning from m/z 115 to Quantification was determined according to the concentrations of a corresponding external standard at 280 (hydroxybensoic acid, flavan-3-ols, hydroxycinnamic acids) and 350 nm (flavonols) and concentrations were expressed as mg per kg FW. Extraction for evaluating the total phenolic content (TPC) was done in the same way as for individual phenolic compounds, but only methanol was used. TPC was determined using the Folin Ciocalteu reagent and spectrophotometrical measurements of absorbance at a wavelength of 765 nm were recorded. TPC was expressed as gallic acid equivalents (GAE) in mg per kg FW. The following standards were used for the quantification of phenolic compounds: gallic acid (Merck, Germany), chlorogenic acid (5-caffeoylquinic acid) (Sigma, USA), (-)-epicatechin and quercetin-3-o-rhamnoside (Fluka Chemie, Switzerland), quercetin-3-o-arabinofuranoside and quercetin-3-o-xyloside (Apin Chemicals, UK) and (+)-catechin (Roth, Germany). A one-way analysis of variance (ANOVA) was carried out to determine differences in disease severity and phenolic contents among the ProCa treatments. The statistical differences between means at a 95% confidence level were calculated using the LSD test. For this purpose the statistic program Statgraphics Plus 4.0. (Manugistics, USA) was used. A statistically significant impact of ProCa on blight severity was expressed at SD1 (Fig. 1). Treated fruits were very mildly infected, each showing only small necrotic spots (severity class 0.7 and 0.8, respectively), that covered less than 3% of the husk surface. Medium infection with separated spots (severity class 2.9), which covered around 10% of the husk surface, was observed on control fruits. Reduced disease severity caused by ProCa applications had been reported for actively growing shoots of pears and young leaves of apple infected by Erwinia amylovora (Costa et al., 2001a; Sabatini et al., 2003; Bazzi et al., 2003b; Roemmelt et al., 2003a), as well as for apple leaves infected by Venturia inaequalis (Costa et al., 2001b; Bazzi et al., 2003a). In walnut tree, the pre-flowering time is known as one of the most important for the early infection of buds by Xaj (Ninot et al., 2002; Chevallier et al., 2010). For this reason, a stronger disease reduction was expected when the ProCa was first used before stigmas emergence (R1) compared to after-flowering treatment,

3 008_COST(Solar)_S :30 Pagina 49 Journal of Plant Pathology (2012), 94 (1, Supplement), S1.47-S1.52 Solar et al. S1.49 Fig. 1. Xanthomonas arboricola pv. juglandis severity (in severity classes) (Means ± SE), assessed on walnuts, cv. Franquette at SD1 and SD 2 after two ProCa treatments (R1, R2) and control (K). at the stage of brown stigmas (R2). Opposite to our expectations, the two different ProCa applications resulted in almost equal reduction in disease severity. It can be concluded that the ProCa treatment, if applied at pre-flowering and at lower concentration (R1), could be used with the same efficiency as the double ProCa concentration, applied after females flowering (R2). The defense reaction caused by ProCa, remained identical throughout the season. Approximately two weeks before maturation (SD2), the ProCa-treated fruits were still less infected with Xaj than the untreated fruits (Fig. 1). In the case of the apple trees and scab disease, Bazzi et al. (2003a) reported that trees sprayed with ProCa need approximately 7-20 days to display lowered scab susceptibility. As the Xaj severity in our study was assessed 15 days (R1) and only five days (R2) after ProCa treatments, respectively, an even stronger reduction in disease development could have been observed a week or ten days later. This is in part confirmed by the lowest infection severity observed at SD2 after R2 ProCa treatment (Fig. 1) and fits with Bazzi et al. (2003a) observations who reported that the resistance lasts for several weeks, once it has built up. Phenolic compounds, which are thought to play an important role in plant defense against different pathogens (Treutter and Feucht, 1990; Benett and Wallsgrove, 1994; Mayr et al., 1997; Halbwirth et al., 2003; Usenik et al., 2004; Mikulic Petkovsek et al., 2009, 2011), were also found in symptomatic walnuts. They were from the group of hydroxycinnamic acids, HC (3-caffeoylquinic and 3-O-p-coumaroylquinic acid), flavonols (quercetin-3-o-arabinoside, quercetin-3-orhamnoside, quercetin-3-o-xyloside), flavanols [catechin, (-)-epicatechin], and gallic acid from the group of hydroxybenzoic acids. When analysing the impact of ProCa on the synthesis of phenolic compounds, a response to treatments was evident in relation to the specific phenolic group and sampling date. Contents up to 14 fold lower were determined for all phenols, except for flavanols at SD2, compared with SD1. A decreasing trend from the beginning of fruit development towards their maturation may be due to the possible competition between the growth of the fruits and the secondary metabolism, which resulted in phenol synthesis (Treuter, 2005). At SD1, walnut husks had statistically significant higher total phenolic content (TPC) after the R2 ProCa treatment, compared to the control (Fig. 2). TPC also increased when apple leaves were treated with ProCa (Mikulic-Petkovsek et al., 2009) whilst a reduced TPC was reported in ProCa-treated apple fruits by the same authors. Furthermore, treated walnuts had more hydroxycinammic acids than the untreated ones (Fig. 3), Fig. 2. Total Phenolic Content in walnuts (ekv. GA g kg -1 ) (Means ± SE), cv. Franquette after two ProCa treatments (R1, R2) and control (K).

4 008_COST(Solar)_S :30 Pagina 50 S1.50 Prohexadione-Ca impact on walnut blight Journal of Plant Pathology (2012), 94 (1, Supplement), S1.47-S1.52 which is in agreement with Roemmelt et al. (2003) and Mikulic-Petkovsek et al. (2009). Increase was only evident immediately after the ProCa application. Later in the season, this was lost and the treated fruits showed lower HC contents than the control (Fig. 3). Flavonoids, the group of phenolic compounds known to be especially important secondary metabolites involved in increasing resistance against various plant diseases (Shirley, 1996; Mayr et al., 1997), reacted differently with respect to their response to ProCa. Quercitin-glycosides (flavonols) decreased after R1 ProCa treatment, whereas the R2 treatment did not affect the synthesis of flavonols (Fig. 3). These results partly correspond to those of Halbwirth et al. (2003) and Roemmelt et al. (2003) who reported that a general decrease of the constitutive flavonoids was observed in apple leaves treated with ProCa. The post-treatement synthesis of flavanols, which are another important group of flavonoids, was also strongly affected by ProCa applications, but in the opposite direction than that of flavonols. As Fig. 3 shows, both R1 and R2 ProCa caused increased flavanol contents compared to the control. Such a trend is in contrast with the results obtained for apple leaves and fruits, in which ProCa caused a significant decrease of flavanols Fig. 3. The content of hydroxycinnamic acids, flavonols, flavanols and gallic acid in walnuts (in mg kg -1 FW) (Means ± SE), cv. Franquette after two ProCa treatments (R1, R2) and control (K).

5 008_COST(Solar)_S :30 Pagina 51 Journal of Plant Pathology (2012), 94 (1, Supplement), S1.47-S1.52 Solar et al. S1.51 (Mikulic-Petkovsek et al., 2009). The increased flavanols observed during this study may have been due to an additional postinfectional synthesis within the walnuts infected with Xaj. As to gallic acid, which is known as the starting material for hydrolysable tannin synthesis (Ossipov et al., 2003), its content in control fruits, as well as in R1 ProCa-treated fruits almost the same (Fig. 3), but it decreased markedly following R2 ProCa application. At SD2 both the R1 and R2 ProCa applications resulted in lower gallic acid amounts compard with the control (Fig. 3). The results of this study show that ProCa treatments lowered the progression of Xaj and reduced the number of necrotic lesions on the green walnut husks, thus indicating that ProCa acts as a signal which modulates walnut resistance against Xaj through the regulation of some phenolic compounds biosynthesis in walnut fruits. Further experiments including in vitro testing of the effects of ProCa on artificially infected plant material are needed to confirm the potential use of ProCa for activating walnut defense responses, and to evaluate its role in walnut production in the framework of sustainable control programmes against Xaj. ACKNOWLEDGEMENTS The authors are grateful to the Slovenian Ministry of Higher Education, Science and Technology, who supported this research, which is a part of their programme Horticulture No P It was conducted within the framework of COST 873. V. Nour acknowledges the COST action to support her Short Term Scientific Mission in Slovenia. 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