Plant Pathology Bulletin 19: 213-224, 2010
Bacillus pumilus 41 k, at 137 M
1 t A i i N t1
*i 2 Ifs ;;-3- k 3, 4
' tilZ7rn''`,---*EVAMPM:61111MME3E.T4Ni/Jiffi fttfglit*;41=P t1M-4frk-T*
" fill37car,--0-E'AZAVAMARkId4 SPViiirEt : [email protected]
H J1)] t'" IA 99 12 /q 2
?,TP,P,. C461 .Y.IPA %MX. 2010. Bacillus pumilus krlig[lICEEfffiltWIA. ftAirfil19: 213-224.
2008 ' RNA-AIL ,t ` rri'AURMTIA'ITNtlitAgclgt6' flMil_416f*IVM4PIV-tfitiA% 1-CftAREI)fth EMtWileZMY,MJ ° AffV4E1gi
allARialifiCYPATA)gft ° f T r RtfL Biolog 11N1B1 Ch/ W--ftlu'a Alitttik,J=JR 16S rDNA try4-AL-yAnttiLRILiTlirit'f}tfi M*P,VrZAL94":_i5
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-
-11N,IM -n-il-NISTJ[rifla-LITMAfiff5-tffiii-YR12.[IiMAEMOMINTi*M.EMItffi 112
tliTO-q1kEPMLAMitliks4.tl-ftVffiA , wane') ,
tif0I,Z01,9AMP °Biolog Z-k.ARFP, Bacillus pumilus '} A
BR c E--.6%fit! IOEM041.1)=1114firiVi}IJ BiologBacillus pumilus B C ' a- 0.140.66,M0F,A,PArk-gW*M42.VighwCh-1,-1W
C15:0 iso C15:0 anteiso Er C17:0 anteiso VI-1*A B. pumi-
lus 2.1,1Plii-fkip-VIMMI_A- 0.663-0.745 IRW3-t;
98.76,-99.21% Galal A'13T 2006 *1±-L-LEASE.'-k-',2A1,91W B. pumilus AIVIIVink,Chlon Cl5,0
iso C15,0 anteiso 017:0 anteiso ' i3f5tA,IArA1,9:1110,71%
° ritA ift,1.4[14ffin2 16S rDNA Ji ^> NCBI f11 SDSC-Biology Workbench 2.
i4f4)*TLY1. VIMIM7Y1-1 B. pumilus 16S rDNA AI1/2}
Yliffinirii9111[Rfitiliii. 99100% fElt1M-A2.[Ii*Igq
irtfift*-94PC I E. LAtinitV/1011A-TRA C. flac-cumfaciens(27) FYI 16S rDNA 11V)-}1.1271. AfflPft%81-83% ° MJ-'71 B. pumilus SE2-
10-f / sE2-10-r"5) PCR l)-}th .1fAMARTF--,MAZ
o-Emtu-p-wt--1. 500 by DNA ap,-1.AtItzt Biolog Vi Ti 16S rDNA I rI1 17}1fi
RW--ttq I -T-74 PCR AN_IMALLA
Atli Bacillus pumilusS3ZE-f*-6--1-1K., Bacillus
pumilus )e-141_1,14 (yam) 2.1E0 °
B. pumilus VVE±.1ili iAtviArirrs]Ntri- tit/Pamm altNICIVOVIM/PAIVO
Klopper WA.fliffl Bacillus spp. *6)1
MPARtil'ItMEtt_flAVAR "6' 17) PAH T 1997
Saleh (Prunus)4g YMEz.f)Lffit persica cv. Balady) iJ
RTft_L-4VM B. pumilus rjfq I EfifiltfAMA (23)
CIE B. pumilus -ft1 .qiEtAPJA-1 ; ,aa.2006*-GalalWORAIIIIM-ftTql1-MIE.EAWAMA "3) ; 2008 54.
REIErt-B. i Milf4ItrZ B. pumilus*M RA (5) , iv-g Font '4A (10) VOA, B. pumilus 111-
TERTTLZ*MAN°WEA,MMViIA,
B. pumilus M2.13 coral-line lethal algal disease (7) ° V141414.Uf B. pumilus
ffil Erwinia aroideae E. phytophthora B. polymyxa
B. subtilis IkEgmrfisilE171RAVYM "3'23)
Galal WArtitETffNirrsJ B. pumilus tyi tiigni41 (13) *RF5VIWATAIAVAPAffiPIMAnilf*glAiYI B. pumilusfolca-,mnigut-±A I EVitArrAiR Atf LPIR.LL P.carotovorum subsp. carotovorum (Pcc) Et. B. cepacia
W-41111ffittfAl-M- 221
1P441-TR*I_LZRMEIP-E111)}2EMyam3 PAS RWEP
yam7 yam9 TX yam15 NIVR ' Ati=1=LIRMEM-±=116/LJ'4 yamll JjZ yam17 4-M-A -A.PiiRMEPfEtfIRYAME,704/}Thitit MEOMNIZAR,,N1 °Galal AtAgi:TffNillf,J B. pumilus 01#11-fr71-1A-1-
-1--TEMTh'tERItt1VIn±.Z.WhiR(13)ri-M,A01,311AVAAti" B. pumilus IVE-F-17-411f4ta,
11/AfEaTiArZPVkilA af-71.-41---MRTE#11-4464
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
222 frMgVArfli 'M 19 a AA 3 ItA 2010
tOftl4P, latigiaTV7VPAI-; iffr-r_,2. B. pumilus
MATRIttfii. Galal 111,3 B. pumilus riqrA-tittt
A B. pumilus MRAIRitriiILLRHIMM-CLE
kremi Fz-s am , WA' t* HINMIT5AfIliRattf:VitiALLIMATPARZ
tAlf(1$117ZAtkil ° EASIAAW ItftMlAng.TvfLA1 OfttlAfLAA
ItL EH rAlin3hM5-64M 1;11.1 ti-
th.--V El rHIMVAT °
qi 3Z la (LITERATURE CITED)
1. Adaskaveg, J. E. and Hine, R. B. 1985. Coppertolerance and zinc sensitivity of Mexican strains ofXanthomonas campestris pv. vesicatoria, causal agentof bacterial spot of pepper. Plant Dis. 69: 993-996.
2. Agriculture and Food Agency. 2002. Crop yields ofthe order query-Yam. Retrieved Jan. 4, 2010, fromAgricultural report resources network on the worldwide web: http://agr.afa.gov.tw/afa/afa_frame.jsp (inChinese)
3. Amusa, N. A., Adegbite, A. A., Muhammed, S., andBaiyewu, R. A. 2003. Yam diseases and its managementin Nigeria. Afr. J. Biotechnol. 12: 497-502.
4. Atu, U. G., Odurukwe, S. 0., and Ogbuji, R. 0. 1983.Root-knot nematode damage to Dioscorea rotundata.Plant Dis. 67: 814-815.
5. Bell, A. A. 2008. Bacillus seed and boll rot of cotton:Symptoms and transmission by Hemiptera. Phytopa-thology 98: S21. (Abstr.)
6. Benhamou, N., Kloepper, J. W., Quadt-Hallman, A.,and Tuzun, S. 1996. Induction of defense-related ultra-structural modifications in pea root tissues inoculatedwith endophytic bacteria. Plant Physiol. 112: 919-929.
7. Cervino, J. M., Littler, M., Littler, D., Poison, S., Gore-au, T. J., Brooks, B., and Smith, G. W. 2005. Identifica-tion of microbes associated with coralline lethal algaldisease and its relationship to glacial ice melt (globalwarming). Phytopathology 95: S120. (Abstr.)
8. Chen, C., Bauske, E. M., Musson, G., Rodriguez-Ka-bana, R., and Kloepper, J. W. 1995. Biological controlof Fusarium wilt on cotton by use of endophytic bacte-ria. Biol. Control 5: 83-91.
9. Chun, W. and Vidaver, A. K. 2001. III Gram-positivebacteria C. Bacillus. Pages 250-259. in: Laboratoryguide for identification of plant pathogenic bacteria. 3rdedition Schaad, N. W., Jones, J. B., and Chun, W. eds.APS, St. Paul, USA. 373 pp.
10. Font, M. I., Bassimba, D. D. M., Cebrian, M. C., Mo-lina, L. M., and Jorda, C. 2009. Junly 1. First report ofBacillus pumilus on Phaseolus vulgaris in Spain. NewDisease Reports 19, 019054. Retrieved November 27,2009, from http://www.bspp.org.uk/publications/new-disease-reports/ndr.php?id=019054
11. Food and Agriculture Organization of United Nation.Crops primary equivalent-Yam in 2005. Retrieved Feb.9, 2010, from FAOSTAT on the world wide web: http://faostat.fao.org/site/609/default.aspx#ancor
12. Food and Agriculture Organization of United Nation.Crops-Yam in 2008. Retrieved Feb. 9, 2010, fromFAOSTAT on the world wide web: http://faostat.fao.org/site/567/default.aspx#ancor
13. Galal, A. A., El-Bana, A. A., and Janse, J. 2006. Bacil-lus pumilus, a new pathogen on mango plants. Egypt. J.Phytopathol. 34: 17-29.
14. Hearon, S. S., Corbett, M. K., Lawson, R. H., Gillaspie,A. G., Jr., and Waterworth, H. E. 1978. Two Flexous-rod Viruses in Disocorea floribunda: symptoms, iden-tification, and ultrastructure. Phytopathology 68: 1137-1146.
15. Isenegger, D. A., Taylor, P. W. J., Mullins, K.,McGregor, G. R., Barlass, M., and Hutchinson, J. F.2003. Molecular detection of a bacterial contaminantBacillus pumilus in symptomless potato plant tissuecultures. Plant Cell Rep. 21: 814-820.
16. Kloepper, J. W., Rodriguez-Ubana, R., Zehnder, G. W.,Murphy, J. F., Sikora, E., and Fernandez, C. 1999. Plantroot-bacterial interactions in biological control of soil-borne diseases and potential extension to systemic andfoliar diseases. Austral. Plant Pathol. 28: 21-26.
17. Kloepper, J. W., Ryu, C.-M., and Zhang, S. 2004.Induced systemic resistance and promotion of plantgrowth by Bacillus spp. Phytopathology 94: 1259-1266.
18. Lebas, B. S. M., Ochoa-Corona, F. M., Elliott, D. R.,Tang, Z., and Alexander, B. J. R. 2005. Partial Char-acterization of a Carla-Like Virus Infecting Yam (Di-oscorea spp.) from China. Plant Dis. 89: 912. (Abstr.)
19. Lipson, D. A. and Schmidt, S. K. 2004. Seasonalchanges in an alpine soil bacterial community in theColorado Rocky Mountains. Appl. Environ. Microbiol.70: 2867-2879.
20. Liu, S. L. 2005. Yam. (Special Crops). Pages 225-228.in: Taiwan agriculture encyclopedia. vol. 1. HarvestFarm Magazine of non-profit organization, Fang ed.Taipei, R.O.C. 404 pp. (in Chinese)
21. Ni, H. E, Hsu, S. L., and Yang, H. R. 2007. Evaluationof different method for inoculation for Pratylenchuscoffeae on yam. J. Taiwan Agric. Res. 56: 99-106. (inChinese)
22. Park, K. S. and Kloepper, J. W. 2000. Activation of
PR-la promoter by rhizobacteria that induce systemicresistance in tobacco against Pseudomonas syringae pv.tabaci. Biol. Control 18: 2-9.
23. Saleh, 0. I., Hung, P. Y., and Hung, J. S. 1997. Bacilluspumilus, the cause of bacterial blotch of immatureBalady peach in Egypt. J. Phytopathology 145: 447-453.
24. Sangoyomi, T. E. and Ekpo, E. J. A. 2002. First reportof Nattrassia mangiferae as a postharvest fungalpathogen of white yam (Dioscorea rotundata) inNigeria. Plant Dis. 86: 919.
25. Sasser, M. 1990. Identification of bacteria through fattyacid analysis. Pages 199-204. in: Methods in phytobac-teriology. Klement, Z., Rudolph, K., and Sands, D. C.eds. Akadetniai Kiad6. Budapest. 568 pp.
LUCEFLAtiXillf-A 223
26. Schaad, N. W. 2001. Initial identification of commongenera. Pages 1-10. in: Laboratory guide for identifica-tion of plant pathogenic bacteria. 3rd edition Schaad, N.W., Jones, J. B., and Chun, W. eds. APS, St. Paul, USA.373 pp.
27. Wang, G. J. 2006. Identifying pathogen of bacterial leafspot disease in the yam. Henan Agricultural University,master's thesis. 55 pp. (in Chinese)
28. Yang, T. C. 2005. Study of incidences and control ofmajor pests on yam, Dioscorea spp. in Hualien area.Bull. Hualien DAIS. 23: 15-29. (in Chinese)
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 [email protected])
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