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European Journal of Applied Sciences – Vol. 10, No. 2
Publication Date: April 25, 2022
DOI:10.14738/aivp.102.12172. Opondo, F. A., K’Owino, I. O., Chepkwony, S. C., Kosgei, V. J. (2022). In Vitro Antibacterial Activity of Essential Oils from Tithonia
Diversifolia Leaves and Flowers Against Ralstonia Solanacearum. European Journal of Applied Sciences, 10(2). 523-539.
Services for Science and Education – United Kingdom
In Vitro Antibacterial Activity of Essential Oils from Tithonia
Diversifolia Leaves and Flowers Against Ralstonia Solanacearum
Florence Atieno Opondo
Department of Chemistry and Biochemistry
School of Sciences and Aerospace, Moi University
P.O.BOX 3900-30100, Eldoret, Kenya
Africa Center of Excellence II in Phytochemicals Textiles and
Renewable Energy (ACEII-PTRE), Moi University
P.O.BOX 3900-30100, Eldoret, Kenya
Isaac Odhiambo K’Owino
Department of Chemistry and Biochemistry
School of Sciences and Aerospace, Moi University
P.O.BOX 3900-30100, Eldoret, Kenya
Africa Center of Excellence II in Phytochemicals Textiles and
Renewable Energy (ACEII-PTRE), Moi University
P.O.BOX 3900-30100, Eldoret, Kenya
Department of Chemistry, Faculty of Science
Masinde Muliro University of Science and Technology
P.O.BOX 190-50100 Kakamega, Kenya
Sarah Cherono Chepkwony
Department of Chemistry and Biochemistry
School of Sciences and Aerospace, Moi University
P.O.BOX 3900-30100, Eldoret, Kenya
Africa Center of Excellence II in Phytochemicals Textiles and
Renewable Energy (ACEII-PTRE), Moi University
P.O.BOX 3900-30100, Eldoret, Kenya
Viola Jepchumba Kosgei
Department of Chemistry and Biochemistry
School of Sciences and Aerospace, Moi University
P.O.BOX 3900-30100, Eldoret, Kenya
Africa Center of Excellence II in Phytochemicals Textiles and
Renewable Energy (ACEII-PTRE), Moi University
P.O.BOX 3900-30100, Eldoret, Kenya
ABSTRACT
Bacterial wilt disease caused by Ralstonia solanacearum is a major constraining
factor in the production of tomatoes in Kenya, leading to an overreliance on
synthetic pesticides. As a result, there is increased research on bio-pesticides as
safer alternatives. The present study, therefore, characterized and evaluated in
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European Journal of Applied Sciences (EJAS) Vol. 10, Issue 2, April-2022
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vitro antibacterial activity of essential oils from Tithonia diversifolia leaves and
flowers against R. solanacearum. Hydrodistilled essential oils were characterized
through Gas Chromatography-Mass spectrometry (GC-MS) while functional groups
were confirmed using Fourier Transform Infrared Spectroscopy (FT-IR).
Antibacterial activity was performed using the disc diffusion method and minimum
inhibitory concentration evaluated using broth dilution. Hydrodistilled leaves of T.
diversifolia yielded 0.18 + 0.08 % (v/w); 0.10 + 0.07 % w/w as compared to 0.15 +
0.09 % (v/w); 0.08 + 0.03 % w/w in the leaves . GC-MS profiling revealed the major
compounds were (Z,Z,Z)-9,12,15- Octadecatrienoic acid ethyl ester(18%), palmitic
acid (16%), spathulenol (12%), Cis- 9,12,15- Octadecatrienoic acid (8.14%),
tetrateracontane (6%) and 1-Octen-3yl-acetate (5.22%) in the leaves ad α-linolenic
acid trimethylester(33%), Z,Z Hexadecadienoic acid(26%), octadecanoic acid
trimethylester (9%), palmitelaidic acid (8.49%), Germacrene D(5.45%), azelaic
acid (5.02%) and caryophyllene oxide (5.00%) in the flowers. Antibacterial activity
showed that T. diversifolia essential oils had mean inhibition zones of 12.61+ 0.22
mm and 11.82 + 0.76 mm from the leaves and flowers respectively, in comparison
to metham sodium which gave inhibition zone of 25.78 ±0.29 mm (p = 0.001). Based
on the results, this study gives credence to T. diversifolia essential oils as viable
antibacterial agents.
Keywords: In vitro; antibacterial activity; Spathulenol; Tithonia diversifolia; Ralstonia
solanacearum
INTRODUCTION
Bacterial wilt caused by soil-borne bacterium Ralstonia solanacearum is one of the most
devastating bacterial disease limiting tomato production in tropical, subtropical, and warm
temperate regions of the World [1-4]. The bacterium is classified as the World’s most
destructive phytopathogenic bacteria causing tomato yield losses due to its lethality,
persistence, wide host range in solanaceous crops, ability to grow endophytically, survive in
soil, broad geographic distribution, and versatile methods of transmission [1-6]. Additionally,
R. solanacearum can survive in plant debris, infected plants host weeds, and spread from one
field to another by irrigation or floodwater, soil, farm equipment, and remaining crops from the
previous seasons, hence it is difficult to manage [3-4]. Despite the availability of several
methods for the management of bacterial wilt disease in tomatoes including chemicals,
biological agents, cultural and physical practices [3], the disease has not been successfully
managed in Sub-Saharan Africa [2,7]. Emerging trends in physical practices for control of R.
solanacearum include solarization, hot water treatment of infected soil, planting in the cold
season, soil fumigation, and soil drenching [3]. Some of the aforementioned methods including
planting tomato, potato and tobacco crops during winter when the temperatures are low and
the bacterium is inactive6, may not be viable in Sub-Saharan Africa where temperatures are
usually high all year round. In addition, wet conditions and moderate temperatures in Sub- Saharan Africa usually favor the survival of the bacteria [7]. As a result, synthetic pesticides
continue to be overused for plant disease management and pest control due to their efficacy,
reliability, rapidity of action, and quick knockdown effect [8-9]. However, control of R.
solanacearum using synthetic pesticides has far-reaching implications such as environmental
pollution, contamination of groundwaters, accumulation of toxic residues in food, and
elimination of non-target organisms [9-12]. Biopesticides on the other hand are less toxic, less
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Opondo, F. A., K’Owino, I. O., Chepkwony, S. C., Kosgei, V. J. (2022). In Vitro Antibacterial Activity of Essential Oils from Tithonia Diversifolia Leaves
and Flowers Against Ralstonia Solanacearum. European Journal of Applied Sciences, 10(2). 523-539.
URL: http://dx.doi.org/10.14738/aivp.102.12172
persistent, environment-friendly, safe to humans and non-target organisms, economically
affordable, highly effective, and target specific [8-13].
Application of biopesticides includes the use of bio-control agents like Bacillus
amyloliquefaciens [5] and Pseudomonas fluorescens [14] and plant extracts like Amomum
nilgiricum [15] for control of R. solanacearum. Furthermore, biopesticides have been appraised
to be less persistent in the environment as they are often degraded rapidly by sunlight, or
moisture, are less phytotoxic, and are consequently potentially less of a threat to the
environment and human health [8]. Additionally, they contain a mixture of compounds, which
can work together synergistically in reducing a pathogen or pest with varying modes of action
[16]. In this context, plant extracts contain Phyto complex of active compounds which could be
contributing to the overall biological effect against bacteria through the collective effect of all
its components, some of which will cooperate and some might modulate, while others will act
on different, distantly connected targets, ultimately generating synergistic activity of the
phytoconstituents [16-17]. Essential oils, in particular, have been demonstrated to be effective
antibacterial agents because their lipophilic characteristics contribute to their being capable of
destroying the cell wall of bacteria [18]. Additionally, they contain a large number of
phytochemicals and hence it is most likely that the reported antibacterial activity of essential
oils is due to the synergistic effect of all the constituents and not attributed to one mechanism
[18]. Therefore, using essential oils as biopesticides could lead to reduced occurrence of
pathogen and pest resistance development. As a result, the use of essential oils has been on the
increase as an emerging, potential, and alternative approach in disease management for R.
solanacearum [12, 19-20]. Essential oils are naturally occurring volatile substances obtained
from a variety of plants including T. diversifolia [21].
T. diversifolia (Hemsl.) A. Gray (Asteraceae) is a shrub and a member of the sunflower family
and is native to North and Central America [21-22]. Although this species is native to the
lowlands of southeastern Mexico and Central America, it has now been naturalized in different
regions of the world including Africa and Asia [22], where they have become an ecological,
agricultural, and economic burden [23].
Despite the negative effects of the invasive shrub, ethno pharmacologically, T. diversifolia has
been exploited in folkloric medicinal practices as well as in remediation of heavy metals from
the soil [21, 23].
In Nigeria, T. diversifolia has been reported to be used by herbal medicine practitioners in the
treatment of menstrual pain, treatment of wounds, and diabetes mellitus [22]. In Mexico where
this plant originates from, it is used to treat sprains, bone fractures, bruises and contusions [22].
In Kenya, T. diversifolia locally known as maruru, maua and amalulu (for Luhya tribe), maua
makech (Luo), amaua amaroro (Kisii) and mula (Kamba) all implying that the plant is bitter to
the taste are effectively used for treatment of snake envenomation [24-25]. Among the Kalenjin
community in Kenya, T. diversifolia is commonly known as mauat ne ng’wan meaning bitter
flowers, and is used to treat diarrhea.
Biological activities of T. diversifolia includes anti-inflammatory, analgesic, antimalarial,
antiviral, antidiabetic, antidiarrhoeal, antimicrobial, antispasmodic, vasorelaxant, cancer- chemo preventive, insecticidal, antiemetic, and antiamoebic properties [21-22, 26-27]. Most of
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the pharmacological activities of T. diversifolia have been attributed to sesquiterpene, lactones,
diterpenes, flavonoids and some chlorogenic acid derivatives present in the leaves of this
species [21]. T. diversifolia essential oils consist of terpenes, terpenoids and lactones [28]. These
compounds have been demonstrated to possess antibacterial, antifungal and insecticidal
activities [23, 28]. In a previous study, Farias et al., [26] demonstrated antimicrobial activity of
T. diversifolia leaf essential oils against Staphylococcus aureus, Escherichia coli, and
Pseudomonas aeruginosa and reported that the observed antimicrobial activity was due to the
presence of the main chemical constituents including α-pinene, Limonene, (Z)-β-ocimene, p- cymen-8-ol, Piperitone, (E)-nerolidol and Spathulenol. In another study, Agboola et al., [28],
evaluated the chemical composition and antimicrobial activity of T. diversifolia essential oils
they reported that secondary metabolites present in the oils including monoterpenes,
sesquiterpenes, alcohols, and aldehydes proved effective against Escherichia coli, Proteus
mirabi, Bacillus megaterium, Klebsiella pneumonia, Bacillus cereus and Streptococcus pyrogens
thereby, unlocking the potential of the oils for bio-pesticide production. In a recent study,
Njuguna et al., [29], investigated the contact toxicity of essential oils from the T. diversifolia
leaves against Thrips tabaci, Bemisia tabaci, and Aphis gosypii and reported that the essential
oils possessed remarkable insecticidal activity which could be employed as a safer alternative
to synthetic pesticides. In this context, the current research evaluated the chemical composition
of T. diversifolia essential oils and investigated their antibacterial potential against R.
solanacearum in vitro, with the aim of developing natural, green, and sustainable biopesticides
for application in the management of bacterial wilt disease in tomato.
MATERIALS AND METHODS
Sample collection and preparation
Fresh leaves and flowers from T. diversifolia were collected from Maseno in Kisumu County GPS
location, 0°02'10.2"S34°45'18.8" E, in Kenya. All the plant samples collected were taken to the
Chemistry laboratory at Moi University. Voucher specimens of the plant species were deposited
at the laboratory of Biological Sciences of Moi University. The fresh samples from T. diversifolia
were then washed using distilled water to remove dust and immediately chopped into small
sizes and placed into 1000 mL clevenger apparatus for hydrodistillation.
Extraction
Essential oils from the fresh leaves and flowers of T. diversifolia were extracted separately via
hydrodistillation using clevenger apparatus according to the methods of Liu et al., [30], as
described by Wanzala et al., [31]. Briefly, 1 kg of freshly chopped T. diversifolia leaves and
flowers were separately weighed and hydrodistilled with 1500mL of distilled water using
clevenger apparatus for 8 hours. After hydrodistillation was completed, the volatile essential
oils were removed from the top of the hydrosol, dried over anhydrous sodium sulfate (Na2SO4),
and stored in sealed amber bottles at 4 °C awaiting chemical and bioassay analyses.
GCMS Analysis of essential oils and solvent extracts
GC-MS analyses was performed with a Clarus 500 GC gas chromatograph (Perkin Elmer Inc.,
USA) coupled with a Clarus 500 MS quadrupole mass spectrometer (Perkin Elmer Inc., USA).
Gas chromatography was carried out on a fused-silica capillary column (Elite-5 ms,
60 m×0.25 mm, 0.25 μm film thickness, Perkin Elmer Inc, USA). The gas chromatograph was
equipped with an electronically controlled split/splitless injection port while the carrier gas
was helium with a constant flow of 1.2 mL/min. The GC oven temperature was set at 200 ͦC for
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Opondo, F. A., K’Owino, I. O., Chepkwony, S. C., Kosgei, V. J. (2022). In Vitro Antibacterial Activity of Essential Oils from Tithonia Diversifolia Leaves
and Flowers Against Ralstonia Solanacearum. European Journal of Applied Sciences, 10(2). 523-539.
URL: http://dx.doi.org/10.14738/aivp.102.12172
4 min and programmed in the range 200 ͦC -330 ̊C at a rate of 5 ̊C /min and finally held constant
at 330 ͦC for 15 min. Ionization was performed in the electron impact mode at 70 eV, while
detection was carried out in scan mode from m/z 35- 700 atomic mass units ( a.m.u). Relative
percentage amounts were obtained directly from GC peak areas while retention time was
recorded in minutes. Components present in the essential oils were identified by matching of
mass spectral data with MS library NIST 08 (NIST/EPA/NIH) and comparing the MS
fragmentation patterns with those reported in the literature.
Fourier Transform Infrared (FT-IR) spectroscopy
The essential oils were characterized to confirm the functional groups in the compounds
identified by GC- MS analysis. The essential oils, from T. diversifolia leaves and flowers, were
analyzed using an Attenuated Total Reflectance- Fourier Transform Infrared (ATR-FTIR)
spectrophotometer, NICOLET 6700 Thermo Scientific 2009-27701 Model. The FT-IR spectra
were recorded in the spectral range 4000 to 500 cm-1 and scanning was performed with a
resolution of 4 cm−1 for 100 scans. The functional groups of compounds present in the essential
oils were determined by comparing the wavenumbers in the spectra with those on an IR
correlation chart and comparison of spectral data with those reported in previous studies.
Bactericidal Activity
Isolation and characterization of Ralstonia solanacearum strains
All experiments were performed using highly virulent R. solanacearum strain race 3 biovar III
which was isolated from ten diseased potato plants from Timboroa, Uasin Gishu County, and
deposited at the Biological sciences Laboratory at Moi University. Collected potato tubers were
sterilized with 1% Sodium Hypochlorite (NaOCl) solution for 2 min, followed by three repeated
washings with distilled water and blot dried according to the methods of Singh as described by
[32]. The plant sections (0.5 cm) were then placed inside test tubes containing distilled water
and then be plated onto 2, 3, 5 triphenyl tetrazolium chloride (Kelman’s TZC agar) medium
(glucose 10 g, peptone 10 g, casein hydrolysate 1 g, agar 18 g, distilled water 1000 ml). An
aliquot of 5 ml TZC solution filter-sterilized was added to autoclaved medium to give a final
concentration of 0.005% v/v, followed by incubation of the plates at 28 ̊C for 48 hours [32].
The virulent colonies in the medium were characterized by dull white color, fluidal, irregularly
round with light pink centers which were further streaked on TZC medium to get pure colonies
of the bacterium. Isolated pure colonies of R. solanacearum were refrigerated at 20 ̊C to
maintain their virulence. To revive an isolate, the stored bacteria were streaked on a TZC agar
medium and well-separated fluidal colonies were selected. Preparation of R. solanacearum
bacterial suspension was performed by pouring sterile distilled water over 24hr old bacterial
growths on nutrient agar slants, and the suspension was adjusted to an optical density (O.D)
0.5 in Spectrophotometer ( Beckham Coulter DU 700) to obtain a bacterial population of 1 x 108
colony-forming unit per milliliter of the suspension (optical density at 600 nm). Profiling of the
pathogen was performed morphologically using culture techniques and biochemical tests
including Gram staining test, Potassium hydroxide test, Catalase oxidase test, Gas production
test, Starch hydrolysis test, and sugar utilization test according to Manual of Systematic
Bacteriology as described by [32].
In vitro disc diffusion experiments
The antibacterial activity of the essential oils from T. diversifolia leaves and flowers were tested
in vitro against R. solanacearum according to the disk diffusion method described by [33].
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Briefly, a single colony of the isolate containing 1x 108 Colony Forming Units (CFU) at 0.5 optical
density measured at 600nm was grown on casamino acid peptone glucose medium (CPG) at 28
°C for 48 hours. Each bacterial suspension (200 μl) was then spread on nutrient agar plates and
5mm diameter disks containing 50 μL of essential oil were placed on the surface of the prepared
agar plates. Sterile 1% dimethyl sulphoxide was used as negative control while Metham sodium
(125 μg/mL) a known soil fumigant [1]. was used as a positive control. The plates were then
incubated at 28°C for 24 hours after which diameters of inhibition zones were measured in
millimeters using Vernier calipers. All experiments were performed in triplicate.
Minimum inhibitory concentration
Minimum inhibitory concentration (MIC) was evaluated using the broth microdilution method
using a 96 well microtiter plate as described by [34], with a few modifications on the synthetic
fumigant used. Briefly, 50μL of essential oil was added from the 1st to the 10th well on each row
and diluted two-fold using 1% DMSO in the concentration range 1.953- 1000 μg/mL. Negative
control (1% DMSO) and positive control (metham sodium) were also prepared in the same
concentration range. Then, 50μL of 1x 10-8 CFU of freshly prepared R. solanacearum colonies
were added to each well followed by incubation at 28 °C for 24 hours. Finally, 50 μL of 0.01%
Tetrazolium chloride medium (TZC) was added to each well followed by incubation at 28 °C
for one hour after which MIC was evaluated by visual observation of the color change of the
Tetrazolium chloride medium.
Statistical analysis
Data on the in vitro antibacterial activities of T. diversifolia essential oils against R. solanacearum
were analyzed statistically using Minitab version 17 software at a 99% confidence interval.
Data from mean inhibition zones from three replicate experiments on the four treatments
bioassayed were analyzed and the standard mean error was computed. The difference between
the means was analyzed using One Way Analysis of Variance (ANOVA). Means of inhibition
diameters were separated using Tukey’s honestly significant difference test [33]. p-values
<0.01 were considered statistically significantly different [35].
RESULTS AND DISCUSSIONS
Extraction of essential oils
Essential oils from leaves and flowers of T.diversifolia, were hydrodistilled using clevenger
apparatus. Data obtained was tabulated as shown below:
Table 1: Percentage yield for essential oils
Plant extracts Part of the plant % yield (Volume/
weight)
% yield (Weight/
weight)
T.diversifolia Leaves 0.18+ 0.08 0.10+ 0.07
Flowers 0.15 + 0.09 0.08 + 0.03
Percentage yield of the essential oils was calculated in volume by weight and weight by weight
(Igwaran et al., 2017) as shown in equations1 and 2 below:
% Essential oil yield (v/w) = !"#$%& "( &)&*+,-# ",# (%#)
0&,12+ "( 3#-*+ )-%3#& (1)
(Equation 1)
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Opondo, F. A., K’Owino, I. O., Chepkwony, S. C., Kosgei, V. J. (2022). In Vitro Antibacterial Activity of Essential Oils from Tithonia Diversifolia Leaves
and Flowers Against Ralstonia Solanacearum. European Journal of Applied Sciences, 10(2). 523-539.
URL: http://dx.doi.org/10.14738/aivp.102.12172
% Essential oil yield (w/w) = 4&,12+ "( &)&*+,-# ",# (1)
56&)2 0&,12+ "( 3#-*+ )-%3#& (1)
(Equation 2)
Leaves of T. diversifolia gave the highest percentage yields of 0.18 + 0.04 % (v/w); 0.10 + 0.02
% w/w as compared to the flowers which yielded 0.15 + 0.07 % (v/w); 0.08 + 0.01 % w/w on
a fresh weight basis, however there was no statistical significant difference between leaves and
flowers essential oils yields (p˃ 0.01). Similar results were obtained by [36], who reported
essential oils yields of 0.18% v/w in T. diversifolia leaves essential oils. Contrary, Moronkola et
al., [37], performed hydrodistillation of air-dried plant material from T. diversifolia and
reported essential oil yields of 0.019 and 0.1% w/w in the leaves and flowers respectively. In
another study, [38], reported essential oils yields of 0.12% v/w while performing
hydrodistillation of air-dried leaves of T. diversifolia. The low yields reported in the leaves and
flowers may be due to the fact that air-dried plant materials were used for hydrodistillation
contrary to the fresh leaves and flowers used in this current research. It is noteworthy that
studies performed using fresh leaves of T. diversifolia leaves and flowers reported higher
essential oils [26,36], suggesting that the sample preparation process is a significant factor that
affects essential oils yields. Other factors known to affect T. diversifolia essential oils yields
include the geographical location, climatic conditions, type of soil, and phenotypic
characteristics of the plant [21,39].
GC MS analysis
GC MS analysis of T. diversifolia essential oils tentatively identified 21 compounds in the leaves
and 8 compounds in the flowers as shown in Tables 2 and 3 respectively. The relative amounts
were based on the relative percentage area computed directly from the area under the GC
spectra (Figure 1 and Figure 2). Compounds were listed according to their elution order on a
non-polar fused-silica capillary column (table 2). GC MS profiling revealed that the essential oil
constituents of the leaves was dominated by fatty acids (74.2%), oxygenated sesquiterpenes
(12%), and oxygenated monoterpenes (10.48%) (Table 4), whereby the major compounds
present were found to be (Z,Z,Z)-9,12,15- Octadecatrienoic acid ethyl ester(18%), palmitic acid
(16%)(1aS,4aS,7R,7aS,7bS)-1,1,7-Trimethyl-4-methylenedecahydro-1H-cyclopropa[e]azulen- 7-ol (spathulenol) (12%), Cis- 9,12,15- Octadecatrienoic acid (8.14%), tetrateracontane (6%)
and 1-Octen-3ylcetate (5.22%).The major chemical constituents in the flowers' essential oils
were fatty acids (86.73%), sesquiterpene hydrocarbons (5.45%), and oxygenated
sesquiterpenes (5%), as evidenced by the presence of α-linolenic acid trimethylester(33%),
Z, Z Hexadecadienoic acid(26%), octadecanoic avid trimethylester (9%), palmitelaidic acid
(8.49%) and S, 1Z, 6Z) -8 Isopropyl-1 methyl-5- methylenecyclodeca-1,6-diene (Germacrene
D)(5.45%) azelaic acid (5.02%) and caryophyllene oxide (5.00%) (Table 3). Minor compounds
assayed in the leaf essential oils included α-pinene (1.17%), β-pinene (1.17%), 1-octen-3-ol
(2.21%), 2-octen-1-ol (1.17%) and bicycloheptane (0.63%). Similar findings were observed by
Farias et al., [26], who reported that α- pinene, Limonene, (Z)-β-ocimene, Piperitone, and
Spathulenol were some of the major chemical constituents identified in T. diversifolia leaves. In
another study, contrary to our results, Wanzala et al., [31], analyzed the aerial parts of T.
diversifolia growing on the southern slopes of Mount Elgon in western Kenya and reported that,
α- pinene, β-pinene, iso caryophyllene, nerolidol, 1-tridecanol, limonene, sabinene, α-copaene,
α-gurjunene, and cyclodecene are pre-dominantly distributed. In a recent study, Njuguna et al.,
[29], while evaluating the qualitative and quantitative profiling of essential oils from air-dried
leaves of T. diversifolia collected from Kandara in Murang’a County, Kenya reported that 3-
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carene was the most abundant compound in the essential oils. Major differences were observed
in the essential oils composition of T. diversifolia growing in the Maseno region, Kisumu County,
Kenya as compared to other regions in Vietnam [38], and Brazil [39], as reported in previous
studies. This could be attributed to environmental conditions, ecological conditions, climatic
factors, geographical distribution, extraction method, sample preparation protocols, and part
of the plant assayed [21, 26, 39].
Figure 1: GC spectrum of T. diversifolia leaf essential oils
20211027 , 27-Oct-2021 + 17:07:12
6.40 11.40 16.40 21.40 26.40 31.40 36.40 41.40 46.40
0 Time
100
%
TDEOL3 02 Scan EI+
TIC
2.10e9
26.14
23.18
12.72
10.07
2.81
6.07 5.27 6.77 10.20
22.46
14.01
25.85
23.39
25.29
34.97
32.36
29.46 28.59
28.06 30.44
34.83 36.22
37.82
39.77
42.21
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Opondo, F. A., K’Owino, I. O., Chepkwony, S. C., Kosgei, V. J. (2022). In Vitro Antibacterial Activity of Essential Oils from Tithonia Diversifolia Leaves
and Flowers Against Ralstonia Solanacearum. European Journal of Applied Sciences, 10(2). 523-539.
URL: http://dx.doi.org/10.14738/aivp.102.12172
Table 2: GC-MS Analysis of compounds present in T. diversifolia leaves essential oils
Peak
no
Retention
time
Compound Relative percentage area
1 5.27 alpha.-Pinene 1.17
2 6.07 1-Octen-3-ol 2.21
3 6.65 beta.-Pinene 0.22
4 6.77 2-Octen-1-ol, (Z)- 3.42
5 10.07 1-Octen-3-yl-acetate 5.22
6 10.20 Bicyclo(3.1.1)heptane-2,3-diol,
2,6,6-trimethyl- 0.63
7 12.72 Spathulenol 12
8 14.01 Azelaic acid bis trimethylester 1
9 22.46 n-Hexadecanoic acid 2
10 23.18 Palmitic acid 16
11 23.39 Palmitic acid, ethylester 7.12
12 25.29 (Z,Z,Z)-9,12,15- Octadecatrienoic
acid methyl ester
2
13 25.85 Cis- 9,12,15- Octadecatrienoic acid 8.14
14 26.14 (Z,Z,Z)-9,12,15- Octadecatrienoic
acid ethyl ester
18
15 28.06 Hexadecanoic acid cyclohexylester 2
16 28.59 Tetracosane 5.94
17 29.48 Heptacosane 2
18 30.44 Hentriacontane 1
19 32.36 Tetratriacontane 1
20 34.97 Tetratetracontane 6
21 37.82 Tetracosanoic acid 2
Figure 2 :GC spectrum for T. diversifolia flowers essential oils
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Table 3: GC-MS analysis of compounds present in T. diversifolia flower essential oils
Retention
time
Retention time Compound Relative percentage
area
1 12.63 Caryophyllene oxide 5
2 13.45 Germacrene D 5.45
3 14.59 Azelaic acid bis trimethylester 5.02
4 16.50 Palmitelaidic acid 8.49
5 16.58 (Z,Z)-Hexadecadienoic acid -
trimethylester
26
6 17.73 α-Linolenic acid, trimethylester 33
7 17.84 Octadecanoic acid trimethyl ester 9
8 25.07 Hexadecanoic acid cyclohexylester 5.22
Table 4: Analysis of chemical constituents present in T. diversifolia leaf and flower essential
oils
Chemical compound group Leaf Area % Flower Area %
Monoterpene hydrocarbons 1.39 -
Oxygenated monoterpenes 10.48 -
Sesquitrepene hydrocarbons - 5.45
Oxygenated sesquiterpenes 12 5
Fatty acids 74.2 86.73
Total 98.07% 97.18%
FTIR analysis
Characterization of T. diversifolia leaf essential oils using FTIR-ATR gave significant broadband
at 3410.87 cm-1 in the region 3450 cm-1 to 3300 cm-1 representing –OH stretching vibrations
of essential oils and two peaks at 2918.78 cm-1 and 2848.07 cm-1 depicting C-H stretching
vibrations (Table 6). There was a significantly strong peak at 1710.63 cm-1 in the leaf essential
oils which is a typical -C=O stretching bands [40], attributed to the presence of spathulenol
which was one of the principal phytoconstituents identified by GC-MS analysis. Other
vibrational frequencies were observed at 1466. 11 cm-1, 1317.82 cm-1, 1183.24 cm-1 and
1020.51 cm-1 as summarized on Table 6 below. In the flowers, significant peaks were observed
at 3375.51 cm-1, 2921.58 cm-1 , 2851.01 cm-1 , 1713.63 cm-1,1195.02 cm-1 and 1018.23 cm-1
(Table 6). Similar results were reported by [41-42].
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Opondo, F. A., K’Owino, I. O., Chepkwony, S. C., Kosgei, V. J. (2022). In Vitro Antibacterial Activity of Essential Oils from Tithonia Diversifolia Leaves
and Flowers Against Ralstonia Solanacearum. European Journal of Applied Sciences, 10(2). 523-539.
URL: http://dx.doi.org/10.14738/aivp.102.12172
Table 6: FTIR analysis of T. diversifolia leaf and flower essential oils
Major absorption bands (cm-1)
T. diversifolia leaves
essential oils
T. diversifolia flowers
essential oils
Functional groups
3410.87 3375.51 -OH group
2918. 78 2921.58 -C-H stretching vibrations
2848.07 2851.01 -C-H stretching vibrations
1710.63 1713.63 -C=O stretching
1466.11 1442.54 -CHbending vibrations
1317.82 1309.47 -CH bending vibrations
1183.24 1195.02 C-O stretching vibrations
1020.51 1018.23 C-O-C stretching vibrations
In vitro Antibacterial activity
Highly virulent R. solanacearum strain race 3 biovar III were isolated from ten diseased potato
plants from Timboroa, Uasin Gishu County, and were characterized using biochemical tests. The
wild R. solanacearum colonies gave positive results for the catalase oxidase test and potassium
hydroxide solubility tests as shown in Figures 5a and 5b below and were found to be gram- negative as depicted in figure 5c and 5d. Similar results were obtained by Khasabulli et al., [32],
who reported that the positive catalase test could be attributed to the presence of catalase
enzyme in R. solanacearum because Gram-negative bacteria undergo aerobic respiratory
metabolism hence the Production of gas bubbles was observed.
a b
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European Journal of Applied Sciences (EJAS) Vol. 10, Issue 2, April-2022
Services for Science and Education – United Kingdom
Figure 5 a) Potassium hydroxide solubility test, b) Catalase oxidase test, c) Gram staining of R.
solanacearum, Magnification x 10, d) Gram staining of R. solanacearum, Magnification x 100
R. solanacearum colonies were identified by their large and elevated size, fluidal nature, and
color [43], characterized by dull white color, fluidal, irregularly round with light pink centers
as shown in Figure 6 a below. The virulent colonies were further steaked on TZC Kelman’s
medium to get pure colonies of the bacterium as depicted in Figure 6 b. Virulent colonies of R.
solanacearum were identified by their large and elevated size, fluidal nature, and if they were
either entirely white, or with a pale red center while the mutant and non-virulent strains of R.
solanacearum were uniformly round and dark red, smaller in size, and butyrous or dry on TZC
medium (Figures 6a and 6b). Similar results were obtained by [32, 43], who reported that the
virulent R. solanacearum colonies were characterized by fluidal whitish with a pink center,
indicating virulent species.
Figure 6: a) Virulent and avirulent colonies of R. solanacearum isolates b) Culturing of R.
solanacearum bacterium on TZC Kelman’s Agar medium
In vitro antibacterial activity of the essential oils from T. diversifolia leaves and flowers against
R. solanacearum was analyzed using the disk diffusion quadrat method. The data obtained was
tabulated as shown in Table 7 and illustrated in Figure 7.
c
d
a
b
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Opondo, F. A., K’Owino, I. O., Chepkwony, S. C., Kosgei, V. J. (2022). In Vitro Antibacterial Activity of Essential Oils from Tithonia Diversifolia Leaves
and Flowers Against Ralstonia Solanacearum. European Journal of Applied Sciences, 10(2). 523-539.
URL: http://dx.doi.org/10.14738/aivp.102.12172
Table 7: In vitro antibacterial activity of T. diversifolia essential oils against R. solanacearum
after 24 hours of incubation at 28°C
SNO Treatment Diameter of inhibition zones
in (mm)
1 1% DMSO 5±0.22c
2 TDEOL 11.82 ±0.47b
3 TDEOF 12.61 ±0.22b
4 METHAM SODIUM 25.78 ±0.29a
Values are the mean of three replicates ± standard error.
Values within columns followed by different letters are significantly different at P ≤ 0.01
according to Tukey’s honestly significant difference test
Figure 7: Antibacterial activity of T. diversifolia essential oils against R. solanacearum in vitro
Mean zone of inhibition results revealed that essential oils from the leaves of T.diversifolia
exhibited the highest antibacterial activity of 12.61 ±0.22 in the leaves, as compared to 11.82
±0.76 in the flowers. The MIC for T. diversifolia essential oils against R. solanacearum was 250
μg/mL in the leaves and 500 μg/mL in the flowers (Table 8) while the MIC for Metham sodium
was 125 μg/mL. Statistical analysis of the antibacterial activity of T. diversifolia essential oils in
comparison to Metham sodium which was the positive control revealed that there was a
statistically significant difference between the antibacterial activity of T. diversifolia leaves in
comparison to metham sodium (p = 0.001). Similarly, a statistically significant difference was
observed between the antibacterial activity of Metham sodium and the antibacterial activity of
T.diversifolia flowers essential oils (p = 0.0009) as shown in table 7. A comparison of bioactivity
between the T. diversifolia leaf and flower essential oils and 1% DMSO which was the negative
control (Table 7), revealed that there was a significant difference in bioactivity with p < 0.01.
The observed antibacterial activity of T. diversifolia essential oils could be attributed to
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Services for Science and Education – United Kingdom
phytochemicals including monoterpene hydrocarbons, oxygenated monoterpenes, and
sesquiterpene Hydrocarbons, oxygenated sesquiterpenes, and fatty acids as depicted by GC-MS
profiling of the major phyto compounds and confirmed by FTIR analysis.
Table 8: Minimum inhibition concentration (MIC) values for T. diversifolia essential oils, DMSO,
and Metham sodium against R. solanacearum after 24 hours of incubation
Treatments Concentration μg/mL
1.953 3.906 7.8185 15.625 31.25 62.5 125 250 500 1000
1% DMSO + + + + + + + + + +
TDLEO + + + + + + + + - -
TDFEO + + + + + + + - - -
METHAM
SODIUM
+ + + + + + - - - -
KEY
+ bacterial growth appears
- no bacterial growth
Essential oils contain monoterpenes, diterpenes, and sesquiterpenes which serve as defense
molecules in different plant parts [19], and hence the bioactivity of T. diversifolia essential oils
against gram-negative. R. solanacearum bacterium in vitro could be attributed to the presence
of the major chemical constituents including (1aS,4aS,7R,7aS,7bS)-1,1,7-Trimethyl-4-
methylenedecahydro-1H-cyclopropa[e]azulen-7-ol, 0Z- Octen-1-ol, (24.22%), (Z,Z,Z)-9,12,15-
Octadecatrienoic acid ethyl ester), Cis- 9,12,15- Octadecatrienoic acid(12%), 1-Octen-3ylcetate
and (S, 1Z, 6Z) -8 Isopropyl-1 methyl-5- methylenecyclodeca-1,6-diene, which could be causing
the synergistic antibacterial effect. Additionally, minor compounds profiled including α-pinene,
β-pinene in the leaves, and caryophyllene oxide in the flowers are known antibacterial agents
[44] and could be responsible for the observed antibacterial activity against R. solanacearum in
vitro. These results are in agreement with a previous study by [26], who reported that the main
compounds found in T. diversifolia essential oils including α-pinene, Limonene, (Z)-β-ocimene,
p-cymen-8-ol, Piperitone, (E)-nerolidol, and Spathulenol could be responsible for the
antimicrobial activity against Staphylococcus aureus, Escherichia coli, and Pseudomonas
aeruginosa. In another study, Li and Yu [45], demonstrated that R. solanacearum is susceptible
to the presence of essential oils in vitro. The findings in this current research revealed that T.
diversifolia leaf and flower essential oils possess remarkable in vitro antibacterial activity
against R. solanacearum and hence, could be used to make viable formulations to combat the
devastating tomato bacterial wilt disease.
CONCLUSION
This study demonstrated that T. diversifolia leaf and flower essential oils possess
phytochemicals including monoterpene hydrocarbons, oxygenated monoterpenes,
sesquiterpene hydrocarbons, oxygenated sesquiterpenes, and fatty acids. Additionally, in vitro
antibacterial activity showed that the profiled phytochemicals possessed remarkable
antibacterial activity against R. solanacearum. Based on the observed antibacterial activity of
the T. diversifolia leaves and flowers essential oils, this study emphasizes the great potential of
these essential oils for commercial applications in the management of phytopathogenic
bacteria and corroborates the extensive use of T. diversifolia in folkloric medicine.
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Opondo, F. A., K’Owino, I. O., Chepkwony, S. C., Kosgei, V. J. (2022). In Vitro Antibacterial Activity of Essential Oils from Tithonia Diversifolia Leaves
and Flowers Against Ralstonia Solanacearum. European Journal of Applied Sciences, 10(2). 523-539.
URL: http://dx.doi.org/10.14738/aivp.102.12172
ACKNOWLEDGMENT
The authors are grateful to the World Bank, through the Africa Center of Excellence II in
Phytochemicals, Textiles and Renewable Energy (ACEII-PTRE), and Moi University for funding
this work. The Authors also acknowledge LERMab for the GC-MS profiling and FT-IR analysis
that was performed in the (LERMAB), research group laboratory, at the University of Lorraine,
France.
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