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Y . B a s h a na n d I . A s s o u l i n e ( 1 9 8 3 ) P h y t o p a r a s i t i c at t ( 3 , 4 ) ;
BACTERIALENRICHMENTTECHNIQUES
FORT
COMPLEMENTARY
DETECTIONOF PSEUDOMONAS
SYRINGAEPY. TOMATO AND
XANTHOMONASCAMPESTRISPV. VESICATORIAIN INFESTEDTOMAT
PEPPER
SEEDS
Y. BASHAN*and I. ASSOULINE**
A schemefor routine seed testing for Xanthomonascampestis pv, \'esicatot
and keudomonas slringae pv, tonato in pepperand tomato seedswasdevelope
The schemeis based on different bacterial enrichment techniques.As few a
1000 and 10-100 colony forming units per gram of seedswere detected using
liquid endchmenttechniqueor leaf enrichmenttechnique,lespectively.Relative
large amounts of saprophyteson the seed surlacesdid not interfere with th
detection of the pathogens.
KEY IIORDS: Bacterial speck of tomato; bacterial scab of pepper; seedbor
palhogens:
phytopathogenic
bacteria.
INTRODUCTION
The production of tomato and pepper seedsfree of the pathogenicPseu
syringaepv. tomato and Xanthomonas campestrispv. t)esicatoia is of great e
importance for both the seed industry and the glowels. It is cornmonly agr
detecting small numbers of phytopathogenic bacteria in seedsis difficult, du
relatively large numbers of saprophytic bacteria which accompanythe patho
the seedsand interfere with the detection, due to their fast growth on selecti
(12, 15). The methods ard procedures developed to oyercome this proble
reviewed
recently(9, l0).
Tomato and p.epperplants which germinatefrom seedsinfested with P.
pv. tomato ajid X. campestris pv. vesicatoria may develop diseasesympto
initiate an epidemic in the field, if the environmental conditions are suitab
5 .8 . 2 0 r .
ReceivedJan. 18, 1983; receivedin final folm Aug. 15, 1983.
* Dept. of Plant Piithology and Microbiology, The Hebrew University of Jerusalem,F
Agriculture,Rehovot.
Presentaddress:Dept. of Plant Genetics,The WeizmannInstitute of Science,Rehovot
** Analyst Ltd., Qiryat WeizmannLeMadah,Rehovot.
Phytoparasiticd11 : 3 4, 1 983
Heat treatment and fermentation for tomato seeds(2, 5) and chlorine t
for pepper seeds(4) are commonly practised in the elimination of these p
However, efficient quality control of the disinfestation plocess dep€nd
methods used to identify the pathogenswithin the host seedsand on their
Recently, a sehsitive method was developed to detect very small numb
syfingae py. tomato and X. campestrispy. vesicatoia in tomato and pepper s
The method is based on the selectiveenrichment of the pathogensinside th
host leaves.Th€ sensitivity of the method is within the range of 10-lO0 ba
gram of seeds.The rnain drawback to this method is that only leaves
absolutely pathogen-freecan be used. An additional difficulty, although
one, is that t}le tomato or pepper plants must be grown at least 6 weeks b
test. Therefore, a conventionallaboratory technique for routine work is stil
In this study, we describecombined bacterial enrichment techniques(leave
media) for routine seed testing for X. campestris p't. vesicatoria ar,d,P. syr
tomato in pepper znd tomato seeds.
MATERIALS AND METHODS
Organisms,Growth Conditions and Inoculum heparation
Pseudomonassyingae py. tomato (PST) (WT-I, WT-2, WT-3, Bet Dag
and ATCC 10852), Xanthomonss campestris py. vesicatoria(XCV) (R-2,
Dagan and ATCC 11633), tomato plants (Lycopersicon esculentum) cv.'V
(susceptible to bacterial speck, ref. l9) and pepper plants (Capsicum ann
'Ma'ol'wele grown
asdescribedpreviously(l).
Cultures used as inoculum were grown on a yeast peptone liquid
containing 0.06 M potassiumphosphatebuffer, pH 6.8, in a shakingbath (100
min) at 30"C for 24 h. The cultures were centrifuged at 12,0009 for 10 min
three times in the same buffer, and finally adjusted to 0.3 absorbanceunit
nm (for PST) and to 0.1 absorbanceunits at 420 nqr (for XCV) h a Junior lI
spectrophotometer,correspondingto l0e colony-forming units (CFU) per ml.
Media
The foUowing media were used:
Nutdent broth (Difco), 8 g per liter distilled water, for inoculum prepara
total bacterialcount.
Nutrient-sodiumdeoxycholate(ND), containing 23 g nutrient agar(Difco) and
sodium deoxycholate per liter (applied after autoclaving),to count XCV from
pepper seedsand leaves.
King-B-Fuchsin-TTc (KFT), containing King-B medium (6), 9 rng of basi
and 1.4 mg triphenyltetrazolium chloride (Sigma), to count PST from toma
and leaves.
188
Phltoparusitica I I :3
2, 5) andchlorinetreatment
dnationof thesepathogens.
n processdependson the
seeds
andon their surfaces.
very smallnumbersof p
matoard pepperseeds
(15).
)athogens
insidetheir living
tngeof 10-100bacteria
per
that only leavesof plants
fficulty, althougha minor
at leasl6 weeksbeforethe
rutinework is still needed.
techniques
(leaves
or liquid
UatoriaandP. syringaepv.
Liquid King-B medium (K), for enrichment of PST.
Phosphatebuffer
sodium deoxycholate (PBD), containing phosph
6.8, with 200 mg sodiumdeoxycholate,was used for enrichmentof X
seeds.
Leaf and SeedDisinfestation
Prior to inoculation,leaveswere washedunder a streamof ta
min, soakedfor 3 min in a 0.57osolution of sodiumhypochlorite(Fru
and washedagainfor 10 min under a streamof tap water. Seedswe
mtn \n 2% of the same solution and washedthree times with sterile
Seedswere placedin sterilepetri dishesand allowedto dry for 3 h in
hood.
SeedInoculation
Surface-disinfested
seedsor commercialseedswere infestedby p
(lOr to lOe cells/ml)undervacuu
seedsin 200 ml bacterialsuspensions
was broken abruptly to favor penetration of the pathogensinto the
PST was used for tomato seedsand XCV for pepperseeds.Seedswe
scribedabove.
2, WT-3,BetDagan134.1
via (XCv) (R.2, R-3, Bet
esculennm)
cv.'VF-198,
:s(&psicumannuum) cv.
Leof Inoculation
After surfacedisinfestationthe l€ayeswere placedasepticallyon 0
plates with their bottom side up. The leaveswere inoculated with a 2
ot previously infested dried seedsin sterile distilled water as describ
petri dishes were sealed with parafilm to prevent drying and cross
andincubatedunder continuouslight (100 Wm') at 28'C.
peptoneliquid medium,
fiakingbath(100 strokes/
,000gfor l0 min, washecl
I absorbance
units at 540
iV)in a JuniorII Coleman
its (CFU)perml.
Liquid Enichment
King
- Seed samples(4000 seedseach) were soaked in the liquid
28"C in Erlenmeyer flasks and shaken vigorously for 4 h in a sha
strokes/min).
toculum preparation and
agar(Difco) and 200 mg
lount XCV from infested
r, 9 mg of basic fuchsin
PST frorn tomato seeds
opota tica 11:34, 1993
Determination of Bacterial Population from Leqves and Seeds
Viable bacterial counts from soaked seed suspensions
were
spreading
0.2 ml of a ten-folddilution (17) with a glassrod on agarpla
KFT media. Total bacterialcountswere performedon nutrient agarp
werecountedafter 48 h of incubationat 28oC.
Colony ldentificatio n
Colonies suspectedof being XCV were typically yellow and m
extract-dextrose-CaCO3
agar (18). Coloniessuspectedof being PST
marginsand produced a typical fluorescent pigment. All PST colonies
negative(&) Saprophytic pseudomonadswere oxidase-positiveand gr
PhytoporasiticaI I : 34, 1983
the parasitic species.After 24 h the saprophytesformed strongly fluorescentco
with smooth edges(15). Suspectedcolonies were tested for pathogenicityas fo
Two plants with 4-6 true leavesfor each isolate were sprayed with a w
(l0e CFU/ml, supplemented
bacterialsuspension
with 0.1 g carborundum,3O
and incubatedseparatelyin partialmist chambers(5 secmist every15 min, at 2
(1, 5).
for PSTor 30:2"C for XCV for 6 or l0 days,respectively
All experimentswererepeatedtwo or threetimesin four replicateseach.
RESULTSAND DISCUSSION
A schemedevelopedto detect XCV and PSTin pepperand tomato seeds
fulfill the following requirements:/4/ lt should b€ sensitiveenoughto detect a
a number as possibleof the phytopathogenicbacteria;/b/ it should requir
technical expertiseor equipment and be relatively inexpensive,and /c/ resultss
b e o b t a i n e dw i t h i na f e w d a y s( 9 , l l , l 3 , 1 4 , l 6 ) .
FiguresI and 2 show that by using the leaf and the liquid enrichment tech
as few as 10-100and 1000 CFU/g seeds,respectively,
were detectable.(Comp
Ieft and the right columns in each pathogen infestation level.) No decreaseoc
in pathogen detection from infested seedswhich had not been previouslydisin
PST colonies were detectable on the KFT diagnosticplates in spite of the fac
largenumbersof saprophyticbacteria(up to IOE CFU/g seeds)developedat th
time. The interference of saprophytic bacteria in detection of XCV was minim
to the supply of antibiotics to either the enrichment or growth medium. The t
yellow pigment of XCV also helped to distinguish it from other saprophytes.S
trends in the resultswere obtained from all isolatestested.
'f
-0151!t!5E!l!E0:
!l
ilu
lr
;,f
3
9l
B1
lLliil
Ert
oL
I
o",r*tarseg
Z t -^^,r^ .. ,,*,.,,.
srenoenvrrs
! ur oenrrrreo
ffi
f. --*,r^
pv y.5,tufo'o AFr€F €
Fr& -1. Detection of Xanthomonat compestis pv, yesicdtoriarn pepper seeds.PBD
incubation medium: ND. count method.
190
Phytoparcsitica I I:34,
stronglyfluorescentcolonies
for pathogenicityas follows:
were sprayedwith a washed
.l g carborundum,
300 grid)
nist everyt5 min, at 22!2" C
(1,s).
,:f
four replicateseach.
,l
rperand tomato seedsshould
ive enoughto detect as small
; r/D/it should require little
rnsive;and /c/ resultsshould
t't
;l
;"t
E'l-l
rF
iquid enrichmenttechniques
re detectable.(Compare the
level.)No decreaseoccurred
beenpreviouslydisinfested.
rtesin spite of the fact tnat
ieeds)developedat the same
n of XCV was minimal due
rowth medium. The typical
r other saprophytes.Similar
a!t!!!!
N. ,.voto.a
gE
pr r&.otano
AFTER €NRTCHVENT
u peppe. seeds.pBD,
,towdritica
I I:34,
1983
.L
SEEDS
OISINFE5TEO
I
illilr
l|l rllI
t
lill
/l[il
urunm seeos
Q e vrw c"
E uNro€NrF
l]]fl e zlrer pv
(loq no ol bocl€no)
TNFESTATIoN
Fr& 2 Detectionof heudomonassyfingaepr. tomatoin tomaloseed
medium:
KFT.countmethod.
In spite of the heavy saprophytic interference, the ten-fold
enrichedsuspensions
used for the detectionof both bacteriasho
or 1O-2. In higher dilutions, the pathogenswere not detectable.Na
bers of saprophytesgiven in Figs. I and 2 were counted by the con
technique.
The sensitivity of the medium enrichment techniqueswas 103
it was found suitable for routine inspection of highly contaminate
ever, in the few caseswhere only small numbers of pathogenicbact
seeds,a leaf enrichment method which is more precise but more
routine work was used.It was found that when seedswere inoculated
containing l0 CFU/ml, the medium enrichment technique did ro
bacterial cells. However, after enrichment of the sam€ suspensionsi
host leaves, both pathogens could be detected easily. The recom
detection of PST and XCV is givenin Fig. 3.
Thirty-eight commercialpepper lots and 53 tomato lots (200 g
by the liquid enrichment method for the presenceof the compatib
sampleswere contaminated with saprophytic bacteda at levelshighe
seeds.Sevenpepper lots and 28 tomato lots wer€ contaminated'wit
pathogen at levels higher than 103 CFU/g seeds.The remaining lo
the leaf enrichment method and it was found that eight pepper lots
lots were contaminatedwith the compatible pathogenat low concent
These enrichment techniques are inexpensive, simple and r
Phytopararitica 1l : 3 4, 198 3
40 ml Kins ts or PBD,
shakentbr 4 h at 28,29"C
Liquid
(not usedfurther)
Only on neglllve
rcsponse
resullwith the liquidenrichDrcnt
method
I
Ten-iblddilutionsin PB o
0.1 ml suspension
sprea
solidagarplates
I
Leal enrichmcnr method
,/\
P c p p c rl e a v e '
lolraio
(XCV)
ND
leirres
(xcv)
(PST)
Pa t h o g e n i c i l y t e
Holnogenized
at 0"C in satine(0.8512
NaCl)
I
I
Ten'folddilutionin phosphite
bufter
I
I
0.1 ml suspcnsion
spfeadon solidagarplates
ND
KI]T
(PST)
(xcv)
P a t h o g e n i c i t ytcst
F{. -t. Flow diagrarn for lhe dctcction of Pseudotnonas ,'i
gae p\. tonato (PS'f)
Xanthomot4s cat pcsttit pv. r.sicdtotia (XCV\ in tomaro and pcpper seedsby bac
enrichment techniques.
been run successfully
in our laboratoriesby non-experttechniciansduring the
years,sincethey were developed.They are thereforerecommendedfor routin
rnspectlon.
ACKNOwLI:DGMIiNTS
The authors thank Dr. M. Tishel and Miss Jacquelyn Singer for critical
of the manuscript;and Dr. C. Icitzman, Agricultural ResearchOrganizat
Dagan,Israel,for local isolatesof the pathogens.
This work was partially sup
by grantNo. 8231026from the IsraelMinistryof Agriculture.
t92
Pb) to paruritica I I : 3 -4
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PhytoparusiticaI I:34,
I983