Int. J. Pure Appl. Si. Tehnol., 19(1) (2013), pp. 37-43 Interntionl Journl of Pure nd Applied Sienes nd Tehnology ISSN 2229-6107 Aville online t www.ijopst.in Reserh Pper Pthogeniity Evlution of Some Lol Isoltes of Entomopthogeni Fungi ginst the Nut Sle Inset Eulenium Tilie L. Lzgeen H. Assf 1, Feyroz R. Hssn 2, * nd Dilshd S. Ahmd 2 1 Generl Diretorte of Agriulture, Duhok, Kurdistn Region, Irq 2 Plnt Protetion Deprtment, Shool of Plnt Prodution, Fulty of Agriulture nd Forestry, Duhok University, Kurdistn Region, Irq * Corresponding uthor, e-mil: (feyrozrh77@yhoo.om) (Reeived: 9-8-13; Aepted: 18-9-13) Astrt: Lortory iossys were onduted to evlute the pthogeniity of Beuveri ssin, B. rongnirtii, Peilomyes frinosus nd Isri Jvnius s ioontrol gents ginst nut sle inset Eulenium tilie L. with onentrtion ( 10 7 ) spores / ml for eh fungus. Results indited tht the four fungi were pthogeni to nut sle inset nymph nd dults with vrying pilities ording to the fungus speies nd exposure durtion. The nymphs instrs were more suseptile thn the dults. The mortlity perentges inresed signifintly with I. jvnius nd B. ssin rehed 100% in oth femle nd mle nymphs fter 10 dys of tretment. Cumultive dult mortlity of 98.47% nd 65.71% ws hieved fter 12 dys from inoultion with I.jvnius nd P. frinuses spore suspension respetively. Keywords: Eulenium tilie, Entomopthogeni fungi, Soft sle insets. Introdution There re out 7800 desried speies of sle insets (Hemipter: Coide ) in the world. Most re importnt pests of griulture, forestry, fruit orhrds, hortiulture nd ornmentl plnts (Ben- Dov et.l. 2009). Akrwi, 2011 mentioned tht in Irq, speilly in Kurdistn region the soft sle inset (nut sle inset) Eulenium tilie L. is the most importnt pest of the fig trees nd uses series dmge y suking the trees sp. Also the ltter uthor dded tht the nut sle inset hve two nymphl instrs esides the pupl stge in se of mles. Shrm nd Dogn (1982) mentioned tht soft sle insets Eulenium tilie is slowly nd grdully uilding up it's popultion nd if left unheked my ttin the sttus of mjor pest.
Int. J. Pure Appl. Si. Tehnol., 19(1) (2013), 37-43 38 Sle insets serete wx from vriety of wx glnds, nd this wx forms protetive oting over the ody whih limits the ttk of nturl enemies nd ts s shield ginst insetiide sprys. Therefore, it is diffiult to ontrol sle insets y pplying hemil pestiides. In ddition, sprying pestiides uses environmentl pollution n e toxi to nturl enemies nd promotes insetiides resistne (Liu et.l. 2011). Biologil ontrol y pplying entomopthogeni fungi is potentilly importnt mens of restriting sle inset infesttions in oth rops nd in the wider nture eosystem. Beuveri speies hve wide host rnge nd, lthough these fungi egn to e used s iologil ontrol gents of soil pests, they re eing developed ginst phylloplne inset pests suh s white flies, thrips or lepidopterns (Mlsm et.l.1997). Reserh on fungl pthology of sle insets is sre proly euse of the rriers to infetion (wx lyer) nd the insets life yle. Evns nd prior (1990) stted tht Asomyetes nd their deuteronyetes nmorphs hve een desried s the most entomopthogeni fungi ttking sle insets (Coide). In Irq, espeilly in Kurdistn region, over the lst dede mny studies hve een done on the entomopthogeni fungi nd their role to ontrol insets. The most ommon inset speies found infested y entomopthogeni fungi in dormnt lotions in Irq ws Eurigster integrieps put. (sunn pest), the highest perentge of it's mortlity ws used y the fungus Beuveri ssin (Bls.) Vuill. ( Assf, 2007 ). Beuveri rongnirtii (So.) peth. ws isolted from sunn pest (Eurygster integrieps) for the first time in Irq (Assf et.l. 2011). Isri Jvnius ws isolted from Irqi soil for the first time t inset hierntion sites (Hssn et.l.2012). The im of this study ws to evlute the pthogeniity of lol isoltes of Beuveri ssin, B. rongnirtii, Peilomyes frinosus nd Isri jvnius to the nut sle inset Eulenium tilie L. under lortory ondition. 2- Mterils nd Methods 2-1 Fungi Inoulums Preprtion Isoltes from Beuveri ssin (BEG11), B. rongnirtii (BEG17), Peilomyes frinosus (PEG12) nd Isri jvnius (IEG20) were otined from the Myology nk / plnt protetion deprtment Fulty of Agriulture nd Forestry, Duhok University. The isoltes were identified in previous studies (Assf, 2007 ; Assf et.l 2011). Isoltes were grown on Potto Dextrose Agr (PDA) for 6-7 dys t 25±1C, nd onentrtion of 1 10 7 spore /ml of wter for eh fungi isolte ws prepred nd used s inoulum. 2-2 Pthogeniity of the Entompthogeni Fungi to Nut Sle Insets Eulenium tilie L. Fig rnhes nturlly infested with Eulenium tilie were olleted from fig trees in Akre region / Duhok government. The rnhes were trnsferred to the plnt protetion deprtment lortory, then divided to three groups; first one mostly infested y femle nymph, the seond mostly infested with mle nymphs nd third group ws mostly infested y the dults. Brnhes (3-5m) with out 30-50 sle insets for eh group were ut. These rnhes with insets were inoulted with 1 10 7 spore/ ml of wter (3ml/ replite) y new prfn spryer (50 ml pity) in ddition to ontrol tretment in four replites inside petri dish (9m) in dimeter, provided y moisture filter pper in ottom. Both sides of the rnhes were wrped up with otton wool soked in wter in order to keep them fresh (Hssn 2003). The experiments were done under lortory onditions t 25±1C nd 59.89% R.H. The mortlity perentge ws reorded fter 3, 5, 8 nd 10 dys, for nymphs nd fter 3,7 nd 12 dys for dults. Cumultive mortlity ounts otined from experiments were orreted for nturl morlity using Aott's formul (Aott, 1925). Dt were sttistilly nlyzed using omplete rndomized Design
Int. J. Pure Appl. Si. Tehnol., 19(1) (2013), 37-43 39 (CRD) with four replitions nd using SAS progrm. The mens were ompred using Dunn's multiple rng test t p 0.05. 3- Results nd Disussions 3-1 Infetive Symptoms Four speies of the entomopthogeni fungi, B. ssin, B. rongnirtii, P. frinosus nd I. jvnius were sujeted to initil ssy on nut sle inset's nymphs nd dults to selet fungi with high pthogeniity ginst nut sle inset Eulenium tilie under lortory onditions. After nut sle insets were inoulted with the four speies of entomopthogeni fungi, no ovious symptoms were found under disseting mirosope within the first 24h. After 48h, sprse myeli ppered ut fter 72h of inoultion, the myeli emerged oviously round the ody mrgin (fig1a ), nd 96h fter inoultion (fig1b) the myeli lmost overed the entire ody of the insets. The myeli hd depleted ll the nutrition within the inset's ody (fig.1c), fter 120h of inoultion leding to the deth of inset. I. jvnius ws the fstest fungus tht ppered effet on the nymphs nd dults, in ddition tht it s myeli ws grown on the rnhes lso (fig1d). Quesd-Morg et.l. (2006), explined tht the effiieny of entomopthogeni fungi egn lerly fter 48h from inoultion nd the hyphe penetrted the integument inside the trhe nd the epithelil nd epiderml ells. 3-2 Pthogeniity of the Entompthogeni Fungi to Nut Sle Insets Eulenium tilie L. Nymphs Pthogeniity studies in tle (1) indited tht the four speies of the entomopthogeni fungi, B. ssin, B. rongnirtii, P. frinosus nd I. jvnius were found to e pthogeni to nut sle nymphs, however there ws vrition in their infetivity ginst the trget inset. The perentge mortlity rnged etween 82.93-100% fter 8 dys on femle nymphs. Generlly the mortlity perentge used y the four fungi tested were signifintly higher thn the nturl mortlity in ontrol tretment. In mle nymphs, the perentge of mortlity ws 3.17% fter three dys of tretment y P. frinosus whih not differ signifintly from ontrol (1.02%), nd 100% fter eight dys of tretment y I. jvnius whih ws signifintly higher thn the other types of entomopthogeni fungi used, in exeption with B. ssin s 85.87% in the sme period. A1 A2
Int. J. Pure Appl. Si. Tehnol., 19(1) (2013), 37-43 40 B C D Fig. 1: Photogrphs of Eulenium tilie L. infeted y Beuveri ssin nd Isri Jvnius oserved under disseting mirosope (A1, A2) Dorsl view of the femle nd mle nymph of nut sle inset, showing the mrgin surrounded y myeli; (B) The B. ssin myeli developed nd overing the dying sle ody; (C) ventrl view, showing B. ssin hyphe minly olonizing the eggs under the shell; (D) Fig rnh overed y the I. jvnius myelim Tle (1): Pthogeniity of the four entomopthogeni fungi to the nut sle inset nymphs: Correted Mortlity % for Nymphs Fungus B. ssin Femle Nymphs Mle Nymphs Dys fter tretment 3 5 8 10 3 5 8 10 10.54 37.26 98.41 100 5.48 33.24 85.87 100.00
Int. J. Pure Appl. Si. Tehnol., 19(1) (2013), 37-43 41 B. rongnirtii 7.20 25.11 82.93 87.21 7.85 19.49 80.43 82.46 P. frinosus 4.83 38.99 88.41 88.93 3.17 14.96 82.00 80.46 I. Jvnius 5.10 58.35 100 100 13.94 45.40 100.00 100.00 Control 3.01 4.87 d 4.87 4.87 1.02 1.02 d 3.82 3.82 * Mens followed y ommon letter within the sme olumn re not signifintly different t the 5% level y DMRT The results showed tht the perentge of mortlity ws inresed with inresing the time of exposing, lso indited tht ll femles nd mles individuls were died in most tretments fter eight dys of exposing to I. jvnius. These results not found in se of B. rongnirtii nd P. frinosus exept with B. ssin in femle nymphs test tht mortlity perentge rehed 98.41%. The lowest perentge of mortlity reorded ws 3.17% in mle nymphs treted with P. frinosus fter three dys of tretment. These vrines mong the mortlity perentge with different fungi types greed with Assf et.l. 2011, nd Brroni nd Hjek,1994 who mentioned tht the effet of entomopthogeni fungi ws differ ording to the type of insets nd the spore suspension onentrtion tht used in eh tretment. There were no signifint differenes etween the mortlity perentge of femles nd mles nymphs in spite of tht the femle nymphs were more sensitive to entomopthogeni fungi I. jvnius, B. ssin, B. rongnirtii nd P. frinosus. Liu et. l. (2011) stted tht the sle insets serete wx from vriety of wx glnds, nd this wx forms protetive oting over the ody whih limits the ttk of nturl enemies nd ts s shield ginst insetiide sprys. 3-3 Pthogeniity of the Entompthogeni Fungi to Nut Sle Insets Eulenium tilie L. Adults: The iossys results on nut sle dults were indited tht mortlity perentge differed ording to the fungus speies. The mortlity of dults ws reorded fter 3, 7 nd 10 dys fter sprying with fungi suspensions. The mximum mortlity perentge reorded for I. jvnius (98.47%) followed y B. ssin (91.52%) ompred with 3.82% in ontrol fter 12 dys of tretment (tle 2). Fungus Tle (2): Pthogeniity of the four entomopthogeni fungi to the nut sle inset dults: Correted Mortlity % for Nymphs Dys fter tretment 3 7 12 B. ssin 1.46 59.97 91.52 B. rongnirtii 0.00 49.17 72.03 P. frinosus 0.00 32.62 65.71 I. Jvnius 2.23 73.90 98.47
Int. J. Pure Appl. Si. Tehnol., 19(1) (2013), 37-43 42 Control 1.70 1.70 d 3.82 * Mens followed y ommon letter within the sme olumn re not signifintly different t the 5% level ydmrt The results in t (2), showed tht B. rongnirtii nd P. frinosus hve lowest effet on the nut sle inset dults with mortlity perentge 72.03 nd 65.71%, respetively fter 12 dys of tretment. I. jvnius hve een reported in severl osions on Lepidopteron nd Coleoptern insets (Shimzu nd Tketuko 2010) s well s on insets in Hemipter (Sorselli et.l. 2008). Hssn et.l.(2012), isolted I. jvnius from diverse soils t inset hierntion sites for the first time in Irq nd proved it s pthogeniity on Hylopterus prumi nd Aphis pomi, under lortory onditions. There were signifint differenes etween the nut sle nymphs nd dults sensitivity to the tested fungi. The verge mortlity perentge/dy tht reorded in nymphs stge ws oviously more thn tht of the dults. Ezz et.l. (2008) stted tht pthogeniity of B. ssin to the soft sle inset Sisseti offee (Homopter: Coide) nymphl stge ws more thn tht of dult femles nd dded tht in the field tril B. ssin redued the popultion of different stges of S. offee. Referenes [1] D.S.A. Akrwi, Some eologil nd iologil studies of Ceroplstes rusi L. nd Eulenium tilie L. (Homopter: Coide) on fig trees in Akre region, M.S. Thesis, College of Agriulture, Slhddin University, (2011), 72. [2] L.H.A. Assf, Eologil study nd evlution of tivity of Beuveri ssin (Bls.) Vuill. nd Peilomyes frinosus (Diks ex Fr.) on some iologil spets of sunn pest on Whet, Ph.D. Thesis, College of Agriulture nd Forestry, Mosul University, (2007), 231. [3] L.H. Assf, R.A. Hleem nd S.K. Aullh, Assoition of entomopthogeni nd other opportunisti fungi with insets in dormnt lotions, Jordn Journl of Biologil Sienes, 4(2) (2011), 87-92. [4] L. Asensio, L.V. Lopez-Llor nd J.A. Lopez-Jimenez, Use of light, snning eletron mirosopy nd iossys to evlute prsitism y entomopthogeni fungi of the red sle inset of plms (Phoenioous mrltti Ckll., 1899), Miron, 36(2) (2005), 169-175. [5] H. Askry, N. Benhmou nd J. Brodeur, Ultrstruturl nd ytohemil hrteriztion of phid invsion y the hyphomyete Vertiillium lenii, J. of Invert-Pthol, 74(1) (1999), 1-13. [6] Y. Ben-Dov, D.R. Miller nd G.A.P. Gison, SleNet: A dtse of the sle insets of the world, United Sttes Deprtment of Agriulture (USDA), (2009), http://www.sel.r.usd.gov/slenet/slenet.htm. [7] H.C. Evns nd C. Prior, Entomopthogeni fungi, In: D. Rosen (Ed.), The Armoured Sle Insets, their Biology, Nturl Enemies nd Control (Vol. B), Elsevier Siene Pulishers, Amsterdm, The Netherlnds, 1990. [8] N.A. Ezz, I.A. Hemeid, H.A. El-Shrwy, O.M.N.I. El-Shn nd E.I. Helmy, Evlution of the entomopthogeni fungus Beuveri ssin (Blsmo) s ioontrol gent ginst the soft sle inset, Sisseti offee (Wlker) (Homopter: Coide), Egyptin Journl of Biologil Control, 18(1) (2008), 75-80. [9] F.R. Hssn, Studies in poplr lef eetle melsom (=Chrysomel) populi L. (Chrysomelide: Coleopter) in Dohuk region, M. S. Thesis, College of Agriulture, Duhok University, (2003), 83. [10] W.A. Hssn, L.H. Assf nd S.K. Adullh, Ourrene of entomopthogeni nd other opportunisti fungi in soil olleted from inset hierntion sites nd evlution of their entomopthogeni potentil, Bulletin Irq Nturl History Museum (in Press), (2012). [11] W. Liu, Y. Xie, J. Xue, Y. Zhng nd X. Zhng, Ultrstruturl nd ytohemil hrteriztion of rown soft sle Cous hesperidum (Hemipter: Coide) infeted y the Leniillium lenii (Asomyot: Hyporeles), Miron, 42(2011), 71-79.
Int. J. Pure Appl. Si. Tehnol., 19(1) (2013), 37-43 43 [12] O. Mlsm, M. Filin, H. Ru diger nd D. Berg, Fungl insetiides, In: T. Anke (Ed.), Fungl Biotehnology, Chpmn & Hll, 1997. [13] E. Quesd-Morg, J.A. Crrso-Diz nd C. Sntigo-Alvrez, Insetiidl nd ntifeedout tivities of proteins rereted y entomopthogeni fungi ginst Spodopter littorlis (Lepidopter: Notuide), J. Applied Entomol., 130(2006), 442-452. [14] J.P. Shrm nd G.S. Dogr, Biology nd sesonl history of plum sle, Eulenium sp.? Tilie (L.) in Himhl Prdesh, Biull. Ent., 23(1982), 74-84.