Factors affecting the vase life of Rosa cultivar 'Sonia': Microbiological and scanning electron microscopic investigations
Factoren die van invloed zijn op het vaasleven van Rosa cultivar 'Sonia': Microbiologisch en raster electronenmicroscopisch onderzoek
CENTRALE LANDB OUW CATALO GU S
0000 0454 3563 Promotor: Dr Ir F. M. Rombouts hoogleraar levensmiddelenmicrobiologie vakgroep levensmiddelentechnologie Henriette M. C. Put
Factors affecting the vase life of Rosa cultivar 'Sonia': Microbiological and scanning electron microscopic investigations
Factoren die van invloed zijn op het vaasleven van Rosa cultivar 'Sonia': Microbiologisch en raster electronenmicroscopisch onderzoek
Proefschrift ter verkrijging van de graad van doctor in de landbouw- en milieuwetenschappen, op gezag van de rector magnificus, dr H. C. van der Plas, in het openbaar te verdedigen op woensdag 15me i 1991 des namiddags te vier uur in de aula van de Landbouwuniverstiteit te Wageningen.
V ' . \) S v -, "-'-, ! Roos (foto Judith Tromp)
BIBLIOTHEEK ,ANDBOU\ UNIVERSDŒJLB WAGENINGEN HUOÔZDl, Hit
STELLINGEN
1. Bij onderzoek naar de invloed van planthormonen op het vaasleven van snijbloemen, dientme n aseptische technieken tehanteren . (ditproefschrift ,hoofdstu k1) .
2. Demicroflor a die zichontwikkel t inhe tvaaswate r van snijbloemenword t bepaald door de ecologische omstandigheden in de vaas en niet door de microflora aanvankelijk aanwezig op destengel . (ditproefschrift ,hoofdstu k2) .
3. De rolva nmicrofungi in snijbloemenvaaswater verdient meer aandacht, (ditproefschrift ,hoofdstu k 1, 2e n4) .
4. Dode-o fgeïnactiveerd emicro-organisme nspele nee nnie tt eonderschatte n rol inhe tvaasleve nva n snijbloemen. (ditproefschrift ,hoofdstu k 1e n5) .
5. Eenroo si see nroos ,i see nroos ,i see nroo s (Stein,E .1931 .In :Befor e the Flowers of Friendship Faded, Faded Friendship Faded). Ditbeteken t ook: Iedereroo sheef thaa r eigenidentiteit ,groeivermogen ,beginbesmetting . Voor statistisch betrouwbaar onderzoek is het noodzakelijk zo homogeen mogelijk uitgangsmateriaal te gebruiken, inhe t bijzonder als voor die onderzoekingen binnen de gegeven tijdsduur van het vaasleven bloemen worden opgeofferd aanperiodie k uit tevoere n analyses. (ditproefschrift ,hoofdstu k3-8) .
6. Deeffecte nva nlag eaantalle nmicro-organismen ,ster kverdund emicrobiël e pectolytische enzymen, en lage concentraties microbiële metabolieten in vaaswater opd ewaterhuishouding ,knopontplooiin g envaasleve nva n Sonia rozen, doen specifieke werkingsmechanismen vermoeden. (ditproefschrift ,hoofdstu k 5, 6e n 7; Halverson, L.J. & Stacey, G. 1986.Microbiol .Rev. , 50, 193-225).
7.Kunstbloeme nverdiene nvoorran g inziekenkamer sva noperatiepatiënte n en intensive care afdelingenva n ziekenhuizen.
8. Het begrip snijbloemvatverstopping door bacteriën wordt menigmaal ten onrechte gebruikt. Dit duidt dan op een verstoorde waterbalans waaraan andere oorzaken ten grondslag liGgen dan bacteriële verstopping van de watertransportbaan van de snijbloem . (ditproefschrift ,hoofdstu k 7; Accati Garibaldi,A . 1983.Act a Horticulturae, 138, 255-260). 9. Microbiologisch onderzoekaa nvaaswate r enstengeldele nva nsnijbloemen , zoals rozen,n a eenvaasleve nva nee nwee ko f langer, geeft geen inzicht in de ontwikkeling van de microflora gedurende de daaraan voorafgaande dagenva nhe tvaasleven ,terwij ljuis tdez ebepalen dzij nvoo rhe tverloo p vanhe t vaasleven. (Aarts,J.F.Th . 1957.Mededelinge n van de LHWageningen , 57(9)1-62).
10. De toepassing van weefselkweekmethoden voor snelle selectie en vermeerdering van siergewassen zowel als van land-, tuin- en bosbouwgewassen, dientbevorder d teworden .
11. -I nd ebijn aeen-en-negenti g jarige geschiedenisva nd eNobelprij svoo r literatuur isvij f maal eenvrou w bekroond. Dit is geenszins eenbewij s dat het literaire oeuvre van vrouwen van minder gehalte is dan datva nmannen . -Ann a Cramer (1873-1968) is niet de enige verzwegen vrouwelijke componist die onze vaderlandse muziekgeschiedenis rijk is.
12. Stat rosapristin a nomine,nomin a nuda tonemus. (Eco,U . 1983. Ilnom e della rosa) Van demen s vanwelee r blijft ons denaam . De geur, de klank, devor m verdwijnen. Naakte namen en het geschreven woord restenons .
Stellingenbehoren d bij het proefschrift: Factors affecting thevas e life ofRos a cultivar 'Sonia': Microbiological and scanning electronmicroscopi c investigations. HenrietteM.C . Put Deventer-Wageningen, mei 1991 Stat rosapristina nomine, nomina nuda tenemus. De roos van weleer bestaat als naam, naakte namen houden we over. Uit: Il nome della rosa (Umberto Eco, 1983)
Voor: Anton
'A rose is a rose is a rose is a rose' Gertruda Stein (1874-1946) Coverplate: Margaretha Roosenboom (1843-1896)Stil l life with roses OilPain t On Canvas, 50X 6 0cm .
References: Hermes,N . 1989.Margareth a Roosenboom In: Bloemen uit de kelder: negen kunstenaressen rond deeeuwwisseling ,40-4 7 Redactie:Oele ,A. , VanRijsingen , M., Vande n Donk, H. Uitgever: Waanders, Zwolle en Gemeentemuseum, Arnhem
Defoto' s bij detitelpagin a en bij detitel s van de hoofdstukken 1,8 e n 9zij n van Judith Tromp. Dezefoto' s vormen een deelva n een fotografisch bloemenproject. Defoto' s bij de titels van de hoofdstukken 2-7zij n van de auteur.
ISBN90-800045-4- 5 CIP-GEGEVENS KONINKLIJKE BIBLIOTHEEK, DEN HAAG
Put. Henriette Maria Christina
Factors affecting the vaselif e of Rosa cultivar 'Sonia': microbiological and scanning electron microscopic investigations / Henriette Maria Christina Put. - Deventer :Vijfsprong . -111 . Proefschrift Wageningen. - Met lit. opg. - Met samenvatting in het Nederlands. ISBN 90-800045-4-5 Trefw.: rozen ;microbiologisc h onderzoek. Uitgave: VIJFSPRONG, vrouwenuitgeverij, Deventer, Nederland Kopijrecht berust bij de auteur H. M.C . Put VOORWOORD
'Sonia'rooskleurig eroo s Ind eochten dva nd e 20*apri l198 3reisd ei kva nDevente rnaa rWageningen , bezochthe tSprenge r Instituutaa nd eHaagsteeg ,e nha dee ngespre kme td e directeurdrs .G.J.H .Rijkenbarg .Ee ntweed egespre kvolgde ,di tkee rme td e afdelingshoofdene ncolleg amicrobioloo gY .d eWitte .Ee nderd egespre kzett e puntjeso pd eovereenkoms tdi ewer dgeslote ntusse nd everpakkingsindustri e Thomassen& Drijver-Verblif aN.V .(TDV )t eDevente re nhe tSprenge rInstituu t (SI)t eWageningen . Indez eovereenkoms twer donde rmee rvastgelegd :"TD Vdetacheer tHenriett e "M.C. Put,adviseu rmicrobiologi ee n-technologi evoo rmaximaa l4 dage npe r "weekbi jhe tS Ivoo rhe tverrichte nva nonderzoe kbinne nhe tkade rva nd e "doelstellingen,e ni nopdrach tva nhe tSI .Gemiddel dza lzi jéé nda gpe rwee k "beschikbaar zijn voor haar TDV-functie. Het dienstverband tussen TDV en "Henrietteblijf tvolledi gva nkracht. " Deduu rva ndez eovereenkoms t strektezic hui tva n1 oktobe r 1983to t1 januari 1987, het bereiken van de SUM-gerechtigde leeftijd (Stichting Uittreden Metaalindustrie). Daaropaansluitend werd een persoonlijke overeenkomstgeslote n tussend edirecteu rva nhe tS Ie nmi jzelve ,teneind e publicatie te realiseren van de resultaten van de uitgevoerde reeksen onderzoekingen.Dez eovereenkoms twer dbeëindig do p1 septembe r1988 .
Geenroze nO Pd e(spoor)wege ntusse nDevente re nWageninge n Wel:lang ewachttijden ,vuil ewachtkamers ,tochtig eperrons ,i nvoll etreine n publicaties lezend, inslaa p sukkelen,ko uvatten. .Doc hoo konderhoudend e autotochtjes met Natalio Gorin van SI Wageningen naar NS Arnhem, en de donderdagsemaaltijde nme tChristi eBerkhols tbi jDiedenoord' sresto . Door het SJ,wer d gekozenvoo r onderzoek naar de relatie tussenmicro - organismen invaaswate re nhe tvaasleve nva nsnijbloemen ,speciaa l 'Sonia' rozen. De resultaten van dit onderzoek zijn vastgelegd in een 7-tal publicatiesdi ed eker nvorme nva ndi tproefschrift .He tonderzoekpla nkwa m totstan dn acirc a3 maande nliteratuuroriëntatie .He tpla nwer dte rdiscussi e gestelde ngeaccepteer ddoo rrespectievelijk :d eafdelin gfysiologi eva nhe t SI (hoofdO.L . Staden), deadviescommissi e 'Slerteeltprodukten'va nhe tS I (voorzitter J.A.M. Brockhoff) en de contactcommissie houdbaarheid van snijbloemen (voorzitterH .Veen) .
Schieteni nd eroo s Bestuderingva nliteratuu rove rsnijbloemen ,vaaswate re nmicro-organismen , wasaanleidin ghe tonderzoekpla nt erichte no penkel eno gweini gbestudeerd e microbiologische factoren in het vaasleven van snijbloemen, en microbiologische methodieken te gebruiken die zelden zijn gebezigd bij onderzoekva nsnijbloeme ne nvaaswater .Di tbetreft :1 .d einitiël ebesmettin g vansnijbloemenstengels ;2 .d ero lva nfung ii nhe tvaasleve nva nsnijbloemen , naast die van bacteriën; 3. het gebruik van gewassen reincultures en gezuiverde of zuivere microblële metabolieten voor de bestudering van mechanismendi eleide nto tee nverkor tvaasleven ;4 .D etoepassin gva nSEM - technieken (Scanning Electron Microscope). Immers, 'verstopping' van xyleemvaten van snijbloemen zou slechts bestaan in de geest van de onderzoeker, tenzijd eonderzoeke rkijk to pd ejuist eplaats ,schrijve nParup s enMolna r (1972), Rasmussen en Carpenter (1974).
Een roos op uw hoed Iederepersoo ndi edirec to f indirectheef tbijgedrage n totd e totstandkoming van dit proefschrift wordt een roos op de hoed gestoken. Dit zijn alle medewerkers vanhe t voormalige Sprenger Instituut. In het bijzonder noem ik: medewerkers van de afdeling CMK (Chemie, Microbiologie en Kwaliteit) waarbij ik was ingelijfd en die mij wegwijs maakten; de technische dienst,bibliotheek , fysiologie, statistiek, directie en personeelszaken. Ikdan kpersone n die tekstenkritisc hbekeke n o.a. (B.Boekestein, U. van Meeteren enH . de Stigter);Murie l Rhodes-Roberts, diemij n Engels beproefde en bijschaafde; Ans van Hardeveld, die menige tekst menig maal invoerde en herinvoerde ind etekstverwerker ;Joh nBosc h (equipment engineeringTDV )voo r het vervaardigen van de vele figuren en grafieken; mijn co-auteurs: Bram Boekestein,Ank eClerkx, Liek eJansen ,To nva nde rMeijden ,Wi mKlo p enFran s Rombouts.
Dewonderlijk e wegenva n een rozegçeel Deure ndoorgebrach t achterd eJeo l3 5C raste relectronenmicroscoo pzij nmi j dierbaar geworden.He tgedul dva nSEM-specialist eAnk eClerkx, strek tmi j tot voorbeeld.D egedeeld e spanninge nd evreugd ebi jhe tzie nva n 'mooie'beelde n en scherpe opnamen, dediscussie s enkeuze s makenvoo rpublicaties , doch ook het wetenschappelijk natafelenveela l leidend tot nieuw nieuwsgierig makend onderzoek. Dit alles onder de bescheiden hoed van het TFDL (Stichting Technische en Fysische Dienst voor de Landbouw) en de £JJ voorman B. Boekestein, is een onlosmakelijk deel geworden van dit proefschrift.
Een roos is een roos, is een roos, is een roos Vijftig rozen,vijfti g signalenva nvreugd e enwaarderin g voor mijn promotor en co-auteur professor dr ir Frans M. Rombouts, die mij stimuleerde en begeleidde op de weg van publiceren naar promoveren. Rozen ook voor Anton, wetenschapper, taalslijper en liefste vriend, die ikhee l ergmis .
Deventer, januari 1991 CONTENTS Page Voorwoord 7 Chapter 1 Introduction 11 1.1. Economic importance 13 1.2. Thecapabilit y ofcu tflower s fora post - harvest vase life 16 1.3. Thefat e ofcu tflower s 18 1.4. Factorswhic hma y influence thevas e life of cut flowers 20 1.5. Different views on the contribution of physiological and microbiological factors in the vase life of cut flowers 24 1.6. Theselectio n ofa greenhous e rose cultivar asmateria l forpost-harves t research 37 1.7 Vase life experiments 40 -Reference s 49 2. Micro-organisms isolated from freshly harvested cutflowe r stems anddevelopin g during thevas e life of: Chrysanthemum, Gerbera and Rosa cultivars. Scientia Horticulturae, 43,129-14 4 (1990). H.M.C.Put . 59 3. Infiltration of Pseudomonas puCida cells, strain 48, into xylem vessels ofcu t .Rosa cv. 'Sonia'. Journal of Applied Microbiology, 64, 197-208 (1988). H.M.C. Put and T. Van derMeijden . 77 Chapter page
4. Theinfiltratio nabilit yo fmicro-organism s Bacillus, Fusarium, Kluyveromyces and Pseudomonas spp.int oxyle mvessel so f Gerbera cv. 'Fleur'an d Rosa cv. 'Sonia'cu tflowers : ascannin gelectro nmicroscop estudy . Journal of Applied Bacteriology, 64,515-53 0 (1988). H.M.C.Pu tan dA.CM .Clerkx . 91 5. Theeffect so nth evas elif eo fcu t Rosa cultivar 'Sonia'o fbacteri aadde dt oth e vasewater . Scientia HorCiculturae, 39, 167-179 (1989).H.M.C .Pu tan dL .Jansen . 109 6. Theeffect so fmicrobia lexopolysaccha - rides(EPS )i nvas ewate ro nth ewate r relationsan dth evas elif eo f Rosa cultivar 'Sonia'. Journal of Applied Bacteriology, 68, 367-384 (1990).H.M.C .Pu tan dW .Klop . 125 7. Theinfluenc eo fpurifie dmicrobia lpecti c enzymeso nth exyle manatomy ,wate ruptak ean d vaselif eo f Rosa cv.'Sonia' . Scientia Horticulture, 38,147-16 0 (1989).H.M.C .Pu t andF.M .Rombouts . 163
8. Theinfluenc eo fA1 2(S04)3adde dt oth evas e fluido nth einfiltratio nabilit yo f Bacillus subtilis cellsint oxyle mvessel so fcu t Rosa flowerscv . 'Sonia':a scannin gelectro n microscopestudy . Submittedfo rpublicatio nin : The Journal of Applied Bacteriology (1991)H.M.C .Put , W.Klop ,A.C.M .Clerk xan dB .Boekestein . 9. Discussion 185 -Summar y 197 -Samenvattin g 200 -Glossar yan dAbbreviation s 207 -Curriculu mvita e 218
10 Chapter 1
Introduction Rosa (Judith Tromp)
12 Chapter1 1.INTRODUCTIO N
Prehistoric paintings and pottery record that cut flowers have long been used as ornaments and evidence for the expression of love, friendship, triumph and sadness. The long history of the art of painting comprises many still life studies of cut flowers, widely regardeda sclassi cexample so fperfec tbeauty . Palladius (4th century a.c.) in his Opus Agriculturae "De Re Rustica" devoted a special section to the study of the keeping quality of cut roses "De rosisviridibu s servandis" (Palladius,Ed . J.M. Gesner, 1781. See Appendix). In recorded medieval mystical experiences, the rose alsoplay s an important role; (1)a s asymbo l of love, beauty, happiness and silence; (2) as a symbol of virginity -th ewhit e rose; (3)a s a symbol ofmartyrdo m -th e red rose (Lejeune,1978) . The secondpar to fth e20t hcentur ybrough tus : (1)th e "flower power" wave; (2) the spring time flowering bulb fields of tulips, daffodils and hyacinths in the Netherlands; (3) the magnificent Keukenhof gardens near Amsterdam; (4) the folklore of the flower- corso; (5) the best-seller "The Name of The Rose" (1983) by the Italianwrite rUmbert oEco ,an dals o linguisticexpression s suchas : beo na be do froses ;n oros ewithou ta thorn .Th ewidesprea dus eo f flowers at weddings and funerals further reflects their ability to demonstrateotherwis eunexpressibl esentiments .
1.1.Economi cimportanc e
SinceWorl dWa rI Ith ehorticultura lproductio no fornamenta lflower s has increased continuously inth eNetherlands ,an d the supply ofcu t flowerst oth eflowe rauction si nth eNetherland s isstil lshowin ga n upwardlin e(se eTabl e 1).
13 Table1 :Th eturnove ro fcu tflower s inTh eNetherland s
Year Millionguilder s
1975 962,- 1980 1.648,- 1985 2.501,- 1987 2.689,- 1988 2.938,- 1989 3.036,- 1990 3.297,-
Source:CBOP ,Commodit yBoar dfo rOrnamenta l Plants;PVS ,Productscha pvoo rsier gewassen,De nHaa g
14 0 -tf r» m CM co CM -* •O O O o o O o o o" o" o" Ol U 1 OM 3. •H u 0 O o o 0 co o o o VO r-l co Ol O X i-l Oi m CM 25 0 10 o CM O u PI U 0 m "O G o ij i-iS - Ha •rl (Vi IM Si 4J m m m in m oo CO CM CO CO 00 o 4) CM co r-> co vo m 0 "^ m o oo m r» 3 4J 4J S5
lil V 1-1 o co o o 0 vo a co CO w o o o o o o o"o u CO I 00
vo 00 oo 8 VO IN g o u I» o \ u 0 CO 00 00 00 CM 00 ^ co r» 0 0 •o -* CM co m m CM CM co CM . 3 8" o » i-l •w o 1-1 o O O O o O O o" o* SO tl r-l ta 00 (Vi 15 Rose cultivars area tpresen t thecu tflowe r forwhic h therei s the greatest demand: this is followed by chrysanthemum, tulip, freesia, carnationan dgerber a cultivars,a sshow ni nTabl e2 . 1.2.Th ecapabilit yo fcu tflower sfo ra post-harves t vase life Factors whichma ycontribut e greatly toth evas e lifeo fcu tflower s are: (1)th eornamenta l valueo fth especie so rth ecultivar ; (2) the cultivation method; (3)th emomen t ofharvesting ; (4)th emetho do f harvesting;an d(5 )post-harves t handling. - The ornamental value of Che species or the cultivar Flower species andcultivar s which posesses less ornamental value,a s wella sa shor t vase life,ar eo fmino r importance forth ecu tflowe r market (Halevy et al., 1978;Halevy , 1989). Fashion trends,however ,ma yals o play asignifican t role in the consumer's preference fora special cut flower cultivar. Forth e commercial launching ofa ne wcu tflowe r cultivar variety orhybri d of, forexampl e tulip, gerbera, orrose , a 'popular' name givent o that flower mayinduc e theconsume r tobu yit . What isi na name ?A rose with anyothe r namema ysmel la sswee t (Shakespeare, 1564-1616) perhaps,bu tno tnecessaril yb echose nfo rpurchase . By modern hydroponic methods and laboratory tissue culture techniques, iti s possible to cultivate andpropagat e newhybrid s very rapidly inhig h numbers. Statistical evaluation ofth eabilit y of new cultivars or hybrids to survive well during vase life is therefore much easier and quicker to ascertain, speeding up introduction toth ecu tflowe r market (Vand ePo l& Breukelaar , 1982; Pierik, 1985; Hickleton et al., 1987; Mor& Zieslin, 1987;an dD e Jong, 1989). - The cultivation method In theNetherlands , most ornamental flowers aregrow n ingreenhouse s with precise regulation oftemperature , humidity, irrigation, light- dark phases,o rsoi l substrate constitutuents. Therefore many flower cultivar canb ecultivate d allth eyea r round anddifficul t climatic conditions circumvented. -Prematur e harvesting As soona sth eflowe r iscu tan dseparate d from themothe r plant,i t 16 obviously no longer receives the dissolved nutritive elements from the mother plant via the mobile sap. The cut flower from then on is entirely dependent on its own reserves, especially those of sugars and starch, which may quickly be exhausted (Paulin et al., 1981; Paulin, 1986). Premature picking of roses may lead to a low petal starch reserve; this is most likely to be an indispensable source of energy for the cut flower to bloom maximally (Berkholst, 1989). With the rose 'Sonia'; insufficient amounts of petal starch were shown to be due to (1) premature picking, and (2) the breakdown of starch after cutting during any long storage periods preceding the vase life. Low storage temperature (2-5°C) did not arrest this starch breakdown after cutting, although storage at higher temperatures (10-15°C) caused petal starch to disappear much more rapidly (Berkholst & Navarro Gonzales, 1989). Thus , practices such as immature cutting, long storage and exposure to high temperatures, are detrimental to the quality of roses and to theirvas e life. - Harvest methods The harvesting equipment, handling methods, and general standards of hygienehav e aprofoun d influence on the final keeping quality of the flowers. Mechanical damage at the cut end of the stem due to rough handling and the application of blunt scissors may lead to blockage of the xylemvessels , thus disturbing thewate r flow to the flower. Dirty equipment including buckets, hands, gloves and cloths, comprise the first exogenic source of microbiological contamination of the flower stem and its cut surface. Thereafter many opportunities formicrobia l contamination of the cut flower may occur. Post-harvest handling: from the grower to the consumer's home During thepost-harves t period, from the point of cutting to the vase in the consumer's home, the methods of handling, packaging, storage and transport throughout the whole commercial chain from grower to retailer markedly affects the final keeping quality of the cut flowers. 17 1.3.Th efat eo fcu tflower s - Theeffects of cutting The flower cutting operation Iso fparamoun t importance insofa ra s it affects thelif e ofth ecu tflower . Forvalidl y interpreting certain aspects ofcu tflowe r physiology, i.e.th ewate r relations and water balance, iti sessentia l tocompar e thebehaviou r of cut flowers with thato fintac t ones underth esam e conditions.Th estud y of De Stigter (1980) shows that this is possible by means of experiments involving single-stem, self-rooted hydroponically-grown roses: simply removing theroo t system turns an intact rose plant intoa notherwis e identicalcu tflower . By using intact-plant behaviour asa reference which showsth e fate of the rooted rose, the specific effects of the cutting operation emerge more conclusively.Tw oaspect s were consideredb yD e Stigter (1980): (1)th e short-term effects; i.e.,th e immediate effectso freplacin g water uptake throughth eroot sb ytha t occurring through theexpose d xylem vessels. This involves theeliminatio no f root resistance andth esubstitutio n ofth eorigina l tracheal sapb y vase water with an artificial chemical composition andmainl y a higher osmotic (orsolute ) potential; ;(2 )th elong-ter m effects:a cut rose shoot with amatur e flowerbu di sa relativel y young plant part; itma yreac h this stage ina matte ro fsi x weeks from axillary bud break.Th eensuin g flowering ofth ecu tros e then takes abouttw o weeks essentially thesam e time asneede d forth eflowerin g of the intact hydroponically grown rose (DeStigte r & Broekhuysen, 1986). Hydroponic production ofcu tflower s forpost-harves t physiological and microbiological studies, haveno tbee n reportedb yothe r workers. Commercial trials, however, arestil l inprogres s (Hickleton et al., 1987). Intact root-grafted roses ona shoot with cutrose s fromth e same shoot were compared by Durkin & Kuc (1966). These authors observed that theintac t flowers lasted atleas t twicea slon g (8-13 days)a sth ecu tflower s (4-10 days)whic h were placed invas e water underth esam e climatic conditionsa sth eroote d roses. - Effects of transport and storage Cut flowers aremainl y transported inth edr ystate . During storage at lowtemperature s theflower s usually arehel d dryan dpile du p 18 horizontally, or more rarely, they are placed upright in buckets or basins filled withwater . Water stress occurs during the period when cut flowers are held in the dry state. Thereafter, recovery of the natural water balance is necessary. The water balance of cut flowers is a result of interrelated processes involving: (1)wate r uptake; (2)wate r transport; (3)wate r loss during day and night periods; (4) the capacity of the flower to retain its water, and (5) competition between flower and leaves. De Stigter (1980a) observed that the long-lasting constancy of water uptake by cut roses in consecutive dark periods sharply contrasted with the rapid decline in water content during the light periods, as shown in Figures 1 and 2. These observations may or may not be compatible with the concept of the causative agent of reduced water uptake, as proposed by many other authors (Rasmussen & Carpenter, 1974; Durkin, 1979). They claim reduced water uptake to be due to vascular occlusions occurring as massive and immobile plugs of solid materials filling xylem vessels. Water stress caused by xylem plugging is a constant non-reversible stagnation of the water transport in cut flowers which disturbs the water balance throughout both day andnight . Flowers like many gerbera and chrysanthemum cultivar show a cavity or hollowness in the center of the stem, which is formed during stem elongation: thus there are two possible pathways for water uptake, one directly through the xylem vessels at the cut surface, and the other indirectly from the cavity of the stem into the adjacent lateral vessels. The stem cavity shows a diameter 5-10 fold longer than the diameter of the xylem vessels, and is filled with water up to the level of the surrounding water in the flower container when trapped air can disappear from the cavity (see Figure 3, Van Meeteren, 1980). Consequently xylem vessel plugging occurring in the lower part of the vessels,belo w the level of the vase water, may not cause a total water stress and a total cessation of water uptake. Water uptake through the second lateral pathway from the inner cavity may occur and circumvent any xylem plugging, as long as transpiration rate is not too high, as is mostly the case with (leafless)Gerbera . 19 - The role of "pulsing" and flower-holding-water solutions "Pulsing" is a procedure wherein flower tissues are infiltrated with certain chemicals directly after harvest, to protect the flower during transport and subsequent storage and obtain optimal longevity during the vase life. Pulsing solutions may consist of: sugars (energy sources and osmotic components); organic acids (pH regulators); germicides; an ethylene inhibitor; mineral salts (improving water uptake), and on occasion growth regulators (phytohormones). In flower-holding-water solutions ethylene inhibitors are not applied. Depending upon the contact time of the pulsing solution and the cut flower, the concentrations of similar applied chemicals in the pulsing solution are much higher than in the cut flower holding water. In addition, inpulsin g and holding water solutions a wetting agent is sometimes applied (Halevy &Mayak , 1981). 1.4 Factorswhic hma y influence thevas e life of cut flowers The vase life of cut flowers which are cultivated, harvested, transported and stored under controlled conditions may be influenced by: (a)physiologica l factors,e.g . cut surfaces andwoundin g of stem xylem vessels, vessel wall changes, xylem vessel cavitation, air embolisms, changed hormone activities depletion of cabohydrates (energy), and (b) microbiological factors, e.g. the composition of the vase water microflora, their multiplication abilities and interactions, the infiltration ability of themicroorganism s into the flower xylem vessels and the toxicity ofmicrobia l metabolites. 1.4.1. Effects ofphvsiolopica l factors Physiological factors affecting the vase life of flowers may be categorised as follows. - Harvesting inlurv of xvlem vessels Injuries of the stem during theharves t may cause mechanical blockage of xylem vessels at the cut surface and higher up the stem, thus 20 disturbing the water relations of the flower (Dixon & Peterson, 1989). Further material from injured cells may act as endogenous elicitors of phytoalexins, providing signals towards which recognition by the plant may induce biochemical activities acting as plant defence mechanisms (Darvill & Albersheim, 1984; Halverson & Stacey, 1986): (see also section 1.5.). - Cavition A disruption of water column continuity in the xylem vessels of an intact plant canb e induced by water stress,usuall y as a consequence of the dehydration of the wood xylem vessels during the winter period. Cavition in cut flower vessels however, can be caused by (1) air-drying during transport and dry storage, and alsob y (2) physical blockage at the cut surface and toapprox . 200m m up the stem. The consequence of cavitation by air-drying is a decline in water conductance up the stem, induced by water stress, although no vessel occlusions are present (Dixon & Peterson, 1989). Water-stress- induced cavitation can alsob e causedb y ageing of the flowers in the vase. Cavitation can be shown by infiltrating a fluorescent tracer (berberine sulfate) through the stems: cavitated vessels do not stain and show no occlusions. Cavitation can be localized and measured by ultrasonic acoustic emissions, and thus be distinguished from water stress caused by the breakage of cellulose fibers in the wood and other diverse forms of stem plugging such as tyloses (Tyree et al., 1984; Tyree & Dixon, 1986, Dixon, 1987; Dixon et al., 1988; Dixon & Peterson, 1989). - Xvlem embolism Air bubbles and dissolved gases may occur in xylem vessels. As xylem pressure becomes increasingly negative the adhesion between water molecules is reduced and vaporization occurs. The maximum pressure difference (AP, in MPa) withstood by a meniscus at an intervessel pore can be calculated from the pore diameter (D, in j*m)an d xylem sap surface tension (T,i nN 2m" 1)usin g the capillary equationA P - 4 (T/D)), which refers to the AP of a pit membrane pore at it's bubble pressure (Sperry & Tyree, 1988). When embolisms occur at pressures less negative than normal, other mechanisms sometimes initiate vaporization, e.g. mechanical shock with air coming out of vessel 21 water,du et orapi dpressur echanges . Theair-seedin ghypothesi si sa thir dexplanatio no fth ecaus eo f xylem embolisms, and this has the most experimental support (Zimmermann, 1983);viz :embolism sma yb etriggere db yai raspirate d intoth exyle m vesselsvi apore si nth ewal lwher ei tadjoi na nai r space.Onc einsid eth exyle mvessel ,th eai rdisrupt sth ecohesio no f the water column which therefore retracts (cavitates), leavinga vessel filled with water, vapor, andai r(Sperr y & Tyree, 1988). Eventually,th evesse lbecome scompletel yair-fille da sai rcome sou t of solution from surrounding water, a phenomenon acceleratedb y higher temperatures, resulting in decreased stem or vessel conductance,a sshow nb yDixo n& Peterso ni ncu trose s (1989). Embolism of adjoining xylem vessels isprevente d as longa s pressure differences across intervessel walls dono texcee dth e surface tensiono fth eair-wate r interfacea tth epore si nth ewall . The largestpore sar eth emos tvulnerabl et oth epenetratio no fair ; such are the pores in thepi t membranes which under normal circumstances facilitate water flow between vessels (seeFigure s4 and 5). Aftera lon g periodo fdr ystorag eo fcu tflowers , rehydration and de-aeration ofxyle m vessels mayb e slow, resulting in sub- optimal conditionsfo ra goo d vase life (DeStigte r& Broekhuysen , 1989). Cavitationan dair-embolis m alonema yno tb eharmfu lfo r the vase lifeo fcu t'Sonia 'roses ,unles s more thanhal fo fth exyle m vessels areblocke d (VanDoor n& Buis , 1985). Repair ofth ewate r relationso fcu tflower s affectedb yembolis man dcavitatio nma yb e enhancedb yusin gde-aerate dvas ewate ro flo wp Ht owhic ha wettin g agentha sbee nadde d (Durkin,1979) . -Pffte rpftysipipgjgfl 1 factçrs affecting the vase life of flowers: e.g. carbohydrates and phytohormon es are mentioned in sub. 1.2 and 1.5 (Table 3) respectively. 1.4.2.Effect so fmicrobiologica lfactor s - Micro-organisms as particles in the vase fluid Apart from toxic andothe r effects towards cut flowers, micro- 22 organisms may act by their mere physical presence and dimensions as particles, small enough to enter xylem vessels passively, along with the transpiration stream ofwater . Ifmicro-organism s are taken up in sufficient quantity, they may plug the vessels especially at the intervessel pores and disturb the water uptake (De Stigter & Broekhuysen, 1986b; Van Doorn et al., 1986). It is therefore important that after harvest cut flowers, are not confronted with deterious water by being placed in dirty buckets or basins with re used water, containing dust, soil particles, plant hairs, plant fragments and dead cells, slime, and viable micro-organisms, which might use the dirty materials as a growth medium. Both micro organisms and other non-identified particles may be taken up passively by the sap stream of the cut flowers, and plug the xylem vessels. - Active infiltration and adhesion of micro-organisms into xvlem vessels Active infiltration of micro-organisms may be possible due to microbial motility, aided by Chemotaxis. The bacterial flagellum does enable the bacterial cell to move actively; Brownian movement will cause only random cell rotation. Movement of flagellate bacteria into a capillary system such as xylem vessels may result in an active movement higher up into thevessel s compared with non-motile bacteria whichmov e up only passively in the sap stream. Motility, taxis, and tropisms are of value in the natural environment of micro-organisms. They are complex systems which have evolved andbee n retained as a consequence ofphenotypi c responses of the organism to the environment (Carlile, 1980; Konings & Veldkamp, 1980). Micro-organisms show taxis or tropisms towards a wide variety of physical and chemical stimuli. A cell may respond to a stimulus when it occupies a physiologically suboptimal position within a concentration gradient. A cell capable of Chemotaxis must be able to detect ('sense') changes in the concentration of a chemo-effector, transmit this sensory information to the locomotory organelles (i.e. flagella), and then make the appropriate response; it must also be able to adapt to the continued presence of the chemo-effector by ceasing to react when the concentration of the chemical reactor becomes uniform. 23 The Infiltration ofmicro-organisms , passively or actively, into the xylem vessels of cut flowers may result in: (1)physica l blockage of the water transport vessels (xylem) at the cut surface and in the open vessels; microorganisms may also block the pit membranes of these vessels, obstructing the water flow through the lateral pit membranes; (2) spontaneous reactions may occur in the vessel cells, disturbing the water relations of thevesse l system; (3) the adhesion of infiltrating micro-organisms to the cut surface, the vesselwalls , and to the pit membranes, may be followed by multiplication, which may seriously disturb the water transport and the water balance of the cut flower. In nature, micro-organisms frequently bind specifically or non- specifically to a substratum or to other cells, by means of specialised microbial components (adhesins) or anchoring structures (fimbriae).Free-living micro-organisms in aquatic habitats often adhere to submerged surfaces such as particles of debris, other organisms, or cut flower stems - sometimes forming biofilms (Marshall, 1980 & 1980a). Adhesion may affect the activity of the adherent micro-organisms since the conditions at a submerged surface differ from those in the bulk aqueous phase: e.g. surfaces of cut flowers may adsorb or excrete (from the phloem) nutrients and stimulatory or inhibitory ions so that the solid-liquid interface may be a significantly more advantageous (rarely disadvantageous) habitat compared with the bulk liquid phase (Woltering, 1987). Furthermore the micro-organisms may be able to detach temporarily by producing an emulsifying agent (emulcyan) which induces cell-surface hydrophobicity, thus permitting detachment and colonization of fresh surfaces (Fletcher, 1979 & 1979a; Fletcher et al., 1980). 1.5. Different views on the contribution of physiological andmicrobiologica l factors in thevas e life of cut flowers - The work of Aarts (1957) Aarts (1957) did not confine himself to study the vase life of only one flower cultivar, but studied simultaneously the behaviour of at least 12 cut flower cultivars. He also investigated the effects of a great number of biocides, bacteriocides as well as fungicides on the 24 vase lifeo fcu t flowers,an dstudie dals o the influence ofa numbe r ofsugar san dsom eothe rflowe rnutrients . Aarts (1957)conclude dthat ,(1 )suga rtogethe rwit ha non-toxi c concentration of a bacteriocide and a fungicide added to the vase fluid significantly increased thevas e lifeo f the flower placed in that fluid, to an extent dependent on the flower cultivar orrace . (2)Bacteri a invas ewate rwer eharmfu ltoward s the flower due toa directpluggin go fth evessel swit hbacteria lparticles ,bu t (3)non - filterable substances also inducedplugging . (4)Eve n inth eabsenc e ofbacteri a inth evas ewater ,vesse lblockag e occurred,probabl ya s aconsequenc eo fsubstance ssecrete db ydisorganize dvascula rcells . Vessel plugging was not always demonstrated microscopically; instead the water conductivity of the vessels was measured as parameterfo rplugging .Th erol eo ffung iwa sno tinvestigated . Unlike Aarts (1957)wh o studied many flower cultivars andman y vase life influencing factors, later scientists mainly studied the vaselif eo fon eflowe rcultivar ,an don eo rtw ovariant so fth evas e fluid. The most studied cut flower cultivars are: Rosa hybrids and Dianthus (carnation). Vase life disturbance phenomena of cut roses are: bentneck ,delaye dbu ddevelopmen t andprematur e fallo fflowe r petals. Dianthus cultivarso nth econtrar yar ever ysensitiv etoward s exogenican dendogeni cethylen e (see later). - The role of phvtohormones Phytohormones or plant hormones are compounds produced by plants which regulate the plants' own metabolic processes such as cell division, seed germination, fruiting, flowering, senescence (Bruinsma, 1983). Such agents include abscisic acid, auxins, cytokinins, ethylene and gibberellins. The phytohormone ethylene probably plays the most important role in the vase life of cut flowers.However ,othe rphytohormone s areals o involvedi nflowe rbu d development and the senescence of cut flowers, although the mechanismsunderlayin g theiractivit y areno talway s clear andther e mayb esubtl e interactions (Schröder,1987 ;Borocho v& Woodson ,1989 ; Zucconi& Bukovac ,1989) . Phytohormones orphytohormone-lik e compounds may alsob eforme d by certain micro-organisms such as Azotobacter, Agrobacterium Pseudomonadsan dman yfungi ,a sshow ni nTabl e3 . 25 co co 3 T3 at ^-1 -.r-l Q c - O e • 00 JD < u •-c 0 6 S ^ • u 4 cd h Vt^O O X v. •e 3 CD CO 3 C O •O co co o —c co (U bt>r4 —4 cd u c •»* o ox < o co u • box "o u »a «[••rftOD-D -U • 3 C c •« • (DO—1C"3C0C OB Xo w-C" oO B ^X cä bO-U Wc O b0>^ O co L4 « fiOO « «Ci O «uu N-a ^-1 C O O M 0) -X - Q.-I CUD C H o.« O B co 40 o —1 CO •H d 4>H ac 4) o C O • w co o co 3 «• a. ca o CD - O-CO-C CDr-1 CHCHX E 0) • O 3 O CO 3 O O bOCM O J3 -C •** CD O J3 £«—1 CO co *-c • • O ÊTJUE bo cj —1 II C OU - O CJ o >v-< «> 0) B >-. CO C X) V* O O %eoi* c cu O0>Mau ) -O CoD ooC xU c^ae«£i<^-ri O W •.-) -r4 3 CU O •cacrij: -bocoo. rfi-cco.3* C0OOO-HUO6O tïcoco CO £. u Q0 3 •o P. bOOï c o C co o co • t4 V • u 3 H -~4 CO CU - > U CO CD > O C e-o o. ü «U-O *z 10 U > T4 o) CU a> u CO O C ~-l COr-C au n U 26 (continuation; 01 ON cd ^-t r» ü m ON 3 •är-HärJ 1 •H 0 cd c c 0 • IM i ta p to ot C i M-H .e 0 o> u a>« o •U u •H eu M oi •°* S 0 o «eu --fi 0 H S p. •- OOO .M ; "~>~ M N'rlO\ Ü u 3 27 - Ethylene Manymicro-organis me.g .filamentou ssoi lfung ian dsom ebacteri ama y produce ethylene (Lynch, 1974;Domsc he t al., 1980;Lee , 1989);bu t other micro-organisms produce inhibitors of ethylene formation (Lieberman, 1979). Therol eo fethylen e inth epostharves t lifeo f roses, isstil l unclear. Reid et al. (1989) studied theethylen e sensitivityo f2 7differen tros ecultivar sb yexogeni ctreatmen twit h ethylene,an dobserve d that severalcultivar swer ever y sensitive( < 100pp bC 2H4a t22°C ). Soni aroses ,th ecultiva ruse di nou r studies, and also often usedb yEuropea n researchers,wa sfoun d torespon d favourably onlyt ohig hconcentration s( >100 0pp bC 2H4)a t22°C . The time courseo fethylen e emanationb yros e petals resembles thato f carnationan dothe rflower san di scompose do fthre edistinc tphases : (1)a lo wstead y state inethylen e production; (2)a naccelerate d rise toa maximum ; and(3 )a declin e init sproductio n (Halevy& Mayak, 1981).A ris ei nethylen eproductio nwa sals oeviden ti nros e flowers during cold storage. Following cold-(stressed) storage,th e ethylene production ofstore d roseswa shighe r than that offres h flowersan dthei rlongevit yshorte r (Faragher et al., 1986).However , ethylene productionb yros e petals islow ,a tmos t 1-5%o ftha t producedb ycarnatio npetal s (Faragher& Mayak ,1984) . Inhibitorso fethylen esynthesi sfaile dt oprolon gth elongevit y of rose flowers despite the reduction in ethylene production. Inhibition of the response of flowers for ethylene by silver thiosulfate (STS)increase dth elongevit yo fcu tros e flowers (Veen, 1983& 1987 ;Faraghe re t al., 1986a)an dprevente d theaccelerate d abscissiono fros epetal scause db yexogenousl y applied ethylene(D e Stigter& Broekhuysen , 1986a). STSshow s also biocldal activityan d mayinactivat evas ewate rmicro-organism s (Reide tal. , 1989a). Stressingo fplan t tissuesb ychemicals ,drought , cold, insect damage, mechanical wounding or cutting may result in increased ethyleneproduction . Stress-inducedethylen ema yassis tth eplan to r flower tocop e with stress conditions. Under drought conditions, ethylene would cause leaf abscissionan dthereb y reduce water loss (Apelbaum& Yang ,1981) . - Wounding of plant tissue bv cutting Woundingo fplant sca nb ecause db ymechanica l (wind)an dphysica l 28 (insect bites, cutting) damage,an da wounde d plant maylos e a part of itsnatura l defence mechanism mediatedb yth eepidermis , although contact between thewounde d part ofth eplan t andth enon-damage d tissues remains.A cutflower , however, isfull y separated fromth e mother plantan dit sroote d system.I ttherefor e isno tclea r whether the wounding reactions ofa cutflowe r arecomparabl e orno twit h those observed ina rooted flowering plant after wounding. Wounding of plant tissue leads to several reactions inth eplant , i.e.(1 ) genetical changes affecting phytoalexin synthesis; (2)biochemica l reactions involving hormone activity; (3)morphologica l changes such as stemvesse lwal l degradation,whic hma yal linterac t reciprocally. - Genetic response to wounding In plants, asi nal lorganisms , theplasm a membrane serves twomai n roles; viz: (1)th etranspor to fsolute s inan dou to fcells ,an d (2) sensory transduction,i.e .th esensin gan dinitiatio no fth ecellula r responses to changing environmental conditions (Sussman & Harper, 1989; Siebertze t al., 1989). - Hormone mediated genetic expression Wounding of plants is responsible for the regulation of a wide variety of genetic products in plants e.g. BAP,th e cytokinin (benzylaminopurine); NAA,th eauxi n (a-naphtalene acetic acid);IAN , indole acetamide; IAA, theauxi n (indole-3-acetic-acid).Auxi n (IAA, indole-3-acetic-acid) levels regulated the expression of a wound- inducible proteinase inhibitor II,-chloramphenicol acetyl transferase (CAT) in gene fusion experiments, JJJ vivo andin ,vitr o (Kernan & Thornburg, 1989). The wound inducible genetic system involves plant defences against microbes andherbivore s (i.e. wounding). This isfollowe db y the induction oftw osmal l gene families (proteinase inhibitor I& II) which encode quantitatively microbial or insect resistance factors; thes e areinduce d froma quiescen t toa nactiv e state after wounding. - Phvtoalexins and their ellcitors Phytoalexins appeara sth eresul to fplant-microb e interactions. They are an important factor in plant disease resistance, being the response ofth eplan t toa ninfectio nb ypathogeni c ornonpathogeni c micro-organisms: bacteria (Billing, 1982) andfung i (Domsch et al., 29 1980). They are a chemically heterogeneous group of low-molecular- weight compounds with antimicrobial properties and are not found in healthy plant tissues; they appear only at the site of an infection. Phytoalexin synthesis can be induced by molecules of abiotic or biotic origin called elicitors. They include oligosaccharides, as shown in Table 4, and there are about 100 known phytoalexins. It is probable, that all plants have the ability to synthesize phytoalexins, which usually are structurally correlated with the plant family or species (Albersheim &Valent , 1978). The presence of an endogenous elicitor in plant cell walls may be themechanis m by which abiotic molecules induce elicitor activity. Thus, plants have the capacity to recognize molecular signals originating from microbes or their own cell walls, and are thus triggered to elicit phytoalexins aspar t of their defense response. The specificity of many plant-microbe interactions implies some mechanism of recognition, one of which may be the exchange of molecular signals between host and microbe. Such signal molecules can have either a positive or a negative effect on the plant microbe interaction. In general, recognition is the culmination of a complex series of events involving genetic and physiological changes. The response to inoculation of avirulen t microbial strainma y require 10 min or up to 2 days to produce immunity in the cells surrounding the inoculation site, and the defense may be equally effective against compatible or incompatible microbes (Sequeira 1973;Kuhn , 1987). Initiation of some host plant defenses may be mediated by the recognition of a signal resulting from the degradation of the plant cell wall, rather than by the direct recognition of a component of the pathogen. For example: endopolygalacturonases digest the plant cell wall and release oligogalacturonides, which can induce phytoalexin synthesis. A systematic study is needed to establish a correlation between pathogenicity and the presence or absence of polygalacturonases. An incompatible race might induce the activity of elicitors with the initiation of a more dramatic defense response. Darvill and Albersheim (1984) suggested that abiotic elicitors might cause cell injury or death with the release of cell wall pectic fragments. These cell wall fragments could be the same, as the endogenous oligosaccharide elicitors. However, it is not known 30 whether cell wall pectic fragments released by cutting injury of flower stems may act as endogenous biotic elicitors. It is also not clear whether biotic and abiotic elicitors in the vase water fluids of cut flowers e.g.: fungal cell wall fragments; microbial enzymes; heavy metals; or detergents may act as signal molecules which elicit phytoalexin synthesis, which may influence the flower development during thevas e life (Collmer, 1987). Elicitors probably act inconcer t with other mitigating factors. For example plant oligogalacturonide cell wall fragments and fungal 0-glucans together act synergistically to induce phytoalexin accumulation in soybean (See later). Many signal molecules have been identified as oligosacharides (Halverson & Stacey, 1986;Nothnage l et al., 1983). Table 4: Elicitors ofphytoalexi n synthesis inplant s BIOTIC ELICITORS: -peptide s from fungal mycelium a. Exogenous - oligosaccharides (/8-glucans)an d chitosan from fungal cellwall s orcultur e filtrates. -polysaccharide s frommicrobia l cell walls -pecti c degrading microbial enzymes (endopolygalacturonases) b. Endogenous - small molecules from plant tissue (dead or injured cells) -plan t cell-wall fatty acids -plan t cell-wall (pectic) polysaccharides -plan t enzymes (oligogalacturonide releasing enzymes; pectic degrading enzymes) -pecti c plant cell-wall fragments released by microbial acitivty - ethylene ABIOTIC ELICITORS: heavy metals autoclaved ribonuclease chloroform detergents 31 - Oligosaccharides and Olipasaccharins By wounding ofplan t leaves Intravascular glycosidases arerelease d which interact specifically with adjacent cell wallst oliberat elo w mol.weight oligosaccharides. These oligosaccharides have been isolated andca nb efe dthroug h thepetiol e ofdetache d leavest o induce the accumulationo fproteinas e inhibitorsI & II .Th e kinetics of the inductiono fbot h familieso fproteinas e inhibitors involvea 4-6 h period following wounding before mRNA begins toaccumulate . This long prelnduction period isunusual . Other wound-inducible systems, such as those following the inoculation ofa virulent microbe orvirus , showa mor e rapid gene activation (Lawton& Lamb , 1987). Naturally occurring plant cell wall carbohydrates exhibiting biological regulatory functions have been termed oligosaccharins. Thus /3-glucans and oligogalacturonides would be classified as endogenous oligosaccharins. Themechanis m by which the0-gluca n enhances phytoalexin synthesisi sno t known. Some reactions are shown within 2-4h afte r treatmento fth e plant (invitr oo ri nvivo )wit h the phytoalexin elicitor. Under assay conditions anactiv e hapta-/?- glucoside elicitor shows a half maximal response of phytoalexin synthesis after using only0. 6ng : (10~8 -10" 10M )o fth eglucosid e (Sharp et al., 1984). This means that very lowconcentration s ofa certain phytoalexin elicitor mayb e needed to elicite a complete physiological reactiono fth eplan t (Rickauer et al., 1989). - Gaps inou rknowledg e ofth emechanism s leading towate r stress andvascula r blockageo fcu t flowers A scientifically literate reader would have considerable difficulty when trying torelat e general conclusions drawn from a biochemical study ofa phytopathogeni c bacterium tothos e drawn from a genetic study ona rust fungus (Gabriel, 1986). Thesam e reader mayhav e similar difficulties when comparing conclusions drawn from physiological studies onth epost-harves t life ofcu tflower s with microbiological data. It is unclear whether (1)plan t hormones (ethylene andindole-3-aceti c acid) produced bymicro-organism si n vase watero ri ncu t flower vessels, influence the vase lifeo f the cut flower, and(2 ) whether microbiological elicitors mayinitiat e 32 vessel wall reactions which reduce the vase life of the flowers. Furthermore, (3)th e role of micro-organisms in,an d the mechanisms ofvascula r blockage ofcu tflowers ,ar eals o controversial. Since water stress is not always directly related to vascular blockage, other phenomena might be theconsequenc e ofa disturbance ofth ewate r relations ofth ecu tflower .Microscopi c examinationsa s well as scanning electron microscopic studies are scarce, and these studies only sometimes indicate vascular blockage bymicro-organisms ; however visualization of vascular blockage may easily be missed(Parups &Molnar , 1972;Sacalis , 1975). - Visualization of vascular blockage "Vascular blockage due to plugging with micro-organisms is only in the imagination of the scientist", concluded Rasmussen & Carpenter (1974). Some 15year s earlier (Aarts, 1957)a swel l as some 15year s later, visualization ofvascula r blockage duet o micro-organisms is still lacking in many studies, notwithstanding conclusions that microbial plugging of xylem vessels ofcu t flowers is the cause of water stress andflowe r senescence (Burdett, 1970;Danserea u &Vines , 1975; Marousky, 1980; VanMeeteren , 1980). The evidence implicating micro-organisms in the vase water as agents reducing the vase life of cut flowers by plugging of the vascular system, isno tconclusive .Va nDoor ne t al. (1986) concluded that an adverse effect on rose flowers was only manifest when the initial bacterial number exceeded 109pe r ml vase water. Zagory & Reid (1986) however, observed that microbial numbers of only 103-10* cfu (colony forming units) per ml vase water was deleterious, the vase lifeo fcarnation s androse sbein g reduced from 14an d6 day st o 7.5 and 4.5 days, respectively. Similar numbers of micro-organisms also reduced thevas e life of Chrysanthemum morifolium Ramat and cultivars of carnation, yet other microbial species isolated from vase water apparently hadn o effect when tested byth e same method, although the number of cfu's at the end of the vase life didno t exceed 105-107pe rm lvas e water. It must be emphasized that the demonstration of the presence of micro-organisms inside stems and the location of the plug by agar plating methods involves both avoidance of contamination with exogenous micro-organisms, and the simultaneous application of 33 methods for the enumeration of different bacterial and fungal species. However, the plating method does not give any information on colonization and effective plugging inside the vascular system (Van Doom et al. , 1990). - Light microscopic observations Aarts (1957), reported plugging at the cut surface of Dahlia stem after 8 days of vase life, and plug formation by amorphic materials in the stem, about 4 cm above the cut surface.Likewise , Lineberger & Steponkus (1976) revealed vascular occlusions due to microbial growth and cell deposition in sections of stems of Rosa hybrida L. cv. Red American Beauty. Microbial occlusions were restricted to the basal 2.5 cm of the stem. In addition, gum deposition was observed, but this always occurred above the vase solution level. Micro-organisms reacted histochemically with the protein stain mercuric bromphenol blue, and the gums were identified with periodic acid-Schiff' s reagent and GLC (gas liquid chromatographic) analysis of the resultant monomers after acid-hydrolysis of the occludingmaterial . Earlier published work of Gilman & Steponkus (1972)however , showed no wound responses of rose xylem after the excision of the cut flower. This is normally accompanied by lignin, tannin, or tylose formation in the cut vessels, and normally is detectable by histochemical-microscopy. - Histochemical microscopy Histochemical studies by Parups & Molnar (1972) of 5-day-old xylem- plugged rose stems showed that the material blocking the vessels of senescing rose stems contained carbohydrates, pectin-, lipid-, and protein-like compounds and some enzymes.Tannins , lignin, and callose were not encountered in the blocked vessels. However, it was not clear whether the plugging materials originated from micro-organisms, microbial particles, microbial metabolites, or from decomposition of thevascula r cellwall s (Molnar & Parups, 1977). - Scanning electron microscopy (SEM) Vascular blockage in the tracheids of cut maidenhair fern fronds was observed by light and electron microscopy and indicated the presence of numerous vascular occlusions in the xylem. No microbial particles were observed. Histochemical studies indicated pectinaceous occluding materials; occlusions were not present in the xylem of freshly-cut 34 fronds or in fronds which had been maintained in vase water supplemented with Ag+ ions. Tannins and lignin were not present. Even when only small parts of a tracheid had been removed, amorphous pectinaceous material was present in the vascular lumen, appearing to reflect a substantial degradation of the primary cell wall in the scalariform pits (Fujino et al., 1983). Ag+ is biocidal and a strong inhibitor of pectlnase and cellulase enzymes. The pectin sources are the primary cell wall, perforation plates, pit membranes and end walls of the vascular system. Wounding of many plant tissues causes increased production of ethylene, which is the hormone-mediated response to the injury. Since Ag+ has been shown to be a highly specific inhibitor of ethylene action, it seems possible that the formation of vascular occlusions in fronds of maidenhair fern might be a response to ethylene produced by the damaged cells. "Gum" formation in plants may occur in response to various physiological or pathological disturbances that induce a break-down of cell walls and cell contents, and stimulate the production of growth regulators and enzymes which might degrade the primary wall forming amorphous gel-like occlusions (VanderMolene t al., 1977). The SEM studies of cut rose stems by Rasmussen & Carpenter (1974) revealed several types of vascular occlusions after 5-10 days in either water or sucrose solutions at 18°C. They found that, (1) the number of tracheids which exhibited blocking at floral senescence was less than 4%; (2) the incidence of blocking was similar at the stem base, water-line and stem neck; (3)breakdow n of secondary tissue in the xylem was observed more frequently than other types of occlusions, but this appeared only after 5 days of vase life; (4) occasionally, blocking materials were found protruding through the pits and end plates of tracheids; (5)som e tracheids in stems held in a 2% sucrose solution developed occlusions with fungal and bacterial growth or slime plugs; (6) the number of occlusions was not influenced by sucrose in vase water, although the occlusions were much larger and observed after a much shorter vase life (3 days) compared with roses placed in water (5-10 days); (7) misleading clogging materials may occasionally come from the phloem at the time ofharvest , orma yb e dried exudate from the cuttingprocess . 35 A serial study involving SEM-examinations of the cut surface of hydroponically-grown Rosa flowers hybrid Tea-rose cv. Sonia (Sweet Promise)showe dtha tth ecu tsurfac ever yquickl ybecam e thesit eo f prolificbacteria l and fungalgrowt h (within2- 3 dayso fvas e life), inspit eo fa lo wleve lo fmicrobia lcontaminatio no fth evas ewate r (i.e. containing only 103-10* cfu of bacteria per ml).Th e water uptakewa sdiminishe d comparedwit h theuptak e inbiocide-containin g vasewater ,bu tther ewa sn otota lblockage ,an dth elongevit yo fth e rose flowers was not decreased dramatically (De Stigter & Broekhuysen, 1986b). Concerning the mechanism of vascular plugging, Durkin & Kuc (1966)conclude d from their experiments with cut rose flowersthat , (1) the actual mechanism for plugging remains obscure; (2) water deficiency isinduce db yvascula rblockage ; (3)vascula rblockag e is not necessarily identicalwit hplugging . In fact, inmos t instances the vascular blockage isno t caused by the accumulations ofmicro organisms inth evascula rsystem ,becaus eexperiment sconducte dunde r completely sterile conditions have not prevented blockage,no rhav e microorganismsbee nrecovere dfro mblocke dstems .Durki n& Ku c (1966) furtherconclude dtha tmicro-organism sd oeffec tth elongevit yo fcu t rose flowers, but such problems only occur after tissue breakdown when the blockage is well advanced. Moreover, it is not widely accepted that xylem conductivity is a good parameter for (1) estimating the water relations of the cut flower; (2) acting as a sensitive indicator for xylem blockage; or (3) indicative of microbialpluggin go fth evessels . As already discussed, neither xylem blockage nor water stress need necessarily be caused by micro-organisms plugging the vessels (Accati, 1980& 1983;Va nAlfe n et al., 1983). For evenwhe nmicro organisms are virtually absent, vascular blockage and water stress may occur,causin g adecrease d vessel conductivity. Thus the causes and the consequences of the vascular plugging phenomena described remainunclear . 36 1.6.Th e selection of a greenhouse rose cultlvar as material for post-harvest research The selection of the rose as material for research is based not only upon the economic importance of rose cultivars in the horticultural production of ornamental plants in the Netherlands; biological scientific and artistic factors have also led to this choice, because: - the rose possesses ahig h grade ofwidely-accepte d beauty, which is evident during all the phases of development from the prime bud to mature flower; - the seven phases of the bud development are well described in the literature and easily differentiated (Berkholst, 1980); - the ideal vase life of roses placed in an environmentally - controlled room (temperature, relative humidity, day/night exposure to light, ethylene washing) can be the basis for the evaluation of vase life; the influence of various factors can easily be recorded by parameters suitable for statistical processing; - cut rose hybrids cultivated in the Netherlands form very low amounts of endogenic ethylene (C2H4)a plant hormone that regulates the flower senescence. Moreover, the cut rose exhibits a low sensitivity to exogenously- and endogenously- produced ethylene (Woltering &Va n Doorn, 1988;Rei d et al., 1989a). - the cut rose isver y sensitive towards microbiological activity in variously-modified vase fluids (Put, 1986); - the starch content of the outer petals of roses gives reliable information concerning undesirable premature picking, unduly long storage periods, or too high storage temperatures, as shown by Berkholst &Navarr o Gonzales (1989); - the vase life of a 'Sonia' rose cut at phase 1-2 placed in a controlled room at 20°C, is normally 8-10 days. Therefore it is possible to finish one test cycle within 8 days, and carry out new tests weekly; - fresh roses, of the same quality can be obtained nearly the whole year around; - the initial microbiological contamination of hygienically picked roses is usually very low, so that with careful handling of the 37 flowers, activities of the initial microbial flora will not interferewit hothe rfactor st ob estudie d (Put,1990) ; atth esam e time the addition of a secondary interfering biocide can be avoided. -Preliminar y investigations These investigationswer e designed toascertai n the influence ofth e stem microflora of Rosa 'Sonia', Gerbera 'Fleur' and Chrysanthemum 'Spider' and their metabolites on the vase life of these» thre e cultivars,b y cross-testing therespectiv emicrobiologica l materials obtained from shake cultures of stem segments, as shown in the scheme of Figure 6, using vase cultures in suspensions containing about 107 cells per ml vase water. The results may be itemised as follows: i Rosa flowerswer emuc hmor e susceptible toa reductio n inwate r uptake causedb ymicro-organism s or theirmetabolite s thanwer e Gerbera- and Chrysanthemum flowers, ii Theeffec to fmicro-organism s (107ml" 1vas ewater )tende d tob e greatertha ntha to fthei rmetabolites , iii No significant differences in water uptake by the respective cultivarsteste dwer eobserved ,whe nth eeffec to faddin gviabl e versus heat-inactivated microbial cells to the vase fluid were compared, iv Whencomparin gth einfluenc eo fth evariou smetaboli ccomponent s of the stem microflora, however, it was shown that the Chrysanthemum microfloracontainin gth ehighes tnumbe ro ffungi , caused the most serious disturbance of thewate r relations and the greatest reduction in the ornamental value of the three cultivarsteste d (Put,1986) . -Method suse d Plant material was used immediately, or stored at 4°C for 24-48h . Flowerswer e cutwit h a stem length of 45 cm, the stems werewipe d with a paper tissue soaked in ethanol (98% v/v) to minimize contamination,an dpu tsingl yint ocylindrica lvase scontainin gresp . 100m l of (1)steril e tapwater ,o r (2)a sterile 1% (w/v)sucros e solution inta pwater , (3)a suspension insteril e tapwate r ofth e micro-organism to be tested, (4)a known concentration of a pectlc 38 enzyme, (5) Al2(SOA)3 solution, (6) the microbial EPS-containing retentate obtained after filtration, (7) the low Mol.wt compound contained in such filtrates. The vases were held at 20°C, 60% r.h. (relative humidity) and with 10.25 W/m2 photoradiation at flower height during a 12 h light/dark photoperiod each 24 h (Zieslin, 1989). Vase life measurements were estimated as follows: every 24 h the water uptake (ml),th e flowering condition or stage of flowering, and the ornamental value up to a maximum of 8 d of vase life was measured. After 2, (3, 5) and 7 d of vase life the microbiological state of the vase water was assessed. SEM preparations were made after 24-48 h ofvas e life, a deliberately short contact time chosen, to exclude microbial colonization and to detect any early response of the host vascular system towards the infiltrating micro-organism, pectic enzyme or microbial EPS. Water conductivity was measured in 3 consecutive stem segments of 5 cm each, after 24 en 48 h of vase life, and microbial EPS was obtained by molecular filtration of the supernatant of a shaken microbial culture. The microbes were removed by centrifuging and pressure filtration. Vascular plugging was also tested by estimating acid fuchsin solution uptake, using 0.5% w/v acid fuchsin in 50% v/v ethanol. A beetroot tissue cube test was devised to demonstrate the ability of micro-organisms to form cell membrane- or cell wall- damaging products. Finally the results were evaluated statistically: analyses of variance were applied to all the measurements and observations of the water uptake, water conductivity, vase life and flower opening of the 'Sonia' roses tested. All test objects involved three replicates, and a few testswer e repeated 2-3 times. - Microbiological factors investigated The initial numbers and types of micro-organisms on stems of freshly harvested cut flower cultivars (Rosa, Gerbera, Chrysanthemum) were determined, and the multiplication ability of the initial stem microflora in vase water was assessed. Further studies assessed the infiltration ability into xylem vessels of pure cultures of stem micro-organisms. The influence of A12(S04)3 in vase fluid on the infiltration of bacteria up the flower stems was determined by means of scanning electron microscopy, and the influence on the vase life 39 and the xylem morphology of microbial factors in vase water was also investigated. This involved vase experiments with suspensions of known numbers of cells in pure cultures, purified microbial pectic enzymes, andmicrobia l exopolysaccharides. 1.7. Vase life experiments The purpose of the microbiological investigations executed by the author was: (1) to clear-up the uncertain role of micro-organisms on the post-harvest physiology of Rosa flowers; and (2) to describe the phenomena concerned therein. 40 APPENDIX I O w> u t* •4 s C D o »Ö lJ < •s i # Ç -9 J3 s h—4 « « S I S ,2 S te *~ " z » «** =a". S S rt S e O vi BOB w ; -S T3 .2 tö «H e E -S < on t—1 > ° «> ° (A S « c e c" ce! S o< I s ° d •s s "& i > H H •H a c u tl I •a « S < Vi .s °* „ 'C •* b •5 .s > s CO 6 " £ 1 §5 S ï -2 0) rt O | g s H* _; 0 -a s u .s f > a .S -c î "l » s* ? > c Ci o g S Ë * « & S " B »• .2 .-S £ «8 Z E •S 3 S 'S » M « »> H Q cd « "S = rt .. J; j. 5 g HH as rt «8 o <# >S <§ < > .Su » « S .2 —; M 4> O •s * s EU u J J S.« ES O. S »î "-0 5 W D. •- "S '•'o8 •J s «s o (S „- o < m fe! s U > < z u d rt •—i 41 APPENDIX II g « 1 i -a .2 4 -is s a . « . * «- s 3 2 Ä !3 h ,<3 B 'Ä » -O S •s l - s •« g £ § s » ; o e ua o £t-° , a£ ~s ^: 2 j,«~ •Bl ' .2M 32a, •i e *i -a S I'' s " s ~ § I S ei *-B '8 £* .s-2.s > r1S s S - ;!»"•• -! 1 è à J | s § § â- é £i * 8- . è g,g J i ï i § •= H -s E««3,>3a§°--E<=2— S1^.2 Sic"»" 3 • 3 § S S t- £ §| Bi ;• 8.S | S £ s £ S || g. SJ S 3 s "siJi sa"--»««*» î.^MïJi la's •J S. « RJoS32.a,*as-s'"S'-s »-«Sa«.6, B — *J -> • -. ^ ** _ *n o' E3 * e-S ï ü Ç* *3 ,« *— r^* i—• .s S ö rt S c ö S 2 1 I « g i1 i § ti j 1H -i i ** s i î 's J1 1„ *. H £ s ., s g g ; - s | g. j. H !. = . 1= o * J! ï » "- f ï * 8 S ï .S F 3â H SSS^B-rE-S 42 160- g 140 c 120 JÏ CJl 'QJ 100 5 JZ eu 80 60- T—i—i 1—i—i—i—r , 1 23456789 10 d°ys Fig. 1.Fres hweigh to fcu t (brokenlines )an dintac trose s(straigh t lines)a tth een do fth e1 4h an d1 0h light :dar kperiods ,expresse d asa percentag e ofth einitia lweight .O nda y8 an d1 0respectively , petals showed symptoms of senescence,an d were removed (DeStigter , 1980). 43 X A Ti 5n en pm 4- ai QH o §• 3- «5 W 2- 1- s QZE on an on an an nu an 1 2 3 4 5 6 7 8 9 10 11 12 days * T 5-, B xz pnq Ol 4- aî o 3- * 2- (O o ^ 1- 1111 ai m I 1 M 1 1 2 3 4 5 6 7 8 9 10 11 12 days •X" = light period p.day # . dark period p.day Fig.2 .Wate r lossan duptak eo fintac t(A )an dcu t(B )roses ,pe rh in light anddark . Surpluses areverticall y hatched, deficitsar e solid black. Column width corresponds toth eduratio n ofligh tan d dark periods: 14h and1 0h respectively. Arrows indicate timeo f petalremoval ,o fFig .1 (D eStigter ,1980) . 44 Cavity of flower stem xylem vessel vase water level Indirect lateral -water uptake through plant stem tissue Vase water Direct water uptake Fig. 3.Th etw oroute so fwate ruptak e ina cu tflowe rwit ha hollo w stem (VanMeeteren , 1980). middle lamella primary wall secondary wall border. pit membrane border Fig. 4.A diagram toillustrat e differences inth estructur e ofpit s in plant xylem vessels. A, a normal pit, B, a bordered pit (from Sperry& Tyree , 1988). 45 EraOUZED FUNCTIONAL Pil membrane intervessei pil Fig. 5. Diagram of wall structure between adjacent xylem vessels showing intervessei pit structure. The porous pit membrane develops from primary cell walls of the two vessels and middle lamella; it is overarched by thick secondary walls to form a pit chamber that opens to the vessel lumen via a pit aperture. When a vessel is embolized, air isprevente d from spreading toadjacen t functional vessels by the capillary force of the air-water meniscus spanning pit membrane pores. If the pressure difference across this meniscus exceeds the force holding it there (which is dependent on pore diameter and sap surface tension), air is aspirated into the functional vessel and it becomes embolized, this is the air seeding mechanism of water stress- induced embolism (from Sperry & Tyree, 1988). 46 > •** ü •o w c o 0) o O +F- 0) CO si t CO end e o c 8 Q- O. +* E m w 03 TJ 0) CS io (D k- £o .w (0 *-» c o E (0 o 0) la 3 •M D ü E o (0 © Fig. 6. A summary of the various experimental procedures used to assess the influence of microbial stem cultures on the vase life of cut flowers (Put, 1986). 47 Chapter1 References Aarts, J.F.Th. 1957. Over de houdbaarheid van snijbloemen. On the keepability ofcu tflowers .Wit henglis hsummary . Mededelingen van de Landbouwhogeschool, Wageningen,5 7(9) ,1-62 . 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Portland, Oregon, USA: Timber Press,47 0pp . 57 Chapter 2 Micro-organisms isolated from freshly harvested cut flower stems and developing during the vase life of: Chrysanthemum, Gerberaan d Rosa cultivars. 59 Rosacv .'Sonia ': stag e2 o fflowe ropenin g (Berkholst,1980 ) 60 Scientia Horticulturae, 43 (1990) 129-144 Elsevier Science Publishers B.V., Amsterdam —Printe d in The Netherlands Micro-organisms from freshly harvested cut flower stemsan d developing duringth e vase life ofchrysanthemum , gerbera and rose cultivars Henriette M. C. Put*, Sprenger Institute, Ministry of Agriculture and Fisheries, P.O. Box 17, 6700 AA Wageningen (The Netherlands) (Accepted for publication 1Septembe r 1989) ABSTRACT Put, H.M.C., 1990. Micro-organisms from freshly harvested cut flower stems and developing during the vaselif e ofchrysanthemum , gerbera and rosecultivars .Scientia Hortic, 43: 129-144. Awid evariet yo fmicro-organisms ,bacteri a and fungi, wasisolate d from freshly harvested cut flower stems and vase contents of Chrysanthemum (Dendranthema grandiflora)cultiva r 'Spider', Gerbera cultivars 'Appelbloesem' and 'Fleur' and Rosacultiva r 'Sonia'. Fungal spp.wer e isolated much more frequently than by other authors. Bacterial genera, present on the stems, were mainly also present in the corresponding vasewater . The dominant initial stem microflora, Enterobacter, Bacillus spp.an d fungi, lost their dominance in the vasewater , which after 3day so fvas elif e showed a predominance of Pseudomonas spp. The longer the vase life, the greater were the changes in the microflora of the vasewater , which later again showed a predominance ofEnterobacter spp. and often also ofBacillus spp. After ? 10day s of vase life, fungal growth increased dramatically in Chrysanthemum and Ger bera vasewater .Th e uniqueecologica l conditions in thevas eflui d and, to alesse rextent , theantago nistic activities of many of the microbial species of the mixed vase flora will have led to the initial predominance ofPseudomonas spp .an d totypica l changesi nth edominan t flora duringth ecours eo f theflowers ' vaselife .Th e microbial load on stemso fcu tRosa wa sfoun d tob emuc h lowertha n those on Chrysanthemuman d Gerbera stems.Th een do fth evas elif e ofth eRosa flower s wascharacterize d by 'normal' senescence symptoms or by weak wilting of leaves and flowers. In Chrysanthemum and Gerbera however an extensive water stress developed. Further studies are required to evaluate the effects of pure cultures of stem micro-organisms on the vase life ofcu t flowers. For the prevention of microbial activity incut-flowe r vasewater , which leadst oa shorter flower vaselife ,goo d commercial practicesar edesirable . Keywords: cut flower; Chrysanthemum Dendranthema;flowe r stem microflora; flower vase micro^ flora; Gerbera;Rosa. Abbreviations:API=Appareil s et Procédés d'Identification; CFU=colony-forming units; MA.=malt extract agar; McK.A.= MacConke y agar; My.A.= mycophy l agar; P.C.A.= plat e count agar; Ps.A. (F.P.) = Pseudomonas selectiv e agar (Fluorescens and Pyocyanin). *Present address: Kerksteeg 4, 7411 EW Deventer, The Netherlands. 61 H.M.C. PUT INTRODUCTION Bacterialpluggin go fxyle mvesse lelement sha sbee nclaime dt ob eth ema jor causeo frapi d senescenceo fcu t flowers (Aarts, 1957).Howeve rthi sphe nomenon has seldom been clearly demonstrated, for in many such studies bacterial numbers have not been adequately assessed and bacterial species causing senescence of cut flowers have rarely been identified (Ford et al., 1961;McClar y andLayne , 1977;va nMeeteren , 1980; Zagoryan dReid , 1986; Put and Jansen, 1989). Fungalpluggin go fxyle mvesse lelement sis ,o nth econtrary ,reporte dt ob e rare (DeStigterandBroekhuysen, 1986). Itha sals obee nsuggeste dtha tsom e (usuallyunidentified ) microbialprod uctsexcrete d intovas ewate rb ypur e ormixe d (usually unidentified) micro bial species can cause vascular plugging (Gentile and Accati, 1981; Accati- Garibaldi, 1983;Put , 1986;Zagor y and Reid, 1986). In addition, it is not clear whether cut-flower vase micro-organisms origi nate from the initial stem flora, or may have been introduced accidentally intoth evases . Therei sals olittl einformatio n availableo nth epotentia lo fth e microflora for multiplication in vasewater . Thepresen tpape rgive sth etype san dnumber so fmicro-organism s isolated from freshly harvested cut flowers. In addition, attempts weremad et o iden tify thedominan t microflora inthei r vasewater . MATERIALS AND METHODS Plantmaterial. - The following plants were studied: Chrysanthemum mori- folium cultivar 'Spider' (Dendranthema grandiflora, Anderson, 1987), Ger beracultivar s'Appelbloesem ' and 'Fleur', andRosa hybri d cultivar 'Sonia'. Harvestof flower cultivars. - The flowers were grown in commercial glas shousesan dharveste dbetwee n Februaryan dOctober .Cu tflowers wer esam pled2- 4 timesan d eachcultiva r washarveste d from twodifferen t glasshouse growers. The harvested flowers were packed into parchment paper, trans ported to the laboratory within 2-3 h and stored at 2-5° C for <48 h. Chry santhemum and Gerberawer ecu ta t < 10c mabov eth esoil .Th elengt ho fth e stem varied between 65 and 75 cm.Rosa flowers were harvested by cutting the stems ~ 1.50 mfro m thesoi la ta lengt h of ~6 5cm . Preparation ofstem samples for plate counts. - Fromeac hcultivar , 10flower s per test wereused . Each test was repeated 2-4 times. The intervals between the tests were ~ 1 month. From each of the 10cu tflower stem s tested,five segments of 4-cm length were cut off from the lower part of the stem and added to 20 ml of sterilized tap water with 0.05%v/ v of Tween 80 (Difco) 62 MICRO-FLORA FROM FLOWER STEMSO F CHRYSANTH, GERBERA, ROSE in a sterile screw-capped bottle. The bottles were shaken for 30 min and su- pernatants, containing only the microflora washed off from the correspond ingflowe r stem,wer estore d in sterilebottle sa t 10°Cfo r amaximu m of 2-4 h,whereafte r microbial analyseso fth esupernatant s werecarrie d out. Preparation ofsamples for estimationof the dominant vase water microflora. - Ten flowers ofeac h of thecultivar s werefurthe r cut off to a stem lengtho f 45c man dpu t singlyint ocylindrica lvase scontainin g 100m lsteril eta pwater , or a sterile 1% w/v sucrose solution in tap water (Aarts, 1957;Chi n and Sa- calis, 1977) . The effect of 1% w/v sucrose in vasewate r on the development ofth e stem microflora wasals o included in thestudies ,a s 1% w/v sucrosei s frequently addedt ovas ewate rt osupplemen tth eenerg yneede dfo r flowering and to positively affect the balance between solution uptake and transpira tion. The depth of the stems immersed in water was ~ 18cm . The height of the water column inth e vaseswa shel d between 8an d 18 cmb yth e addition of sterilized tap water. Protection against contamination was achieved by sealing a plastic film on the top of each vase (Parafilm M; American Can Co.). The vases werehel d at 20±2°C in an atmosphere of 60%relativ e hu midity and lighting at 10.25 W m-2 photoradiation at flower height during alternating 12h light/dar k periods.Sample s from the vaseswer etake n asep- tically each third dayo f vaselif e until flowering senescence. Microbiological analyseswer ecarrie d outwithi n 2-4 hafte r sampling. Media. - The media used were nutrient agar (Difco, 1984; 0001 ) or plate count agar (Difco, 1984;047 9 at 28°C), and/or both agar media to which lOOjUgml -1 ofpimarici n (Mycopharm, Delft, TheNetherlands ) wereadde d to suppress fungal growth. Enterobacteriaceae were counted at 37° C on MacConkey agar (Difco, 1984;0075) ,Pseudomonas species weregrow n on Pseudomonas selective agar (Oxoid, 1983;C M 457) at 28°C, as well as on PseudomonasF (fluorescens) andP (pyocyanin) medium (Difco, 1984;044 8 and0449) .Fung iwer eenumerate d at25° Co nmal textrac taga r (Difco, 1984; 0112) or mycophyl agar (BBL,Divisio n ofbioquest ; 11452,Becto n Dickin son and Co.,Cockeysville , MD 21030,U.S.A. ) to which 50n%ml -1 ofglob - enicol Na succinate (Mycopharm, Delft) and 10fig ml -1 of polymyxin B- sulphate (Pfizer, SA, Brussels) were added to suppress bacterial growth (Smith et al., 1952; Kirchman et al, 1982;Reasone r and Geldreich, 1985; Gould etal. , 1985). Microbialcounts of stem flora and vase water flora. - Onemillilitr eo rdecima l dilutions to a maximum of 10-6 ml of the supernatants of the shaken stem samples and vase water samples were pour plated using the different plate count and (selective) agarmedia .Surfac e spreadplate swer eals omad e from the samedilution s usinga Spira l Plate Count Machine (Model C,Do n Whi- 63 H.M.C. PUT tley Scientific Ltd., Shipley, West Yorkshire). MacConkey agar plates were incubated at 37±1°C. The remaining bacterial plates were incubated at 28±2°C for 2-3 days. This incubation temperature was used to obtain an almost representative and rapid colony formation of psychrotrophic aswel l as mesophilic flora. For fungal growth, plates were incubated aerobically at 25± 2° Cfo r amaximu m of 10days ,whereafte r the number of colony-form ingunit s (CFU) wa sassesse d and the numbers ofbacteri a and fungi per 10- cm stem and per millilitre of vase water were calculated (Tan et al., 1983; Taylore tal. , 1983). Isolation andpurification ofmicro-organisms. - Colonieswer eexamine dvis ually and representatives of all the different colonial types were randomly isolated from both the selective and non-selective media. The randomly se lected colonies were examined microscopically. Cultures were then purified priort o further classification. Identificationof micro-organisms. - The final pure cultures, ~350 strains, were grouped on the basis of a preliminary identification by conventional methods (Buchanan and Gibbons, 1974), using the recommended time/ temperature conditions. After allocation to the main groups (bacteria, moulds or yeasts), thebac teria were further characterized using Gram staining, tests for motility and the presence of oxidase, oxygen requirement as revealed by the API M-me- dium (5012) and fermentative activity as revealed by the API OF medium (5011) (API system S.A. La Balme les Grottes 38390, Montalieu Vercieu, France). Further identification wasachieve d usingcommerciall y available identifi cation systems (Fung and Cox, 1981;Fun g et al., 1984). The OXI/FERM tube II (F. Hoffmann-La Roche and Co. Ltd., Diagnostica, CH-4002 Basle, Switzerland) wasuse d for the identification of oxidase-positive Gram-nega tiverod s (Stanieretal., 1966).Th epresenc eo fPseudomonas aeruginosa wa s confirmed by growth at 42.5°C . Enterobacteriaceae were identified by the Enterotube II method of F. Hoffmann-La Roche (Leclerc, 1962; Ewing and Fife, 1972;Dietsch , 1981;Co xe t al., 1983).Th e presenceo fEscherichia coli (and pathogenic Enterobacteriaceae) was confirmed by growth at 42.5°C ; selective biochemical and/or serological tests were also carried out. When inconclusive results were obtained, the API systems 20 NE or 20 B (API- system SA, La Balme les Grottes 38390, Montalieu, Versieu, France) were applied (Lampean d van der Reyden, 1984). ForBacillus strains,th e Enter otubeI I (La Roche) and API 50C H systemswer eused ,i ncombinatio n with classicaltest s for the hydrolysis or decomposition of casein,gelati n and leci thin (Smith etal. , 1952; Gordon etal. , 1973;Berkele yan dGoodfellow , 1981; Gil et al., 1986). The formation of bacterial polysaccharides, dextran and 64 MICRO-FLORA FROM FLOWER STEMS OFCHRYSANTH , GERBERA, ROSE levan was detected on API sucrose agar code 5013 (Facklam, 1977). The identification of most ofth eBacillus strains wascross-teste d byth e APIRe search Laboratoriesa t Montalieu using acombinatio n ofAP I 50C H and 20 E (Logan and Berkeley, 1981, 1984). Fungal strains were identified at the Centraal Bureau voor Schimmelcul tures (C.B.S.), Baarn, The Netherlands (Alexopoulos and Mims, 1979; Domsch et al., 1980;Kreger-va n Rij, 1984). Additional testswer eapplie d tosevera lstrain so fsom eo fth ebacteria l and fungal species,whic h owing to their ability to multiply in vase water, might have had a significant role in the reduction of the vase life of cut flowers. These tests were the decomposition of pectin by the method of Wieringa (1956 ) orPerombelo n and Hyman (1986),th ehydrolysi so fcarboxy-methyl - cellulose (CMC) as described by Skerman (1959) and Robson and Cham- bliss (1984),th eformatio n ofexopolysaccharide so nsucros eaga r (API) and theminimu mtemperatur e forgrowt ha t 5-25°Ci nsucros e (0.1%w/v ) flower stemextrac t broth (0. 1% w/v) . Microscopic observations of the vase water. - Thin smears of the vase water samples incubated at 20° Cwer efixe d and Gram stained. The fungi-contain ing samples were also examined using cotton blue lactophenol. In addition, preparations from vasewate r samplestake n duringth e course ofth e incuba tion werestudie d byphase-contras t microscopy. RESULTS Microbialcounts on stems. - The number of viable bacteria and fungi on freshly cut flower stemsrange d from < 10—104 CFUpe r 10-cmlengt ho fste m (Table 1 ). For Chrysanthemum,6 0 and 30%o f the 40 flower stems tested contained > 102 viable bacteria and fungi, respectively, per 10c m of stem, whereas 45 and 52%o f the 40 Gerbera flower stems tested contained >10 2 viable bacteria and fungi, respectively, per 10c m of stem. However only 4 and20 %o fth e5 0Rosa stem steste dcontaine d > 102viabl ebacteri aan d fungi, respectively, per 10c m of stem. Moreover, it was observed that for 86 and 56%o fth e 50Rosa stem stested , < 10 viablebacteri a and fungi, respectively, were isolated per 10-cm length of stem.Thu s the microbial load on stemso f cut Rosawa smuc h lower than that onth e stems ofth e Chrysanthemum and Gerbera cultivars tested. Microbialcounts ofvase water. - (1 ) Chrysanthemum and Gerbera cultivars. Duringth e first 6day so f vaselife , bacterial numbers exceeded aleve l of 106 CFUml ~' vasewate rsevera ltimes .Maximu m numberso fCF Ucounte dwer e ^ 108bacteri a and > 107fung i ml"' vasewate r (Table 2). (2) Rosa'Sonia' . During the first 6 days of vase life, bacterial numbers remained < 106CF U 65 H.M.C. PUT TABLE 1 Estimation of the numbers of micro-organisms on 20 or 50 stems of freshly harvested cut flow ers. For bacteria on plate count agar, incubated for 2-3 days at 28°C ; for fungi on malt extract agar incubated for 6-10 days at 25° C Flower cultivar Micro Percentages of stem segments of 10c m length Actual no. organisms showing plate count numbers of: of stems 0 1-10 10'--102 102--103 103-104 Chrysanthemum 'Spider'-white Bacteria 10 10 10 50 20 20 Fungi 20 20 30 30 <5 20 Chrysanthemum 'Spider'-yellow Bacteria 20 10 20 50 <5 20 Fungi 10 30 30 20 10 20 Gerbera 'Appelbloesem' Bacteria 10 20 20 50 <5 20 Fungi 10 10 10 60 10 20 Gerbera 'Fleur' Bacteria 10 25 25 40 <5 20 Fungi 15 20 30 30 5 20 Rosa 'Sonia' Bacteria 16 70 10 4 <2 50 Fungi 8 48 24 12 8 50 ml~ ' vasewate r (Tabl e2 ) .Durin gth eprim esenescenc ephas eo fth e' Sonia ' flowers, the number of CFU counted seldom exceeded 106 ml-1 and fungal countsremaine d <5 x 104ml - ' vasewater . Identificationof stem microflora (Table 3). - The predominant bacteria iso lated from freshly harvested cut flower stems were species of the generaEn terobacter (E.agglomérons and E. cloacae)an d Bacillus (B.subtilis, B. lich- eniformis, B. cereus). Pseudomonas spp. were isolated occasionally. The predominant mould genera were Cladosporium, Fusarium,Pénicillium, Mu- coran dRhizopus. It was furthermore observed that low initial numbers of micro-organisms ona nindividua l stem mainlyconsiste d ofa wid evariet y ofth e flora. Identificationof vase water microflora. - Duringth efirs t 3-6 dayso fvas elife , Pseudomonascepacia, Pseudomonasfluorescens an dPseudomonas putida re placed the initially dominant Enterobacteran d Bacillus stem flora. However after >6 days of vase life, Enterobacter spp. again became more dominant 66 MICRO-FLORA FROM FLOWER STEMS OF CHRYSANTH, GERBERA, ROSE •O ^to tu •" '"> vu \0 00 X ^ tu C < U t o o o o O vo O o Ü o vO (N O O O CS <§ oo > Ü o ra oo ta O O 00 o * — — o — O oo X X «5 3 X x- tN 't o. i^ 00 A " S i D. T3 *; « I » I>f l I X o o CA O O O oo r- tN « — — — O o X X ? A\ A\ A\ 1 XXX. * x x "O VI o VS c 1/1 oo ^O w-i A\ '\ £ CD CL, TABLE 3 Identification of the microflora from freshly harvested cut flower stems (s) and present in the vase water (v) after 3-12 days ofvas e life Micro-organism Flower cultivar Chrysanthemum Gerbera Rosa white/yellow 'Appelbloesem' 'Sonia^ 'Spider' and 'Fleur' s V s V s V Gram-negative rods Acinetobacter sp.* + Achromobacter sp.* + Alcaligenessp. * Chromobacterium violaceum Citrobacter freundii + + C.var . amalonaticus — + Enterobacter E. agglomérons + + + + + + E. cloacae + + + + + E. gergovinae + E. sakazaki + Enterobacter sp.* + Erwinia Er. amylovorum* + + Er.herbicola* + + Flavobacterium sp.* + Pseudomonas P. aeruginosa + — - P.cepacia + — — P.fluorescens - + + P. maltophilia + - — + P. putida + + + + P. putrefaciens + P. stutzeri + P. vesicularis + - - Pseudomonas sp.* _ + _ Gram-positive rods Bacillus B. cereus (lecithinase— ) + + + + B. cereus (lecithinas e+ ) + B. circulons B. licheniformis + B. mycoides + + B. polymyxa + + + + + B. subtilis + + + + B. subtilis var. niger + + + + B. thiaminolyticus -I- 68 MICRO-FLORA FROM FLOWER STEMS OF CHRYSANTH, GERBERA, ROSE TABLE 3 (continued) Micro-organism Flower cultivar Chrysanthemum Gerbera Rosa white/yellow 'Appelbloesem' 'Sonia' 'Spider' and 'Fleur' Corynebacteria* - + Gram-positive cocci Streptococcus S. lactisgroup * - Fungi Acremonium alternatum Link: Fr. + Aureobasidium pullulans (de Bary) Arnoud + + A. pullulans var.* - + Botrytis cinerea Pers.: Fr. — + Botrytis sp.* OI- - Cladosporium cladosporides Fres.) de Vries + C.sphaerospermum Penz. + Drechslera sorokiniana (Sacc.) de Vries — - - - + Fusarium solani — F. tabacinum var. + + - + plectosphaerella cucumerina (Lindfors) W. Gans + F. oxysporum* - + + - + Mucor hiemalis (Wehmer) - + + - + M. plumbeus Bon - + - - M. racemosus — Pénicillium brevicompactum (Dierckx) + + P. brevicompactum atyp.1 * + P. brevicompactum atyp. II* + + + + P.purpurogenum Stoll - P.variable + + + Pénicillium sp.* + Rhizopus stolonifer (Ehrenb. ex Link) &Vuil l + + Rhizopus sp.* + + Trichoderma pseudokoningii Rifai — 69 H.M.C. PUT TABLE3 (continued) Micro-organism Flower cultivar Chrysanthemum Gerbera Rosa white/yellow 'Appelbloesem' 'Sonia' apiaer and 'Fleur' s V s V s »V Verticillium albo-atrum (Reinke and Berthold) - - + - - - V. brevicompactum + - - - - + V. psalliotae Treschow - - + - - - Yeasts Candida albicans — + — + — - C.famata - + — - - - Candida sp.* — + — + + + Kluyveromyces marxianus — — — + - - Rhodotorula mucilaginosa — + — — — - R. rubravar .I * — + — + — + R. rubravar .II * — — — + — - Saccharomyces* + - - - - + *Not further identified, somecharacteristic s differ from the official descriptions ofth e charac teristicsoftha t genuso rspecies . (E. agglomerans andE. cloacae).Furthermore , Bacillusspp. , Erwinia and alsonon-fermentin g Gram-negative bacteria predominated later. Fungal activity in vase water was low until after >6 days ofvas e lifeo f Chrysanthemum and Gerbera cultivars; Cladosporium,Fusarium, Pénicil liumspp .an dals osom eyeas tgener awer emos t frequently isolated{Candida andRhodotorula ) . Sucrose (1% w/v) added toth evas e water initially induced acertai nin crease (about afacto r of 10-50) inmicrobia l multiplication rates and prob ably also a shift inth epredominanc e ofcarbohydrate-dependen t microbial species;afte r ~6 day so fvas elif e these differences becameles smarked . Additionaltests on the identified(dominant) flora. - The resultso fth e addi tionaltest sindicate dth e following. (1 ) SomeBacillus spp. ,som ePseudomonas spp. ,Kluyveromyces strain san d allth eisolate d mould strainshydrolyse d pectin. (2) Adhesive capsular slime was formed byBacillus spp. , Pseudomonas spp.,som eEnterobacter spp .an dth eRhodotorula strains . (3) Some Bacillusspp. , some Pseudomonasspp .an d some fungus spp. formed exopolysaccharides. 70 MICRO-FLORA FROM FLOWER STEMS OF CHRYSANTH, GERBERA, ROSE (4) The isolated Fusariumoxysporum and Botrytiscinerea strains belong to phytopathogenic genera (Domsch et al., 1980); they were shown to pro duce plant cell membrane-injuring compounds (beetroot test, Put andKlop , 1990). (5 ) Cellulase was of minor importance due to the slow production of this enzyme. DISCUSSION Enumeration and identification ofcut-flower stem microflora. - Most of the microbial species isolated from the cut flower stems are normal inhabitants ofth euppe rlaye ro fagricultura l soil (DoCarmo-Sousa , 1969; Buchanan and Gibbons, 1974;Domsc h et al., 1980) and thenon-soi l micro-organismswer e probably introduced by other sources of exogenous contamination (Ford et al., 1961; McClary and Layne, 1977;Heni s and Bashan, 1986). Some ofth e microbial species isolated, e.g. Enterobacter agglomérons and Erwinia amy- lovora, are commonly associated with plants (Lasko and Starr, 1970; Ver- donck et al., 1987).Pseudomonas cepacia and Bacilluspolymyxa are phyto- phagens, but the majority are common soil saprophytes or epiphytic constituents of the phyllosphere. In the phyllosphere of herbaceous plants, yeastsa swel la sfilamentous fung i areusuall yfound , although in lowernum berscompare d with bacteria (Last and Price, 1969;Buchana n and Gibbons, 1974;Davenport , 1976;Domsc h etal. , 1980;Kreger-va n Rij, 1984). The numbers of micro-organisms on Rosa stems were found to be much lower than those on Chrysanthemum and Gerbera stems (Table 1), which probably reflects their distance from the soil.Furthermore , the hairy epider mis of Gerbera (van Meeteren, 1980) and the numerous tiny side leaves of the Chrysanthemum stem (Henisan d Bashan, 1986) probabl y facilitated the adherence of soil micro-organisms to the stems. Comparison of data pre sented here with data reported by other authors could not be made because published data concerningcut-flowe r stem microflora areno t available. Enumeration of vase water microflora. - Multiplication of micro-organisms in the vase water containing the cut flowers was relatively slow (Table 2). The microflora initially present on the stem of freshly harvested cut flowers maygerminat e (spores) and multiply in vasewater , but exhibitsa compara tively long generation time due to the nutrient-poor vase water growth me dium.Th eadditio n of 1% w/v sucroseinitiall y induced anincreas ei nmicro bial multiplication rates and a shift in the predominance of carbohydrate- dependent microbial species,whic h later became less marked, probably due to the leakage of growth-promoting compounds such as carbohydrates and proteins from phloem vesselsan d injured plant cells (Kooy etal. , 1982; Wol tering, 1987). The competition between the different microbial genera and speciesmigh t then havebee n ofgreate r influence than the ability ofth eindi vidual microbial cellt o multiply in tap water or sucrose-tap water, enriched with compounds leakingou t ofcu tflower vessels . 71 H.M.C. PUT Identificationof vase watermicroflora. - Most ofth emicrobia lgener apresen t onth e stems wereals opresen t in the vasewater , but some ofthe m were not. Equally,man y ofth e microbial speciesdevelopin gi nth evas ewate rwer enot alwaysdetecte d onth e corresponding stems. Thesefinding s indicate: (1 )th edistributio n ofth emicro-organism so nth e stems is not homogeneous, so the microbial species present on 10c m of the lower parts ofth e stems,use d for plate counts and identification ofth e stem flora, will not have been true replicates ofth e microflora on the upper parts ofth e stems,washe d off byth e vase fluid; (2) the selectivity ofth e isolation method used may have failed, comparing single populations of micro-orga nisms adhering to a flower stem and a competitive mixed flora of the vase fluids. The fact that Pseudomonas spp. were more frequently isolated from cut- flower vasewater stha nEnterobacter spp .ma yb ebecaus emos tPseudomonas spp.d ono t requireorgani cgrowt h factors and canmultipl y readilyi n diluted aqueous media (Kooy et al., 1982). Enterobacteriaceae often have morede manding growth requirements so their multiplication may have accelerated after the leakage of carbohydrates (glucose, fructose, sucrose), proteins and growthfactor s from thedisrupte dplan ttissue s (Koningsan dVeldkamp , 1980; Zimmermann, 1983;Woltering , 1987). E. agglomerans (Erwinia herbicola) showed yellow-pigmented colonieso n plate count agar and may have been confused with other yellow-pigmented Gram-negative organisms (e.g.Pseudomonas, Flavobacterium) by other au thors, and thus may not always have been recognized (Leclerc, 1962; Gra ham, 1964;Graha m and Hopkins, 1967;Lamp e and van der Reyden, 1984; Verdoncke t al., 1987). The predominance and shift in predominance of certain microbial genera in vasewater , e.g.Bacillus, Enterobacter, Pseudomonasan d fungi, duringth e course of vase life, which were mainly not correlated with the specific host plant, indicate that micro-organisms developing in the cut-flower vasefluid are more a function of the unique ecological conditions in the vase than of the micro-organisms initially present onth estems . Theeffects ofthe vase microflora onthe vase lifeof the flowers. - Many ofth e stem micro-organisms might cause a decreased vase life and a rapid senes cence of cut flowers, especially if they are able to multiply in vase water to > 106 ml-1 because: (1) microbial propagules may then be able to cause physical pluggingo f the xylem vessels and disturbance of water uptake (Put and Jansen, 1989); (2) toxicmicrobia l compounds mayb eexcrete d into the vase water and accelerate senescence (Lasko and Starr, 1970; Accati-Gari- baldi, 1983;Put , 1986; Zagory and Reid, 1986; Leary et al., 1986; Put and Klop, 1990; Put and Rombouts, 1989); (3) it is uncertain whether or not sound cut flowers can be affected pathologically by fungi during their fairly short vase life; although Fusariumoxysporum, whic h multiplied in the vase 72 MICRO-FLORA FROM FLOWER STEMS OF CHRYSANTH, GERBERA, ROSE water, induced wilt symptoms in pot-grown carnations within 4 weeks after soil,roo t or stem inoculation (Demmink et al., 1987;Baaye n et al., 1988). Publisheddata concerning vase waterflora. - It wasshow ntha t the materials and methodsuse dwer emainl ydifferen t and hardly comparable,a sshow nb y the following examples. (1 ) Dansereau and Vines (197 5) reported on the vase water and xylem fluid flora ofsnapdrago n cultivar 'Tampica'; Marousky (1980), in hisbacte riological in vivo and in vitro vasewate r experiments, used snapdragon cul tivar 'Oklahoma' andgladiolu scultiva r 'White Friendship'. (2) Gardner et al. (1982) identified the xylem-residing bacteria in roots ofFlorid a citrustrees . (3) Accati-Garibaldi (1983) analysed vase water of carnation cultivar 'Siva' flowers. (4) Sampleswer emainl ytake n from vasewate ro fsenesce d flowers (Aarts, 1957;Danserea u and Vines, 1975;Accati-Garibaldi , 1983;Zagor y and Reid, 1986). (5) Only small numbers of representative colonies (25-70) were isolated andidentifie d byFor de tal . (1961),McClar yan d Layne (1974),Zagor yan d Reid (1986) and deWitt ean d van Doorn (1988). (6) Gardner et al. (1982) isolated randomly 850 colonies and identified 550pur eculture s (seeite m (2) above). (7) Numbers ofisolate d colonieswer eno t given byAart s (1957) andAc cati-Garibaldi (1983). (8) Human pathogenic micro-organisms were not found in the present study.Author swh oreporte d the isolation ofsuc hbacteri a (Ford etal. , 1961; McClary and Layne, 1977) isolated them from cut-flower vase water or the flower containers,whic h wereno t treated aseptically. (9) Plate counts of fungi were only reported by Zagory and Reid (1986) and data on the initial microflora of cut flower stems were not reported pre viously. Inth e present study, lownumber s of fungi were found onth estems , many different species were identified and their distribution was not corre latedwit hth e specific host plant. Furtherresearch. - The results presented here are very preliminary from a horticultural standpoint. Further research isneede d to reveal the role which micro-organismsma ypla yi nth emechanism sleadin gt oa shorte rflowe r vase life. Therefore much more data need tob e available on the initial microflora onstem so f freshly harvested cut flower cultivars,th eabilit y ofpur e cultures ofth e main stem and vasewate r microflora to multiply in vasewate r needs to beassesse d aswel la sth e effects ofpur eculture s ofthes e micro-organisms onth ewate ruptak e and vaselif e ofth ecu t flower cultivarsconcerned .Thes e effects may consist of mechanical plugging of xylem vessels, physiological changeso fth ewate r relationshipso fth ecu t flower, morphologicalchange so f 73 H.M.C. PUT the xylem vessel structure following elicitor or enzymatic activities and (phyto)toxicit yo fth e microbial vasewate rculture . ACKNOWLEDGEMENTS Theautho ri sgratefu l toDr .Murie lE . Rhodes-Roberts,Universit yo fWales , Aberystwyth, Great Britain, for critical review ofth emanuscript . The author also thanks Ans van Hardeveld for text registration. The flower project was supported, inpart ,b yth etechnica ldirecto r ofThomasse n &Drijver-Verblif a N.V., packaging industry, Deventer, The Netherlands, and by secondment (1984-1987) ofth e author at the former Sprenger Institute, Wageningen. REFERENCES Aarts, J.F.Th., 1957. Over de houdbaarheid van snijbloemen. (On the keepability of cut flow ers.) Meded. Landbouwhogesch. Wageningen, 57: 1-62 (in Dutch with an English summary). Accati-Garibaldi, E., 1983. Postharvest physiology of mediterranean carnations: partial char acterization of a bacterial metabolite inducing wilt of flowers. Acta Hortic, 138: 225-260. Alexopoulos, C.J. and Mims, C.W., 1979. Introductory Mycology. 3rd Edn. Wiley, New York, 632 pp. Anderson, N.O., 1987. Reclassification of the genus Chrysanthemum L. HortScience, 22: 313. Baayen, R.P., Elgersma, D.M., Demmink, J.F. and Sparnaaij, L.D., 1988. 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Van Meeteren, U., 1980. Water Relations and Keeping Quality of Cut Gerbera Flowers. Ph.D. Thesis, University of Agriculture, Wageningen, The Netherlands, 78 pp. Verdonck, L., Mergaert, J., Rijckaert, C, Swings, J., Kersters, K. and De Ley, J., 1987. Genus Erwinia: Numerical analysis of phenotypic features. Int. J. Syst. Bacteriol., 37: 4-18. Wieringa, K.T., 1956. The micro-organisms decomposing pectic substances in the dew retting process of the flax. Neth. J. Agric. Sei., 4: 204-209. Woltering, E.J., 1987. The effects of leakage of substances from mechanically wounded rose stems on bacterial growth and flower quality. Scientia Hortic, 33: 125-136. Zagory, D. and Reid, M.S., 1986. Role of vase solution micro-organisms in the life of cut flow ers. J. Am. Soc Hortic. Sei., Ill: 154-158. Zimmermann, M.H., 1983. Xylem Structure and Ascent of Sap. Springer Verlag, Berlin, 143 pp. 76 Chapter 3 Infiltration of Pseudomonasputida cells ,strai n 48, into xylemvessel s of cut Rosa cv. 'Sonia'. 77 Roea cv. 'Sonia': stage 3 of flower opening (Berkholst, 1980) 78 Infiltration ofPseudomonas putida cells, strain 48,int o xylem vessels ofcu t Rosa cv. 'Sonia' HENRIETTE M.C. PUT*| & T. VAN DER MEYDENJ *Carl C. Conway Laboratories, Thomassen &Drijver-Verblifa N.V., PO Box 103, 7400 GB Deventer and ^Sprenger Instituut, The Ministry of Agriculture and Fisheries, PO Box 17,6700 AA Wageningen, The Netherlands Received 16 March 1987,revised 17September 1987 and accepted 9 October 1987 PUT, H.M.C. &VA N DER MEYDEN, T. 1988. Infiltration ofPseudomonas putida cells, strain 48,int o xylem vessels ofcu t Rosacv .'Sonia' . Journal ofApplied Bacte riology 64, 197-208. Pseudomonas putida cells were unable to pass theinter-vesse l pitmembrane s ofth e xylem system ofcu t roses (Rosahybrida cv. 'Sonia'). It was further shown that(1 ) the number of bacteria which infiltrated into the xylem vessels decreased with increased distance between the cutting point andsamplin g point; (2)th e number of bacteria which infiltrated into theope n xylem vessels increased with time andwit h increasing numbers ofPseudomona s cells; (3)onl y amino r part ofth e Pseudomonas cells homogeneously suspended in thevas e solution was able to infiltrate intoth e xylem vesselso fth ecu t roses upt oa distanc e from the cutting point of >1 cm;an d (4)eve n low levels ofinfiltrate d Pseudomonas cells could bedemonstrate d by mea surements ofth e water conductivity ofste m segments. More research is needed to reveal which mechanisms (e.g. gumnosis) might have contributed, directly orindi rectly,t oth e prevention offurthe r infiltration ofbacteria l particles into the cut open vascular system ofth e Rosa cultivar. Good keeping quality ofharveste d cutflowers i s sen 1986). It is not known if micro-organisms essential for increasing production of The can penetrate into the xylem system using the Netherlands flower industry. Roses are oneo f inter-vessel membrane pathway of cut flowers the most important economic flower crops, but like roses and this paper reports on such an their post-harvest life is limited by several investigation. factors (Aarts 1957). For example, cut flowers have a shorter life than uncut flowers even when the conditions are optimal (Stigter 1980). Rose life may be further shortened by bacteria and their products which may infiltrate from vase Materials and Methods water into thecu t flower xylem, causing loss of water flow, wilting and flower senescence (Put 1986). The xylem consists of numerous short PLANT MATERIAL vessels (2-5-50 ^m wide and 2-250 mm long) Plants of Rosa hybrida cv.'Sonia ' were supplied with inter-vessel pit membranes containing cap by local glasshouse growers. The roses were illary holes of about 50nm , which areto o small harvested between 15 February and 15 March for bacteria to pass through (Van Alfen et al. 1986 at flowering stage 1-2 (Berkholst 1980). 1983; Zimmermann 1983; Stigter & Broekhuy- They were wrapped in parchment-like paper, transported to thelaboratory , stored at 4°C for f Address for correspondence: Kerksteeg 4, 7411 24—48h and the experiments were then carried EWDeventer , TheNetherlands . out immediately. 79 Henriette M. C.Put andT. van der Meyden MICRO-ORGANISMS the Mariotte tube to the upper end of the stem segment was 60 cm. A piece of soft plastic Pseudomonas putida strain 48 was isolated from tubing was attached to each T-piece for connec the vase water of cut roses and identified by the tion to a single stem segment (Mariotte, E. Oxy-ferm Tube II Test method (F. Hoffmann- Dyon 1620-Paris 1684). La Roche & Co. A.G., Diagnostics CH-40002, Triplicate samples of roses were taken from Basel, Switzerland). This strain does not form the vase water and three consecutive stem sec phytotoxic compounds or produce pectinolytic tions of 5 cm each were cut immediately under or cellulolytic enzymes. water from the basal end upwards and inserted onto the manifold in an upside-down position. The water solution was then allowed to flow VASE-LIFE EVALUATION OF Rosa through the segments for 60 min to purge air in CV.'SONIA' the lines and to obtain an equilibrated water The rose stems were cut aseptically to a stan flow. Thereafter the lower end of each stem dard length of 45 cm and each was placed in a segment was connected to a plastic tube of cylindrical vase calibrated up to 100 ml. All known weight. The water flow through the seg leaves except the upper three were removed ments was assessed by weighing of the tubes from the rose stems. The vases contained sterile after 30, 60 and 90 min of water flow respec tap water (control) or a freshly prepared water- tively. The average water conduction per 30 min washed suspension of Pseudomonas putida strain was then calculated (Durkin 1979). Conductivity 48 containing a known number of viable bac was defined as the reciprocal value to resistivity, teria per ml. Protection against contamination i.e. resistance to flow, expressed per transverse- was obtained by placing a plastic film on the sectional area of 5 cm stem length (Fig. 1; top of each vase (Parafilm 'M', American Can Gilman & Steponkus 1972; Chin & Sacalis Co). Each test consisted of comparable tripli 1977). cates and was repeated three times. Observations were made of the condition, the Vase-life was evaluated in a room maintained ornamental value and the flowering stage of the at a temperature of 20° ± PC, a relative humid roses (Fig. 2; Berkholst 1980)a t 24 h intervals. ity (RH) of 60 + 2% and with a 4000 lux light (10 W/m2) with a 12 h photoperiod during each 24 h. At the conclusion of each experiment the ste rility of the control vase solutions was tested as well as the purity and the number of Ps. putida cells in the test vase suspensions. In addition, SAMPLING OF STEM SEGMENTS OF ROSES the water conductivity of stems of roses held in FOR BACTERIAL COUNTS a vase of water which initially contained Roses were removed from their vases and the 104-108 Ps. putida cells/ml was measured after outer part of the stem was carefully disinfected 24 h of vase-life. The water uptake was mea with ethanol (98%,v/v) . They were then cut into sured at 24 h intervals by reading the water four segments, each 5 cm long. From points 0, column of the calibrated cylindrical vases in ml. 5, 10 and 15 cm respectively, 1c m was cut and To exclude any microbial contamination the the epidermis and cortex of the stem particles flowers were not taken out of their vases for were peeled off to avoid possible introduction of manipulations, such as measurement of the micro-organisms attached to the outer epider water evaporation of the flower. mis of the stem. The xylem part of each stem The apparatus for measuring water conduc sample was placed in a disposable pre-sterilized tivity consisted of an overhead 10 1 bottle filled screw-capped polystyrene tube, 16 x 125 mm with glass-distilled water and connected by (Falcon Bioquest, Los Angeles, USA), contain medical plastic tubing to a glass manifold of 24 ing 5 ml of sterile tap water plus 005% v/v of pieces. A Mariotte tube was inserted through a Tween-80 (BDH). After vortex mixing for 1 min, rubber stopper into the 10 1bottl e to maintain decimal dilutions were made in sterile tap water. constant water pressure. The distance or height A laminar flow cabinet was used for aseptic of the water column measured from the base of manipulations. 80 Pseudomonas sp.infiltration intoxylem vessels 60 cm ,^HfiniTOTîro i r—Ster n * segment 5OTV k it 14 h " . !• "• " "f •' \J\J\J\J\J\JKJ\J\J\J\J\J Fig. 1. Apparatus designed to measure xylem conductivity (Chin & Sacalis 1977).a , Bottle containing 101 sterile glass-distilled water; b, rubber stopper; c, Mariotte tube; d, base of the Mariotte tube; e, upper end of the upside-down-inserted stem segment of 5c m length; f, plastic tube of known weight to collect water flowing through the stem segment;g ,glas smanifol d with 12 connectors;h ,connecto r ofsof t medical plastictubing . SPIRAL PLATE COUNT METHOD TO ASSESS for 48 h. In the calculation of the number of THE NUMBER OF BACTERIA INFILTRATED bacteria in the sample tested, plates with INTO XYLEM VESSELS OF ROSES between 20 and 500 colonies were used (Silley Viable counts were made on Plate Count Agar 1985). The water capacity of 1c m of stem xylem (Difco), using a Spiral Plate Count Machine vessel of roses was 005 ± 0003 g. Thus the (model C, Don Whitley, Shipley, West York ratio of water capacity of stem segment (1 cm) shire, UK). The plates were incubated at 28°C to vase water (1 ml) is 1: 005 . 10 Days of development 20°C i 8 Stages of flower opening Supreme bloom Fig. 2. Stagesi n theflower openin g and development ofornamenta l value ofRosa cultivars (Berkholst 1980). The ornamental value isnegativ ewhe n the acceptability ofth eflowe r in thevas ei svisuall y decreased to <50%. 81 Henriette M. C. Put and T. van der Meyden Vase-life (d) hi i i i 6l 8r II Ô5E O 0-1 cm . L5-6av IJOHI J5-I6ctn, Vase solution Stem sample Fig.3 . Bacterial infiltration of xylem vessels of roses held in sterilized tap water during 1-6 d of vase-life. The averagewate r capacity ofxyle mvesse l segments of 1 cmste m length was00 5 ml ± 0003 ml. STATISTICAL EVALUATION INFILTRATION OF Ps. putida CELLS INTO THE XYLEM SYSTEM OF CUT ROSES Results of water uptake, water conduction, vase- life and flower opening were subjected to an After 24 h ofvase-life analysis of variance. In addition, Least Signifi cant Difference values were calculated. A prob The numbers of Pseudomonas cells infiltrated ability level of significance of P < 005 was used. into the first cm of the xylem vessels, with a water-holding capacity of ca 005 ml, after 24 h of vase-life was a maximum of five times higher than the numbers of Pseudomonas cells initially Results and Discussion present in the vase water. The infiltration into the xylem vessels decreased progressively as the distance from the cutting point to the sampling MULTIPLICATION OF Ps. putida CELLS point increased (Fig. 4). IN VASE WATER In control vases initially filled with sterile tap Plate counts of vase water after 1-6 d of vase- water the numbers of cfu on plate count agar life showed: (1) no significant multiplication of were < 1/cm after 24 h of vase-life. Further up Ps. putida cells at the highest initial cell concen the stems plate counts were also < 1 cfu/cm of trations of > 107/ml of vase water and 2-4 d of stem (see Fig. 3). vase-life; (2) that at the lowest initial Pseudo monas numbers tested, 5 x 105/ml of vase After >1-6 d ofvase-life water, multiplication yielded ca 107/ml after 6 d of vase-life; and (3) that in non-inoculated During the vase-life of the roses, i.e. as long as control vases, bacterial counts were up to the ornamental value of the roses remained 5 x 103/ml of vase water after 6 d of vase-life acceptable, the numbers of Pseudomonas cells (Fig. 3). infiltrated into the xylem vessels increased with 82 Pseudomonas sp. infiltration intoxylem vessels Vase solution no. |,l M l I l7| |,i M i i l7| 1,11111 l7| |,I I 1 1I 17 O) — w O E O.S. o 5- -o o Q. a. r- 0-1 cm 5-6 cm .JO-llcmm 15-16 cm Vase solution Stem sample Fig.4 . The number of infiltrated cells of Pseudomonas putida strain 48 at four different levels of the xylem vessel system of cut roses after 24 h of vase-life when the initial number of Ps.putida cells was min. 5 x 105 and max. 5 x 108 cells/ml of vase water. The average water capacity of xylem vessel segments of 1c m length was 005 + 0003ml . time and with increasing numbers of Pseudo Table 1. Infiltration of Pseudomonas putida cells into xylem vessels of roses during 1-3 d of vase-life and an monas cells initially suspended in the vase 7 water. initial number of 5 x 10 Pseudomonas cells/ml of vasewate r When the number of Pseudomonas cells ini tially suspended in the vase water was ca Ps. putida 5 x 107/ml, the density of Pseudomonas cells (cfu) per cm of stem sample infiltrated into xylem vessels of the rose was ca 3 Vase-life (d) 5 x 10 cfu/cm at 15 cm stem height, at the end Stem sample(cm) * of the vase-life of 3 d (Table 1). The lower the initial cell numbers in the vase 0-1 106 107 water tested, the longer the vase-life and also 5-6 105 105 the longer the time taken for micro-organisms 10-11 103 10* 2 to infiltrate further up the stem. Vase water ini 15-16 10 5x 10 3 5 tially containing ca 10 Pseudomonas cells/ml * Water holding capacity of 1 cm stem: after a vase-life of 6 d showed infiltration at 005 ± 0003ml . 15 cm stem height up to a maximum of 102 Pseudomonas cells/cm of stem. WATER RELATIONS AND VASE-LIFE OF CUT In control vases, initially filled with sterile tap 'SONIA' ROSE HELD IN VASE WATER water, the number of microbial cells (cfu) infil CONTAINING Ps. putida CELLS trated from the vase water into the xylem vessels after 6 d of vase-life was a maximum of Increasing initial numbers of Ps. putida cells per 102/cm of stem at a stem height of 15-16 cm ml of vase water showed: (1) decreasing water (Fig. 3). uptake; (2) decreasing vase-life; (3) decreasing 83 Henriette M. C. Put and T. van der Meyden Table2 . The influence of the number of Pseudomonas putida cells/ml vase water on the water uptake (ml/d) of Rosa cv. 'Sonia' Vase-life (d) Log no of Ps putida 12 3 4 5 6 7 8 cells/m 1vas e water Water uptake (ml/d) 70* 3-5* 2 7 12-5 80 4-5* 3* 6 15-5 13 8 6-5* 3* 5 18-5 20-5 16-5 11-5 8-5 4 20-5 21-5 23 19 16 0(Control ) 18-5 19-5 29-5 20 15 10 * End ofth evase-life . Mean valueso fthre etriplicat etests . flower opening; and (4) decreasing water con Table 3. The influence of the initial number of ductivity of the respective roses. Pseudomonas putida cells per ml of vase water on the vase-life ofRosa cv.'Sonia' * Log no. of Ps. putida WATER UPTAKE, VASE-LIFE AND cells/ml vase water Average vase-life(d) * FLOWER OPENING Control The amount of water taken up by roses in a 5 7 sterile vase solution increased during the first 5-5 5-6 6 5 few days and gradually decreased each suc 6-5 3-4 ceeding day until the end of the vase-life at full 7 3 flower opening. 7-5 1-2 The amount of water absorbed by roses in 1 vase water containing Ps. putida cells at 105- * The end of the vase-life is the day at which the 108/ml decreased abruptly to significantly lower ornamental value of the Rosa flower in the vase is levels 1-2 d before the end of the vase-life visually decreased to <50%, compared with the normal bud development shown in Fig. 2 (Berkholst (Table 2). No significant differences in the daily 1980). uptake of vase water were noticed, however, in the period preceding the abrupt drop of water uptake. The end of the vase-life of the roses was char initial numbers of Ps. putida cells per ml of vase acterized by wilting of leaves and petals fol water; (2) the highest water flow reduction in lowed by 'bent-neck' of the flower stalk stem segments cut from the base of the stem; (3 ) (Table 3). the same tendency for water flow reduction, Initial numbers of > 106 Ps. putida cells/ml although to a lower extent, as the base segment of vase water caused a vase-life reduction of in stem segments cut at 5 and 10 cm stem height respectively; and (4) an initial number as low as >50% when compared with control roses held 4 in sterile tap water. The flower opening also 10 Ps. putida cells/ml of vase water resulted in remained < 50% of the normal development of a significantly lower water conductivity at three the flower (Berkholst 1980). levels of the stem when compared with stem seg ments of roses held in sterile tap water (Table 4). An initial number of 104 Ps. putida cells/ml of vase solution, however, caused only a slight reduction in the vase-life, and a normal course of water uptake and flower development. SCANNING ELECTRON MICROSCOPE OBSERVATIONS Scanning electron microscope observations of WATER CONDUCTIVITY the cut surface and longitudinal sections of the Water flow through three consecutive isolated 5 stem of 'Sonia' roses held for 24 h in vase water cm stem segments of 'Sonia' roses showed: (1) a containing 106 Ps. putida cells/ml showed a decreased water flow capacity at increasing markedly decreasing number of Pseudomonas 84 Pseudomonas sp. infiltration intoxylem vessels Table 4. The influence of the initial number of Pseudomonas putida cells/ml of vase solution on the water conductivity of stemso fRosa cv. 'Sonia' after 24h vas elife * Water conductivity/30 min (ml)* Stem segments (cm) Log no. of Ps. putida cells/ml vase water 0-5 5-10 10-15 8 0-01" 0-36' 0-41* 7 0-30*" 0-39"" 0-31"" 6 0-31*" 0-41'" 0-475*"c 5 0-58" O_95.bc 110bcd 4 l-37c l-29c 1-57" 0 (Control) 2-34" 2-08" 2-32« Average 0-815 0-915 103 LSD value 0-54 0-63 0-50 * Mean values in a column followed by different letters are significantly different, P <005 . LSD, Least significant difference. cells at increasing stem height up to 8 cm VASCULAR PLUGGING BY Ps. putida CELLS (Figs 5a, 5b, 6a and 6b). Scanning electron microscope observations showed no vascular plugging when vase solu tions contained ca 106 Ps. putida cells/ml (Figs 5 XYLEM VESSEL LENGTH DISTRIBUTION and 6). Xylem vessels of stems can be considered as Vase water containing > 106 Ps. putida cells/ parallel lines of different lengths (Fig. 7; Zim ml showed wilting and 'bent-neck' before vascu mermann 1983). A dextran blue solution in lar occlusions by clotting or colonization of water (mol. wt 106) was used by Stigter & infiltrated Pseudomonas cells might have Broekhuysen (1986) to show that the minimum occurred except at the base of the stem as xylem vessel length of a cut rose is 1-2 mm shown in Fig. 4. while the maximum varies between 15 and 30 The extent of decreased conductivity (Table 4) cm. The greater the distance from the cutting of rose stem segments at increasing initial point to the point of the stem tested, the lower numbers of Pseudomonas cells in vase water, the number of open vessels and the less after 24 h of vase-life, cannot have been the sole opportunity there is for bacteria to infiltrate result of vacular plugging by Ps. putida cells. into the xylem vessel system. The number of Therefore the number of infiltrated Pseudo open vessels in the cutting surface of 'Sonia' monas cells was too low, and the time (24 h) roses is about 1500. was too short, for mass colonization of infil The curved shape of the xylem vessels and the trated cells. Some other factors must have been spreading of the diameter of the individual involved. vessels are factors that influence the 'normal' The extent of decreased conductivity of stems water flow resistance. of 'Sonia' roses when the vase solution con Microbial particles flushing up into the xylem tained 10* Ps. putida cells/ml indicated, more vessels by the uptake of water may be attached over, that the vascular water transport system is to the vessel wall and initiate a complex defence very sensitive, complicated, and may involve mechanism. This may result in a decreased several as yet unknown factors. water conductivity. Consequently other factors should have The limit of the ascent of Pseudomonas cells played an additional role in vascular plugging into the xylem vessels was shown to be lower of Rosa xylem vessels, and the decreased water than the maximum length of open vessels conductivity (Table 4). (Peresse 1974). (1) The micro-climate of the cutting surface of 85 Henriette M. C. Put and T. van der Meyden a) Fig. 5. a, Scanning electron micrograph (SEM) of the cut surface of Rosa cv. 'Sonia' held for 24 h in a vase solution containing ca 106 Pseudomonas putida cells/ml. The cut surface shows many bacterial rod-forming cells, entering the xylem vessels. Mucoid and amorphic materials are also shown. Bar marker represents 1/mi ; b, SEM of the cut surface of Rosa cv. 'Sonia' held for 24 h in sterilized vase water. The cut surface and vessel entrance are clean. No bacterial cells or mucoid materials are shown. Bar marker represents 10 pm. 86 Pseudomonas sp.infiltration intoxylem vessels a) b) Fig.6 . a, Scanning electron micrograph (SEM) of a longitudinal section of a xylem vessel of Rosa cv. 'Sonia' at 2c m from the cutting point, after 24h of vase-life in vase water containing ca 106 Pseudomonas putida cells/ml. Many bacterial rods and somemucoi d or amorphic materials are shown in the xylem vesselan d around thevesse l pits.Th e number ofbacteria l cells observed in the vessel ismuc h lower than observed on thecuttin g surface ofth e roses. Bar marker represents 1 /mi; b,SE M of a longitudinal section of a xylem vessel of Rosa cv.'Sonia ' at 8 cm from the cutting point,afte r 24h ofvase-lif e in vase water containing ca10 6Ps. putida cells/ml.A few rod-forming bacterial cellsan d somemucoi d or amorphic material are observed in the vessel and around the vascular pits.Ba r marker represents 1 /an. 87 Henriette M. C.Put andT. van der Meyden defence against the infiltration ofbacteria l cells into the vascular system, ort o ethylene-induced rapid senescence ofth e Rosa flowers (Zagory& Reid 1986). Moreover, vascular plugging by pectinaceous gels cannot have been caused by enzyme activ ity of the Ps.putida strain. Thedecrease d water conductivity, however, measured upt o >1 5 cm from the cutting point, might have been caused by occluding materials produced directly or indirectly by xylem cells. Histochemical studies are needed to detect such significant xylem Length of stem segment element occlusions. Fig.7 . Diagram of xylem vessel length distribution Much more research is needed to reveal (Zimmermann 1983). Assuccessiv e segments are cut which currently unknown mechanisms, other off at B,C , D, E,F an d Gmor e and more vessels are cut open at both ends. Thenumbe r ofope n vessels than gumnosis, could have ledt oth e prevention increases asth este m segment isshortened . of the spontaneous flushing up of bacterial cells with thevas e water into thecut , open xylem vessels. the roses may have stimulated the rapid multi Henriette M.C. Puti s oh detachment at the plication of bacteria and blockage of the vessels Sprenger Institute, Wageningen. Tonva n der on thecuttin g surface of roses placed in vase Meyden is a trainee from the Horticulture water with high initial numbers of Pseudomonas College at Utrecht. Scanning electron micro cells (> 106/ml, see Fig. 4). graphs were prepared by the Technical and (2) Pseudomonas cells, flushing up intoth e Physical Research Service, Wageningen (Dr. A. xylem vessels by the uptake ofvas e water,ma y Boekestein &Ank e Clerkx). become attached toth e vessel wall and initiate the release, directly or indirectly, of plugging compounds (gums), resulting in a markedly References decreased water conductivity after 24h of vase- life, as shown in Table 4 (Parups & Molnar AARTS, J.F.T. 1957 Over de houdbaarheid van snij 1972; Van Alfen et al. 1983; Vandermolen bloemen. Onth ekeepin g quality ofcu flowers.t I n Mededelingen van de Landbouwhogeschool te 1983). Wageningen, Vol. 54(9). pp.1-62 . Wageningen: H. (3) The production of plugging compounds, Veenman &Zn . directly or indirectly, by multiplying Pseudo ALBERSHEIM, P. & ANDERSON-PROUTY, A.J. 1975 Car monas cells should be taken into account bohydrates, proteins, cell surface, andth ebiochem istry of pathogenesis. Annual Review ofPlant (Anderson 1984). Physiology 26, 31-52. (4) Low mol. wt bacterial or vascular cell ALBERSHEIM, P. & DARVILL, A.G. 1985 Oligosac wall compounds flushing upward with the sap charins.Scientific American 253(3), 44-50. stream might act as elicitors, initiating the ANDERSON,A.J . 1984Difference s between lipopolysac- charide composition ofplan t pathogenic and sap release of compounds which might have con rophytic Pseudomonas species. Appliedand tributed toth edefenc e ofbacteria l infiltration as Environmental Microbiology 48, 31-35. well ast oth e rapid senescence ofth e cut roses BERKHOLST, C.E.M. 1980 Thewate r relations of cut (Albersheim & Anderson-Prouty 1975; Fujno et roses.Bedrijfsontwikkeling 11,332-338 . al. 1983; Vandermolen et al. 1983; Albersheim CHIN, C. & SACALIS,J.N . 1977Metabolis m of sucrose in cutroses .II .Movemen t and inversion ofsucros e & Darvill 1985). absorbed bycu t rose stems.Journal ofthe American The Ps.putida strain used inth e experiments Societyfor Horticultural Science 102, 537-540. is notphytopathogeni c anddoe s not produce DURKIN, DJ. 1979 Some characteristics of waterflow pectin-degrading enzymes. Rosa cultivars are through isolated rose stem segments.Journal of the American Societyfor Horticultural Science 104, 777- not sensitive to the plant hormone ethylene. 783. Consequently the elicitor function ofplan t cell FUJNO, D.W., REID, M.S.& VANDERMOLEN, G.E. 1983 wall oligosaccharins could not have ledt o Identification ofvascula r blockages inrachide so f Pseudomonas sp.infiltration intoxylem vessels cut maiden hair (Adiantum Raddianum) fronds. STIGTER, H.C.M. DE198 0 Water balance ofcu t and Scientia Horticulturae 21, 381-388. intact 'Sonia' rose plants. Zentralblatt für Pflan- GILMAN, K.F. & STEPONKUS, P.L. 1972 Vascular zenphysiology 99,131-140. blockage in cut roses. Journal of the American STIGTER, H.C.M. DE & BROEKHUYSEN, A.G.M. 1986 Societyfor Horticultural Science 97, 662-667. Experimentally induced plugging ofcu t rose xylem PARUPS, E.V. & MOLNAR, J.M. 1972 Histochemical by particulate or macromolecular matter. Acta Hor study of xylem blockage incu t roses. Journal of the ticulturae 181,365-370 . American Society for Horticultural Science 97, 532— VAN ALFEN, N.K., MCMILLAN, B.D., TURNER, V. & 534. HESS, W.M. 1983 Role of pit membranes in PERESSE, M. 1974Modification s cytologiques dans les macromolecule-induced wilt ofplants . Plant Physi cylème de tiges d'oeillets expérimentalement infec ology 73, 1020-1023. tées, au cours de l'installation du Phialophorecin- VANDERMOLEN, G.E., LABAVITCH, J.M., STRAND, L.L. erescens (Wr.) van Beyma. Phytopathologische & DEVAY, J.E. 1983Pathogen-induce d vascular Zeitschrift 79, 35^16. gels: ethylene as a host intermediate. Physiologia PUT, H.M.C. 1986 Investigations into the influence of Plantarum 59, 573-580. the microflora from stems ofcu t flowers on thevas e ZAGORY, D.& REID, M.S. 1986Rol e ofvas e solution life ofRosa 'Sonia', Gerbera'Fleur' , and Chrysanthe micro-organisms inth elif e ofcu t flowers. Journalof mum 'Spider'. Acta Horticulturae 181, 415-418. the American Society for Horticultural Science 111, SILLEY, P. 1985 Evaluation of total-count samplers 154-158. against the traditional pour plate method forenu ZIMMERMANN, M.H. 1983 Xylem Structure and the meration of total viable counts of bacteria in a Ascent of Sap, Springer series IIV Wood Sciences. process water-system. Letters in Applied Micro ed. Timmell T.E. Vol. 11. pp. 1-148. Berlin: biology 1, 41-43. Springer-Verlag. 89 Chapter 4 The infiltration ability of micro-organisms Bacillus, Fusarium, Kluyveromyces and Pseudomonas spp.int o xylemvessel s of Gerbera cv. 'Fleur' and Rosacv .'Sonia 'cu t flowers: ascannin g electron microscope study. 91 Rosacv .'Sonia' :stag e4 o fflowe ropenin g (Berkholst,1980 ) 92 The infiltration ability of micro-organisms Bacillus, Fusarium, Kluyveromyces and Pseudomonas spp. into xylem vessels of Gerbera cv. 'Fleur' and Rosa cv. 'Sonia' cut flowers : a scanning electron microscope study HENRIETTE M.C. PUT* & ANKE CM. CLERKXf Carl C. Conway Laboratories, Thomassen &Drijver-Verblifa NV, PO Box 103, 7400 GB Deventer, The Netherlands. On detachment at Sprenger Institute, Ministry of Agriculture and Fisheries, PO Box 17,6700 AA Wageningen, The Netherlands and ^Department of Electron Microscopy, Technical and Physical Engineering Research Service, PO Box 356, 6700 AJ Wageningen, The Netherlands Received 7September 1987,revised 1 February 1988and accepted 6 February 1988 PUT, H.M.C. & CLERKX, A.CM. 1988. The infiltration ability of micro organisms Bacillus, Fusarium, Kluyveromyces and Pseudomonas spp. into xylem vessels of Gerbera cv. 'Fleur' and Rosacv . 'Sonia' cutflowers: a scanning electron microscope study.Journal of Applied Bacteriology64 ,515-530 . Scanning electron microscope (SEM) observations have been made of transverse and longitudinal sections of xylem vessels of cutflowers o f Gerbera cv. 'Fleur' and cut Rosa cv.'Sonia ' after a maximum of2 4h vaselife . Vase waters used were:steril e tap water; sterile tap water plus suspensions of single pure cultures of Bacillus poly- myxa, B.subtilis, Fusarium oxysporum microspores, segments of young mycelium of F.oxysporum, Kluyveromyces marxianus, or Pseudomonas putida, up to a maximum of 106an d 5 x 107cells/m l ofvas ewater . The results of the SEM observations showed that only a small fraction of the microbial cells entered into the vascular system with the normal intake of vase water;mos t microbial cells remained attached to the submerged cut surface whilea small fraction of the initially attached microbes were sometimes liberated into the surrounding vase water. The SEM figures give the impression that adhesion on to the cut stem surfaces of the bacterial cells {Bacillus and Pseudomonas spp.) by their capsular materials may have occurred, although the cut surface possibly acted as a filter, trapping cells because of the upward pressure of the transpiration stream into the vessels.Fe w attached yeast cellswer efound , but Fusarium microconidia formed cell clusters on the cut surface, partially blocking the xylem vessel entrance; Fusarium mycelium covered the cut surfaces as another filter layer through which onlysmal l mycelial particlescoul d enter thevascula r system. The extent ofinfiltratio n ofmicrobia l cellsint o the xylem vessel system depended upon the number of microbial cells/ml of vase water (up to a certain maximal number)an d on theshap ean d sizeo fth eindividua l cellsan d thewidt h ofth exyle m vessels. The end of the vase life of Gerbera and Rosaflowers wa s marked by an extensive water stress in the xylem vessels so that the water intake necessary for a normal flower development was insufficient and wilting ensued. The water deficit may have been enhanced by secondary biochemical and physiological interactions between the microbes and the plant tissues, which may also have affected the infil tration ofmicro-organisms . In a previously published paper (Put & Van der vessels of cut Rosa cv. 'Sonia', it was shown Meijden 1988) concerning the infiltration of that: (i) in roses Ps. putida cells were unable to Pseudomonas putida cells strain 48 into xylem pass the pit membranes between the xylem * Corresponding author. Present address: Kerk- vessels, (ii) The number of Pseudomonas cells steeg4 ,741 1E WDeventer ,Th eNetherlands . which infiltrated into xylem vessels decreased 93 H. M. C. Put and A. C. M. Clerkx with increasing distance between the cutting stem segments measured after only 24 h of vase point and the sampling point. This may have life, SEM was applied to study the infiltration been partially due to the decreasing number of ability into xylem vessels of micro-organisms open vessels at increasing stem height, (iii) The (bacteria and fungi) added to the vase water. infiltration of Pseudomonas cells increased when Further investigations concerning the influ contact time and the number of Pseudomonas ence of microbial cells on the water relations of cells/ml of vase water increased, although only a flower cultivars other than roses were desirable minor fraction of the pseudomonads suspended to assess host-specific phenomena, so compara in the vase water was able to infiltrate into the ble SEM investigations have also been made of xylem vessels of the roses up to a distance of the xylem vessels of Gerberacv . 'Fleur', for com > 10m m from the cutting point. parison with roses; the Gerbera cultivar was It is not yet known, however, whether vase chosen because of: (i) the biological and ana micro-organisms enter the xylem system of cut tomical differences between the hollow-stemmed flowers with the water flow like any other bio Gerbera and solid-stemmed Rosa, and (ii) the logical inert particle of the same shape and size, popularity of Gerbera as an ornamental cut or whether biological activities complicate the flower in practice. mechanism of infiltration of micro-organisms Factors which may affect the infiltration of from cut flower vase water into xylem vessels micro-organisms into a vascular system are: (i) (Burdett 1970;Rasmusse n & Carpenter 1974). movement effected by bacterial flagella; (ii) the In a study of the effects on the vase life of cut day (light) - night (dark) period; (iii) orthokin- flowers of Rosa cv. 'Sonia' of suspensions of esis due to chemicals, e.g. Escherichia coli shows Bacillus, Enterobacter, or Pseudomonas spp. a positive Chemotaxis towards oxygen, amino added to the vase water, Put & Jansen (1988) acids and sugars, but fatty acids are a signal for found that: (i) the vase life of the roses was cells to escape 'overcrowded' conditions; (iv) markedly decreased when decimal dilutions microbial interactions such as antagonism, com (108-104/ml) of water-washed viable or heat- petition and synergism among the natural stem killed cells of each of the separate bacterial microflora; (v) small sub-communities formed species were added to the vase water; (ii) the during the vase life on the stem as well as on the initial numbers of either viable or heat-killed cut surface (Ewing & Fife 1972; Bowen 1980; bacterial cells added/ml of vase water influenced Carlile 1980; Halverson & Stacey 1986; Stigter the vase life; (iii) different bacterial strains & Broekhuysen 1986). exerted very similar effects, notwithstanding Little is known about the environmental their morphological and biochemical differ ecology of the parent plant compared with the ences, (iv) vascular plugging caused by infiltrat ecology of the vase life of cut flowers. The ing bacterial cells (e.g. Pseudomonas) could not phenotypic responses of micro-organisms to the alone have lead to the decreased water conduc environmental change from living aerially on a tivity of Rosa stem segments as measured up to rooted plant stem compared with living on an a distance of 10-15 cm from the cut surface, individual submerged cut flower stem are because the number of bacterial cells infiltrated unknown, as are the interactions of the natural into the xylem vessel was too low (Put & Van microbes in their new environment (Konings & der Meijden 1988). Veldkamp 1980). The microbial strategies with Phenomena which might be responsible for respect to competitive utilization of various interactions between the vascular system of the energy sources and the concentrations thereof cut roses and the viable or heat-killed cells are have rarely been studied. still unknown; they might be typical for cut In this paper the results are given of a series roses, or for cut flowers in general when they of investigations into movements of micro are in vase water containing certain critical organisms into the stem xylem vessels of two numbers of bacteria. cultivars of cut flowers : Gerbera cv. 'Fleur' and To gather more background cytological infor Rosa cv. 'Sonia' by SEM observations. To mation on the relationships between the exclude any competitive activity due to vase numbers of viable or heat-killed bacterial cells water micro-organisms, pure cultures were added to the vase water of Rosa flowers and the studied using aseptic techniques throughout as resultant decreased water conductivity of their far as possible. 94 Microbial infiltration into cut flower xylem vessels Materials and Methods Yeast :Kluyveromyce s marxianus PLANT MATERIAL The same procedure was used as for bacteria except that the plating medium was Mycophyl Gerbera cv. 'Fleur' and Rosa hybrida cv. 'Sonia' agar (BBL) and incubation was at 25°C for 2-3 were supplied by local glasshouse growers. The days. roses were harvested at flowering stage 1-2 (Berkholst 1980) and the gerberas were taken from the plant when the stamens of about two Mould: Fusarium oxysporum circles of bisexual disc florets were ripe. The A mixture of spores and mycelium segments harvested flowers were wrapped in parchment was inoculated on Potato Dextrose agar (Difco) paper, transported to the laboratory, stored for in Erlenmeyer flasks. They were incubated at 24-48 h at 4°C, before the experiments were 25°C for 3 d (harvest of mycelium) and for 10 d carried out. (harvest of microconidia). The spores or mycelium were washed off into sterile 005% MICRO-ORGANISMS Tween 80 solution in tap water by gentle shaking, followed by Vortex mixing, centri- The micro-organisms used were Bacillus fuging, washing (twice) and dilution in sterile polymyxa-21S; Bacillus subtilis-207 ; Pseudo tap water to give ca 5 x 106 microspores or monas putida-4$; Fusarium oxysporum-1, Kluy- mycelium particles per ml. Storage was at 2-5 veromyces marxianus-120. These five test °C. Mycelium particles and conidial numbers strains were originally isolated from stems of cut were assessed microscopically followed by plate flowers. Identification of the bacteria was done counting of decimal dilutions of the mould sus by API-20E, 50CH or 20NE systems (API pensions on Potato Dextrose agar (Difco) and systems, La Balme Les Grottes 38390, Monta- incubation at 25°C for 2-4 d. lieu, France). Mould and yeast strains were identified by the CBS (Centraal Bureau voor Schimmelcultures) at Baarn and Delft, The Netherlands. The five strains showed their char VASE LIFE acteristic differences in morphology as well as in Flowers were cut with a stem length of 45 cm, their pathogenic and metabolic properties. the stems were wiped with a paper tissue soaked in ethanol 98% v/v to minimize airborne con tamination, and put singly into cylindrical vases PREPARATIONS OF VASE WATER containing either 100 ml of sterile tap water, a SUSPENSIONS sterile 1% (w/v) sucrose solution in tap water, or a suspension in sterile tap water of the micro Bacteria organism to be tested. Each variable tested con One to three isolated colonies of a 24 h old sisted of triplicates. streak plate culture on Nutrient agar (Difco) The vases were held at 20°C, 60% r.h. were suspended in 1 ml of sterile tap water, (relative humidity) and 10-25 W/m2 photoradia- mixed on a Vortex mixer at maximum speed tion at flower height during a 12 h light/dark and ca 0-2 ml was inoculated on Nutrient agar photoperiod each 24 h. Every 24 h of the vase (Difco) in 500 ml Erlenmeyer flasks. The cul life, observations were made of: the water tures were incubated at 28°C for 24 h. The uptake (ml), the flowering condition of the gerb growth was then washed off with sterile 005% eras (Van Meeteren 1980), the flowering stage of Tween 80 (Difco) in tap water, centrifuged at the roses (Berkholst 1980) and the ornamental 10°C and 10000 g (Beekman J-21 C), value up to a maximum of 7 d of vase life. After resuspended in sterile tap water, Vortex-mixed, 2, 3, 5 and 7 d of the vase life the microbio recentrifuged and diluted in sterile tap water to logical state of the vase water was assessed by give ca 5 x 106/ml of Ps. putida and B. subtilis the streaking of vase water samples on agar and ca 5 x 107/ml for B. polymyxa cells. Cell plates. Scanning electron microscope prep concentrations were assessed microscopically, arations were made after a maximum of 24 h followed by plate counting of decimal dilutions vase life, a deliberately short contact time, to on Plate Count agar (Difco). exclude colonization and to detect early 95 H. M. C.Put andA. C.M. Clerkx responses of the host vascular system towards the infiltrating micro-organisms (Peresse 1974; Van Alfen et al. 1983). PREPARATION OF STEM SEGMENTS FOR SEM OBSERVATIONS Pieces of the stem about 10 mm long were freeze-fractured and subsequently fixed over night in 3% glutaraldehyde (in cacodylate buffer 0-1 mol/1; pH = 7-2), washed twice in buffer and then dehydrated through a graded series of ethanol/water (10% v/v to 100%) leaving the specimens in each solution for 10 min. The Fig. 2. Gerbera cv. 'Fleur', held in sterile vase solu tion. Longitudinal section near cutting point. Barrep specimens were then critical-point dried and resents 100 fim. mounted with aluminium adhesive tape and silver paint on aluminium specimen tables. Finally, the specimens were coated with gold Xylem vesseldiameter and examined in a scanning electron microsope (Jeol 35C) at 15 kV accelerating voltage. Scanning electron microscope examinations showed that the xylem vessel diameter of Gerbera cv. 'Fleur' was wider (varying between 20-100 urn) than the xylem vessel diameter of Results Rosa cv. 'Sonia', which varied between 2-5—35 fim (compare Figs 1an d 11). SEM OBSERVATIONS Flowers in vasewater to which suspensionsof Flowers insterile vasewater pure cultures of micro-organisms wereadded No micro-organisms were observed in SEM All the SEM observations are summarized in preparations of the cut surfaces and the xylem Table 1(a) Gerbera and (b) Rosa and Figs 1-18 vessels of surface-disinfected stems from Gerbera have been selected to illustrate some of the cv. 'Fleur' and Rosa cv. 'Sonia' up to a results obtained. maximum of 300 mm stem height (Figs 1an d 2, It was predictable that infiltration of micro 11an d 12). organisms into the wider xylem vessels of Gerbera might have been easier compared with roses. More microbial cells were indeed observed at longer distances from the cutting point of the Gerbera stems than were observed in SEM preparations of stem samples of roses (see Table la and b). Bacillus subtilis-214 cells(c a 106/»iOadded to the vase water of Gerbera cv. 'Fleur' Blockage of cut xylem vessels by B. subtilis cells was not shown. Most Bacillus cells infiltrated into the xylem vessels were single ones, but some small cell clusters were observed. A few single cells and cell clusters infiltrated up to a Fig. 1. Gerbera cv. 'Fleur', held in sterile vase solu tion, 0 h. Transverse section of cutting surface. Bar distance of 50 mm from the cutting point. represents 100jim . Around the cell clusters amorphous mucoid 96 Microbial infiltration intocut flower xylem vessels Table 1. (a) A summary of the scanning electron microscope observations of xylem vessels of Gerbera cv. 'Fleur' Distance from cutting point to Micro-organisms sampling SEM added to vase Vase Stem point SEM fig. water life (h) section (mm) observations no. None 0\ 10 50 24 0 10 50 No micro-organisms 0 10 50 24 0 10 50/ Bacillus 24 T 0 Free Bacillus cells subtilis-214 observed; blockage of ca5 x 106/ml xylem vessels by Bacillus cells was rare 24 T 10 Relatively high numbers of Bacillus cells 24 T 50 Relatively high numbersiof Bacillus cells 24 L 0 Bacillus cells in open xylem vessels 24 L 10 Relatively high numbers of infiltrated Bacillus cells 24 L 50 Some vessels with relatively high numbers of Bacillus cells; other vessels few or no cells Fusarium 24 T 0 Clusters of conidia oxysporum-1 blocking (some) vessels micro-conidia 10 Some vessels blocked ca 106/ml by clusters of conidia T 50 A few conidia infiltrated L 0 Some vessels blocked by conidia L 10 Relatively high numbers of infiltrated conidia L 50* Relative low number of infiltrated conidia Fusarium T 0 Mycelium partly covering oxysporum-1 the cut surface mycelium particles 10 I Few mycelium particles ca 3 x 106/ml infiltrating into the 50 ( vessel system up to 100 mm height (continued) 97 H. M. C.Put andA. C.M. Clerkx Table 1. (continued) Distance from cutting point to Micro-organisms sampling SEM added to vase Vase Stem point SEM fig. water life (h) section (mm) observations no. Infiltration of mycelium particles predominant in the widest non-blocked 10 } vessels; relatively high numbers of small mycelium particles up to 10 mm height (Deposit formed L 50* Few small mycelium after 24 h) particles Kluyveromyces 24 T 0 Clusters of yeast cells, marxianus-120 partly blocking the open vessel ends ca 5 x 106/ml T 10 Low numbers of yeast cells T 50 A few yeast cells L 0 Large numbers of yeast cells infiltrated into cut-open vessels L 10 Low numbers of infiltrated 10 yeast cells (Deposit formed L 50* Few or no yeast cells after 24 h) T, transverse section; L, longitudinal section. * No micro-organisms were observed at sampling points 150 and 300m m from the cutting point. Table 1. (b) A summary of the scanning electron microscope observations of xylem vessels of Rosa cv. 'Sonia' Distance from cutting point to Micro-organisms Vase sampling SEM added to vase life Stem point SEM «g- water (h) section (mm) observations no. None 0 T 0-80 j 11 4 T 0 8 T 0 } No micro-organisms 16 T 0 24 T 0 ' 4 L 10 } 4 L 20 4 L 80 \ No micro-organisms 24 L 50 12 24 L 50*' 98 Microbial infiltration intocut flower xylem vessels Table 1 (b)(continued) Distance from cutting point to Micro-organisms sampling SEM added to vase Vase Stem point SEM fig. water life (h) section (mm) observations no. Pseudomonas 4 T Cut surface half covered putida-48 with bacteria 8 T ca 106/ml 16 T 0 1 24 T o Slightly more bacteria 4 T 10 Many fewer bacteria than 4 T 20 at sampling point 0 4 T 80 | 24 T 10 1 Slightly more bacteria 24 T 20 than after 4 h 24 T 80 I 4 L 10 I A few bacterial cells ca 20/001 mm2 4 L 20 I ca 10/001 mm2 4 L 80 I 24 10 Slightly more bacteria than after 4 h; 24 20 ca 100/001 mm2 13 24 80* ca 30/001 mm2 Bacillus Surface almost covered with 14 polymyxa-218 Bacillus cells; blockage of open vessels ca 5 x 107/ml 24 Surface almost covered with Bacillus cells; blockage of open vessels 10 Numerous infiltrated 15 Bacillus cells although lower numbers than observed at point 0 24 L 10f Slightly more infiltrated Bacillus cells Fusarium 4 T 0 Xylem surface partly oxysporum-1 covered with conidia micro-condia 24 T 0 Slightly more conidia; ca 106/ml some conidia clusters 4 or 24 L 5 Relatively numerous 16 conidia in the vessel system 4 L 10 Few conidia in the vessel system 24 L 5 Relatively numerous conidia in the vessel system 24 L 10 A few conidia present, ca 25/005 mm2 24 L 50t A few conidia present, ca 5/005 mm2 (continued) 99 H. M. C.Put andA. C. M. Clerkx Table 1 (b)(continued) Distance from cutting point to Micro-organisms Vase sampling SEM added to vase life Stem point SEM fig. water (h) section (mm) observations no. Fusarium 4 T 0 Mycelium on cutting oxysporum-1 surface partly blocking mycelium particles open vessels ca 3 x 107ml 24 T 0 Thicker mycelium on 17 cutting surface 4 L 10 A fewsmal l mycelial particles 24 L 10 Slightly more small mycelium particles 24 L 50;10 0 A fewmyceliu m particles (Deposit formed 24 L 150* One mycelium particle in vase after only (artefact?) 24 h) Kluyveromyces 4 T 0 Surface partly covered with 18 marxianus12 0 yeast cells, blockingo f ca 5 x 106/ml open xylem vessels 24 T 0 As at 4 h 4 T/L 5 Yeast cells fewer than on thecuttin g surface 24 5 Yeast numbers slightly increased 24 10 A fewyeas t cells (Deposit formed 24 50t Sporadic yeast cells in vase after 24 h) T,transvers e section; L,longitudina l section. * No micro-organisms observed at samplingpoint s >15 0 mm. f No micro-organisms observed at sampling points >10 0mm . Fig.3 . Gerbera cv.'Fleur' ,hel d inBacillus subtilis sus Fig.4 . Gerbera cv.'Fleur' , held in Bacillus subtilis sus pension, 24h . Transverse section, cutting surface. Bar pension, 24 h. Longitudinal section, 50 mm from represents 1pm. cutting point. Bar represents 1 /mi. 100 Microbial infiltration intocut flower xylem vessels Fig. 5. Gerbera cv. 'Fleur', held in Fusarium oxyspo- Fig. 8. Gerbera cv. 'Fleur', he'd in Fusarium oxyspo- rum conidia suspension, 24 h. Transverse section, rum mycelium, 24 h. Transverse section, cutting cutting surface. Bar represents 10/im . surface. Bar represents 10 fira. Fig. 6. Gerbera cv. 'Fleur', held in Fusarium oxyspo- Fig. 9. Gerbera cv. 'Fleur', held in Kluyveromyces rum conidiconidia suspensionsuspension, i2t4 uh. i^uuguuuLongitudinai l section, marxianus suspension, 24 h. Longtiudinal section, near near cuttin**: g point:_*. BaT» r representI».«.»»..*s »10 1A0A /an ...« cutting point. Bar represents 100 pm. Fig. 7. Gerbera cv. 'Fleur', held in Fusarium oxyspo- Fig. 10. Gerbera cv. 'Fleur', held in Kluyveromyces rum conidia suspension, 24 h. Longitudinal section, 50 marxianus suspension, 24 h. Longitudinal section, 10 •nm from cutting point. Bar represents 10/mi . mm from cutting point. Bar represents 10 pm. 101 H. M. C.Put andA. C.M. Clerkx Fig. 11.Rosa cv .'Sonia' , held in sterile tap water, 24 Fig. 14. Rosa cv. 'Sonia', held in Bacillus polymyxa h. Transverse section, 10m m from cutting point. Bar suspension, 4 h. Transverse section, cutting surface. represents 100/im . Barrepresent s 10 /an. Fig. 12. Rosacv . 'Sonia', held in sterile tap water, 24 Fig. 15. Rosa cv. 'Sonia', held in Bacillus polymyxa h. Longitudinal section, SOm m from cutting point. suspension, 4 h. Longitudinal section, 10 mm from Barrepresent s 100/on . cutting point. Barrepresent s 1 /an. Fig. 13. Rosa cv. 'Sonia' held in Pseudomonas putida Fig. 16. Rosacv .'Sonia' , held in Fusarium oxysporum suspension, 24 h. Longitudinal section, 20 mm from conidia suspension, 24 h. Longitudinal section, near cutting point. Bar represents 1 /an (Put & Van der cutting point. Barrepresent s 10/an . Meijden 1988). 102 Microbial infiltration intocut flower xylem vessels At ca 50 mm distance from the cutting point an occasional single rod-shaped cell was observed. The roses showed wilting symptoms after 24 h of vase life. Fusarium oxysporum-7 microconidia (ca 106/m/) added to the vase water of Gerbera cv. 'Fleur' and Rosa cv. 'Sonia' The cut surface of both cultivars was partly covered with small chains of conidia, attached together as well as to the cut surface, locally Fig. 17. Rosacv .'Sonia' , held in Fusarium oxysporum blocking the xylem vessel entrance and causing mycelium suspension, 24h .Transvers e section,cuttin g wilting of the Rosa flowers within 24 h. A rela surface. Bar represents 100/an . tively large number of microconidia infiltrated into the xylem vessels also caused vessel block age. Attachment of conidia to the xylem vessel walls was not clearly shown, nor was slime for mation evident around the conidial cells. Some of the microconidia germinated, and germ tube penetration into the vessel wall or pit mem branes, may have occurred. Up to a distance of 50 mm from the cutting point of the Gerbera, many infiltrated conidia were observed, while at the same distance from the cutting point of the Rosa flowers only a few microconidia were evident. Fusarium oxysporum-7 mycelium particles 6 Fig. 18. Rosa cv.'Sonia' , held in Kluyveromyces marx- (ca 3 x 10 /mOadded to the vase water of ianus suspension, 24 h. Transverse section, cutting Gerbera cv. 'Fleur' and Rosa cv. 'Sonia' surface. Bar represents 10 j«n. Much of the cut surface of the stems was covered with a network of mycelium after 4 h of material was shown and probably aided the vase life. The initially thin mycelial layer became attachment of these cell clusters to the xylem thicker during the course of the vase life, walls. causing increased blockage of the xylem vessels; this enabled only the smallest mycelial particles to infiltrate into the widest non-blocked vessels Bacillus polymyxa-218 cells(c a 5 x 107m/) up to maximum of 150 mm stem height. Attach added to the vase water of Rosa cv. 'Sonia' ment of mycelium particles to the vessel walls was not clearly shown. After 24 h of vase life Blockage of a relatively large fraction of the weak wilting of the roses was observed. xylem vessels of the cut surface was shown. Relatively large numbers of predominantly single rods were observed at ca 10 mm distance Kluyveromyces marxianus-120cells from the cutting point, after 4 h of vase life. The (ca 5 x lO6/™/)added to the vase water of number of infiltrated Bacillus cells was slightly Gerbera cv. 'Fleur' and Rosa cv. 'Sonia' increased between 4-24 h of vase life. In addi tion, some small clusters were observed, they The cutting surface of both flower cultivars was were surrounded by amorphous mucoid partly covered with yeast cells loosely attached material, and were attached to the vessel wall. to the surface and they blocked the xylem 103 H. M. C.Put andA. C.M. Clerkx vessels. Infiltration of the xylem vessels by The number and the size of micro-organisms several single yeast cells was clearly observed. added to the vase water Complete blockage of the infiltrated Rosa The greater the number or the size of micro vessels was evident, but the wider Gerbera organisms initially suspended in vase water, the vessels were not totally blocked by infiltrating shorter was the vase life of the cut flowers yeast cells after 24 h of vase life. The number of (compare: B. polymyxa 5 x 107/ml vase water yeast cells in the xylem vessels decreased at and B. subtilis 5 x 106/ml and Ps. putida and F. increasing distance from the cutting point. oxysporum 106/ml vase water; K. marxianus and Single infiltrated yeast cells were occasionally B. subtilis 5 x 106/ml vase water) (see Table 2). observed, not attached to the xylem vessels up A detailed study by Put & Jansen of this pheno to a maximum of 50 mm stem height. Vascular menon is in progress to supplement these cyto- attachment of yeast cells was not shown. After logical observations. 24 h of vase life, weak wilting of the roses was observed. The microflora of the vase water when sterilized tap water wasused Pseudomonas putida-48 cells(c a 106/ml) added to the vase water of Rosa cv. 'Sonia' After 24 h of vase life in sterilized tap water, the number of viable micro-organisms was < 10/ml On the cut surface of the rose stems a thin layer of vase water. During the course of the vase life, of bacterial cells had developed after 4 h of vase some micro-organisms (notwithstanding the life. The cells were surrounded by granular aseptic handling of the flowers) remained viable mucoid material which probably facilitated the and attached to the outer epidermis of the stem. attachment of the bacteria to the transverse These were washed off into the vase water and surface structures of the cut stems. subsequently multiplied, to an extent deter After 24 h of vase life more cells were mined by their growth factor requirements and attached to the cut surface and they blocked a the availability thereof in the vase water. After 7 small fraction of the open vessels. Infiltration of d of vase life the numbers of these micro bacterial cells into the xylem vessels adjacent to organisms were: 103 (minimal) and 106 the cut surface was shown, but these vessels (maximal) cfu of bacteria, and < 102 (minimal) were not blocked. The greater the distance from and 104 (maximal) cfu of fungi/ml of vase water. the cutting and sampling points, the lower was the number of infiltrated cells observed. Up to a distance of 80 mm a few single cells and some small cell clusters surrounded by granular Discussion material were evident. Attachment of these bac terial cells to the vessel surface may have The vase life of cut flowers was decreased by occurred. The first symptoms of wilting were microbial cells in the vase water when they visible after 3-4 d of vase life. plugged the cut vessels so much that the normal water demand for flower development was inhibited (see Table 2). Fungal mycelium had the strongest vessel-plugging capacity compared VASE LIFE with single microbial cells and spores (see Figs 5 and 8; 16 and 17 and Stigter 1981). Stigter & Water uptake Broekhuysen (1986) observed in their model The water uptake during the first 24 h of the system that macromolecules (dextran-D 1 x 3 vase life was significantly lower when the cut urn) suspended in vase water enter cut xylem flowers were placed in microbial suspensions vessels of 'Sonia' roses along with the transpira than when they were placed in sterilized tap tion stream of water and plug the stem when ca water (Table 2). It was also shown that the 10 mg of the dextran particles has been taken ornamental value of the cut flowers became up by the stem. The harmful effects on the critical when the uptake of vase water was flower being more acute with higher, and more decreased to <3 ml/d for Gerbera cv. 'Fleur' chronically with lower concentrations and size and < 6ml/ d for Rosa cv. 'Sonia'. of macromolecular materials in the vase water. 104 Microbial infiltration intocut flower xylem vessels >. 5« Ivt M hi 'S o O 00 03 3 > u a O «o 4> M « 3 o3 . k* 00 OO " 'Sta 00 6 M O a ôt-l u '(gM o 8 4c> 3 tC _g V J= IS H «o '-3 «•< « 2a H ö 2 âlî v « » S " 3 &- fi *3 Te >s 105 H. M. C.Put andA. C.M. Clerkx The vase life of sound Gerberaan d Rosa Biochemical studies are essential to explain the flowers is long enough (8 and 14d respectively) role of any polymers involved in adhesion, and for colonization by infiltrated micro-organisms, in this context the phenomenon of signal although it isprobabl y too short for any signifi exchange in plant-microbe interactions should cant Phytotoxin production and activity as beconsidere d (Halverson &Stace y 1986). might occur in whole plants. Formation of Infiltration of micro-organisms into a single microbial metabolic compounds, e.g. poly xylem vessel might occur up to the maximum saccharides, microbial enzymes and toxic com length ofan y cut vessel (Put &Va n der Meijden pounds, responsible for plugging, swelling and 1988); the penetration of intervessel pit mem destruction of the vascular system as a result of brane barriers by infiltrating micro-organisms, vascular gelation and degradation, (Fujino et al. however, might be mechanically inhibited, 1983;Harlin g &Taylo r 1985;Pu t & Rombouts although some plant pathogenic moulds 1988) may have reduced the vase life of the cut (Fusarium) have energy reserves sufficient for flowers studied here (see Table 2) compared both spore germination and germ tube penetrat with their natural state. This merits further ion into non-damaged host plants (Carlile 1970; investigations. Harlinget al. 1984;Harlin g& Taylo r 1985). Formation of toxic components by infiltrat Further SEM preparations of rose and carna ing potentially phytopathogenic Fusarium and tion stem segments made after 1 week of vase Bacillus cells was slight or nil, and compared life in vase water containing the natural with the non-toxic micro-organisms tested, no developing vasewate r microflora (ca 107 cfu/ml) serious or typical necroticdisease s ofth e flowers showed coccoid and rod-shaped micro were observed during the vase life (Table 2,Fig s organisms. They formed clusters, which were 3-8, 14-17). Surprisingly, no morphological or naked or surrounded by granular, amorphous anatomical deviations of the xylem vessel mucoid material. The micro-organisms covered systems of Rosaan d Gerbera, due to infiltrating much of the cut surface and also infiltrated into micro-organisms were found here,i n contrast to the xylem vessels. Some small microbial cell the observations of Zimmermann (1983) and clusters were localized, attached around the Put &Rombout s(1988) . vesselpit s (Van Doom 1985). The cellular reactions of the plant xylem to The work of Dansereau & Vines (1975) the invading microbial cellssugges t only general showed that many microbial species (bacteria stress responses in accordance with the findings and fungi) were present in the vase water con of Put & Jansen (1988). In their studies it was taining cut Antirrhinum, but only two bacterial shown that relatively low numbers of bacteria species were isolated from the internal vascular added to the vase water may infiltrate into the xylem of their cut stems; they were identified as xylem vessels,causin g a relatively large decrease Flavobacterium lutescens and Pseudomonas mar- of the water conductivity of the Rosa stems, ginalis, and because of their known antagonistic irrespective of the potential pathogenicity of the activity they may have prevented the infiltration bacterial strain tested(Billin g1982) . and multiplication of the remaining micro In SEM preparations, Bacillusan dPseudo organisms present in the vasewater . monascell s showed adherence to the vascular Antagonistic micro-organisms in the soil, e.g. xylem walls of Gerbera and Rosa stems, but Pseudomonas spp., Trichoderma spp. and Glio- Fusarium microconidia and Kluyveromyces cells cladium spp., can cause a delay and/or a remained unattached, possibly due to the adher decrease in the appearance or development of ent capacity of bacterial capsules, their cell wilt symptoms in rooted host plants (Lynch & envelopes or adhesive pili(Roger s1979) . Ebben 1986). Further electron microscopy is required to Xylem-residing micro-organisms may also understand precisely how micro-organisms play a role in the vase life of cut flowers. Thus adhere to cut surfaces or to xylem walls far, xylem-residing micro-organisms have been (Fletcher 1979). Much more information is also isolated only from xylem vessels of Florida needed to reveal the chemical and physical citrus trees by Gardner et al. (1982). About properties of the surfaces of both the bacterial, 102-10* bacteria/g xylem vessel were found and fungal and the xylem wall surfaces to which and thirteen genera of bacteria were isolated. micro-organisms either do or do not adhere. Pseudomonas, Enterobacter, Bacillus and Cory mb Microbial infiltration into cut flower xylem vessels nebacterium were most frequent, and some of CARLILE, M.J. 1970Th e photoresponses of fungi. In these resident bacteria were potentially phyto- Photobiology ofMicro-organisms ed . Halldal, P. pp. 309-344. London: Wiley. pathogenic. CARLILE, M.J. 1980 Positioning mechanism - the role Scanning electron microscope observations of motility, taxis and tropism inth elif e of micro are not sensitive enough to assess the visually organisms. InContemporary Microbial Ecologyed . low numbers of either xylem-residing micro Ellwood, D.C., Hedger, J.N.,Latham , M.J., Lynch, organisms or infiltrated micro-organisms far J.M. & Slater, J.H. pp. 55-74 London: Academic Press. from the cut ends of the xylem vessels of cut DANSEREAU, B. & VINES, H.M. 1975 In-stem move flowers. The plate count method asdescribe db y ment isolation andidentificatio n of two bacteria Put & Van derMeijde n (1988) should therefore and their antibiotic sensivity. Acta Horticulturae 41, be used as this gives fairly reproducible data 183-195. even at lowlevel s of infiltrated microbial cells. EWING, W.M. & FIFE, M.A. 1972 Enterobacter agglomérons(Beyerinck ) Comb. nov. (the Herbicola- This method is not applicable, however, to cut Lathyri Bacteria). International Journal ofSystem flowers with hollow stems like Gerbera (Van atic Bacteriology 22,4-11. Meeteren 1980), unless prior disinfection ofth e FLETCHER, M. 1979 The attachment ofbacteri a to sur hollow stem area is possible. faces in aquatic environments. In Adhesion of Micro-organisms to Surfaces ed. Ellwood,D.C. , From the SEM observations reported here Melling, J.& Rutter, P.pp . 87-108. London: Aca some interesting additional information was demic Press. obtained on theinfiltratio n ability of pure cul FUJINO, D.W., REID, M.S.& VANDERMOLEN, G.E. 1983 tures of micro-organisms into thexyle m vessels Identification of vascular blockage in rachides of cut maidenhair (Adiantum raddianum) fronds. Scien- of cut flowers, but much more multidisciplinary tia Horticulturae, 21,381-388 . research is needed to reveal precisely which GARDNER, J.M., FEEDMAN, A.W. & ZABLOTOWICZ, factors may berelate d (directly or indirectly) to R.M. 1982 Identity and behavior ofxyle m residing the mechanism of xylem water-stress, and the bacteria in rough lemon roots of Florida citrus adhesion and plugging of xylem vessels by trees. Applied & Environmental Microbiology 43, 1335-1342. microbial cells (and/or their metabolites), HALVERSON, L.I. & STACEY, G. 1986 Signal exchange leading to decreased water uptake and conse in plant microbe interactions. Microbiological quent shorter vase life ofcu t flowers. Reviews 50, 195-225. HARLING, R., TAYLOR, G.S. & CHARLTON, W.A. 1984 Xylem vessel regeneration incarnatio n in response The authors wish to thank Dr A. Boekestein to infection by Fusarium oxysporumf. sp. dianthi. (TFDL, Wageningen) and Dr M.E. Rhodes- Netherlands Journalof Plant Pathology 90, 173-176. HARLING, R. & TAYLOR, G.S. 1985 A light microscope Roberts (University College of Wales, study ofresistan t and susceptible carnations infect Aberystwyth) for reading and commenting on ed with Fusarium oxysporumf. sp. dianthi.Canadian the manuscript. The authors thank also MrF . Journalof Botany, 63, 638-646. Thiel (TFDL, Wageningen) and Dr H.M. de KONINGS, W.N. & VELDKAMP, H. 1980 Phenotypic Stigter (CABO, Wageningen) for their critical responses to environmental change. In Contempo rary Microbial Ecology ed. Ellwood, D.C, Hedger, discussions through the course of the SEM J.N., Lantham, M.J.,Lynch , J.M.& Slater , J.H.pp . observations. 161-192. London: Academic Press. LYNCH, J.M. & EBBEN, M.H. 1986 The use of micro organisms to control plant disease. Journal of References Applied Bacteriology 61. Symposium Supplement ed. Bateson, M., Benham, C.L.& Skinner , F.A. pp. BERKHOLST, C.E.M. 1980 De waterhuishouding van 115S-126S. afgesneden rozen. Bedrijfsontwikkeling 11, 332-336. MEETEREN, U.VA N 1980Water relations and keeping BILLING, E. 1982 Entry and establishment of patho quality ofcut gerbera flowers. Dissertatie. Wagen genic bacteria in plant tissues. In Bacteriaand ingen, Landbouwhogeschool. Plants ed. Rhodes-Roberts, M.E.& Skinner, F.A. PERESSE, M. 1974 Modification cytologique dans le pp. 51-70. London: Academic Press. xylème detige s d'ouillets expérimentalement infec BOWEN, G.D. 1980 Misconceptions concepts and tées, au cours del'installatio n du Philophora cenere- approaches in rhizosphere biology. In Contempo scens (Wr.) van Beyma. Phytopathologische rary Microbial Ecology ed. Ellwood, D.C., Hedger, Zeitschrift 79, 35-46. J.N., Lathman, M.J.,Lynch , J.M. &Slater , J.H.pp . PUT, H.M.C. & ROMBOUTS, F.M. 1988Th e influence 283-304. London: Academic Press. of purified pectic enzymes on xylem anatomy, water BURDETT, A.N. 1970Th e cause of bent neck in cut uptake andvas e life of Rosa cv. 'Sonia'. Scientia roses. Journalof Horticultural Science 95,427-431 . Horticulturae (in press). 107 H. M. C.Put andA. C.M. Clerkx PUT, H.M.C. & VAN DER MEIJDEN, T. 1988 The infil intact 'Sonia' rose plants. Zeitschrift fur Pflan tration of Pseudomonas putida cells strain 48 into zenphysiologie 99, 131-140. xylem vessels of cut Rosa cv. 'Sonia'. Journal of STIGTER, H.C.M. DE & BROEKHUYSEN, A.G.M. 1986 Applied Bacteriology 64,197-208. Role of stem cut-surface in cut rose performance. RASMUSSEN, H.P. & CARPENTER, W.J. 1974 Changes in Acta Horticulturae 181, 359-364. the vascular morphology of cut rose stems: a scan VAN ALFEN, N.K., MCMILLAN, B.N., TURNER, V. & ning electron. Microscopy study. Journal of the HESS, W.M. 1983 Role of pit membranes in American Society for Horticultural Science 99, 454- macromolecule-induced wilt of plants. Plant Physi 459. ology 73,1020-1023. ROGERS, H.J. 1979 Adhesion of micro-organisms to VAN DOORN, 1985 Annual Report ofthe Sprenger Insti surfaces. Some general considerations of the roleo f tute Wageningen: Th e Netherlands. the envelope. In Adhesion of Micro-organisms to ZIMMERMANN, MJ . 1983 Xylem structure and the surfaces ed.Ellwood , D.C., Melling, J. & Rutter, D. ascent of sap. ed. Timell, T.E. Berlin: Springer- pp. 29-56. London: Academic Press. Verlag. STIGTER, H.C.M. DE 1981 Water balance of cut and 108 Chapter 5 The effects of thevas e life ofcu t Rosacultiva r *Sonia'o f bacteria added to thevas e water. 109 Rosa cv. 'Sonia': stage 5 of flower opening (Berkholst, 1980) 110 ScientiaHorticulturae, 39 (1989) 167-179 Elsevier Science Publishers B.V., Amsterdam —Printe d in The Netherlands The Effects on the Vase Life of Cut Rosa Cultivar 'Sonia' of Bacteria Added to the Vase Water HENRIETTE M.C. PUT and LIEKE JANSEN Sprenger Institute, P.O.Box 17, 6700AA Wageningen (The Netherlands) (Accepted for publication 24Octobe r 1988) ABSTRACT Put, H.M.C. and Jansen, L., 1989. The effects on the vase life of cut Rosa cultivar 'Sonia' of bacteria added to the vase water.Scientia Hortic, 39:167-179. Amarke d decrease inth e vaselif eo fcu tRosa cultiva r 'Sonia' wasobserve dwhe n water-washed viable or heat-inactivated cells of either Bacillus subtilis, Enterobacter agglomerans, Pseudo monasfluorescens orP. putida wereadde dt oth e vasewater .Th e concentrations ofviabl eo r heat- killedbacteria l cellsinfluence d the vase life,bu t different bacterial strains exerted similar effects. High initial numbers of bacterial cells added to the vase water reduced the water conductivity, shortenedth evas elif ean ddelaye dth eonse t offlower-bu d development. Similarnumber so fviabl e cellsadde d to the vasewate r (< 107-> 0 5cell sml "' ) decrease d thevas e life ofth e rosesmor e than did heat-inactivated cells.Water-conductivit y measurement was a rapid, reproducible method of assessingth e effects ofbacteri a after only 24 h of vase life. Vascular plugging caused by bacterial cellswa s not the solecaus e of decreased water conductivity in the stem segments of the roses. Keywords:Bacillus subtilis; Enterobacter agglomerans;Pseudomonas fluorescens; Pseudomonas putida; Rosa cultivar 'Sonia'; vase life. Abréviations: API= Appareil s et Procédés d'Identification, La Balme Les Grottes -38390 , Mon- talieu Vercieu (France); LSD= leas t significant difference; RH= relativ e humidity; SEM=scanning electron microscope. INTRODUCTION The dominant microflora offreshl y cut flower stemso f Chrysanthemum cul tivar 'Spider', Gerbera cultivars 'Appelbloesem' and 'Fleur', and Rosa cultivar 'Sonia' havebee n found to showtypica l cultivar-related differences, whichwer e alsoobserve dbetwee nth e stem flora ofth e wholeplan t andwhe nth ecu t stems 'Present address: Kerksteeg 4, 7411E W Deventer, The Netherlands. Ill wereplace d invas ewate r (H.M.C .Put ,unpublishe d data, 1989) .Th e bacterial genera Bacillus and Enterobacter were dominant on freshly harvested Rosa stems,bu t they lostthei r dominance in the vasewater .Pseudomonas spp.mul tiplied more rapidly in the nutrient-poor vase water. Bacterialpluggin go fxyle mvesse lelement s hasbee nclaime dt ob eth e major cause of rapid senescence ofcu t flowers (Aarts, 1957).However , in many such studies, microbial numbers have not been adequately assessed (Zagory and Reid, 1986) . Bacterial strains used to study cut-flower senescence have rarely been identified (Gardner et al., 1982; Accati-Garibaldi, 1983) . It has also been suggested that some (usually unidentified) microbial products excreted into vase water cause vascular plugging and cut-flower senescence (Gentile and Accati, 1981; Put, 1986;Zagor y and Reid, 1986). Experiments toelucidat e the roleo fcertai n (phytopathogenic and non-phy- topathogenic) bacterial species, associated with fresh Rosa stems during the course ofth e vase life of Rosa flowers, have been carried out. Known numbers of pure cultures of bacteria were added to vases of cut Rosa flowers to assess their effects on the vase life (cf. Durkin and Kuc, 1966; Burdett, 1970; Line- berger and Steponkus, 1976;Zimmermann , 1983) . The purpose of this study was to elucidate the role which viable and inacti vated bacterial cells play in the phenomena leading to xylem vessel plugging, disturbance of the water relations and vase life of cut flowers. MATERIALS AND METHODS Plant material. - Rosa hybrida 'Sonia' was supplied by local greenhouse grow ers. The roses were harvested at flowering stage 1 (Berkholst, 1980) and wrapped in parchment paper, transported to the laboratory and stored at 4°C for 24-48 h prior to carrying out the experiments. Bacterialstrains. - Fivebacteria l strainswer etested ,viz .Bacillus subtilis Strains 207 and 214, Enterobacter agglomérons Strain 41;Pseudomonas fluorescens Strain 52,P. putida Strain 48. They were isolated from freshly harvested Rosa stems and from vase water containing Rosaflowers . Identification was carried out by conventional methods. In addition, commercially available identification systems were used. Oxi/ Ferm tube II (La Roche1) was used for the identification of oxidase-positive Gram-negative rods (Pseudomonas) and Enterotube II (La Roche) for oxi- dase-negative Gram-negative rods (Enterobacteriaceae). For Bacillus strains (Gram-positive aerobic sporeforming rods) Enterotube II (La Roche) and API 50 CH were applied in combination with tests for the hydrolysis of: casein, gelatin and lecithin. The formation of bacterial polysaccharides, dextran and 'F. Hoffmann-La Roche &Co .Ltd. , Diagnostica, CH-4002 Basle, Switzerland. 112 levan,wa s detected in API sucrose agar code 5013.Stoc k cultures were main tained on nutrient agar and stored at 5°C. Vase watersuspensions. - Washedbacteria l cells,grow n oneithe r nutrient agar (Difco) or sucrose agar (API), were aseptically suspended in sterile tap water to give 108cell s ml-1 (measured turbidimetrically). Cell concentrations were also counted microscopically (Roser et al., 1987) and by plate counting on plate count agar (Difco) incubated aerobically at 28°C for 48 h. Each suspension was divided; one aliquot was stored at 5° C (< 20 h) , the second was heat-treated at 85° C for 10min . Just prior to the vase life evalua tion, spiral plates of the suspensions were made on plate count agar (Difco) and incubated aerobically at 28C Cfo r 48 h. Vaselife evaluation. - The Rosastem swer e cut aseptically toa standard length of 45 cm. All except the 3 upper leaves were removed. Each rose was placed singly in a sterilized cylindrical vase calibrated to 100ml .Th e vases contained sterile tap water (control) or a freshly prepared bacterial suspension diluted to give 108, 107, 106, 105 or 104 viable or heat-inactivated cells ml-1 of vase water. Each dilution was replicated 6 times. Protection against air contami nation was obtained by sealing plastic "Parafilm M" on the top of each vase. The vases were maintained at 20°C±1°C, 60% RH±2% and 10.25 W m"2 photoradiation with a 12-h photoperiod. Waterconductivity ofcut flowers. - Water-conducting ability ofcut-flowe r stems was assessed by an apparatus described by Put and van der Meijden (198 8) . Triplicate samples of roses were taken from the vase after 24 h of vase life in water or in abacteria l suspension. Three consecutive stem sections,o f 5c m each, were cut off from the basal end upwards and inserted on to the manifold ofth e apparatus in an inverted position. The water flow through the segments was assessed after 30,6 0an d 90 min ofwate r flow. The average water conduc tivity in ml per 30 min wasthe n calculated (Durkin, 1979). Conductivity was defined as the reciprocal value of the resistance to water flow through xylem vessels of flowers, expressed per transverse-sectional area of 5-cm length. The higher the water conductivity (ml per 30 min), the lower the water-flow resis tance of the xylem vessels (Gilman and Steponkus, 1972; Chin and Sacalis, 1977;Pu t and van der Meijden, 1988). Water uptake by cut flowers. - Water uptake by cut roses in sealed cylinders containing water or a bacterial suspension was measured at 24-h intervals by recording the water level in the calibrated cylindrical vases. The vase fluid levelswer ehel dbetwee n 50an d 100m lb yasepti cadditio n ofth e corresponding suspension. 113 Vase lifeof cut flowers. - Vase life evaluation ofth e cut flowers wasassesse d by visually observing: (1) the condition of the flower and leaves; (2) the flower bud development (Berkholst, 1980); (3) the ornamental value.Th e ornamen tal value is negative when the acceptability ofth e flower in the vase is visually decreased to <50% , compared with the 'normal' bud development of cut Rosa flowers placed in sterile tap water. Negative features in ornamental value are considered to be: wilting of leaves and petals; bent neck; desiccation; discol oration, chlorosis, necrosis. Acid-fuehsin test to detect plugged vascular tissues. - When the ornamental value of the roses was decreased to < 50%, the roses were removed and placed for 30mi n in vases containing an acid-fuchsin solution of 0.5% w/v in 50% v/ v ethanol. The epidermis and cortex ofth e stems werepeele d off and plugging was revealed by the extent and height of the red coloration of the vessels. Statistical evaluation. - Analyses ofvarianc ewer eapplie dt oth e measurements and observations of the water conductivity, vase life and flower opening, and mean values were compared. Aprobabilit y level ofP < 0.0 5 was used. RESULTS Effect of the concentration of the bacterial cellsadded to vase water. - When viableo rheat-inactivate dbacteria lcell so fpur eculture so fth e5 bacteria l strains were added to the vase water, there was a marked reduction in water conduc tivity through the xylem (Table 1), vase life (Table 2) and flower bud devel opment (Table 3), compared with cut roses placed in sterilized tap water. It was furthermore shown that the higher the number of bacteria initially added ml~ 1vas ewater ,th e lowerth e water conductivity (m lpe r 30mi n) ,th e shorter the vase life (days) and the more inhibited the flower opening. Effects of the addition of viable vs. heat-inactivated bacterial cells to the vase water. - Results with the 5bacteria l strains were mainly similar. When aver aging the results of the 5 strains, the water conductivity through the xylem vessels (Fig. 1) ,th e vase life (Fig. 2) an d the flower bud development (Fig.3 ) of cut roses was decreased to a greater extent by viable bacterial cells than by the addition to vase water of the same concentration of inactivated cells. The greatest difference between the effects ofviabl evs .inactivate d bacterial cells were found when the initially suspended bacterial cell numbers varied between > 105an d < 107ml -1 vase water. The least differences between the effects of viable vs. inactivated bacterial cells on the water relations and vase life of cut roses were exerted when > 107 or 104viabl e orheat-inactivate d cells,respectively ,wer eadde d ml-1 vase water. 114 ^ 00 0Î Tf cc O O 00 W O IN lO (O iß •e -* N (N (N 6 Ó d •a «g 'E « o. 2 ___ H C^ 00 U5 O O Oi I> OCOTfCCOCCCOTfTtiOcnHWCNiO o, co ÖÖÖÖÖÖÖÖÖÖÖrHi-ï^HiJ -—- '-P •~ 3 CQtfeatSeQEGCGtQiOCQCdcäU^^ HOiUN^HTffllOOINeOt-^N^ o om^ONcocoiotûio^t-HTC'fl' ÖÖÖÖÖÖÖÖÖÖÖÖI-H^HI-H > co Ü CU 03 • •—s CU > cso 0) aj « <0 S en ^ w U T3 41 tl O "0 TJ 03 T3 O lOiflt-HCÎJOO^OOHOOO^W» HHißNN^HOcoHcoconio >> CO ÖÖÖC>Ö--HI-HI-HC^CNCNI-H«-H^H -w S3 > (4-1 CU o o CM O & 0> CN t— I—1 S - t> CN co OS 00 t> -3 *H 1-H ^H O O o o c CU CNiOtCCO'^'^'^iC^rH^jHCOCON J3 OCOMINlNcNCOtDCÎlOîOHCÎiCnOS -i~» 0P)n &H CO ©©OOOOOOOO^^HI-HI—it—4 % -S- Sti o CU i* es JS J3 0) 1-H i-t 00 tO O) CO GO f- CN CN e CU -*-> a CN CO a HNOiOOOt- O O 00 CN CN oööoööcJööö l-H i-H l-H CM CN §• co v 1-H CO CN co CO 1-H CT> •^3 CM CN g 2 £ B 1—1 CN 1-H 1-H O o 3 U T3 i§ T3 CN co 00 rH *«H U 3'g 1-H tO SD TO CN O oq co OÖÖÖÖÖÖÖ-HÖ f-t l-H CN TO CN tu o >> .O as as S l o >0) O CO CS £ ü £ 0 ÇJ -Q -o V CU ,-U^ügo"0"fl"Dci)cUoti) OaßißOißOOCOOOlßeOO© ^iO^COO5COCq^05CDi>iNO) ,3 *> HHHCNH(N(NC>JN(N(NINC>i :d o ^3 CN O) 1-H CN l-H l-H O •c »OOOlNOlNiCHCOINiOO^^^Tt «1 ^^t^(N^(X3COCOHtUCOCOiß(Nt> oq co ÖÖÖÖÖÖÖÖI-HÖI-HI-HI-HCNI-H a "O c ^ eesê ißOißiOOißißOißißOißißOaßißOißiOOiß » »s | I I I I I I I I I I I I I I I I I I 1 I I S «I » u OiOOOißOOißOOtßOOiOOOißOOiOO S.B CO co -*3 •—- co Ä"S S CD o as .-s3 a« 'äs <»-. 3 « o T3 'S -S s •c° o -«J e m V H o af H o _. a. 1 1 IH ^H o .g S O CO lH m S 3 G Q s *5 S c bc -§ -2 o co I1I ES £.3 as .3 11 2 J 115 TABLE2 The influence on the vase life (days) of Rosa 'Sonia' of the numbers of viable (v) or heat-inactivated (i) bacterialcell s (5bacteria lstrains ) addedt oth evas ewate r Logn bacterial cells Bacterial strain aaaea mi oi vase water B. subtilis B. subtilis E. agglomérons P. flu orescens P. putida Strain 207 Strain 214 Strain 41 Strain 52 Strain 48 V i V i V i V i v i 8 1.0° 2.5b 1.0" 2.5ab 1.5a 1.5" 1.0" 0.5' 1.5 7 2.5b 4.5C 2.0ab 3.0"bc 1.5" 1.5" 2.0b 1.0' 3.5 6 3.0" 5.5cd 2.5ab 5.0cd 2.0* 3.5b 2.5b 1.0" 4.5 5 5.5cd 6.5de 3.5* 6.0de 4.5bc 5.5cd 5.5C 6.5d 6.0 4 6.5de 7.0e 6.0de 7.0* 5.5"1 6.0d 7.5e 7.5e 7.5 None 7.0e 7.5e 7.5e 7.5e 8.0 LSD 1.4 2.3 1.0 0.9 - - =No ttested . Meanvalue spe r bacterialstrai n followed bydifferen t superscripts aresignificantl y different, P<0.05. TABLE3 The influence on the flower bud development of Rosa 'Sonia' of the numbers of viable (v) or heat- inactivated (i) bacterial cells (5bacteria lstrains ) addedt oth evas ewate r Logn bacteria l cells Bacterial strain addedml "' ofvas e water B. subtilis B.subtilis E. agglomérons P.fluorescens P. putida Strain 207 Strain 214 Strain 41 Strain 52 Strain 48 8 1.5" 2.5al 2.0" 2.0* 1.5" 2.0" <0.5 0.5" 2.0 7 2.0" 3.5bc 2.0" 3.0"b 3.5cd 3.0^ 1.0" 1.0" 3.0 6 2.0" 5.0* 2.5*b 6.0C 4.5de 5.0ef 1.5"b 2.5b 4.0 5 4.5cd 6.0e 3.5b 6.5cd 5.0ef 6.0fg 5.5C 5.5C 5.5 4 4.5cd 7.5f 7.0cd 7.5d 6.5g 6.5* 7.0d 7.0d 7.5 None 7.5f 7.5d 8.0h 7.0d 8.0 LSD 1.3 1.0 1.1 1.1 -= No t tested. Mean valuespe r bacterialstrain ,followe d bydifferen t superscripts aresignificantl y different, P<0.05. No effect on the vase life and flower bud development was exhibited when 104 inactivated cells ml-1 vase water were added, compared with the roses placed in sterilized tap water. 116 STEM SEGMENT STEM SEGMENT STEM SEGMENT 0- 5 cm 5_10cm I0.l5cm 2.00 1.50- 1.50 1.00 O O z o ü 0.50 -0.50 0.00 "1 1 I I I I l i I I I 8 7 6 5 4 40 8 7 6 5 4 *0 8 7 6 5 4 io Log N bacterial cells / ml Fig. 1.Th e influence ofth e numbers ofviabl e or heat-inactivated bacterial cells (m l~ ' ) added to the vase water, on the water conductivity of stem segments of Rosa 'Sonia' after 24h of vase life. O =viabl e cells (v); •= heat inactivated cells (i); *= contro l (c) value for sterilized tap water. The values shown are means of 3triplicat e tests ofth e 5differen t bacterial strains. Water conductivity of consecutive stem segments. - The lowest water conduc tivity was measured at the basal end ofth e Rosa stem when the highest initial numbers ofbacteri a were added to the vase water. The two consecutive tested segments of the corresponding rose stems showed a slightly increased water conductivity (Table 1an d Fig. 1). Vasewater uptake (ml per 24 h). - The mean values of the daily water uptake of the individual roses when the same concentration of either viable or heat- inactivated bacteria were added to the vase water showed that the higher the concentration ofbacteri a initially added ml-1 ofvas ewater ,th e lowerth e water uptake (ml day-1, Fig. 4). Lower also was the uptake from vase water with viable cells than from vase water with the same initial concentration of inac tivated cells. Besides, it was found that after > 2 days of vase life the water uptakedecreased ,an dthi sdecreas ewa smor eextensiv ewhe nviabl ecells ,rathe r 117 CO 5 5 o Log N bacterial cells added per mL vase water Fig.2 .Th e influence ofth e numbers of bacterial cells (m l~ ' ) added to the vase water,o n the vase life of Rosa 'Sonia'. O =viabl e cells (v); # = heat inactivated cells (i); *=control (c) value for sterilized tap water. The values shown are means of triplicate tests of the 5 different bacterial strains. than an equal concentration of inactivated bacterial cells, were added to the vasewater . The highest water uptake wasmeasure d when roseswer eplace d in sterilized tap water. Acid-fuchsin test to detect vascularplugging. - The higher the initial concen tration of bacterial cells ml-1 of vase water, the lower the infiltration ability ofth e acid fuchsin solution into the xylem vessels of the roses.Initia l concen trations of 108bacteri a ml-1 vasewate r showedentirel y blockedvessels .Fuch - sin-red infiltration was observed in a few vessels at 107 ml-1 viable cell con centrations. A few more vessels were coloured when the same number of inactivated bacterial cellswer eadded .Initia l concentrations of 106viabl e ml"1 showed a nearly half-blocked vessel system. The non-blocked vessels coloured up to the flower head and the leaves. The vessel colouration ability of fuchsin red increased further at initial concentrations of 105 and 104 bacterial cells ml-1 vasewater . Smalldifference s remained visiblebetwee nequa l initial num bers of viable and inactivated cells.O f alltreatment s rosesplace d in sterilized tap water showed the highest colour intensity through the xylem system of stem, leaves and into the flower petals. 118 o _J >UJ '<° Log N bacterial cells added per mL vase water Fig.3 .Th e influence ofth e numbers ofbacteria l cells (ml-1) added to the vasewater , on the bud development of Rosa 'Sonia'. O =viabl e cells (v); •= heat inactivated cells (i); *=control (c) value for sterilized tap water. The values shown are means of triplicate tests of the 5 different bacterial strains. DISCUSSION Viableo rheat-inactivate d water-washedcell so fth e5 bacteria l strains caused a marked decrease in the vase life of the roses. The initial number ofbacteri a added affected the decrease ofth e vase life of the roses, but surprisingly the individual bacterial strains behaved very simi larly (Table 2), notwithstanding their differences in: (1) cell shape; (2) cell motility; (3) slime production on agar media; (4) formation of a cellular cap sule; (5) production of levan from sucrose; (6) ability to decompose pectin or cellulose; (7) the opportunistic pathogenicity of the different strains tested (Anderson, 1984;Halsal l and Gibson, 1985;Pu t and Rombouts, 1989). Water-washed suspensions of strains of many other bacterial species, iso lated from flower stems, showed the same phenomena regarding the effects of the initial bacterial cell load added to the vase water on the vase life of cut roses. These observations (not detailed here) applied to strains of: Bacillus cereus;B. cereusvar . mycoides;B. circulons;B. licheniformis;B. polymyxa; En terobacter cloaca; E. sahazaki; Pseudomonas aeruginosa; P. cepacia; P. maltophilia. Initial numbers of 108viabl e or heat-inactivated bacterial cells ml-1 added 119 18 > •o IU < H Q. 3 10 BC UI *~ ft S ui »< 6 > "V-IO6 VASE LIFE (DAYS) Fig.4 .Th e influence ofth e numberso fbacteria l cells (ml- ') adde d toth e vasewater , onth e daily water uptake of Rosa 'Sonia'. The values shown are means of triplicate tests of the 5 different bacterial strains. *=control (c) values for sterilized tap water; 0=viable cells (v) 108 ml-1; • =hea t inactivated cells (i) 108ml_1; A=viable cells (v) 107ml_1; A= heat inactivated cells (i) lO'ml-1; V=viable cells (v) 106ml-1; •= heat inactivated cells (i) 106ml_1; D=viable cells (v) 105ml -';•=heat-inactivated cells (i) 106ml -1; O=viable cells (v) 104ml_1; •=heat- inactivated cells (i) 104 ml-1. to vase water caused xylem element occlusions at the basal end of the stem; this was seen in SEM observations (Put and Clerkx, 1988). The water con ductivity data and blockage of infiltration of acid fuchsin reported here con firm these observations (Table 1an d Fig. 1) . Initial numbers of 107viabl e or heat-inactivated bacterial cells ml-1 of vase water,however , showedonl ya slightl yincrease d xylemwate r conductivity and vase life of the roses compared with 10-fold higher initial bacterial numbers. SEM observations and the acid-fuchsin test indicated that the xylem vessels were partially blocked by bacterial cells.Thi s suggests that in addition to the physical-mechanical blockage of the xylem vessels, there were some non-spe cifiable secondary reactions between the infiltrating bacteria and the vascular 120 VASE LIFE (DAYS) Fig. 5. The maximal vase life (days) plotted against the maximum flower bud development of Rosa 'Sonia' placed in water initially containing decimal dilutions of 108-104 ml-1 of cells of a bacterial strain. The results are a summation ofth e results using the 5 strains separately. O =vi able cells (v); •= heat-inactivated cells; *=control (c) value for sterilized tap water. water-transport system. These might have contributed to the observed water stress, wilting and premature senescence of the roses. At lower initial numbers (> 105-< 107 ml-1) of bacteria added to the vase water, the differences in the effects of viable and heat-inactivated bacterial cells on the vase life of 'Sonia' roses became significant (Table 2an d Fig. 2). Thus, not only inert bacterial propagules, but also bacterial metabolic activi tiesprobabl y played a significant role in the course ofth e vase life of the roses (Gentile and Accati, 1981;Accati-Garibaldi , 1983;Zagor y and Reid, 1986; Put and van der Meijden, 1988). Areduce d water conductivity of Rosa stems measured after 24h of vase life can implicate a much later observable decrease of the ornamental value of the flowers. To reveal whether a reduced water conductivity is caused by viable and/or non-viable bacteria, a microscopic counting method should be combined with a plate-count method. 121 It is not known why the infiltration of viable or heat-inactivated bacterial cells into the xylem vessels resulted in water stress measured as decreased water conductivity. The similarities between the reactions of different bacte rial strains tested (Fig. 5) suggests that these reactions were merely non-spe cific stress responses (Konings and Veldkamp, 1980; Halverson and Stacey, 1986). Initial numbers of 104 bacteria ml-1 added to the vase water only slightly influenced the water relations or decreased the vase life of Rosa flowers. For maximum longevity of Rosa flowers their handling and storage should ideally beunde r virtually aseptic conditions.Thi sca nb eestimate dwithi n ashor t time after harvest by rapid methods such as stem conductivity measurements or direct microscopic counts of bacteria in the flower vase water (Roser et al., 1987) and in the xylem vessels (Put and van der Meijden, 1988). ACKNOWLEDGEMENTS The authors wish to thank Dr. M.E. Rhodes-Roberts, Department of Biol ogyan d Microbiology, University ofWales ,Aberystwyth , Gt.Britain , for read ingan d commenting on the manuscript. The authors thank also Ans van Har- develd for text registration, Hanna Lagerwerf for statistical treatment of the results and Peter Schrijver for the design of the figures. REFERENCES Aarts,J.F.T. , 1957.O nth e keepability ofcu tflowers. Meded . Landbouwhogesch. Wageningen,57 : 1-62 (Dutch with English summary). Accati-Garibaldi, E., 1983.Postharves t physiology of mediterranean carnations: partial charac terization of a bacterial metabolite inducing wilt offlowers. Act a Hortic, 138:255-260 . Anderson, A.J., i984. Differences between lipopolysaccharide composition of plant pathogenic and saprophytic Pseudomonas species.Appl .Environ . Microbiol., 48: 31-35. Berkholst, C.E.M., 1980.Th e water relations of cut roses.Bedrijfsontwikkeling , 11:331-336 . Burdett, A.N., 1970.Th e cause of bent neck in cut roses.J . Am. Soc. Hortic. Sei., 95: 427-431. Chin, C.an d Sakalis,J.N. , 1977.Metabolis m of sucrose in cut roses.II . Movement and inversion of sucrose absorbed by cut rose stems.J . Am. Soc.Hortic . Sei., 102:537-540 . Durkin,J. , 1979.Effec t of millipore filtration, citric acid and sucroseo n pedunclewate r potential of cut roseflower. J . Am. Soc.Hortic . Sei., 104: 860-863. Durkin,D .an d Kuc,R. , 1966. Vascular blockagean d senescenceo fth e cutros eflower. J . Am.Soc . Hortic. Sei., 89:683-687 . Gardner, J.M., Feedman, A.W. and Zablotowicz, R.M., 1982.Identit y and behavior of xylemres- idingbacteri a in roughlemo nroot so fFlorid acitru strees .Appl .Environ . Microbiol., 43:1335- 1342. Gentile, A.an d Accati, E., 1981. Partial characterization of a bacterial metabolite responsible for causing wiltingo f carnation flowers. Riv. Ortoflorafruttic. Ital., 65:257-263 . Gilman, K.F. and Steponkus, P.L., 1972. Vascular blockage in cut flowers. J. Am. Soc. Hortic. Sei.,97 :662-667 . Halsall, D.M. and Gibson, A.H., 1985.Cellulos e decomposition and associated nitrogen fixation 122 bymixe dculture s ofCellulomonas gelida an dAzospirillum specieso rBacillus macerans. Appl. Environ. Microbiol., 50:1021-1026. Halverson, L.I. and Stacey, G., 1986. Signal exchange in plant microbe interactions. Microbiol. Rev.,50 :195-225 . Konings,W.N .an d Veldkamp, H., 1980.Phenotypi c responsest o environmental change.In :D.C . Ellwood, J.N. Hedger, M.J. Lantham, J.M. Lynch and J.H. Slager (Editors), Contemporary Microbial Ecology, Academic Press, London, pp. 161-192. Lineberger, D.R. and Steponkus, P.L., 1976. Identification and localization ofvascula r occlusions in cut roses.J . Am. Soc. Hortic. Sei., 105:246-250 . Put, H.M.C., 1986. Investigations into the influence of the microflora from stems of cut flowers on the vase life of Rosa cv. Sonia, Gerbera cv. Fleur, and Chrysanthemum cv. Spider. Acta Hortic, 181:415-418 . Put, H.M.C, and Clerkx, A.C.M., 1988.Th e infiltration ability of micro-organisms:Bacillus, Fu sarium, Kluyueromyces and Pseudomonas spp. into xylem vessels of Gerberacv . Fleur and Rosa cv.Soni a cut flowers. Ascannin g electron microscope study.J . Appl.Bacteriol. ,64 :515 - 530. Put, H.M.C. and van der Meijden, T., 1988.Th e infiltration ofPseudomonas putida cells, strain 48, into xylem vessels of cut Rosa cv. 'Sonia'.J . Appl. Bacteriol., 64: 197-208. Put, H.M.C. and Rombouts, F.M., 1988. The influence of purified microbial pectic enzymes on the xylem anatomy, water uptake and vase life of Rosa cultivar 'Sonia'. Scientia Hortic, 38: 147-160. Roser, D.J., Bavor, H.J. and McKersie, S.A., 1987.Applicatio n of most-probable-number statis tics to direct enumeration of micro-organisms. Appl.Environ . Microbiol., 53: 1327-1332. Zagory, D. and Reid, M.S., 1986.Rol e of vase solution micro-organisms in the life of cut flowers. J. Am. Soc. Hortic. Sei., Ill: 154-158. Zimmermann, M.H., 1983.Xyle mstructur e andth e ascento fsap .In :T.E . Timell (Editor) , Series in Wood Science. Springer Verlag,Berlin , 143pp . 123 Chapter 6 Theeffect s of microbial exopolysaccharides (EPS) in vasewate r on the water relations and thevas elif e of Rosa cv. 'Sonia'. 125 Rosa cv. 'Sonia': stage 6 of flower opening (Berkholst, 1980) 126 The effects of microbial exopolysaccharides (EPS)i nvas e water on the water relations and the vase life ofRosa cv. Sonia HENRIETTE M.C. PUT* & W. KLOP Sprenger Institute, Ministry of Agriculture and Fisheries, PO Box 17, 6700 AA Wageningen, The Netherlands Accepted 26 June 1989 PUT, H.M.C. & KLOP, W. 1990.Th e effects of microbial exopolysaccharides (EPS) in vase water on the water relations and the vase life of Rosa cv. Sonia. Journal of Applied Bacteriology 68,367-384 . Pure cultures of five microbial species were used to test the formation of exo polysaccharides (EPS) when grown in agitated sucrose (5% w/v)containin g liquid cultures. These test species were isolated from stems offreshl y harvested cut flowers (Chrysanthemum, Gerberaan dRosa) orfro m thevas e water ofthes e flower cultivars. The partial conversion of sucrose into other saccharides was demonstrated by HPLC andcolorimetri c analysis. The final polymeric character ofth e newly formed saccharides was investigated. SEM preparations of xylem vessels of Rosa main tained in EPS-containing vase water showed blockage, disorganization and injury of the vessel structure. EPS were shown not to pass the xylem pit membranes. Recovery from the first symptoms ofdisturbe d water flow (wilting) duet o EPS was possible in young flowers bycuttin g offth e blocked part ofth e stem (15-20cm.Th e higher the microbial conversion rate of sucrose into polysaccharides, the more dis turbed were the water relations of the roses placed in the EPS-containing fluid, as was demonstrated byth edecreas e of: (1)wate r conductivity of Rosa stem segments (ml/30 min); (2) water uptake (ml/d); (3) Rosa vase life (d);an d(4 ) flowerbu ddevel opment. Bacterial EPS(presumabl y levans and dextrans) could be concentrated in the retentate by molecular filtration with a cut-off level of 1000 0 Da.Filtrate s did not cause Rosa xylem blockage and'bent-neck ' ofth e flower stems, but still may be toxic to roses. Twosimpl e methods were also used for diagnostic investigations: (1) the beetroot tissue cube test todetec t microbial products causing injury ofth e plant cell membranes, (2)th e acid fuchsin test, to show the extent and location of Rosa xylem vessel occlusion. In general, the vase life of cut flowers is absorb water and maintain turgidity (Halevy & enhanced by a sucrose addition (up to 0135 Mayak 1979; Paulin 1980; Paulin et al, 1981; mol/1) in the vase water (Aarts 1957; Chin & Paulin & Jamain 1982). These effects may be Sakalis 1977; Durkin 1979),althoug h vase life of reversed, however, by enhancement of bacterial cut roses such as cv. Sonia is negatively corre growth which may be sucrose-mediated. lated with bacterial growth and bacterial Many of the microbial species isolated from numbers in the vase water (Put 1986; Put & cut flower stems and cut flower vase water can Jansen 1989). produce extracellular polysaccharides as cap Exogenously added sucrose contributes to sules attached to the outer cell walls or as slime cell metabolism as an energy source, increases released to the environment, or both the osmotic value, and enhances the ability of (Sutherland 1977). the tissue in which the sucrose accumulates to An increasing resistance to water flow in vascular tissue is the major cause of water stress * Corresponding author. Present address: Kerk- (wilting) in cut flowers (Aarts 1957). Purified steeg 4, 7411 EW Deventer, The Netherlands. bacterial polysaccharides induced wilting of 127 Henriette M. C.Put and W. Klop sunflower and tomato stem cuttings (Corey & MICRO-ORGANISMS Starr 1957), and Hodgson et al. (1949) found Five microbial species were tested: Bacillus sub- that the effectiveness of artificially synthesized tilis strains 207 & 214; Pseudomonas fluorescens polymers correlated with the molecular size. strain 52; Ps. putida strain 48; Botrytis cinerea In alfalfa plants (Medicago sativa L.) inter strain 196; Fusarium oxysporum strain 07. These ference by dextran of vascular conductance was organisms were isolated from stems of freshly directly correlated with the molecular weight harvested cut flowers of Chrysanthemum, (mol.wt < 250000 rarely affected vascular flow). Gerbera or Rosa, or from vase water containing However, different sections of the vascular these cut flowers. system showed different susceptibilities to plug ging by dextrans of known mol. wt (Van Alfen & Allard-Turner 1979). Macromolecules prob VASE WATER PREPARATION ably disrupt vascular flow by accumulating on pit membranes (Van Alfen et al. 1983), so the Shaken cultures frequency of pit membrane occurrence in the water flow pathway may thus be an important Bacteria: the growth of a 24-48 h old streak wilting determinant. plate culture on Nutrient Agar (Difco) incu In most of the work reported by other bated at 28°C was suspended in 20 ml sterile tap authors, purified bacterial polysaccharides (see water and vortex-mixed at maximum speed. Corey & Starr 1957) or artificially synthesized After mixing, 2-5 ml of the suspension was polymers were applied to elucidate the mecha added to each of a series of 500 ml screw- nisms of phytopathogenicity. The effects of capped bottles containing 250 ml of sterilized polysaccharides on ornamental cut flowers, 5% w/v sucrose solution in tap water. Five ml however, have rarely been studied (Hodgson et of a clear supernatant fluid of a sterilized al. 1949; Van Alfen & Turner 1975; Van Alfen mashed Rosa stem suspension (5% w/v) was & Allard-Turner 1979; Van Alfen et al. 1983;d e added to each bottle. Stigter & Broekhuysen 1986; Neumann 1987). Fungi: growth was harvested from 2-4 d old In the present work, molecular filtrates and streak plate culture on Malt Extract Agar retentates of supernatant fluids of pure cultures (Difco), incubated at 25°C. The harvesting and of several micro-organisms grown in an agitated inoculation of the fungus cultures into the sucrose-containing medium were used as a vase bottles was as described for bacteria. medium for cut roses cv. Sonia. The micro All inoculated bottles were shaken at 60 organisms tested originated from stems of cut rev/min and an amplitude of 20 cm during ca 3 flowers or from cut flower vase water. weeks at 20 + 1°C to obtain maximum poly The aim of our investigations was to reveal a saccharide formation. possible correlation between vase life, water flow and the quantity of microbial poly Static cultures saccharides in the vase water. The medium used in static cultures consisted of glucose 1-3% w/v, or sucrose 1% w/v, and a clear supernatant fluid of a sterilized mashed Materials and Methods Rosa stem suspension (0-1% w/v) in tap water. The microbial inoculum was the same as for the PLANT MATERIAL shaken cultures. Incubation was at 20 + 1°C for Rosa hybrida cv. Sonia was supplied by local 2-3 d. The single pure cultures thus obtained greenhouse growers. The roses were harvested were used as controls, simulating microbial vase at flowering stage 1 (Berkholst 1980) and water. Young, static cultures in media with a wrapped in parchment paper, transported to the relatively low carbohydrate content will show laboratory and stored at 4°C for 24—48 h before little or no microbial exopolysaccharide forma doing the experiments. In addition some com tion. Pectolytic enzymes may or may not be parative tests were done with Chrysanthemum formed. Fungus cultures were slightly agitated cv. Daymark and Gerberacv . Rebecca. to prevent surface growth. 128 Microbial exopolysaccharides in vase water Harvest of microbial products corrected for this (GF). Therefore the average degree of polymerization of the mixture of sac The grown pure cultures were centrifugea at charides, other than glucose, fructose or sucrose 10°C and 10000 g (Beekman J-21C). The super is given by: natant fluid was decanted, and microbial cells and mycelium particles removed by pressure fil El - (GF + S) DP' = (1) tration through a 0-22 fim sterile disposable El - (GF + E2) filter unit (Millipak-60, MPGL-06-Hz). The Glucose, fructose and sucrose were determined sterile filtrate was stored at ca 5°C. in aliquots of culture liquids by a Waters Associates liquid Chromatograph equipped with a 6000A pump, U6K injector and R401 RI Molecular filtration detector. The sugars were separated on a A Pellicon recirculating cassette system for Lichrosorb-10-NH2 column (Chrompack, Vhss 'molecular sieving' was used with a nominal ingen, The Netherlands) with ethyl acetate- cut-off level of mol. wt 10000 Da (Millipore, a acetone-water 300 :55 5 :150 (v/v) at a flow rate PT packet 10K NMWL, retentate separator of 1-3 ml/min. Column and detector tem 10/PK and pump-motor 23 OV 50/60 HZ at 10 perature was 30°C. PSI). When the retentate could not be recovered Absorption value (El or E2) with the phenol- aseptically, an additional filtration through a sulphuric acid method were converted to molar 0-22 p.m.Millipa k sterile disposable filter unit concentration (mol/1), with a calibration curve was applied. Sterilized tap water was added to for sucrose and the appropriate dilution factor. get equal volumes of filtrate and retentate. Storage of sterile filtrates and retentates was at SCANNING ELECTRON MICROSCOPE 2-5°C up to a maximum of 60h . OBSERVATION (SEM) SEM preparations were made after 24 h of vase ANALYSIS OF CULTURE LIQUIDS life, as described by Put & Clerkx (1988). The Rosa stem segments studied were from vase Assayfor the presence of polysaccharide water containing added filtrate or retentate of culture fluids of: B. subtilis, F. oxysporum and The total concentration of saccharides (as Ps.fluorescens. anhydrohexose) in culture liquids was estimated by the phenol-sulphuric acid method (Dubois et al. 1956), wherein glycosidic linkages are split VASE LIFE EVALUATION and the reducing groups formed react to give The vases contained 100 ml of sterile tap water, coloured compounds, which absorb at 488 nm sucrose 1% w/v in sterile tap water (control), or (absorption El). When the reaction mixture was decimal dilutions in sterile tap water (maximal incubated for 20mi n at 80°C before cooling, the 10"3 ) of the retentate or the nitrate of one of the molar absorptions for glucose, fructose and microbial vase water supernatant fluids. anhydrohexose from the sucrose value are The vase life of the roses was assessed as nearly equal. In a parallel test, existing reducing described by Put & van der Meijden (1988), and endgroups were first reduced to alcohol groups consisted of: (1) water conductivity measure with NaBH4 solution, followed by the phenol ments of stem segments (ml/30 min); (2) deter and sulphuric acid reaction. The resulting minations of water uptake (ml/d); (3) visual absorption, E2, is less than El and the average observations of the flower bud development degree of polymerization, DP', is given by, (Berkholst 1980); and (4) assessment of the DP' = E1/(E1 - E2) (Timell 1960). Because any ornamental value of the flowers. remaining sucrose gives equal contributions to El and E2 (for pure sucrose El = E2) the mea sured absorptions have to be corrected for the ACID FUCHSIN TEST TO DETECT PLUGGED contribution of sucrose, (S). Glucose and fruc VASCULAR TISSUES tose, when present, contribute only to El thus At the end of the vase life, when the ornamental lowering the measured DP'; therefore El is also value of the roses had decreased to < 50%, the 129 Henriette M. C.Put and W. Klop roses were removed and placed for 30 min in is given in Table 1. The molar concentrations vases containing an acid fuchsin solution of pertaining to culture supernatant fluids (SN) are 0-5% w/v in 50% v/v ethanol. The epidermis in accord with the molar balance of sucrose and cortex of the stems were then peeled off, conversion: GF + S + P + SM = 0-292 mol/1 and plugging was revealed by the extent and where SM is metabolized sucrose. height of the red coloration of the vessels It appears that the percentage conversions of (Parups & Molnar 1972). sucrose into polysaccharides by B. subtilis, Ps. fluorescens, Ps. putida and Bot. cinerea are 51-61%, 14-41%, 31% and 22% respectively. BEETROOT TISSUE CUBE TEST Results for the supernatant fluid of F. oxyspo- To demonstrate the ability of micro-organisms rum are not available, but the molar fractions of to form cell-membrane- or cell-wall-damaging polysaccharides in the retentates Rl and R2 products, raw beetroot was peeled and cut into account for partial conversion of sucrose into cubes of ca 7 mm and immediately washed in polysaccharides. running tap water until the washwater remained It was also observed that shaken cultures colourless; they were then dried with paper inoculated with the same mother culture did not tissue and transferred singly into sterile screw- show significantly different EPS production. capped polystyrene 15 x 45 mm tubes. Five ml Replicate cultures, however, sometimes produc of the supernatant fluid, retentate or filtrate of a ed different quantities of EPS as is shown by Ps. culture of the strain to be tested was added to fluorescens in Table 1; the toxicity of molecular each test tube, which was then shaken gently filtrates (B. subtilis; Ps. fluorescens and F. before static maintenance at room temperature. oxysporum) was also different. The colour intensity of the fluid (indicative of cell membrane damage) was examined after 30mi n and 4h at 20°C and compared visually Average degreeof polymerization with a control tube containing sterile tap water Relative standard deviations for El, E2 and for (see Plate 1). the lower saccharides were 4% and 8% respec tively. The absolute standard deviation in the numerator and denominator of equation (1) was PECTINASE TEST calculated using a standard procedure. The The method described by Wieringa (1956) was denominator was considered to be near zero used to demonstrate any possible formation of when its numeric value was less than the calcu pectic enzymes in the different microbial cul lated absolute standard deviation. In that case tures used. only the lower limit for the DP' value is given in Table 1. High DP' values (2-12) were also measured in STATISTICAL EVALUATION supernatant fluids of shaken cultures of B. Analyses of variance was applied to all the mea licheniformis and B. polymyxa while a moderate surements and observations of the water uptake, DP'-value of 1-5-2 was measured in culture water conductivity, vase life, and flower opening retentates of Enterobacter agglomérons and of the Rosa flowers. Kluyveromyces marxianus (results not pre sented). Results Molecular filtration The proportion of polysaccharides, P/El, with MICROBIAL EXOPOLYSACCHARIDE (EPS) B. subtilis, strain 214, was 70% for both super FORMATION IN SHAKEN CULTURES natant fluid and its retentate. The latter, however, had a significantly higher average Sucrose conversion degree of polymerization than the original The composition of the supernatant fluids and supernatant fluid (Table 1). With Ps. putida the some retentates after molecular filtration of DP' values were barely different, but the reten supernatant fluids of pure cultures of six strains tate had a higher proportion of P (50%) than 130 Plate 1. The beetroot tissue cube test, to assess cell-membrane damage due to supernatant fluids of microbial broth cultures: (1) control, in sterile tap water, —; (2) no cell-membrane damage, -; (3) weak cell membrane damage, w; (4) cell-membrane damage, + ;(5 ) cell membrane damage +/+ + ;(6 ) cell-membrane damage + +. Only a slightly increased colour-intensity occurred at 4h compare d with 30mi na t20°C . 131 Plate 2. Showing the ornamental value of Rosa cv. 'Sonia' after 2 d of vase life with various additives from a culture supernatant fluid ofF. oxysporum-01 added to the vase water. F"1 = plus molecular filtrate diluted 10"'; F~2 = plus molecular filtrate diluted 10"2; R~' = plus retentate diluted 10"'; R~2 = plus retentate diluted 10"2; W= insterilize d tap water. Plate 3. Showing the leaves of Rosa cv. 'Sonia' after 2 d of vase life, with various additives prepared from a culture supernatant fluid of F. oxysporum-07 added to the vase water. F0 = plus molecular filtrate; R0 = plus retentate;W = insterilize d tap water. 132 Microbial exopolysaccharides in vase water Table 1. Conversion of sucrose by pure cultures of micro-organisms. Results for saccharides, including denomina tor (D) of equation 1 (see text) are expressed as mol/1 anhydrohexose Total saccharides Polysaccharides Average Micro-organism Glucose + Before After (= Nominator of Denominator degree of with strain no. Fructose Sucrose reduction reduction of equation 1) of equation 1 polymerization and sample type GF S El E2 P D DP' Bacillus subtilis strain 207 SN 0058 <0001 0-237 0052 0179 (0-010) 0127 (0010) 1-4 (01) strain 214 SN 0018 0047 0-214 0175 0149 (0009) 0021 (0011) 7-1 (3-7) R* 0071 <0-00'1 0-243 0169 0172 (0011) 0003 (0013) >12 Pseudomonas fluorescens SN1« 0015 0058 0194 0173 0121 (0009) 0-006 (0010) >11 strain 52 SN2* 0024 <0-00 1 0103 0050 0079 (0-004) 0029 (0005) 2-7 (0-6) SN3» 0127 <0-00 1 0183 0051 0042 (0013) 0005 (0013) >2 Ps. putida strain 48 SN 0137 0-009 0-237 0110 0091 (0012) -001 (0013) >6 R 0089 <0-00 1 0177 0098 0088 (0010) -001 (0011) >7 Botrytis cinerea strain 196 SN 0108 0-008 0179 0070 0063 (0010) 0001 (0010) >5 Fusarium oxysporum strain 07 Rl 0082 0056 0-214 0126 0076 (0012) 0-006 (0012) >5 R2 0094 <0O01 0198 0100 0104 (0011) 0004 (0012) >8 SN, culture supernatant fluid; R, retentate of supernatant fluid. * Highly viscous. P = El - GF - S, D = (El - GF - S) - (E2 - S) = El - GF - E2, DP' = P/D (see text for further details). Data between brackets are absolute standard deviations. the supernatant (38%). The four retentates The higher the conversion of sucrose into shown in Table 1 did not contain glucose, so polysaccharides, the greater was the disturbance GF here refers to fructose; the high proportions of the water relations of the roses; thi s was mea are inexplicable, Because no filtrates were sured as decreases in the water conductivity, the analysed and no constant volume of retentate water uptake, and the vase life of the Rosa was obtained, a comparison of the saccharide flowers. concentrations of SN and R was not possible. The influence of the specific culture additives on the water relations and the vase life of Rosa flowers are as follows. MICROBIAL EPS FORMATION IN STATIC CULTURES No EPS was found, except in the supernatant Bacillus subtilis strains 207 &214 fluid of a stationary culture of Ps. fluorescens-52 The EPS production and polymerization in which contained a very low concentration of shaken cultures of B. subtilis was so high that EPS. even retentate dilutions up to 10"2 (strain 214) decreased the vase life of Rosa as a consequence of xylem occlusions. Wilting, shrivelling, 'bent- WATER RELATIONS AND VASE LIFE OF neck', as well as chlorotic spots on the leaves 'SONIA' ROSES IN EPS-CONTAINING VASE were evident, but at the end of the vase life even WATER necrotic spots. The 10"3 retentate dilution The water conductivity values of the roses mea showed only minor visual deviations compared sured after 24 h of vase life as influenced by the with the control roses (strain 214).Se e Fig. la-c. composition of the vase water used, are set out The non-diluted Bacillus culture filtrate pro in Figs la-5a. The water uptake (ml/d) from moted flower wilting, shrivelling and at the end these vase waters is given in Figs lb-5b. In of the vase life leaf chlorosis and necrotic spots, addition the vase life and the flower opening and sometimes also 'bent-neck' were evident. shown by these roses (Berkholst 1980) are The non-diluted retentate of the static cultures shown in Figs lc-5c. caused a decrease of the vase life of about 50%. 133 Henriette M. C.Put and W. Klop Diluted retentate, however, showed no visual disturbance of the water uptake of the Rosa differences compared with roses in sterilized tap flowers, but also in the accelerated senescence. water. The end of the vase life, when retentate or fil trate was added to the vase fluid, was marked by flower wilting, 'bent-neck' and leaf chlorosis Pseudomonas fluorescens strain 52 alongside the veins. In addition, leaf necrosis The deviations and disturbance of the water and dehydration of the leaves was observed pre relations and the vase life of Rosa flowers in ceding the 'bent-neck' phenomenon. Only a vases supplemented with Ps. fluorescens-52 slightly reduced ornamental life was shown shaken culture retentate were (but to a some when roses were subjected to supernatants of a what lesser extent) similar to those induced by young static culture of F. oxysporum-CYJ. B. subtilis retentate. At the end of the vase life, flower shrivelling and 'bent-neck' were observed. The undiluted filtrate also caused some flower Botrytis cinerea strain 196 wilting. After two days of vase life the non- diluted retentate of the Ps. fluorescens-52 static Roses in vase fluids with a concentrated molecu culture promoted an acute 'toxic' reaction lar retentate supplement from shaken cultures towards the flowers, shown by a fall in the vase of Bot. cinerea-196 showed xylem vessel fluid uptake, and 'bent-neck' and shrivelling of occlusions, disturbance of the water relations and a decreased vase life, even when retentate the flowers, although the EPS content of the 2 static culture was very low, indeed. The static dilutions up to 10" were added. To a signifi culture filtrate caused only a slightly decreased cantly lesser extent, the culture filtrate caused vase life. See Fig. 2a-c. decreased water conductivity, water uptake and vase life (Fig. 5a-c). At the end of the vase life, in both types of vase fluids the roses showed Pseudomonas putida strain 48 flower wilting and shrivelling and chlorotic as well as necrotic spots on the leaves. Supernatant A heavy disturbance of the water relations fluids of young static cultures, however, only (wilting and 'bent-neck') of Rosa flowers was slightly reduced the vase life of'Sonia' roses. caused by shaken culture retentates of Ps. putida, even at 10"1 dilution, although to a lesser extent than caused by the non-diluted retentate. Non-diluted filtrates of these shaken PECTINASE PRODUCTION IN MICROBIAL cultures caused a slightly decreased vase life CULTURES (Figs 3a-c), as did also the supernatant fluids of No pectinase activity was observed in shaken static cultures of this strain. cultures (with 5% w/v sucrose) of the six micro bial strains tested, nor in the young static Fusarium oxysporum strain 07 control cultures (with 1% w/v sucrose) of these microbial strains. In older glucose broth cul Vase fluids with additions from shaken cultures tures which were inoculated with washed cells of F. oxysporum-07 caused the most significant obtained from a pectin-containing growth effects on the vase life of the Rosa flowers com medium, pectinase activity was positive (B. sub pared with the other microbial strains tested tilis, Ps fluorescens, F. oxysporum, Bot. cinerea) (see Plates 2 and 3 facing p. 371). Besides xylem or variable (Ps putida) (Put & Rombouts 1989). vessel occlusions and the consequences thereof Thus microbial pectinase could not have played on the water relations of the roses, the flower a role in the vase life of the roses. bud development was accelerated significantly, compared with control roses (Fig. 4a-c). This phenomenon might have been initiated by ethylene production by the Fusarium strain or BEETROOT TISSUE CUBE TEST elicited thereby (Albersheim et al. 1977). It is The tissue-permeability damaging reaction of not yet clear whether low molecular weight supernatant fluids of the shaken cultures tested compounds collected in the filtrate might against cubes of beetroot tissue was variable have played a certain role, not only in the (Ps. putida) or strongly positive (remaining 134 Ilff §1* 8 •- 'S « -5 M « .>° Î .a s 1 2 I as .s 72 Ä " S «> ^ 8n °3 2•» S 35 VS S « . • o a O KWWWWWWj » KWWN X * « c« J- g M t*VVV\\\\\VWM •a's*s « * ^ T5S I _L _|_ _L _L _L 2 s O) CO t^ ID m "d" rO CM — -- - ä° « (0861 |S|og>)jaa) 6uiuado-jaMO|j jo S&DJS (P ) 8ÜI SSDA 'S » tS ï ö * £ E a S 3 S «5 « s e -S s ^ - o S 2 8 g3 1 ilÊ88.|8 S S " " ^. ° e 3 0.2-Ä8 I BEafsfS o g-SÄ SJ=3 Sg£-3§ *- c -a ~ a.v a .o ^ fi « *s <« .5 in o 3 gS »w « S (p/|iu) pinii asDA jo 3HD4dn •g*s sa-s g s 3V .a'S Sre " -A ëfi» ~.SS ^ - Sj- *s O « 3 « £ g ï 3 . 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C. Put and W. Klop strains). After molecular filtration of these cul a part of the xylem vessels by amorphous tures the retentate was shown to give a some material. Diluted retentates also showed, what stronger beetroot-damaging reaction. The although to a lesser extent, similar aberrations, filtrates, however, were beetroot negative (Ps. compared with cross-section images of the putida), weakly positive (Bot. cinerea), positive control roses placed in sterilized tap water. In (B. subtilis) or strongly positive (Ps. fluorescens addition it was found that the F. oxysporum and F. oxysporum) in their ability to damage retentate injured and partly deformed (Figs 6 beetroot tissue. Static culture supernatant fluids and 7) the vessel structure of the Rosa stems. almost always showed no effects on beetroot In non-diluted filtrates of B. subtilis or Ps. fluo tissue permeability. rescens, the rose stem vessels showed no or only slight morphological deviation. Fusarium oxy sporum filtrate, however, caused heavy disorga ACID FUCHSIN TEST nization of the vascular structure, even when present at lower concentrations. EPS-containing culture retentate The greater the disturbance of the water rela tions of the roses, the greater was the reduction Discussion in the vase life of the roses, and the ability of the fuchsin red to infiltrate into the Rosa xylem THEROL EO FSUCROS EI NROS A VASE vessels was also markedly reduced, to values WATER mainly from 1-2 mm up to 2-3 cm upwards into the vessels. Roses placed in diluted reten- In commercial practice, 2-10 d may elapse tates were equally blocked (or to a somewhat between the harvest of the flowers and the com lesser extent compared with the non-diluted mencement of their ornamental vase life. During retentate) although a few vessels remained open, the pre-ornamental phase, plant sucrose is used as observed by their red coloration. Further by the cut flower as a source for respiratory dilutions showed a decreased vessel blockage: energy, because complete flower development is the dye penetrated ca 12-25 cm up the stem. energy-requiring. Sucrose interaction with plant hormones, i.e. ethylene, abscisic acid and cyto kines, moderates the programmed flowering Culture filtrate process, and sugars added to vase water con When culture filtrates were present in the vase tribute towards the osmotic adjustment of the water a minor (with F. oxysporum) or no (with developing flowers (Halevy & Mayak 1979) as the remaining strains) vessel blockage was well as to cell metabolism (Paulin et al. 1981 observed, even when the filtrate was shown to and Paulin & Jamain 1982).Th e general protec be toxic to roses. tive effect of sugar on membrane integrity also maintains mitochondrial structure and function (Halevy & Mayak 1981). An exogenous supple CONTROL ment of sucrose (or glucose) during the post- Roses placed in sterilized tap water, or sucrose harvest life of cut flowers such as Rosa is thus 1% w/v or filter-sterilized mashed Rosa stem advantageous to obtain mature flowers. Fur suspension (0-1% w/v) showed no vessel block thermore, sucrose plays an important role in the age and a fuchsin red coloration of xylem uptake, transport and storage of carbohydrates vessels due to fuchsin uptake was observed even in leaves and petals of cut Rosa flowers in the flower petals and the leaves. (summarized diagrammatically in Fig. 8). Exter nally applied sugars are first accumulated in rose leaves and only then translocated from the SCANNING ELECTRON MICROSCOPE leaves to the flowers (Paulin 1980). Normally OBSERVATIONS relative high concentrations of chemical solu tions are used for pulsing cut flowers, interme When F. oxysporum-01 retentate was present in diate for bud-opening, and low concentrations the vase water, at one cm upwards from the cut for holding solutions (Halevy & Mayak 1981). surface of the rose stems, the SEM images of the The supplied sugar may also reduce naturally cross sections showed occlusion and blocking of 140 Microbial exopolysaccharides in vase water Fig. 6. Rosa cv. 'Sonia', held in sterilized tap water 1d . (a) Longitudinal section from near the cutting point. Bar represent 100 /an. (b) Transverse section from near (ca. 1 cm) the cut surface. Bar represents 10 /an. (c) Detail showing pit openings in a xylem vessel. Bar represents 10pm. (SEM microphotographs, TFDL, Wageningen, The Netherlands. 141 Henriette M. C. Put and W. Klop Y'iy- • \f ;l s|.;/j», . », f ir'î!,,* ^i„..» .,.. :h. -.,.-5' • .<> .V-. •t •asfe^^^Jffiffl^« Fig. 7. Rosa cv. 'Sonia' after 1d in vase fluid containing some of the filter retentate of a culture supernatant fluid F. oxysporum-01, diluted 10' '. (a) Transverse section ca 10 mm from the cut surface of the stem. Bar represents 100 jim. (b) Detail of (a). Bar represents 10/an . (c) Longitudinal section of the stem, near the point of cutting. Bar represents 100/i m (SEM microphotographs, TDL, Wageningen, The Netherlands). 142 Microbial exopolysaccharides in vase water EPS-producing micro-organisms. A12(S04)3, 50-100 fig/ml, has been used in many preserv ative formulation for pulsing and holding solu tions for cut roses. It contributes to a lowering of the pH, but does not generally contribute to a lowering of the microbial activity in the vase fluid to below an acceptable level. Ag- thiosulphate (STS) is a more effective germicide. It acts as inhibitor of ethylene production of many cut flower cultivar, but is not successful for pulsing of roses (Halevy & Mayak 1979; Veen 1986, 1987). In our experiments the roses were tested under strictly hygienic conditions within 1-2 d after the harvest. Therefore, the carbohydrate reserve of the cut flower may have been optimal and not dependent on exogenous sucrose sup plementation for flowering. POLYSACCHARIDE FORMATION Among the different kinds of bacterial exo- Fig.8 . Diagram representing (semiquantitatively by polysaccharides constituting the EPS of cap means of arrow thickness) carbohydrate transport in sules or slime are ß(2 -> 6)-linked poly-D- thefloral branc h of'Carina'-ros e supplied with asolu tion of glucose or sucrose (G = glucose; F = fructose; fructans (levans). In bacterial cultures they may S = sucrose),afte r Paulin etal. (1981).Whe n the floral be synthesized from the fructose moiety of branch of a rose was supplied with a solution of 016 sucrose, present in the medium. Reports of mol/1 glucose or 008 mol/1 sucrose the level of levan-forming Bacillus and Pseudomonas species C(arbon) rose at the same rate in both cases.Glucos e are mentioned by Sutherland (1977, 1979) and was immediately transformed into sucrose in the stem axis. Sucrose migrated to the leaves and flower and by Powell (1979). Most of the organisms used in was equally distributed between the ovary and the our experiments are known commonly to form petals. In the flower, sucrose is immediately hydro- exopolysaccharides (possibly levans) (Wilkinson lysed, in the leaves its hydrolysis occurs more slowly. 1958,1977; Gorin & Spencer 1968;Gova n et al. A strong isomeraseactivit y occurred in thepetals . 1979; Powell 1979; Anderson 1984; Marques et al 1986;Neuman n 1987). The culture liquids of our test organisms con occurring starch hydrolysis and lipid degrada tained a high proportion of saccharides (other tion in cut Rosa stems held in water (Molnar & than glucose, fructose or sucrose) with an Parups 1977, Paulin 1980). The first post- average degree of polymerization above 2. harvest addition of sucrose is mainly given in Although nothing is known about the distribu the greenhouse directly after cutting when the tion of monomers, oligomers and polymers in a roses are placed in basins with circulating water, saccharide solution with DP' = 2, (as an but other metabolic sugars like glucose and example), two distribution limits are possible in fructose are similarly effective (Halevy & Mayak such a solution: (1) The whole fraction consists 1981, Paulin et al. 1981, Paulin & Jamain 1982). of dimers (other than sucrose); (2) the anhy- In these basins the first contact of the cut drohexose units are equally distributed between flower with microbial EPS or EPS-producing monomers and high polymers. Moreover, some micro-organisms may also occur. culture liquids with a DP' above 2 were highly Further handling of the Rosa flowers (at the viscous, and may be interpreted as indicating point of sale, during transport, storage and at exopolysaccharide synthesis from sucrose. the retail market), involves further opportunities In our experiments the DP value as well as for the flowers to contact microbial EPS and the quantity of EPS produced, was found to 14.1 Henriette M. C. Put and W. Klop depend on the microbial species, the microbial Moreover, cytochemical studies show that not strain and the physiological state of the culture only microbial EPS but also other plant-derived used. The quantity of EPS in most shaken cul carbohydrates such as pectins material may be tures was so high that even retentate dilutions coloured by ruthenium red (Parups & Molnar of 10"2 used as vase fluid caused blockage of 1972). Rosa xylem vessels, as is shown in Figs 1-5, Plate 2. EFFECTS OF THE INFILTRATION OF MICROBIAL COMPOUNDS ON XYLEM CELL WALLS THE INFLUENCE OF MICROBIAL EPS ON THE WATER RELATIONS OF ROSES It is noteworthy that cytotoxic polysaccharides may be released by partial hydrolysis of plant The greater the microbial conversion of sucrose cell walls by enzymes. Thus, in addition to into polysaccharides, the more disturbed were diverting xylem flow, xylem-borne polymers the water relations of the roses placed in the might directly induce senescence of leaf cells EPS-containing fluids. EPS taken up with the (Neumann 1987). The apparent absence of sap stream by cut Rosa flowers, does block visible xylem blockage by plant polysaccharides the open xylem vessels at the base. The smaller reported by Van Alphen & Turner (1975) and the DP' value or the lower the EPS concentra Neumann (1987) suggests that complex hor tion (Table 1), the higher did the EPS infiltrate monal regulation may affect polysaccharide upwards into the open xylem vessels. The concentrations in specific sections of the xylem microbial EPS produced could not have passed system. To reveal the occurrence of this pheno the capillary holes in the pit membranes (0-02- menon, further research is needed (Halverson & 0-10 um diameter) nor would it cause mechani Stacey 1986). cal rupture of the membranes (Van Alfen & Allard-Turner 1979; Halevy & Mayak 1981; Neumann 1987). The highest point which could THE EFFECTS UPON ROSA VASE LIFE OF have been reached by infiltration of EPS up the LOW MOLECULAR WEIGHT MICROBIAL stems was shown to be the longest open vessel COMPOUNDS IN VASE FLUIDS (ca 25 cm according to de Stigter & Broekhuy- Elicitation of a significantly decreased vase life sen 1986). (accelerated bud opening) of Sonia roses was It is not clear, however, if microbial EPS infil caused by the uptake of culture filtrates of B. trated into partially blocked Rosa xylem vessels; subtilis-207; Bot. cinerea-196 and F. oxysporum- it might have blocked the pit membranes in 07. Although no physical plugging of xylem these vessels and thus also inhibited the sap vessels was found, the water conductivity stream (Zimmermann 1983). decreased significantly. By cutting-off ca 15- In addition, it was observed that EPS- 25c m of the stem, no reversal of visual symp containing culture retentates of Bacillus-214 and toms occurred if the roses were replaced in PseudomonasAS & 52 disturbed to a consider sterilized tap water. Hence eventual low molecu able extent the vase life of Chrysanthemum cv. lar weight toxic microbial compounds in the 'Daymark' and Gerbera cv. 'Rebecca' (results vase fluid may have been flushed up in the sap not reported). stream through the pit membranes. LOCALISATION OF XYLEM OCCLUSIONS CONSEQUENCES OF MICROBIAL COMPOUNDS IN VASE FLUIDS FOR THE With the acid fuchsin test, xylem occlusions POSTHARVEST HANDLING OF CUT ROSES were easily rendered visible to the naked eye. This, however, does not give any information Many of the micro-organisms coming into about the nature and chemical structure of the contact with cut flowers and their vase water occluding material. are able to excrete high molecular weight com Cytochemical staining methods such as the pounds (exopolysaccharides) and also low use of aqueous ruthenium red (0-02% w/v) molecular weight toxins (or toxoids) which can involve microscopic examination (Peresse 1974; adversely affect the longevity of the flowers Rasmussen & Carpenter 1974; Roth 1977). (Accati 1980; Accati-Garibaldi 1983). Thus 144 Microbial exopolysaccharides in vase water contact of cut flowers with micro-organisms GORIN, P.A.J. & SPENCER, J.F.T. 1968 Structural should be prevented, and conditions shouldb e chemistry of fungal polysaccharides. Advances in such that microbial growth isminimal . Carbohydrate Chemistry 23, 367-417. GOVAN, J.R.W., FYFE, J.A.M. & MCMILLAR, C. 1979 The instability of mucoid Pseudomonas aeruginosa : The authors wish to thank Dr M.E. Rhodes- Fluctuation test and improved stability of the Roberts (University of Wales, Aberystwyth) for mucoid form in shaken culture. Journal of General reading andcommentin g on the manuscript. Microbiology 110,229-232. The cut flower project was supported, inpart , HALEVY, A.H. & MAYAK, S. 1979 Senescence and postharvest physiology of cut flowers. Part 1.I n by the technical direction of Thomassen & Horticultural Reviews, Vol. 1, ed. Janick, J. pp. 204- Drijver-Verblifa N.V. Packaging Industry, 236. Westport, Connecticut, USA: AVI. Deventer, The Netherlands and by secondment HALEVY, A.H. & MAYAK, S. 1981 Senescence and (1984-1987) of the author H.M.C.P. atth e postharvest physiology of cut flowers. Part 2.I n Horticultural Reviews Vol. 3, ed. Janick, J. pp. Sprenger Institute, Wageningen. Theauthor s 59-143. Westport, Connecticut, USA: AVI. acknowledge thetechnica l assistance of John HALVERSON, L.J. & STACEY, G. 1986 Signal exchange Bosch,Ank eClerkx ,An sva n Hardeveld, Hanna in plant-microbe interactions. Microbiological Lagerwerf, Ton van derMeijde n and Willem Reviews 50, 193-225. Spekking. HODGSON, R., PETERSON, W.H. & RIKER, A. 1949 The toxicity of polysaccharides and other large mol ecules to tomato cuttings. Phytopathology 39, 47-62. MARQUES, A.M., ESTANOL, I., ALSINA, J.M., FUSTE, C, References SIMON-PUJOL, D., GUINEA, J. & COUGREGADO, F. 1986 Production andrheologica l properties of the AARTS, J.F.T. 1957O n the keeping quality ofcu t extracellular polysaccharide synthesized by Pseudo flowers. Mededelingen vande Landbouw Hogeschool monas sp. strain EPS-5028. Applied and Environ Wageningen 54, 1-62. mental Microbiology 52, 1221-1223. AccATi, E. 1980 The role of bacterial metabolites) in MOLNAR, J.M. & PARUPS, E.V. 1977 A histochemical affecting water uptake by carnation flowers. Acta study of starch, lipids andcertai n enzymes in sene- Horticulturae 113, 137-143. scing rose stems. Canadian Journal of Botany 55, ACCATI GARIBALDI, E. 1983 Post-harvest physiology 617-624. of mediterranean carnations: partial character NEUMANN, P.M. 1987 Sequential leaf senescencean d ization of a bacterial metabolite inducing wilt of correlative controlled increase in xylem flow resist flowers. Acta Horticulturae, 138, 255-260. ance. Plant Physiology 83, 941-944. ALBERSHEIM, P., AYERS, A.R., VALENT, B.S., EBEL, J., PARUPS, E.V. & MOLNAR, J.M. 1972 Histochemical HAHN, M., WOLPERT, J. & CARLSON, R. 1977 Plants study of xylem blockage in cut roses. Journal of the interact with microbial polysaccharides. Journal of American Society for Horticultural Science 97, 532- Supramolecular Structure 6, 599-616. 534. ANDERSON, A.J. 1984 Differences between lipopolysac- PAULIN, A. 1980 Influence d'une alimentation con charide compositions of plant pathogenic and sap tinue avec une solution glucosée surl'évolutio n des rophytic Pseudomonas species. Applied and glucides et des protéines dans les divers organes de Environmental Microbiology 48, 31-35. la rose coupée (Rosa hybrida cv. Carina). Physiol- BERKHOLST, C.E.M. 1980 The water relations of cut ogia Plantarum 49, 55-61. roses. Bedrijfsontwikkeling 11, 331-336. PAULIN, A., SOUTER, D. & BUREAU, J.M. 1981 Evolu CHIN, C.& SAKALIS, J.M. 1977 Metabolism of sucrose tion comparée des glucides dans lesdiver s organes in cutroses . II.Movemen t and inversion of sucrose de la rose coupée alimentée temporairement avec absorbed bycu t rose stems. Journal of the American une solution glucose ou de saccharose. Physiologie Society for Horticultural Science 102, 537-540. Végétale 19, 59-76. COREY, R.R.& STARR, M.P. 1957 Colony types of PAULIN, A. & JAMAIN, C. 1982 Development of flowers Xanthomonas phaseole. Journal of Bacteriology 74, and changes in various sugars during opening in cut 137-140. carnations. Journal of the American Society for Hor DE STIGTER, H.C.M. & BROEKHUYSEN, A.G.M. 1986 ticultural Science 107,258-261. Experimentally induced plugging of cut-rose xylem PERESSE, M. 1974 Modifications cytologiques dans le by particulate ormacromolecula r matter. Acta Hor xylème de tiges d'oeillets expérimentalement infec ticulturae 181, 365-370. tées, au cours de l'installation du Phialophora cin- DUBOIS, M., GILLES, K.A., HAMILTON, J.K. & SMITH, erescens (Wr) van Beyma. Phytopathologisches F. 1956 Colorimetric method for determination of Zeitschrift 79, 35^46. sugars and related substances. Analytical Chemistry POWELL, D.A. 1979 Structure, solution properties and 28, 350-356. biological interactions ofsom e microbial extracellu DURKIN, J. 1979 Effect of Millipore filtration, citric lar polysaccharides. In: Microbial Polysaccharides acid and sucrose on peduncle water potential of cut and Polysaccharases ed. Berkeley, R.C.W., Gooday, rose flowers. Journal of the American Society for G.W. & Ellwood, D.C. pp. 117-160. London: Aca Horticultural Sciences 104, 860-863. demic Press. 145 Henriette M.C. Put and W. Klop PUT, H.M.C. 1986 Investigations into the influence of saccharides: control of synthesis and acetylation. In the microflora from stems of cut flowers on the vase Microbial Polysaccharides and Polysaccharasesed . life of Rosa cv. 'Sonia', Gerbera cv. 'Fleur',an d Berkeley, R.C.W., Gooday, G.W. &Ellwood , D.C. Chrysanthemum cv. 'Spider'. Acta Horticulturae 181, pp. 1-34. London: Academic Press. 415^18. TIMELL, T.E. 1960 Determination of the degree of PUT, H.M.C. & CLERKX, A.C.M. 1988 The infiltration polymerization ofreducin g pentose and hexose oli ability of micro-organisms Bacillus,Fusarium, Kluy- gosaccharides. Svensk Papperstidning 63,668-671. veromyces,an d Pseudomonasspp . into xylem vessels VAN ALFEN, N.K. & ALLARD-TURNER, W. 1979 Sus of Gerbera cv. 'Fleur' and Rosa cv. 'Sonia'cu t ceptibility of plants to vascular disruption by flowers: a scanning electron microscope study. macromolecules. Plant Physiology 63, 1072-1075. Journal of Applied Bacteriology, 64, 515-530. VAN ALFEN, N.K. & TURNER, W. 1975 Changes in Pur, H.M.C. &JANSEN , H.W. 1989 The influenceo n alfalfa stem conductance induced by Corynebacter- the vase life of cut Rosa cv. Sonia of bacteria added iuminsidiosum toxin . Plant Physiology 55, 559-561. to the vase water. Scientia Horticulturae 39, 167— VAN ALFEN, N.K., MCMILLAN, B.D., TURNER, V. & 179. HESS, W.M. 1983 Role of pit membranes in PUT, H.M.C. & ROMBOUTS, F.M. 1989 The influence macromolecule-induced wilt ofplants . Plant Physi of purified microbial pectic enzymes onth e xylem ology 73, 1020-1023. anatomy, water uptake and vase life of Rosacv . VEEN, H. 1986. Atheoretica l model for anti-ethylene Sonia. Scientia Horticulturae 38, 147-160. effects of silver-thiosulphate and 2,5-norbornadiene. PUT, H.M.C. &VA N DER MEUDEN, T. 1988 The infil Acta Horticulturae 181,129-134. tration ability of Pseudomonas putida cells strain 48 VEEN, H. 1987 Use of inhibitors of ethylene action. into xylem vessels of cut Rosa cv. 'Sonia'. Journal of Acta Horticulturae 201,213-222 . Applied Bacteriology 64,197-208. WIERINGA, K.T. 1956 The micro-organisms decompo RASMUSSEN, H.P. & CARPENTER, W.J. 1974 Changes in sing pectic substances in the dew retting process of vascular morphology of cut Rosa stems: A scanning the flax. Netherlands Journal of Agricultural Science electron microscope study. Journal of the American 4,204-209. Societyfor Horticultural Science 99, 454—459. WILKINSON, J.F. 1958 The extracellular poly ROTH, I.L. 1977 Physical structure of surface carbo saccharides ofbacteria . Bacteriological Reviews 22, hydrates. In Surface Carbohydrates of the Pro- 46-73. caryotic Cell ed. Sutherland, I.W. pp. 5-26. London: WILKINSON, S.G. 1977 Composition and structureo f Academic Press. bacterial lipopolysaccharides. In: Surface Carbo SUTHERLAND, I.W. 1977 Bacterial exopolysaccharides, hydrates of the Prokaryotic Cell ed. Sutherland, I. their nature and production. In Surface Carbo pp. 97-176. London: Academic Press. hydrates of the Prokaryotic Cell ed. Sutherland, I.W. ZIMMERMANN, M.H. 1983 Xylem Structure and the pp. 27-96. London:Academi c Press. Ascent ofSap, Series in wood science, ed.Timell , SUTHERLAND, I.W. 1979 Microbial exopoly T.E. 254 pp. Berlin: Springer Verlag. 146 Chapter 7 Theinfluenc e of purified microbial pecticenzyme s on the xylem anatomy, water uptake and vaselif e of Rosacultiva r 'Sonia'. 147 Rosacv .'Sonia' :stag e7 o fflowe ropenin g (Berkholst,1980 ) 148 The Influence of Purified Microbial Pectic Enzymes on the Xylem Anatomy, Water Uptake and Vase Life of Rosa cultivar *Sonia ' HENRIETTE M.C.PUT 1*an d FRANK M. ROMBOUTS2 Sprenger Institute, Ministry ofAgriculture and Fisheries, P.O.Box 17, 6700AA Wageningen (The Netherlands) agricultural University, Department ofFood Science, De Dreijen 12,6703 BC Wageningen (The Netherlands) (Accepted for publication 20Jun e 1988) ABSTRACT Put, H.M.C. and Rombouts, F.M., 1989.Th e influence of purified microbial pectic enzymes on the xylem anatomy, water uptake and vase life of Rosa cultivar 'Sonia'. Scientia Hortic., 38: 147-160. Thispape r describes macroscopican d microscopic symptoms following the addition of purified pectic enzymes (pectate lyase of Pseudomonas fluorescens and polygalacturonase of Kluyvero- mycesfragilis ) t o the vasewate r containing cut flowers ofRosa cultivar 'Sonia'. These symptoms were: decreased water uptake; slightly decreased water conductivity after 24 h and 48 h of vase life; decreased flower-bud development; a deep red colouration of desiccated flower petal edges; chlorotic spots on the leaves,whic h became necrotic in the center ofth e spots.Scannin g electron microscope (SEM) observations ofth e stem xylem vessel system after 40 h vase life showed: (1) degradation ofwal lstructure s causingdisorde r and looseparticle s in the vessels; (2) injury ofth e vesselpi t structure; (3) release d spiralvessels .Thi spromote d desiccation ofth eflower petal s and leaves, but not vessel blockage or wilting. The structural abnormalities apparently were not the reason for the reduced water uptake caused by plugging of the xylem vessels, as was shown by water conductivity experiments after 24an d 48h of vase life. Keywords:cu tflowers; pecti c enzymes;roses ;vas e life; xylem vessel structure degradation. Abbreviations: M=molar (mol 1_1); mol.wt.=molecular weight; RH= relativ e humidity; SEM= scannin g electron microscope;U=units ; W=watt. INTRODUCTION Microbial contamination and microbial products have been implicated as causes of reduced vase life ofcu t flowers (Aarts, 1957;Mastalerz , 1977;Accat i et al., 1980) . However,vascula r pluggingals o mayhav ebee n causedb y cutting *Present address:Kerkstee g 4,741 1E W Deventer, The Netherlands. 149 injury, enzymatic activity or endogenous ethylene formation (Abelis, 1973; Fujino et al., 1983;Vandermole n et al., 1983).I n studying the influence of the microflora on the stems of cut flowers on the vase life ofRosa 'Sonia', Gerbera 'Fleur' and Chrysanthemum 'Spider' (Put , 1986) ,i twa sobserve dtha t not only microbial occlusions,bu t alsomicrobia lproduct s (e.g.enzymes) ,ma y decrease the vase life of cut flowers, which must receive a minimal amount of water (and carbohydrates) to remain alive and turgid (Conrado et al., 1980). A major factor contributing to the rapid senescence of cut Rosa flowers ap pears to be the blockage of water-conducting vessels of the xylem, which ac counts for the lack of turgor in the flower pedicel, causing "bent neck" and wilting of petals (Parups and Molnar, 1972; Halevy and Mayak, 1981). His- tochemical studies of xylemblockag e incu t roses furthermore showedtha t the occluding material stained positively with ruthenium red, indicating pectina- ceous material and other high mol.wt. polysaccharides (Lineberger and Ste- ponkus, 1976; van Alfen and Allard-Turner, 1979; Vandermolen et al., 1983; Zimmermann, 1983;Neumann , 1987). The major cell-wall constituent of herbaceous plants which influences the water-holdingcapacit y ofth e intactplan t ispecti n (Domsch and Gams,196 9) . A number of pectolytic enzymes are responsible for the process of pectin deg radation (Codner, 1971), and soil is a large reservoir of pectolytic microorga nisms; > 106pectolyti c microorganisms g_1 soil can be counted. The phyllos- phere ofhealth y plants also harbours awid evariet y ofpectolyti cbacteri a with a preponderance ofPseudomonas, Bacillus andFlavobacterium and the rhizos- phere also contains abundant aerobic pectolytic microorganisms (Rombouts and Pilnik, 1980). It is not surprising that many plant pathogens secrete pectin-degrading en zymes because the cell wall is a barrier which must be breached by most mi crobial pathogens (Domsch et al., 1980). It is possible that these pectin-de grading enzymes may activate host enzymes that release phytoalexins, thus acting as an endogenous elicitor (Halverson and Stacey, 1986). Phytoalexin accumulation can also occur in the absence of exogenous materials when the tissuei swounde do rwhe n someo fth e cellsar ekilled ,e.g .b ycuttin go rpruning . Toprov etha t anyenzym eproduce db ya specifi c microorganism really causes the disease of the plant or decreases the vase life of cut flowers, and that this is not due solely to chance contaminant(s) , it was thought to be desirable to testth e influence ofsingl epurifie d enzymes.Moreover ,b yth e addition ofpuri fied pectic enzymes to vase water, the side effects of microbial products other than pectic enzymes can be avoided and therefore it is possible to study spe cifically the effect of added pectic enzymes on the vase life of cut flowers. The resultso fa serieso fexperiment s withpurifie d microbial pectic enzymes added to the vase water containing cut Rosa flowers are given in this paper. 150 MATERIALS AND METHODS Plant material. - Rosa hybrida 'Sonia' flowers were supplied by local green house growers. The roses were harvested between 15 February and 15 March 1986 at flowering Stage 1-2 (Berkholst, 1980), wrapped in parchment paper, transported to the laboratory, stored at 4°C for 24-48 h and the experiments then carried out. Pectic enzymes. - The enzymes tested were: pectate lyase from Pseudomonas fluorescens and polygalacturonase from Kluyveromyces fragilis (cf. Rombouts and Pilnik, 1980). These enzymes were produced and purified according to Rombouts et al. (1978) and Phaff (1966), respectively. The enzyme prepara tions contained: 50 U ml-1 of pectate lyase and 20U ml-1 of polygalacturon ase, measured by the methods of Rombouts et al. (1978) and Phaff (1966), respectively. The pectate lyase was diluted in 1/80 M sterile Tris-HCl buffer at pH 7.5.Th e polygalacturonase was diluted in 1/80 M sterile citrate buffer atp H 4.5.Hea t sterilized non-chlorinated Ca2+-containingta pwate r wasuse d for preparation of the buffered dilutions. Vasewater. - Dilutions containing: 0 (control), 1,0.5 , 0.1,0.0 5 U of buffered pectate lyase ml-1 of sterile vase water and 1, 0.5, 0.1,0.0 5 U of buffered po lygalacturonase ml-1 of sterile vase water wereprepare d under aseptic condi tions. Freshly-harvested 'Sonia' roses were cut off to a stem length of 45 cm and put singly into cylindrical vases calibrated up to 100ml ,containin g 40 ml of sterile vase water. Each of the dilutions were at least tested in triplicate. The vaseswer e held at 20± 1°C ,6 0± 2% RH (relative humidity) and 10.25W m-2 photoradiation at flower height during a 12-hphotoperio d each 24h . Pro tection against air contamination was obtained by sealinga plasti c film on the top of each vase (Parafilm "M", American Can Cy.). Vaselife. - Every 24 h observations were made on the water uptake (ml), the floweringstag e (Fig.1 ;Berkholst , 1980) an d the ornamental valueo fth e roses. days of development 20°C 8 stages of flower opening 1cmJ 1suprem e bloom Fig. 1.Stage s in the flower openingan d development of ornamental value ofRosa cultivars (Ber kholst, 1980) 151 When the ornamental acceptability of the flower in the vase was visually de creasedt o < 50% ofth e "normal" flower-bud development in sterilevas e water, the ornamental value was negative. Negative features in ornamental value are considered to be:wilting , drying, curling and discolouration of leaves and pet als;ben t neck;chloroti c and necrotic spotso nth e leaves;retardatio n offlower- bud development. After 24 and 48 h the water conductivity of the stems was measured according to the method of Chin and Sacalis (197 7) . The apparatus usedconsiste do fa n overhead 10-1bottl e filled with sterileglass-distille d water, connected by medical plastic tubing to a glass-manifold of 2X1 2 pieees (Fig. 2) . AMariott e tube was inserted through a rubber stopper into the 10-1bottl e to establish constant water pressure. The distance or height of the water col umn measured from the base ofth e Mariotte tube toth e upper end ofth e stem segment was 60cm .A piec e of soft plastic tubingwa sattache d toeac h T-piece for connection to a single stem segment. 60 cm \J\J\J\J\J\J\JKJ\J\JVjyj Fig. 2. Apparatus designed to measure xylem conductivity (Chin and Sacalis, 1977). (a) Bottle containing 101steril e glass-distilled water; (b) rubber stopper; (c) Mariotte tube; (d) baseo f the Mariotte tube; (e) upper end of the (upside down) .inserted stem segment of 5 cm length; (f) plastictub eo fknow nweigh tt ocollec twate rflowin gthroug hth e stemsegment ; (g) glass manifold with 12connector s (h) of soft medical plastic tubing. 152 TABLE 1 The influence ofth e addition ofpurifie d polygalacturonase in buffered vase water on (1) stageo f flower development and (2) ornamental value of cut flowers of'Sonia'; + =normal; ±=accept- able; -=unacceptable (see Fig. 1) Days ofvas e life 0 1 2 3 4 5 6 7 8 9 Control: citrate buffer 1/80 Mp H 4.5 (1) 1 2 3 5 6 6 7 7 8 8 (2) + + + + + + + + + Plus polygalacturonase lUml"1 (1) 1 2 3 4 4 4 (2) + + ± + - - 0.5 U ml"1 (1) 1 2 3 4 4 4 (2) + + ± + + - 0.1 U ml"1 (1) 1 2 3 4 4 4 5 5 (2) + + + + + + ± - Values are means of 6replication s and are rounded off. Significant differences of flower develop ment and vaselif e areassociate dwit h addition ofpolygalacturonase : 1 U,0. 5U an d —0. 1 U ml~l vase water (P<0.05). Triplicate samples of roses were taken from the vase water after 24 and 48 h vase life. Immediately thereafter 3 consecutive stem sections of 5 cm each were cut off from the basal end upwards and inserted onto the manifold in an upside down position. The water solution was allowedt o flow through the seg ments (i.e.th e natural direction) fo r 60mi n topurg e air inth e linesan d obtain an equilibrated water flow. Thereupon the water was collected in plastic tubes ofknow n weight.Th e water flow through the segments wasassesse db yweigh ingth e tubes after 30,6 0an d 90mi n ofwate r flow. The average water conduc tivity 30min -1 wasthe n calculated (Durkin, 1979).Conductivit ywa s defined as the reciprocal value to the resistance to flow, expressed per transverse- sectional area-1 of 5-cm stem length (Burdett, 1970;Gilma n and Steponkus, 1972;Pu t and van der Meijden, 1988). Microscopicand macroscopic observations. - After 40 h vase life SEM prepa rations of transverse and longitudinal sections of the stem and xylem vessels weremad eb yth e Technical and Physical Engineering Research Service, Man- sholtlaan 12,Wageningen , The Netherlands (Dr. Ir. A. Boekestein and Anke Clerkx). Statistical treatment ofdata. - Separation of mean valueswa sdon eb y analysis ofvarianc e (ANOVA) followed bya tes tfo r least-significant differences (LSD) at a probability level of P< 0.05 . A computer programme of the Lawes Agri- 153 VASE LIFE (DAYS) OF 'SONIA' Fig.3 .Th e influence of the addition ofpurifie d pectate lyase to buffered vase water on the flower openingo f cut roses.Vas ewater : o, 1/8 0 M Tris buffer at pH 7.5; •, pluspectat e lyase lUml-1; A,plu spectat e lyase 0.5U ml-1; A,plu s pectate lyase 0.1 U ml-1; *,en d ofth e vase life. cultural Trust (Rothamsted Experimental Station, Herts,England ) was used, carried out by the Statistical Department of the Sprenger Institute, Wagen ingen, The Netherlands. RESULTS Vaselife observations. - The vase life of the control roses was 9± 1days . After the addition of 1an d 0.5 U ml-1 of buffered (pH 7.5) pectase lyase or 1.0 or 0.5 U ml-1 of buffered (pH 4.5) polygalacturonase to the vase water, the vase lifewa sdecrease d to 3± 1 days,whil e 0.1U ml~ x ofeithe r pectic enzyme added separately to the vasewate r resulted in a decreased vase life of 6± 1day s (Ta ble 1,Fig .3) . However, no effect was shown when 0.05 U ml-1 of either of the pectic enzymes was added to the vase water. Both pectic enzymes showed similar effects on the vase life of the roses, so detailed data concerningonl ypectat e lyasear epresente d here.Durin gth e first 36 h of the vase life no effects of the enzyme treatment were visible on the flowering stage of the roses, which developed from Stage 1+ to Stage 4. Also the water-uptake level (Fig. 4) and the water-conductivity values (Table 2) wereno t significantly lesscompare dwit h the controlvases .However ,afte r >2 days of vase life the uptake of water decreased compared with the control; at the sametim e the outer petals ofth e flowers showed signs of inadequate water 154 => 14. _l »O u 12 Z O £ H < M o- lo VASE LIFE (DAYS) OF 'SONIA' Fig.4 .Th e influence ofth e addition ofpurifie d pectate lyaset o buffered vasewate r on the uptake ofwate r by cut roses.Vas ewater : o, 1/80 MTri s buffer at pH 7.5; •, pluspectat e lyase 1 U ml-1; A,plu spectat e lyase0. 5 U ml-1; A,plu s pectate lyase 0.1U ml-1; *,en d ofth e vase life. uptake by the curling, drying and dark-red colouring of the petal edges. In addition, the leaves showed water-deficiency symptoms, at first by chlorotic spots, which became larger and a few days later became partly necrotic (Fig. 5). Scanning electron microscope observations. - Transverse and longitudinal sec tions of control roses showed "clear" xylem vessels and normal pit structures (Figs. 6 and 7), but numerous loose vessel fragments (Fig. 8), loose spiral xylemvessel s (Fig.9 ) and injured pits (Fig.1 0) wer e seen in the xylem vessels of enzyme-treated cut roses after 40 h ofvas e life. DISCUSSION When purified pectolytic enzymes, viz. pectate lyase of Pseudomonas fluo- rescens and polygalacturonase of Kluyveromyces fragilis, were added to buff ered vase water of cut 'Sonia' roses,thei r water relations were disturbed. The causethereo f mayhav ebee n duet oenzymati cdegradatio no fth e pectin-linked structures ofth e xylemvessel sa sobserve d in SEMpreparations .Neithe r wilt ing nor "bent necks" of rose stems were evident, yet desiccation of petals and leaves and inhibition of flower-bud development occurred. 155 TABLE 2 Water conductivity (ml 30 min ~' ) of stems of 'Sonia' after 24 and 48 h of vase life in buffered vasewate r with 1U o fpecti c enzymes added ml-1 Vase water Vase life Distance from the ba se of (h) the stem of the stem segment tested (cm) Enzyme added Buffer 1/80M 0-5 5-10 10-15 (pH) Control: nopecti c enzyme 7.5 24 1.85 2.20 3.00 added 48 2.25 3.45 3.30 Plus pectate lyase: 1 U ml-1 7.5 24 2.25 1.60 2.30 48 0.90 1.65 1.50 Control:n o pectic enzyme 4.5 24 3.60 2.45 2.70 added 48 2.35 2.90 2.75 Pluspolygalacturonase : 1 U 4.5 24 3.25 2.30 2.60 ml"1 48 2.90 3.45 2.50 Control:sterilize d tap water. 7.2 24 2.80 3.90 3.11 Nopecti c enzyme added 48 2.50 3.10 3.35 Valuesar e means of 9replications . Asignifican t effect on the water conductivity isobserve d after addition ofpectat e lyase and 48h of vase life (P< 0.0 5) . It isconspicuou s that apronounce d ability to decompose pectin is in almost allpecti c enzyme-producing microorganisms linked with a high variability be tween different strains of the same microbial species (Rombouts and Pilnik, 1980) .Polygalacturonas e production by certain microbial strains (bacteria and fungi ) may be remarkably stimulated by maintaining the microorganism in a pectin-containing culture medium (Nyeste and Hollo, 1962; Verhoeff and Warren, 1972) ; othe r factors may also affect pectin enzyme production or ac tivity. Bacillus circulons,B. polymyxa, B.pumilis, B. sphericus, B. subtilis, and Pseudomonas fluorescens isolatedb yu sfro m flower stemsproduce d only slight amounts of endopectate lyase when grown in/on plain yeast extract media (Difco). In our experiments, pectic-enzyme production of these strains could greatly be stimulated by addition of pectin or pectate to the growth medium. Nevertheless, only cell-free culture liquids obtained from the post-stationary growth phase of these Bacillus strains showed a markedly increased endopec tate lyase activity. Pectic enzyme-producing fungi isolated from flower stems, on the contrary, maintained their enzyme activity, even after transfer into plain malt extract agar (Difco). The symptoms causedb y the addition ofpurifie d pectic enzymest o the vase watercontainin gcu tflower so f'Sonia 'differe d from thoseobserve dwhe n water- washed microorganisms or microbial products, e.g. polysaccharides, and phy- 156 Fig. 5. The influence of purified pectate lyase added to buffered vase water on the ornamental value ofcu t roses after 3day s ofvas e life. Pect IE=plus pectate lyase 1 U ml-1 in Tris buffer pH 7.5;p H 7.5= 1/80 M Tris buffer. totoxins, were suspended or mixed with vase water (van Alfen and Allard- Turner, 1979;Neumann , 1987; Put and van der Meijden, 1988). Washed mi crobial cells of Bacillus, Enterobacter, Kluyveromyces and Pseudomonas spp. addedt ovas ewate r wereshow n to infiltrate intoth e cut xylemvessel so froses ; this resulted in blockage of the water-transport system, decreased water up take, decreased water conductivity and "bent-neck" flower stems within 24- 48h vase life (Put and van der Meijden, 1988;Pu t and Jansen, 1988). Micro bial polysaccharides (mol.wt.> 10000 ) in the vase water caused a sudden ar rest of the flower development of the roses, "bent-neck" and total blockage of the water transport system within 24 h (H.M.C. Put and W. Klop, personal observations, 1988).However , it isno t yet clear whether the interaction of the microbialpecti c enzymes and theplan t cell-wall carbohydrates ofth e cut roses induced the release of plant cell-wall oligosaccharins, resulting in the elicita- tion of: (1) host defence responses as shown in many intact plant; (2) abnor malgrowth ; (3) morphogeneti c changesinduce db ydisturbe d growth hormone- regulating mechanisms (Albersheim and Valent, 1978;Halverso n and Stacey, 157 Fig.6 .Longitudina l section of xylemvessel sof'Sonia ' near the base ofth e stem. Cut flowers were held in sterile vase water for 40 h and the vessel structure was normal. Bar mark represents 100 /an. Fig. 7.Longitudina l section made at 5c m from the base of the stem of 'Sonia' cut flowers held in sterile vase water for 40 h. Anorma l xylem vessel pit structure is shown. Bar mark represents 10 /mi. 158 Fig. 8. Longitudinal section made at 2-10 mm distance from the base of the stem of 'Sonia' cut flowers held in a buffered sterile pectate lyase solution of 1U ml-1 for 40 h. The vessel structure issee n to bedegradated . Bar mark represents 100 /an. M 19W Fig. 9. Longitudinal section made at 2-10 mm distance from the base of the stem of 'Sonia' cut flowers heldi n abuffere d sterilepectat e lyasesolutio n of 1U ml-1 for 40h .Loos espira l fragments ofvessel s are evident. Bar mark represents 100 /an. 159 Fig. 10. Longitudinal section made at 10 mm distance from the base of the stem of 'Sonia' cut flowers heldi na buffere d sterilepectat elyas esolutio no f 1U ml~l for4 0h .Th exyle mpi t structure isdamage dan d manyloos evesse lfragment s andmucoi dmaterial sar eshown .Ba r mark represents 1 /an. 1986; Zagory and Reid, 1986). The many ways in which microorganisms, through their pectic enzymes, may interfere with plant xylem cells, are very complicated, and their mechanisms are far from clear (Rombouts and Pilnik, 1980;Billing , 1987). Further experiments are needed to reveal whether other purified microbial pectic enzymes cause similar symptoms during the vase life of Rosa and other cut flowers. ACKNOWLEDGEMENTS The authors wish to thank Dr. M.E. Rhodes-Roberts for reading and com menting on the manuscript. The cut flower project was supported, in part, by the technical director of Thomassen & Drijver-Verblifa N.V., packaging in dustry, Deventer, The Netherlands and by secondment (1984-1987) of the author (H.M.C. Put) at the Sprenger Institute, Wageningen. The authors ac knowledge the technical assistance of Lieke Jansen, Ans van Hardeveld and Peter Schrijver. 160 REFERENCES Aarts, J.F.T., 1957.O n the keepability of cut flowers. 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Zimmermann, M.H., 1983.Xyle mstructur e andth e ascento fsap .In :T.E .Timel l (Editor), Series in Wood Science. Springer, Berlin, 143pp . 162 Chapter 8 Theinfluenc e of A12(S04 ) 3adde d to thevas efluid o n the infiltration abilityo f Bacillussubtilis cellsint o xylemvessel s of cut Rosacv ."Sonia' :a scannin gelectro n microscope study. 163 The heart of a fully blooming Rosa (Photograph: Judith Tromp) 164 Chapter 8 Theinfluenc e of A12(S04)3 added to the vasefluid o n the infiltration ability ofBacillus subtiliscell sint o xylemvessel s of cut Rosacv . 'Sonia': ascannin gelectro n microscope study. Henriette M.C .Put* ) and W.Klop ,forme r Sprenger Institute, Ministry of Agriculture and Fisheries,P.O . Box 17,670 0 AAWageningen , TheNetherlands ;Ank eC . M. Clerkx and A.Boekestein , Department of Electron Microscopy, Technical and Physical Engineering Research Service, P.O.Bo x356 ,670 0A J Wageningen,Th eNetherlands . Submitted for publication in The Journalof Applied Bacteriology (Januaryh 1991) *) Corresponding author Present Adress, Kerksteeg 4, 7411 EW Deventer, The Netherlands 165 ABSTRACT Put.H.M.C. .Klop .W. .Clerkx .A.C.M .& Boekestein .A . 1991.Th eInfluenc e ofA1 2(S0,)3adde dt oth evas eflui do nth einfiltratio nabilit yo f Bacillis subtllis cells into xylem vessels of cut Rosa cv. 'Sonia': a scanning electronmicroscop estudy . TheJourna lo fApplie dBacteriolog y (submitted). 1 Aluminiumsulphate ,A1 2(S0«)3.18H20,50 0/i gml" adde dt odeionize dwate r(1. 5 mMAl 3+)result si na slightl yopalescen tsolutio nwit ha p Ho fapprox ,4 ,du e tohydrolysis .Th einsolubl eA1(0H) 3form sa glass ycolloida lsolutio na tp H 4.O n standing atroo m temperature,th eA1(0H) 3 flocculates tosmal lwhit e flocky deposit. The quantity of precipitated Al(0H)3 is increased by increasing of the pH from pH 4 - 5.5. Al3+ ions react easily with some inorganican dorgani canioni csubstance se.g .bacteria lcel lwal lpeptides , substanceso fdamage d stemcell san d stemxyle m cellwal lcompound so fcu t Rosa flowersimmerse dint ovas efluid scontainin gAl_ 3+an d Bacillus subcilis cells. The Al-compounds formed, aggregate and flocculate. The Bacillus/ aluminiumcompound scontribut et oa nincreas eo fflock ydeposit so nth ecu t surfacean dope nxyle mvessel so fth eroses ,preventin gth einfiltratio no f the Bacillus cellswhic hi sdemonstrate db ycryo-SE Mobservation san dX-ra y microanalyseso fpiece so fth este mtissue . The aluminium sulphate effect in Rosa vase fluid consists of three factors: (1)a nalmos tbiostati c (orver y weakbiocidal )activit y towards Bacillus subcilis cells; (2) preventing of Bacillus subtills cells to infiltrate(an dplug )int oope nxyle mvessels ;(3 )slightl ydecreasin go fth e daily water uptake as well as the Rosa bud development, extending the longevity (ornamentalvalue )o fth eflowers . Cryo-SEMobservation sshowe dtha tth ealuminiu msulphat ean dth e Bacillus subtilis cells added to the vase fluids did not affect the xylem ultra structuralmorpholog yo fth erose swithi n2 day so fvas elife . Shorttitle :Cryo-SE Mobservation so f Rosa flowerxyle mvessel s Keywords: aluminium sulphate, Bacillus subcilis; infiltration ability; Cryo-SEM; Rosa cv. 'Sonia';xyle mvessels . 166 INTRODUCTION In supplying cut flowers with sugar as source for their energy, it is necessary toad d simultaneously agermicid e toth evas e solutions inorde rt o inhibitmicrobia l growth (Aarts,195 7an dPaulin , 1971). For this purpose 8- hydroxyquinoline-citrate (HQC) as well as aluminium sulphate have found widespread use in post-harvest handling of cut flowers (Halevy & Mayak, 1981). Sometimes these chemicals are used together with calcium nitrate (Goszczynska et al., 1989). Intended as vase-solution disinfectants, both Al2(S04)3 and HQC are also reported tohav evariou s importantphysiologica l effects onth ecu tflowers . They lower the pH of the vase solution and suppress the growth of non- acidophilic bacteria (Aarts, 1957). In addition the Al3+ ions affect the stomatal status by diminishing stomate opening and subsequently decreasing water evaporation (Schnabl& Ziegler ,1975 ;Schnabl ,1976) .D eStigte r (1981) however, observed that the ornamental value and longevity of Al3+-treated roses were only slightly better compared to the non-treated controls, although their water loss was reduced by stomatal closure. Similar observationswer ereporte db yHalev y& Maya k (1981). Theseauthor s found that the effect of Al3+ on cut roses was attributed to the lowering of the rose petal pH and stabilizing the anthocyanins, while also acidifying the vase water, and thus reducing bacterial growth and improving water uptake. A certainreductio no fth enumbe ro fviabl ebacteri a infiltratedint oth elowe r part of the xylem vessels of roses, treated with aluminium sulphate, was shown by Van Doom et al. (1990) when the number of cfu (colony forming units)wer e assessed at the 7th day of thevas e life. Therefore,ou rstud ywa saime da tth eeffect so fAl 3+an d Bacillus subtilis cells added tovas ewate r of roseswit h respect to: (1)th exyle m morphology of the roses; (2) the infiltration ability of Al3+ with the sap stream; (3) the infiltration of bacterial cells into the xylem system; (4) the multiplication abilityo fbacteria lcell sadde dt oth evas e fluidan d (5)th e water uptake of the flowers. For thispurpos e acry opreparatio nmetho d ofste m specimens for scanning electron microscope observations has been used, as well as X-ray microanalysis of stem pieces in order to investigate the infiltration of bacterial cells andAl 3+ intoxyle mvessels . 167 MATERIALS AND METHODS - Plant material Rosa hybrids cv. 'Sonia' were supplied by a local greenhouse grower. The roses were harvested at flower stage 1-2 (Berkholst, 1980). The experiments were started the same day, within 4h after harvest. - Micro-organisms Bacillus subtllls, was inoculated onNutrien t agar (Oxoid), incubated for4 8 h at 32°C, washed off into sterile deionized water, mixed and diluted in sterile deionized water to a cell concentration of ca. SxlOa perml . Cell concentrations were assessed microscopically, and by plate counting on Plate Count agar (Oxoid). The suspensions were stored at -20°C. -Vas e life evaluation Flowerswer e cutwit ha stem length of4 5c m and aste m edge of45° ,usin g an anatomic razor blade and aseptical handling. The stems were wiped off with paper tissue soaked in ethanol 98% (v/v) to minimize contamination, and placed singly into sterile cylindrical vases containing the respective vase fluids tob e tested: (1)steril edeionize dwater ; (2)steril edeionize dwate r towhic hwa s added 5xl06o r 5xl07Bacillu s subtilis cellspe rml ; (3)steril e deionizedwate r towhic hwa s added 500p gA1 2(S0A)3.18H20 (Merck)pe rml ; (4) sterile deionized water to which was added 500 pg A12(S04)3.18H20 per ml as well as Bacillus subtilis cells 5xl06o r 5xl07pe rml . Thevas e fluidwa s adjusted top H 5an dcompare d with the effects atp H 4 (normal pH after addition of Aluminiumsulphate). Each modification was measured in triplicate. The vases were incubated at 20°C, 60% r.h. (relative humidity) and 10.25 W/m2 photoradiation at flower height during a 12 h light/dark photoperiod each 24h . Every 24h of thevas e life, observations were made of: thewate r uptake (ml),th e flowering stage and the ornamental value (Berkholst, 1980) up to a vase life of 4 days. After 0, 2 & 4 d of vase life the microbiological state ofwel l mixed samples of the vase fluids was assessed by counting of cfu (colony forming units) on Plate Count agar (Oxoid), as wella sb y directmicroscopi c counts ofGram-staine dsmear so fth evas e fluid samples by amodifie d Breed smear technique. Scanning electron microscope preparations were made after 48 h of vase 168 life (see below). -Cryo-SE M observations andX-ra y microanalysis Pieces of rose stem of 1 cmwer e taken at the first cm, and at 5, 10 and 20 cm from the cutting point respectively. The pieces were mounted on brass stubs and subsequently frozen innitroge n slush. Specimens were transferred to the preparation chamber of a Hexland -Oxfor d CT 1000/CP2000 system and freeze-fractured. The specimens were etched for ca. 30 min at 190 K (temperature of anticontaminator was 90 K). Finally the specimens were sputtered with gold and examined ina Philips SEM 535 at ca. 110K at 15k V accelerating voltage (Boekestein & Henstra, 1990). For X-ray microanalysis the EDAX 9900 system was used. Analysis conditions: 100 live sec. counting time, 15 kV accelerating voltage, spot 500 nm. For X-ray microanalysis specimens were coated with carbon. Each stem piece wasmeasure d fivetimes , at different points (Boekestein &Henstra , 1990). RESULTS AND DISCUSSIONS - Precipitation ofAl-compound s 3+ A12(S04)3 added to deionized water (1.5 mM Al ) results in a slightly opalescent solutionwit h a pH of about 4 due tohydrolysi s and formation of colloidal A1(0H)3. In a sterile buffered solution of pH 5-5.5 at 20°C, insoluble colloidal A1(0H)3 is flocculated to a white loose deposit. The quantity ofprecipitate d A1(0H)3 is raised with increasingpH . During and shortly after the addition of the non-buffered suspension of Bacillus subtilis cells toth ealuminiu m sulphate solution,th ep H increased from 4 to 4.5 and a precipitate of loose flocky material was formed. After buffering of the aluminium sulphate/Bacillus suspension at pH 5-5.5, the quantity of the flockymateria l increased further,probabl yb y the formation ofmor e insoluble A1(0H)3. 3+ 2 Al and S04 " ions can easily penetrate the capillary holes of the intervesselxyle mpi twalls ,an d thus infiltrate togetherwit h the sapstream upwards into the vessels as shown by X-ray micro-analysis. Obviously the flocky Al-containing material, is not able to pass these intervessel capillary holes. These Al-compounds are therefore only found on the cut surface and in the openxyle mvessels . Concerning the nature of the flocky material, it should be taken into 169 account that Al3+ ions may form insoluble compounds with negative inorganic or organic ions, e.g. (1)phosphates ; leaking out of disrupted plant cells; (2)peptide s (peptidoglycan) of the Bacillus cell wall (Rogers, 1970; Dawes & Sutherland, 1976); (3) organic anionic vascular material. The compounds formed may not be able to pass the intervessel capillary pit holes, or to enter with the sap stream into thevascula r system of the cut roses. -Th e influence of Al2(SO^)3 on thewate r uptake andvas e life Aseptically handled roses placed in Al3+ containing vase fluid showed a slightly slower bud development according to the Berkholst criteria (Berkholst, 1980) and slightly lower daily water uptake compared with non- treated control roses (see Table1) . Our observations confirm the results of previous observations of De Stigter (1981), who studied the effects of aluminium sulphate on the water balance ofcu t 'Sonia'roses ;h ereporte d that thewate rbalanc e ofcu t roses was improved by the addition ofAl-sulphat e + glucose to thevas e fluid. The Al-treated roses gave only slightly better outgrowth and flowering than the (tap) water controls. De Stigter (1981) concluded that: (1) glucose had a physiological effect; (2)glucos e +antimicrobia l agents (8-HQS or aluminium sulphate)interacte dwit hsom ephysiologica lprocesse so fth ecu troses ,suc h that the effects were different; (3) the results differed from the Al3+ effects reported by Schnabl & Ziegler (1975) and Schnabl (1976); (4) the mechanism(s) related to the interaction of antimicrobials and the water balance ofth erose swer eunknown .Th eantimicrobia l activityo fbot hagents , A12(S04)3 and8-HQS , however,wa sno t testedan dp Ho fth evas e fluidswa sno t measured. -Anti-microbia l activity of A12(S04)3 Our observations indicate that the Al-compound added to vase water was of less importance as a germicide thanwa s the accompanying lowering of the pH of the vase fluid. Only acidophilic and acidotolerant micro-organisms are able to survive ormultipl y in theAl-containin gvas e fluid at orbelo w pH 4 (Kushner, 1971). Inou robservation s awea kbiocida lactivit ywa sshow n towards B. subtilis cells atS p H 4,bu t no effect oronl y asligh t biostatic activitywa s found atp Hi 5 (Table1) . Itwa s furthermore found that in the vases treated with the Al-compound, 170 the Bacillus cellsdi dno tsta ysuspende dhomogeneously .I nth esupernatan t ca.10 * cfuml" 1o f Bacillus cellswer eenumerate dafte r1- 2d o fvas elife , whilei nth edeposi tca .10 7cf uml" 1wer erecorded .I nth ewel lmixe dsample , however,ca .10 5cf uml" 1wer ecounte d (Table 1). Theseobservation smigh tsugges t thatth emultiplicatio n abilityo fth e viable Bacillus cellswa sdecrease db yinteractio no fth ecell s (cellwall ) withA1(0H) 3resultin gi nco-precipitatio no fthes ecell so nth ecu tsurfac e ofth eroses ,an dgravitationa ldepositio no fcel laggregate sa tth ebotto m ofth evase . -Cryo-SE Mobservation so fcu t Rosa stemsafte r4 8h o fvas elif e Resultssummarize d inTabl e2 meri tfurthe rcomment s: ..Sterilize dvas ewate r Cryo-SEMmicrograp hobject sma ysuffe rles sfro mpreparatio nartefact san d showedmor e relevant structuraldetail scompare dwit h thoseretaine di n SEMobject s fixedwit hglutaraldehyd e (Fraser& Gilmour ,198 6an dPu t& Clerkx,1988) .Cryo-SE Mimage so fth evascula rxyle mcel lwal lshowe dgoo d detailedstructure s (Plates1-4) . ..A1 2(S04)3 insterilize dvas ewate r Athi nwhit e granularlaye rwa sforme do nth ecu tsurfac epartl ycoverin g thesmalle r (S 1/im )xyle mvessels ,a sshow ni nPlat e5 . In the adjoining openvessels ,a t increasing distance from the cutting point,decreasin gamount so fAl-flock swer eobserved . .. B. subtilis cellsadde dt osterilize dvas ewate r Cryo-SEMimage sconfirme dpreviousl yreporte dobservation s(Clerk x et al., 1989), that the cut surface of Rosa stems canac t asa coarse-threade d filter.Onl ya certai nfractio no fth e Bacillus cellswer einfiltrate db y thesa pstrea mhighe ru pint oth eope nxyle mvessel s (Plates 7& 8) .Th e remainingfractio nwa sattache dt oth ecu tsurfac e(Plat e6 )o radhere do n the lower part of the vessels, subsequently partly blocking the water uptake (Plate9) . .. B. subtilis andAl 2(S04)3 insterilize dvas ewate r Cryo-SEMimage so fth ecu tsurfac ean do fste mspecimen s5 c mu pint oth e Rosa stem (Plates 10-14) show that the Bacillus subtilis cells are surroundedb ya nAl-complex ,thickenin gth ecells ,causin gthe mt oadher e toth ecu tsurface .Th evascula rxyle m systemwa spartiall yblocke dan d thusaffectin g thewate ruptak ecompare dwit h B. subtilis insterilize d 171 water. SEM observations also show that amuc h smaller fraction of theAl - complexed B. subtilis cellswa sabl e toente rwit h thesapstrea mhighe ru p into the openvessels , compared with non-Al-treated Bacillus cells. It is not yet known, however, whether other microbial genera and species e.g. Gram-negative bacteria, yeasts, fungalmyceliu m and sporesmigh t react similarly as aconsequenc e of the addition ofAl-sulphat e to thevas e water of roses. -X-ra y microanalysis No aluminium was detected on the cut cross section of roses placed in deionized water (Fig.1) . Aluminiumwa sabundantl ypresen t onth ecu tcros ssectio n (Fig.2 )and , to a much lesser extent, in the first cm of the xylem vessels of the stem ofa rose immersed in an aluminium sulphate containing vase fluid of pH 5 (Fig. 3). Yet, only in one out of five points measured at 5 cm from the cutting point, a small aluminium peak was observed, while at 20 cm from the cutting point, no aluminium was detectable. These observations reveal that most of the A1(0H)3 flocks formed at pH 5 and precipitated in the vase fluid, were not taken up by the sap stream of the rose, but remained as a filtered layer attached to the cut surface. Rose stems immersed in an aluminium sulphate containing vase fluid of pH 4, on the contrary, showed inth exyle mvessels , evenhighe r aluminium peaks as far as 10 cm from the cutting point, compared with these detected on 5 different points of the cut cross section (Fig. 4).A t pH 4, the A1(0H)3 is a glassy colloid, slightly flocculated by the formation of very small particles which easily entered the xylem system of roses. They may also be able to pass the capillary holes in the pit membranes. It should be noted, however, thatw ehav e localized andanalyze d only limitedAl-concentrations ; + lowerconcentration s especially ofth eAl 3 ma yb e transportedhighe ru p into the flower stem. Other elements like P andK , are frequently found inbiologica l tissues, but their concentrations mayvary . Speak s canb e attributed to the Al2(SOt)3 added to the vase fluid. - The role of flower 'preservatives' Chemical compounds added to vase fluids as "flower preservatives" are - mainly chosen and evaluated for a specific property, although they may show 172 furthereffect sa sa consequenc eo finteraction .E.g . (1)Co 2+ improvesth e waterbalanc eo frose sbu ti sals ofungicida lan di sa nethylen e(C 2H^)binde r (Reddy, 1988), (2) Ag+ is an ethylene binder but has also germicidal properties (Veen, 1987), (3)Al 3+ atp H £ 4 isbiocida l and decreases the infiltration of microbial cells into the xylem system as shown in our investigations,bu tals oslightl yimprove sth ewate rbalanc eo fcu t'Sonia ' roses,a sals oobserve db yD eStigte r (1980& 1981) , (4)however ,a tp H5 - 5.5colloida lA1(0H) 3ma ymainl yac ta sa physica lfilte rlaye ro nth este m crosssection ,blockin gbacteria linfiltratio nan dals oslightl ydecreasin g the water uptake. A typical phenomenon unfolded by SEM, X-ray and microbiological examinations is presented in this paper. These further effects of antimicrobial agents added to the vase water of roses, need furtherelucidation ,an dshoul db etake nint oaccoun ti nth edevelopnmen to f themethodolog yuse dfo rthei revaluation . ACKNOWLEDGEMENT The author wishes to thank Professor Dr. Ir. F.M. Rombouts (Agricultural University,Wageningen ,Th eNetherlands )an dDr .Murie lE .Rhodes-Roberts , (UniversityColleg eo fWales ,Aberystwyth )fo rreadin gan dcommentin go nth e manuscript. The author thanks also Ans van Hardeveld and Ir. Christy Berkholstfo rtechnica lassistance . 173 REFERENCES Aarts,J.F.T .195 7O nth ekeepin gqualit y ofcu t flowers. Mededelingen van de landbouw Hogeschool Wageningen, 5â,1-62 . Berkholst, C.E.M. 1980 De waterhuishouding van afgesneden rozen. Bedrijfsontwikkeling, 11,332-336 . Boekestein,B .& Henstra ,S .199 0Cryo-SEM :zich to pbevrore nleven . Natuur en Techniek, 58.696-711 . Clerkx, A.C.M., Boekestein, A. & Put, H.M.C. 1990 Scanning electron microscopyo fth este mo fcu tflower so f Rosa cv. 'Sonia'an d Gerbera cv. 'Fleur'. Acta Horticulture, 261.97-105 . Dawes, I.W. & Sutherland, I.W. 1976 Microbial Physiology. Basic Microbiology Vol. 4. Ed. Wilkinson, J.F. London: Blackwell Scientific Publications.18 5pp . De Stigter, H.C.M. 1980 Water-balance aspects of cut and intact 'Sonia' rose plants and effects of glucose, 8-hydroxyquinoline sulfate and aluminiumsulfate . Acta Horticulturae, 113.97-107 . De Stigter,H.C.M . 1981Effect s ofglucos ewit h 8-hydroxyquinoline sulfate or aluminium sulfate on the water balance of cut 'Sonia' roses. Zeitschrift für Pflanzenphysiologie, ÎQI,95-105 . Fraser, T.W. & Gilmour,A . 1986 Scanning electron microscopy preparation methods: their influence on the morphology and fibril formation in Pseudomonas fragi (ATCC4973) .Journa l of Applied Bacteriology, 60,527 - 533. Goszczynska, D.M., Michalczuk, B. & Rudnicki, R.M. 1989 The effect of floralpreservativ e enrichedwit h calciumnitrat eo nkeepin gqualit yo f 'Sonia'roses . Acta Horticulturae, gel, 281-286. Halevy,A.H .& Mayak ,S .198 1Senescenc ean dpostharves tphysiolog y ofcu t flowers -Par t2 . Horticultural Reviews, 3,59-143 . Kushner, D.J. 1971 Influence of solutes and ions on micro-organisms. In: Inhibition and Destruction of the Microbial Cell. pp.259-284 .Ed .Hugo , W.B.London :Academi cPress .81 9pp . Paulin,A. , 1971 Influence de lacompositio n de la solutionnutritiv e sur lateneu r endiver sacide s aminés librese te nammonia c despétale sd e rosescoupées . Annales de Technologie Agricoles, 20,283-303 . 174 Put, H.M.C. & Clerkx, A.C.M. 1988 The infiltration ability of microorganisms Bacillus, Fusarium, Kluyveromyces and Pseudomonas spp.int o xylemvessel so f Gerbera cv. 'Fleur'an d Rosa cv. 'Sonia'cu tflowers :a scanningelectro nmicroscop estudy . Journal of Applied Bacteriology, 64. 515-530. Reddy,T.V . 1988Mod e of action of cobalt extending thevas e life ofcu t roses. Scientia HorCiculturae, 3_£,303-313 . Rogers,H.J . 1970 Bacterial growth and the cell envelope. Bacteriological Reviews, 34,194-214 . Schnabl, H. & Ziegler, H. 1975 Über die Wirkung von Aluminium-ionen auf die Stomatabewegung von Vicia faba Epidermen. Zeitschrift für Pflanzenphysiologie, 24,394-403 . Schnabl, H. 1976 Der Einflusz von Aluminium ionen auf den Stärke metabolismus von Vica faba - Epidermen. Zeitschrift für Pflanzenphysiologie, 21, 167-173. VanDoorn ,W.G. ,D eWitte ,Y .& Perik ,R.J.J . 1990Effec to fantibacteria l compoundso nth enumbe ro fbacteri ai nstem so fcu tros eflowers . Journal of Appplied Bacteriology, ££.117-122 . Veen, H. 1987 Use of inhibitors of ethylene action. Acta Hortlculturae, 201.213-222 . Zieslin,N . 1989 Postharvest control ofvas e life and senescence of rose flowers. Acta Horticulturae, gel, 257-264. 175 Table ITh einfluenc e onth ebacteria l activity andth eornamenta l valueo f 1 8 7 the roses ofA1 2(S0J3.18H20 (500/i gml" )an d B. subtilis cells (5xl0 -5xl0 ml"1)adde d tosterilize d deionizedvas e water of Rosa cv.Soni a Vase PH Vase Bacterialcoun t Water Stage fluid life ml"1vas e water uptake ofbu d (days) cfu Micros ml/d devel copic opment contents (*) Control-sterile 5.5 0 <5 0 . 0 1-2 deionized vase 2 <5 0 - 50 5 water 4 <5 0 - 40 7" 5.5 0 <5 0 - 0 1-2 2 <5 0 - 45 5 4 <5 0 - 35 6-7 . 2)+ Al 2(SO,)3 5.5 0 <5 0 0 1-2 2 <5 0 - 35 3-4 4 <5 0 - 30 6-7 0 <5 0 - 0 1-2 2 <5 0 - 35 4-5 4 <5 0 - 30 6-7 3)+ B. subtilis 5.5 0 5xl06 ca.105 0 1-2 5xl0ml"6 1 2 107 ca.107 45 3 4 Sxaio7 5xl07-108 20 4-5 + B. subtilis 4 0 5xl07 ca.107 0 1-2 5xl0ml"7 1 2 ilO7 ca.107 45 3 4 2:5xl07 ca.5xl0 7 20 6" 6 6 4)+ A1 2(S0A)3 5 0 5xl0 ca.10 0 1-2 + B. subtilis 2 5xl06 ca.108 35 5 5xl0ml"6 1 4 ca.107 ca.107 25 6-7 7 + A12(S04)3 4 0 5X10' ca.10 0 1-2 + B. subtilis 2 ca.108 ca.106 30 4 5xl0ml"7 1 4 ca.105 ca.106 25 6-7 cfu colony forming units * Berkholst,198 0 -- n obacteria l cells observed 176 TableI ICryo-SE Mobservation so fxyle mvessel so fstem so fRos eflower safte r 48h o fvas elif eI nvas efluid scontaining :sterilize ddeionize dwate r (1)t o whichwa sadded : (2)A1 2(S0«)3; (3) B. subtilis cells;(4 )A1 2(S04)3an d B. subitilis cells Vase Sampling point fluid Cuttingpoin t Plate 50m mfro mth e Plate no. cuttingpoin t no. Code PH (1) normal;diam .o f 1,2,3 normaldefine dimage s 4 5.5 vesselsfro m oflongitudina l <0.5-5 0p m sectiono fth exyle m (2) Al-compoundvisible , Afe wwhit eflock s bothmacroscopical y inth evessel , ando ncryo-SE M normalvesse l images,a sa thi n ultrastructure. whitegranula rlaye r onth ecu tsurfac e anda sthi nwhit e flocksi nth e vessels. 5.5 Thickwhit eflock y Thinwhit eflock s layero nth ecu t Normalvesse lultr a surface. structure. (3) Onth ecu tsurfac e Somevessel splugge d 7,8 5.5 afe w Bacillus cells, with Bacillus cells. inth eadjacen tves - Normalvesse lultra - sesnon ,a fe wo r structure. pluggedwit h Bacillus cells. Imagesa sa tp H5. 5 Imagesa sa tp H5. 5 (4) Oncu tsurface s 10,11 Afe wwhit eflock s Bacillus cellsobscu ofAl-compound . redb yAl-flocks ;i n Nobacteria lcell s adjacentvessel s observed. adecreasin gnumbe r Normalvesse lultra - of Bacillus andAl - structure. flocks. Atca .0. 1m mfro m 12,13, Afe wwhit eflock s thecuttin gpoin t 14 ofAl-compoun d many Bacillus cells andsporadi csom e amdAl-flock sar e Bacillus cells. shown.A t1-1. 5m m Normalvesse lultra - onlya fe w Bacillus structure. andthi nAl-flock s observed 177 2.00 4.00 6.00 8.00 OC N T 0 .O 0 K E V iOeV/ch A E D A X Flg. 1. Energy-dispersive X-ray spectrum ofa xyle mvesse l inth e first cm of the stem of a Rosa flower held insterilize d water ofp H 5.5. a.oo 4.00 6.00 8.00 0 C N T 0.0ÛKEV IOeV/ch A E D A X Fie. 2. Energy-dispersive X-ray spectrum of axyle mvesse l close to the cut surface of a Rosa stemhel d in flocculated A12(S04)3 solution of pH 5.5. 178 8.00 4.00 6.00 8.00 0C N T 0 .0 O K E V iOeV/ch A ED A X Flg.3 . Energy-dispersiveX-ra yspectru mo fa xyle mvesse li nth efirs tc m ofth este mo fa Rosa flowerhel di nflocculate dA1 2(S04)3solutio n ofp H5.5 . A1K« S K« K K« l/OT*wL I . .. 1 i i ^Vv*Y* '**1/it*y**rr*w** '*> **-V> u , 4.00 6.00 a.oo e.oo 0 CN T 0 .0 0 K E V IOeV/ch A E0 A X Fie. 4. Energy-dispersiveX-ra yspectru mo fa xyle mvesse la tapprox .1 0c m from the cutting point of a Rosa flowerhel d ina clea r A12(S04)3 solutiono fp H4 . 179 o 5 o 5 S g s ; 9I S 3 . ? C u O 3 ü C -7 C 181 /'*/# iin 182 183 Chapter 9 Discussion 185 Rosa (Judith Tromp) 186 Chapter9 DISCUSSION Inpaleontology.i tha sbee n exerted that the lifeo fwil d rosesma yb ea t leastsevent ymillio nyear sold .Breedin go froses ,originate sprobabl yi n China,an di salread ypractice dfo rabou t500 0(Shepherd , 1954). InChines e culturalhistory , theros ei sconsidere d tob eo fgrea tvalue .Mos t ofth e European Rosa cultivarsdescen dfro ma numbe ro fabou teigh tAsia ncultivars . Therose ,a sw ekno w this flower today,U s the 'GretaGarbo 'amon gth e ornamental flower cultivars. Its beauty is fascinating, its behaviour is capricious.Fo rth evas elif eo frose si smarke db ya ninconstanc ywhic hi s difficultt obecom eacquainte dwith ,an dwhic hi sstil lmor edifficul tt oge t controlon . Postharvest microbiology of ornamental flowers and flower vasefluids , particularlyo froses ,appear st ob ea fiel dwhic hi shardl ystudied ,i nspit e ofth efac ttha tth evas elif eo fflower sca nb edramaticall y influencedb y micro-organisms (Aarts,1957 ;Zieslin ,1989) . Thepurpos eo fth eresearc hpresente di nthi sthesi sis :t orevea lfactor s ofth evas elif eo froses ,i nwhic hmicro-organism spla ya dominan trole . Theinitia lnumbe ro fmicro-organism so ncu tflowe rstem s Theactivit yo fmicro-organism sca nb eth emai ncaus eo frapi dsenescenc eo f cutflowers ,a salread ypostulate db yAart s (1957).However ,littl ei sknow n concerningth einitia lmicroflor ao nstem so ffreshl yharveste dcu tflowers . Aserie so fInvestigation sint oth emicrobia lloa do nstem so ffreshl ypicke d Chrysanthemum, Gerbera, and Rosa cultivarsshowe dtha tth enumber so fviabl e micro-organismsenumerate da scf u(colon yformin gunits )o nth estem so f Rosa flowersar ever ylow ,an dmuc hlowe rtha nthos eo n Chrysanthemum and Gerbera stems,probabl yreflectin gthei rdistanc efro mth esoi lwhe nharvested ,bu t possiblyals odu et oth eglossynes so fth e Rosa stemepidermis .Moreover ,th e numeroustin ysid eleave so fth e Chrysanthemum steman dth ehair yepidermi s ofth e Gerbera probablyfacilitat eth eadherenc eo fepiphyti can dsoi lmicro organismsont oth estem s (Chapter2) . Identificationo fcu tflowe rste mmicroflor a Awid evariet yo fmicrobia lspecies ,bacteri aa swel la sfilamentou sfung ian d 187 yeasts are Isolated from stems of freshly harvested Rosa, Gerbera and Chrysanthemum flowers.Thes emicrobia lspecie sar eidentifie da snorma lsoi l saprophyteso ra sepiphyti cconstituent so fth ephyllosphere ,an dsom efung i areknow na sphyhtopathogenic : Botrytis cinerea andFusariu m solani. Yeasts as well as filamentous fungi are usually isolated in lower numbers than bacteria (Chapter2) . Incontras t thevas ewate ro fcu tflower sha sa mor ediversifie dflora . Microbialspecie sno toriginatin gfro msoi lo rth eflowe rphyllospher emus t thereforehav ebee nintroduce dint oth eecosyste mo fth eflowe rste man dth e flower vase fluid after harvest, e.g. by microbial contamination of the harvestmaterials ,th ewate ran dth econtainer sused . Multiplicationo fcu tflowe rste mmicroflor a Thecu tflowe rstem ,th evas eflui dan dth eflowe rste mimmerse di nth evas e orcontainer ,represen ta necosyste mi nwhic hcertai nmicrobia lspecie ssho w anoptima labilit yfo rmultiplication .Althoug hmos to fth emicrobia lgener a presento nth ecu tstem swer eals opresen ti nth evas ewater ,competitio nan d antagonistic activitiesbetwee nth edifferen tmicrobia lgener aan dspecies , aswel la secologica lcondition si nth evas efluid ,poo ri nnutrients ,lead s to the observed initial dominance of Pseudomonas species, not requiring organic growthfactor s (Chapter 2). InChapte r2 i ti sals o showntha tth e initialpredominanc e of Pseudomonas species isfollowe db yth epredominanc e ofmor egrowt hfactor srequirin g Enterobacter and Bacillus speciesan dfro m theno nals ofunga lgrowt hwa sstimulate da swel la sgrowt ho fothe rbacteri a e.g.mor edemandin g Pseudomonas species. The evolution of the microflora ofvas e water during the vase lifeo f flowersha ss ofa rbee nstudie donl yfragmentaril y(D eStigte r& Broekhuysen , 1986; Woltering, 1987). However, to elucidate the influence of microbial factorso nth evas elif eo fcu tflowers ,i ti sindispensabl et ohav eknowledg e oftypes ,number san dmultiplicatio nabilit yo fth emicroflor a- bacteri aa s wella sfung i- durin gth ewhol ecours eo fth evas elife . Thechoic eo fth eros e Theusuall yver ylo winitia lmicrobia lcontaminatio no fhygienicall ypicke d greenhouse roses, makes this flower cultivar very appropriate for investigationsint oth einfluenc eo fmicrobiologica lfactor so nit svas elife . Withcarefu lhandlin go fth eflowers :(1 )activitie so fth einitia lmicrobia l 188 flora canb eminimized , (2)th eadditio n ofbiocide s to thevas e fluid canb e avoided and therewith their physiological activities, (3) suppletion of a nutritive sugar is not necessary for anorma l bud development. Added sugar influences themultiplicatio n ofmicro-organism s and also the formationo fcertai nmetaboli cproduct swhic hca ninterfer e -physiologicall y and microbiologically -wit h the factors tob e studied (Chapter1.6) . The methodology The methods used in the consecutively executed investigations were standardized in order to make possible: (1) to check the reproducibility of the results obtained, (2) to apply statistical evaluation of the various measurements, visual observations and data, (3) to compare data and observations ofth edifferen ttes tseries , (4)t ob eabl et oattribut e certain observationst ospecifi cphenomen ao fcu tflowe rvas elife ,an devent sleadin g to aprematur e senescence. Standardization ofth eplan tmateria lwa srealize db ypickin g ofth erose s at the same stage ofbu d development (Berkholst, 1980). The cut flowers were stored formax .4 8h at4° Cbefor euse .Th e flowerswer e cut toa ste m length of4 5cm ,an dal lexcep tth e3 uppe r leaveswer eremove dbefor e therose swer e placed singly into cylindrical vases containing 100m l of thevas e fluid to be tested. All microbiological factors i.e. initial numbers of microbial cells, microbialmetabolite s (highmol .w tan dlo wmol .w tcompounds ,pecti c enzymes) were tested intriplicate s and inserie so fa tleas t three decimaldilutions . All the (vase) materials were sterilized. Pure cultures were used, and microbial products sucha senzyme swer e thoroughly purifiedbefor e use.Vas e fluids were prepared from sterilized deaerated tapwater . Incubation of the vases was at controlled environmental conditions. Observations weremad e in2 4h intervals. Scanning electronmicroscop e (SEM) studies, macro colour photographs, and conductivity measurements were made after 24 and 48 h ofvas e life. A rose is a rose, is a rose, is a rose. In other words, roses although picked at the same time, in the same stage, at the same greenhouse are individual biological entities. Thus, it should be taken into account that from roses used after 24 h and 48 h of vase life for SEM preparations or conductivitymeasurements ,onl yspeculation sca nb emad eabou tho wth ecours e of their vase life might have been in case these flowers were held in their vases. 189 Vessel plusping Bacterialpluggin g ofxyle mvesse lelement sha sbee nclaime d tob e themajo r cause of rapid senescence ofcu t flowers (Aarts, 1957). This study confirms these claimswit h observations described inChapter s 3,4 and 5. Vessel plugging by micro-organisms causes water stress. However, water stress does not always need the consequence of microbial vessel plugging, although micro-organisms -viabl e as well as inactivated ones - do play an important role inth ephenomen a related tomicrobia l vessel plugging and cut flower water stress. In Chapter 5, it is shown thatviabl e aswel l ashea t inactivated water- washed bacterial cells prepared from pure cultures of different strains and specieso f Bacillus, Enterobacter, and Pseudomonas, addedi ndecima ldilution s to the vase fluid, caused a marked decrease in the vase life of roses. The initialnumbe r ofbacteri a added affected the decrease of thevas e life,bu t the individualbacteria l strainsbehave dver y similarly,notwithstandin g the differences inthei rindividua lpropertie ssuc has :cel lshape ,cel lmotility , slimeproduction ,capsula r formation,leva nproduction ,enzym eactivit yan d- opportunistic -pathogenicity . Initialnumbe ro f10 8viabl eo rhea tinactivate dbacteria l cellsml" 1adde d toth evas eflui do frose scause dxyle melemen tocclusion smerel ya tth ebasa l endo fth estem ,a sshow nb ySE Mobservations ,wate rconductivit ydat aan dth e fuchsinre dtes t (Chapters4 an d 5). A tlowe rinitia lbacteria lnumber s added to the vase fluid, the differences in the effects on the vase life of the roses of viable and inactivated cells became significant, although the influence of inactivated bacterial cells on the vase life of the roses remained an important factor. The increasing differences of the effects of viableversu sinactivate dbacteria lcell sinitiall yadde di ndecima ldilution s toth evas efluid so fth eroses ,ca nb erelate dt oth emultiplicatio ncapacit y of theviabl e bacterial species, and to specific metabolic compounds formed duringgrowth ,e.g .pecti c enzymes,lo wmol .w ttoxi ccompound san dhig h mol. wtvascula r plugging compounds. To revealwhethe r areduce dwate r conductivity iscause db yviabl e and/or by inert microbial propagules, or by microbial metabolites, a microscopic counting method should be combined with plate count methods, to assess cfu (colony forming units) ofbacteri a and fungi in thevas e fluid. In addition thebee troo ttissu ecub etest ,t oasses scell-membran edamage ,suc ha scause d by the activity ofpecti c enzymes,a swel l asquantitativ e chemical analysis 190 ofmicrobia l metabolic compounds (EPS)shoul db e applied (Chapters 6an d7) . Finally, by the acid fuchsin test,xyle m plugging canb e localized as shown in Chapters 5 and 6. With exception of the quantitative chemical analysis., these methods are easy to introduce inroutin e examinations. The role ofbioti c elicitors Microbial propagules, microbial metabolic compounds and flower vessel cell contents or fragments,ma y act asbioti c elicitors. These elicitors may act alone or in combination (Halverson & Stacey, 1986; Chapter 1.5). These elicitors,take nu pb y thesa pstream ,ma y initiatea stil lunknow nserie s of physiological reactions,whic hma y disturb thewate r relations and the vase life of the roses (Hahn et al., 1989). Asa resul to fphysica ldamage ,cuttin go fth eflowe rstem ,th eplant' sow n elicitorsma yb eforme da tth esid eo fth ecuttin gwound .Thes eelicitor sare : plant hormones, cell wall fragments and enzymes, which may reinforce the physiologicalreaction selicite db ymicro-organisms .Lo wnumber so fbacteria l propagules alone (10*-108 ml"1) added to the vase fluid, could not by themselves have caused the extent ofwate r conductivity loss measured after 24 h and 48 h of vase life. These observations indicate that some still unknown physiological reactions of the water transport system, elicited by micro-organisms,ma y also play a role (Chapter5) . In this thesis no direct experiments were caried out to show if any of thesemechanisms ,concernin gbioti c elicitors,wer e involved.However , iti s tempting to ascribe some of the unexplained phenomena to such mechanisms. There is clearly a need for further research at this point (Hahn et al., 1989a). SEM (scanning electronmicroscope ) observations The microbiological techniques and additional analytical methods used in Chapters 3-8,a swel l as theresult s obtainedwit h SEMobservation s allow to conclude that: the cut surface of a flower, such as a rose, can act as a coarse threaded filter (Figure 1).Onl y a small fraction ofmicrobia l cells andhig hmol .w tcompound senter sfro mth evas ewate r intoth evascula r system with the transpiration stream. Most of the micro-organisms and high mol. wt EPS (exopolysaccharides) remains attached to the submerged cut surface, obstructing xylem vessels andblockin g water uptake (Chapters 4 and6) . This phenomenon was quantified by investigations done with Pseudomonas putida cell strain 48 (Chapter 3). Althoug h the number of bacteria that 191 infiltratedint oope nxyle mvessel sincrease dwit htim ean dincreasin gnumber s of Pseudomonas cells added to thevas e fluid, and decreased with increasing distance between cutting point and sampling point, the total load of infiltrated Pseudomonas cellswa smuc hlowe r thancalculate db y thevolum eo f vaseflui d takenup ,a sshow ni nFigure s4 ,5 an d6 o fChapte r2 .Thi s finding suggests that the bacteria themselves, when deposited at the cut surface of the flower stem, may act as a filter preventing otherbacteri a from entering thevessels . Furtherobstructio no fxyle mvessel sa tth ecu tsurfac edepend son : (1)th e number ofvessel s and their diameter. Gerbera cv. 'Fleur'vessel s are wider (20-100 /im) than the xylem vessels of 'Sonia' roses (2.5-35 /im), (2) the number and the size of microbial cells added to the vase fluid. Pseudomonas cells are0. 5 x 1.5 /iman d Kluyveromyces cellsmeasur e approx. 3x 10/im ,(3 ) thecapacit y offunga lconidi ae.g .Fusariu m oxysporum toagglutinat e and,o f certain bacterial species e.g. Bacillus polymyxa, Bacillus subtilis as well as Pseudomonas putida, tofor mcel lcluster sb yadhesio n ofcapsula r material onto thecu t surface, (4)th ecapacit yo ffilamentou s fungi tofor m mycelium, covering the cut surface as another filter-layer through which only small mycelium particles and microbial cells could enter the vascular system with the sap stream (Chapter 4, Clerkx, Boekestein & Put, 1990), (5) the ability of micro-organisms to form adhesive high mol. wt compounds e.g. EPS (exopolysaccharides) as already mentioned above, and the amounts thereof (Chapter6) . Light microscope and scanning electron microscope observations are essential methods to reveal the role ofmicro-organism s inwate r stress and vessel plugging of cut flowers during their vase life, as shown in Chapters 3, 4, 6, 7 and 8. Besides, it can be concluded that cryoSEM images are more defined than SEM images obtained after chemical fixation by means of glutaraldehyde.CryoSE M images of thevascula r xylem cellwal l show afiner , moredetaile d structure thanSE M imageso fchemicall y fixed specimen (Chapter 8, Figures 1-4; Chapter 4, Figures 11-13; Fraser & Gilmour, 1988). The role of pectic enzymes Purifiedpectolyti cenzymes ,suc has :pectat elyas eo f Pseudomonas fluorescens and polygalacturonase of Kluyveromyces fragllls, added to a buffered vase fluid,distur bth evas elif eo f 'Sonia'roses .However ,pecti cenzyme s invas e fluids need a certain lapse of time to show their typical effects on vessel 192 morphology,wate rrelation san dvas elif eo f Rosa flowers.Th esymptom sar e verytypica lI na smuc h theseenzyme scaus eneithe rwilting ,no rben tneck , butdesiccatio no fpetal san dleaves ,an dinhibitio no fflowe rbu ddevelopmen t (Chapter7) . SEMfigure ssho wtha tpecti cenzyme scaus edegradatio no fth epectin-linke d structure of the Rosa xylem vessels. This may result inbreakag e of the capillarity of thepi tmembranes ,causin gwate r stress and also increased vulnerabilityfo rxyle mcavitatio nan dembolis m (Sperry& Tyree ,1988 ;Dixo n & Peterson,1989) . Iti sobviou stha tpecti cenzyme sadde dt oth evas eflui do f Rosa flowers inlo wconcentration s may cause strong effectso n thevas e lifeo froses . Besidesth eeffect so nxyle mvesse lmorpholog yan dwate rrelation so froses , it is possible that in breakdown of xylem cell wall components, higher oligogalacturonicaci dcompound sar eformed .Thes eso-calle d'oligosaccharins ' areknow nt ohav ea physiologica leffec to nth eplant .Oligosaccharin sma yac t as endogenous biotic elicitors or signalmolecule s elicitinghos t defence responses, or abnormal growth (Chapter 1.5; Albersheim & Valent, 1978; Halverson & Stacy, 1986;Rickauer , 1989). As with the phytohormones, it remains tob e determinedho w certain 'oligosaccharins' control growth and development of plants (Darvill et al., 1989). It is not known whether oligosaccharins affect thevas e lifeo froses ,no rwhethe r the effectso f pecticenzyme sobserve d inChapte r 7,wer emediate db yoligosaccharins .A n intriguing field for further investigations (Albersheim & Darvill,1985 ; Cervonee tal. , 1989). Aluminiumsulphat ei nvas ewate r Aluminiumsulphat eshow shydrolysi si nvas ewater ,lowerin gth ep Han dformin g analuminiu m hydroxyde colloid,whic h reacts easily withbacteria lcells , materialso fwounde dste mcells ,an dxyle mvesse lwall so fth erose ssubmerge d inth evas efluid . Bacterium-aluminiumcomplexe sdevelo pa flock ydeposi twhic hadhere sont o thecu tsurfac eo froses ,obstructin ginfiltratio no fbacteria lcell sint oth e open xylem vessels. This phenomenon is clearly demonstrated by cryoSEM observations,photomicrographs , and X-ray microanalysis of stem specimens (Figure2) . The aluminium sulphate effect on Rosa flowers invas e fluid containing bacterialcells ,i scause db ythre efactors :(1 )a biostati cactivit ytoward s 193 bacterialcells ,(2 )obstructin go fbacteri at oinfiltrat eInto ,an dplu gope n xylemvessels , (3)maintainin g of the daily water uptake and therewith the Rosa bud development, extending the longevity of the flowers (Chapter8) . Bioeides in flower vasewater It isno t surprising that thenegativ e effectswhic hmicro-organism s invas e fluids may have on the vase life of ornamental flowers, have led to the additiono fbiocide s toflowe rvas efluids .A napplicatio nwhic h ispractice d lucratively. However, little isknow n concerning the initial microbial load ofvas e fluids,an d of themicrobia l species tob e inactivated. Also little isknow n concerning the sensitivity of the diverse microbial species,whic h can multiply in vase fluids, towards biocides normally added, as well as effects on thephysiolog y ofth ecu t flower,exerte db y thebiocide s (Aarts, 1957). Inthi scontext , thedevelopin go fa mode l system totes tth ebiocida l and biostatic capacity ofcertai ncompound s isnecessary . Typical differences in sensitivity towardsbiocida lcompound so fbacteria ,fungi ,an dth ecu tflowe r itself, shouldb e taken into account indevelopin g awidel y applicable test model. Methods as described and normalized for biocides to be used in food hygiene practice, can be a basic example (Council of Europe, Strasbourg, 1987). The use of biocides is only one element in a more general approach to maintain the quality ofcu t flowers.A s infoo d technology thismor e general approach could be laid down in codes of hygienic practice. Good hygienic practices, from the greenhouse handling toth ebeginnin g of thevas e life at thebuyer' s home, should provide thebasi s forhig h quality flowers with an extended vase life. Stat rosa pristina nomine (Eco, 1983). 194 REFERENCES Aarts.J.F.Th .1957 .Ove rd ehoudbaarhei dva nsnijbloemen . On the keepability of cut flowers. Netherlands,wit henglis hsummary .Mededelinge nva nd e Landbouwhogeschool,Wageningen ,57(9) ,1-62 . Albersheim,P .& Darvill,A.G . 1985.Oligosaccharins . Scientific American, 253,44-50 . Albersheim, P. & Valent, B.S. 1978.Host-pathoge n interactions inplants . Plants, when exposed to oligosaccharides of fungal origin, defend themselvesb yaccumulatin gantibiotics . The Journal of Cell Biology, 78, 627-643. Berkholst, C.E.M. 1980. The water relations of cut roses. Bedrijfsontwikkeling, 78,331-336 . Cervone,F. , DeLorenzo ,6. , Salvi,6. , Bergmann, C, Hahn,M.G. , Ito,Y. , Darvill, A. & Albersheim, P. 1989. Release of phytoalexin ellcitor- active oligogalacturonides by microbial pectic enzymes. In: Signal Molecules in Plants and Plant-Microbe Interactions. 85-90. Ed. Lugtenberg,F.J.J. ,Berlin :Springe rVerlag ,42 5pp .Nat oserie sH :Cel l Biology,vol .36 . Clerkx, A.CM., Boekestein, A. & Put, H.M.C. 1989. Scanning electron microscopy ofth este mo fcu t flowerso f Rosa cv. 'Sonia'an d Gerbera cv. 'Fleur'. Acta Horticulturae, 261,97-105 . Council of Europe, Strasbourg. 1987. Test methods for the antimicrobial activity of disinfectants in food hygiene. Partial agreement in the socialan dpubli chealt hfield .1-35 . Darvill, A.G., Albersheim, P., Bucheli, P., Doares, S., Doubrava, N., Eberhard,S. ,Golling ,D.J. ,Hahn ,M.G. ,Marfà-Riera ,V. ,York ,U.S .& Mohnen, D. 1989. Oligosaccharins - plant regulatory molecules. In: Signal Molecules in Plants and Plant-Microbe Interactions, 41-48.Ed . Lugtenberg,F.J.J .Berlin :Springe rVerlag ,42 5pp .Nat oserie sH :Cel l Biology,vol .36 . DeStigter ,H.C.M .& Broekhuysen ,A.G.M . 1986.Rol eo fste mcut-surfac ei n cut-roseperformance . Acta Horticulturae, 181,359-364 . Dixon,M.A .& Peterson ,C.A .1989 .A re-examinatio no fste mblockag e incu t roses. Scientia Horticulturae, 38,227-285 . Eco,U .1983 .I Inom edell arosa .Turino ;Guili oEnaudi . 195 Fraser, T.W. & Gllmour,A . 1986.Scannin g electronmicroscop y preparation methods: their influence on the morphology and fibril formation in Pseudomonas fragi (ATCC4973) . Journal of Applied Bacteriology, 60,527 - 533. Hahn,M.G. , Cheong, J.J., Birberg,W. ,Fügedi , P., Filoti,Ä , Garegg, P., Hong,N. ,Nakahara ,Y .& Ogawa ,T .1989 .Elicitatio no fphytoalexin sb y synthetic oligoglucosides, synthetic oligogalacturonides and their derivatives. In: Signal Molecules in Plants and Plant-Microbe Interactions, 91-98.Ed .Lugtenberg ,F.J.J .Berlin :Springer-Verlag ,42 5 pp.Nat oserie sH :Cel lbiology ,vol .36 . Hahn,M.G. ,Bucheli ,P. ,Cervone ,F. ,Doares ,S.H. ,O'Neill ,R.A. ,Darvill , A. & Albersheim, P. 1989a. The roles of cell wall constitutents in plant-pathogeninteractions .In : Plant-Microbe Interactions, vol.3 .Ed s Nester,E .& Kosuge ,T .Ne wYork :McGra wHill . Halverson, L.J. & Stacey, G. 1986. Signal exchange in plant-microbe interactions. Microbiological Reviews, 50,193-225 . Rickauer, M., Fournier, J. & Esquerré-Tugayé, M.T. 1989. Induction of proteinaseinhibitor si ntobacc ocel lsuspensio ncultur eb yelicitor so f Phytophtera parasitica var. nicotiniae. Plant Physiology, 90,1065-1070 . Shepherd,R.E .1954 . History of the Rose. NewYork :Th eMacMilla nCy . Sperry, J.S. & Tyree, M.T. 1988.Mechanis m ofwate r stress-induced xylem embolism. Plant Physiology. 88,581-587 . Woltering, E.J. 1987. The effects of leakage of substances from mechanicallywounde dros estem so nbacteria lgrowt han dflowe rquality . Scientia Horticulture, 33,129-136 . Zieslin,N . 1989.Postharves t control ofvas e life and senescence ofros e flowers. Acta Horticulturae, 261-264. 196 SUMMARY Thepaper scompile di nthi sthesi scompris ea serie so fsuccessivel yexecute d investigations into the role of micro-organisms in xylem plugging, and disturbanceo fth ewate rrelation san dth evas elif eo fcu tflowers .Fo rthi s purpose Rosa hybrids cultivar 'Sonia' (the hybrid tea-rose Rosa cultivar 'SweetPromise' )wa sselected . Chapter 1comprise s an introduction intoth e economic importance ofcu t flowersan dth ecapabilit yo fcu tflower sfo rpos tharves tlife .I naddition , abrie frevie w isgive no fliteratur e onphysiologica l andmicrobiologica l factors which can influence the vase life of cut flowers. Gaps in our knowledgeo fth emechanism s leadingt ovascula rblockag eo fcu tflower sar e discussed. Anoutlin e isgive n of: (1)th e selectiono f agreenhous e Rosa cultivara splan tmateria lt ostudy ,(2 )th eexperimenta lmethod sapplied ,(3 ) theresult so fpreliminar yinvestigations ,an d(4 )th emicrobiologica lfactor s investigated. Chapter2 show stha tstem so ffreshl ycu tflower scontai na wid evariet y andlo wnumber so fmicrobia lspecies .Th einitia lmicrobia lloa do nstem so f Rosa flowers was found tob e much lower than those on Chrysanthemum and Gerbera stems.Thei rdistributio no nth eflowe rste mi sno thomogeneous . Theste mflora ,predominantl y Enterobacter, Bacillus andfunga lspecies , lost its dominance in the vase fluid. The vase water showed an initial predominance of Pseudomonas species which do not require organic growth factors. Inth ecours e of thevas e life Enterobacter and Bacillus species becamedominant .Thes ebacteria lgener arequir eorgani cnutrient san dspecia l growthfactor sfo rthei rmultiplication . Fungal growth was shown at a later stage, mainly in vase fluids of Chrysanthemum and Gerbera flowers,whic hhav ea muc hlonge rvas elif ean da higherinitia lnumbe ro ffung io nthei rstem stha n Rosa flowerstems . Chapters3 ,4 an d5 demonstrat etha tth eexten to finfiltratio no fviabl e microbialcell sint oth exyle mvesse lsyste mo f Rosa and Gerbera cutflower s dependsupo nth enumbe ro fmicrobia lcell spe rm linitiall yadde dt oth evas e fluid,th eshap ean dth esiz eo fth eindividua lmicrobia lcell san dth ewidt h ofth exyle mvessels . 197 ScanningElectro nMicroscopi c(SEM )observation sa swel la sth eassessmen t of the number of bacteria infiltrated into .Rosaxyle m vessels showed in additionthat :(1 )th enumbe ro fbacteri awhic hinfiltrate dint oxyle mvessel s increasedwit htim eand ,(2 )thi snumbe rincrease dwit hincreasin gnumber so f bacteria initiallyadde d toth evas e fluid, (3)thi snumbe rdecrease dwit h increasingdistanc ebetwee ncuttin gpoin tan dsamplin gpoint ,(4 )onl ya mino r parto fth ebacteria lan dfunga lcell ssuspende d inth evas eflui dwas *abl e to infiltrate into thexyle m vessels of the flowers,a majo r part ofth e microbialcell sremaine dattache dt oth ecu tsurface ,(5 )eve nlo wnumber so f infiltratedmicrobia lcell scause da significantl ydecrease dconductivit yo f stemsegments ,(6 )a simila rwate rconductivit ydecreasin gphenomeno no fste m segmentswa sobserve dwhe nlo wnumber so fheat-inactivate dmicrobia lcell so r lowconcentration s ofmicrobia l EPS (exopolysaccharides)wer e added toth e vasefluid ,a sshow ni nChapter s5 an d6 . Chapter 7doe ssho wtha tpurifie dmicrobia lpecti c enzymesadde dt oth e vaseflui do f Rosa flowerscaus ea rapidl ydecreasin guptak eo fvas ewater , promoting desiccationo fth eflowe rpetal san d leaves.Thi sphenomeno nma y havebee ndu et oenzymati cdegradatio no fth epectin-linke dstructure so fth e xylem vessels as observed in SEM preparations. The SEM figures show: (1) degradationo fth exyle mvesse lwal lstructure , (2)loos eparticle s inth e vessels, (3)release dspira lvessels ,an d (4)injur yo fvesse lpits . Theconsequence sfo rth eornamenta lvalu eo fth erose so fvesse lpluggin g and flower desiccation were easy to assess by means of macroscopic observations.Sinc epecti c enzymes inextremel y lowconcentration s exerta dramaticeffec to ncu tflowers ,i ti slikel ytha tsom eo fth eproduct so fth e enzymaticactivit yelici tspecifi cresponse si nth eflowers ,furthe raffectin g theirvas elife . Chapter 8 covers the results of an electron microscopic study, using cryoSEM techniques and X-ray microanalysis to demonstrate the effects of aluminiumsulphat eadde dt oth evas eflui do f Rosa flowerso nth eabilit yo f bacteriae.g . Bacillus subtilis cellst oinfiltrat eint oth exyle mvessel so f the Rosa flowers.Th ebiocida lactivit yo faluminiu m sulphateadde d toth e vaseflui dwa snegligible .Aluminiu msulphat eprobabl yact sa sa flocculatin g agent,i nwhic hbacteria lcell sar eembedded .Indeed ,Al 3+ ionsreac teasil y 198 withinorgani can dorgani canioni csubstance se.g . Bacillus subtilis cellwal l peptides,substance so fwounde dste mcell san dste mcel lwal lcompound so fcu t .Rosaflowers .Th eBacillus/aluminiu mcomplexe scontribut et oth eformatio no f flockydeposit swhic hadher eont oth ecu tsurfac ean dth eadjacen tope nxyle m vesselwal lo fth eroses .Th eflocculate dmateria lma yfor ma filte rbe da t thecu tsurfac eo fth eflowe rstem ,hardl ydecreasin gth ewate ruptake ,bu t stronglyobstructin gth eembedde d Bacillus cellst oente rth evascula rsyste m ofth eroses ,a sclearl yshow nb ycryoSE Mfigures . 199 SAMENVATTING Dit proefschrift is samengesteld uit een inleidend hoofdstuk en zeven publicaties(hoofdstu k2-8) .Dez epublicatie szij nd eneersla gva nee nreek s onderzoekingen naar de rol van micro-organismen in de verstoring vanhe t vaasleven van gesneden bloemen. De hybride Rosa cultivar 'Sonia' is het geselecteerduitgangsmateriaa lvoo rdez eonderzoekingen . Hoofdstuk1 beva tee noverzich tva nhe teconomisc hbelan gva nsnijbloeme n inrelati eto the tvermoge nva nsnijbloeme nto tontplooiin ge ntoenam eva nd e sierwaardetijden she tvaasleven . Vervolgens is een beknopt overzicht gegeven van relevante literatuur betrekking hebbend op fysiologische en microbiologische factoren die het vaaslevenva nsnijbloeme nkunne nbeïnvloeden .Witt evlekke ni nonz ekenni sva n mechanismendi ekunne nleide nto tvatverstoppin ge nverkortin gva nhe tvaas leven van gesneden bloemen, worden besproken. Enkele kanttekeningen zijn gemaaktbij : (1)d eselecti eva nee nkasroo scultiva r 'Sonia'al suitgangs materiaalvoo rd ereek sonderzoekingen ,(2 )d egebruikt eanalyse-methoden ,(3 ) deresultate nva noriënteren donderzoek ,(4 )d ebestudeerd emicrobiologisch e factoren. Hoofdstuk 2. Op de stengels vanver s geoogste snijbloemen zijngering e aantallen geteld van verschillende soorten micro-organismen. De initiële microbiologischebesmettin gva nrozenstengel sblee kvee lgeringe rda ndi eva n Chrysanthemum en Gerbera stengels.D esoorte ne naantalle nzij nnie thomogee n verdeeldove rd ebloemstengels . Dominerende stengel micro-organismen - Enterobacter, Bacillus en schimmelsoorten - verliezen hun dominantie in het vaaswater waarin aanvankelijk Pseudomonas soortendomineren .Dez e Pseudomonas soortenhebbe n geenorganisch egroeifactore nnodig .I nee nlate rstadiu mva nhe tvaasleve n worden Enterobacter en Bacillus soortendominant .Dez ebacterië nhebbe nmeer , enhoger econcentratie snutriënte nnodi gvoo rvermenigvuldiging . Hetecologisc hmilie uva np iant estenge lsi nvaaswate rka ncompetiti etusse n de diverse microbengenera en species hebben bevorderd. Lekkage van groeistoffenui td eaangesnede nbloemstengel ska nteven shebbe nbijgedrage n totverschuivin gva nd edominant emicroflora .I nee nno glate rstadiu mva nhe t 200 vaasleven isoo kvermenigvuldigin gva nfung iwaargenomen ,voora l invaaswate r van Chrysanthemum en Gerbera. Beide cultivarshebbe n een langervaasleve n en eenhoger e initiële besmetting met fungi dan Rosa cultivars. Hoofdstuk 3. 4 en 5 laten zien dat de mate van infiltratie van levende micro-organismen in de xyleemvaten van gesneden Rosa en Gerbera stengels afhangtva nd eaantalle ncelle nva nmicro-organisme ndi eaanvankelij k aanhe t vaaswaterzij ntoegevoegd .Oo kd evor me nd egroott eva nd eindividuel e cellen spelen een rol, zowel als dewijdt e en de spreiding van de doorsnedenva nd e xyleemvaten. Rasterelectronenmlcroscopisch ewaarneminge n (SEM)zowe lal stellinge nva n bacteriën die zijn geïnfiltreerd in de xyleemvaten van Rosa stengels, laten zien: (1)d e aantallen ind exyleemvate n geïnfiltreerde bacteriecellen nemen toe met de toename van de contacttijd van stengel en vaaswater, (2) de aantallengeïnfiltreerd ebacteriecelle nneme nto eafhankelij kva nd eaantalle n bacteriëndi eaanvankelij kaa nhe tvaaswate rzij ntoegevoegd , (3)d eaantalle n geïnfiltreerde bacteriecellen nemen afbi j de toename van de afstand tussen het stengel-snijvlak enstengel-monsterpunt , (4)ee nklei ndee lva nd e inhe t vaaswatergesuspendeerd emicro-organisme nworde nme td evochtstroo m opgenomen in het xyleemsysteem van de roos; een groot deel van de vaaswater micro organismen zal zich hechten aan het stengelsnijvlak, (5) reeds door geringe aantallen geïnfiltreerde micro-organismen kan de geleidbaarheid van het xyleem-watertransport in belangrijke mate afnemen, (6) overeenkomstige verschijnselenva nafnam eva nd ewatergeleidbaarhei dva n Rosa stengelsegmenten zijn gemeten na inoculatie van het vaaswater met geringe aantallen geïnactiveerde microben-cellen, en bij lage concentraties microbiële exopolysacchariden, zoals is aangetoond ind ehoofdstukke n 5e n6 . Beidehoofdstukke n (5e n6 )late nzie nda tgeïnactiveerd emicro-organisme n en verbindingen met een hoog moleculair gewicht zoals exopolysacchariden (EPS), zich in vaaswater gedragen als inerte deeltjes die met de sapstroom wordenopgenome ndoo rd eplant .Dez einert edeeltje sverstoppe ni nzeker emat e de watertransportbaan, verstoren de waterhuishouding en verkorten het vaasleven van de roos. Micro-organismen, noch de geteste hoog moleculaire EPS-verbindingen zijn in staat de pitmembranen van de xyleemvaten te passeren via de capillaire 201 openingen met een doorsnede van ca. 50n m (De Stigter & Broekhuysen, 1986) enveroorzake n eenverstoppin g van deze caplllairen. Het vatverstoppend vermogen vanmicrobe n en microbiëel EPS is aangetoond door: (1) de fuchsine-rood test, (2) de watergeleidbaarheid test van stengeldelen, (3)electronenmicroscopisch e waarnemingen. Met de sapstroom worden ook laag-moleculaire microbiële metabolieten opgenomendoo rd eplant .Dez emetaboliete nkunne nd epitmembrane npassere ne n binnendringen inhoge rgelege nplant- ,blad- ,e nbloemdelen .Dez emetaboliete n kunnen toxisch zijnvoo rd egesnede nbloe m end esierwaard e doenafnemen ,he t vaasleven verstorend, zoals inhoofdstu k 6 is aangetoond. Hoofdstuk 7toon t aan dat gezuiverde pectolytische enzymen: pectaat lyase van Pseudomonas fluoresceins enpolygalacturonas eva n Kluyveromyces fragilis, toegevoegd aan het vaaswater van rozen, de vochtopname vrij plotseling vrij snelka ndoe nverminderen .Hierdoo rword tbevorder d datblad - en bloembladen verdrogen. De oorzaak van deze verschijnselen kan mogelijk worden toegeschrevenaa nenzymatisch eafbraa kva npectine-verbindinge ndi estevighei d verlenen aan de xyleem-vatwand-structuur. SEM-preparaten tonen aan: (1) aantastingva nd estructuu rva nxyleemvaten , (2)aantastin gva npitstructuren , (3)loss e deeltjes ind e xyleemvaten, (4)loss e spiraalvaten. Het onderscheid tussen vatverstopping en bloemverdroging en de gevolgen ervan voor de sierwaarde van gesneden rozen kan macroscopisch worden vastgesteld. Extreem lageconcentratie s pectolytische enzymen invaaswate r veroorzaken reeds een dramatisch effect op de sierwaarde van de gesneden bloemen. Deze verschijnselen wijzen op de mogelijkheid dat bepaalde producten van plantecelwand-pectolyse (oligosaccharinen)ee nprikke l (elicitor)kunne nzij n tot het optreden van specifieke reacties die het vaasleven van rozen verstoren. Inhoofdstu k8 i see ncryo-SE M techniektoegepast , gecombineerd met radio micro-analyses. Met deze methoden zijn de effecten bestudeerd van aluminiumsulfaat en Bacillus subtil is cellen invaaswate r ophe tvermoge nva n Bacillus subtilis cellen tot infiltratie enverstoppin g van xyleemvaten van gesneden rozen. Bij pH £ 4, werkt het aluminiumsulfaat als een uitvlokkingsagens, dat 202 bacterlecellenomhult .D egeflocculeerd e deeltjesvorme nee nfilterbe do phe t snijvlak van de bloem. Hierdoor neemt de wateropname in geringe mate af, terwijld einfiltrati eva nd eomhuld ebacterlecelle ni nbelangrijk emat eword t verhinderd en daarmee tevens de mate van vatverstopping, verstoring van de waterbalans enverkortin g vanhe t vaasleven. 203 Fig. 1. Lieke Jansen observing Rosa flower bud development and ornamental value. 204 Fig. 2. Ton Van der Meijden measuring water conductivity of Rosa cv. 'Sonia' stem segments. 3.Molecula r filtration of amicrobia l culture by a Millipore Pellico n recirculating cassette system; Henriette Put at the bench. 205 Glossary and abbreviations ABA: abscisic acid, abscisic acid: a phytohormone, which acts as growth inhibitor, as an inhibitor of germination, and as an accelerator of leaf abscission, abiotic: non-living; ofnon-biologica l origin. adaptation:changes(s )i na norganism ,o rpopulatio no forganisms ,b ymean s of which the organism(s) become more suited to prevailing environmental conditions. adhesion: in nature, micro-organisms often bind specifically or non- specifically to a substratum or other cells,adhesio n canb e mediated by specialized microbial components orstructures . aerob:a norganis mwhic hha sth eabilit yt ogro wi nth epresenc eo foxygen , aerobic: refers toa nenvironmen t inwhic h oxygen ispresen t ata partia l pressure similar to that in air; or, having the characteristics of aerobe(s). aerobiosis: the state or conditions inwhic h oxygen ispresent ; or, life in the presence ofair . anaerobe: an organism which has the ability to grow in the absence of oxygen; in nature, normally grow - or can only grow - in anaerobic habitats. antibiotic: anyumicrobia l compound which, in low concentrations (/tg/ml) caninhibi to rkil l (susceptible)micro-organisms ;o rcurrently ,refer st o natural,semi-syntheti can dwholl ysyntheti canti-microbia lcompound swhic h are effective at low concentrations. aseptic technique;precautionar ymeasure s taken topreven t thecontamina tion of cultures or plants by extraneous micro-organisms, auxins: any ofa clas s ofphytohormone swhic hpromot e stem elongation and play important roles inman y other plant processes. - bacterium (bacteria); prokaryotic micro-organisms: aheterogeneou s group ofusuall ysingl ecelle dorganisms ;mos thav ea characteristi c typeo fcel l wall; free living, occurring in almost every conceivable environment - sometimes under extreme physical conditions. bactericidal:bacteriocidal ;abl et okil la tleas tsom etype so fbacteria , bacteristaic:bacteriostatic ; ablet oinhibi tth egrowt han d reproduction of at least some types ofbacteria . biofilm: a film of micro-organisms, usually embedded in extracellular polymer,whic h adheres to surfaces submerged in, or subjected to aquatic environments;biofilm s can frequently cause fouling. 207 Glossary and abbreviations Brownlanmovement : randommovement smad eb ysmal l (ca.1 /im )particles ,o r organisms freely suspended in a fluid medium. callose: a linear (l-*3)-£-D-glucan which occurs associated with sieve platesan dsiev e tubes inhighe rplants .Callos ema yb edeposite d inplan t cellwall s inrespons e towoundin g and invasionb y pathogens. capsule: (bacterial) a layer of material external tobu t contiguous with the cell wall; includes any polysaccharide and/or protein surface layer: macrocapsules;microcapsules ;slim elayers .Th ecapsula rcompositio nvarie s with species, growth condition etc. (mycological) structures analogous to bacterial capsules occur in many fungia sgel-lik e ormucilaginou s layerssurroundin ghypa eo ryeas tcells , may diffuse into the surrounding medium. cavitation:wate rcolumn sbreakin gi nxyle mvessels ;a structura lbreakdow n or disruption of water columnm continuity causing a continued decline in xylem vessel conductance (Tyree & Dixon, 1984; Dixon & Peterson, 1989). CBS; Centraal Bureau voor Schimmelcultures, Oosterstraat 1, Baarn, The Netherlands.Yeas t Division CBS,Julianalaa n 67A, Delft, TheNetherlands . Chemotaxis: a taxis inwhic h the stimulus is a cocnentration gradient or aparticula r chemical (chemo-effector); cells make ane t movement towards higher concentrations ofsom e types ofchemo-effecto r (chemo-attrractant) whichma yo rma yno tb e anutrient , andawa y fromother s (chemorepellent)I chlorine: (asantimicorbia lagent) :chlorin e isa neffectiv emicro-bicida l agent used e.g. for the disinfection of water supplies. It is a strong oxidizing agent, it can react directly with certain groups in cells, chlorine refers to C12, hypochlorous acid and other active chlorine compounds. chloramines:exer tantimicrobia lactivit yb ydecomposin g slowly toreleas e chlorine; their activity ismuc h less rapid than that of hypochlorites; organic chloramines are less toxic and less irritating to biological tissues (human, animal, plants). countingmethods : (a)th e totalnumbe r oflivin gan ddea d cells ina give n volume or areao f asampl e isterme d the 'totalcel lcount 'whic h usually referst osingle-celle dorganisms ,bacteria ,spore so ryeasts .A tota lcel l count may be determined by microscopy or other instruments (coulter counter,turbidimeter) ;specifi ctype sca nb ecounte db y immunofluorescence microscopy; liquid samples containing small numbers can be membrane- filtered and the stained cells counted in situ; (b) viable cell count 208 Glossary and abbreviations refers toth enumbe r oflivin g cellspe rvolum eo rare a ina give n sample. Theyca nobviousl y includeonl ythos eorganism swhic har edetectabl eb yth e particular method used. A viable cell count may be determined by: direct examination in a counting chamber, using avita l stain; statistical by a multiple tubemethod ; orb y colonycount sexpresse d asCFU , colony forming units, developed on a solid medium on incubation; each colony developing froma singl eviabl ecell .Th eselectivit yo fth emediu man dth econdition s of incubation may significantly affect the number of viable cells which give rise tocolonies . Some types ofcell s tend tofor m clumps or to grow inchain s or filaments or inmicrocolonies ; inthes e cases CFU canb e any entity capable of giving rise to a single colony. cultivar: (cv.)a commercia l orcultivate d 'variety'o fa give nspecie s of plant or fungus. culture: (1)a liquid or solidmediu m ono rwithi nha s grown apopulatio n ofparticula rtype(s )o fmicro-organism sa sth eresul to fprio r inoculation and incubationo f thatmedium ; (2)th eproces s ofpreparin ga culture ; (3) to encourage growth of particular micro-organisms under controlled conditions. cytokinins: phytohormones which stimulate metabolism and cell division; theyar e6-N-substitute d adenineswhic har esynthesize dmainl ya tth eroo t apex and translocated via thexylem . Certainmicro-organism s may produce similar cytokinins. Da: dalton ;Atomi c Mass Unit. disinfectant:an ychemica lagen tuse dfo rdisinfection ;disinfectant swhic h are non-injurious to human, animal of plant tisseus may also be used as antiseptics or preservatives. disinfection: the destruction, inactivation or removal of those micro organisms likely to cause infection or other undesirable effects of the tissue orproduc t tob e disinfected. DP; degree of polymerizaton. electron microscopy: microscopy inwhic h an electron beam interacts with a specimen and subsequently contributes, directly or indirectly to the formation of an image of the specimen. (EM);(SEM) ; (CryoSEM). SEM:scannin gelectro nmicroscopy :electron spas sthroug hth especimen ,an d theenergie so fth eemergen telectron sar emeasure di na nanalyse ran duse d to construct an image of the specimen. CryoSEM: the specimen is cooled to very low temperatures without prior 209 Glossary and abbreviations dehydration,suc htha tth ewate ri nth especime nform sa 'glass;like'soli d withoutundergoin g crystallization, andca nb euse d toconstruc t animage , elicitors: molecules capable of inducing phytoalexin accumulation in plants. Blotic elicitors may be derived from or secreted by the plant- invading micro-organisms.Abioti c elicitors are ofnon-biologica l origin. Both types of elicitors may act indirectly by releasing phytoalexins or other endogenous elicitors fromplant s e.g. someplan t cellwal l polysac charides (Halverson & Stacey, 1986). embolism: air bubbles in xylem vessels. Numerous mechanisms have been proposed for how water stress causes embolism: e.g. (1) xylem pressure becoming increasingly negative; (2)mechanica l shock; (3)ai r coming out of vessel water solutions; (4) the air-seeding hypothesis (Zimmermann, 1983, Sperry & Tyree, 1988). endophytic bacteria: those bacteria inhabiting the leaf intercellular spaces or substomatal cavities. They are usually pathogenic (Henis & Bashan, 1986). Extensive exchanges between epiphytic and endophytic populations, especially through stomata, may occur, dependent on the microbiological conditions inside and outside the leaf. epiphytic bacteria: those bacteria that can be removed from above-ground plant parts by washing, they may be residents and casually occurring bacteria. They can be either pathogenic or saprophytic (Henis & Bashan, 1986). EPS; extracellular polysaccharide. ethylene: a phytohormone which, in higher plants, can induce e.g. germination, flowering, senescence. Ethyelene is also produced by plant tissues in response to stress, damage, wounding, infection by pathogens, or toplan t tissue hypersensitivity. filament: an elongated bacterial or fungal cell, one inwhic h the length exceeds thewidt hb y ca. 10time s ormore ; asheathe d orunsheathe d chain ofmicrobia l cells. Filamentous fungi. fimbriae: (fimbrium) thin, proteinaceous, chromosome- or plasmid-encoded filaments which extend from the cell surface of some types of microbial cell, occurring in'larg e numbers, they are typically able to facilitate cell-cello rcell-substratu madhesio n (functionallydistinc tfro mpil ian d flagella). flag eHum : athread-lik eappendag eresponsibl efo rmotilit yi nth emajorit y ofmotil e bacteria. 210 Glossary and abbreviations 5 fungus (fungi): unicellular, multicellular or coenocytic, heterotrophic, eukaryotic micro-organisms which do not contain chlorophyll and which characteristically form a rigid cell wall containing chitin and/or cellulose. Fungi are widespread innature . germicide: any (bio)chemicalagen t inhibitingmicrobia lmultiplicatio n or inactivatingmicrobia lvaibility; an ydisinfectant ,antiseptic ,sterilant , biocide. gibberellins; a class ofphytohormone swhic h are synthezised e.g. in the leaveso fplant san dwhic hregulate sste m elongationsee dgerminatio netc . - hybridization: (inplants ) the formation of ahybri d organism by a cross between genetically dissimilar organisms. - hydroponic cultures: plan t cultureswhic h aregrow n innutrien t solutions as an alternative for soil grown cultures (De Stichter, 1986). - hypha: (hyphae)i nman ymycelia l fungian d insom ebacteria :a branche do r unbranched filament,man yo fwhic htogethe rconstitut e thevegetativ e form of the organism and (in some species) form the sterile portion of a fruiting body: amas s ofvegetativ e hyphae isreferre d to as mycelium, IAA: indole 3-acetic acid aphotohormone . See:auxins . incubation: themaintenanc e ofinoculate dmedi ao rothe rtype so fmateria l at a particulate ambient temperature for a period of time to provide conditions suitable forgrowth . Specialized incubationpermit s control of humidity, light, radiation etc. infection:th einitia l entryo fa (pathogenic)micro-organis m intoa host ; or a culture which isno t handled aspectically. isolation: anyprocedur e inwhic h agive n species oforganism , present in a particular sample or environment, is obtained inpur e culture. - macroscopic: visible to theunaide d eye. - medium: (microbiol.)an yliqui do rsoli dpreparatio nmad especiall yfo rth e growth, storage or transport ofmicro-organisms . selective medium: amediu m which allows or encourages the growth of some type(s) of organisms inpreferenc e toothers . - microbicidal: able tokil l at least some types of micro-organisms. - microbistatic:abl et oinhibi tth egrowt han dreproductio no fa tleas tsom e type(s) ofmicro-organisms . microflora: (1)i na microbiologica l context:th etota lo fmicro-organism s normallyassociate dwit ha give nenvironmen t orlocation ; (2)i na broade r biological context: the microscopic plants and 'plant-like' organisms 211 Glossary and abbreviations (bacteria, fungi, algae etc.) normally present in given environment or location. - Mol.wt: Molecular weight. - motility (microbiol.) locomotion, i.e., anactiv eproces s inwhic h acel l ororganis mmove sfro mplac e toplac eb yexpendin generg yt oexer t force(s) directly against the surrounding medium or contiguous substratum. It enables an organism to respond to certain environmental stimuli (see taxis). mould: an imprecise term for any fungus which forms a visible layer of mycelium and/or spores on the surface of a product, which does not form macroscopic fruiting bodies. mucoid: viscous, slimy, mucus-like. The term is often applied to the colonies of certain capsulated bacteria (micro-organisms). mucopeptide: any peptide containing mucopolysaccharide (peptidoglycan). mucopolysaccharide: (glycosaminoglycan)a polysaccharid e whichcontain sa high proportion of amino sugars. mucoprotein: aprotei n covalently linked to amino sugars. mycelium: a group ormas s of discrete hyphae: the form of the vegetative thallus inman y types of fungi and incertai n bacteria. necrosis:localize d deathan ddegeneratio no ftissue s ina livin g organism - due to an infection or injury (plant leave necrosis). ornamental value: a visual parameter of cut flower bud development; expressed in %, compared with the 'normal' bud development of the cut flower when handled aseptically and placed in sterile tap water (or saccharose 1%w/v) . - parasite; parasitism: symbiosis, in which an organism (the parasite) benefits at the expense of the other (thehost) , theparasit e obtains its nutrients from the host, and the association is detrimental to the host (commensalism). Flantparasite: the plant as 'host'. - pascal (Pa):th e SIuni t ofpressure ; it is equal to 1 newton/m2. 1 mmHg - 1 torr - 133.3 Pa- 0.133 kPa (1Ba r - 1at m - 101.325kPa) . pathogen: any micro-organism which, by direct interaction with another organism, causes disease in that organism (phytopatogenic, plantpathoge- nic). pecticenzyme s:(pectinases ):enzyme swhic hdegrad epectins ,pectinase sar e produced by awid e variety of micro-organisms. - pectins: the major component of thepecti c polysaccharides, occurring in 212 Glossary and abbreviations plant cell walls, and intercellular regions ofplants . - pectinolytic (pectolytic): able to degrade pectins. - pellicle: acontinuou s or fragmentary film, ora ma t oforganisms , formed at the surface of a liquid culture by certain bacteria or fungi. - phytoalexin: smalllipolyti cmolecule stha thav eantibioti c activitiesan d are formed as arespon s tomicrobia l (elicitor)attac k ofplant s (Albers- heim &Valent , 1978;Albershei m & Darvill, 1985; Cervone et al., 1989). -phyotalexi n 'de novo': slaat op aanmaak. - pili (pilus): filamentous or elongated proteineaceous structures which extend from the cell surface inbacteri a (See: fimbriae). pit connection:a structur e locatedbetwee ntw oadjacen t cells (xylemcel l walls), connected by a continuous cytoplasmic (pit)membrane. - plasmodesma (plasmodesmata): a fine channel in a plant cell wall (or a fungal septum), through which cytoplasma can pass. prokaryote: synonymous with bacteria; micro-organisms in which the chromosomes are not separated from the cytoplasm by a membrane. eukaryote:micro-organism s (e.g.fungi ,algae )i nwhic hth echromosome sar e separated from the cytoplamsb y amembrane . - pulsing: a procedure or pretreatment of cut flowers, directly after harvest, to load flower tissues with certain chemicals (Halevy et al., 1978). phytohormones:planthormones ;endogenou scompound ssynthesize da ton esit e andtransporte dt oanothe rsit eo fth eplan twher ethe yexer ta physiologi cal effect. - oligosaccharins: naturally occurring plant cell wall carbohydrates exhibiting biological regulatory functions (Halverson & Stacey, 1986). race:physiologica l race (mycology); anon-specifi c designationwhic hma y refer e.g. to a strain or avariety . safety cabinet: sterile cabinet; (laminar flow) cabinet within which microbiological work is carried out so as topreven t contamination (air, personnel etc.). secundary metabolism: metabolism which isno t essential for,an dplay sn o part in, growth; it commonly occurs maximally under conditions of restricted growth or absence of growth. senescence: an irreversible process ofgradua l degeneration and disinter- gration of plant tissue, eventually leading to the death of the organism (Bruinsma, 1983). 213 Glossary and abbreviations signalexchange :i nplant-microb einteraction ;involve sth erecognitio nan d exchange of specificmolecule s by thehos t ormicrob e orbot h that elicit (or trigger) physiological, or biochemical responses that affect the development of the plant-microbe interaction (Halverson & Stacey, 1986). signal molecules: may be DNA, RNA, protein, lipid or polysaccharides, signals that initiate the process and, signals that maintain and control the interactions (Halverson & Stacey, 1986). silver: antimicrobial agent; aheavy metalwhic h inelementa l or compound form, is typically microbistatic in low concentrations; the functional antimicrobial moiety is the silver ion, which also may be microbicidal, depending on theAg + concentration used. smoot-roughvariations : (S--R)i nman ytype so fbacteria ;a chang ei ncell - surface composition which occurs spontaneously during invitr o or inviv o growth. S - smooth, glossy colonies; may be capsulated containing lipopolysaccharides; R - rough, dull colonies; may be non-capsulated, missing lipopolysaccharides in the cellwall . species:microbiology; oneo fth esmalle rtaxonomi egrouping swhic hdispla y ahig hdegre eo fmutua lsimilarit ypopulation swhic har ecapabl eo fgeneti c interchange and/or interbreeding, and which are evolving together in a common pattern. spore:a differentiate d formo fa micro-organis mwhic hma ybe : specialized fordissemination ; orproduce d inrespons e toenvironmenta l conditions;o r produced during an asexual or sexual reproductive process. sporicide: any chemical agent which inactivates microbial spores, staining: inmicrobiology : any process which imparts colour, opacity, or electron density, contrast, or confers the ability to fluoresce to parts ofa specime n -prio r toexaminatio nb ymicroscop e orelectro nmicroscope , sterile:refer s toth econditio no fa nobject , ora nenvironment , whichi s free of all living cells, all viable spores and all viruses (sterilant, sterilizer,sterilization) . strain: an organism, or population of organisms, regarded as being genetically similar or identical. symbiosis: any stable condition inwhic h two different organisms (symbi- onts) live inclos e physical association; or form a close association to their mutual benefit (mutualism). taxonomy: the science of biological classification, i.e. grouping of organisms according to their similarities (systematics)use d as ake y for 214 Glossary and abbreviations the identification of organisms, permits naming (nomenclature). taxis: Chemotaxis: a locomotive response to an external stimulus (air, light, chemical),exhibitin gb ycertai nmicro-organism s inwhic h direction of locomotion is related to the direction or oriention of the stimulus (kinesis,phototaxis , tropism, Carlile, 1980). toxic: poisonous, harmful (toxin). toxigenic: (toxinogenic):refer s to an organism which can produce one or more toxins. toxin: (microbiological):an y microbial product or component which, when present at low concentrations in cells or (plant)tissues of a higher (multicellular) organism, can cause injury by interferring with the structure or functional integrity of those cells or tissues. tropism: a response to a directional stimulus exhibiting by bending or growth of an organism (or part of it; stem or bud of a flower) in an orientation dictated by that of the stimulus (chemotropism; geotropism; phototropism, theotropism, etc.). transport systems:system swhic hpermi t theuptak eo refflu xo fsubstance s (nutrients, ions) across membranes, which are otherwise impermeable to those substances. tween: any of a range ofnon-ioni c surfactants which are polyoxyalkylene derivatives of fatty acid esters of sorbitan (Tween 20;40 ; 60;80) . tylose: (plant pathology); aballoon-lik e outgrowth from xylem parenchym cell which expands into, and blocks, the lumen of a xylem vessel or a tracheid; occurs as a respond towoundin g or infection (wilt syndrom due to Fusarium, Gilman & Steponkus, 1972). - vascular wilt: (plant pathology); any ofvariou s plant diseases inwhic h infection of thevascula r systemb y afunga l orbacteria lpathoge n results in wilting (flaccidity) of the plant. Pathogenic factors involved are: gummy polysaccharides; tylose, toxins, vessel blockage by the pathogen itself. - vase life: the potential longevity (days)o f the cut flower at the final consumer's home (Halevy &Mayak , 1979). - vase life end-point: the first signo fwilting , or thetota l deatho f the cut flower (Halevy &Kofranek , 1977). - water activity: (a„); the fraction of 'free' or 'available* water in a given substrate. - water conductivity: incu tflowers ; thereciproca lvalu eo fth e resistance 215 Glossaryan dabbreviation s towate rflo wthroug hxyle mvessel so fflowers ,expresse da sm lwate rpe r 30 min per transverse-sectional area of 5 cm vessel length (Gilman & Steponkus,1972 ;Chi n& Sacalis ,1977 ;Pu t& Va nde rMeijden ,1988) . - waterrelation s: i ncu tflower s; th ebalanc ebetwee nth ecapacit yo fth e flower for:wate r uptake,wate r transport, and transpiration (Halevy & Mayak,1981 ;Va nMeeteren ,1980) . yeasts:a non-taxonomica lcategor yo ffung idefine di nterm so fmorphologi cal and physiological criteria. The typical yeast is an unicellular saprotroph which can metabolize carbohydrates and in which asexual reproductionoccur sb ybudding ,o rsexua lb yascospor eformation . References Albersheim, P. &Valent ,B.S . 1978.Host-pathoge n interactions inplants . Plants, when exposed to oligosaccharides of fungal origin, defend themselvesb yaccumulatin gantibiotics .Journa lo fCel lBiology ,78 ,627 - 643. Albersheim,P .&Darvill ,A.G .1985 .Oligosaccharins .Scientifi cAmerican ,25 3 (3),44-50 . Bruinsma,1983 .Hormona lregulatio no fsenescence ,aging ,fadin gan dripening . In: Post-harvest physiology and crop preservation, pp. 141-163.Ed .M . Lieberman.Ne wYork :Plenu mPress . Carlile,M.J .1980 ,Positionin gmechanism s- Th erol eo fmotility ,taxi san d tropismi nth elif eo fmicro-organisms .In :Contemporar ymicrobia lecology . pp. 55-74.Eds .D.C .Ellwood ,J.N .Hedger ,M.J .Latham ,J.M .Lync h& J.H . Slater.London :Academi cPress . Cervone,F. ,D eLorenzo ,G. , Salvi,G. ,Bergmann ,C. ,Hahn ,M.G. ,Ito ,Y. , Darvill,A .& Albersheim ,P .1989 .Releas eo fphytoalexi nelicitor-activ e ologogalacturonidesb ymicrobia lpecti cenzymes .In :Signa lmolecule si n plantsan dplant-microb e interactions,pp .85-90 .Ed .Lugtenburg ,B.J.J . Berlin:Springe rVerla g& Nat oScientifi cAffair sDivision . Chin,C .& Sacalis ,J.N .1977 .Metabolis mo fsucros ei ncu trose sII .Movemen t and inversion of sucrose absorbed by cut rose flowers.Journa l of the AmericanSociet yfo rHorticultura lScience ,102 ,537-540 . De Stigter, H.C.M. Water balance of cut and intact Sonia rose plants. Zentralblattfü rPflanzenphysiology ,99 ,131-140 . 216 Glossaryan dabbreviation s Dixon,M.A .& Peterson ,C.A . 1989.A re-examinatio no fste mblockag e incu t roses.Scienti aHorticulturae ,38 ,277-285 . Gilman,K.F .& Steponkus ,P.L .1972 .Vascula rblockag ei ncu troses .Journa l ofth eAmerica nSociet yfo rHorticultura lScience ,97 ,662-667 . Halevy,A.H. ,Byrne ,T.G. ,Kofranek ,A.M. ,Farnham ,D.S. ,Thompson ,J.F . & Hardenburg, R.E. 1978. Evaluation of postharvest handling methods for transcontinental truckshipmen to fcarnation ,chrysanthemums ,an droses . Journalo fth eAmerica nSociet yfo rHorticultura lScience ,103 ,151-155 . Halevy, A.H. & Mayak, 1981. Senescence and postharvest physiology ofcu t flowers. Part II. Horticultural Reviews, 3, 59-143. Ed. Janick J., Westport.USA :AV IPubl .Cy .Inc . Halverson, L.J. & Stacey, G. 1986. Signal exchange in plant-microbe interactions.Microbiologica lReviews ,50 ,193-225 . Henis,Y .& Bashan ,Y .1986 .Epiphyti csurviva lo fbacteria llea fpathogens . In:Microbiolog yo fth ephyllosphere ,pp .252-268 .Ed .Fokkema ,N.J .& Va n denHeuvel ,J .Cambridge :Universit yPress . Put, H.M.C. & Van der Meijden, T. 1988. The infiltration ability of Pseudomonas putida cellsstrai n4 8int oxyle mvessel so fcu t Rosa cv.'Sonia' .Journa l ofApplie dBacteriology . 64,197-208 . Singleton, P. & Salnsbury, D. 1988. Eds. Dictionary of Microbiology and MolecularBiology .Ne wYork :Joh nWile y& Sons .101 9pp .Genera lreferenc e (whenno treferre dt oothe rauthors) . Sperry,J.S .& Tyree ,M.T .1988 .Mechganism so fwate rstress-induce dxyle m embolism.Plan tPhysiology ,88 ,581-587 . Tyree,M.T .& Dixon ,M.A .1986 .Wate rstress-induce dcavitatio nan dembolis m insom ewood yplants .Physiolog yPlantarum ,66 ,397-405 . VanMeeteren ,U . 1980.Wate r relations andkeepin g quality of cutgerber a flowers.Wageningen :Agricultura lUniversity ,7 8pp . Zimmerman,M.H . 1983.Xyle m structure and the ascent of sap.Timell ,T.E . (Ed.)Berlin :Springe rVerlag .14 3pp . 217 CURRICULUM VITAE Dit proefschrift Is vervaardigd door Henriette Maria Christina Put. Zij is geboren op de eerste september 1925 de De Rijp (NH)ui t een Friese vader en eenAmsterdams e moeder, als derde kind en tweede dochter. Haar schoolopleidingen ontving zij in De Rijp (NH) en in Bergen (NH). Zi j studeerde inAmsterdam , Lille (France)e n Oeiras (Portugal). Zijwerkt e achtereenvolgens bij: -Nederland s Instituut voor Volksvoeding, J.D. Meijerplein, Amsterdam. 1947-1950 . Isoleren, identificeren,,conserveren , toetsenva n het vitamine B2, B6 en B12 producerend vermogen vanmicro - organismen. Onderzoekproject in opdracht van de CSM (Centrale Suiker Maatschappij), Amsterdam. -Central e SuikerMaatschappij ,Gorinchem . 1950-1953 . Chefproductie - enkwaliteitscontrol e laboratorium. . Pilotplan t productie vanvitamin e B6 enB 12 langs microbiëleweg , -Thomasse n & Drijver-Verblifa N.V., Deventer. Research & Development 1953-1990 . Hoofd van de sectie voedingsmiddelenmicrobiologie 1953-1983 . Onderzoek naar de relatie tussen hitteconservering vanvoedingsmiddele n en overlevingskans van micro- organismen. . Tussen 1961 en 1987verschene n 20publicaties . . Onderzoek naar de reïnfectiekansva nmicro-organisme n in door middel vanhitt e geconserveerde verpakte voedingsmiddelen; biofysische aspecten. Hierover verschenen twee publicaties, envertalinge n ervan inhe t Japans en Frans. .Adviseu r voedingsmiddelenmicrobiologie en -technologie 1953-1987 . Rapporten, lezingen en lessen. Deelname aan o.a. .Adviescommissi e inzake antibiotica invoedings middelen (Gezondheidsraad, Min.WVC) ,1975-1980 . . Werkgroep Codex Alimentarius (FAO/WHO); Code of hygienic practice for low-acid canned foods,1977-1986 . .Werkgroe p FAO/WHO Can Damage -visua l inspection -; CFPRA, Campden,U K (microbiology panel)1983-1986 . . Werkgroep verpakte hittegeconserveerde voedings middelen (Min.WVC) ;advie s Nederlandse wetgeving, 1981-1984.Sporenwerkgroe p Nederland, èinds1972 . - Sprenger Instituut,Wageningen . Gedetacheerd namens TDV Deventer. 1983-1987 Deventer,januari 1991 218 mmsNw^^M4*w w _L i _i_ _L _|_ J_ _L _L O o>oor^