Evaluation of the resistance of few citrus rootstocks to alkalinity by applying a faste test of secreening

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1 Afrin Journl of Agriulturl Reserh Vol. 6(4), pp , 18 Februry, 2011 Avilble online t ISSN X 2011 Ademi Journls Full Length Reserh Pper Evlution of the resistne of few itrus rootstoks to lklinity by pplying fste test of sereening Benyhi H. 1 *, Beniken L. 1, Omri F. Z. 1, Benzzouze A. 1, Hndji N. 1, Mstef Y. 2 nd Olitrult P. 3 1 Institut Ntionl de l Reherhe Agronomique, Unité de Reherhe Améliortion et Conservtion des ressoures Phytogénétiques, Lbortoire d méliortion des grumes, BP : 257, kenitr, Mroo. 2 Lbortoire de Physiologie Végétle, Fulté des Sienes Dhr Elmhrz, Université Sidi Mohmed Ben Abdelh, Fès, Mroo. 3 CIRAD, Unité de Reherhe Ameliortion d espèes à multiplition végéttive, TA50/PS4 Boulevrd De L Lironde, Montpellier, Frne. Aepted 12 Jnury, 2011 Alklinity of Moron soils is the mjor bioti onstrint on itrus prodution re. The best hoie of itrus rootstoks dequte nd resistnt is better solution to void this problem. The im of this study is to develop fst test of itrus rootstoks sreening towrds lklinity. The lkline stress ws pplied on ten itrus rootstoks two month old, using irrigtion with Hoglnd nd Arnon solution dded with 1 g CCO 3 /L nd djusted t vrious ph levels 6, 7 nd 9. Observtions onerned symptoms inidene nd severity of iron hlorosis fter two months of rering. Results permitted to lssify Ponirus trifolit nd Flying drgon s the most sensitive to lklinity stresses, wheres, Citrus volkmerin nd Citrus mrophyll were resistnt. These onlusions re equivlent with those obtined with old itrus rootstoks in field trils. Keys words: Citrus, rootstoks, lklinity, sreening. INTRODUCTION It is estimted tht 20 to 50% of fruit trees in the Mediterrnen Bsin suffer from iron defiieny (Jegger et l., 2000). The mjor use of iron defiieny in these regions is due to high onentrtions of lium nd birbonte in soils. High onentrtions of birbonte usully ffet metboli proesses t the leves nd roots nd limit the vilbility of iron for the plnt (Mengel, 1995), whih leds to the development of symptoms of iron hlorosis. Among fruits, iron hlorosis uses signifint loss in performne (Pestn et l., 2003), delyed ripening of fruit ffets the qulity of fruits like peh (Snz et l., 1997) nd itrus (Pestn et l., 2001; Ptriio et l., 2007). In itrus fruit, the problem of soil lklinity n be overomed by hoosing resistnt rootstok (Albrigo nd Dvies, 1994; Pestn et l., 2001). In Moroo, lklinity of the soil is serous problem for Citrus growers (Benyhi, 1998). The sour ornge rootstoks, lthough it is dpted to the lreous soils in Moroo, this inresed sensitivity to tristez virus, whih lls into question its use. The serh of rootstoks lterntives, remins long-term strtegy. The development of rpid sreening tests to identify soures of resistne to this onstrint re of mjor importne to the improvement progrm of rootstoks itrus from INRA Moroo nd more generlly, for ny suh projet in the Mediterrnen Bsin. The objetive of this work is the development of fst sreening test, relible nd reproduible to lklinity for routine evlution of olletion of rootstoks of itrus to INRA, to identify new soures of tolerne. These rootstoks will be used lter in geneti improvement progrmmes s well, by onventionl methods s the tools of plnt biotehnology. MATERIALS AND METHODS Plnting mteril *Corresponding uthor. E-mil: hmidbenyhi2002@yhoo.fr. Ten vrieties of rootstoks were used (Tble 1). We hve hosen

2 Benyhi et l. 781 Tble 1. List of rootstoks used. Root-stoks Resistne to lklinity Citrnge Troyer (CT) bigrdier (Big) Citrus volkmerin (CV) Rootstoks resistnt (7) Citrnge Crrizo (CC) Rootstoks sensitive (7) Mndrnier Cléopâtre (MC) Citrus mrophyll (CM) Citrumelo (CML) Soh jhli (SJ) Ponirus trifolit (PT) Porte-greffe sensitive (7) Ponirus trifolit flying drgon (FD) two rootstok referenes whose hrteristis of sensitivity or resistne to the lklinity re known, to estimte without exessive risk of error the degree of resistne or suseptibility of the other rootstoks. The seed me from the olletion of rootstoks of itrus INRA El Menzeh. Seedlings The seedlings of different rootstoks were mde in plsti trys with dimension of 53 x 53 m, ontining mixture of pet nd snd sterile (Blker nd MDonld, 1986). The trys re pled in plsti tunnel t tempertures between 25 nd 30 C. Irrigtion ws done everydy with wter, nd one every two weeks with 300 ml of nutrient solution of Hoglnd nd Arnon hlf onentrted (Zekri, 1991). Two months fter plnting, young seedlings of eh uniform rootstok were olleted seprtely nd trnsplnted into plsti pots of 15 m deep nd 8 m in dimeter, filled with mixture of sterile snd-pet. Seedlings were irrigted dily with the nutrient solution of hlf onentrted Hoglnd solution for 1 month t the rte of 100 ml per pot representing the field pity of the soil used. Level of lklinity For the level of lklinity we used the method of Abbdi (1998), whih is to use the solution of Hoglnd hlf onentrted with CCO 3 dded to 1 g/l nd djusted to ph 7, 8 nd 9. The plnts ontrol were irrigted with hlf onentrted Hoglnd solution without CCO 3 nd whose ph ws djusted to the vlue of 6. The vlues of ph used were hosen, tking into ount the ph of soil nd wter for irrigtion in itrus orhrds in Moroo. In prts of the Ghrb nd Berkne of ph, in the rnge of 8 is observed by Benyhi (1998). ph 9 represents extreme lklinity while ph 6 represents the optimum onditions for the growth of itrus fruits. Applition of lkline stress After the breeding period of 1 month, 18 seedlings eh of the rootstok, ording returned n experimentl split-plot with three replitions nd on the bsis of two plnts by repetition. The ph ftor ws pled in lrge prels nd the rootstok ftor in smll prels. The plnts were irrigted three times week with lkline solutions. The test ws rried out for two months in greenhouse, whose temperture rnges between 25 nd 35 C. Determintion of resistne to rootstoks to lklinity Inidene of iron hlorosis This is the number of plnts showing iron hlorosis on the totl number of plnts subjeted to stress. Severity of symptoms of iron hlorosis To estimte the severity of hlorosis, we used rting sle from 0 to 5 (Romer et l., 1991; Snz et l., 1997). With 0 plnts showing no sign of iron hlorosis with 5 plnts showing totl hlorosis with flling leves. RESULTS Inidene of iron hlorosis The symptoms of hlorosis hve emerged fter the seond week of tretment (Figure 1). The inidene or frequeny of plnts showing symptoms of hlorosis vries from rootstok to nother nd depending on the ph of the solution for irrigtion. Sttistil nlyses of the vrible inidene of iron hlorosis shows signifint effets of the ftors door rootstoks nd ph. By ontrst, the intertion between the rootstok nd the ph is not signifint. The omprison of the verge inidene of iron hlorosis for different rootstoks n distinguish 4 groups of rootstoks sttistilly different (Figure 2). Group 1. Inludes flying drgon, Ponirus trifolit nd Crrizo itrnge for whih the frequenies of plnts ffeted re the highest, rnging from 60 to 80%. Group 2. Consisting of Citrumelo nd Soh jhli ffeting hlorosis of the order of 50%. Group 3. Contins door rootstoks tht hve n impt on the order of 35%. They re Citrus volkmerin, itrnge Troyer, the mndrin Cleoptr nd Citrus mrophyll. Group 4. Contins only the ornge s the lest ffeted with n inidene of 5%.

3 782 Afr. J. Agri. Res. C.troyer C.volkmerin P.trifolit Sohjhli Figure 1. Symptoms of iron hlorosis on leves of rootstoks itrus irrigted by irrigtion solution with ph = Inidene of hlorosis b b d 0 CC CML FD PT CV SJ Big CM CT MC porte-greffes Rootstoks Figure 2. Averge effet of iron hlorosis mong rootstoks. All ph onfused. The rootstoks followed by the sme letter did not differ signifintly t the 5% threshold (Test Dunn). Severity of iron hlorosis The nlysis of the vrine of severities symptoms of hlorosis shows very highly signifint ftor rootstoks nd ph. In ddition, highly signifint intertion between ftors ph nd the rootstok ws observed. Thus, the inrese in the ph of the solution leds to n inrese in the severity of iron hlorosis, but with differentil behvior of rootstoks (Tble2). Aording to Tble 2, flying drgon nd P. trifolit

4 Benyhi et l. 783 Tble 2. Effet of ph of the solution on irrigtion. Severities of iron hlorosis reorded on different rootstoks. Porte-greffes ph of the irrigtion solution Citrnge Crrizo 1 b 1.66 b 1.93 b Citrumelo 0.8 b 1.33 b 1.5 b Flying drgon Ponirus trifolit Citrus volkmerin 1.16 b 1.16 b 1.33 b Soh jhli 0.33 b 0.93 b 2 b Bigrdier 0 0 d 0.33 C. mrophyll 0.5 b Citrnge Troyer 0.16 b M. Cleoptre 0.16 b The rootstoks followed by the sme letter did not differ signifintly t the 5% threshold (Test Dunn). Tble 3. Resistne (R) nd sensitivity (S) of rootstoks to lklinity. Root-stoks F SEV Clssifition ording to Albrigo nd Dvies (1994) Flying drgon S S? C. volkmerin RM RM R bigrdier R R R Citrnge Troyer R R R Citrnge Crrizo S MR S M. Cléopâtre R R R Soh jhli RM RM? C. mrophyll R R R Ponirus trifolit S S S Citrumelo S RM? F : Frequeny of symptoms of hlorosis; SEV : Symptom severity of hlorosis; R= Resistnt; RM= Modertely resistnt; S= sensitive. rriers re trnsplnts who showed high levels of hlorosis used t different ph. The degree of hlorosis is respetively 3.5 nd 3.66 t ph 9. The sour ornge hs shown good resistne to ph 8 nd 9, followed by C. mrophyll whose index hlorosis t ph 9 is only The mndriner Cleoptr too, with hints of 0.33 nd 0.5 respetively t ph 8 nd 9, hs been resistnt to lklinity. C. volkmerin showed resistne eptble to lklinity, ompred to P. trifolit nd flying drgon. Citrumelo hs engged in ondut similr to tht of Crrizo itrnge. The itrnge Troyer showed high level of tolerne, muh higher thn Crrizo itrnge t ph 8 nd 9. Evlution of resistne to rootstoks to lklinity The lssifition of rootstoks in terms of tolerne or suseptibility to iron hlorosis vries depending on the riterion used for some rootstoks, while it is the sme for others (Tble 3). Thus, flying drgon rnked s sensitive to the frequeny of the severity of hlorosis. For the mjority of rootstoks, our results re similr to those of Albrigo nd Dvis (1994). DISCUSSION The ddition of 1 g/l of solution CCO 3 irrigtion djusted t different ph uses iron hlorosis on leves of rootstoks itrus. Abbdi (1998) mesured the tivity of the helte redutse nd showed tht lklinity in the presene of CCO 3 in smll quntities indued derese in tivity of this enzyme in the suseptible rootstoks nd onsequently redution of supplied iron. Thus, the onset of symptoms of hlorosis is probbly the result of n iron defiieny. The intensity of the iron hlorosis on the rootstoks used vries in frequeny nd severity. Flying drgon nd P. trifolit were very sensitive to lklinity. Albrigo nd Dvies (1994) hve lso lssified these two rootstoks s sensitive. P. trifolit is sensitive to lklinity, nd it ffets the yield nd qulity of fruit

5 784 Afr. J. Agri. Res. vrieties grfted (Durón, 1999; Zekri, 1995; Alv nd Tuker, 1999). The sour ornge showed high tolerne in terms of intensity of the symptoms of iron hlorosis. This result is well orrelted with observtions in orhrds. Indeed, it is renowned for its good behviour in lreous soils in the region of Souss in Moroo. C. volkmerin hs shown intermedite tolerne. This root-stok hs shown promise in the region of Souss, in terms of tolerne nd performne (Ait et l., 2004). In ddition, Cstel nd Gmitter, (1999) rnked this rootstok s modertely tolernt to lklinity. In Venezuél, C. volkmerin ws widely used in lreous soils s the rootstok for replement to sour ornge (Jkson, 1999). In Florid, C. volkmerin presented good behviour in terms of resistne to lklinity (Cstle et l., 1993; Cstle et l., 1997). The mndrnier Cleoptr presented good level of tolerne to lklinity in our study. This root-stok ws lso rnked by Albrigo nd Dvies (1994) s tolernt to lklinity but Cstle et l. (1993) sees it s moderte nd tht in Florid, Cleoptr mndrin presents n intermedite tolerne (Huthison, 1982). It is possible tht these differenes in outomes re relted to the use of different genotypes of Mndrins Cleoptr. Studies on the moleulr mrkings indeed identified geneti polymorphism between different essions of Mndrins Cleoptr in olletions (Luro, om; pers). The itrnge Troyer, lthough it is hybride of Ponirus, it demonstrted in this study high degree of tolerne for lklinity. Reupero nd Russo (1988) rnked this rootstok s n intermediry between the sour ornge ornge nd P. trifolit. In the end, Mribel et l. (2005) rnked these rootstoks s resistnt to hlorosis. The hnge in behvior of itrnge in the vrious studies ould be onneted with the onditions of experimenttion nd the homogeneity of the plnt mteril used. C. mrophyll hs shown good resistne to iron hlorosis, whih is onsistent with the lssifition of Albrigo nd Dvies (1994). The results from our test seedlings ged three months re onsistent with the results obtined in ptients to rootstoks older. This proves the effetiveness of our test, whih is fster. It n therefore be generlized to ssess, in limited spe, mny genotypes or olletion of hybrid breeding progrms. In the longer term, it will be neessry to seek other physiologil riteri, to retrieve the genes responsible for tolerne to rootstoks. In this sense, the study of the tivity nd regultion of ertin enzymes involved in iron metbolism ould be promising pproh for the identifition of rootstoks with degrees of tolerne best meet the shortfll in iron. REFERENCES Proeedings Ntionl Symposium on Minerl Nutrition of Plnts. Edition of University of Mdrid, ISBN X, pp Ait Hddou MM, Jquemend C, Benzzouze A, Afellh M, Benyhi H, Kbbj T, Sriri I, Ezzoubir D, Lmsetef Y (2004). Effet of the rootstok on some gronomi trits of lementines nules nd nour in the souss vley in south moroo. Congres interntionl de Citriulture. Agdir Fevrier 2004 Mroo. Albrigo LG, Dvies FS (1994). CITRUS. CAB. p Alv A, Tuker DH (1999). Soils nd nutrition. In: Citrus Helth Mngement. Timmer, L. W., L. W. Dunn (eds.). The Amerin Phytopthologil Soiety Press. St. Pul Minnesot. USA. pp: Benyhi H (1998). Effet of slinity on the development of Phytophthor diseses on itrus in Moroo. Thesis.University Cdi Ayyd, Fulty of Siene Semlli Mrrkeh, P Blker NS, MDonld JD (1986). The role of slinity in development of Phytophthor root rot of Citrus. Phytopthology 76: Cstle WS, Tuker DPH, Krezdorn AH, Youtsey CO (1993). Roostoks for Florid itrus. Univ. Fl. Publ. SP-42. Cstle WS, Gmitter FG (1999), Rootstok nd sion seletion: In: Citrus Helth Mngement. Timmer LW, Dunn LW (eds.). The Amerin Phytopthologil Soiety Press. St. PulMinnesot. USA. p Durón N (1999). Estblishment oforhrds: In: Citrus for Northwestern Mexio. Reserh nd Ntionl Forest Agriulturl. SAGAR. Mexio. pp: Huthison DJ (1982), Influene of rootstok on the performne of Vleni sweet ornge. In Grierson W. (ed) Pro. Int. So. Citriulture, Citriulture, Vol. II, Orlndo, Fl. Intern. So. Jkson LK (1999). Citrus ultivtion. In: Citrus Helth Mngement.Timmer, L. W. nd L. W. Dunn. (eds.) pp: The Amerin Phytopthologil Soiety Press. St. PulMinnesot. USA. p. 197 Jegger B, Goldbh H, Sommer K (2000). Relese from lime indued iron hlorosis by CULTAN in fruit trees nd its hrteristion by nlysis. At Hort. 531: Mribel P, Amrilis de V, Jvier A, Eugenio AF (2005). Differentil tolerne to iron defiieny of itrus rootstoks grown in nutrient solution. Si. Horti. 104: Mengel K (1995). Iron vilbility in plnt tissues iron hlorosis on lreous soils. In: Abdı, J. (Ed.), Iron Nutrition in Soils nd Plnts. Kluwer Ademi Publishers, Dordreht, The Netherlnds, pp Ptriio R, Bln I, Cstro M, Frniso R, de l Grz R, Guillermo M, Flores JD, Ethevers B (2007). Evlution of different iron ompounds in hloroti Itlin lemon trees (Citrus lemon). Plnt Physiology nd Biohemistry xx 1e5. Pestn M, de Vrennes A, Fri EA (2003). Dignosis nd orretion of iron hlorosis in fruit trees: review. Pestn M, Correi PJ, de Vrennes A, Abdı J, Fri EA (2001). Effetiveness of different folir pplitions to ontrol iron hlorosis in ornge trees grown on lreous soil. J. Plnt Nutr., 24(4 5): Reupero Reforgito G, Russo F (1988). A tril of rootstoks for lementine 'Comune' in Itly. Interntionl Citrus Congress (6th : 1988 : Tel Aviv, Isrel) : Mrgrf. 1: Romer FJ, Alántr E, de l Gurdi MD (1991).Chrteriztion of the tolerne to iron hlorosis in different peh rootstoks grown in nutrient solution. II. Iron stress response mehnisms. In: Chen, Y., nd Hdr, Y. (eds.). Iron nutrition nd intertions in plnts. Dordreht, Netherlnds: Kluwer Ademi Publishers. p Snz M, Psul J, Mhın J (1997). Prognosis nd orretion of iron hlorosis in peh trees: Influene on fruit qulity. J. Plnt Nutr., 20(11): Zekri M (1991). Effets of NCl on growth nd physiology of sour ornge nd Cleoptr mndrin seedlings. Si. Hort., 47: Zekri M (1995). 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