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Identification of bacteria in the vase water of roses, and the effect of the isolated strains on water uptake
Scientia Horticulturae, 35 (1988) 285-291 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
285
Identification of B a c t e r i a in the Vase Water of Roses, and the Effect of the Isolated Strains on Water U p t a k e YKE DE WITTE and WOUTER G. VAN DOORN
Sprenger Institute, P.O. Box 17, 6700 AA Wageningen (The Netherlands) (Accepted for publication 23 November 1987)
ABSTRACT De Witte, Y. and van Doorn, W.G., 1988. Identification of bacteria in the vase water of roses, and the effect of the isolated strains on water uptake. Scientia Hortic., 35: 285-291. The bacteria that were predominant in the vase water of 'Sonia' roses were isolated and identified. All strains belonged to the genus Pseudomonas or were Alcaligenes faecalis. The isolated strains were grown in pure culture and then added singly to autoclaved water at 105 and 107 cfu m1-1. Water uptake of 'Sonia' roses was not affected at 105 cfu m1-1, but at 107 cfu ml -~ water uptake was inhibited from the first day onward. Eight tested bacterial strains all reduced water uptake to the same degree. The isolated bacterial strains showed no pectolytic activity. Vascular blockage by these strains was therefore apparently not due to pectolytic breakdown of xylem cell walls. Keywords: bacteria; cut rose flowers; vase water; water uptake. Abbreviations: cfu, colony forming units.
INTRODUCTION
The vase life of cut roses is often very short because of stem bending just below the flower. Xylem blockage is one of the main reasons for stem bending (Mayak et al., 1974). Blockage of the xylem could be due to air emboli, physiological processes or bacteria (Aarts, 1957; Durkin, 1980). When a mixed population of bacteria originating from rose stems was added to vase water, the uptake of water was inhibited only at bacterial concentrations higher than 107 cfu m1-1 (Van Doorn et al., 1986). Zagory and Reid (1986) showed that two bacterial strains isolated from carnation vase solutions did not reduce vase life of cut roses at a concentration of 10s cfu ml-1, but at the same concentration another strain reduced vase life by 2 days. In the present experiment the predominant bacteria from the vase water of
0304-4238/88/$03.50
© 1988 Elsevier Science Publishers B.V.
286 roses were identified, and the effect of the isolated strains on water uptake of roses was determined. In order to assess the role of the exogenous concentrations of bacteria on the vase life of roses, the total concentration of bacteria in vase water was measured. MATERIALSAND METHODS Bacteria. - Ten rose flowers (Rosa hybrida, cultivar 'Sonia') were placed in a bottle with tap water for 6 days at 20°C, 60% RH and 15/~mol m -2 s -1 photosynthetically active radiation at flower level. The vase water was stirred and 0.1-ml samples were plated (spatule method) on 2-decimal dilution series of the following media: Plate Count Agar (Oxoid, 1980); Glutamic acid-Starch-Phenolred Agar, to which penicillin and pimaricin were added (Kielwein, 1969); Tryptone Soya Broth with 12 g l-1 Oxoid Agar No. 3 and 2 mg l-1 crystal violet (Oxoid, 1980; Mossel and Tamminga, 1980); Tryptone Soya Broth with 12 g 1-~ Oxoid Agar No. 3, covered by a 5-mm layer of Violet Red Bile Glucose Agar (Oxoid, 1980; Mossel and Tamminga, 1980). The four different plate types are used to select aerobic bacteria, Aeromonas and Pseudomonas species, Enterobacteriaceae plus Pseudomonas species (suppression of Gram positive bacteria), and Enterobacteriaceae, respectively (Oxoid, 1980; Kielwein, 1969; Mossel and Tamminga, 1980). The plates were incubated for 2 days at 30 ° C. The colonies were visually inspected and 39 single colonies were selected, based on colour and colony form, and replaced on Plate Count Agar until pure cultures were obtained. Colonies were obtained from all mentioned types of plates, except from the last one where virtually no growth occurred. The isolates were tested for Gram stain reaction and oxidase reaction (according to Gerhardt et al., 1981) and for motility according to the method of Van Riemsdijk (1949). The capacity of the strains to degrade pectin (method according to Mossel and Tamminga, 1980), glucose, starch and gelatin (Gerhardt et al., 1981, gelatin test with HgC12HN03) was also determined. The isolated bacteria were further characterized on the O X I / F E R M system (Oberhofer, 1979). Pure culture isolates of 8 bacterial strains were incubated on Plate Count Agar for 3 days at 30 ° C. Individual strains were washed off the agar with sterile 0.85% NaC1 solution and the resulting bacterial suspensions were adjusted to 105 and 107 cfu m1-1. The vase water of cut roses was regularly sampled to determine the total number of bacteria. Five roses were individually kept in 100 ml tap water, that was not renewed, at 20°C, 60% RH and 15 zmol m -2 s -1 photosynthetically
287
active radiation at flower level. In one series the flower preservative Chrysal (Bendien, Naarden, The Netherlands) was added to the vase water, and in another series the preservative Aadural (Aagrunol, Groningen, The Netherlands) was added at the recommended concentrations. Experiments included 4 replications. Water samples were placed on Plate Count Agar using the spiral plate method (model C spiral count machine, Don Whitley Scientific Instruments, Yorkshire, Great Britain). P l a n t s . - 'Sonia' roses were obtained from a commercial grower, transported
dry to the laboratory and stored in tap water at 5 °C until the start of the experiments. Before placing the roses in the vase, leaves were removed from each stem until only the uppermost five leaves remained. The stems were recut in air (removing about 5 cm) and immediately placed in the vase solution. Roses were placed individually in autoclaved 150-ml bottles with 100 ml of an appropriate bacterial suspension. Roses in sterile tap water served as controls. Water uptake was determined by daily weighing. Water uptake data for Days 1, 2 and 3 were subjected to analysis of variance and F-test. RESULTS
The predominant bacterial strains from 6-day vase-water were isolated, and were classified as belonging to 7 species. Six species belonged to the genus P s e u d o m o n a s and one to the genus A l c a l i g e n e s (Table 1 ). The ability of each strain to degrade glucose, starch, pectins and gelatin is also given in Table 1. TABLE
1
Bacterial strains isolated from6-day-old Oxydase
Gram
vase-water of 'Sonia' roses. +, positive; -, Motility
test
Alcaligenes faecalis Pseudomonas aeruginosa-1 P. aeruginosa-2 P. cepacia-1 P. cepacia-2 P. maltophilia P. putida P. stutzeri P. vesicularis-1 P. vesicularis-2
Colour on Plate
negative
D e g r a d a t i o n of."
Count Agar Starch
Gelatin
Pectin
Glucose
+
-
+
Grey-white
-
+
-
- (*)
+ +
-
+ -
White, fluorescent
+
-
-
+
White, fluorescent
-
+
-
+
+
-
+
Yellow
-
-
-
+
+
-
+
Yellow
+
+
-
- (*)
-
-
+
Yellow
-
+
-
+ / -
+
-
+
White, fluorescent
-
-
-
+
+
-
+
White, fluorescent
-
+
-
+
+
-
+
Yellow
-
+
-
+
+
-
+
Yellow
+
-
-
+
(*) n o a c i d f o r m e d f r o m glucose.
288 TABLE 2 Water uptake (ml per day per flower) of 'Sonia' roses with initial vase solution bacterial populations of 105 cfu ml - 1, or about 10° cfu ml - 1 (water control). All data are the means of 10 replications
Control
Alcaligenes [aecalis Pseudomonas aeruginosa-1 P. aeruginosa-2 P. cepacia-1 P. maltophilia P. putida P. stutzeri P. vesicularis-1
Day 1
Day 2
Day 3
37 40 38 38 36 38 40 33 39
32 26 28 31 26 27 27 26 26
24 21 21 20 19 17 19 18 21
w a t e r uptake (g per f l o w e r per day) 40
32
"
~
o
~
control
24
8
0
I 1
I 2
• P. cepacia-1 • P. aeruginosa~ 1 P aeruginosa- 2 P stutzeri I 3
I
I
I
1
2
3
R putida IR vesicularis- 1 A. faecalis R maltophilia days
Fig. 1. The effect of isolated bacterial strains (P. =Pseudomonas, A. =Alcaligenes) on water uptake of 'Sonia' roses. 107 cfu ml-1 vase water were added at the start of the experiment. All data are the means of 10 replications. None of the isolated strains degraded pectin. Based on the chemical charact e r i s t i c s ( T a b l e 1 ), t w o d i f f e r e n t s t r a i n s w e r e f o u n d in P. aeruginosa, P. cepacia a n d P. vesicularis. W h e n a d d e d to t h e v a s e w a t e r a t 105 m l - 1 , n o n e o f t h e i s o l a t e d s t r a i n s h a d a n effect o n w a t e r u p t a k e o f ' S o n i a ' r o s e s a f t e r 1 day, as c o m p a r e d to a w a t e r c o n t r o l ( T a b l e 2). O n l y t h e w a t e r - u p t a k e v a l u e for t h e first d a y w a s considered, since o t h e r o r g a n i s m s m a y s u b s e q u e n t l y develop, a n d b a c t e r i a l c o n c e n t r a t i o n s will p r o b a b l y increase. A t 107 b a c t e r i a m l - 1 , all o f t h e s t r a i n s r e d u c e d w a t e r u p t a k e b y t h e first d a y (Fig. 1 ). T h e d i f f e r e n c e s a m o n g t h e s t r a i n s w e r e n o t s t a t i s t i c a l l y s i g n i f i c a n t (P(F) >0.05).
289 n u m b e r of v i a b l e b a c t e r i a p e r ml.
106 1°54 /
"~.
control
10 10 3
102
~
1 10
|__•
•
,
.
I~
Chrysal , Aodural
,
,
,
,
~
0
2
4
6
8
,
10 d a y s
Fig. 2. The number of cfu of bacteria in the vase water of cut roses. The flower preservatives Chrysal and Aadural were added in the commercially recommended dose (11 and 15 g l - 1, respectively) at the start of vase life.
The population of viable bacteria in uninoculated vase water of roses reached a level of 10~ cfu m l - 1 by the third day of vase life, and declined slightly thereafter (Fig. 2 ). The flower preservatives Chrysal and Aadural prevented development of high bacterial numbers. DISCUSSION
The isolated strains inhibited water uptake of rose flowers at 107 cfu ml-1, but not at 105 cfu m l - 1. This is in accordance with the effects of a mixed culture of bacteria, also originating from rose vase water (Van Doorn et al., 1986). It is also consistent with the effect of two isolated strains tested against 'Cara Mia' roses at 106 cfu m1-1, showing no effect on vase life (Zagory and Reid, 1986). The latter authors, however, found a fluorescent Pseudomonas species from carnation vase water that reduced vase life of roses at 106 cfu ml-1. Since we made 39 isolations from the vase water, it is likely that the majority of the strains present in the water were included in our study. The isolates were made from plates that contained the highest dilution, and thus represent bacterial strains that were quantitatively dominant. We apparently did not isolate the fluorescent Pseudomonas of Zagory and Reid (1986). The dominant flora may vary because the initial species composition in the vase water will depend on that of the stem surface. The surface flora in turn will depend on the conditions of cultivation (Dickinson and Preece, 1976). The similarity of the effect of the various bacterial strains on water uptake was contrary to expectation. A strain of P. cepacia has been found to elicit the hypersensitive reaction in the tobacco leaf, an indicator for potential phytopathogenesis, whereas P. putida did not show this reaction (Gardner et al.,
290
1982). Furthermore, visual inspection of the bacterial colonies on Plate Count Agar showed extensive slime (extracellular polysaccharide) production in some strains and less in others. The role of extracellular polysaccharides in vascular blockage is not known, but may be important. Whatever the mechanism by which bacteria cause vascular blockage may be, the hypothesis put forward by Burdett (1970) that bacteria produce pectic enzymes which digest cell walls and thereby cause vessel plugging seems unlikely because neither of the isolates in the present study was found to show pectolytic activity. The flora isolated from the vase water of 'Sonia' roses was similar to that reportedly found by others in flower vase water. P. aeruginosa occurred in 90% of tested hospital flower vases, while P. cepacia occurred in only 4% (Taplin and Mertz, 1973; McClary and Layne, 1977). In containers at wholesale flower distribution centers, a number of Pseudomonas species were also found, as well as A lcaligenes faecalis (Ford et al., 1961 ). These papers mention several species not found in the present experiment, whereas we isolated P. maltophilia, P. putida, P. stutzeri and P. vesicularis, which were not mentioned in the above studies. The tested flower preservatives prevented development of high bacterial numbers in vase water. Their effectiveness against bent necks in roses may therefore be partially due to the presence of an effective bactericide. The concentration of bacteria in uninoculated vase water; without a flower preservative, reached levels slightly higher than 105 cfu ml -1 by the third day of vase life (Fig. 2). Taplin and Mertz (1973) found, also after 3 days of vase life, a concentration of 10 ~ cfu m1-1, and lower concentrations earlier. Marousky (1976) sampled the vase-water from 'Forever Yours' roses after 3 days and found 1.5 × l0 s cfu ml-1. This indicates that, under some conditions, the exogenous concentration of bacteria may account for vascular blockage in roses, from about the third day of vase life onward. However, blockage and bent neck are often already observed on the first day of vase life. The exogenous concentration of bacteria may be too low to account for such early blockage. We suggest that air emboli a n d / o r the endogenous number of bacteria in stems may be a cause of bent neck observed early in vase life. ACKNOWLEDGEMENTS
We thank Peter Haghuis for skilful technical assistance and Devon Zagory for critically reading the manuscript. REFERENCES Aarts, J.F.T., 1957. Over de houdbaarheid van snijbloemen. Meded. Landbouwhogesch. Wageningen, 57(9): 1-62.
291 Burdett, A.N., 1970. The cause of bent neck in cut roses. J. Am. Soc. Hortic. Sci., 95: 427-431. Dickinson, C.H. and Preece, T.F., 1976. Microbiology of Aerial Plant Surfaces. Academic Press, London, 669 pp. Durkin, D.J., 1980. Factors affecting hydration of cut flowers. Acta Hortic., 113: 109-117. Ford, H.E., Clark, D.T. and Stinson, R.F., 1961. Bacteria associated with cut flower containers. Proc. Am. Soc. Hortic. Sci., 77: 635-636. Gardner, J.M., Feldman, A.W. and Zablotowicz, R.M., 1982. Identity and behavior of xylemresiding bacteria in rough lemon roots of Florida citrus trees. App|. Environ. Microbiol., 43: 1335-1342. Gerhardt, P., Murray, R.G.E., Costilow, R.N., Nester, E.N., Wood, W.A., Kireg, N.R. and Philips, G.B., 1981. Manual of Methods for General Bacteriology. American Society of Microbiology, Washington, 524 pp. Kielwein, G., 1969. Ein Nfihrboden zur selektiven Ztichtung yon Pseudomonaden und Aeromonaden. Arch. Lebensmittelhyg., 20:131-133. Marousky, F.J., 1976. Control of bacteria in vase water and quality of cut flowers as influenced by sodium dichloroisocyanurate, 1,3-dichloro-5,5-dimethylhydantoin, and sucrose. USDA Agricultural Research Service. Publication S-115, 14 pp. Mayak, S., Halevy, A.H., Sagie, S., Bar-Joseph, A. and Bravdo, B., 1974. The water balance of cut rose flowers. Physiol. Plant., 31: 15-22. McClary, C.L. and Layne, J.S., 1977. Flower vase water and ornamental potted plants as reservoirs for gram-negative pathogenic bacteria. Dev. Ind. Microbiol., 18: 731-739. Mossel, D.A.A. and Tamminga, S.K., 1980. Methoden voor het Microbiologisch Onderzoek van Levensmiddelen. Noordervliet, Zeist, The Netherlands, 322 pp. Oberhofer, T.R., 1979. Comparison of the AP120E and the OXI/FERM systems in identification of non-fermentative and oxidase-positive fermentative bacteria. J. Clin. Microbiol., 9: 220226. Oxoid, 1980. The Oxoid Manual of Culture Media, Ingredients and other Laboratory Services. 4th edn. Oxoid Ltd., Basingstoke, Great Britain, 310 pp. Taplin, D. and Mertz, P.M., 1973. Flower vases in hospitals as reservoirs of pathogens. Lancet, 7841: 1279-1281. Van Doorn, W.G., Buis, H.C.E.M. and de Witte, Y., 1986. Effect of exogenous bacterial concentrations on water relations of cut rose flowers. II. Bacteria in the vase solution. Acta Hortic., 181: 463-465. Van Riemsdijk, M., 1949. Laboratory Manual in Bacteriology and Serology. Swets and Zeitlinger, Amsterdam, 367 pp. Zagory, D. and Reid, M.S., 1986. Role of vase solution microorganisms in the life of cut flowers. J. Am. Soc. Hortic. Sci., 111: 154-158.
285
Identification of B a c t e r i a in the Vase Water of Roses, and the Effect of the Isolated Strains on Water U p t a k e YKE DE WITTE and WOUTER G. VAN DOORN
Sprenger Institute, P.O. Box 17, 6700 AA Wageningen (The Netherlands) (Accepted for publication 23 November 1987)
ABSTRACT De Witte, Y. and van Doorn, W.G., 1988. Identification of bacteria in the vase water of roses, and the effect of the isolated strains on water uptake. Scientia Hortic., 35: 285-291. The bacteria that were predominant in the vase water of 'Sonia' roses were isolated and identified. All strains belonged to the genus Pseudomonas or were Alcaligenes faecalis. The isolated strains were grown in pure culture and then added singly to autoclaved water at 105 and 107 cfu m1-1. Water uptake of 'Sonia' roses was not affected at 105 cfu m1-1, but at 107 cfu ml -~ water uptake was inhibited from the first day onward. Eight tested bacterial strains all reduced water uptake to the same degree. The isolated bacterial strains showed no pectolytic activity. Vascular blockage by these strains was therefore apparently not due to pectolytic breakdown of xylem cell walls. Keywords: bacteria; cut rose flowers; vase water; water uptake. Abbreviations: cfu, colony forming units.
INTRODUCTION
The vase life of cut roses is often very short because of stem bending just below the flower. Xylem blockage is one of the main reasons for stem bending (Mayak et al., 1974). Blockage of the xylem could be due to air emboli, physiological processes or bacteria (Aarts, 1957; Durkin, 1980). When a mixed population of bacteria originating from rose stems was added to vase water, the uptake of water was inhibited only at bacterial concentrations higher than 107 cfu m1-1 (Van Doorn et al., 1986). Zagory and Reid (1986) showed that two bacterial strains isolated from carnation vase solutions did not reduce vase life of cut roses at a concentration of 10s cfu ml-1, but at the same concentration another strain reduced vase life by 2 days. In the present experiment the predominant bacteria from the vase water of
0304-4238/88/$03.50
© 1988 Elsevier Science Publishers B.V.
286 roses were identified, and the effect of the isolated strains on water uptake of roses was determined. In order to assess the role of the exogenous concentrations of bacteria on the vase life of roses, the total concentration of bacteria in vase water was measured. MATERIALSAND METHODS Bacteria. - Ten rose flowers (Rosa hybrida, cultivar 'Sonia') were placed in a bottle with tap water for 6 days at 20°C, 60% RH and 15/~mol m -2 s -1 photosynthetically active radiation at flower level. The vase water was stirred and 0.1-ml samples were plated (spatule method) on 2-decimal dilution series of the following media: Plate Count Agar (Oxoid, 1980); Glutamic acid-Starch-Phenolred Agar, to which penicillin and pimaricin were added (Kielwein, 1969); Tryptone Soya Broth with 12 g l-1 Oxoid Agar No. 3 and 2 mg l-1 crystal violet (Oxoid, 1980; Mossel and Tamminga, 1980); Tryptone Soya Broth with 12 g 1-~ Oxoid Agar No. 3, covered by a 5-mm layer of Violet Red Bile Glucose Agar (Oxoid, 1980; Mossel and Tamminga, 1980). The four different plate types are used to select aerobic bacteria, Aeromonas and Pseudomonas species, Enterobacteriaceae plus Pseudomonas species (suppression of Gram positive bacteria), and Enterobacteriaceae, respectively (Oxoid, 1980; Kielwein, 1969; Mossel and Tamminga, 1980). The plates were incubated for 2 days at 30 ° C. The colonies were visually inspected and 39 single colonies were selected, based on colour and colony form, and replaced on Plate Count Agar until pure cultures were obtained. Colonies were obtained from all mentioned types of plates, except from the last one where virtually no growth occurred. The isolates were tested for Gram stain reaction and oxidase reaction (according to Gerhardt et al., 1981) and for motility according to the method of Van Riemsdijk (1949). The capacity of the strains to degrade pectin (method according to Mossel and Tamminga, 1980), glucose, starch and gelatin (Gerhardt et al., 1981, gelatin test with HgC12HN03) was also determined. The isolated bacteria were further characterized on the O X I / F E R M system (Oberhofer, 1979). Pure culture isolates of 8 bacterial strains were incubated on Plate Count Agar for 3 days at 30 ° C. Individual strains were washed off the agar with sterile 0.85% NaC1 solution and the resulting bacterial suspensions were adjusted to 105 and 107 cfu m1-1. The vase water of cut roses was regularly sampled to determine the total number of bacteria. Five roses were individually kept in 100 ml tap water, that was not renewed, at 20°C, 60% RH and 15 zmol m -2 s -1 photosynthetically
287
active radiation at flower level. In one series the flower preservative Chrysal (Bendien, Naarden, The Netherlands) was added to the vase water, and in another series the preservative Aadural (Aagrunol, Groningen, The Netherlands) was added at the recommended concentrations. Experiments included 4 replications. Water samples were placed on Plate Count Agar using the spiral plate method (model C spiral count machine, Don Whitley Scientific Instruments, Yorkshire, Great Britain). P l a n t s . - 'Sonia' roses were obtained from a commercial grower, transported
dry to the laboratory and stored in tap water at 5 °C until the start of the experiments. Before placing the roses in the vase, leaves were removed from each stem until only the uppermost five leaves remained. The stems were recut in air (removing about 5 cm) and immediately placed in the vase solution. Roses were placed individually in autoclaved 150-ml bottles with 100 ml of an appropriate bacterial suspension. Roses in sterile tap water served as controls. Water uptake was determined by daily weighing. Water uptake data for Days 1, 2 and 3 were subjected to analysis of variance and F-test. RESULTS
The predominant bacterial strains from 6-day vase-water were isolated, and were classified as belonging to 7 species. Six species belonged to the genus P s e u d o m o n a s and one to the genus A l c a l i g e n e s (Table 1 ). The ability of each strain to degrade glucose, starch, pectins and gelatin is also given in Table 1. TABLE
1
Bacterial strains isolated from6-day-old Oxydase
Gram
vase-water of 'Sonia' roses. +, positive; -, Motility
test
Alcaligenes faecalis Pseudomonas aeruginosa-1 P. aeruginosa-2 P. cepacia-1 P. cepacia-2 P. maltophilia P. putida P. stutzeri P. vesicularis-1 P. vesicularis-2
Colour on Plate
negative
D e g r a d a t i o n of."
Count Agar Starch
Gelatin
Pectin
Glucose
+
-
+
Grey-white
-
+
-
- (*)
+ +
-
+ -
White, fluorescent
+
-
-
+
White, fluorescent
-
+
-
+
+
-
+
Yellow
-
-
-
+
+
-
+
Yellow
+
+
-
- (*)
-
-
+
Yellow
-
+
-
+ / -
+
-
+
White, fluorescent
-
-
-
+
+
-
+
White, fluorescent
-
+
-
+
+
-
+
Yellow
-
+
-
+
+
-
+
Yellow
+
-
-
+
(*) n o a c i d f o r m e d f r o m glucose.
288 TABLE 2 Water uptake (ml per day per flower) of 'Sonia' roses with initial vase solution bacterial populations of 105 cfu ml - 1, or about 10° cfu ml - 1 (water control). All data are the means of 10 replications
Control
Alcaligenes [aecalis Pseudomonas aeruginosa-1 P. aeruginosa-2 P. cepacia-1 P. maltophilia P. putida P. stutzeri P. vesicularis-1
Day 1
Day 2
Day 3
37 40 38 38 36 38 40 33 39
32 26 28 31 26 27 27 26 26
24 21 21 20 19 17 19 18 21
w a t e r uptake (g per f l o w e r per day) 40
32
"
~
o
~
control
24
8
0
I 1
I 2
• P. cepacia-1 • P. aeruginosa~ 1 P aeruginosa- 2 P stutzeri I 3
I
I
I
1
2
3
R putida IR vesicularis- 1 A. faecalis R maltophilia days
Fig. 1. The effect of isolated bacterial strains (P. =Pseudomonas, A. =Alcaligenes) on water uptake of 'Sonia' roses. 107 cfu ml-1 vase water were added at the start of the experiment. All data are the means of 10 replications. None of the isolated strains degraded pectin. Based on the chemical charact e r i s t i c s ( T a b l e 1 ), t w o d i f f e r e n t s t r a i n s w e r e f o u n d in P. aeruginosa, P. cepacia a n d P. vesicularis. W h e n a d d e d to t h e v a s e w a t e r a t 105 m l - 1 , n o n e o f t h e i s o l a t e d s t r a i n s h a d a n effect o n w a t e r u p t a k e o f ' S o n i a ' r o s e s a f t e r 1 day, as c o m p a r e d to a w a t e r c o n t r o l ( T a b l e 2). O n l y t h e w a t e r - u p t a k e v a l u e for t h e first d a y w a s considered, since o t h e r o r g a n i s m s m a y s u b s e q u e n t l y develop, a n d b a c t e r i a l c o n c e n t r a t i o n s will p r o b a b l y increase. A t 107 b a c t e r i a m l - 1 , all o f t h e s t r a i n s r e d u c e d w a t e r u p t a k e b y t h e first d a y (Fig. 1 ). T h e d i f f e r e n c e s a m o n g t h e s t r a i n s w e r e n o t s t a t i s t i c a l l y s i g n i f i c a n t (P(F) >0.05).
289 n u m b e r of v i a b l e b a c t e r i a p e r ml.
106 1°54 /
"~.
control
10 10 3
102
~
1 10
|__•
•
,
.
I~
Chrysal , Aodural
,
,
,
,
~
0
2
4
6
8
,
10 d a y s
Fig. 2. The number of cfu of bacteria in the vase water of cut roses. The flower preservatives Chrysal and Aadural were added in the commercially recommended dose (11 and 15 g l - 1, respectively) at the start of vase life.
The population of viable bacteria in uninoculated vase water of roses reached a level of 10~ cfu m l - 1 by the third day of vase life, and declined slightly thereafter (Fig. 2 ). The flower preservatives Chrysal and Aadural prevented development of high bacterial numbers. DISCUSSION
The isolated strains inhibited water uptake of rose flowers at 107 cfu ml-1, but not at 105 cfu m l - 1. This is in accordance with the effects of a mixed culture of bacteria, also originating from rose vase water (Van Doorn et al., 1986). It is also consistent with the effect of two isolated strains tested against 'Cara Mia' roses at 106 cfu m1-1, showing no effect on vase life (Zagory and Reid, 1986). The latter authors, however, found a fluorescent Pseudomonas species from carnation vase water that reduced vase life of roses at 106 cfu ml-1. Since we made 39 isolations from the vase water, it is likely that the majority of the strains present in the water were included in our study. The isolates were made from plates that contained the highest dilution, and thus represent bacterial strains that were quantitatively dominant. We apparently did not isolate the fluorescent Pseudomonas of Zagory and Reid (1986). The dominant flora may vary because the initial species composition in the vase water will depend on that of the stem surface. The surface flora in turn will depend on the conditions of cultivation (Dickinson and Preece, 1976). The similarity of the effect of the various bacterial strains on water uptake was contrary to expectation. A strain of P. cepacia has been found to elicit the hypersensitive reaction in the tobacco leaf, an indicator for potential phytopathogenesis, whereas P. putida did not show this reaction (Gardner et al.,
290
1982). Furthermore, visual inspection of the bacterial colonies on Plate Count Agar showed extensive slime (extracellular polysaccharide) production in some strains and less in others. The role of extracellular polysaccharides in vascular blockage is not known, but may be important. Whatever the mechanism by which bacteria cause vascular blockage may be, the hypothesis put forward by Burdett (1970) that bacteria produce pectic enzymes which digest cell walls and thereby cause vessel plugging seems unlikely because neither of the isolates in the present study was found to show pectolytic activity. The flora isolated from the vase water of 'Sonia' roses was similar to that reportedly found by others in flower vase water. P. aeruginosa occurred in 90% of tested hospital flower vases, while P. cepacia occurred in only 4% (Taplin and Mertz, 1973; McClary and Layne, 1977). In containers at wholesale flower distribution centers, a number of Pseudomonas species were also found, as well as A lcaligenes faecalis (Ford et al., 1961 ). These papers mention several species not found in the present experiment, whereas we isolated P. maltophilia, P. putida, P. stutzeri and P. vesicularis, which were not mentioned in the above studies. The tested flower preservatives prevented development of high bacterial numbers in vase water. Their effectiveness against bent necks in roses may therefore be partially due to the presence of an effective bactericide. The concentration of bacteria in uninoculated vase water; without a flower preservative, reached levels slightly higher than 105 cfu ml -1 by the third day of vase life (Fig. 2). Taplin and Mertz (1973) found, also after 3 days of vase life, a concentration of 10 ~ cfu m1-1, and lower concentrations earlier. Marousky (1976) sampled the vase-water from 'Forever Yours' roses after 3 days and found 1.5 × l0 s cfu ml-1. This indicates that, under some conditions, the exogenous concentration of bacteria may account for vascular blockage in roses, from about the third day of vase life onward. However, blockage and bent neck are often already observed on the first day of vase life. The exogenous concentration of bacteria may be too low to account for such early blockage. We suggest that air emboli a n d / o r the endogenous number of bacteria in stems may be a cause of bent neck observed early in vase life. ACKNOWLEDGEMENTS
We thank Peter Haghuis for skilful technical assistance and Devon Zagory for critically reading the manuscript. REFERENCES Aarts, J.F.T., 1957. Over de houdbaarheid van snijbloemen. Meded. Landbouwhogesch. Wageningen, 57(9): 1-62.
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