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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 REFERENCES l. fc Ldilutionsin PB or PBD: uspenslon spreaclon lr Plales 5. 6. ) V) \./ KFT (PST) P a t h o g e n i c i l yt c s l 1. 8. 9. 10. ll. 12. t4. 15. pv.ronaro(PST)and )perseeds by bacterial 16. 11. .ansduring the last 2 Ldedfor routine seed 18. 19. 20. er for critical review h Organization, Bet i pa ially support€d t s i t i c a1 1 : 3 4 , 1 9 8 3 Barhan, Y., Okon, Y. and Heds, Y. (1978) Infection studies of Pseudomo causalagentof bactelialspeckof lomato. Prl toparasitica& 135-143. Chambers,S.C.and Merriman,P.R. (1975) Pelennationand conttol cf Pseudom rn yictoria. Autt, J, agric, Res, 26.. 651463. Crossan,D.F. and Morehart, A.L. (1964) Isolation of Xanthomonasresicatonaf of &psicum d huutu. Phytopdtholog! 54t 358-359. Dernpsey,A.W. and Walker, J.T. (1973) Efficacy of calcium and sodium hypo seedtteatment of pepper,Hottscience 8| 328-329. 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Dit.64: 851-852. Yunis, H., Bashan,Y,, Okon, Y. and Henis, Y, (1980) Weather dependence and control of bacterial speck of tomato causedby Pseudomonastono 64t 931-939. PhytoparusiticaI I:34, I983