Induction of systemic acquired resistance against Leaf blight of lupine (Lupinus termis Forsk) caused by Bacillus megaterium pv lupine

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1 Interntionl Journl of Agriulturl Sienes ISSN: Vol. 3 (7), pp , September, Avilble online t Interntionl Sholrs Journls Full Length Reserh Pper Indution of systemi quired resistne ginst Lef blight of lupine (Lupinus termis Forsk) used by Billus megterium pv lupine Abdel-Monim M. F. 1*, Gbr M. R. 2, El-Gntiry S. M. 1, Sht M. N. 2 nd El-Bn A. A. 2 1 Legume Diseses Res. Dep., Plnt Pthology Res. Inst., Agri. Res. Cen., Giz, Egypt. 2 Plnt Pthology Deprtment, Fulty of Agriulture, El-Mini University, Mini, Egypt. Aepted 12 June, 2013 Four hemil induers viz. Benzoi, feruli, mli, nd oxli ids t different onentrtions (50, 100 nd 200 ppm) were pplied on lupine plnts in the frme of lef blight used by Billus megterium mngement study. All the tested hemils lerly retrded disese development on lupine plnts treted with hemil induers (espeilly in se of higher onentrtion, 200 ppm) ompred with the untreted plnts. Oxli nd mli ids were the most effetive hemil induers s they gretly redued disese severity, while feruli nd benzoi ids were the lest effetive ompounds. Also, the in vitro effet of these hemils on bteril growth ws wek or moderte t higher onentrtion when ompred with the ontrol. Assessment of peroxidse, polyphenoloxidse, phenyllnine mmoni lyse nd phenol ompound ontents in lupine treted plnts indited gret inrese in their tivities in hemil induers treted plnts. Lupine plnts treted with oxli nd mli ids reorded the highest of ll the tested enzymes nd totl phenol ompounds, while feruli nd benzoi id gve the lowest ones in this respet. Key words: Chemil induers, lef blight, lupine, indue resistne, oxidtive enzyme, phenol ontents. INTRODUCTION Lupine (Lupinus termis Forsk) is one of the oldest field rops grown in Egypt. Lupine is used s fodder rop nd green mnure for sndy nd poor soils to relim new lnds. The seeds of lupine ontin gret rtio of proteins, fibers nd rbohydrtes. It is lso used for medil nd industril purposes (Mknikiene, 2001). Lupine plnts re prone to mny diseses whih ttk the folir prts using downy mildew, rust nd lef blight diseses (Pulitz nd Cote, 1991; Yng nd Sweetinghm, 2002, Abdel-Monim et l., 2012). Suh diseses re destrutive nd use gret redution in seed yield nd qulity (Osmn et l., 1986; Muller et l., 1999). Hosford (1982) found tht form of Billus megterium uses white to very light tn blothing nd streking of whet (Tritium estivum L.) leves. The nme B. megterium de Bry 1884 pv. erelis pv. Nov. is proposed for this pthovr; nd the hlopthotype strin is WB 28. *Corresponding uthor. E-mil: fowzy_2008@yhoo.om Tel , Fx: There is no dequte hemil ontrol for suh disese. However, mngement prties, suh s the use of bteri-free seed, rottion nd ploughing of infested strw, hs not been studied until now in this disese. Limited suess hs been hieved with pplition of fungiides, suh s Bordeux mixture, opper oxyhloride, opper sulphte, nd ntibiotis (Settler, 1989 nd Shwrtz & Glvez, 1989). Cost, potentil hemil residues, nd resistne mong bteril strins re the known drwbks of hemil pplitions. Thus, the use of resistnt lupine ultivrs to Billus megterium is eonomilly nd tehnilly the most prtil method for effetive mngement of lef blight (Abdel-Monim, 2008). A new tehnology for plnt disese ontrol is bsed on the tivtion of the plnt s own defense system with the id of low moleulr weight syntheti moleules tht indue systemi quired resistne in plnts ginst wide rnge of mirobil pthogens. The systemi quired resistne (SAR) is n importnt omponent of plnt defense ginst diseses where initil infetion provides systemi resistne to subsequent infetion by

2 Abdel-Monim et l. 579 vriety of bteril, fungl nd virl pthogens (Gffney et l., 1993). Indued resistne depends on reognition of pthogen or stress by the plnt. This genertes sde of events, eventully leding to the expression of defense mehnisms, whih inlude physil brriers, metbolites nd proteins tht interfere with spred of the invding miroorgnism. In reent yers, lrge number of hemils, (viz., benzoi, feruli, oxli nd mli ids et.) whih mimi the funtion of the pthogen in indution of systemi quired resistne hve been reported (Ryls et l., 1996; Rgb et l., 2009). Indued resistne is expressed lolly t the site of infetion or systemilly t the sites remotely loted from the initil infetion. The indued systemi resistne (ISR) sensitizes the plnt to respond rpidly fter tretment. These responses inlude tivtion of peroxidse, polyphenoloxidse nd phenyllnine mmoni lyse nd phenols (Pnin et l., 2005; Rgb et l., 2009; Abdel Al et l., 2012). This reserh imed t studying the effet of benzoi, feruli, oxli nd mli ids s hemil induers on lupine plnts ginst lef blight disese nd identifying some ssoited biohemil hnges in treted plnts. MATERIAL AND METHODS In vitro effet of hemil induers on bteril growth In this study two pthogeni bteril isoltes (B2 nd B6) of B. megterium isolted from lupine plnts tht used lef blight previously isolted by Abdel-Monim (2008) nd four hemil induers, i.e., benzoi id (C 7 H 6 O 2 ), feluli id (C 10 H 10 O 4 ), mli id (C 4 H 6 O 5 ) nd oxli id (C 2 H 2 O 4 ) tht re known to funtion s ntioxidnts were used. Stok solutions ontining different hemil induers to be tested were prepred nd volumes of whih were inorported into nutrient gr medi to obtin onentrtions of 50, 100 nd 200 ppm. The hydrogen onentrtions of the prepred medi were djusted to ph 7 before utolving. Sterilized medi were then poured in sterile Petri dishes nd left to solidify. Pltes were then inoulted by sreening film of ell suspensions of seleted bteril isoltes onto the surfe of the nutrient gr medium. Pltes ontining nutrient gr medi without ny hemils were similrly inoulted with the sme bteril isoltes to be tken for omprison. All pltes were inubted for 48 h t 27 ºC. The inhibitory effet of the forementioned hemils on two bteril isoltes ws evluted fter 48 h by lulting the derese in olony numbers of the subjeted isoltes t ll onentrtions s ompred with the ontrol tretment (Grhm nd Leite, 2004). Effet of hemil induers on disese resistne development under greenhouse onditions The effiy of the forementioned hemil to be used s induer for resistne in lupine plnts ginst B. megterium (isoltes B2 nd B6) ws studied. This ws omplished by sprying folir lupine plnts (30-dy-old) with the hemil solutions t 50, 100 nd 200 ppm onentrtions. Treted plnts were then inoulted with two bteril isoltes fter 24 h. from plnt sprying with hemil induers. The inoulted plnts were overed with plsti bg for 24 hr. There were 3 replites for eh tretment nd eh ws sown with 8 seeds. Untreted plnts with ny hemil were similrly inoulted with the sme pthogens to be used for ontrol tretment. All experiment units were irrigted s needed nd mnure s usul with mmonium sulphte. The plnts were kept under dily observtion. After the inubtion period (15 dys), disese severity ws evluted using the disese index (DI) s follows: 0 = no symptoms, 1=1 9%, 2=10 24%, 3=25-49%, 4=50-74%, nd 5=75-100% of blighted lef re. The men of disese index (DI) nd disese severity index (DSI) for eh replite ws lulted by the formul suggested by Liu et l. (1995) s follows: d DSI 100 d mx n Where, d is the disese rting of eh plnt, d mx is the mximum disese rting, nd n is the totl number of plnts exmined in eh replite. Effet of lupine folir tretment with hemil induers on oxidtive enzymes Ativities of polyphenol oxidse, peroxidse nd phenyllnine mmoni lyse enzymes were studied in tissue extrts of lupine plnts treted with hemil induers nd untreted plnts. Preprtion of tissue extrts This ws rried out by grounding 0.5 g fresh tissue in preooled mortr ontining 10 ml solution of 10 mm potssium phosphte buffer, ph 7.0, 1% (W/V) polyvinylpyrrilidon (PVP), nd 0.1% (V/V) triton x 100 (Croft et l., 1990). The homogente ws entrifuged for 25 min t 8000 rpm nd 4 o C nd the superntnt ws used for ssy immeditely. Enzyme determintion Polyphenoloxidse tivity ws ssyed in the prepred extrts s reported by Jennings et l. (1969). The retion mixture onsisted of 0.5 ml tissue extrt, 1.5 ml of 25 mm itrte phosphte buffer, ph 6.0, plus 0.5 ml proline (5 mg proline / ml buffer solution) nd 0.5 ml tehol (2 mg tehol /ml buffer solution). Ativity of PPOX (define) ws determined olorimetrilly t 485 nm nd then expressed s enzyme unit mg -1 protein min -1 Peroxidse tivity ws determined with guiol s n enzyme substrte using modified proedure of Mehly nd Chne (1954) s desribes by Moershbher et l. (1988). The retion mixture onsisted of 3 ml of phosp-

3 580 Int. J. Agri. Si. phosphte buffer (0.1 M, ph 5.8) ontining guiol (40 mm) nd 200 µl rude enzymes extrt. The retion ws strted by dding 50 µl H 2 O 2 solution (250 µl H 2 O 2 /10 ml distilled wter). The tivity ws expressed s inrese in bsorbne t 470 nm mg -1 protein min -1 Phenyllnine mmoni lyse tivity ws mesured using modified method of Green et l. (1975). The retion mixture onsisted of 1.5 ml of L-phenyllnine (2 mm in borte buffer ph 8.8) nd 0.5 ml rude extrt. The retion mixture ws inubted t 40 o C for 2 h. The tivity ws expressed s inrese in bsorbne t 290 nm mg -1 protein min -1. The ontrol tretment ontined intivted enzyme by boiling for 10 min. All olorimetril mesurements were onduted using Milton Roy Spetrophotometer (Milton Roy spetroni 1201). Protein ssy Protein of extrted smples ws determined by the method of Brdford (1976) using bovine serum lbumin (BSA) s stndrd solution. Effet of lupine folir tretment with hemil induers on phenol ontents Totl phenols were ssyed by method of Mhdevn nd Sridhr (1982). Well expnded lupine leves were dethed, ir dried t lbortory, rushed nd extrted in 95% ethnol (1:20 w/v). One miroliter of Folin Demis regent ws dded to 1 ml extrt. After 3 min, 1 ml of sturted sodium rbonte solution ws dded to the retion mixture nd mixed thoroughly. After 1 h, the developing blue olour ws mesured olorimetrilly t 735 nm ginst blnk. Phenol onentrtion ws lulted using stndrd urve prepred with tehol. Sttistil nlysis All experiments were performed twie. Anlyses of vrine were rried out using MSTAT-C, 1991 progrm version Lest signifint differene (LSD) ws employed to test for signifint differene between tretments t P 0.05 (Gomez nd Gomez, 1984). RESULTS Effet of hemil induers on bteril growth in vitro Dt in Tble (1) show tht ll tested hemil induers hd no or little effet on growth of the two bteril isoltes B2 nd B6. However, the lowest tested onentrtion of some hemil induers (50 ppm) stimulted the growth of B. megterium isolte B2, but the highest onentrtion (200 ppm) exhibited little inhibited growth of both bteril isoltes. Effet of hemil induers on disese severity under greenhouse onditions All the tested hemils signifintly redued the disese severity used by either bteril isoltes B2 or B6 but with slight vrition (Tble 2). Benzoi nd oxli ids were the most tive ompounds retrding disese development espeilly when pplied through plnt spring method t higher onentrtion (200 ppm). The mens of disese severity in plnts treted with solutions of the forementioned ompounds (200 ppm) were 8.5 nd 10% for isolte B2 nd 11.5 nd 15.5%, for isolte B6, respetively. On the other hnd, feruli id nd mli id reoded the lowest ones in this respet. Effet of hemil induers on some oxidtive enzymes Chnges of the tivities of some oxidtive enzymes peroxidse, polyphenol oxidse, nd phenyllnine mmoni lyse were known to ply gret role in plnt defense mehnism ginst the invding mirobes ws investigted. Peroxidse tivity All the tested hemil induers inresed peroxidse tivity in folige tissues s ompred with untreted ones (Fig. 1). Oxli id nd mli id reorded the highest POX tivity, wheres reorded nd enzyme unit mg -1 protein min.-1 ompred with enzyme unit mg -1 protein min -1 in ontrol. While benzoi id nd feruli id reoded the lowest ones. Polyphenoloxidse tivity In generl, signifint inrese in the tivity of PPO (define) ws observed in lupine plnts treted with ny hemil induers more thn ontrol tretment (Fig. 2). PPO umulted more mrkedly in plnts treted with oxli nd mli id (0.524 nd enzyme unit mg -1 protein min -1 ) thn the plnts treted with feruli id nd benzoi id (0.430 nd enzyme unit mg -1 protein min -1 ). Phenyllnine mmoni lyse tivity Dt in Figure 3 show tht the levels of PAL tivity in

4 Abdel-Monim et l. 581 Tble 1. Effet of hemil induers on growth of two Billus megterium isoltes B2 nd B6 pthogeni to lupine folige. Tretments Benzoi id Feruli id Oxli id Mli id Conen. (ppm) B. megterium isoltes B2 B6 Number of olony / plte Inhibition (%) Number of olony / plte Inhibition (%) Men Men Men Men Control LSD t 0.05 for: Tretments (A) = 2.53 Conentrtions (B) = 1.79 Isoltes (C) = 1.23 Tble 2. Effet of hemil induers on lupine folige (v. Bldy) ginst to Billus megterium isoltes B2 nd B6 under greenhouse onditions Conen. (ppm) Disese severity used by B. megterium (%) Isolte B2 Isolte B6 Tretments Disese severity Protetion (%) Disese severity Protetion (%) (%) (%) Benzoi id Men Feruli id Men Oxli id Men Mli id Men Control LSD t 0.05 for: Tretments (A) = 1.32 Conentrtions (B) = 2.28 Isoltes (C) = 1.33 Intertion (AxBxC) = 5.22

5 582 Int. J. Agri. Si. b d Figure 1. Peroxidse (POX) tivity in extrts prepred from folir lupine tissues treted with hemil induers s ompression untreted tissue. Different letters indite signifint differenes mong tretments ording to lest signifint differene test (P = 0.05). b b Fig. 2. Polyphenol oxidse (PPOX) tivity in extrts prepred from folir lupine tissues treted with hemil induers s ompression untreted tissue. Different letters indite signifint differenes mong tretments ording to lest signifint differene test (P = 0.05). treted plnts with ny tested hemil induers were highly inresed thn in untreted plnts (ontrol). On the other hnd, dt show tht PAL tivity ws highly Totl phenol ontent The totl phenols were mesured in lupine plnts treted with hemil induers (Fig. 4). It is obvious tht ll hemil induers show signifint role to umultion signifint inrese in treted plnts with oxli id nd mli id thn plnts treted with feruli id nd benzoi id. of phenoli ompounds thn untreted plnts. However, oxli id nd mli id pre-treted lupine plnts hllenge inoulted with the pthogen showed rpid inrese in the umultion of phenol ompounds. While lupine plnts treted with feruli id nd benzoi id reorded the lowest inresed of umultion of totl phenol ompounds.

6 Abdel-Monim et l. 583 b d e Figure 3. Phenyllnine mmoni lyse (PAL) tivity in extrts prepred from folir lupine tissues treted with hemil induers s ompression untreted tissue. Different letters indite signifint differenes mong tretments ording to lest signifint differene test (P = 0.05). b b Figure 4. Totl phenol ompounds (TPC) ontent in extrts prepred from folir lupine tissues treted with hemil induers s ompression untreted tissue. Different letters indite signifint differenes mong tretments ording to lest signifint differene test (P = 0.05). DISCUSSION Indued resistne in plnts treted with hemils is now in urrent use in disese mngement. These ompounds re essible nd not hrmful to the environment. During the present work four hemil induer ompounds were tested for their potentility s resistne induers in the treted plnts. From whih four

7 584 Int. J. Agri. Si. ompounds nmely benzoi, feruli, mli nd oxli ids proved the most tive induers for disese resistne in lupine plnts ginst bteril lef blight used by Billus megterium pv. lupini. However, lupine plnts treted with solutions of the four hemil induers hieved gret protetion ginst this disese under greenhouse onditions. Suh resistnt induers ginst plnt diseses ws first reported by White (1979) who reported sliylte tretment to use drmti redution in TMV lesion number in tobo leves. Subsequently, mny reports onerning the use of these hemils s resistne induers hve been published (Abdel-Kreem et l., 2004; Obrdovi et l., 2005; Hill et l., 2006; Mggie et l., 2006; Hssn et l, 2007 ). Indued resistne hieved in plnts treted with induer ompounds ppers to be the result of severl mehnisms, inluding synthesis of protein inhibitors, relese of oxidtive enzymes nd inrese of totl phenol ompounds nd probbly other mehnisms (Niholson, 1992). In the present study, ssessment of the oxidtive enzymes tht is, peroxidse, polyphenyl oxidse nd phenyllnine mmoni lyse nd totl phenoli ompounds were performed in helthy folige lupine tissues treted with the forementioned four hemil induers t onentrtion 200 ppm. The results obtined showed gret inrese in ll tested enzymes in helthy plnt folige treted with the four hemils over those of the ontrol untreted plnts. This simple experiment rried out in helthy treted tissues prt of ny pthogen invsion lerly indites strit response of the inrese of oxidtive enzymes nd totl polyphenols with pplition of hemil induers ompounds nd this in turn uses the resistne to be indued. Polyphenoloxidse nd peroxidse re nturlly present in plnt tissues. Polyphenoloxidse onverts polyphenoli ompounds to the oxidized form quinines whih re thought to be inhibitory to pthogens invsion. Also it ws found tht higher peroxidse tivity ws usully more tive in resistnt ultivrs. On the other hnd, inrese in PAL enzyme due to the hemil tretment ws found to be orrelted with inrese in polyphenol umultion (Chen et l., 1993; Milosevi & Slusrenko, 1996 nd Fize et l., 2004). REFERENCES Abdel Al AE, Abd-El-Kder, Dwlt A, Khedr MA, Khlif MMA (2012). Indution of resistne in sesme plnts ginst hrol rot disese by some hemil induers. Zgzig J. Agri. Res., 39: Abdel-Kreem FI, El-Mougy NS, El-Gml NG, Ftouh YO (2004). Indution of resistne in sqush plnts ginst powdery mildew nd Alternri lef spot disese using hemil induers s protetive or therpeuti tretments. Egypt. J. Phytopthol., 32: Abdel-Monim MF (2008). Studies on some folir nd root lupine diseses with speil referene to indued resistne. Ph.D. thesis, F. of Agri., Mini Univ. Abdel-Moni MF, Gbr MR, El-Gntiry SM, Sht MN, El- Bn AA (2012). Billus megterium, new pthogen on lupine plnts in Egypt. J. Bteriol. Res. 4: Brdford MM (1976). A rpid nd sensitive method for the quntiztion of mirogrm quntities of protein utilizing the priniple of protein- dye bending. Anl. Biohem., 22: Chen Z, Silv H, Klessing D (1993). Ative oxygen speies in the indution of plnt systemi quired resistne by sliyli id. Siene, 262: Croft KPC, Voisey CR, Slusrenko AJ (1990). Mehnism of hypersensitive ell ollpse. Correltion of inresed lipoxygense tivity with membrne dmge in leves of Phseolus vulgris L. inoulted with n virulent re of Pseudomons syringe pv. phseoliol. Physiol. Mol. Plnd Pthol., 36: Fize M, Fize L, Koike N, Ishizk M, Ishii H (2004). Aibenzolr-S-methyl-indued resistne to Jpnese per sb is ssoited with potentition of multiple defense responses. Phytopthology, 94: Gffney T, Friedrih L, Vernooij B, Negrotto D, Nye G, Uknes S, Wrd E, Kessmnn H, Ryls J (1993). Requirement of sliyli-id for the indution of systemi quired-resistne. Siene, 261: Gomez KA, Gomez AA (1984). Sttistil proedures for Agriulturl Reserh. A. Lviley. Intersiene publition. New York, pp Grhm, JH, Leite Jr RP (2004). Lk of ontrol of itry nker by indued systemi resistne ompounds. Plnt Dis., 88: Green NE, Hdwiger LA, Grhm SC (1975). Phenyllnine mmoni lyse, tyrosine mmoni lyse nd lignin in whet inoulted with Erysiphe grmins f.sp. tritii. Phytopthology, 65: Hssn MEM, Abdel-Rhmn SS, El-Abbs IH, Mikhil MS (2007). Chnges in peroxidse tivity due to resistne indued ginst fb ben hoolte spot disese. Egypt. J. Phytopthol., (1): Hill AA, Nd MGA, Zky WH (2006). Indued resistne ginst Slerotini slerotiorum disese in some Umbelliferous mediinl plnts s possible nd effetive ontrol. Egypt. J. Phytopthol., 34: Hosford RM Jr (1982). White bloth in whet inited by Billus megterium pv. erelis. Phytopthology, 72: Jennings PH, Brmmn, BL, Zsheile FP (1969). Peroxidse nd polyphenol oxidse tivity ssoited with Helminthosporium lef spot of mize. Phytopthology, 59: Liu L, Kloepper JW, Tuzun S (1995). Introdution of systemi resistne in uumber ginst fusrium wilt by plnt growth-promoting rhizobteri. Plytopthology, 85: Mehly AC, Chne B (1954). The ssy of tlses nd peroxidse. In: methods of biohemil nlysis.

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