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1 Res. Plant Dis. 16(2) : (2010) Research in Plant Disease The Krean Sciety f Plant Pathlgy š «³ «Á y 1 Á Á wá * wû w yw, 1 š w Selectin f Bicntrl Agents against Phytphthra Blight f Pepper and Its Rt Clnizatin Ability Li-Jing Zhang, Hng-Zhng Shi, Jing-Jing Wang, Shu-Xian ChangG and Shun-Shan Shen* Cllege f Plant Prtectin, Henan Agricultural University, Zhengzhu , China 1 Xinyang Agricultural Cllege, Xinyang , China (Received n April 9, 2010; Accepted n June 23, 2010) Fur prmising bicntrl agents against Phytphthra capsici were selected frm 507 bacterial islates cllected frm rhizsphere sils and rts f pepper plants. In vitr experiment, these fur bicntrl agents inhibited mycelial grwth, germinatin f cystspres, and frmatin f zsprangia and zspres f Phytphthra capsici. In the pt experiment, the fur bicntrl agents shwed cntrl efficiency higher than 70%. In greenhuse experiment, the islates G28-6 gave the cntrl value f 79.4%. These fur bicntrl agents successfully clnized in the ppulatin density beynd 10 5 cfu/g n rts f pepper in vitr. The islates G28-6 was identified as Pseudmnas aurantiacas based n its cultural, mrphlgical, and bichemical characterizatin and 16S rrna gene sequence analysis. Keywrds : Bicntrl agent, Phytphthra blight f pepper, Rt clnizatin š w ƒw ƒ w w w š. š š ƒ j wù x š»w w w š (Yin, 2007). ù»w Á û w ³ x, d q, m ü» k. x š m z m q j» w w» w š (Weller, 1988). y ey w ƒ ƒ w š. w ù y w š y š. w *Crrespnding authr Phne) , Fax) ) shen0426@hanmail.net Agrbacterium radibacter K84, w Bacillus subtilis w Quantum4000 Kdiak x š, Pseudmnas spp. w m ³ w y ƒ š (Keer, 1980; Siddiqui, 2003; Mena, 2007). w, g w ¼w q Trichderma harzianum Gustafsn z w y, w ¼w ³ Glicladium virens Garace z w Gligard t xy (Spadar, 2005). ¼w w š y w w š y (Yin, 2007). ¼w «³ m ¼w «³ «ù w ÿ. p, m yw k, «, ¼w «³ j ùkü (Zhang, 2000). x w ¼ w «

2 s ¼w z {w w ƒ., «³ w m j ¼w ³ w ù, y y enw j š w ü ³ w j ƒ š (Shen, 2005). š w «³ w š ³ w z «k w y ƒ sƒw. š ³. x š ³ x w y w ³. «³. wû š s w š «m w m sq «³ w. «³ š ³ w ¼wz PDK(Ptat Dextrse Brth 20 g, Peptne 10 g, Agar 15 g, 1000 ml) P. capsici e w z, ³ ³ (inhibitin zne) j» ¼wz ƒ w ³ 1 w. ¼w ³ š ³ w z. In vitr ³ P. capsici ûx, û, vûs w z w. V8A(V8 juice 100 ml, CaCO 3 1 g, Agar 15 g, 900 ml) š ³ crker brer(8 mm) ü Petri plate»š ¼w ³ xk (10 cfu/ml) ³ ƒ ¼ ƒw 8 25 C xÿ 16 w z û x x w w ¼w ³ š û x w z w š, ³ x š ³ û ¼w ³ xk ƒw 4C 30 wš 25 C 30 w w z, x w wš û û Ë» û w û w z w š, š ³ vûs xk ¼w ³ xk y ww slide glass w z 25 C j l w 2 w. ¼w ³ š z. m q w 50 w š (t : œ 16-A) ¼w³ xk 1 e w Pt( 10 cm) š š ³ xk (10 4 /ml, š «³ «159 5ml/ ) w z w. 7 z š w ƒ y w. yw (80%Manczeb 1000 ) w w. sp x z ƒ w G28-6³ w š w š z x w. 50 š G28-6xk 1 e w w w z ƒ w š 60 š w. yw (80%Manczeb 1000 ) ew w. «d. ¼w ³ «w» w Duble Layer Filter Paper(DLF) w. DLF ³w Petri plate Filter paper w š ³ 4.5 ml ƒw z ¼w ³ xk (10 cfu/ml) w e k š š 8 Filter paper z 28 C w ù ó 1cm üš w 0.1 ml/l MgSO 4 w 1/10 TSA w. w z 28 C j l 2 w x clny w «³ y w. ¼w ³. G28-6³ v w xk p Bergey's manual f Systematic Bacterilgy» w w š G S rdna» NCBI BLAST searchw w. PCR lignucletide primer 27f (AGA GTT TGA TCM TGG CTC AG) 1492r(TAC GGH TAC CTT ACG ACTT) w š denaturatin, annealing š extensin 92 C, 55 C š 72 C 30cycle ww š Sequencing vectr pbluescript II SK(+), cmpliment bacteria DH5α w. š ¼w ³. wû š s w š «m l ³ 507³ w, š ³ e w ¼w ³ G28-6, G28-5, I7-6 H14-1³ w (Table 1, Fig. 1). š ³ w z. In vitr ¼ w ³ š ³ w z w, G28-6, G28-5, I7-6 H14-1 ³ ³ w ³ û x, š vûs z w,

3 160 Á y Á Á wá Table 1. Inhibitin f mycelial grwth f Phytphthra capsici by the selected bacterial islates n PDK medium Islates Inhibitin zne (mm) G ±0.17 G ±0.14 I7-6 13±0.14 H ±0.18 Fig. 2. Inhibitin f zspres germinatin f Phytphthra capsici by the selected 4 islates. Errr bars represent the standard deviatin. Fig. 1. Inhibitin f mycellial grwth f Phytphthra capsici by the selected bacterial islates n PDK medium. Table 2. Inhibitin f zsprangia frmatin f Phytphthra capsici by the selected 4 islates Islates Rate f zsprangia frmatin Inhibitin rate G c G bc I bc H b Cntrl (DW) a - *Data fllwed by the same letter in each clumn are nt significantly different based n Ducan's multiple range test (P=0.05). Table 3. Inhibitin f zspre frmatin f Phytphthra capsici by the selected 4 islates Islates Rate f zspre frmatin Inhibitin rate G c* G bc I bc H bc Cntrl (DW) a - *Data fllwed by the same letter in each clumn are nt significantly different based n Ducan's multiple range test (P=0.05). G28-6 I7-6 z ƒ w (Table 2, 3, Fig. 2). š ³ ³ ù ùs k m w» ùs w û x wš û w ù x w m w» w e w 1. y û x 2 w w ü m t (Hwang, 1995). š š» û w. x ¼w ³ š ³ ù x w ¼w ³ ƒ š. š z. Pt x 4³ 70% š z, p G % ƒ x œ yw 80% Manczeb z ƒ w (Table 4). Pt x z ƒ w G28-6³ š w š z w, 79.4% ƒ yw ƒ 48.4% { z (Table 5). G28-6 pt w š x w w š ¼w ƒ ùkþ. «. In vitr DLF ¼w ³ «d w, 4³ w «(Table 6). m «m w

4 š «³ «161 Table 4. Effect f bicntrl f Phytphthra blight f pepper by selected 4 islates in pts Islates Disease incidence Cntrl efficiency G c 90.0 G b 70.0 I bc 80.0 H b 70.0 Chemical 30.0 b 70.0 Cntrl a - *Data fllwed by the same letter in each clumn are nt significantly different based n Ducan's multiple range test (P=0.05). Table 5. Effect f bicntrl f Phytphthra blight f pepper by G28-6 in green huse Islates Disease incidence Cntrl efficiency G c 79.4 Chemical 10.5 b 48.4 Cntrl 20.3 a - *Data fllwed by the same letter in each clumn are nt significantly different based n Ducan's multiple range test (P=0.05). Table 6. The clnizatin f selected 4 islates n the rt f pepper Islates Rt clnizatin density (cfu/cm rt) G G I H œ š w (Zhang, 2000). š ¼w «³ G28-6, G28-5, I7-6 H14-1 z š w š, sp x z ƒ w G28-6 w x š» «m š w w š z w ( x ). ¼w ³. š w ¼w ³ G28-6³ 16S rrna gene» w Pseudmnas aurantiaca 99% ùkþš, xk p p Bergey's manual f Systematic Bacterilgy» w w (Table 7)., G28-6³ Pseudmnas aurantiaca w. Table 7. Cmparisn f the characteristics f antagnistic bacterial islate G28-6 Characteristics P. aurantiaca G28-6 Gram reactin Cell frm straight rd straight rd Flagellum Single flagella Single flagella Cell size µm, µm µm, µm Mbility + + Aerbic grwth + + Pigment + + Grwth in NaCl 2% + + 5% + + 7% + + Gelatinase + + Starch hydrlysis - - Nitrus acid dexidatin reactin - - Nitrate dexidatin reactin + + Glucse xidizes + + Chitinase + wû š s w š «m ³ 507³ w, š ³ ¼wz ƒ w G28-6, G28-5, I7-6 H14-1³ w. In vitr ³ š ³ ³, û x, š v ûs x w w š, Pt x ³ 70% z ùkþš, sp x z ƒ w G28-6³ š w 79.4% ƒ œ yw 80% Manczeb { z. 4³ in vitr w «. š w ¼w ³ G28-6³ 16S rrna gene» xk, p Bergey's manual f Systematic Bacterilgy» w Pseudmnas aurantiaca w. wû w» z ( , ) w w.

5 162 Á y Á Á wá š x Hwang, B. K. and Kim, C. H Phytphthra blight f pepper and its cntrl in Krea. Plant Dis. 79: Keer, A Bilgical cntrl f crwn gall thrugh prductin f agrcin 84. Plant Dis. 64: Mena vilante, H. G. and Olalde Prtugal, V Alteratin f tmat fruit quality by rt inculatin with plant grwth prmting rhizbacteria(pgpr): Bacillus subtilis BEB-13bs. Scientia Hrticulturae 113: Shen, S. S., Chi, O. H., Park, S. H. and Park, C. S Rt Clnizing and Bicntrl Cmpetency f Serratia plymuthica A21-4 against Phytphthra Blight f Pepper. Plant Pathl. J. 21: Siddiqui, I. A. and Shaukat, S. S Cmbinatin f Pseudmnas aeruginsa and Pchnia chlamydspria fr cntrl f rt infecting fungi in tmat. J. Phytpathlgy 151: Spadar, D. and Gullin, M. L Imprving the efficacy f bicntrl agnets against silbrne pathgens. Crp Prtectin 24: Weller, D. M Bilgical cntrl f sil-brne plant pathgens in te rhizsphere with bacteria. Annu. Rev. Phytphthlgy. 96: Yin, J. F., Zhang, W. H., Li, J. Q., Li, Y. H., Hu, H. L. and Zhu, X. Y Screening and antagnistic mechanism f bicntrl agents against Phytphthra blight f pepper. Acta Phytpathlgica Sinica. 37: Zhang, B. X., Zhang, P. and Chen, X. B Factrs affecting clnizatin f intrduced micrganisms n plant rts. Chinese Jurnal f Applied Eclgy. 11: