Increased Phenolic Content in Apple Leaves Infected with the Apple Scab Pathogen
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007_TESTO_667_049 22-02-2008 17:58 Pagina 49 Journal of Plant Pathology (2008), 90 (1), 49-55 Edizioni ETS Pisa, 2008 49 INCREASED PHENOLIC CONTENT IN APPLE LEAVES INFECTED WITH THE APPLE SCAB PATHOGEN M. Mikulic Petkovsˇek, F. Stampar and R. Veberic Department of Agronomy, Chair of Fruit Growing, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, Ljubljana, Slovenia SUMMARY chemicals belong to the phenolic group (Grayer and Kokubun, 2001), which are involved in the natural de- Phenolic compounds were measured in leaf tissues of fense reactions of apples against various diseases, being apple cvs Jonagold and Golden Delicious, healthy and toxic to pathogens. These compounds are produced infected by Venturia inaequalis. Leaves were sampled and accumulate at a faster rate after infection (Picinelli from May to September 2005 and analyzed by high per- et al., 1995; Usenik et al., 2004; Treutter, 2005). formance liquid chromatography. Hydroxycinnamic Phenolics, flavanols in particular, play a role in the acids detected were chlorogenic, caffeic, ferulic and p- resistance of apples to V. inaequalis (Treutter and coumaric. In addition, the presence was ascertained of Feucht, 1990a, 1990b). The ability to accumulate fla- the dihydrochalcone phloridzin and the flavonoids epi- vanols after infection in the tissue surrounding infected catechin, catechin, rutin and quercitrin. Total phenolics sites is a differential property of susceptible and resist- were determined with the Folin-Ciocalteu method. In- ant cultivars (Treutter and Feucht, 1990b), and consti- fection by V. inequalis caused an accumulation of phe- tutes additional evidence that these compounds are in- nolic compounds in infected leaves with a 1.4 to 6.2- volved in the defense mechanism against apple scab fold increase of flavonols, a 2 to 6-fold increase of (Mayr et al., 1995). In resistant cultivars, phenolics, chlorogenic acid and a 1.4 to 2.4-fold increase of the such as chlorogenic, caffeic and ferulic acids (Mikulic Folin-Ciocalteu values. Petkovsˇek et al., 2003), accumulate at a faster rate than in the susceptible ones (Picinelli et al., 1995; Usenik et Key words: apple scab, Malus domestica, phenolic al., 2004; Treutter, 2005). compounds, Venturia inaequalis. As to the connection between resistance and phenolic content, the information is somewhat contradictory. Picinelli et al. (1995) reported that the content of chloro- INTRODUCTION genic acid, coumaric acid derivatives and flavanols was generally higher in the resistant than in the susceptible Scab caused by the fungus Venturia inaequalis (Cooke) apple varieties. However, Sierotzki and Gessler (1993) G. Winter is a widespread disease of apple (Malus domes- came to the conclusion that there is no positive correla- tica Borkh.). Growers try to counteract the impact of this tion between resistance and pre-formed flavan-3-ols in disease with fungicide treatments, the use of resistant or the relationship between Malus x domestica and Venturia tolerant genotypes, or through the induction of defense inaequalis. mechanisms by treating plants with suitable triggering Phloridzin was shown to play an important role in agents (e.g. prohexadione-Ca) (Mayr et al., 1995; Hrazdi- host resistance to V. inaequalis. It was suggested that na et al., 1997; Rademacher et al., 1999). phloridzin is hydrolysed in vivo by various fungi (V. in- Defense strategies of plants against pathogens are aequalis included) to create phloretin, which, in turn, is several, including the production of antifungal chemi- degraded to phloroglucinol, phloretic acid and p-hy- cals, which are either pre-formed (i.e. already present in droxybenzoic acid, which inhibit the development of V. plant tissue in different amounts) or induced following inaequalis (Hamauzu, 2006). infection (e.g. de novo synthesized phytoalexins) (Gray- The phenolic derivatives can react and oxidize pro- er and Kokubun, 2001). In both groups of chemicals, teins, thus causing the loss of enzyme function and re- those synthesized in secondary metabolism are especial- stricting the viability of aggressors. They can also be de- ly interesting. Many of the phytoalexins or pre-formed posited inside cell walls as an important first line of de- fense against infection (Schwalb and Feucht, 1999). Pre- vious studies (Mayr et al., 1997; Michalek et al., 1999) Corresponding author: R. Veberic Fax: +386.1.4231088 showed that, for a successful protection, rapid biosyn- E-mail: [email protected] thesis of flavanols starting from phenylalanine was nec- 007_TESTO_667_049 22-02-2008 17:58 Pagina 50 50 Phenolic content in apples infected by V. inaequalis Journal of Plant Pathology (2008), 90 (1), 49-55 essary. The inhibition of the enzyme phenylalanine-am- extraction, healthy or infected leaves were frozen in liq- monia-lyase (PAL) led to severe scab symptoms because uid nitrogen and stored at –20 °C. there was a reduced flavonol accumulation. Further- Leaf sampling was made every 20 days. Sampling at more, earlier investigations had shown that growth-pro- Ljubljana began on June 2nd (25 days after full bloom), moting N nutrition reduce flavonoid accumulation in at Maribor on May 27th (24 days after full bloom) and the leaves, thus increasing susceptibility to scab (Rüh- was completed on September 12th. mann et al., 2002; Leser and Treutter, 2005; Strissel et al., 2005). Extraction and determination of phenolic com- The way in which orchards are managed can influ- pounds. Frozen leaves were lyophilized and ground in a ence the amount of phenolics, as shown by Veberic et mortar. For each treatment, 5 groups of 10 leaves per al. (2005), who reported that organically grown apples group were extracted as described by Colaric et al. had somewhat higher amounts of phenolics as com- (2005) with some modifications. The fine powder (50 pared with traditionally grown apples. These authors mg) was extracted with methanol (20 ml) containing concluded that this is probably because organically 1% 2,6-di-tert-butyl-4-methylphenol (BHT) for 30 min grown apples face more stressing conditions, for syn- in a cooled water bath using sonication. BHT was thetic fertilizers and pesticides are not used. added to the samples to prevent oxidation. It did not in- According to previous studies, we hypothesized that terfere with the extracted phenols during the subse- infected leaves have a higher content of certain phenolic quent HPLC analysis, because it was eluted at the end compounds compared with healthy leaves. As already of the gradient or in the equilibration phase between mentioned, several previously published papers ad- the two analyses. After centrifuging at 10,000 rpm for dressed this subject only in part. Therefore, we tried to 10 min at 4°C, the supernatant was filtered through a compare the differences in the content of single phenolic 0.45 µm membrane filter (Macherey-Nagel, Düren, compounds, as well as of total phenolics in healthy leaves Germany) prior to injection into HPLC. and in V. inaequalis-infected leaves of cvs Jonagold and Phenolic compounds were analyzed using the Ther- Golden Delicious over a whole growing season. Golden mo Finnigan Surveyor HPLC system with a diode array Delicious was grown in two different locations. detector at 280 and 350 nm. Hydroxycinnamic acids (chlorogenic, p-coumaric, ferulic and caffeic) and the monomeric flavan 3-ols (catechin, epicatechin) were de- MATERIALS AND METHODS tected at 280 nm, whereas rutin, phloridzin and quercetin-3-rhamnoside (quercitrin) were estimated at Plant material and growing conditions. Experiment 350 nm. Spectra were also recorded between 200 and was carried out in 2005 using leaves from six-year-old 400 nm. The column used was Phenomenex Gemini C18 apple trees grafted on M9 rootstocks, growing in Ljubl- (150x4.60 mm) operated at 25°C. The elution solvents jana (central Slovenia) and Maribor (north-eastern were A (aqueous 0.01 M phosphoric acid) and B (100% Slovenia). The orchards were managed following the methanol). The samples were eluted according to the guidelines for integrated production. Despite the use of linear gradient described by Escarpa and Gonzales fungicides, weak scab symptoms were present on the (2000) with slight changes: 5% B initially; 50% B for 10 leaves, consisting of small velvety lesions with olive min; 70% B for 5 min; 80% B for 5 min and, finally, green colour, and undefined margins. Samples were col- 100% B for 10 min. The injection amount was 10 µl, lected from cv. Golden Delicious in both locations and and the flow-rate was 1 ml/min. Identification of com- cv. Jonagold in Maribor. pounds was achieved through a comparison of retention In 2005, weather parameters (temperatures and rain- times and spectra, as well as with spiking by standards. fall) were favourable to the development of scab. Aver- Concentrations of phenolic compounds were calculated age temperatures during the growing season were ap- from the peak areas and expressed as mg/100 g dry proximately 1 to 1.5°C higher than the long term aver- weight or mg/g dry weight (d. wt). age (1961-1990). Rainfalls in May and June were some- what lower than the long term average but, in other Determination of total phenolic content. Extraction months, the limit of long term rainfall average was wide- of leaf samples for determining total phenolics was done ly exceeded in both locations. as described above, but without BHT addition. The total Ten fully developed healthy or infected leaves were phenolic content (TPC) of extracts was assessed using collected from each tree. The leaves were sampled from the Folin-Ciocalteau phenol reagent method (Singleton five trees at different dates for each cultivar. The 3rd or and Rossi, 1965). To 100 µml of extracts [diluted 1: 5 the 4th fully developed leaf from annual shoots was tak- (v/v) with MeOH], 6 ml of bidistilled water and 500 µml en for analysis. Infected leaves showed young green of Folin-Ciocalteau reagent were added; after resting be- sporulating lesions, which were excised together with a tween 8 sec and 8 min at room temperature, 1.5 ml of narrow strip (1–2 mm) of surrounding tissues.