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S. Diab et al.
STUDIESOF INFECTIONWITHXANTH
PV. VESICATORIA,
CAUSALAGENTO
IN ISRA
OF PEPPER
S. DIAB.* Y. BASHAN** and
Xanthomonascampestrispv. vesicatoria,
the caus
per, wasisolatedin severalregionsin Israel.When
ticed, pathogengrowth was enhancedby high te
inoculumconcentrationof 106 colony-formingu
for symptomsto developon plants.Pre-inoculat
the leavesby rubbing them with carborundump
dilutedwax solvents,markedlyincreaseddiseases
Pre-inoculation
conditioningat two differentrelativ
or R.H. ( 407o)did not affect diseaseseverity.Y
affected following infection than older leaves.D
severalisolatesoI X. campestrispv. vesicatoia.
KEY I|ORDS: Epidemiology;Capsicumannum; i
campestispv. vesicatoria;
bacterialspot of pepper
I
INTRODUCTIO
i
Bacterialscab(leaf spot) of pepper is caus
vesicatorio(4)
andresultsin cropdamagein Israelma
The first infection symptomsto appearon
scablikelesions.These lesionssubsequentlybec
centerand a smallchlorotic zone. In heavilyinfect
tion which resultsin fruit damagedue to sun exp
thedaytimetemperatures
in the summersometim
i
hrblication of the Agricultural Research Organization. Re
f o r mJ u n e 1 5 , 1 9 8 2 . N o . 5 2 3 - E , 1 9 8 2 s e r i e s .
* Dept. of Plant Pathology and Microbiology, The Hebre
Agriculture, Rehovot.
** Div. of Plant Pathology, ARO, T.he Volcani Center, Bet
I 0:3, I 982
Phytoparasitica
also be found in relativelywarm winters(day temperature2
the world it usually developsat an intermediate tempera
under high relativehumidity (R.H.) (4, 5,9, 10, l2). St
preliminary field observations,have shown that bacterialsc
a-weeksprinkle-irrigatedfields, in which the plant leavesre
becauseof the prevailingarid conditions(temp. ) 30oc;R
tions are usually adversefor multiplication of bacterialpa
leaves(8, I 5).
The purpose of this study was to explore the fac
pepper by X. campeslnspv. vesicatoria,in order to establi
further studies.
MATERIALS AND METHODS
Twenty strainsof Xarethomonas
campestrispv.vesica
fectedpepperplantsin the summerfrom four areas,name
Lakhishregion,the JordanValley, and the Jerichoarea.Th
nutrient agar(Difco) platessupplemented
with 0.15 g/liter
pathogenswere kept on nutrient agar slopesat room te
transferred to fresh medium weekly. To prevent loss of
leaveswere inoculatedwith the pathogenand the bacteriaw
a month according to the leaf enrichmentmethod (6, 14\
leavescontaininga viablepathogenpopulationwerekept d
g e l( 1 ) .
An isolate of X. campesfnspv. vesicatoia from the
most of the infection studies.Bacterialpopulationswere
viously(3).
Pepperplants cv.'Ma'or', susceptible
to bacterialsca
ments.The experimentswere carriedout in humid chamb
of polyethylenebags,placedeither in a fully controlleden
(30' t 2"C, 16 h light, 75 Wlm2, 8 h darkness)or phytot
[3 secmist every30 min, 30o + 2"C,daylight(3)]. Seedsw
Co.,Haifa.Experiments
wereconductedtwo or threetimesin
fashion with ten replicates.Other susceptiblecultivars(
Naharia)wereusedalso.
Growth conditions,inoculum preparationand inocu
(2, I l).
described
elsewhere
After inoculation,plantswere further irrigatedby dri
foliagedry.
Growth of bacteriain liquid mediumwasmonitoredin
equippedwith side arms,eachcontaining25 ml nutrient br
were incubatedat temperatures
rangingliom l5o to 37oCo
tl
184
':Itj ':T1in drYmost or the
dav
ii"]
mp.) 30"C:R.H.< 40Zo).
Thesecondi
of bacterial
pathogens
on the surface
of
:plorethe factorsaffecting
infectionof
:derto establish
a system
thatwill enable
overthe leaf; 2 = 6-10 scabs;3 = more than I 1
rpread
wereexamined8 days after inoculation; four plants
number
of scabsper leaf was countedseparatelyand th
considered
asthe disease
index of the plant.
RESULTS
) METHODS
Ttspv.vesicatoriawereisoiated
from in.
:.areas,
namely,the yizre,el Valley,
the
'icho
area.The bacteriawereisolated
on
t 0.15gilitersodiumdeoxycholate.
The
temperature(20o_40oc)and
1r.t:or
lossof pathogenicity,the pepper
I:1t
nebacteria
werereisolateaat leastonce
thod(6, l4). Also lyophilized
infected
werekeptdry in glassboxes
with silica
,ia from the Jericho
TABLE 1
EFFECT OF INOCULUM CONCENTRATIONO
CAMPESTRISPV, VESICATORI,4ON BACTERIA
IN PEPPERPLANTS
(Disease
indiceswererecorded8 daysafte
after 14
and defoliationpercentage
area was usedin
rtionswerecountedas described
pre-
lacterialscab,wereusedin
all experi_
n i d c h a m b e (r 2
s 0x 30 x 40 cm) made
ntrolledenvironnlent
growth chamber
r).orphytotron,or in a mist
chamber
3)j. Seeds
wereobtainedfrom Hazera
tree_
timesin a completelyrandomized
cultivars(California, Zohar,
Zahov_
and inoculationprocedureswere
atedby drip irrigation,which
left
Inoculum
concentration
(CFUlmt)
0
10,
103
as
10"
105
10u
10'
10'
the
onitoredin 100-mlErlenmeyer
flasks
nutrientbroth.The inoculated
flasks
'
to 37oCon a rotary shaker,
and turphytoparasiticoI 0:3, gg2
I
Effectof inoculum concentration of X. campestrispu.
symptoms
in pepper
Pepperplantsweresprayedto run-offwith pathoge
levels
between 102 and l0E colony-formingunits (C
sprayed
with sterilewater.Diseaseindex was determin
humidchamber.Pepperplantsreact to stressby defolia
fore,percentage
of defoliationand day of appearance
o
Theoptimum level for efficient artificial inoculatio
106CFU/ml (Table l). Scabsymptomswhichdevelope
in naturallyinfested fields. Percentageof defoliation
infection
could be achievedat very low inoculumlevel
periodsof time and with a lesserseverity.
longer
I
I
I
Disease
index
Defoliation
(%)
(0-3)
0e*
0 . 0 4 8e
0 . 1 4 7e
0.52 d
1.48 c
2.36 b
2.64 a
2.72 a
0d
0d
0d
3 . 0c
7 . 5c
14.',7
b
1 9 . 1b
3 8 . 3a
* Numbers followed by different letters in the same colu
at P = 0.05.
Phytoparasitica 1 0: 3, I 982
Effect of mechanicar and chemicar pretreatments
on diseaseseverity
Prior to pepper infection by spraying
rot crui*r1 the fol
rreatmentswere carried out. pepper leaves "ntrr
were rubbed with carborund
(300 grid), or punctured
fu:
n.rar.r, or sprayed with d'uted
w
l,l
e.g. chloroform, acetoneand
NaOH (O.l%, O.lVo and0.001 N, respective
to facilitate penetration. control prants
were-sprayedwith sterile water
wax solvents,or they were mechanically
wounded, or not p;;";;;.
L
were transferredto a humid chamberor
to a mist chamber.
Each of the pretreatmentssignificantly
increasedthe number of sca
leaf and the percentageof defoliation (rig.
r, A and B). However,x.' cam
@
TREATM
z
o
!
iod
a
s
RH: too o/.
RH <40 ./.
of differenrpretrearments
on disease
severity
!rt;j:.!j:::,
of bacterial scab of pepper
causedby Xanthomoiar;;;;;:
tris pv. vesicatoria.A. Mechanicalpretreatments.
S. Ol._i..t
pretreatments. C. Environmental
pretreatments- Hatched
areasrefer to defoliation. Different letters
at tops of
-aiirrr.n".
columns
jyth^*^ _the same graph) indicate significait
(P= 0.0s).
r86
Phytopalasitica I 0
s on diseaseseveity
vith 106 CFU/ml, the following
pre.
re rubbed with carborunOumpowder
or sprayedwith diluted wax
solvent,
6 and 0.001 N, respectively),
in order
prayedwith sterilewater or
with the
ded,or not pretreated.Later,
plants
. chamber.
;reased
the number of scablesionsper
\ and B). However,X. campestris
pv.
ttesicatoia
wascapableof infectingpepperplantsalsowithout m
pretreatments,
providedthe plants were later subjectedto per
Nodifferencein diseaseseveritywasobservedbetweenincubatio
and
a mistchamber.
he-infectionrelative humidity and diseaseseveity
Pepperplants were maintainedat R.H. = 4Vo in contr
R.H.= 1007oin humid chambersmade of polyethylenebags
previously,and incubatedin a humid chamberor in
described
407,R.H. for 8 days.control plants were sprayedwith sterile
under
thesameconditions.
plantsto different R.H. conditionsprior to inoc
Subjecting
0nthe numberof lesionsper leaf or on the percentof defolia
keptat R.H. = 100%after inoculationdid show disease
symptom
incubated
under40% R.H. for 8 daysshowedno disease
symptom
Effectof method of injection on diseaseseverity
Two methodsof injectionwereexamined:(a) Direct injecti
suspension
into leaves,using a hypodermicneedle;and (D) slo
bacterial
suspension
into the stem(2). Inoculumlevelswere l0a
halfof the plantswere incubatedfor 7 days in a growth chamb
thesecond
half at R.H. = 100%.control plantswereeitherspray
pensions
or with sterilewater until run-off, or were injectedwi
experiment
showed that injection of 104 cFU x. campestis
pepper
leavesafter a 9-day period of incubationat R.H. I 4W
resulted
in localizednecrosis.
Injectionof the samedoseinto tlle
symptoms
on leaves.However,sprayingof leaf surfaces
with the s
incubation
at R.H. = 100%resultedin a diseaseindex of 0.2. In
higher
bacterialconcentrations
(10e CFU/rnl) causedeither sev
p l a n tw
s e r ei n c u b a t e da t R . H . < 4 0 % ) o r p l a n t c o l l a p s e
(at R.H
intothe stemdid not resultin any apparentsymptoms.Howev
withthe samesuspension
led to severedisease(diseaseindex =
incubata
e tdR . H .= l j W c .
on disease
severity
nthomonascampes_
ments.B. Chemical
9atments.Hatched
at topsof columns
rificant difference
Phytoparasitica I 0 :3, I gg2
Infectionof pepper plants with different isolates
Twenty summer strains of X. campesftispv. vesicatoru
regions
in Israel,were testedfor their ability to initiate bacteri
significant
differenceswere tbund among the isolatestested.Is
theYizre'elValley, the Lakhishregion,the Jerichoareaand the J
a disease
indexof 1.42, | .29, 1.53 and I .8, respectively.
Effectof leaf ageat time of infection on diseaseseverity
Pepperplants with four, six or ten mature leavescorresp
3 monthsafter seeding,respectively,
were inoculatedwith 106 C
eitherincubatedlbr 8 daysin a humid chamberat R.H. = l)Wo
Phytoparasitica
I 0:3, I 982
Diseaseindex for matureinoculatedlea.res
and for young leaves(wh
to full size during the incubation period) was monitored. It was found
pestris pv. vesicatoria developedbetter on young leavesof pepper
developmentand disease
severityincreased
concomitantly.
TABLE 2
DISEASEINDEX OF YOUNC LEAVES AND MATURE LEAV
ON THE SAME PEPPERPLANT INOCULATEDWITH
XANTHOMONAS CAMPESTRIS PV. VESICATORIA
(Experiment repeatedtwo times, with three replicates.)
Plant age
(months)
Diseaseindex (0-3)
Youngleaves
1.5
2
3
Matur
2 . 6 2a *
2 . 3 9a
2 . 5 5a * *
0.
0.
0
+ Numbers followed by different letters
in the samecolumn differ signifi
atP= 0.05.
| | Incubation in petri dishes.
,af
1
^'uI
-c
td
U
=
el
F
z
tr
<r.
EI
!''f
ttz
u
z
U
10
?0
30
/.0
l0
I E M P E R A T U R E( O C )
20
30
Fig. 2. Genetation time (left) and lag time (right) of Xanthomonascampestrispv
liquid culture.
188
Phytoparasi
development
ungleaves(which developed dnease
ttre effect of incubation temperature on the growth of X. c
. It wasfound that X. cam- I
:s of pepperand that leaf
'TATURELEAVES
,ATEDWITH
SICATORIA
:e replicates.)
ndex(0-3)
Mature leaves
0 . 9 7b
0 . 4 8b
0 . 1 2c
\?
Uo
4
U
(r)
umn differ significantly
I
o
Fig. 3. Effect of different temperatureson diseaseseverity of
bacterialscabof peppercausedby Xanthomonascampestrispv.
vesicatoria.Different letters at tops of columns indicate significant difference(P = 0.05).
DISCUSSION
AO
nascampestrispv. vesicatoriain
PhytoparasiticaI 0 :3, I 982
Pepper
is one of the most importantcropsof the Israeliexportveg
andbacterial
scabofpeppercauses
seriousdamageto the fruit. To date,n
sive
studyhasbeenreportedof the factorsaffectinginfectionby this dis
a fewstudiesof artificialinoculationunder different conditionsare av
13,17).Wehaveshownthat in order to obtainclearscabsymptomsund
mentalconditions it is necessaryto use a high concentration of X. c
vesicatorio
isolates.However,some symptoms(diseaseindex = 0.05) cou
tained
with a low inoculum doseof the pathogen.Although disease
wa
factors
whichcommonly favor bacterialinfections,suchas tissuedama
theisolates
were also capableof causingsymptomswhen sprayedon
pepper
plants,incubatedunder high R.H., but which had not beensub
highR.H.treatment.
Theresultsof this study alsoconfirmedpreviouswork (7, 10, l6)
of
scab tomato and chili, suggesting
that X. csmpestis pv. vesicatoriais m
youngleaves,
attack
which developto full sizeafter infection.
Phytoparasitica
10:3, I 982
Information in the literature concerning optimal tem
development indicated that symptoms of bacterial scab deve
m e d i a t et e m p e r a t u r e( s2 2 ' - 2 7 " C )( 4 , 5 , 7 , 9 , 1 0 , 1 2 ) . T h e d a t ap
indicated that Israeli isolatescausedthe most markeddiseasesy
tron conditionsat temperatures
of z'7o-32oc,and even at36o
diseaseseverity.
A direct significantcorrelationbetweenratio of diseaseinde
time of bacteriainliquidcultureson the one hand,and tempera
was obtainedin the temperaturerangeof l5o-32oc (y = 0.04
Above thesetemperatures
(32o-37oc)there was no correlatio
whether the Israeli isolatesof X. campeslrrspv. vesicatoriaor
isolatesandbecameadaptedto the local warm conditions or whet
ACKNOWLEDGMENTS
This work was partially supportedby grant No. 823/026 f
Researchorganization,Ministry of Agriculture,Israel,and by gra
the United States-Israel Binational Agricultural Researchand
(BARD).
REFERENCES
t.
4.
6.
7.
8.
9.
10.
tl
190
Bashar,Y., Diab, S. and okon, Y. (1982) Survivalofxazthomonas
in pepperseedsand roots,in symptomless
and dry leavesin n
soil. P/. So/ 54 (in press).
Bashan,Y., okon, Y. and Henis,Y. (1978) Infectionstudiesof pser
agentofbacterial speckoftomato. Phytoparasitica
6: 135-14
Devash,Y., okon, Y. and Henis, Y. (1980) survivat of pseudom
seeds.Phytopath. Z. 99 I 7S-l 85.
Doidge,Z.M. (1921) A tomato canket.Ann. appl.Biol. 7: 407430.
Gardner,M.w. andKendrick,J.B. (1921)Bacterialspotof tomato.J. a
Henis, Y., Okon, Y., Sharon,E. and Bashan,y. (1980) Detecti
phytopathogenic bacteria using the host as an enrichmentme
vi (abstr.).
Leben, C. (1963) Multiplication of Xanthomonas vesicatoia on t
pathology 53:778-781.
Leben,C., Daft, G.C. and Schmitthenner,A.F. (196g) Bacterialblig
tion levels of Pseudomonasglycinea in relation to sympto
pathology58: I 143-1145.
Morton, D.J. (1966) Bacterialspot developmentin excisedpepp
severaltemperattrrcs.Phytopathology 56 : I I 94-l I 95.
Nayudu,M.V. and Walker,J.C.(1960)Bacterialspot of tomato asinf
and by the ageand nutrition of the host.phytopathotogy50:
Phy
Okon,Y., Bashan,Y. and Henis,Y. (1978) Studiesofbacterials
heudomonastomato.Proc.4th Int. Conf.Pl. Path.Bacr. (A
Peterson,G.H. (1963) Survivalof Xanthomonasvesicatoriain
plants.Phytopathology 53 : 76 5-'167 .
Schnathorst,W.C. (1966) Unalteredspecificity in severalXan
passagethrough Phaseolusvulgaris.Phytopathology 56: 58{
Sharon,E., Okon, Y., Bashan,Y. and Henis,Y. (1981) Leaf e
detectingsmallnumbersof phytopathogenicbacteriain see
ioof disease
indexin plants/generation
of vegetables.
Phytoparasitica
9: 250 (abstr.).
d, andtemperature
on the other hand, 1 5 . Shaw,L. (1935) Intercellularhumidity in relation to fire-blights
-32'C(Y = 0.04x - 0.652; =
pear.Mem. ComellAgric. Exp. Stn l8l: 340.
r
96.6,).
ts no correlation.It is not yet known
t0.
shekhawat,P.S. and chakravarti,B.P. (1976) Factorsaffecting
leaf spot of chillies causedby Xanthomonasvesicato
v. vesicotoriooriginated from foreiqn
393402.
nditionsor whetherthey are endemi.
t1 Vakili, N.G. (1967) Importanceof woundsin bacterialspot (Xa
tomatoesin the field.Prrytopathology57: 1099-1103.
t 8 . Volcani,Z. (1962) Bacterialspot diseaseof tomatoesand pepp
46:175.
1 9 . Yunis,H., Bashan,Y., Okon, Y. and Henis,Y. (1980) Two sourc
speckof tomato causedby Pseudomonas
tomato.Pl. Dis.6
ng optimal temperaturesfor disease
terialscabdevelopedmainly at inter.
), l2). The datapresented
in this work
narkeddisease
symptomsunder phyto.
and evenat 36oCplantsshowedhirh
INTS
: No. 823/026from the Agricultural
rael,andby grantNo. I-214-g0from
Research
and DevelopmentFund
>f.Xanthomonas
campestrispv. vesicatoria
td dry leaves
in nonhostplants and in the
n studiesof Pseudomonastomato. causal
tsitica6:135-143.
val of Pseudomonastomoto in soil and
iol.7:407430.
rt of tomato../.agric.Res.2l: 123-156.
(1980)Detectionof small numbers
of
t endchmentmedium. appl. Bact. 49:
"I.
,esicatoiaon tomato seedlings.phyto.
8) Bacterialblight of soybeans:popula_
ion to symptom development.phyto.
excisedpepper and tomato leayesat
- 11 9 5 .
f tomatoasinfluencedby temperature
Dathology
50: 360-364.
PhytoparasiticaI 0:3, I 9g2
Phytoparasitica
I0 :3, I 982