OPCE-STR.vp Acta Bot. Croat. 69 (2), 155–162, 2010 CODEN: ABCRA 25 ISSN 0365–0588 Detrimental effect of quercetin on phytoplasma-infected Catharanthus roseus (L.) G. Don shoots grown in vitro MIRNA ]URKOVI]-PERICA*, MARIN JE@I] University of Zagreb, Faculty of Science, Departmen of Biology, Maruli}ev trg 9a, 10000 Zagreb, Croatia Quercetin is known to possess antimicrobial activity against bacteria, fungi, and viruses. The activity of this flavonoid against phytoplasmas, non-cultivable plant pathogenic bac- teria that cause numerous plant diseases, has never been examined before. The aim of this research was to examine the effect of different concentrations of quercetin (10 mM, 100 mM and 1 mM) on 'Candidatus Phytoplasma asteris' and on phytoplasma-infected peri- winkle shoots grown in vitro. The addition of quercetin neither supported the growth of the shoots nor induced the remission of symptoms in the infected plants. On the contrary, addition of quercetin induced browning of leaves and the appearance of black spots on the leaves of treated infected and non-infected shoots. It also had no curative effect against the pathogen. Phytoplasma presence was confirmed by nested PCR in infected shoots treated with quercetin through three subcultures. Key words: phytoplasma, bacteria, antimicrobial activity, flavonoid, quercetin, peri- winkle, plant tissue culture Introduction Flavonoids, natural substances present in all vascular plants, possess an antimicrobial activity against different bacteria, fungi, and viruses (HARBORNE and WILLIAMS 2000, CUSHNIE and LAMB 2005). When the antibacterial properties of flavonoids are discussed, most of the data come from in vitro studies of human pathogenic bacteria. Those studies re- vealed that many different flavonoids, including quercetin, inhibit bacterial growth. How- ever, different studies on the same bacterial species produced ambiguous results, possibly due to the different inoculum sizes and different assays used (disk diffusion assay, broth macrodilution assay, broth microdilution assay, agar well diffusion assay, agar dilution as- say) as well as to different diffusion rates of various flavonoids, formation of precipitates or salts and structural alternations of flavonoids inside the applied inoculums. Whether flavonoids act as bacteriostatic or bactericidal agents is under debate because some re- search groups have shown that flavonoids reduce the number of colony-forming units, while others speculate that this is the consequence of the formation of bacterial aggregates under flavonoid-exposure rather than bactericidal effect (CUSHNIE and LAMB 2005). Several ACTA BOT. CROAT. 69 (2), 2010 155 * Corresponding author, e-mail: mirna@botanic.hr U:\ACTA BOTANICA\Acta-Botan 2-10\334 Curkovic Perica.vp 11. listopad 2010 11:21:00 Color profile: Disabled Composite 150 lpi at 45 degrees hypotheses have been proposed for the mechanisms of the antibacterial action of flavo- noids: intereference with DNA synthesis (PLAPER et al. 2003), inhibition of membrane function and inhibition of energy metabolism (CUSHNIE and LAMB 2005). Data on flavonoid activity against plant pathogens encompass mostly antifungal and antiviral activity. Flavonoids are able to suppress fungal infection or inhibit spore germina- tion, thus protecting plants from fungal attacks (HARBORNE and WILLIAMS 2000). Their antiphytoviral activity encompasses reduction of virus infectivity by interfering with initia- tion of virus infection (RUSAK et al. 1997), weakening of interactions among coat-protein subunits (VERMA 1973, MALHOTRA et al. 1996) or altering interactions between viral nu- cleic acid and capside proteins (FRENCH et al. 1991). Activity of quercetin against phytoplasmas, plant pathogenic bacteria, has not hitherto been tested. Phytoplasmas are wall-less prokaryotes that have a two-host cycle involving plants and insect vectors (CHRISTENSEN et al. 2005). These uncultivable bacteria are the main cause of many economically important diseases infecting several hundred plant spe- cies worldwide. Different techniques and treatments have been applied in attempts to cure diseases caused by different phytoplasma species: treatments with tetracyclines, b-amino- -butyric acid, polyamines, terpenes, auxines, tissue culture and/or heat or hot water treat- ment (]URKOVI] PERICA and [ERUGA MUSI] 2005, ]URKOVI] PERICA 2008a). The aim of this research was to test whether quercetin treatment could eliminate phytoplasmas from the host or at least induce recovery of phytoplasma-infected plants. Material and methods Material and plant tissue culture methods Catharanthus roseus (L.) G. Don shoots infected with 'Candidatus Phytoplasma asteris' (strain HYDB), belonging to the 16SrI-B subgroup, were grown in vitro on MS (MURA- SHIGE and SKOOG 1962) basal nutrient medium supplemented with 100 mgL–1 myo- -inositol, 1 g L–1 casein hydrolysate, 30 g L–1 sucrose, 9 g L–1 agar and 0.5 mg L–1 benzyl- aminopurine (BA). In such a system, phytoplasmas are present in a high titer and all shoots express symptoms of infection (proliferations, internode shortening, stunting). Phyto- plasma-infected shoots were obtained from Phytoplasmology Laboratory of the University of Bologna (IRPCM 2004). Healthy in vitro-grown C. roseus shoots were also included in experiments as controls. Each shoot was grown in a test tube (20 ´ 150 mm) filled with 15 mL of MS nutrient medium. The pH of the medium was adjusted to 5.7 before autoclaving at 118 kPa and 120 °C for 20 min. In order to test the effect of quercetin (Sigma) on 'Ca. P. asteris' – infected and healthy shoots, they were transferred to the medium mentioned above, which did not contain agar. This medium was, after autoclaving, supplemented with 10 mM, 100 mM or 1 mM of quercetin. Quercetin stock solutions were prepared in ethanol. Therefore, in the control ex- periment, ethanol without quercetin was also added to the medium in order to rule out the possibility of the antiphytoplasmal activity of the alcohol. Untreated infected and non-in- fected controls were also transferred to liquid medium. The cultures were incubated at 22±2 °C under a 16-h photoperiod and subcultured in a 3 week-culture period. Twenty-four infected C. roseus shoots per treatment, positive control (untreated infected shoots on the medium with BA), non-infected treated shoots, and negative control (non-infected shoots 156 ACTA BOT. CROAT. 69 (2), 2010 ]URKOVI]-PERICA M., JE@I] M. U:\ACTA BOTANICA\Acta-Botan 2-10\334 Curkovic Perica.vp 11. listopad 2010 11:21:00 Color profile: Disabled Composite 150 lpi at 45 degrees on the medium with BA) were included in the experiment (Tab. 1). Shoots were placed on paper bridges with their stems 2–3 mm immerged in the liquid medium Weight (g) and length (cm) were measured on 16 shoots per treatment to insure availability of plant mate- rial for phytoplasma detection. Measurements were performed at the beginning and at the end of the first three subcultures. Fresh weight increase and shoot length increase were cal- culated as a ratio of the final and the initial fresh weight or shoot length, respectively. Mean values, analysis of variance and Duncan's test were used for analysis and interpretation of the data. Phytoplasma detection Eight samples (0.5 g each) of randomly chosen infected shoots treated with different concentrations of quercetin, positive control and non-infected shoots were taken after the 3rd subculture in order to be tested for the presence of 'Ca. P. asteris'. Exceptions were in- fected shoots grown on 1 mM quercetin, which started to decay in the first subculture and were therefore tested after the first subculture. The procedure for phytoplasma detection, including total nucleic acid isolation, PCR amplification and product analysis was previ- ously described (]URKOVI] PERICA et al. 2007; ]URKOVI] PERICA 2008a, b). Also, serial di- lutions of total DNA (i.e. 20, 10 and 5 ng µL–1) were used as templates in PCR to determine a possible lower titer of phytoplasma in the treated shoots. Direct PCR assays were per- formed using universal phytoplasma primer pair R16F1/R0 (LEE et al. 1995), for the ampli- fication of highly conserved 16S rDNA. The amplification products were diluted and reamplified in the first nested PCR with primers R16F2n/R2 (GUNDERSEN and LEE 1996). The additional second nested PCRs were performed using R16(I)F1/R1 (LEE et al. 1994) only for the shoots treated with 1 mM of quercetin. Results Shoot length and fresh weight increase were used as parameters for measuring the ef- fect of quercetin on 'Ca. P. asteris'-infected and non-infected C. roseus shoots (Tab. 1). None of the used quercetin concentrations showed any beneficialeffect on phytoplasma-in- fected plants. On the contrary, quercetin (100 µM and 1000 µM) added to the medium re- sulted in increased severity of symptoms, accompanied by leaf yellowing and browning, and emergence of black spots on leaf edges. Healthy periwinkle shoots treated with quercetin (100 µM) also expressed stunting accompanied by leaf yellowing and slight browning. At the highest concentration of quercetin (1 mM), plants infected with 'Ca. P. asteris' started to decay shortly after the transfer to the quercetin-supplemented medium, during the first subculture. Shoot elongation and fresh weight increase of both, infected and non-infected plants, treated with quercetin (100 mM or more), were lower than those of un- treated controls (Tab. 1), and there was no remission of symptoms in infected plants. After 'Ca. P. asteris'-infected C. roseus shoots were grown through three subcultures on media with different quercetin concentrations, molecular analyses using consecutive PCR reac- tions with R16F1/R0 and R16F2n/R2 primer pairs showed the presence of 'Ca. P. asteris' in all of the tested samples on media supplemented with 10 mM or 100 mM quercetin and in positive control (Fig. 1). When total DNA in the concentration of 20 ng µL–1 or 10 ng µL–1 was used as template, all samples were positive in the direct PCR, but PCR using template ACTA BOT. CROAT. 69 (2), 2010 157 EFFECT OF QUERCETIN ON PHYTOPLASMA U:\ACTA BOTANICA\Acta-Botan 2-10\334 Curkovic Perica.vp 11. listopad 2010 11:21:00 Color profile: Disabled Composite 150 lpi at 45 degrees 158 ACTA BOT. CROAT. 69 (2), 2010 ]URKOVI]-PERICA M., JE@I] M. Tab. 1. Effect of different quercetin concentrations on shoot elongation and fresh weight increase on phytoplasma-infected Catharanthus roseus shoots in vitro. Substance and concentration Shoot elongation1 Fresh weight increase1 Phytoplasma infected Quercetin2 (10 mM) Quercetin2 (100 mM) Quercetin2 (1000 mM) EtOH (1‰) control 1.16±0.13 c 1.01±0.07 d decay 1.19±0.11 c 1.21±0.12 c 1.22±0.1 c 1.04±0.06 d decay 1.33±0.23 c 1.39±0.27 c non-infected Quercetin2 (100 mM) EtOH (1‰) control 1.48±0.2 b 1.82±0.31 a 1.86±0.45 a 2.48±0.26 b 3.16±0.59 a 3.2±0.48 a 1 Mean ± standard deviation. Shoot elongation and fresh weight increase were calculated as the ratio of the final and the initial shoot length or fresh weight, respectively. Means labeled with the identical letters are not significantly different at the 95% level of confidence. 2 Quercetin stock solution was prepared in ethanol (1‰ final concentration in the medium Fig. 1. Agarose gel (1%) electrophoresis of nested PCR amplification products of phytoplasma 16S rDNA obtained using primer pair R16F1/R0. M – Molecular weight marker fX174 BsuRI digested; BA – 'Ca. P. asteris' –infected Catharanthus roseus shoot from the medium supple- mented with: 2.2 mM 6-benzylaminopurine. 1 – PCR positive control, 2 – 10 mM quercetin, 3 – 100 mM quercetin, 4 – ethanol; 5 – healthy C. roseus shoot from the medium supple- mented with: 2,2 mM BA, 6 – 100 mM quercetin, 7 – ethanol, 8 – water control. U:\ACTA BOTANICA\Acta-Botan 2-10\334 Curkovic Perica.vp 11. listopad 2010 11:21:03 Color profile: Disabled Composite 150 lpi at 45 degrees in the concentration of 5 ng µL–1 DNA did not amplify visible PCR product in samples treated with 10 mM or 100 mM quercetin (Fig. 2). However, phytoplasma presence was again confirmed in all samples in nested PCR (Fig. 3). For the shoots treated with 1 mM quercetin an additional second nested PCR, using the primer pair R16(I)F1/R1, was per- formed after the first subculture. Phytoplasma was detected in seven out of eight tested samples, although shoots were already decaying. Therefore, the fact that phytoplasma was detected in the second nested PCR or was not detected at all in one shoot is irrelevant since this high quercetin concentration (1 mM) also caused the decay of the host. Discussion Reports on quercetin toxicity in plants are quite ambiguous, and while some researchers found no deleterious effects of quercetin in the concentration range of 10–1000 µM on Arabidopsis plants (REIGOSA and PAZOS-MALVIDO 2007), others found that quercetin in the concentration of 100 µM and 333 µM was toxic to the same species (PARVEZ et al. 2004). BASILE et al. (2000) found out that quercetin inhibits seed germination of Raphanus sativus. In our experiments, quercetin supplemented to the medium in the concentration of 100 mM, and higher, caused leaf yellowing and browning in both non-infected and infected periwin- kle shoots. The experience from other experimental systems shows that the mode of appli- cation can greatly influence the outcome of quercetin treatment (CUSHNIE and LAMB 2005). ACTA BOT. CROAT. 69 (2), 2010 159 EFFECT OF QUERCETIN ON PHYTOPLASMA Fig. 2. Agarose gel (1%) electrophoresis of direct PCR amplification products of phytoplasma 16S rDNA obtained using primer pair R16F0/R1. M – Molecular weight marker 1kb ladder (Frementas); BA – 'Ca. P. asteris' –infected Catharanthus roseus shoot from the medium supplemented with: 2.2 mM 6-benzylaminopurine. 1 – PCR positive control, 2 – 10 mM quercetin, 3 – 100 mM quercetin, 4 – ethanol, 5 – water control. DNA concentration is indi- cated by the letter a=20 ng mL–1, b=10 ng mL–1, c=5 ng mL–1 U:\ACTA BOTANICA\Acta-Botan 2-10\334 Curkovic Perica.vp 11. listopad 2010 11:21:04 Color profile: Disabled Composite 150 lpi at 45 degrees Since in our experiment quercetin was supplied to plants via liquid medium, which would allow better uptake of this flavonoid through the plant's vascular system, it is quite plausi- ble that concentrations used in this experiment could have been toxic to healthy and in- fected plants. Although quercetin treatment proved to have bacteriostatic or bactericidal effect against many bacteria (CUSHNIE and LAMB 2005), the titer of phytoplasma was only slightly affected in the shoots treated with 10 mM or 100 mM quercetin. Moreover, the severity of the symptoms increased in treated phytoplasma-infected plants. The undesirable effect of quercetin treatment, resulting in the stunting of non-infected and infected plants and in in- creased severity of symptoms in infected plants, might be explained by several hypothe- sized mechanisms. First of all, quercetin may interfere with auxin transport through plants by binding to auxin transport membrane protein complexes (MURPHY at al. 2000) thus dis- turbing the balance of plant growth regulators in healthy and infected shoots. Phytoplasmas also interfere with auxin transport in plants (ALDAGHI et al. 2009). The addition of quercetin might have increased severity of symptoms in phytoplasma-infected shoots by additionally interfering with auxin transport. Periwinkle shoots infected by 'Ca. P. asteris' exhibit symptoms like proliferations, leaf curling and internode shortening. However, stunting was even more pronounced in quercetin-treated than in non-treated infected plants, moreover leaf browning and appearance of black spots on the leaf edges appeared on quercetin treated plants. 160 ACTA BOT. CROAT. 69 (2), 2010 ]URKOVI]-PERICA M., JE@I] M. Fig. 3. Agarose gel (1%) electrophoresis of nested PCR amplification products of phytoplasma 16S rDNA obtained using primer pair R16F2/R2. M – Molecular weight marker 1kb ladder (Frementas); BA – 'Ca. P. asteris' –infected Catharanthus roseus shoot from the medium supplemented with: 2.2 mM 6-benzylaminopurine. 1 – PCR positive control, 2 – 10 mM quercetin, 3 – 100 mM quercetin, 4 – ethanol, 5 – water control. DNA concentration is indi- cated by the letter a=20 ng mL–1, b=10 ng mL–1, c=5 ng mL–1 U:\ACTA BOTANICA\Acta-Botan 2-10\334 Curkovic Perica.vp 11. listopad 2010 11:21:06 Color profile: Disabled Composite 150 lpi at 45 degrees SPENCER et al. (2003) have showed that quercetin, when present in tissue culture media in concentrations higher than 10 µM, actually promoted oxidative damage of cells. In our experiment, healthy and infected plants performed worse on quercetin- than on control me- dium, with phytoplasma-infected shoots showing greater susceptibility to potentially toxic effects of quercetin. The results presented in this paper do not reveal the mechanism by which quercetin in- creased severity of the symptoms in phytoplasma–infected periwinkles, but it is possible that its negative effect is a consequence of several mechanisms. The supplement of quercetin had no curative effect on 'Ca. P. asteris'. Although quercetin slightly reduced phytoplasma titer in C. roseus tissues, its' detrimental effect on plantlets outweighs the pos- sible benefits. The addition of quercetin made symptoms of phytoplasma infection even worse, probably due to synergistic effect of phytoplasma and quercetin as an auxin trans- port inhibitor. Acknowledgements We gratefully acknowledge Professor Assunta Bertaccini for phytoplasma reference strains. References ALDAGHI, M., MASSART, S., BERTACCINI, A., JIJAKLI, M. H., LEPOIVRE, P., 2009: Identifica- tion of host genes potentially implicated in the Malus pulmila and 'Candidatus Phyto- plasma mali' interactions. Proceedings 21 ICVF Conference, Neustadt, 43–44. BASILE, A., SORBO, S., GIORDANO, S., RICCIARDI, L., FERRARA, S., MONTESANO, D., CASTALDO COBIANCHI, R., VUOTTO, M. L., FERRARA, L., 2000: Antibacterial and allelopathic activity of extract from Castanea sativa leaves. Fitoterapia 71, 110–116. CHRISTENSEN, N. M., AXELSEN, K. B., NICOLAISEM, M., SCHULTZ, A., 2005: Phytoplasmas and their interactions with hosts. Trends in Plant Science 10, 526–535. CUSHNIE, T. P. T., LAMB, A. J. 2005: Antimicrobial activity of flavonoids. International Journal of Antimicrobial Agents 26, 343–335. ]URKOVI] PERICA, M., 2008a: Auxin-treatment induces recovery of phytoplasma-infected periwinkle. Journal of Applied Microbiology 105, 1826–1834. ]URKOVI]-PERICA, M., 2008b: Effect of indole-3-butyric acid on rooting of phytoplasma- -recovered and healthy periwinkles (Catharanthus roseus (L) G. Don). Croatica Chem- ica Acta 81, 641–646. ]URKOVI] PERICA, M., [ERUGA MUSI], M. 2005: Effect of b-aminobutyric acid on phyto- plasma infected Catharanthus roseus shoots. Journal of Plant Diseases and Protection 112, 544–549. ]URKOVI] PERICA, M., LEPEDU[, H., [ERUGA MUSI], M., 2007: Effect of indole-3-butyric acid on phytoplasmas in infected Catharantus roseus shoots grown in vitro. FEMS Mi- crobiology Letters 286, 171–177. ACTA BOT. CROAT. 69 (2), 2010 161 EFFECT OF QUERCETIN ON PHYTOPLASMA U:\ACTA BOTANICA\Acta-Botan 2-10\334 Curkovic Perica.vp 11. listopad 2010 11:21:06 Color profile: Disabled Composite 150 lpi at 45 degrees FRENCH, C. J., ELDER, M., LEGGETT, F., IBRAHIM, R. K., TOWERS, G. H. N., 1991: Flavonoids inhibit infectivity of tobacco mosaic virus. Canadian Journal of Plant Pathology 13, 1–68. GUNDERSEN, D. E., LEE, I.-M., 1996: Ultrasensitive detection of phytoplasmas by nested- -PCR assays using two universal primer pairs. Phytopathologia Mediterranea 35, 144–151. HARBORNE, J. B., WILLIAMS, C. B., 2000: Advances in flavonoid research since 1992. Phytochemistry 55, 481–504. IRPCM Phytoplasma/Spiroplasma Working Team–Phytoplasma taxonomy group, 2004: 'Candidatus Phytoplasma', a taxon for the wall-less, non-helical prokaryotes that colo- nize plant phloem and insects. International Journal of Systematic and Evolutionary Microbiology 54, 1243–1255. LEE, I.-M., GUNDERSEN, D. E., HAMMOND, R. W., DAVIS, R. E., 1994: Use of mycoplasma- like organism (MLO) group-specific oligonucleotide primers for nested-PCR assays to detect mixed MLO-infections in a single host plant. Phytopathology 84, 559–566. LEE. I.-M., BERTACCINI, A., VIBIO, M., GUNDERSEN, D. E., 1995, Detection of multiple phytoplasmas in perennial fruit trees with decline symptoms in Italy. Phytopathology 85, 728–735. MALHOTRA, B., ONYILAGHA, J. C., BOHM, B. A., TOWERS, G. H. N., JAMES, D., HARBORNE, J. B., FRENCH, C. J., 1996: Inhibition of tomato ringspot virus by flavonoids. Phyto- chemistry 43, 1271–1276. MURASHIGE, T., SKOOG, F., 1962: A revised medium for rapid growth and bioassays with tobacco tissue cultures. Plant Physiology 15, 473–497. MURPHY, A., PEER, W. A., TAIZ, L., 2000: Regulation of auxin transport by aminopeptidas- es and endogenous flavonoids. Planta 211, 315–324. PARVEZ, M. M., TOMITA-YOKATANI, K., FUJII, Y., KONISHI, T., IWASHINA, T., 2004: Effects of quercetin and its seven derivates on the growth of Arabidopsis thaliana and Neuro- spora crassa. Biochemical Systematics and Ecology 32, 631–635. PLAPER, A., GOLOB, M., HAFNER, I., OBLAK, M., SOLMAJER, T., JERALA, R., 2003: Charac- terization of quercetin binding site on DNA gyrase. Biochemical and Biophysical Re- search Communications 306, 530–536. REIGOSA, M. J., PAZOS-MALVIDO, E., 2007: Phytotoxic effects of 21 plant secondary meta- bolites on Arabidopsis thaliana germination and root growth. Journal of Chemical Ecology 33, 1456–1466. RUSAK, G., KRAJA^I], M., PLE[E, N., 1997: Inhibition of tomato bushy stunt virus infection using a quercetagetin flavonoid isolated from Centaruea rupestris L. Antiviral Re- search 36, 125–129. SPENCER, J. P. E., KUHNLE, G. G. C., WILLIAMS, R. J., RICE-EVANS, C., 2003: Intracellular metabolism and bioactivity of quercetin and its in vivo metabolites. Biochemical Jour- nal 372, 173–181. VERMA, V. S., 1973: Study on the effect of flavonoids on the infectivity of potato virus X. Zentralblatt für Bakteriologie, Parasitenkunde, Infektionskrankheiten und Hygiene. Zweite naturwissenschaftliche Abt.: Allgemeine, landwirtschaftliche und technische Mikrobiologie 128, 467–472. 162 ACTA BOT. CROAT. 69 (2), 2010 ]URKOVI]-PERICA M., JE@I] M. U:\ACTA BOTANICA\Acta-Botan 2-10\334 Curkovic Perica.vp 11. listopad 2010 11:21:06 Color profile: Disabled Composite 150 lpi at 45 degrees