Selection of biocides for use in floral preservatives
Michael Knee *
Department of Horticulture and Crop Science,The Ohio State Uni6ersity,Columbus,OH43210,USA
Received 22 March 1999; accepted 30 September 1999
Abstract
The effects of concentrations of various biocides, in a solution containing 0.2 g l−1citric acid and 10 g l−1glucose,
were tested on cut roses (Rosa hybridaL., ‘Classy’),Alstroemeria pelegrinaL. and carnations (Dianthus caryophyllus
L.). Compounds were evaluated for their effects on flower life (time to decline in fresh weight), maximum gain in fresh weight, solution uptake from 4 to 6 days, resistance to water flow in the stem, stem respiration and solution absorbance. Longer flower life and higher gain in fresh weight of roses were observed with a concentration of 0.05 g l−1 than with higher concentrations of most biocides. At this concentration, bromopropanediol, Dantogard and
thiabendazole did not prevent a rise in stem resistance to water flow, or solution absorbance. Aluminium sulphate up to 0.8 g l−1was also ineffective in these respects. Stem respiration was inhibited by sodium benzoate,
hydroxyquino-line citrate (HQC), Isocil and Physan-20. Principal component analysis on the rose data indicated that the best treatments were 0.05 g l−1benzoate, cetylpyridinium chloride, Isocil and Physan-20, 0.05 and 0.2 g l−1
dichloroiso-cyanuric acid, and 0.2 and 0.8 g l−1HQC. Tests with carnation and Alstroemeriaindicated that HQC, Isocil and
Physan most consistently promoted fresh weight increase and maintenance. © 2000 Elsevier Science B.V. All rights reserved.
Keywords:Biocides; Cut flowers; Phytotoxicity; Vase life; Water relations
www.elsevier.com/locate/postharvbio
1. Introduction
A major cause of deterioration in cut flowers is blockage of xylem vessels by microorganisms that accumulate in the vase solution or in the vessels themselves. When the stem is blocked, continuing transpiration by the leaves results in net loss of water by flower and stem tissues. In roses, this often leads to the ‘bent neck’ disorder (van Doorn and Perik, 1990). For many years, floral
preserva-tives have been acidified and have usually in-cluded biocides to inhibit bacterial proliferation (Nowak and Rudnicki, 1990). There is limited information in the literature on the comparative effectiveness of different biocides, and no clearly established criteria for selection of particular com-pounds. van Doorn et al. (1989) related the effec-tiveness of biocides to maintenance of the hydraulic conductance of rose stems. van Doorn et al. (1990) compared the inhibition of bacterial multiplication in rose stems by a number of com-pounds with their phytotoxic effects on foliage. They concluded that none of the compounds had
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E-mail address:[email protected] (M. Knee)
‘‘a consistent and high anti-bacterial effect at concentrations that are not toxic to flowers’’. However, they tested the compounds in solution in plain water and did not determine whether they would extend flower life when included in a preservative formulation. This research deter-mines the effectiveness of different biocides, in-cluded in a preservative formulation, for rose, carnation andAlstroemeria.
2. Materials and methods
Roses (Rosa hybrida L. ‘Classy’), Alstroemeria pelegrina L.(‘Sunburst hybrids’), and carnations (Dianthus caryophyllus, unknown white cultivar) were obtained from a local wholesaler. The stems were cut to 40 cm and any leaves that would contact solution were removed before standing in citric acid solution (0.5 g l−1) for 1 – 2 h.
The biocides were obtained from Sigma (St. Louis, MO), unless otherwise specified, and tested at 0.8, 0.2 and 0.05 g l−1. Isocil (Lonza Inc.,
Fairlawn, NJ) and Physan 20 (Maril Products Inc., Tustin, CA) were obtained as liquid formula-tions and were made up by volume to give 0.2 and 0.05 g l−1
of active ingredients (11.5 g l−1
5-chloro-2-methyl-4-isothiazolin-3-one and 3.5 g l−1 2-methyl-4-isothiazolin-3-one in Isocil; 100 g
l−1
n-alkyl [C12– C18] dimethyl benzyl ammonium
chloride, and 100 g l−1 n-alkyl [C
12 and C14]
dimethyl ethylbenzyl ammonium chloride in Physan-20). Dantogard (1,3-dimethyl-5,5-dimethylhydantoin) was obtained from Lonza Inc.
The standard preservative solution contained 10 g l−1 glucose and 2 g l−1 citric acid. This was
obtained by dilution of a 20× stock solution, which had been adjusted to pH 3 with KOH. Appropriate concentrations of the different bio-cides were dissolved in this solution. The stock solution came to pH 3.3 – 3.4 after dilution; pH was not affected by the biocides except for alu-minium sulphate, which required addition of KOH to restore it to pH 3.3. For roses, three replicate flowers were tested for each concentra-tion of biocide. One replicate of each treatment was placed in 125 ml solution made up with
distilled water, one replicate in tap water and one in ‘synthetic tap water’ (STW). STW was made up from 0.0202 g l−1 KNO
Rule et al. (1986)). The experiments involved four separate batches of roses. For eight of the bio-cides, single roses were placed in each concentra-tion of each biocide, and the experiment was repeated with different batches of roses and differ-ent kinds of water. In the fourth experimdiffer-ent, three roses from a single batch were tested with each concentration of the three biocides (Dantogard, Isocil and Physan-20), but the solution for each of the roses was made up with a different kind of water; additional roses in 0.2 g l−1
hydroxyquino-line citrate were included for comparison with similar treatments in the first three experiments. ForAlstroemeriaand carnation, all solutions were made up in STW. Further flowers in water and in citrate – glucose without biocide were included as controls in all experiments.
The flowers were kept in a room at 20°C with 12 h fluorescent light at 10mmol m−2s−1and 12 h dark. Flowers and flasks were weighed at 2-day intervals. Flower opening was expressed as the maximum percentage gain in fresh weight. Flower life of roses and Alstroemeria was defined as the time when the weight of a flower fell to 90% of its maximum observed weight. For carnation, the limit was set at 98% of maximum weight. The time was calculated by interpolation between the first weight below the threshold and the previous weight. Solution uptake by flowers was estimated from the change in weight of flasks corrected for evaporation by subtracting the weight change in equivalent flasks without roses.
pressure required to sustain flow at 0.1 ml h−1,
which was the approximate rate of solution up-take observed for intact roses at the beginning of the experiment. After this test, the stem segment was enclosed in a 50 ml Erlenmeyer flask with a rubber septum seal for 2 – 3 h. At the end of this time, CO2 produced by tissue respiration was
measured by gas chromatography on a column of Porapak N and with a thermal conductivity detec-tor (Knee, 1995). Microbial growth in flasks was assessed by measuring the increase in absorbance at 400, 500 and 600 nm over 12 days, and calcu-lating the mean of these values.
Data were analyzed using GLM and PRIN-COMP procedures in SAS Version 6.12 (SAS Institute Inc., Cary, NC).
3. Results
One type of water was included in each of the first three experiments with roses, so effects of water were confounded with variation between batches of flowers. In the fourth experiment, all three types of water were represented with a single batch of flowers. Length of life, solution uptake and stem respiration were lower in tap water than in distilled or synthetic tap water (Table 1). Resis-tance to water flow and solution absorbance were lower in distilled water than in the other two types. Analysis of variance was performed on data for treatments that were common to all experiments, water, glucose – citrate and 0.2 g l−1
hydroxyquinoline citrate (HQC). Average values
of all variables varied from one experiment to another (PB0.05), but comparison of the fourth experiment with the first three showed no signifi-cant differences. The results from all experiments were treated as a common data set. Because of the consequent lack of balance in the design, the GLM procedure in SAS was used and ‘experi-ment’ was declared as a class variable in the analysis.
Maximum weight gain, respiration, of basal stem segments and solution absorbance were higher for roses in citrate – glucose solution with-out biocide than in plain water, but decline in fresh weight, solution uptake and stem resistance to water flow were similar in citrate – glucose and water (Table 2). Biocides delayed the decline in fresh weight of roses, with the exception of alu-minium sulphate, benzalkonium chloride, and bromopropanediol (Table 2). Biocides did not increase the gain in fresh weight and a number of them actually inhibited it. At the beginning of experiments, flowers took up solutions rapidly (0.3 ml g−1day−1), independent of composition;
by 4 – 6 days, rates were declining, particularly in aluminium sulphate, bromopropanediol, cetylpyridinium chloride (CPC) and Isocil. At the end of the experiments, resistance to water flow in basal stem segments and solution absorbance was high in the absence of biocide and in aluminium sulphate, bromopropanediol, Dantogard, and thi-abendazole. Respiration of stem segments was low in sodium benzoate, HQC, Isocil and Physan-20 (Table 2). As concentration of biocide in-creased, the delay in decline in weight, resistance
Table 1
Effect of water type on mean values of life (time to decline in fresh weight), maximum gain in fresh weight, solution uptake from 4 to 6 days, resistance to water flow in the stem, stem respiration and solution absorbance of ‘Classy’ roses kept in various solutions in experiment 4a
Water Life (days) Weight gain (%) Uptake Resistance (kPa) Respiration Absorbance (ml g−1day−1) (ml g−1−1)
8.89 9.55 0.174 12.3 43.7
Distilled 0.056
7.07 7.42 0.124
Tap 28.9 25.4 0.157
0.171
P 0.054 0.033 0.003 0.101
Table 2
Effects of biocide on life (time to decline in fresh weight), maximum gain in fresh weight, solution uptake from 4 to 6 days, resistance to water flow in the stem, stem respiration and solution absorbance of ‘Classy’ rosesa
Solution Life (days) Weight gain (%) Uptake (ml g−1 Resistance (kPa) Respiration (ml Absorbance g−1h−1)
day−1)
2.67 0.149 29.5
Aluminium sulphate 5.84 193 0.126
5.51 3.96 0.116 0.45
Benzalkonium chlo- 41.9 0.040
ride
4.20 0.127
Sodium benzoate 6.78 0.56 10.5 0.047
3.03 0.095 31.6
5.66 103
Bromopropanediol 0.163
6.57 2.35 0.113
Cetyl pyridinium 0.50 77.4 0.025
chloride
7.04 0.132
Dantogard 7.45 41.5 67.5 0.094
6.29 0.155
DICA 8.28 0.50 76.2 0.026
8.16 0.199 11.6
9.01 113
HQC 0.057
7.33 0.105
Isocil 8.16 0.20 15.0 0.004
9.53 0.164 0.55
8.41 27.9
Physan-20 0.037
5.57 0.177 12.1
Thiabendazole 6.45 131 0.081
8.26 0.171 57.6
7.01 139
Citrate–glucose 0.227
5.02 0.195 65.2
Water 6.99 43 0.015
0.019 0.0325 5.29
0.52 13.4
S.E. (n=9) 0.0275
aMean data are shown for each biocide. S.E., standard error from residual term in GLM.
Table 3
Probability of null hypotheses, according to GLM, for effects of biocide, concentration and their interaction on life (time to decline in fresh weight), maximum gain in fresh weight, solution uptake from 4 to 6 days, resistance to water flow in the stem, stem respiration and solution absorbance of ‘Classy’ rosesa
Wt gain Uptake
Effect Life Resistance Respiration Absorbance
0.0034 0.0002 0.0001
Biocide 0.0003 0.0001 0.309
0.189 0.0001 0.0032
0.0001 0.0001
Concentration 0.309
0.349 0.0715 0.486 0.131
Interaction 0.0188 0.428
aData shown in Tables 2 and 4. Results for water and citrate–glucose were excluded from the analysis.
Table 4
Effect of biocide concentration on life (time to decline in fresh weight), maximum gain in fresh weight, solution uptake from 4 to 6 days, resistance to water flow in the stem, stem respiration and solution absorbance of ‘Classy’ rosesa
Concentration (g Life (days) Weight gain (%) Uptake (ml g−1 Resistance (kPa) Respiration (ml g−1 Absorbance h−1)
day−1) l−1)
8.26 0.171 57.62 139.0 0.279
0 7.01
5.67 0.186 24.09
7.73 124.3
0.05 0.109
4.66 0.143 9.10 92.6 0.084
0.20 6.81
4.06 0.096 10.48
5.96 55.4
0.80 0.078
0.567 0.0108 3.056
S.E. (n=27) 0.303 7.75 0.0159
Table 5
Correlation matrix (n=36) for life (time to decline in fresh weight), maximum gain in fresh weight, solution uptake from 4 to 6 days, resistance to water flow in the stem, stem respiration and solution absorbance of ‘Classy’ roses
Weight gain Uptake Resistance
Life Respiration
Absorbance −0.112 0.168 0.062 0.633 0.452
−0.045 0.452
to water flow, stem respiration, and solution ab-sorbance tended to decrease (Tables 3 and 4). Biocides tended to diminish weight gain by com-parison with citrate – glucose, although the differ-ence between concentrations from 0.05 to 0.8 g l−1
was not significant (Tables 3 and 4).
Flower life, maximum weight gain and solution uptake were all correlated; resistance to water flow, stem respiration and solution absorbance were also correlated (Table 5). Between these two groups of variables, only respiration and solution uptake were correlated. The first two components in principal component analysis accounted for 72.8% of the variance. The first component (40% of variance) showed highest weighting by flower life, weight gain, and solution uptake; the second showed negative weighting with flower life and positive weighting with respiration, resistance and absorbance (Table 6). A plot of component 2 on component 1 showed an inverse linear relationship for concentrations of benzalkonium chloride, sodium benzoate, CPC, dichloroisocyanuric acid (DICA), Isocil and Physan-20 (Fig. 1). Aluminium sulphate, bromo-propanediol, Dantogard and thiabendazole were clustered around points representing water and citrate – glucose, without biocide. Data for HQC were distributed perpendicular to the general trend of other data (Fig. 1). A cluster of points with high values of component 1 and low values of component 2 included 0.05 g l−1
benzoate, CPC, Isocil and Physan-20, 0.05 and 0.2 g l−1DICA, and 0.2 and
0.8 g l−1HQC. The third component in the analysis
accounted for a further 13.7% of the variance but, when plotted against the first two components, did not reveal any obvious trends.
Concentrations of particular biocides that main-tained fresh weight of roses for at least 8 days were
also tested with Alstroemeriaand carnations. The biocides generally promoted higher gain and longer retention of fresh weight than water or citrate – glu-cose (Tables 7 and 8). The statistical analysis was also run without the controls, so that differences between biocides could be tested (Table 8). There was no significant difference in the effects of biocides on flower life. The main effect of type of biocide on weight gain was not significant, but there was a significant interaction with flower type. This indicates that flowers responded differently to indi-vidual biocides. DICA inhibited weight gain in rose and Alstroemeria, but not in carnation; CPC was inhibitory in rose, and Dantogard in Alstroemeria only.
4. Discussion
This research confirms earlier observations that water composition can affect longevity of cut flowers (Rule et al., 1986). It seems that tap water can have adverse effects on roses (shorter life,
Table 6
Contribution of original variables (time to decline in fresh weight, maximum gain in fresh weight, solution uptake from 4 to 6 days, resistance to water flow in the stem, stem respiration and solution absorbance) to the first two eigenvectors in principal component analysis
Component 1 Component 2
Life 0.435 −0.456
Weight gain 0.440 −0.336
Fig. 1. The first two eigen vectors from principal component analysis of six variables (time to decline in fresh weight, maximum gain in fresh weight, solution uptake from 4 to 6 days, resistance to water flow in the stem, stem respiration and solution absorbance) for roses in different biocides. C, Citrate glucose; W, water; als, aluminium sulphate; bac, benzalkonium chloride; ben, sodium benzoate; bro, bromopropanediol; cpc, cetyl pyridinium chloride; dan, Dantogard; dic, dichloroisocyanuric acid; hqc, hydroxyquino-line citrate; iso, Isocil; phy, Physan-20; tbz, thiabendazole. For each biocide, the smallest point represents 0.05 g l−1, the intermediate point is 0.2 g l−1and the largest is 0.8 g l−1.
Table 7
Effect of biocide on flower life (time to decline in fresh weight) and maximum gain in fresh weight forAlstreomeria, carnation and rose
Concentration
Biocide Alstroemeria Carnation Rose
(g l−1)
Life (days) Gain (%) Life (days) Gain (%) Life (days) Gain (%)
0.05 20.4 19.8
Cetyl pyridinium chlo- 22.0 19.0 9.5 5.1
ride
18.3 14.3 22.0
Dantogard 0.05 18.2 8.0 10.3
19.3 15.5 21.7 22.8 8.5 5.1
DICA 0.2
20.4 18.9 20.7
0.2 15.7
HQC 9.4 9.1
0.05
Isocil 18.7 19.8 21.4 21.4 9.6 8.7
0.05
Physan 17.7 18.8 21.6 23.8 9.4 10.0
17.8 18.3 16.9 15.4
Citrate–glucose 7.0 8.3
Water 16.7 12.7 18.8 10.2 7.0 5.0
0.46 1.90 1.30 1.94 1.33 2.26
S.E. (n=3)
lower stem respiration) that were not reproduced by the synthetic tap water used. One possible damaging constituent of tap water is fluoride, and since this was not included in the synthetic mix-ture, it may account for the difference (Lohr and
Pearson-Mims, 1990).
that these compounds are also phytotoxic. Stem respiration was reduced by several compounds, particularly at high concentrations. (High respira-tion does not necessarily mean that compounds were non-toxic because a dead stem could be colonized by microorganisms.) Early in the vase life of roses, many of the compounds inhibited flower opening (gain in fresh weight) and this was associated with a tendency to earlier decline in fresh weight. Because of these phytotoxic effects, long flower life was not necessarily associated with low resistance to water flow in the stem or low microbial accumulation. Sodium benzoate was effective in both respects but did not greatly extend flower life. Bromopropanediol, and thi-abendazole maintained lower resistance and solu-tion absorbance at 0.2 or 0.8 g l−1than at 0.05 g
l−1, but flower life and weight gain were higher at
the lower concentration.
Interestingly, rate of solution uptake was not correlated with resistance to water flow in the stem. This suggests that transpiration was main-tained even when the vessel elements were par-tially blocked; presumably, this led to a lower xylem water potential than when resistance was low and petal cells were then unable to maintain turgor. Environmental conditions, or compounds that restrict transpiration, could alleviate the problem. Although rate of solution uptake was correlated with flower life, Isocil prolonged flower life with low uptake; since it also maintained low resistance, it may have inhibited transpiration.
Principal component analysis differentiated be-tween effective (benzalkonium chloride, sodium benzoate, CPC, DICA, Isocil and Physan-20) and ineffective biocides (aluminium sulphate, bromo-propanediol, Dantogard and thiabendazole).
Treatments in the cluster with high component 1 but low component 2 are regarded as optimal. For most of the effective biocides, long flower life, high weight gain and solution uptake were associ-ated with low respiration, resistance to water flow and solution absorbance. Hydroxyquinoline cit-rate is one of the most commonly used biocides, but it seems to act differently from the other effective biocides tested because it did not con-form to this pattern. It is surprising that Physan-20 appeared to be superior to benzalkonium chloride because the compounds are chemically similar. Perhaps there were minor ingredients in benzalkonium chloride that adversely affected flower life, or the presence of ethylbenzalkonium chloride in Physan-20 rendered it less phytotoxic. Several biocides promoted flower life or gain in fresh weight in Alstroemeria and carnations as well as in roses. None of the six biocides tested inhibited weight gain in carnation, whereas three of them were inhibitory to Alstroemeria or rose. This could be related to differences in the rate of solution uptake; rose and Alstroemeria took up solution at a rate fivefold higher than carnation, so that higher concentrations would have accumu-lated in their tissues. It is well known that flower opening is promoted by sugars supplied through the stem, but vase life may not be extended be-cause the sugar encourages multiplication of bac-teria, which eventually block xylem vessels. Biocides can prevent this from occurring, al-though they may all be to some extent phytotoxic (van Doorn et al. 1990). Nevertheless, a preserva-tive including a suitable biocide can improve flower opening and prolong life beyond plain water or a sugar solution. The most generally effective and safest biocides appeared to be hy-droxyquinoline citrate, Isocil and Physan.
Table 8
Probabilities of null hypotheses for effects of flower type and biocide on flower life (time to decline in fresh weight) and gain in fresh weight, with and without data for water and citrate–glucose controls included in the analysis
All data Without controls
Effect
Flower life Weight gain Flower life Weight gain
Flower type 0.0001 0.0001 0.0001 0.0001
Biocide 0.0007 0.0003 0.134 0.264
0.025 0.030
0.762 0.708
Acknowledgements
Thanks are due to Smithers Oasis Inc., Kent, OH for financial support of this work and Nathan Wolke for technical assistance. Salaries and re-search support were provided by state and federal funds appropriated to the Ohio Agricultural Re-search and Development Center, The Ohio State University.
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