IDENTIFICATION AND PATHOGENICITY OF Botryosphaeria parva ASSOCIATED WITH GRAPEVINE DECLINE IN KURDISTAN REGION IRAQ

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1 ACTA AGROBOTANICA Vol. 65 (1): IDENTIFICATION AND PATHOGENICITY OF Botryospheri prv ASSOCIATED WITH GRAPEVINE DECLINE IN KURDISTAN REGION IRAQ 1 Red A. Hleem, 2 Smir K. Abdullh nd 3 Jldet M.S. Jubrel 1 Deprtment of Plnt Protection, Fculty of Agriculture nd Forestry, University of Duhok, Irq 2 Deprtment of Biology, Fculty of Science, University of Zkho, Irq 3 Scientific Reserch Center, University of Duhok, Irq e-mil: smer_bdlh@yhoo.com Received: Abstrct During survey on fungi ssocited with decline symptoms on grpevine cultivrs growing in Kurdistn region of Irq, severl isoltes of Botryospheri species were encountered. All isoltes were identified s Botryospheri prv Pennycook nd Smuels. Pthogenicity test for isolte DKI 1 ws performed on two cultivrs, Tefi nd Rshmew. Under greenhouse conditions, one-yer grpe rooted cuttings were inoculted with the pthogen isolte by two methods, injecting the spore suspension into the green shoots nd by rtificil inocultion of wounded shoots with mycelil mt. The highest cnker length (15.0 mm) ws produced fter four months on the shoots of the Tife cultivr rtificilly inoculted with mycelil mt of the pthogen. Under field conditions, two methods of inocultion were dopted, wounding the green shoots nd drilling hole in the rms of mture vine, followed by inocultion with mycelil mt. The highest cnker length (11.17 mm) ws obtined fter 5 months on wounded shoots of the Rshmew cultivr nd with significnt difference from the Tefi cultivr. The pthogen cused reduction in fresh nd dry weight of green shoots nd roots compred with the non-inoculted control. This is the first report on B. prv in Irq. Key words: Botryospheri prv, grpevine, Irq INTRODUCTION Botryospheri spp., the cusl gents of blck ded rm, hve lso been ssocited with young grpevine showing decline symptoms (Lrignon et l. 2001; Phillips, 2002; Vn Niekerk et l. 2004, 2006). Recent hve studies identified Botryospheri species s importnt grpevine pthogens cpble to cuse stem cnker, wood streking, cne bleching, nd bud necrosis in ll mjor viticulture regions throughout the world (Vn Niekerk et l. 2004; Tylor et l. 2005; Urbez-Torres et l. 2006). Botryospheri species hve been isolted from grpevine from cross the world. This study is the first registrtion of Botryospheri spp. in Irq. A symptom tht is often ssocited with Botryospheri species is bud mortlity, which leds directly to yield reduction. Bud mortlity is often the result of young shoots being infected by the fungus erly in the seson (Phillips, 1998; Lrignon et l. 2001). A symptom closely resembling the wedge-shped necrosis is rch-shped lesions leding to brown internl necrosis, which cn be seen in cross-sectioned rms nd trunks (C stillo-pndo et l. 2001). Vn Niekerk et l. (2004) conducted in vitro nd in vivo pthogenicity studies of 21 isoltes, representing eight different species of Botryospheri, on green shoots nd mture cnes. The results reveled tht B.ustrlis, B.prv, B.ribis nd B.stevensii were mong the most virulent species. All species were successfully re-isolted from the respective lesions nd thus should be considered s potentil pthogens of grpevine. The im of this work ws to isolte, identify nd determine the pthogenicity of Botryospheri prv on grpevine cultivrs with different methods of inocultion under greenhouse nd field conditions of Irq. MATERIALS AND METHODS Isoltion of the pthogen Smples from different loctions of Duhok governorte were collected. Botryospheri species were isolted from complete vine tissues, from cne (brk

2 72 Red A. Hleem, Smir K. Abdullh, Jldet M.S. Jubrel nd wood), bud, trunk or rm (brk nd wood), leves, nd berries. Plnt tissue ws surfce-sterilized by plcing in 70% ethnol for 30 s, 1% NOCl for 1 min nd gin in 70% ethnol for 30 s before drying under lminr-flow hood s described by Vn Niekerk et l. (2004). Smll pieces of tissue were tken from the mrgin between necrotic nd pprently helthy tissue nd plted onto 2% Potto Dextrose Agr (PDA) with 0.25 mg/ml chlormphenicol. Hyphe growing out from the tissue pieces were subcultured onto fresh PDA pltes nd incubted t 25±2 o C (Vn Niekerk et l. 2004). Seven isoltes from different loctions were used for morphologicl chrcteriztion. In order to enhnce sporultion, cultures were plced on 2% wter gr contining utoclved grpevine wood chips nd incubted t 25 o C under intermittent light (12 h) (Luque et l. 2005). Isoltes were exmined weekly for formtion of pycnidi nd conidi. Isolted fungi were grown on PDA nd MEA t 25 o C in drkness or under NUV + fluores cent illumintion with 12-h photoperiod (Philips TLD18W/33) for dys until culture sporulted. Botryospheri isoltes were identified bsed on chrcters of the nmorph in culture nd on nturl substrtes (Phil- lips, 2002; Denmn et l. 2000; Crous et l. 2006; Urbez-Torres et l. 2006). Pthogenicity test in the greenhouse Rooted cuttings of two Vitis vinjfer L. cultivrs, Reshmew nd Tefi, were plnted in pots contining 20 kg of sterilized sndy lom soil (3:1) in greenhouse. When they were well estblished nd the shoots bore more thn five nodes, the fourth internode from the tip of ech shoot ws inoculted by B. prv (isolte DKI 1). At the time of inocultion, these internodes were green but no longer succulent (Philips, 1998). The green shoots were inoculted using two methods: 1) by wounding shoots (8 mm) with sterilized shrp blde nd colonized gr plug, bout 4 mm in dimeter cut from the mrgin of seven-dy-old culture of B. prv, ws plced in the wound nd covered with prfilm (Vn Niekerk et l. 2004); 2) by injecting the spore suspension ( spores ml -1 ) to the green shoot. For the control tretment, the shoots were injured with no inocultion. The length of cnker ws mesured fter 2 nd 4 months fter inocultion. Fresh nd dry weight of green shoots nd roots were mesured fter 4 months of inocultion. The experiment ws rrnged in completely rndomized design. There were three replictes nd ech replicte hd three plnts (P hillips, 1998). Pthogenicity test in the field For this tril, the sme set of B. prv ws used s in the greenhouse experiment. Inocultions were mde in vineyrd on 15-yer-old grpevine plnts of two cultivrs, Reshmew nd Tefi, in Duhok governorte, Kurdistn region of Irq, by pplying colonized gr plug cut from the mrgin of 7-dy-old culture to the wounds (8 mm) on the brk, nd then the inoculted wounds were wrpped with Prfilm. Inocultions were lso mde in mture wood by pouring the rm (drilling hole (4 mm wide nd 15 mm deep) into the rm of the vine). A colonized gr plug cut from 1-week-old culture of B. prv ws plced in the wound. The wound ws seled nd covered with Prfilm. The length of cnker ws ssessed fter 2.5 nd 5 months (Vn Niekerk et l. 2004). The lyout of the tril ws rndomized complete block design with three replictes; 3 shoots were inoculted in ech replicte. Dt ws sttisticlly nlyzed using SAS/ STAT (SAS/STAT, 1999). RESULTS AND DISCUSSION Phenotypicl chrcteriztion Bsed on morphologicl chrcters, ll isoltes of Botryospheri species were identified s Botryospheri prv Pennycook nd Smuels (nmorph: Neofusicoccum prvum (Pennycook & Smuels) Crous, Slippers & A. J. L. Phllips (Fig. 1A-F). The colony growth reched dimeter of 90 mm on PDA nd MEA fter 10 dys of incubtion. Colonies on PDA formed bundnt e ril mycelium tht ws initilly white but turned drk-olivceous fter 5-6 dys t 25 o C. The reverse side ws lmost blck in older cultures. Pycnidi usully were ggregted nd ppered fter dys in cultures incubted under blck light with 12-hour photoperiod nd ppered on the moist cne brk fter 30 dys under room temperture. Conidiophores were reduced to conidiogenous cells. Conidiogenous cells were hyline, holoblstic forming conidi t their tips. Conidi were hyline, guttulte, thin-wlled, non-septte, smooth, fusiform to ellipsoidl with subobtuse pex nd truncte or rounded bse, often with minute bsl frill. Conidi frequently become olivceous or light brown nd develop 1 or 2 sept with drker middle cell. Mesurements of conidil dimensions for our isoltes, s shown in Tble 1 were in line with those reported by Pennycook nd Smuels (1985); Phillips (2002); Slippers et l. (2005), nd Urbez-Torres et l. (2006). Pthogenicity test in the greenhouse The greenhouse pthogenicity test produced obvious cnker on young shoots inoculted by the pthogen by two methods (Fig. 2A-B). The results presented s cnker length in Tble 2 show tht B. prv produced cnker on both cultivrs, Tefi nd Rshmew. The highest cnker ppered on the cultivr Tefi; it reched mm nd 15.0 mm in length fter two nd four months of inocultion, respectively.

3 Identifiction nd pthogenicity of Botryospheri prv ssocited with grpevine decline in Kurdistn region Irq 73 The results presented in Fig. 3 represent the interction between pthogen nd grpevine cultivrs. The results showed obvious cnker on young shoots of both cultivrs ws cused by the fungus. However, there were no obvious symptoms observed on the leves of both cultivrs. This result is in greement with those of Lrignon et l. (2001). Bsed on the speed of wood coloniztion, cv. Tefi ws rted more susceptible to B. prv thn cv. Rshmew. Philips (1998) showed tht symptoms of Botryospheri spp. usully develop slowly, nd severe symptoms become visible only in grpevines tht re 8 or more yers old. In inocultion of one-yer-old Tefi rooted cutting, the cnker length incresed from 5.83 to 7.00 mm by the wounding method nd from 3.00 to 8.57 mm by the injecting method fter 2 nd 4 month of infection by B. prv, respectively (Fig. 4). The results in Tble (3) show tht B. prv decresed the fresh nd dry weight of shoot nd root in the Tefi cultivr, with no significnt differences from the control tretment. The results from cuttings obtined in this work showed tht the most importnt symptom to evlute Botryospheri pthogenicity ws vsculr discolortion, typicl symptom ssocited with plnts infected by vsculr fungi (Snds et l. 1997; Hrrington et l. 2000), which my be ssocited with oxidtion nd trnsloction of some brekdown products of plnt cells ttcked by fungl enzymes (Agrios, 2005). However, four months my hve been insufficient for symptom expression, s pointed out by Hlleen et l. (2007). Pthogenicity test in the field The pthogenic fungus B. prv cused drk brown cnker on young rtificilly wounded shoots nd blck internl discolortion visible in the cross- -sectioned mture rm of 15-yer-old grpevine inoculted by drilling hole (Fig. 2 C-D). The pthogen ws re-isolted from inoculted prts. The results in Tble (4) show tht B. prv produced the highest cnker length reching mm in the Rshmew cultivr, which ws inoculted by wounding the shoots, fter 5 months under inocultion. This tretment ws significntly different from other tretments. Inocultion by drilling hole in the mture rm gve the lowest vlue for the cultivrs Tefi nd Rshmew. The interction between the pthogenic fungus nd inocultion methods (Fig. 5) indicted tht wounding the green shoots with the mycelium of B. prv cused significnt increse in cnker length which reched mm fter 5 months of inocultion. This result is lso in greement with other studies in which B. prv is identified s n importnt grpevine pthogen cusing severe lesions on green shoots nd mture wood (Svocchi et l. 2007; Vn Niekerk et l. 2004, 2006). Tble 1. Conidil dimensions of Botryospheri prv from Kurdistn region, Irq, isolted during this study nd comprison with those reported from previous studies Botryospheri prv in this study Botryospheri prv from previous studies B. prv isoltes Conidil size (μm) Men ±SD (μm) Conidil size (μm) Source of dt DKI 1 DKI 2 DKI 3 (7.5-) (13-) (13-) ± 0.4 * 5.6 ± ± ± ± ± 1 (11 )14 18 ( 23) 5 7( 10) Pennycook nd Smuels, 1985 DKI 4 (10-) ± ± 0.5 (12 )15 20 (4 ) 4.5 6( 7.5) Phillips, 2002 DKI5 (10-) ± ± 0.3 DKI 6 (11-) ± ± 0.6 DKI 7 (19-) ± ± Slippers et l (-10) (5) 7 9 Urbez-Torres, 2006 * Dt re mens of forty conidi per isolte ± SD

4 74 Red A. Hleem, Smir K. Abdullh, Jldet M.S. Jubrel Fig. 1. A) Colony of Botryospheri prv on MEA-left, PDA-right, fter 10 dys of incubtion t 25 o C. B) Pycnidi of B. prv on the surfce of grpevine cne brk. C-D) Conidiogenous cells hyline, holoblstic forming conidi t their tips dischrged from mture pycnidium. E) Conidiogenous cells nd immture conidi. F) Mture conidi. Scle brs: F= 20 μm, E = 10 μm.

5 Identifiction nd pthogenicity of Botryospheri prv ssocited with grpevine decline in Kurdistn region Irq 75 Fig. 2. Symptoms ssocited with B. prv on infected grpevine: A- Drk brown, discolortion cnker on young shoot inoculted by wounding, nd B) Drk brown, discolortion cnker on young shoot inoculted by the injecting of spore suspension, under greenhouse condition; C) Drk brown cnker on young shoots wounded rtificilly under field conditions; D) Blck internl discolortion visible, in cross-sectioned rms inoculted by drilling hole; E) Blck streking in the longitudinl section of young shoot inoculted by wounding of 15-yer-old Rshmew plnt under field conditions.

6 76 Red A. Hleem, Smir K. Abdullh, Jldet M.S. Jubrel Cnker length (mm) c Tefi cv Control 8 c Rshmew cv. B. prv b Fig. 3. Effect of pthogenic fungus nd grpevine cultivrs on cnker length under greenhouse conditions. Cnker length (mm) b 7 b Wounding shoot Inocultion method Two months 3 c 8.57 Injecting shoot Four months Fig. 4. Effect of inocultion method on the cnker length (mm) fter 2 nd 4 months of inocultion 12 Cnker length (mm) b months 5 months Pouring rm Wounding shoot Fig. 5. Effect of inocultion method on disese development under field conditions fter 2.5 nd 5 months.

7 Identifiction nd pthogenicity of Botryospheri prv ssocited with grpevine decline in Kurdistn region Irq 77 Tble 2. Effect of pthogenic fungus nd grpevine cultivrs on cnker length (mm) fter 2 nd 4 months of inocultion under greenhouse conditions Tretment cv. Tefi cv. Rshmew 2 months 4 months 2 months 4 months Control 8.00 * d 8.00 d 8.00 d 8.00 d Inoculted with Botryospheri prv b c b *Mens followed by different letters re significntly different bsed on Duncn s Multiple Rnge test (P=0.05) Tble 3. Effect of isolted fungus on shoots nd root of Tefi nd Rshmew cultivrs Tretment Shoots Root fresh weight (g) dry weight (g) fresh weight (g) Dry weight (g) Control Inoculted with B. prv cv. Tefi 97.04* b b b cv. Rshmew cv. Tefi b b b cv. Rshmew b * Mens followed by different letters re significntly different bsed on Duncn s Multiple Rnge test (P=0.05). Tble 4. Effect of inocultion method on cnker length (mm) of Botryospheri prv in the grpevine cultivr fter inocultion under field conditions Cultivrs Inocultion method fter 2.5 months Inocultion method fter 5 months Wounding shoot Drilling mture rm Wounding shoot Drilling mture rm Tefi 5.27 *b 4.50 b 11.06* 4.67 b Rshmew b b * Mens followed by different letters in ech period re significntly different bsed on Duncn s Multiple Rnge test (P=0.05). REFERENCES A g r i o s G. N., Plnt Pthology, 5th edn. Sn Diego, CA, USA, Elsevier. Acdemic Press. Cstillo-Pndo M., Somers A., Green C.D., P r i e s t M., S r i s k n t h d e s M., Fungi ssocited with diebck of Semillon grpevines in the Hunter Vlley of New South Wles. Aust. Plnt Pth. 30: Crous P.W., Slippers B., Wingfield M.J., Rheeder J., Mrsm W.F.O., Phillips A.J.L., Alves A., Burgess T., Brber P., Groen ew l d J. Z., Phylogenetic linege in the Botryosphericee. Studies in Mycology, 55: Denmn S., Crous PW., Tylor J.E., Kng J-C., P s c o e I., W i n g f i e l d M. J., An overview of the txonomic history of Botryospheri, nd re-evlution of its nmorphs bsed on morphology nd ITS rdna phylogeny. Studies in Mycology, 45: H l l e e n F., M o s t e r t L., C r o u s P.W., Pthogenicity testing of lesser- known vsculr fungi of grpevines. Aust. Plnt Pthol.36: Hrrington T.C., Steimel J., Workneh F., Y n g X. B., Moleculr identifiction of fungi ssocited with vsculr discolortion of soyben in the north centrl United Sttes. Plnt Dis. 84: Lrignon P., Fulchie R., Cere L., Dubos B., Observtions on blck ded rm in French vineyrds. Phytopthol. Mediterr. 40: Luque J., Mrtos S., Phillips A.J.L., Botryospheri viticol sp. nov. on grpevines: new species with Dothiorell nmorph. Mycologi, 97: P e n n y c o o k S. R., S m u e l s G. J., Botryospheri nd Fusicoccum species ssocited with ripe fruit

8 78 Red A. Hleem, Smir K. Abdullh, Jldet M.S. Jubrel rot of Actinidi delicios (Kiwifruit) in New Zelnd. Mycotxon, 24: P h i l l i p s A. J. L., Botryospheri dothide nd other fungi ssocited with Excoriose nd diebck of grpevines in Portugl. J. Phytopthology, 146: P h i l l i p s A. J. L., Botryospheri species ssocited with diseses of grpevines in Portugl. Phytopthol. Mediterr. 41:3-18. Snds D.C., Ford E.J., Miller R.V., Slly B.K., McCrthy M.K., Anderson T.W., Wever M.B., Morgn C.T., Pilgerm A.L., D r l i n g t o n L. C., Chrcteriztion of vsculr wilt of Erythroxylum coc cused by Fusrium oxysporum f. sp. erythroxyli form specilis nov. Plnt Dis. 81: SAS/STAT SAS/STAT User s Guide, Version 8.2, 1st printing. Vol. 2. SAS Institute Inc, SAS Cmpus Drive, Cry, North Crolin. Svocchi S., Steel C.C., Stodrt B.J., Som e r s A., Pthogenicity of Botryospheri species isolted from declining grpevines in subtropicl regions of Estern Austrli. Vitis. 46:32. Slippers B., Johnson G.I., Crous P.W., Couthino A.T., Wingfield B.D., Wingfield J.M., Phylogenetic nd morphologicl re-evlution of the Botryospheri species cusing diseses on Mngifer indic. Mycologi, 97: Tylor A., Hrdy G.E.S.J., Wood P., Burgess T., Identifiction nd pthogenicity of Botryospheri species ssocited with grpevine decline in Western Austrli. Aust. Plnt Pthol. 34: Úrbez-Torres J.R., Levitt G.M., Voegel T.M., G u b l e r W. D., Identifiction nd distribution of Botryospheri spp. ssocited with grpevine cnkers in Cliforni. Plnt Dis. 90: Vn Niekerk J.M., Crous P.W., Groenewld J.Z., F o u r i e P. H., H l l e e n F., DNA phylogeny, morphology nd pthogenicity of Botryospheri species on grpevines. Mycologi, 96: Vn Niekerk J.M., Fourie P.H., Hlleen F., C r o u s P.W., Botryospheri spp. s grpevine trunk disese pthogens. Phytopthol. Mediterr. 45: S54. Identyfikcj i ptogeniczność grzybów Botryospheri prv związnych z zmierniem winorośli w regionie Kurdystnu w Irku Streszczenie W trkcie prowdzeni bdń nd grzybmi związnymi z symptommi zmierni gtunków winorośli rosnących w regionie Kurdystn w Irku ntrfiono n kilk izoltów gtunków z rodzju Botryospheri. Wszystkie izolty oznczono jko Botryospheri prv Pennycook i Smuels. N dwóch odminch, Tefi i Rshmew, wykonno test ptogeniczności dl izoltu DKI 1. W wrunkch szklrniowych jednoroczne ukorzenione sdzonki winorośli inokulowno izoltem ptogen przy użyciu dwóch metod: wstrzykując zwiesinę zrodników grzyb w zielone pędy orz poprzez sztuczną inokulcję nciętych pędów z pomocą grzybni. Nekroz o njwiększej długości (15,0 mm) powstł po czterech miesiącch n pędch odminy Tife sztucznie inokulownych grzybnią ptogen. W wrunkch polowych przyjęto dwie metody inokulcji: ncięcie zielonych pędów orz wywiercenie dziurki w głęzich dojrzłej winorośli, po czym dokonno inokulcji grzybnią. Nekrozę o njwiększej długości (11,17 mm) uzyskno po 5 miesiącch n nciętych pędch odminy Rshmew, przy czym różnic w stosunku do odminy Tefi był istotn. Ptogen spowodowł zmniejszenie świeżej i suchej msy pędów zielonych i korzeni w porównniu z nieokulowną kontrolą. Jest to pierwsze doniesienie n temt grzyb B. prv w Irku.