MANAGEMENT OF FUSARIUM OXYSPORUM F. SP. CAPSICI BY LEAF EXTRACT OF EUCALYPTUS CITRIODORA

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1 Pk. J. Bot., 47(3): , MANAGEMENT OF FUSARIUM OXYSPORUM F. SP. CAPSICI BY LEAF EXTRACT OF EUCALYPTUS CITRIODORA SHAZIA SHAFIQUE *, MUHAMMAD ASIF AND SOBIYA SHAFIQUE Institute of Agriculturl Sciences, University of the Punjb, Lhore 54590, Pkistn * Corresponding uthor s e-mil: shzi.igs@pu.edu.pk Abstrct Fusrium wilt of chili (Cpsicum nnuum L.) is n importnt disese in Pkistn tht cuses significnt yield losses. In the present study, pthogenicity test ws conducted using four strins of Fusrium oxysporum f.sp. cpsici nd ten chili vrieties. It reveled tht strin B ws the most pthogenic strin nd vriety sky red ws the most susceptible while vriety Anchl ws the most resistnt ginst F. oxysporum strin B. Antifungl biossys were conducted to find out ntimycotic effect of extrcts of fruit, brk nd leves of Euclyptus citriodor (Hook.) ginst F. oxysporum. Ten concentrtions (0, 1.0, 1.5, 2.0, 2.5 nd 5%) of methnolic extrcts of ech plnt prt were employed ginst the trget pthogen. Lef extrct imprted the mximum (up to 98%) nd significnt suppression in fungl growth while fruit nd brk extrcts proved less effective exhibiting only 50 60% reduction in fungl mycelil growth. The work concludes tht methnolic extrct of leves of E. citriodor hve potentil to restrin the disstrous effects of the pthogenic fungus s the plnt extrcts of Euclyptus conferred bout 85% disese control in chilli plnts with significntly high intensity of defense relted enzymes under pthogenic stress. Key words: Antifungl biossys, Cpsicum nnuum, Fusrium oxysporum, Euclyptus citriodor, Methnolic extrct. Introduction Chili (Cpsicum nnuum L.) is n importnt vegetble nd spice crop belonging to the nightshde fmily Solncee, cultivted in bout 130 countries, mostly in the tropicl regions of the world (Anonymous, 2003). These re full of vitmin C nd vitmin A contining bet-crotenoids which re potent ntioxidnt. It is very high in potssium, mgnesium, clcium, nd iron (Serr et l., 2002). Mjor producers of chili include Asi, Ltin Americ, Afric, Europe, nd North Americ (Anonymous, 2003b). In Pkistn; chili production hs gret contribution in its economy (Khlid et l., 2000). Its totl re under cultivtion is bout hectre, with totl nnul production of 115,000 tons (Govt. of Pk., 2001). Worldwide yield of chili is now decresed up to 50%. There re mny biotic nd biotic fctors which re decresing chili production. Among these fctors the most devstting re fungl diseses which lower the yield per cre nnully. Chili plnts re lible to ttck by severl fungl diseses mong which dmping-off, root rots nd wilt re widespred (Abd, 1994). The chili wilt hs been found s the most frequently encountered disese problem (Skggs et l., 2000; Siddiqui & Akhtr, 2007). Wilt of chili is fungl disese cused by the Fusrium oxysporum lso known s Fusrium wilt. In Pkistn Fusrium wilt of chili cuses 15 to 20% yield losses in dry res during the lst few yers (Siddiqui & Akhtr, 2007). In order to reduce the economic loss nd to protect the crop from pthogen different control methods re used. One very common method is to use resistnt cultivr. Besides, soil fumignts nd solriztion re prcticed to minimize pthogens in soil. Crbendzim nd Bordeux mixture re lso the most commonly used chemicls but incresing use of pesticides in pst hs led to severl problems, such s environmentl degrdtion, helth hzrds, pest resistnce nd decrese in popultion of beneficil insects (Groenewld, 2005). Thus it is universl requisite to implement the prctice of sustinble griculture, using strtegies tht re environment-friendly nd less dmging to soil nd wter resources. Biologicl nd environmentl innocuous substitutes, such s biocontrol gents, nturl plnt metbolites, nd culturl methods, re being explored for possible use in integrted disese mngement pltforms nd to improve the foreign exchnge on the bsis of the efficcy of plnt extrct. Plnts extrcts nd essentil oils show ntifungl ctivity ginst lrge no of fungl diseses (Shfique & Shfique, 2012; Zheer et l., 2012; Jvid & Iqbl, 2014; Jvid & Ruf, 2015). The plnts re rich in wide vriety of secondry metbolites such s tnnins, terpenoids, lkloids, flvonoids, glycosides, etc., which hve been found In vitro to hve ntimicrobil properties (Dhnuker et l., 2000). Numerous studies conducted in Pkistn reveled wide spectrum prospects of using extrcts of plnts for biologicl control of pthogenic fungi (Bjw et l., 2001; Ahmd & Abdelglie, 2005; Shfique & Shfique 2012; Zheer et l., 2012). By keeping ll these intriccies in view it is suggested tht biologicl control using plnt extrcts provides n effective nd overll fungl mngement progrm, nd represents n lterntive to relince on fungicides. Therefore, the present study ws conducted to find the effective control of Fusrium wilt of chili through Euclptus citriodor fruit, brk nd leves methnol extrcts. Euclyptus Citriodor lso clled lemon-scented euclyptus is diverse genus of fmily Myrtcee. Of the more thn 600 species tht comprise this genus, most re endemic to Austrli (Mrzoug et l., 2011). The ntives of Austrli trditionlly use euclyptus leves to hel wounds nd fungl infections. In recent times, medicinl ssets of the extrcts hve been engrossed. In supplement, the trees hve been known to fbricte severl nturl substnces hving ntgonistic ctivities ginst severl microorgnisms (Lee, 2007).

2 1178 SHAZIA SHAFIQUE ET AL., Mteril nd Method Selection of Chili Vrieties: A totl of 10 chili vrieties nmely Golden hot, Revivl, Hot chili, Sim hot, Sky red, P6, Neelm, Sitr, Anchl nd Kundri were selected which were cquired from NARC, nd Fruit nd Vegetbles Mrket of Allm Iqbl town, Lhore, Pkistn. Procurement nd mintennce of cultures: Four pure cultures of Fusrium oxysproum f.sp. cpsici were isolted from the disesed chilli plnts on PCNB medium nd lbeled s F. oxysproum A, F. oxysproum B, F. oxysproum C nd F. oxysproum D. They were mintined on 2% mlt extrct gr (MEA) medium, sub cultured monthly nd stored t 4ºC. Pthogenicity Test: Pthogenicity test ws performed ccording to modified procedure of Grogn et l. (1975). Pots of 8cm dimeter nd 11cm deep were filled with 350 g soil per pot tht ws sterilized t 45 C for 24 h in hot ir oven. Conidil suspension of 4 x 10 5 conidi ml -1 ws prepred by using the protocol of French & Hebert (1982). Three seeds of selected vrieties were sown in pots nd thinned to one plnt per pot fter 20 dys. One month old chili plnts were spryed with djusted spore suspension. Control received sme quntity of distilled wter. The plnts were kept covered with polythene bgs for 48 hours. Greenhouse temperture rnged from 28 C to 30 C nd wtered when required. Ech tretment ws replicted five times. Disese rting scle ws developed for the estimtion of disese severity. Disese incidence ws observed s the symptoms ppered on the plnt nd disese index ws clculted using formul: Are of Plnt Prt Affected DiseseSeverity = x 100 Totl Are Disese Index = Number of plnts in Prticulr Ctegory Totl Number of Plnts x 100 The isolte cusing mximum disese severity ws selected s the Most Virulent/pthogenic Isolte nd selected for further experimenttion. Moreover, chili vrieties were lso evluted for their individul resistnce ginst fungl pthogen on the bsis of disese index nd screened s the representtive ones for subsequent studies. Phytochemicl Control of the Most Pthogenic Isolte. Synthesis of methnolic plnt extrct: Fresh smples of leves, brk nd fruits of E. citriodor were collected from Quid-e-Azm cmpus, University of the Punjb, Lhore in December, The plnt mterils were wshed, dried nd grinded to mke powder. Then methnolic extrct of ech prt ws prepred by using the protocol of Jvid & Munir (2012). Hundred grms (100 g) of dried powder of ech plnt prt ws soked in 1000 ml methnol for one month. After 1 month mterils were filtered through muslin cloth followed by filter pper filtrtion nd evported under vccum in rotry evportor t 45 C. Then methnolic extrcts (16.2 g) of ech prt were dissolved in sterilized wter to prepre 60 ml of stock solutions of ech plnt prt seprtely. Antifungl biossys: Aqueous solution of ech plnt prt of methnolic residues ws prepred in sterilized distilled wter to mke the finl concentrtion of 0.6 g ml -1. Methnol residues were first dissolved in dimethoxy sulfoxide (DMSO) nd then sterilized distilled wter ws dded to obtin the finl concentrtion of 0.6 g per ml. To check the bioctivity of extrct 10 concentrtions viz., 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 nd 5.0% of the orgnic solvent residue solution were formed. In 100 ml flsks, 55 ml mlt extrct broth ws sterilized by utoclving t 121 C nd cooled t room temperture. Ten concentrtions viz. 5.0, 4.5, 4.0, 3.5, 3.0, 2.5, 2.0, 1.5 nd 1.0 g 100 ml -1 were prepred by dding 5.0, 4.5, 4.0, 3.5, 3.0, 2.5, 2.0, 1.5 nd 1.0 ml stock solutions, nd 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 nd 4.0 ml sterilized distilled wter, respectively to ech flsk to mke the totl volume up to 60 ml, while control received 5 ml of distilled wter. The 60 ml medium of ech tretment ws divided into four equl potions in 100 ml conicl flsks to serve s replictes. An inoculum of 0.3 ml of djusted conidil suspension (4 x 10 5 conidi ml -1 ) ws given to ll tretments nd incubted t 28±2 C for 10 dys. There were four replictes of ech tretment (Jvid & Munir, 2012). Dried mycelil biomss of ll tretments ws determined fter 10 dys ccording to (Bjw et l., 2006). Percentge of fungl biomss inhibition in response to ll concentrtions of the extrcts over control ws deliberted by pplying the following formul: Growth in control - Growth in tretme nt Growth inhibition (%) = 100 Growth in control Sttisticl nlysis: The differences mong the tretment mens were compred by using Duncn s Multiple Rnge Test (DMRT) (Steel & Torrie, 1980). All the dt were nlyzed by nlysis of vrince (ANOVA) using computer softwre COSTAT followed by Duncn s Multiple Rnge (DMR) test to seprte the tretment mens. Results Pthogenicity ssy: Pthogenicity test ws executed using one month old chili plnts to determine the pthogenicity of 4 strins of Fusrium oxysporum on ten different vrieties of chili plnt. Infection nd visible chrcteristic symptoms were evident fter 10 dys of inocultion (Tbles 1 & 2). Symptoms of wilt disese observed on different vrieties of chili plnts were found to be vried with different strins of F. oxysporum (Fig. 1). Symptoms were strted to develop from the lef tips nd long the mrgins of the lef petiole. Strin B of F. oxysporum ws proved to be the most pthogenic s it induced symptoms within 7 dys. Initilly the plnts displyed yellowing on the leves tht ws turned into chlorosis nd necrosis; slowly it ws converted to wilting nd eventully the deth of the whole plnt. The severely infected plnts showed the stunted growth. Dt nlysis reveled tht ll the strins of F. oxysporum demonstrted mximum disese severity in sky red. On the other hnd

3 MANAGEMENT OF FUSARIUM OXYSPORUM LEAF EXTRACT 1179 Sitr, Anchl nd Kundri exhibited less severity (30-35%) ginst the sme pthogen (Fig. 1). F. oxysporum strin B induced mximum disese index of bout 50% in Sky red (Fig. 2). By the evlution of percentge infected re, the vriety Sky red hving the ccession ws proved the most susceptible nd Anchl ws most resistnt ginst Fusrium oxysporum. Tble 1. Disese rting scle exhibiting disese severity. Percentge infected re score Sttus % 1-2 Highly resistnt % 3-4 Resistnt % 5-6 Modertely susceptible % 7-8 Susceptible % 9-10 Highly susceptible Thus the disese progress curve ws plotted for two chili vrieties i.e., Sky red nd Anchl (Fig. 3). This disese progress curve scertined Sky red (vriety ) s the most susceptible nd Anchl s the most resistnt vriety. As Sky red exhibited mximum percentge disese incidence so this susceptible vriety ws used for further lb experimenttion. Phytochemicl control of Fusrium oxysporum: F. oxysporum strin B induced mximum pthogenicity in chili plnts so it ws selected for subsequent studies. The methnol extrcts of leves, brk nd fruit of E. citriodor were used to evlute the ntifungl potentil ginst the trget pthogen. Antifungl ctivity of lef extrct of E. citriodor: The In vitro ntifungl potentil of methnolic Lef Extrct of E. citriodor ws recorded ginst F. oxysporum strin B. The employed concentrtions of lef extrct significntly decresed the fungl biomss s compred to control tretment. The pttern of grdully lower production of biomss in response to incresing concentrtions of methnolic extrct ws reltively more shrp (98 to 99%) in 4-5% concentrtions. Arrest in biomss production ws lest in the lowest concentrtion (1%) but it ws still significnt in comprison to control s it induced bout 47% suppression in biomss. Among ll the employed concentrtions, the most promising effect on the growth of fungl biomss ws observed t the highest concentrtion (5%) s it cused mximum reduction of 99% in fungl biomss (Fig. 4). Tble 2. Pictoril representtion of disese rting scle on the bsis of symptom development. Key scle Disese description Disese severity 0 No symptom 0 1 Yellowing strt t the mrgin of the leves 10 2 Wilting will strt on the whole chili plnt 30 3 Plnt show reduced vigor nd growth 50 4 Whole plnt is infected necrosis on ll the tissues 70 5 Complete deth nd diebck of the plnt 100

4 1180 SHAZIA SHAFIQUE ET AL., Disese severity Fig. 1. Disese severity of different strins of Fusrium oxysporum ginst the selected chili vrieties. Verticl brs indicte stndrd error of mens of three replictes. Vlues with different letters show significnt difference (p 0.05) s determined by Duncn s multiple rnge (DMR) test. cd b b cd bc bc Fig. 2. Disese index of selected Fusrium oxysporum strin B with reference to ll chili vrieties. Verticl brs indicte stndrd error of mens of three replictes. Vlues with different letters show significnt difference (p 0.05) s determined by Duncn s multiple rnge (DMR) test. Fig. 4. Effect of different concentrtions of methnolic lef extrct of Euclyptus citriodor on growth of Fusrium oxysporum strin B. Verticl brs indicte stndrd error of mens of three replictes. Vlues with different letters show significnt difference (p 0.05) s determined by Duncn s multiple rnge (DMR) test. Fig. 3. Disese progress curve of the most resistnt nd the most susceptible chili vrieties. Fig. 5. Effect of different concentrtions of methnolic brk extrct of Euclyptus citriodor on growth of Fusrium oxysporum strin B. Verticl brs indicte stndrd error of mens of three replictes. Vlues with different letters show significnt difference (p 0.05) s determined by Duncn s multiple rnge (DMR) test.

5 MANAGEMENT OF FUSARIUM OXYSPORUM LEAF EXTRACT 1181 Fig. 6. Effect of different concentrtions of methnolic fruit extrct of Euclyptus citriodor on growth of Fusrium oxysporum strin B. Verticl brs indicte stndrd error of mens of three replictes. Vlues with different letters show significnt difference (p 0.05) s determined by Duncn s multiple rnge (DMR) test. Antifungl ctivity of brk extrct of E. citriodor: The growth ssys of F. oxysporum strin B in terms of dry biomss production ginst exposure to methnolic brk extrct of E. citriodor reveled n excessive interference s presented in Fig. 5. Control tretment ws used to compre the difference between the tretments. It ws evident from the results tht with incresing the extrct concentrtion; the significnt reduction in biomss ws more visible with n errtic pttern. The 1% concentrtion depicted less negtive effect on biomss inhibition (12.63% decline) but significntly lower thn control. The tretments % were comprtively insignificnt mong themselves s these inclined bout 20-36% cesstion in fungl growth. Among ll the concentrtions 4.5 nd 5% concentrtions were proved to be the most effective ginst the fungl pthogen s these persuded round 57.8 nd 60% subjugtion in dry biomss production, respectively. Antifungl ctivity of fruit extrct of E. citriodor: In generl the methnolic fruit extrct of E. citriodor ws found to be less effective in terms of its ntimycotic potentil ginst test fungl growth thn lef extrcts. A vrible response of dry biomss production in F. oxysporum strin B ws recorded to methnol fruit extrct of E. citriodor in different concentrtions (Fig. 6). All the tretments presented piercing nd significnt imprisonment in dry biomss production s compred to control but the suppression rte mong the concentrtions ws negligible. There ws bout % inhibition in fungl growth due to ll the employed concentrtions. Among the three plnt prts of E. citriodor tested, performnce of lef extrct ws the best in suppressing the growth of trget pthogen followed by fruit nd brk extrcts. Discussion In the present study, pthogenecity test ws performed to determine the pthogenic potentil of four strins of F. oxysporum. The results depicted tht ll the strins hd the potency to induce wilt s ll of the strins cused more or less typicl symptoms on plnts. Among these, F. oxysporum strin B induced mximum chrcteristic symptoms of wilt nd plnts died within few dys of inocultion. These results were found in greement with the work conducted by Mhmood (2010) who reported the sme trend of disese development in tomto by A. lternt. To evlute the ntifungl potentil of methnol extrcts of leves, brk nd fruit of E. citriodor, ten different concentrtions of extrct of ech plnt prt exhibited mrkedly vrible ntgonistic ctivities ginst the pthogen In vitro conditions. The reltive intensity of this effect however vried with the extrct type, s well s the prticulr concentrtion of the extrct employed. There re other evidences showing similr effects ginst known pthogenic fungi (Khn et l., 1998; Shfique & Shfique, 2008; Brk et l., 2011), which further confirm the presence of ntifungl gents in the test species. Similrly, Shfique et l. (2006) reported 60% reduction in incidence of Alternri lternt on whet due to queous lef extrct of C. lbum. In contemporry studies, Bjw et l. (2007) evluted ntifungl ctivity of queous nd n-hexne shoot extrcts of Aloe ver ginst few pthogenic species viz., Alternri lternt, A. citri nd A. tenuissim. They reported tht the inhibitory effect ws found to be vrible with the pplied concentrtions nd cused significnt inhibition in biomss production of test fungi. Presently, mong the three plnt prts of E. citriodor tested, lef extrct ws evidenced s the best in subduing the growth of F. oxysporum up to 98 to 99% followed by fruit nd brk extrcts. The difference in the fungicidl ctivity of the extrcts of different prts of E. citriodor could probbly be due to the different types nd/or different mounts of chemicl constituents in the vried types of extrcts. A highperformnce liquid chromtogrphy nlysis by Kowthr et l. (2011) showed tht the following cids; cffeic, ferulic, coumric, benzoic, vnelic, chlorogenic, nd hydroxybenzoic were present in Euclyptus extrcts which my be responsible for the fungicidl ctivity of the plnt. Another phytochemicl nlysis of the lef extrcts reveled the presence of nthrquinones, crdic glycosides, flvonoids, sponins nd tnnins (Bhgt, 2012). Among the vrious components of euclyptus, 1, 8-cineole is the most importnt one nd, in fct, chrcteristic compound of the genus Euclyptus, nd is lrgely responsible for vriety of its pesticide properties (Duke, 2004). Su et l. (2006) demonstrted the ntifungl ctivity of essentil oils from Euclyptus grndis, E. cmldulensis, nd E. citriodor ginst the mildew nd wood rot fungi viz. Aspergillus clvtus, A. niger, Chetomium globosum, Cldosporium cldosporioides, Myrothecium verrucri, Penicillium citrinum, Trichoderm viride, Trmetes versicolor, Phnerochete chrysosporium, Pheolus schweinitzii nd Lenzites sulphureus. Bsed on the study, the uthors opined tht essentil oil from E. citriodor could be n excellent choice s wood preservtive nd preservtion of lether goods nd wood rtifcts. In similr kind of reserch work conducted by Suleimn & coworkers (2008) they mentioned tht vegettive growth of Fusrium sp. t different concentrtions of Senn lt extrct were generlly low compred to control. All the lef extrcts were effective in the reduction of the incidence of soil-borne pthogenic fungi tested except extrcts from spermint tht did not give

6 1182 SHAZIA SHAFIQUE ET AL., significnt reduction of the fungi when compred with untreted control seedlings. This result indictes tht the lef extrcts probbly hve some fungicidl properties tht inhibit the growth of the soil-borne fungi. Thus the evidence obtined from present study dvocted tht plnt metbolites of Euclyptus reduced the growth of pthogen up to 98% nd cted s defense wepons s 85% disese control ws observed in protective ssys. Further the high intensity of defense relted enzymes indicted higher expression of plnt defense metbolism under pthogenic stress. References Abd, K.A Fungi ssocited with root rot of pepper nd some fctors ffecting disese incidence. Proc. 7 Th Conf. Phytopthol. Ghzz, Ahmed, S.M. nd S.A.M. Abdelgleil Antifungl ctivity of extrcts nd sesquiterpene lctones from Mgnoli grndiflor L. (Mgnolicee). Int. J. Agric. Biol., 4: Anonymous Vegetbles: Annul summry. Online.United Sttes Deprtment of Agriculture, Ntionl Agriculturl Sttistics Service, Vg 1-2(03). 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