BIOCHEMICAL RESPONSES OF PEACH LEAVES INFECTED WITH TAPHRINA DEFORMANS BERK/TUL.

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1 ACTA UNIVERSITATIS AGRICULTURAE ET SILVICULTURAE MENDELIANAE BRUNENSIS Volume Numer 3, BIOCHEMICAL RESPONSES OF PEACH LEAVES INFECTED WITH TAPHRINA DEFORMANS BERK/TUL. Lyuk Kolev-Vlkov 1, Neshk Piperkov 2, Veselin Petrov 1, Andon Vssilev 1 1 Deprtment of Plnt Physiology nd Biochemistry, Agriculturl University of Plovdiv, Bulgri 2 Deprtment of Phytopthology, Agriculturl University of Plovdiv, Bulgri Astrct KOLEVA VALKOVA LYUBKA, PIPERKOVA NESHKA, PETROV VESELIN, VASSILEV ANDON Biochemicl Responses of Pech Leves Infected with Tphrin Deformns Berk/Tul. Act Universittis Agriculture et Silviculture Mendeline Brunensis, 65(3): The phytopthogenic fungus Tphrin deformns cusing the so clled lef curl disese in pech trees leds to severe yield losses due to the development of lef hypertrophy nd susequent necrosis nd scission. Becuse of its economic importnce, the moleculr mechnisms underlying the onset nd progression of the disese re of considerle interest to the griculturl science. In this study vrious iochemicl prmeters, including the ctivities of the ntioxidnt enzymes guicol peroxidse, syringldzine peroxidse nd ctlse, totl polyphenols nd nthocynin content, concentrtion of free proline, ntirdicl ctivity nd quntity of plstid pigments, were chrcterized. All these were mesured in oth leves with cler symptoms nd distlly situted leves from the sme plnt tht show no signs of the infection. The results demonstrte tht the pthogen induces considerle iochemicl chnges concerning enzymtic nd non enzymtic elements of the plnt defense nd ntioxidnt systems. Moreover, it seems tht the fungus provokes systemic response detectle even in the tissues without oservle symptoms. Keywords: ntioxidnts, enzyme ctivity, lef curl, Prunus persic, Tphrin deformns INTRODUCTION The lef curl disese in pech (Prunus persic L. Btsch), cused y the phytopthogenic fungus Tphrin deformns Berk/Tul., is n economiclly importnt condition, which results in significnt yield losses in the regions with temperte climte. In yers chrcterized with humid nd cool spring seson the indequte pest mngement often leds to epiphytotic outursts of the disese. Its typicl symptoms include premture senescence, necrosis nd scission of the ffected hypertrophic leves. This, in turn, ffects negtively the development nd resistnce of the entire plnt towrds dverse environmentl conditions. The emryonic infection of pech leves with Tphrin deformns cuses drmtic cytohistologicl chnges, hypertrophy nd hyperplsi, tht mnifest s chlorosis, thickening nd deformtion of leves, shoots nd fruit, nd ultimte necrosis of the infected orgns (Cporli, 1964). Tphrin deformns Berk/Tul. is itrophic, dimorphic, monocyclic phytopthogenic fungus (Mix, 1935). It is le to ffect not only individul leves, ut lso systemiclly infect young shoots, which most often necrotize nd die s result. In some cses the symptoms of the disese mnifest t lter stges, in the period of June July, which suggests summer infections with the pthogen (Agrios, 2005). However, such lte symptoms my e lso the consequence of systemiclly developed prsitizing myceli in the young shoots, which cuses lef deformtion y temperture decrese nd humidity increse during cool nd dmp summer sesons. The fungus hyphe re entirely intrcellulr nd otin nutrients from djcent cells, which determine the ility for systemic 871

2 872 Lyuk Kolev-Vlkov, Neshk Piperkov, Veselin Petrov, Andon Vssilev development of the pthogen (Mehrotr nd Aggrwl, 2013). In norml conditions, pthogen ttcks trigger oth ctive nd pssive defensive mechnisms in plnts. The ctive protection includes de novo synthesis of proteins, minly pthogenesis relted (PR) proteins, nd is regulted y n intricte network of signling pthwys. A prominent clss mong them is clss III plnt peroxidses. They prticipte in numerous physiologicl processes, including the hypersensitive response (HR), which induces progrmmed cell deth PCD in order to limit the systemic spred of the invder (Almgro et l., 2008). At the sme time, pssive defenses count minly on creting nd supporting physicl nd chemicl rriers which hinder the ccess to susceptile tissues. However, the estlishment of the contct etween the host nd the pthogen, provokes complex system of interctions. With their elicitor, chemicl nd toxic effects, the pthogenic orgnisms in mny cses inhiit the dequte plnt cell responses towrds the ensuing iotic stress. The ltter results in physiologicl, iochemicl, morphologicl nd ultrstructurl chnges in the host plnt (Prk, 2006). An importnt spect of the considerle current interest on the topic relted to the lef curl disese is the determintion of the physiologicl nd iochemicl sttus of the infected leves, which provides informtion for the extent of the iotic stress. Therefore, the im of the present work ws to investigte some iochemicl mrkers of the enzymtic nd non enzymtic defense in pech during iotic stress, provoked y Tphrin deformns. MATERIALS AND METHODS Plnt mteril nd growing conditions: The smples for nlyses were tken from helthy nd nturlly infected with Tphrin deformns pech trees (Prunus persic L. Btsch) of the vriety Fyette, in the end of April 2014 nd 2015, in the region of Plovdiv, Bulgri. The following vrints were studied: 1. helthy leves of control non infected plnts; 2. red curled leves, rich in nthocynins, of infected plnts; 3. chlorotic curled leves of infected plnts; 4. distlly situted leves, without symptoms of the disese, of infected plnts. Anlyses: The plnt mteril ws hrvested in the period of pek development of the disese. Ech vrint ws tested in mixed smples in 4 repetitions. Antioxidnt enzymes: The ctivities of three ntioxidnt enzymes were mesured. These were guicol peroxidse, syringldzine peroxidse nd ctlse. To otin the enzyme extrct, 1 grm of fresh plnt mteril ws homogenized in 5 ml ice cold 0.1 M Tris HCl (ph 7.8) extrction uffer contining 1 mm DTT nd 1mM EDTA. After tht the smples were centrifuged t 13,500 g (4 ºC for 10 min). The superntnt ws used to determine the enzymtic ctivities spectrophotometriclly on UV/VIS spectrophotometer Phro 300, ccording to the methodology of Mocquot et l. (1996). Ctlse (CAT) (EC ): The ctivity of this ntioxidnt enzyme ws mesured y the method of Aei (1984) t 240 nm wvelength. The rection mixture in the cuvette contined: 2,4 ml 0.1M KH 2PO 4 (ph 7.0), 500 μl 5 mm H 2O 2 nd 100 μl enzyme extrct. Syringldzine peroxidse (SPOD) (EC ): This group of peroxidses is chrcteristic for the poplst nd is le to use syringldzine s the electron donor. SPOD prticiptes in the processes of lignifiction. SPOD ctivity ws mesured t 550 nm, following the method of Imerty et l. (1985) in qurtz cuvette contining the following rection mixture: 2.55 ml 0.1 M Tris HCl uffer (ph 7.5), 300 µl 10 mm H 2O 2, 50 µl 3.5 mm syringldzine nd 100 µl enzyme extrct. Guicol peroxidse (GPOD) (EC ): The ctivity of this group of enzymes ws mesured t 436 nm ccording to Bergmeyer (1974). The rection mixture in the cuvette contined: 2.3 ml 0.1 M KH 2PO 4 (ph 7.0), 300 µl 5 mm H 2O 2, 300 µl guicol nd 100 µl enzyme extrct. Totl phenolics content: The totl mount of phenolic compounds in the plnt extrcts ws determined with the regent of Folin Cioclteu (Wtermn nd Mole, 1994) following the methodology of Singleton et l. (1999), with slight modifictions. The smples (1 g of fresh lef mteril) were ground with qurtz snd nd 10 ml 60 % cidic methnol, nd sumerged in n ultrsound th for 15 min. The homogenized mteril ws then trnsferred to suitle tues, which were crefully seled nd left for 15 hours in the drk t room temperture for extrction. During the incution period, the tues were periodiclly stirred. Afterwrds, the tues were centrifuged nd the superntnt, which ws used for the mesurement of totl phenolics, nthocynins nd ntirdicl ctivity, ws crefully collected in new clen tues. For the determintion of totl phenolics were mixed 40 µl of extrct, 3,160 µl distilled wter, 200 µl Folin Cioclteu regent nd fter minute were dded 600 µl 20 % NCO 3. The test tues were left for 2 hours t room temperture for the rection to occur. After tht the extinction t 765 nm wvelength. Totl phenolics were clculted s gllic cid equivlents (GAE) using stndrd curve nd re presented s mg/g fresh weight. The stndrd curve ws prepred with gllic cid (Sigm Aldrich, St. Louis, MO) in the rnge mg/l. Totl nthocynin content: The mesurement of the mount of monomeric nthocynins ws performed with the ph differentil method (Guisti nd Wrolstd, 2001). The plnt extrcts with cidic methnol (s descried in the procedure for phenolics mesurement) were diluted with uffer (0.025 mol/l potssium chloride), djusted to ph = 1 with HCl, nd nother uffer (0.4 mol/l sodium cette) with ph = 4.5. Ech smple ws diluted to certin extent with the first uffer with ph = 1

3 Biochemicl Responses of Pech Leves Infected with Tphrin Deformns Berk/Tul. 873 (which gives the vlue DF) nd the sorption ws mesured t 520 nm nd 700 nm. A second liquot of ech smple ws diluted to the sme extent with the second uffer with ph = 4.5 nd gin the extinction t 520 nm n 700 nm ws mesured. To clculte totl nthocynin content, the sorption vlues were used in the following formul: A = (A λ520 A λ700) ph 1.0 (A λ520 A λ700) ph 4.5 TA = (A*MW*DF*1000)/ ε*1 where: the molr extinction coefficient (ε) for cynidin 3 glucoside = 26,900 (M 1 cm 1 ), the moleculr weight (MW) of cynidin 3 glucoside = g/mol nd the dilution fctor (DF) demonstrtes the times dilution of the smple. The results (TA totl nthocynins) re expressed s mg cynidin 3 glucoside chloride/100 g fresh plnt mteril used. Antirdicl ctivity: This prmeter ws mesured with 2,2 diphenyl 1 picrylhydrzyl (DPPH) (Bet et l. 2007) in extrcts otined in the wy descried ove for totl phenolics. The incution mixture contined 100 µl extrct nd 3.9 ml mol/l DPPH (0.06 µmol/l). The extrct sorption ws determined t 515 nm t min 0 nd 30 from c c A) c B) САТ c C) 0 1: Activity of ntioxidnt enzymes in pech leves: vrint 1 control (helthy leves from non infected plnts), vrint 2 infected leves with nthocynin colortion, vrint 3 chlorotic infected leves, vrint 4 distlly situted leves of infected plnts, with no evident symptoms. A) Guicol peroxidse, B) Syringldzine peroxidse, C) Ctlse. The sme letters ove the rs indicte lck of sttisticl significnce t P < 0,05

4 874 Lyuk Kolev-Vlkov, Neshk Piperkov, Veselin Petrov, Andon Vssilev the initil mixture of the components. A prllel lnk smple ws tested, which contined distilled wter insted of extrct. The ntirdicl ctivity is expressed s % decolortion nd is equl to: (1 (A 30min/A 0min) 100). Photosynthetic pigments content: The concentrtion of pigments ws determined spectrophotometriclly y the method of Lichtenhlter (1987). Free proline concentrtion: The proline content ws lso mesured spectrophotometriclly y the method of Btes et l. (1973) fter n extrction with 3 % sulfoslicylic cid. Sttisticl nlysis: All generted dt were tested for sttisticl significnce with the SPSS 13 softwre, using the one wy ANOVA test (for P < 0.05). A Tukey s test for min comprison t 95 % confidentil level ws pplied sed on the ANOVA results. RESULTS The ctivity of the ntioxidnt enzymes ctlse, guicol nd syringldzine peroxidses in helthy nd infected with Tphrin deformns pech leves is presented on Fig. 1. A considerle increse in the ctivity of guicol peroxidse ws oserved in vrints 2 (3.89 U/g FW) nd 3 (1.79 U/g FW) in comprison to the control (0.46 U/g FW), which my e due to role of the enzyme in the defensive mechnisms ginst the pthogen (Fig. 1A). The nlysis of syringldzine peroxidse demonstrted significnt ugmenttion of the enzyme ctivity in ll infected vrints in comprison to the control (0.02 U/g FW) (Fig. 1 B). In the leves with clerly oservle symptoms (vrints 2 nd 3) the vlues of the enzyme ctivity reched 0.44 U/g FW, while in the distlly situted leves without symptoms they were 0.27 U/g FW, which ws still 10 times higher thn the controls. Together, the increse in the ctivity of oth peroxidses suggests their involvement in the defensive system of the host plnt ginst the fungl infection. Interestingly, the mesurement of ctlse ctivity (shown on Fig. 1C) demonstrted n opposite trend: in this cse the highest vlues were oserved in the helthy leves (vrint U/g FW), followed y the leves of infected plnts with no symptoms of the disese (vrint U/g FW). In the other 2 vrints, in which the symptoms were mnifested, the enzyme ctivity ws significntly lower (vrint U/g FW nd vrint U/g FW). This phenomenon cn e explined with the existing competition etween ctlse nd peroxidses for the sme sustrte hydrogen peroxide. T. I summrizes the results of the nlyses of totl polyphenols content, nthocynin quntity nd ntirdicl ctivity. Leves infected with Tphrin deformns hd decresed mounts of polyphenol compounds, which ws more prominent in the vrints with mnifested symptoms (T. I, vrint mg nd vrint mg) in comprison to the control (vrint mg). These results re correlted with the ones otined for the ctivity of syringldzine peroxidse. In vrints 2 nd 3, chrcterized with the highest peroxidse ctivity, were detected the lowest concentrtions of totl polyphenols. This is not surprising since phenolic compounds I: Quntities of totl polyphenols, nthocynins nd ntirdicl ctivity in control (helthy) nd infected pech leves with Tphrin deformns. Vrint 1 control (helthy leves), vrint 2 infected leves with nthocynin colortion, vrint 3 chlorotic infected leves, vrint 4 leves of infected plnts without symptoms. Vrints Totl polyphenols mggae/g FW Anthocynins s cynidin 3 glucoside mg/ 100g FW Antirdicl ctivity %DPPH/g FW 1 110,11 0,175d 12,76 (100 %) 2 48,51c 9,018 8,05 (61 %)c 3 69,84 0,335c 12,94 (101 %) 4 103,98 1,334 15,67 (123 %) All the vlues re presented s men. The dt in the columns followed y the sme letter (,, c) re not significnt for P < 0,05 II: Plstid pigments content in helthy nd infected with Tphrin deformns pech leves. Vrint 1 control (helthy leves), vrint 2 infected leves with nthocynin colortion, vrint 3 chlorotic infected leves, vrint 4 leves of infected plnts without symptoms. Vrint Chl. а Chl. Crotenoids Chlorophyll / Chlorophylls (а+)/crotenoids 1 2,65 0,81 1,33 3,28 2,6 2 2,04 0,88 1,03 2,32 2,83 3 1,76c 0,84 0,89c 2,1 2,91 4 2,16 0,70c 1,05 3,1 2,82 All the vlues re presented s men. The dt in the columns followed y the sme letter (,, c) re not significnt for P < 0,05

5 Biochemicl Responses of Pech Leves Infected with Tphrin Deformns Berk/Tul. 875 re the sustrte of syringldzine peroxidse. In the experiment with nthocynin quntifiction, s expected the most undnt levels were detected in vrint 2 (9.01 mg/ 100 g FW). In the leves infected with Tphrin deformns the ntirdicl ctivity ws lso ltered. It ws considerly reduced, mostly in vrint 2 (61 % of the control), in which were detected the lowest mounts of polyphenols. It must e noted, tht the ltter re known to neutrlize free rdicls. In the other vrints the ntirdicl ctivity ws enhnced. In T. II the quntities of plstid pigments, s well s their rtio, re presented. In the infected leves it ws oserved significnt reduction of chlorophyll (with 23, 34 nd 19 % for vrints 2, 3 nd 4, respectively) nd crotenoids (with 23, 33 nd 23 % for vrints 2, 3 nd 4, respectively). The chlrophyll content ws ffected to lesser extent. Finlly, the undnce of nother importnt ntistress primry metolite free proline, ws lso tested nd the results re presented on Fig. 2. Proline is n α mino cid with crucil role for the mintennce of the wter potentil of plnt cells. In pech leves infected with Tphrin deformns n increse in proline concentrtion ws detected, ut only in the vrints with cler disese symptoms (vrints 2 nd % nd 119 % of the control, respectively). DISCUSSION We expnd on the previous studies on the topic y nlyzing vrious iochemicl prmeters not only in smples from leves with clerly oservle symptoms (vrints 2 nd 3), ut lso from tissues which do not show ny defects (vrint 4). The dt of the experiments indicte tht in pech tree leves the ctivity of some ntioxidnt enzymes nd the concentrtion of some of the non enzymtic components of the plnt defense system re influenced y the infection with Tphrin deformns. Similr conclusions were drwn lso y other uthors studying the lef curl disese in pech (Cionu, 2012). The most potent guicol peroxidse ws mesured in infected leves with ccumulted nthocynins (Fig. 1A, vrint 2), followed y the chlorotic infected leves (Fig. 1A, vrint 3). A similr result ws oserved with syringldzine peroxidse s well. This my e n indiction of synergistic effects of oth enzymes in the response to the pthogen. Moreover, the much higher ctivity of syringldzine peroxidse in the infected orgns cn e linked to the role of the enzyme in the oxidtion of phenolic compounds to quinones, which re more toxic for the pthogens (Myer, 2006). Thus, y the upregultion of syringldzine peroxidse ctivity, the host plnt cn limit or even hlt the spred of the invder. This enzyme ws more ctive even in the leves which did not mnifest ny symptoms (Fig. 1B, vrint 4). The reported reduced ntirdicl ctivity in vrint 2 (T. I) is in ccordnce with the dt, otined for the totl polyphenols content, which is lso significntly lower in infected leves. The decrese in totl polyphenols in cell wlls wekens them nd mkes them more susceptile to the influence of the pthogen. As result, the ffected cells re more prone to hypertrophy (Semerdjiev et l., 2014). The induced iosynthesis of nthocynins, found in ll infected leves (even those which re distl nd without symptoms) cn e lso considered s prt of the defensive systems of the host (T. I). The resons for nthocynin ccumultion only in some of the infected leves re not yet fully elucidted. Aprt from their ntioxidnt properties, nthocynins re lso crohydrte reserve, since they exist in glycosylted form (most often glucose). It cn e presumed tht their iosynthesis is stimulted y regultory molecules relesed from the pthogens, which use the crohydrtes s nutrient source or structurl elements (Petit nd Schneider, 1983). 2: Free proline content in helthy nd infected with Tphrin deformns leves of pech trees. Vrint 1 control (helthy leves from non infected plnts), vrint 2 infected leves with nthocynin colortion, vrint 3 chlorotic infected leves, vrint 4 distlly situted leves of infected plnts, with no evident symptoms. The sme letters ove the rs indicte lck of sttisticl significnce t P < 0,05.

6 876 Lyuk Kolev-Vlkov, Neshk Piperkov, Veselin Petrov, Andon Vssilev The considerly lowered ctlse ctivity, found in the infected leves cn e relted to their more ctive peroxidses since the primry sustrte of ctlse is hydrogen peroxide ut it is consumed y the competing peroxidses. Other uthors (Buonurio nd Montlini, 1993) hve lso reported correltion etween reduced ctlse nd incresed peroxidse ctivities, which is in ccordnce with our results. In nother study, it ws demonstrted tht ctlse ws induced in infected pech levels, ut only in the initil phse of the disese. With the dvncement of the infection ctlse ctivity grdully diminished (Cionu, 2012). The impct of Tphrin deformns on infected leves cuses significnt decrese of the quntity of plstid pigments. This effect ws oserved in numer of other studies with the sme pthogen. Rggy (1966, 1967), for exmple, reported tht chlorophyll concentrtions in the curled leves were lowered y 30 % to 50 % in comprison to controls, while ccording to Nikole (2009) the reduction of this prmeter mounts to 53,62 %. Similrly, in the vriety Redhven Montlini nd Buonurio (1986) documented difference of 30 % etween infected nd helthy leves. They found tht the decresed chlorophyll content ws not cused y elevted chlorophyllse ctivity, s is the cse with some virus infections, nd Nicole nd Mitre (2009) suggested tht the reson is the inhiited iosynthesis of the pigments. Moreover, other uthors like Mrte nd Ggiulo (1972), Syrop (1975), Bssi et l. (1984) nd Hung et l. (1993) presumed tht in curled leves the numer of chloroplsts could e severely reduced nd the remining chloroplsts chrcterized with dmged structure of oth the thylkoid network nd the grne. In our study, the nlysis of the ultrstructure of mesophyll cells of leves infected with Tphrin deformns confirm the hypothesis tht chloroplsts degenerte with the progression of the disese (unpulished results). In the genome of Tphrin deformns re found genes, responsile for the iosynthesis of compounds, relted to the pthogenesis proteses, which llow the digestion of plnt tissues; secondry metolites, which fcilitte the interctions of the fungl pthogen with the environment, including the host plnt; nd hormones which re responsile for the typicl hypertrophy nd hyperplsi symptoms (Cisse et l., 2013). Therefore, the structurl chnges in the ffected leves re relted to this hormonl dislnce provoked y the pthogen. In prticulr, the enhnced cellulr growth nd wter content re in correltion with n incresed concentrtion of uxins, while the stimulted cellulr division nd other growth nomlies re influenced y elevted cytokinin ctivity. Indeed, it hs een shown tht the cytokinin ctivity nd the levels of indol 3 cetic cid nd tryptophn re higher, respectively with 81 % nd 65 %, in leves infected y Tphrin deformns (Szirki et l., 1975, Ymd et l., 1990). The significnt ccumultion of uxins nd cytokinins in these leves proly induces their ility to ttrct photossimiltes. According to Goodmn et l. (1986), the photossimiltes re redirected from helthy leves, which show higher CO 2 fixtion, to infected ones in order to prtilly compenste for their crohydrte shortge. Finlly, the mesurement of the concentrtion of free proline indicted tht it ws incresed in the infected leves with oservle symptoms (Fig. 2). This positively correltes with the strong hydrtion of the dmged cells (Piperkov nd Vsilev, 2000). Similr results were otined lso y Rggy (1967), who worked with pech trees infected with the sme pthogen. He oserved elevtion of the levels of the free mino cids proline, ornithine nd glycine during the incution period of the infection. This phenomenon could e explined y the presence of chemicls (elicitors) relesed y the fungus, which stimulte the iosynthesis of such compounds or y incresed protein degrdtion in the cells of the host plnt. The second hypothesis ws supported y the fct tht the totl protein content in infected leves ws lso reduced (Rggy, 1967). In nother study of the sme uthor (Rggy, 1987) it ws shown tht the wter content of infected leves is from 2,4 up to 9,6 times higher thn in helthy leves. It is well known tht free proline serves s n osmolite nd is ccumulted in response to wter stress: wter deficit, slt stress, low temperture stress, wterlogging conditions (Olgun et l., 2008, Hyt et l., 2012). In plnts ttcked y Tphrin deformns, the higher proline content my e cused y chemicl stimuli secreted y the pthogen nd/or the hyperhydrted sttus of the ffected cells. CONCLUSION The nlyzed iochemicl mrkers confirm tht the curled lef disese cused y Tphrin deformns induces serious chnges in the iochemicl sttus of the infected plnts, which re detectle not only in the tissues with oservle symptoms, ut lso in distlly situted ones. These chnges include the elevtion of the ctivity of ntioxidnt enzymes (peroxidses); reduced polyphenols content nd plstid pigments; ltertions of ntirdicl ctivity, nthocynin nd free proline concentrtions. On one hnd these results suggest systemic chrcter of the pthogen infection nd development nd on the other hnd demonstrte tht the entire plnt goes in stte of lert y inducing its defensive systems.

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