Res. Plant Dis. 16(2) : 163-169 (2010) Research in Plant Disease The Korean Society of Plant Pathology t q Pantoea agglomerans 59-4» w ½»*Á«Á wáy Á x Á yáw Á 1 Á 1 Á 2 w», 1, 2 Elucidation of Mode of Action of Pantoea agglomerans 59-4 for Controlling Garlic Blue Mold Yong-Ki Kim*, Mi-Kyung Kwon, Wan-Hae Yeh, Sung-Jun Hong, Hyung Jin Jee, Jong-Ho Park, Eun-Jung Han, Kyung-Seok Park 1, Sang-Yeob Lee 1 and Seong-Don Lee 2 Department of Organic Agriculture, Division of Agro-Food Safety, NAAS, RDA, Suwon 441-707, Korea 1 Department of Agricultural Microbiology, Division of Agricultural Biology, NAAS, RDA, Suwon 441-707, Korea 2 Department of Research Coordination, Division of Research Policy Bureau, RDA, Suwon 441-707, Korea (Received on May 3, 2010; Accepted on May 18, 2010) To screen for potential biocontrol agents against postharvest disease of garlics caused by Penicillium hirsutum, a total of 1292 isolates were isolated from the rhizoshere or rhizoplane of Allium species. Among them, S59-4 isolate was selected as a potential biocontrol agent by in vivo wounded garlic bulb assay. The isolate was identified as Pantoea agglomerans (Pa59-4) through Biolog system. Pa59-4 did not inhibit the mycelial growth of P. hirsutum in dual-culture with P. hirsutum on tryptic soy agar. In order to elucidate mode of action of Pa59-4 on biological control, nutrient competition between Pa59-4 and P. hirsutum was investigated by the simple method using tissue culture plates with cylinder inserts containing defusing membrane reported by Janisiewicz et al.g(2000). The results showed that Pa59-4 effectively suppressed spore germination and mycelial growth of blue mold in the low concentration (0.5%) of garlic juice, but it did not suppress those of blue mold in the high concentration (5%) of garlic juice. This result suggests that the mechanism in biocontrol of garlic blue mold by Pa 59-4 may be involved in nutrient competition with P. hirsutumgon garlic bulbs. KeywordsG: Garlic blue mold, Nutrient competition, Pantoea agglomerans 59-4, Postharvest disease w w» w w w. ü 1980 l š j vw w w³y ¼w³ w w ww w ƒ w ƒ ³ w³y ³ ù» w q w z wš x y w y w w w» w w ƒ š š. (2006) Bacillus valimortis *Corresponding author Phone) +82-31-290-0554, Fax) +82-31-290-0507 Email) yongki@rda.go.kr EXTN-1 w ƒ ³ w w wš w š šw. ü w w w, ³w m ù ƒ, q w w ƒ, w w w. w w w ³ e ¼w³ w š, ƒ t w ¼w³ w (Janisiewicz, 2000). ³(Penicillium hirsutum) sww ³ kƒ (necrotrophic) w ³ s w p v w. p
164 ½»Á«Á wáy Á x Á yáw Á Á Á w ³ œ w ƒ z q. ¾ w ¼w³ w» w (Stockwell, 2002; Wright, 2001; Pusey, 2008), w (Han, 2000, Wilson, 1994), lipopolysaccharide(kochchi, 2006; Tsukioka, 1997) w š ù, w³ w w y š (Janisiewicz, 2000; Meziane, 2006; Poppe, 2003). ù q Allium «l w t q ³ q x w w wš, w³ y k w š, œ w t q» w. x ³. t q s ƒ x q s v z potato dextrose agar(pda) w ³ w 20 o C w» 5 w. ¼ q ³ PDA w x ³ w. ³ PDA 14 w z 20 ml ³ ƒwš š ³ s z wš hemacytometer w w. q «, tv. t q ³ w ¼w³ w» w m, q «, q t l ³ ³ w. m m 20 g 200 ml ƒw 150 rpm kw z w. q w w z ƒ w 20 ml ³ ƒw wš w. q tv ƒ w j» 2ml ³ ƒw wš w. z w ƒƒ w tryptic soy agar (TSA) (Difco Laboratories, Detroit) ƒƒ w g w. ¼w³. ¼w³ e r Fig. 1. Microplate and cylinder used for testing nutrient competition between Pa59-4 and Penicillium hirsutum, a causing agent of postharvest decay of garlics. w w w. 2001 w ³y ¼w³ w» w t q ³ TSA e w ³ x w w w. t q ³ PDA 7 w z 10 6 s /ml xkw 7 mm paper disc vr qw w š, paper disc vrqw z w w³y w (Fig. 1). 2002 l w³y w w ù w j w» w r ww w w ( w r w). Ë ¼ z 1% ùp 1 t ³wš ³ 3 ü š g. 2mm, ¾ 1mm 2 üš e ¼w³ xk w. 30 z Penicillium hirsutum xk e w 20 o C w» 7 wš x» ¼w³ w. 1 r 3 3, 2 r 10 3 x ww. «z w sƒw. z (Suppressive effect, %)= {( )/ } 100 ¼w³. ¼w w ù r x w ³ ³ w w q w, ¼w³ w ƒƒ Biolog GN plates
(Biolog, Inc., Hayward, CA) wš, ¼ w³ w Biolog GP plates w k w w. ¼ w³ w Bartholomew Michell w Walts 7.5% malachite green 0.25% safranine w ü s y w. ¼w³ w³». w ¼w³ Pa59-4 w ³y» w w, TSA 1/10 TSA 9cm rp 20 ml w z ³ ¼w³ Pa59-4 e ( t q ³ wš ƒ ƒx ¼ w³ xk paper disc e )w t q ³ w Pa59-4 x w. w. Pa59-4 w w» w Janisiewicz (2000) w j v p t q ³ s w (Fig. 1). ¼w³ w z w» w j v p (24 ; Costor, Corning Inc.,, ) (Millicell-CM; Millipore Corp., d d, ) w. e s l p p vl( j» 0.45 µm)ƒ s p spacerƒ ù sww j v p w» w w. mw crystal violet ü š j v p ù w y w. ¼w³ PA59-4 w y w 10% 1% ¼w³ xk (1.0 10 cfu/ml) v ƒ 8 w 5% 0.5% w j v p well., 0.5% 5% w w. t q ³ t q Pantoea agglomerans 59-4» w 165 s xk (2 10 5 s /ml) j v p ü ew 0.4 ml w. ³ ¼ w³ sww j v p 25 C 24 48 j w z o well l Éü tissue paper üš membrane scapel ü. ü membrane lactophenol- cotton blue wš qw Ÿwx s ³ w. š q «. t q w» w 2001 l 2002 ¾ 2 q «l t 1,292 w (Table 1). 2002 r w mw w ¼w³ qù «l. t q ¼w³. 2002 w 158³ 89³ in vitro w³ mw, 69³ r mw. w³ mw w ¼w³ w³ j» w 10 mm ³ ƒ 94.4% (Table 2), r mw w ¼w³ 23.2% 10 mm ùkþ 76.8% ¼w³ x w ù 8 mm x w ùkû (Table 3). In vitro t q ³ w w³ in vivo z ƒ û ƒ. ƒ w w ù swwš» w³y w w ü Table 1. Isolation of effective microorganisms from rhizosphere and rhizoplane of Allium species collected in the field Crop Isolation part No. of isolates No. of selected isolates 2001 2002 Subtotal 2001 2002 2003 Garlic Soil 51 434 485 4 45 1 Root 105 501 606 17 111 0 Onion Soil 66 0 66 1 0 0 Root 108 0 108 3 0 0 Others - 25 2 27 3 2 0 Total - 355 937 1,292 28 158 1
166 ½»Á«Á wáy Á x Á yáw Á Á Á Table 2. Inhibitory activity of antagonistic microorganisms selected through dual-culturing of microorganisms isolated from rhizosphere and rhizoplane of Allium species, and Penicillium hirsutum, causing storage decay of garlic DiameterG(mm) of inhibition zone <8 8~10.0 10.1~13.0 13.1~15.0 >15.1 Total No. of isolates 1 4 62 14 8 89 Proportion 1.1 4.5 69.7 15.7 9.0 100 Table 3. Inhibitory activity of antagonistic microorganisms selected through bioassay on garlic cloves by co-inoculation of microorganisms isolated from rhizosphere and rhizoplane of Allium species, and Penicillium hirsutum, causing storage decay of garlic DiameterG(mm) of inhibition zone <8 8~10.0 10.1~13.0 13.1~15.0 >15.1 Total No. of isolates 53 0 0 1 15 69 Proportion 76.8 0 0 1.5 21.7 100 Table 4. Classification of effective microorganisms according to antifungal activity range to Penicillium hirsutum on garlic cloves No. of antagonists at different suppressive effectg(%) <10 10.1~30 30.1~50 50.1~70 >70 Total No. of isolates 37 11 9 2 99 a 158 Proportion (%) 23.4 7.0 5.7 1.3 62.6 100 a Twenty nine isolates caused damage to garlic. w w ³ w w». w³y» w ³ j»ƒ 10.1~13.0 mm ³ ³ s w, r mw w ³ x ù û y w ùkþ. w³y» w ü w³ y w ³ r mw ¼w³ w w³y ¼w³ sww w w ù w w wì w» ƒ. ³ w w w w ù w w. In vitro w³ w³y ¼w³ 89 w t q ³ s y x w w w³y ³ 24³ y w, 63 ³ s gš, 1³ s gš, 1³ s x j ùkû. t q yw ¼w ³ w» w w³ r w ¼w³ 158 œ w r z sƒw (Table 4). 158³ 99 ³ 70% w ùkû, 99³ 29³ w ùkü t q w» š w sƒ. t q z 99³ 29³ w 70³ ƒ š 3 z w x S5-1, S31-8, S38-9 S59-4³ ƒ w z ùk þ (Table 5). ¼w³. r t q w w z 4 ¼w³ 2001 x z 5 ¼w³ w ³w yw p w. ³ w w w z ³ Biolog GN plate, ³ Biolg GP plate w (k, ) w w, z 4 ¼w³ ƒ ƒ S31-8 Klebsiella terrigena, S38-9 Pseudomonas fluorescens, S59-4 Pantoea agglomerans, S5-1 (Table 6). P. agglomerans y w» wš,
Table 5. Suppressive effect of isolated microorganisms on disease development caused by Penicillium hirsutum on garlic cloves t q Pantoea agglomerans 59-4» w 167 Isolate Suppressive effectg(%) a 1st trial 2nd trial 3rd trial Remarks R14-13 21.0 - - R16-13 100G(decayed) - - R14-2 36.0 - - SC1 5.0 - - SC2 42.0 - - R6-15 31.0 - - S5-1 > 80 83 90 Selected S10-2 > 80 83 83 S10-4 > 80 100 83 S14-4 > 80 83 83 S21-2 > 80 83 83 S29-1 > 80 83 83 S31-8 > 80 100 100 Selected S38-9 > 80 100 100 Selected S44-1 > 80 100 83 S47-1 > 80 83 83 S52-1 > 80 83 83 S52-4 > 80 83 83 S59-4 > 80 83 100 Selected J6-1 > 80 83 83 J9-4 > 80 83 83 J25-4 > 80 83 83 J25-5 > 80 83 83 J31-5 > 80 83 83 J50-8 > 80 100 83 a Suppressive effect (%) = {(radii of infected leaions of untreated control samples radii of infected lesions of treated samples)/radii of infected lesions of untreated control samples} 100. Fig. 2. Antifungal activity test by dual-culturing between Pa59-4 and Penicillium hirsutum on ten times-diluted TSA media. Inhibition zones were not formed between Pa59-4 and P. hirsutum. w Penicillium expansum, Botrytis cinerea, Rhizopus stolonifer w» w» w (Nunes, 2001; 2002). w³y. w ¼w³ Pa59-4ƒ t q ³ w w³y ƒ š» w t w w TSA š TSA 1/ 10 w ³ Pa59-4 e w x w, w ¼w³ Pa59-4 x œ w x w (Fig. 2). ¼w³ Pa59-4 t q ³ w w³y q. Stockwell (2002) y P. agglomerans strain Eh252 in vitro w w³ w y ³ x w šw. Wright (2001) P. agglomerans strain Eh252ƒ in vitro w y ³ w ƒ w Table 6. Identification of the nine effective bacteria selected as control agents against Penicillium decay according to the Biolog Microstation System for bacterial identification Isolate Identification Similarity Closest species R14-2 Bacillus sp. * - Bacillus subtilis R14-13 Pseudomonas fluorescens 0.596 - R16-13 Bacillus subtilis * 0.501 SC-1 Bacillus licheniformis * 0.560 - SC-2 Bacillus sp. * - Bacillus amyloliquefaciens S5-1 unidentified - - S31-8 Klebsiella terrigena 0.522 - S38-9 Pseudomonas fluorescens 0.606 - S59-4 Pantoea agglomerans 0.646 - * Endospore formation was confirmed by Walt's method.
168 김용기 권미경 예완해 홍성준 지형진 박종호 한은정 박경석 이상엽 이승돈 산한다고 보고하였다. 그러나 본 시험에서 시험에 공시한 길항균 Pa59-4는 모든 배지 상에서 마늘푸른곰팡이병균 에 대한 항균활성을 보이지 않았으므로 푸른곰팡이병균 의 생장을 억제하는 항생물질을 생성하지 않는 것으로 판 단되었다. 영양원 경합여부 조사 PFTE 멤브레인 필터를 이용하 여 마늘푸른곰팡이병균과 Pa59-4의 nutrient competition을 조사한 결과, 0.5%와 5% 마늘주스에서는 모두 발아가 잘 되었으나 마늘주스에 PA59-4를 혼합하였을 경우에는 0.5% 마늘주스에서는 발아가 되지 않았고 5%에서는 마늘 주 스만 처리하였을 때 보다 발아가 현저하게 억제되었다 (Table 7과 Fig. 3). Pa59-4는 항균활성에 의하여 푸른곰 팡이병균을 억제하지 않는 것은 항균활성 검정결과 확인 된 바 있고, Pa59-4가 영양원인 마늘주스를 적게 첨가해 주었을 때 푸른곰팡이병균의 포자발아와 균사생장을 현 저하게 억제시킨 반면 마늘주스를 많이 첨가해 주었을 때 에는 억제효과가 현저하게 저하되었고 길항균 Pa59-4와. Table 7. Suppression of conidial germination of Penicillium hirsutum by nutrient competition with PA59-4a Treatment Sporulation of Penicillium hirsutumb Water 0.5% garlic juice +++ 5% garlic juice +++ Water + Pa59-4 0.5% garlic juice + Pa59-4 5% garlic juice + Pa59-4 ++ Inoculum concentration of Penicillium hirsutum was 10 spores/ml. Sporulation rate of Penicillium hirsutum was measured by observing spore germination and mycelial growth with three replications. Germination rate:, no germination; +, 10% ; ++, 11~50%; +++, 51%. a b 5 함께 접종하였을 때 발아되지 않았던 병원균 포자를 신 선한 5% 마늘 쥬스에 접종했을 때에는 다시 잘 발아 되 었던(자료생략) 것으로 보아 Pa59-4의 마늘 푸른곰팡이병 억제효과는 nutrient competition(영양원 경합)에 의한 것 으로 판명되었다. Poppe 등(2003)은 감귤 푸른곰팡이병에 높은 방제효과를 보이는 P. agglomerans가 본 시험에 공 시한 Pa59-4와 같이 영양원 경합에 의해 병 진전을 억제 한다고 보고한 바 있다. Castoria 등(2001)은 사과저장 중 에 발생하는 B. cinerea, P. expansum, R. stolonifer와 A. niger에 대한 억제효과가 우수한 Aureobasidium pullulans (LS-30)가 병원균에 대한 영양원 경합능력도 우수하고, 병 원균 균사를 분해할 수 있는 exochitinase와 1,3-glucanase 를 분비한다고 보고하였다. Calvente 등(1999)에 따르면 사과저장 중 부패를 일으키는 푸른곰팡이병에 억제효과 가 우수한 Rhodotorula glutinis는 Rhodotorulic acid라는 siderophore를 생성함으로써 발병을 억제시킨다고 한다. 그러나 본 시험에 공시한 길항균 Pa59-4는 병원균에 대 한 영양원 경합능력은 우수하나 대치배양에 의한 항균성 검정시 저지원이 전혀 형성되지 않은 것으로 보아 병원 균 균사를 분해할 수 있는 효소를 생산하거나 직접 침입 할 수 있는 능력은 없는 것으로 판단된다. Han 등(2000) 은 근권에서 유도저항성을 일으키는 능력이 있는 미생물 을 간편하고 신속하게 선발하는 방법에 대한 연구 결과 를 발표하면서 본 시험에서 공시한 균과 동일한 종인 P. agglomerans strain E278Ar을 근권에 처리할 경우 무에 저항성을 유도하여 잎에 나타나는 무 세균성검은점무늬 병을 방제할 수 있다고 보고한 바 있다. 따라서 본 시험 에서 선발한 Pa 59-4를 근권에 처리했을 때 저항성을 유 도하는지 여부를 오이를 가지고 시험하였는데, 유도저항 성을 일으킨다는 결과를 얻지는 못하였다(자료 생략). Feng Fig. 3. Germination of the conidia on the membranes cut from the cylinders that were inserted for 24 h into wells containing, water (A), garlic juice at 0.5% (B), garlic juice at 5% (C), water + Pa59-4 (D), 0.5% garlic juice + Pa59-4 (E), 5% garlic juice + Pa59-4 (F), for an additional 24 h.
(2006) ü w P. agglomerans YS19 š wš Ÿw j šw, Pa59-4³ w w mƒ v w. t q Pantoea agglomerans 59-4» w 169 j t q w» w q q «l 1,292³ w. w ³ t q w w³ r w t q x w S59-4³ w, Biolog system w w Pantoea agglomerans 59-4(Pa59-4). w ¼w³ Pa59-4» w» w TSA ³ e w w³y. ¼w³ Pa59-4 ³ w Janisiewicz(2000) w, ³ ¼w³ Pa59-4 wì w, ¼w³ Pa59-4 ³ s ³ x w, ¼w³ Pa59-4 wì w ³ s w 5% w ùkù ¼w³ Pa59-4³» w w y., ¼w³ Pa59-4 q w ¼w³ q. š x Calvente, V., Benuzzi, D. and de Tosetti, M. I. S. 1999. Antagonistic action of siderophore from Rhdotorula glutinis upon the postharvest pathogen Penicillium expansum. Int. Biodeter. Biodegr. 43: 167-172. Castoria, R., De Curtis, F., Lima, D. G., Caputo, L., Pacifico, S. and De Cicco, V. 2001. Aureobasidium pullulans(ls-30) an antagonist of postharvest pathogens of fruits: study on its modes of action. Postharvest Biol. Technol. 22: 7-17. Feng, Y., Shen, D. and Song, W. 2006. Rice endophyte Pantoea agglomerans YS19 promotes host plant growth and affects allocations of host photosynthates. J. Appl. Microbiol. 100: 938-945. Han, D. Y., Coplin, D. L., Bauer, W. D. and Hoitink, H. A. J. 2000. A rapid bioassay for screening rhizosphere microorganisms their ability to induce systemic resistance. Phytopathology 90: 327-332. Janisiewicz, W. J., Tworkoski, T. J. and Sharer, C. 2000. Characterizing the mechanism of biological control of postharvest diseases on fruits with a simple method to study competition for nutrients. Phytopathology 90: 1196-1200. Kohchi, C., Inagawa, H., Nishizawa, T., Yamaguchi, T., Nagai, S. and Soma, G. I. 2006. Application of lipopolysaccharide derived from Pantoea agglomerans(ip-pa1) for health care based on macrophage network theory. J. Biosci. Bioeng. 102: 485-496. Meziane, H., Gavriel, S., Ismailov, Z., Chet, I., Chernin, L. and Hfte, M. 2006. Control of green and blue mold on orange fruit by Serratia plymuthica strains IC14 and IC1270 and putative modes of action. Postharvest Biol. Biochem. 39: 125-133. Nunes, C., Usall, J., Teixido, N. and Vinas, I. 2001. Biological control of postharvest pear diseases using Pantoea agglomerans CPA-2. Int. J. Food Microbiol. 70: 53-61. Nunes, C., Usall, J., Teixido, N., Fons, E. and Vinas, I. 2002. Postharvest biological control by Pantoea agglomerans (CPA- 2) on golden delicious apples. J. Appl. Microbiol. 92: 247-255. Park, K., Diby, P. and Yeh, W. H. 2006. Bacillus vallismortis EXTN-1-mediated growth promotion and disease suppression in rice. Plant Pathol. J. 22: 278-282. Poppe, L., Vanhoutte, S. and Hofte, M. 2003. Modes of action of Pantoea agglomerans CPA-2, an antagonist of postharvest pathogens on fruits. Eur. J. Plant Pathol. 109: 963-973. Pusey, P. L., Stockwell, V. O. and Rudell, D. P. 2008. Antibiosis and acidification by Pantoea agglomerans strain E325 may contribute to suppression of Erwinia amylovora. Phytopathology 98: 1136-1143. Stockwell, V. O., Johnson, K. B., Sugar, D. and Loper, J. E. 2002. Antibiosis contributes to biological control of fire blight by Pantoea agglomerans strain Eh252 in orchards. Phytopathology 92: 1202-1209. Tsukioka, D., Nishizawa, T., Miyase, T., Achiwa, K., Suda, T., Soma, G. I. and Mizuno, D. 1997. Structural characterization of lipid A obtained from Pantoea agglomerans lipopolysaccharide. FEMS Microbiol. Lett. 149: 239-244. Wilson, C. L., Ghaouth, A., Chalutz, E. E., Droby, S., Stevens, C., Lu, Y. and Arul, J. 1994. Potential of induced resistance to control postharvest diseases of fruits. Plant Dis. 78: 837-844. Wright, S. A. I., Zumoff, C. H., Schneider, L. and Beer, S. V. 2001. Pantoea agglomerans strain EH318 produces two antibiotics that inhibit Erwinia amylovora in vitro. Appl. Environ. Microbiol. 67: 284-292.