Plant Pathology Bulletin 19: 213-224, 2010

Bacillus pumilus 41 k, at 137 M

1 t A i i N t1

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?,TP,P,. C461 .Y.IPA %MX. 2010. Bacillus pumilus krlig[lICEEfffiltWIA. ftAirfil19: 213-224.

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Bacillus pumiltts AMPITIN I E./2AM 5[11s1EIM'ItWM ° B. pumilus CVP IAA4.S±tfhli.AM 4HAA"-K=fkRITARIAliEj7 61,11g 10 AMI B. pumilus -ffiti"471f[1$1.1E/1\ MUMMA

MITA --igtttitf.: ZAILIM O'IttVA'fLAA Alia _ETE4.11, PI

tiaN 411:16'1.',V4102fAl$117;&WilikkRY.AfthMffil ° itLf.tftWW2C' Bacillus pumilus LU

°

utinizzij : Lf1 tfArA Bacillus tumulus .74.4.0MkIt

W (Chinese yam lAcommon yam) eN1-,84=1 (Di-

oscoreaceae) -2T,RJM (Dioscorea spp.) 24'1V44-1fdtilMIME MANUA (FAO) nt"rf4 (http://www.fao.

org/docrep/t0207e/t0207e01.htm) P'," n/#4:tnA-hTRPT4-r-Al ° 111 ,41411;firfJ/Flin

V'MU- 3E* O'iff5ftfli7 PI ° Y311Vii

RrilAiliriTMCII Mfb: ilf*RWI4 °tiAljTA)]]2afiLLAIVU4A_V. (tuber) '

1Pi'M 1-1/AN-.IF 1113' MR HEAL f!"1-114'/})-3

° 2Ahl/Jiat A`VrtgiV: Mt linArAVEjRN AW141-ME Tiffif5Afli WI RfUlftrYn'4 ° Oill'aR2jtArL471fIU)v,

(We) (D. alata) V ( iA) (D. batatas) El Tc

LA (D. japonica) (1[7,i) (D. alata L. var. pur-

purea) 1W4111* (OWIEFTX) (D. doryophora) MaltM (D. japonica Thunb. var. pseudojaponica) (20)

l_11M5 ETEMV-IVA,Z- 2008

V,Itd.AIITA 5 THE IPetit'DjAMM4r-Iii(fOt`:Rf110_ x r MA tIMW */AI "I) , +HA

qH1,m 6let KIRH Lqrgot-ra (12) ° 1V4/.1\N

fk."PjA ° 1991 *.i1RHIRA)),T °

2009 *fittagfaean 600 (2) Vfltin'A'&1"friVIAVLA---#nt6 1.8 AIM

CINA-41,V.'AVAaiittifiin-Pii§

klfk-AntR, gt-MitA'AVM4A-A-(anthracnose : Colletotrichum spp.) QTM-' (dry

black rot disease 01#, : Botryodiplodia theobromae)

-t-ikE-1-41,9A (leaf spot or wilting disease A),9; Scle-

214 fAVO-if_V4-0-fil 19 AA 3 WI 2010

rotium rolfsii) 1RA (soft rot AIR; : Rhizopus spp.)

Aff_YliA (Rhizoctonia solani) (3) VtarCiii (Nattrassiamangiferae) (24) ioi (Phompsis spp.) (28)4 4-A-/VA

V Yam Mosaic Virus (YMV) )3( Carla-Like Virus (`')

Dioscorea Latent Virus (DLV) Dioscorea Greenband-

ing Virus (DGBV) ffi 1 Eril,TA4, "4' WIVIIJUtil44,;75,M1 (Meloidogyne spp.)14' 21) akiliA1PilAIMVA

(leaf spot AR; : Curtobacterium flaccumfaciens pv.

dioscorea) (27) PAR (wet rot AR; : Erwinia caroto-vora)(3)4- °

2008 fJLAMIA-tkitiM EMtItOt-A-YcI-Or.4 ;t1-&1.0713ZRIgA4AnAM,AMM,--'2.41t,AtIhf-11)MVAIJS'M'AP. °

trif4thma

trAnCLItlk A-U-1-).R.Mgz.LAT:4tffiIVaNISNALZ4

NSILITM.rtgiVE Viz 75% Atri*ffi AMA , 0.1%VION (NaOC1) Ca M# ' g)111,1417.Knit ' JR10ArtEztfAiA,VrilffsAIVA-M-E,,ti-71K-VM,T,1(4ArylEarzain'VP--,A,15-11ffiR-1,,Pifkl% (potato dextroseagar PDA) VRV 30°CVLAttlig- ' MOM PDA TtN±. Va. 3PDA v-lifft 4°C tgmeoThi

MtlifICZ;1-VyMtuifif'4214-Eitif PDA

1 5 COR-Cf-k-Attk pH 7.5 1E (0.5%

phosphotungstic acid 0.1% bovine serum) g1=J Hitach -7000 111,J4-AAI-TVIAAN1-), 20 4,11.1tMall

tgkiR yam3 yam7 yamll yam15 yam17

IlitIUMW}A;(1. PDA V[1:11. 30°C TjA"-A- 4ff-1.1-r_tf_TtIL,1--AV (26) MMA'*'. (KOH) g11.1,14

KJEJTfifilfflA (VOL/WC-AU ' 0/F test) M 61-

(arginine dihydrolase) itirt 55 °C T2.IKnHAV (gelatin) AILfitli ' (starch) 17}1WitiE

)-] (oxidase) 01't 10% NaC1 21KtiEli ' AILt-AhriScj (catalase) '1=tijiut (tyrosinase)

AMIt 2_th-1 (production of levan) (pecto-

134i0 'AMA WOJMi K A 4Mt

Biolog

rj- yam3 yam7 yamll yam15 TSC yam17 41i"

BUG (biolog universal growth medium, me-

dium 36 g, Bacto agar 15 g) tA-Att_LI,Er& 1-)j

NfR/211-VfitarEir4V IF buffer (0.4% NaC1, 0.03%pluronic F-68, 0.01% gellan gum) IT ' M-0A.3MA20% T Biolog GP2 amparMTUJUX 150 yl

ERTE:Pez `RY 30°C -MA 16-24 hrWARitt*RNM,NAMWV4AtIVABiologGP2 'OP* (Biolog 6.1N) 1tLY4 1-Artt4fiV

OMMMR5iNg ;.

yam3 yam7 yamll yam157 yam17TSA (BBL tryptic soy broth 15 g, agar

30°C

iTOW1"4 -4 /gl1

IMEi-k(4,M-iN" (Nicotiana ta-

20 g) , >ACMiP

IP Sasser (")ffiat,',72:h-iPtFC1-111Y1B

g ,

(1)bacum.) W-1( ' ° illZatiPTLAMIta IL (saponification) : VIIIA 1 ml reagent 1 (NaOH 15 g,)1EALMMitIL'C-}VJMNA yaml° yam17 °

KNARIMMArritql4bliMIA Weft/nil ilti,t5PR,AWMAIMMITRTAYMMWMM±( k1[I35)T:),TIca1 n,y1u_m*murmgym.tm&manimg-x,m-it5EaAMIIIM03.4%/*AffEdx-

APAJOYS

Mi1-5( yam3 yam7 yamll yam15 yam17 4r10.1=41-10tkRMTMV.V-thigPDA , 30°C tifrn'Ot

methanol 50 ml, ddH2O 50 ml) 5-10 sec 100°C71(

' :6- 5 min AIWA 5-10 sec 100°C7f4i- 25 min ; (2)Ti

MIL (methylation) : 1-1fiX 2 ml reagent 2 (6 N NaOH 67

ml, methanol 55 ml) ma 5-10 sec 80°C710-'6 10 min ,

(r6;71011) ; (3)VR. (extraction) : MA 1.25ml reagent 3 (85% Hexane 50 ml, methyl-tert butyl ether

50 ml) 10 mitt

710,4 ; (4)MA (base wash) : MA 3 ml reagent 4 (NaOH

1.08 g, ddH2O 90 ml) ±T '=J 5 min

WrIA,TLILtheAROITIE, F-T# 2/3

mron GCGC-MS (HP 6890N, USA) a1-if-1,ffi 9 Aft 1), MIDI Sher-

lock® Microbial Identification System (MIS) z614*-57}

tifkLY4MT °

tutz yam 1 -20'4 20 Arif411F51#341-114 'J1-JJ-J

Bacillus Er92. B. amyloliquefaciens B ( ablA1L.4-ftl,1V g"1) Ez. B. cereus /thuringiensis A (111EAVI1Atifliffi)

fffAT1V i1q-1JUA1IniriM 100°C 10 min Nit)LOA

10,000 rpm 1 min [MR_E.MA DNA J& 2.5 ,u1 1/F

A DNA SE2-10-f / SE2-10-r (1ELq I 4 tr.f111§

sequence characterized amplified region [SCAR] -2.h-A

, f B. purrillus t; i 117 "PCR AK--t ° MO 8.0 ftwi PCR 1-

&111eM ; 5.0 mM dNTPs 1.0 units/ill Pro Taq DNA poly-

merase (Protech Technology, ROC) .1),R 2.5 ,u1 10 X J)

taftez (50 mM Tris-HCI pH 9.0, 15 mM MgC12, 50 mM

KC1, 1.0% Triton X-100) ;,1N.511StlfplA 25 ,u1 ° PCR

94°C a 1 min 94°C 30 sec

58°C 1 min 72°C 1 min 35 frirrUN

72°C 5 min 1 11:11)6IN ° 1I '14111161A Z)=-5-}91 U 1.5%

agarose (1 X TAE buffer) Z-Cil<l}}tfi (100 V) Gen-

100 DNA ladder (GeneMark Technology, ROC) a

IRAJJAUL Lk' (ethidium bromide 0.5 yg/ml)

, MACES °

16S rDNA

yam3 TX yamll NA *A#_±.2.*--n4DNeasy plant mini kit (QIAGEN, Hilden, Germany)

ITV4.-Mil DNA 16S rDNA universal primers

f8-27/r1510 "9) A L ' PCR *111M 1 1 1 ftieff,) 1.0 pm

PCR ; 5.0 mM dNTPs 2.5 units/

t1 RealTaq DNA polymerase (Real Biotech Corporation,

ROC) 1JR 5.0 pi 10 X rliJPCR&I.taffieZ '

MA so al ° PCR 94°C WI 5 min '

ig_fr- 94°C 30 sec 55°C 30 sec 72°C 140 sec 30 El

111-1 NAttf 72°C 5 min 1 illfInEM ° tgligtkZ&M

1,111J-',l 1.0~1.5% agarose Ifi1YZii<1} C ,114-C12.-Rkt_g

(sequencing) NCBI (National Cen-

ter for Biotechnology Information

fn SDSC-Biology Workbench ())1141`1V-LZI.1

. BLAST PIA (Basic Local

Alignment Search Tool) Ifi14-FT;Alal °

LIAti-glt,1WfliA- 215

kka5V1i-lAR

fVNIurtiNAvArift.*.[A1X. briAA`Vi,i,T.M7.K

.114Milk" )5n* nvk s LIAR416111M7.Kit IA 75% NAMPA ilifitarfnvii -KAAMPEP ° lurTffift* yam3 yam7 yam9yam 1 1 yam15 yam17 .1-j Pectobacterium

Erwinia) carotovorum subsp. carotovorum Pos17 Vz. Bur-

kholderia cepacia (BCRC13208 ; MnAT.P11) liThVlfrA

71-F411 1CTINCILArinibitAktrffl-M4r1i4JE ,44'12 1 tift4RIM[Vf °

P181t-ttilig

.Ifnignfnft.na (paper disc diffusionmethod) "' ^ NA fir-AJI_ElliJk:MisAiti.MMR74-Tp-1*

° r7 n1 108 cfu /ml

0.1 ml ' iM 0.8% 7.Kfilt (water agar) FP ' -1=4M. NA

; 111 -SAYill1tflhV'Hi2ft1 1)}9111R 0.14

ml ir2JWaLiAtli 13 mm n0,19241E1. -N,11111 C 3 11]--,

kr'Z'AMI1193. Ati-)sil',1,11,M71(2.i1MCISAPIPAL1 4

7-i.-12L-FIA A 2-3 ' N.111111q11 t/P ° if yarnl -17

17 1 ffi1 fifftgA71 10 fiM*11411-F ;

MSGT (streptomycin + tetracycline APAYWY,*-, 10.0% SP) NAM (streptomycin ANA

APL-fa 12.5% SL) .,1''4.1,4,-f; (thio-

phanate methyl + streptomycin

21; , 68.8% WP) (kasugamycin ai7,15)iwax 11511Lii 2.0% WP) ;

.tWILPI (copper oxychloride --ApAfsignq, 85.0% WP) 1L 17 (copper hydroxide

-1?-6 A711 kocide chemical corporation 77.0% WP)

(tribasic copper sulfate ag,V5414.27.12% SC) ;

it (mancozeb VAMMt- 200 ' 80.0%

WP) ; ire A f j,krjigrjt (kasugamycin + copper oxy-chloride 0771-AfaktzT ' 81.3% WP) ;

Ith%.04*T1112.P`X (oxolinic acid' '

) 1L4m-` , 20% WP) ' °

gni

zL

131114AlffitiMMORIMI1 1 Zt --01115TNIT.1

FR- MI A-TACO: 1, 2) 'INA-ZA-t"PileZMY,-AJ ° ftilikrqYAfififff4ZUFMA

216 IMARV-gfl 19 3 AA 2010

5-MaCtORME1±1:1V-Ps,fftilT6111.NaVAPMATA'LMMA-M,ATftMMAA,2,&MCAMiaWffott$110-LUAIIMRAATveal* , MA-MI 48 hr710:111 Agl R+ANIJIN'LIMA

it*nZMllagfE1301-11-RiANt ; 1-a-N 3; 4 *&7101MAritiitiCf4

; 5 ioww-giErAf)ozir,-ffi.

N ; (1-3) fElttrynAgRNIM

TANIJNILLIM-111-A-M-1 AfilM3MAUIPAAram ; 1118 **M8VIITF,illnii (ter , 4)

°

Mgt-6MPDA JgAM_LINTL,t M111-A-m-moittn-n

E-N-)U-V2A4 1,'C KOH ailigitMAffinAMMEkit' TEM ifft1M-FilIN , 1) itiR 5

1211-JAMIJNITV, Pst.lti-T-11,e1MPTan2, 3, 4)

IIAREAtivfliAZATA 1 , 2 (1: W3 L ; 2 : 141 LIA) ; 3 , [11NT

(Jd 3 itIVON Bacillus pumilus ; 4Fig. 1. Symptoms of bacterial leaf spot of yam in Taiwan. 1, Early symptoms of yam leaf. 2, Field symptom showed dark le-sions and necrosis of yam leaf. 3, Leaf symptoms were shown one week and 4, two weeks after inoculation.

ilAINAltAfliA 217

4

Bacillus pumilus 2..VA-AXTPAMA, (TEM) WW--, 1' A yam3 Ii1n2.1fflitT* ; 2' Atfi-A 5 ,/&&IZP[i0199tf`1+ (iliffiffitri) ; 3 ' ' 4 'Fig. 2. TEM micrographs of Bacillus pumilus isolated from yam in Taiwan. 1. Peritrichous flagella; 2. Oval shape of en-dospore (arrow); 3. formation of terminal endospore; and 4. formation of subterminal endospore.

--0MWM1111MftXMAMIRIILMJATable 1. Physiological and biochemical characteristics of Bacillus pumilus isolated from yam

CharacteristicBacillus pumilus Strains

from yam'Bacillus pumilus2

Curtobacterium flaccumfacienspv. dioscorea3

KOH test G (+)4 G ( +) G ( +)Spore forming + +0/F test 0/F 0/F 0/FOxidase - - -Growth at 55°C + + ND

Catalase + + +Arginine dihydrolase -Gelatin hydrolysis + + ND

Pectolytic activity + + NDTyrosinase - - ND

Starch hydrolysis - +Levan - -Growth in 10% NaCI + + NDTabacco HR + +Acid from:

Cellobiose + - NDGlucose + +Lactose V- - ND

Maltose V- - ND

Mannitol + + NDa, -Methyl-Glucoside V- - ND

Sucrose + + +Trehalose + + ND

Five isolates: yam 3, 7, 11, 15 and 17 were used.2 Data were cited from Saleh, et al., 1997 (23).

3 Data of Curtobacterium flaccumfaciens pv. dioscorea were cited from Wang, 2006 (27).

4 G (+): Gram positive; 0: oxidative; F: formation; +: positive reaction; -: indicated negative reaction; V-: 21-79% negative.

218 fift./VMA"-4-`4-0T1.1 M19# M 3 01 2010

M1 .tfltiLt4ill(VIALEzfl -f) Mt-MA RAM]

Tft-trffi +.:R*IL64 Vt, , wfA10% NaC1 hL 55°C 0+AN13}Afitljvz711ffjCellobiose Glucose Mannitol Sucrose Ez Trehalose

VEM.t.1 (-A-)

Biolog

Biolog 714BiritIATIVAffif yam3 yam15

yam17 04 B. pumilus C 2.fli1Llftffli 0.66 0.64 0.17 ;

yamll yam26 gh:s1 B. pumilus B 2ilifVfg5 0.140.38 ; yam7 B. licheniformis 2. MLitt 0.19 °

WOfiltGC-MS Arr-f/J}tfi & yam Mt cnran

C1®0 ;so C15:0 iso C15:0 anteiso C16:0 iso C16,0

C17:0 iso C17:0 anteiso ' Effif4-14146iA MIDI Sherlock®(MIS) erTPATr, yam3 yam7

yamll yam15 yam17 IIIIVI../-7iV15 0.735 0.663

0.729 0.737 Vz. 0.745 n-Pr' B. pumilus 24R4-1

A 98.76-99.21 % °

ANIUtf,30fiB. pumilus I f SE2-10-f / SE2-10-r

PCR fttfi ' M*W-i-lti42. 20 naMACit'InJ.%i7 tffritirffiJC/1\e'VA 500 by DNA am fi

B. amyloliquefaciens B (11ux r3f9( tfL-4.--TNf-M) Ez B.

cereus /thuringiensis A (1(11AVIWEI/RIJIt) 1=111,11111HO

At.(117.)

16S rDNArDNA k:VEt

.1,;(11111kI" 16S rDNA AW-ft2AITIN- f8-271r1510 X17 PCR /7M111iE0 R yam3 yamllY1.1*/1\5 1,404 Ez 1,339 by 2. DNA `-f)'1. M1ILA-&4_

TT DNA nififfii4N1- BLAST fil'AfTNCBI U SDSC-Biology Workbench tA-rt-it-*141+-1=1

fFIIMP1 yam3 yamll -ffinfA B. pumi-lus (isolate EI-44-7 Ai strain BOH116 GenBank accession

number AJ494734 AY94753) A 16S rDNAIiiV)-)4XrFlia 99100% frIPPIt ; rfri yam3 yamll1*-9 C. flaccumfaciens 16S rDNA (isolate SAFR-001&. strain 2PO4PE GenBank accession numberAY167859 7 t EU977762) A16S rDNAtiltin

1 1

Mit% 81-83% °

CO

ECO

CO

as

CO

CO

CO

Cl

Xr-

1 + +X`r?

cn 01- -71-

X X Xtin tr) ts-cri 2 tri+ Z ± + I

r" c--,-- X XX 00 kr)Cl .--1 4

X

±C;X

4 MX x

+ i00 4-'47' I I6 enX X

71-

kr)X

`O

X CO

cf.)

onCl e, a.)

X X ,-:, ='4) :

+ s4 + i + 4. It5 ,-,.= u. a,

X x ,,,(-1 -ur, _ +,---

CUU'---1

tr, to .4 I.)M 4 kr? ..,,-,X X .4 ... CO

in X =Cl a; u

c.-,

.-1- +c.,-,

sa,z + I + o.4

z,..,

c-i X cn . - tv ct t)

c,iX-1.

2 0°) ox w-,

'Le? c.,, z t4),-,-, ,.= E ,9

o ,-,,,

o b 4..;.' 'u.,a.) :-7-1 7:S

...-.= I.)

8ct a!.?s

.-c7 '-?.O

,,, o o=a , ;- -c,$:: _0 - --o - -F,, ',73,.. ,.. - "72,31,--, 1.., = -00 ---- CD 150t., .--, 8 = 'El cz 74 5, G 0 ,.._ ,o i.. =._,z,

qs...>..) c..) cL,a -..., a ..c)

7,-' ,c2-mC..) NM

iliKeififfiltWilA 219

4 9 10 11 1_ 13 14 15 16 17 18 19 20 21 22 23 M

500 by

111E-. PCR -TV SE2-10-f/ SE2-10-r 91fff M 100 by DNA marker ; 1 negativecontrol ; 2 Bacillus cereus/thuringiensis A (110AWM/3/A) ; 3 B. amyloliquefaciens B (RNOIL4-TV-Vi) ;4-23 yam 1-20 (LL14-aiiiffitt %An B. pumilus) °Fig. 3. Identification of Bacillus pumilus by using PCR with primer pair SE2-10-f/ SE2-10-r. Lane M, 100 by DNA marker;lane 1, negative control, lane 2, Bacillus cereus/thuringiensis A, lane 3, B. amyloliquefaciens B, lanes 4-23, B. pumilusstrains yam1-20 isolated from yam.

X=-. 11+i ITI Bacillus pumilusTable 3. The fatty acid components of yam-isolated Bacillus pumilus strains

Fatty acid yam3 yam7 yamll yam15 yam17 Reference'

13:0 iso 0.28 0.28 0.29 0.24 0.43 0.92

12:0 iso 30H 0.03 0.03 0.04 0.04 0.10 ND'14:0 iso 1.31 1.41 1.30 1.14 1.75 ND

14:0 0.48 0.29 0.44 0.30 0.66 0.87

15:0 iso 50.82 50.7 50.68 52.98 53.01 61.15

15:0 anteiso 23.35 21.71 24.91 20.43 26.38 16.95

16:1 w7c alcohol 0.36 0.95 0.38 0.93 0.51 0.90

16:0 iso 4.13 4.21 4.24 3.58 3.39 2.47

16:0 anteiso 0.05 0.08 0.04 0.08 0.04 ND

16:1 wile 0.30 0.55 0.32 0.67 0.54 ND

16:0 1.94 1.42 1.87 1.40 1.66 2.30

15:0 iso 30H 0.15 0.15 0.13 0.15 0.13 ND

15:020H 0.04 0.05 0.04 0.05 0.08 ND

17:1 iso wl0c 0.85 2.53 1.00 2.84 0.96 NDSum In Feature 4(17:1 anteiso B/iso 1)

0.31 0.72 0.31 0.72 0.34 ND

17:0 iso 7.71 8.93 8.27 8.84 5.66 6.04

17:0 anteiso 5.29 5.04 5.20 4.89 3.66 2.56

16:0 iso 30H 0.04 0.04 0.04 0.04 0.06 ND

18:1 w9c 0.07 0.05 0.03 0.05 0.08 ND

Sim Index 0.735 0.663 0.729 0.737 0.745 ND

Result from date base B. pumilus B. pumilus B. pumilus B. pumilus B. pumilus B. pumilus

I Data are combined by referring to Gala!, et al., 2006 (13)2 ND: not detectable.

220 V41M-IVW-*Til 19S M` 3 tril 2010

tkIXtlEiTgaLti-.4-ft-mm*rm-natm±Luk-ri,v5t-1-±ErgA,

Rron P. carotovorum subsp. carotovorum Pos17

(Pcc) Ez B. cepacia BCRC13208 (Bc) McIATEJJ.LLISZ

Rjatit,p§'.iiik$ B. pumilus Pcc Bc HMV,MRtEMV_EMRil,VtNIRa;WAAn RI-MAAM,yamMailnX±TARTAg

yamli a yam17 -ARIAyam3 yam7 yam9 7X yaml5 4-4it_LV

niA ; ffiVittql42. yam itiMfcnaablmaiAblt7m1

RiglEPR Pcc Bc ; IV17101akAtIlffl4 yam7 yam9 yaml5 *-R-RPRIth ,

yam3 yamll R yam17 MAU]

°

MMEAlitfAIA 17 illicrffiti-n1=J 10 fi-4111a--Rftfll

AftAZWIT 711#34i1PARTRF-J7'1Ulfialli-AT (P

, AffaliZtT2A1i1P54X-'*4ki, ARM*MOJA ZWILMAINLT 0-1 AMMAN** °

(Pcc) agiC2.AhtltUdiAlF61 6 hr P91J11

DSift-Tf01 ZalEg--..-1-4100/9.7f-4,AftiMiZtitTMAitiTt (KOH iiALR) fat2ntrwraw.Fug

it Cl , A.04*M.Fklalt-i-21xtrAAgin-ffirrl °Shaad teMAINITIM-}IMAMM.EUAltriti

coryneform bacteria (Arthrobacter Clavibacter Curto-

bacterium Rhodococcus) Streptomyces Bacillus RClostridium 4IN fliZIAINEPANtATill,31tAtClostridium Er.. Bacillus , kft--71-;( gt1 LfPlINIAWS_

aCIPARIPAM-RMITifk.fiAltltWA, Bacillus WrI/A-it ° MPki Chun 9.,1 Vidaver(9)

YM Bacillus WittVATINTTC)}A : Group I RN1- 10)MLIPat , I lit'.--IMTM19 itr

5.1-;/x)Aavzi.m WarifiraftlfREN.CrffiUWVW; Group II foNtTeTIVI13/_-_ftniaat

ExitTNTIAliWirdixPlxVIATA`h tWAVAK4j,EN MINN ; Group III Ar9t-TN inAt Tv ,T

YAVRHIN--"X'AE5-AVA 1-1-41MAMETA °

,Ii_FL,Zili<ft-.0-4ffilrThAgZ1LIMAILfift[iLLV ge.N11140-

RAI1010 Group I ° HtliftW,[11MEOLV;IIIMItINAWM IMILITittA Curto-bacterium flaccumfaciens pv. dioscorea -111.1%), k.U1114-

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ffil Erwinia aroideae E. phytophthora B. polymyxa

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VA-PrRAWI"THWITVWMMAMtR2NMATable 4. Growth inhibition of Bacillus pumilus by various agrochemicals at different concentrations

Chemical Dilution foldNo. of strains inhibited/

No. of strains testedInhibition zone(cm in diam.)

Streptomycin 1500 17/17 0.47-0.90

(12.5% SL) 1000 17/17 0.53-1.07

500 17/17 0.73-1.20

Streptomycine 2000 17/17 1.20-2.13

+ Tetracyclin 1000 17/17 1.50-2.53

(10.0% SP) 500 17/17 1.77-2.87

Thiophanate methyl 1500 17/17 0.80-1.37

+ Streptomycin 1000 17/17 1.00-1.50

(68.8% WP) 500 17/17 1.17-1.73

Kasugamycin 400 4/17 0.03-0.20

(2.0% WP) 200 13/17 0.07-0.53

100 17/17 0.23-0.83

Kasugamycin 1500 16/17 0.20-0.90

+ Copper oxychloride 1000 16/17 0.37-1.00

(81.3% WP) 750 16/17 0.40-1.07

Copper oxychloride 750 16/17 0.33-1.00

(85.0% WP) 500 16/17 0.40-1.07

250 16/17 0.50-1.23

Copper hydroxide 1000 16/17 0.13-1.13

(77.0% WP) 750 16/17 0.33-1.23

500 16/17 0.43-1.33

Tribasic copper sulfate 1000 17/17 0.10-0.90

(27.12% SC) 500 17/17 0.27-1.07

250 17/17 0.50-1.20

Oxolinic acid 2000 17/17 2.23-3.57

(20.0% WP) 1000 17/17 2.23-3.73

500 17/17 2.33-3.83

Mancozeb 1000 17/17 0.70-1.63

(80.0% WP) 500 17/17 0.83-2.03

250 17/17 1.17-2.20

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th.--V El rHIMVAT °

qi 3Z la (LITERATURE CITED)

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224 tOMMT,V*Tli 19 'ffi 3 Vi 2010

AbstractHseu, S. H.', Lai, W. C.' , Hung, Y. H.2, and Deng, T. C.3' 4. 2010. Yam bacterial leaf spot caused by

Bacillus pumilus. Plant Pathol. Bull. 19: 213-224. (' Department of Plant Protection, Fengshan Tropical

Horticultural Experiment Branch, Taiwan Agricultural Research Institute. Wenshan Rd. Fengshan,

Kaohsiung, 83052, Taiwan, R.O.C.; 2 Department of Biotechnology, Asia University, Wufeng, Taiwan;

3Plant Pathology Division, Agricultural Research Institute, Council of Agriculture, Wufeng, Taichung,

Taiwan, R.O.C.; 4Corresponding author, E-mail tcde@tari.gov.tw)

In 2008, a new, moderately sever bacterial leaf spot disease was found on yam (Dioscorea spp.)

leaves in commercial plantations throughout the central and northern Taiwan. At early stage, infected

leaves showed brownish, water-soaked lesions. Later, the lesions turned black with yellow halos. In

severe cases, the diseased leaves become wilted and eventually fall. The causal agent of the disease

was isolated and identified to be Bacillus pumilus based on the results obtained from Biolog, fatty acid

analysis, polymerase chain reaction with specific primers, and 16S rDNA sequencing. The pathogen is

capable of digesting pectate on CVP medium, macerating potato and yam tubers, but it cannot macerate

the petioles of Chinese cabbage (Brassica rapa). Disk diffusion tests were conducted and showed that

oxolinic acid has better control efficacy in comparison with the other 9 commercial pesticides. This is

the first report of Bacillus pumilus that causes yam leaf spot disease in the world.

Keywords: Dioscorea spp., leaf spot, Bacillus pumilus, agrochemical screening