The effect of herbal essences in preservative solution, on quantitative, vase life,
bacteria-induced stem xylem blockage growing ”Lisianthus var ECHO “
*Farzane Pourianejad, Ministry of Jihad-e- agriculture,
[email protected]
Abstract
In this study the effect of herbal essences as antibacterial components in
preservative solution of Lisianthus Var ECHO (Eustoma grandiflorum) was
investigated. The test was done with application of preservative solution. Cut
flowers were treated with different concentrations of Thymus vulgaris, Peppermint
and Lavandula essences in addition to Sucrose 2.5%. The results showed there is the
longest time in vase life with Thymus in 50 ppm (15.6 days) and the control
treatment showed the shortest vase life (11.6 days). Also Thymus with 50 ppm has
the highest effect on relative fresh weight and solution uptake. After culturing the
bacteria-induced stem xylem blockage, which extracted from the end of stem, in NA
medium with various concentrations of herbal essences, the result, showed that in pure
concentration (100%) inhibition was completed and in various concentration of herbal
essence the bacterial population was reduced.
Key Words:
Thymus, Peppermint, Lavandula, preservative solution, essence, Microorganism
Introduction:
Lisianthus is one of the best ten cut flowers in the world. Culture of this cut flowers was
increased in Asia especially China in three years before (VBN, 2007). The Eustoma
grandiflorum variety is planted as cut and pot flower. Decreases in carbohydrates and
Microorganisms growth in stem xylem and preservative solution are main factors on
decreasing of cut flowers vase life. So adding antimicrobial factors in to preservative
solution and also sucrose usage, as carbohydrate source is necessary. Sucrose is in oftenprotective solutions formulation but other metabolic sugars like as Glucose and Fructose
have the same effect. The roles of sugars in vase life of cut flowers are known. Glucose
uptake from the solution is accumulated in petals tissue and so osmotic potential is
improved and increase the carbohydrates for respiration and growth. This will facilitate
the opening of florets and dilay senecence(Sarkka 2004). Using Sucrose in preservative
soloution caused increase in vase life, stimulating opening flowers, improving color and size
of flowers(Reid,2000; Bhattacharjee, 2005; Ichimura, 2007, Elgimabi,2009).
Using Sucrose 4% as a treatment of preservative solution in Orchid flower caused increase
in vase life and content of solution absorption and delay in disappearance of the color of the
petals (Chandran, et al., 2007). Using Sucrose in cut flower solutions caused delay in
ethylene biosynthesis, decrease in ethylene sensitivity and increase in vase life (Kumar pun,
et al., 2003). Sucrose in preservative solution prevents wilting in Rose var Sonia and results
slow decrease in wet weight (Ichimura, et al., 2003).
Using Sucrose 2% in preservative solution of wallflower caused increase in Inflorescence,
vase life and opening floret (Arab, 2005). Using Sucrose 4% as dominant treatment has
positive effect in vase life of Rose Var Bacara and helps to improvement of petals color
(Pompodakis and Joce 2003).
Essences are aromatic components that due to not induce fat, can produce soap. Some of
these components have attractive properties and the others have repellent properties.
Dominant compound or active ingredient in most essential oils are terpenes(Baghalian and
Naghdi Badi, 2000). The mechanism of the essential oils action as follows: Due to joint and
overlapping of various combinations of essential oils, Wall, and cell membrane of pathogens
degrade and permeability and ion leakag of cells increases. Due to decomposition lipids in
the cell wall, mitochondria, and proteins of membrane and also clotting of cytoplasm, and
depletion of proton motive force, the damaged cells are undergo cell death by essential
oils(Burt, 2004). Also using Zataria multiflora and Thymus vulgaris with SNP prevent
growth of bacteria in preservative solution,Vascular obstruction and dcrease wet wight and
caused increase vase life(Solgi, 2009).
So in this study the effect of different concentrations of essential oils Thymus vulgaris,
Lavand, Pepermint were examined by application preservative solution with sucrose 2.5%
in Lisianthus var Echo for determining suitable essential oil and concentration for increase
vase life and quality of the cut flowers.
Materials and Methods:
The flowers harvested from green house in stage that the buds colored and two of them
were opened. They were transferred to the laboratory quickly. In the laboratory the
flowers stem was cut to 45 cm to coordinate and reduce the different in the size and after
that in the water the end of them cut 5 cm again and omit the shoots too.
Treatments:
1) Distilled water 2) Thymus 10, 25,50,100 ppm 3) Lavand 10, 25, 50 ppm, 4)
Peppermint 10, 25, 50 ppm
At the first the required amount of each essential oil was weighed and then it resolved in
Methanol 80% and was completed in 1000 cc. After that the flowers were immersed in
container with 10-liter capacity, which half of it was the treatment solution for an hour and
then they were removed from the solution and were kept in room temperature in the
laboratory. The solution was dried on the petals and stem’s flowers and then they put on
the Sucrose 10% for 24 hours. Then until the end of vase life they were placed in distilled
water, distilled water was changed every 2 days interval.
Traits are as follows:
 Vase life: Since 50% of florets were faded the vase life was ended (Moon soo,
2001).
 Relative fresh weight: fresh weight of flowers was measured exactly after
transferring to the laboratory by scale (0 day), and then it was measured in (1st,
3rd, 5th, 7th, 10th.)
 Content of water uptake by the flower: it was measured in 0. 1st, 3rd, 5th, 7th, and 10th

by the canter.
Content of Chlorophyll in the leaf: it was measured in 0, 1st, 4th, 6th, 8th, and 12th. For
this purpose 0.1 g of the third leaves harvested in each plot, Quite worn and
chlorophyll was extracted by 5 ml of 80% acetone and after 24 hours was read by
Spectrofotometer in 663, 640, and 645 nm wavelength (Lichtenthaler, 1987). Finally
the content of Chlorophyll was calculated using the following formula:
chlorophyll a =)0/0127×A663( -)0/00269×A640(
chlorophyll b=)0/0229×A645( -)0/00468×A663(
chlorophyll a+b=)0/0202×A645(+)0/00802×A663(
The content of TSS in shoot:
To determine the amount of TSS in the stem, remove 1g of the end of stem and pulverized in
a mortar. After crushing completely the extract was obtain. Then Digital ESR read the
extract Brix degree in 0, 1st, 3rd, 5th, 7th, and 10th (Hettiarachchi & Balas, 2005).
The content of water of petals and leaf:
At the first 2 g of leaf, 2 g of petal was weight and then put in the Oven in 60 C for 72
hours to dry completely. The dry weight was measured in 0, 1st, 3rd, 5th, 7th, and 10th and
was calculated using the following formula (Slavick, 1979):
RWC=
The content of petal Anthocyanin:
To measure the anthocyanin according to Bariola, et al (1999) method, 0.1 g of fresh
petal weighted and pulverized in a mortar. For extracting anthocyanin 5mLit of extract
solution contain methanol and HCL 1% was added in to every sample. Samples were put
in the Falcon and maintain in 4 C one night. Finally the absorption was read in 530 and
657 nm in 0, 1st, 3rd, 5th, 7th, and 10th and calculated using follow formula:
Anthocyanin=D530
−
0.24 D657
Soft rot test:
At the beginning and the end of the test samples was prepared from the end of the end of
the stem. Then the viscous liquid extracted from the stem end of cut flowers (Lysianthus
Var Echo) was cultured on the medium (Nutrient Agar) and after 48 hours pure bacteria
tested on the tomato to identify the crush ability of tested bacteria.
Evaluate the amount of prevention:
Inhibition of natural compounds, essential oils, were evaluated against positive and
negative warm bacteries by Aromatogram method. For this perpose wells with diameter of
2 mm were prepared on solid medium And 4 microliter quantities of oil were added into
each well. After two hours, when the oil was absorbed, Bacterial suspensions were sprayed
uniformly on medium and for 48 hours the Petri dishes were put in Incubator. Then the
diameter of zone inhibition around the wells were measured with a ruler and measure.
Results and discition:
Effects of treatments on Vase life:
Effects of treatments with different concentrations caused increase in vase life in Lysianthus
Var Echo compare with control(table 1). Among the essential oils, Thymus essence 50ppm
has the best effect on vase life (15.6 days) and the control has the lowest vase life (11 days).
In lower concentrations the vase life was higher and with increasing the concentration of
essences specially peppermint (50 ppm) and thymus (100 ppm) vase life was decreased.
Sucrose 25% as source of carbohydrates and herbal essences as antimicrobial components
in preservative solution caused increasing in vase life.
Table 1: effects of various herbal essences on vase life of Lysianthus Var Echo
Treatment
Control
Thymus 10 ppm
Thymus 25 ppm
Thymus 50 ppm
Thymus 100 ppm
Lavand 10 ppm
Lavand 25 ppm
Lavand 50 ppm
Pepermint 10 ppm
Pepermint 25 ppm
Pepermint 50 ppm
Vase life(days)
d11
abc14/53
ab15/33
a15/6
c13/66
ab15/23
ab14/86
ab15
ab15/43
ab14/83
bc14/33
Effects of herbal essence on relative fresh weight:
According to the results (Fig 1) relative fresh weight to 3rd fixed approximately. But after
that it was decrease, so that in 10th it had lowest content. In various treatment with different
concentrations, Thyme with 50 ppm had the highest and control the lowest effect on
relative fresh weight (Fig 2).
120
ab
ab
a
b
relative fresh weight
100
c
d
80
60
40
20
0
0
1st
3rd
5th
7th
Time
Fig 1 : Effect of time on relative fresh weight on Lysianthus cut flower Var Echo
10th
a
100
ab
relative fresh weight
95
abc
ab
ab
bc
90
85
ab
ab
bc
bc
c
80
75
essence
Fig 2: Effect of herbal essence on relative fresh weight on Lysianthus cut flower
Var Echo
Effect of herbal essence on content of solution absorption:
The content of solution absorption fixed until 3rd day but after that started to decrease so
that in the 10th day it had the lowest level. The highest absorption was seen with Thyme
50ppm and Lavandula 25 ppm and the solution uptake was also decreased. While the
lowest content seen in control treatment which caused presence of antimicrobial
components in essentia oils which delaied stem xylem blockage(Fig 3).
250
Content of solution absorption(cc)
240
230
ab
ab
ab
a…
a
ab
ab
ab
ab
220
control
Thyme 10
abc
a…
a… a…
b…
Thyme 25
g…
g… f…
d…
g…a…
210
Thyme 50
c…
200
g…
g…
h
hi
ij
j
Thtme 100‫آ‬
Lavandula 10
Lavandula 25
Lavandula 50
Pepermint 10
190
Pepermint 25
Peremint 50
180
1st
3rd
5th
Time (Day) 7th
10th
Effect of Time and herbal essence on content of chlorophyll:
In the effect of time on content of chlorophyll, it was fix until 5th day approximately and
after that starting to decrease in the 10th day it had the lowest level because of leaf
senescence (Fig 4). Effect of herbal essence on chlorophyll showed that there is significant
difference between control and Thyme 50ppm(Fig 5). There isn’t any significant different
between other treatments. Various essential oils protect of chlorophyll higher than control
treatment.
0.045
a
chlorophyll(m gr/fresh weight)
0.04
ab
0.035
ab
ab
bc
0.03
c
0.025
0.02
0.015
0.01
0.005
0
0
1st
3rd
5th
7th
10th
Time (day)
Fig 4: effect of time on chlorophyll in Lysianthus Var Echo
chlorophyl( mgr/fresh weight)
0.035
a
0.03
0.025
b
ab
ab
ab
ab
ab
ab
0.02
0.015
0.01
0.005
0
Treatments
Fig 5: Effect of herbal essence on chlorophyll in Lysianthus var Echo
ab
ab
ab
Effect of herbal essence on TSS in stem:
In the interaction effect of time and essential oil on the TSS, control treatment in 0 day had
the highest level and in the 10th day had the highest effect on the TSS, which caused in
absence of sucrose.
In all of treatments carbohydrate replaced with sucrose in respiration process and prevent
analysis other cell materials and delay senescence. Thyme 10 ppm has the highest effect in 0
and 10th day on TSS. Thyme 10 ppm had highest significant difference than other
treatments and thyme 100 ppm had lowest effect. Highest concentration of essential oils
prevent sucrose uptake (Fig 6).
8
7
TSS(%)
6
5
4
3
f…k
f…k
ab
abc
a…g a…i
b…j
b…i
d…k
f…k
g…k
h…k
c…k
h…k
ijk
a…d
a…d
a…h
a…i
a…h
a…h
a…g
a…i f…k
b…j
2
control
a
k
1
abc
abcd
a…h
Thyme 10
b…j
c…k
g…k
Thyme 50
jk
Lavand 10
k
k
Thyme 25
Theme 100
Lavand 25
Lavand 50
0
pepermint 10
0
1st
3rd
5th
Time(Day)
7th
10
pepermint 25
pepermint 50
Effect of essential oil and time on water content of petals:
The effect of time on water content in petals showed that it had highest level in 0 day and
lowest level in 10th day (Fig 7). Between treatments control treatments had the lowest effect
and Lavandula 10 ppm highest effect on water content of petals (Fig 8). Thyme 10 ppm,
25ppm, 50ppm and peppermint 10ppm and Lavandula 25 ppm had positive effect on water
content of petals.
Water content of petals(gr)
18
16
14
12
10
8
6
4
2
0
a
a
a
b
b
c
0
1st
3rd
5th
7th
10th
Time (day)
Fig 7: Effect of Time on water content of petals on Lysianthus Var Echo
Water content of petals(gr)
16
ab
14
12
c
ab
a
ab
bc
ab
abc
ab
abc
bc
10
8
6
4
2
0
Time(Day)
Fig 8: Effect of herbal essence on water content of petals in Lysianthus Var Echo
Interaction effect of time and herbal essence on water content of leaf:
According to the results (Fig 9) Lavandula 10ppm in 0 day had the highest effect on water
content of leaf (16.83) and decreasing Leaf water content was very slow and in 10th day was
lowest than other treatments. Due to the positive effects of essential oils and sucrose to
maintain leaf water relations in leaves that caused freshness and vitality.
water content of leaf(gr)
control
18
16
14
12
10
8
6
4
2
0
a
b
b…e
b…e
b…f
b…e
b…g
b…h
b…h
c…i b…i
c…ic…i
c…i
c…i
b…g
b…h
b…i
b…ib…i
d…i
f…i
b…i
c…i
c…i
d…i
f…i
hi
c…i
d…id…i
d…i
f…i
f…
hi
ghi
0
1st
3rd
5th
Time(Day)
7th
10th
Thyme
10
Thyme
25
Thyme
50
Thyme
100
Lavand
10
Lavand
25
Lavand
50
pepermi
nt 10
pepermi
nt 25
Fig 9:Interaction effect of water content of leaf on Lysianthus Var Echo
Effect of herbal essence on Anthocyanin of petals:
The content of anthocyanin was highest since 0 day to 5th and after that started to decrease
and in 10th day it had the lowest level. Actually reduce in anthocyanin levels of petals is one
of effects of cut flower senescence. Between various treatments of essential oils on
anthocyanin didn’t show any significant difference.
Results and discution of Soft rot test:
Dickeya chrysanthemi and Pectobacterium carotovorum as bacterial soft rot on potato
slices were tested. After posetine result different oncentration of essential oils were
investigated. In pure concentration of every three essentioal oils( Thym, Lavandula, and
Pepermint) on midum culture contain bacterias Resulted in inhibition of bacterial growth
and other essential oils concentration resulted reduceing in the microbial population.
This result atributed to the presence of components such as Thymol and Carvacrol in
essential oils which had antimicrobial properties that prevent of vascular occlusion , reduce
of absorption content and so reduce of relative fresh weight and it makes the process
slower. This results were consistent with Sangi et al (2008), Tzortzakis, N.G. (2007),
Martinez (2005), and Ziedan(2008).
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