Antifungal activities of Rosemary (Rosmarinus officinalis) and Eucalyptus (Eucalyptus globulus) essential oils evaluated, suppressed the mycelial growth of postharvest pathogenic fungus Colletotrichum sp. The result obtained with the Agar well diffusion method at 75% and 100% concentration of the essential oil were negligible, with the Agar incorporation method the essential oils had effect on the fungus. Eucalyptus essential oil having the highest inhibition of 100% on the mycelial growth of Colletotrichum sp followed by Rosemary essential oil which had 95.24% inhibition on the growth of the fungus Colletotrichum sp, The GC-MS analysis result of the essential oils shows that in Rosemary: Eucalyptol (1,8-Cineole) (32.83%) and Camphor (24.17%) are the main constituents and in Eucalyptus: Eucalyptol (1,8-Cineole) (26.67%) and Terpinen-4-ol (25.08%) are the main constituents, which could be responsible for the antifungal activities of the different essential oils. These essential oils could be used as possible biofungicides as an alternative to synthetic fungicides against pathogenic fungi on tomato fruits.

1. Characterization and In vitro antifungal potential of Rosmarinus officinalis and Eucalyptus globulus essential oils on phytopathogen
Colletotrichum sp.
WJM
Characterization and In vitro antifungal potential of
Rosmarinus officinalis and Eucalyptus globulus
essential oils on phytopathogen Colletotrichum sp.
1*
Osaro-Matthew Ruth Chiamaka, 2
Azubike-Izah Faith Omonigho, 1
Ofoegbulam Amarachi
Helen
1*
Department of Microbiology, Michael Okpara University of Agriculture Umudike, Nigeria.
2
Department of Biochemistry, Michael Okpara University of Agriculture Umudike. Nigeria.
Antifungal activities of Rosemary (Rosmarinus officinalis) and Eucalyptus (Eucalyptus
globulus) essential oils evaluated, suppressed the mycelial growth of postharvest
pathogenic fungus Colletotrichum sp. The result obtained with the Agar well diffusion
method at 75% and 100% concentration of the essential oil were negligible, with the Agar
incorporation method the essential oils had effect on the fungus. Eucalyptus essential oil
having the highest inhibition of 100% on the mycelial growth of Colletotrichum sp followed
by Rosemary essential oil which had 95.24% inhibition on the growth of the fungus
Colletotrichum sp, The GC-MS analysis result of the essential oils shows that in Rosemary:
Eucalyptol (1,8-Cineole) (32.83%) and Camphor (24.17%) are the main constituents and in
Eucalyptus: Eucalyptol (1,8-Cineole) (26.67%) and Terpinen-4-ol (25.08%) are the main
constituents, which could be responsible for the antifungal activities of the different
essential oils. These essential oils could be used as possible biofungicides as an alternative
to synthetic fungicides against pathogenic fungi on tomato fruits.
Keywords: Antifungal, Colletotrichum sp, Eucalyptus globulus, Rosmarinus officinalis, GC-MS analysis
INTRODUCTION
Fruits are important to human being. They give the
body the required vitamins, fats, minerals and oils for
human growth and development (Eni et al., 2010).
However, trait to fruit existence include: climate
change, pest and phytopathogens.
Spoilage is any change in the nature of food in which
the food becomes less palatable or toxic, this maybe
change in taste, smell, appearance, or texture. Hence,
the urgent need to mitigate fruit spoilage (Barth et al.,
2009).
Colletotrichum is a phytopathogen, which induced
anthracnose an economic important disease in fruit
crops, due to the losses incurred. In tomato fruits,
anthracnose affects the fruit, by producing circular rot
that sunken and turns black as they grow in size (Wani,
2011; Hyde et al., 2009). In commercial farming with
frequent fungicide applications, losses range from 10 to
25% of the harvested fruit. When there are no good
management measures, losses can be 80 or even
100% of the harvest (Cannon et al., 2012; Agrios,
2005). This is therefore, a serious crop disease,
discouraging farmers and resulting to economic loss.
*Corresponding author: Mrs. Osaro-Matthew Ruth
Chiamaka, Department of Microbiology, Michael
Okpara University of Agriculture Umudike, Nigeria. Tel.:
+2347035385253. Email: mrc.osaro@mouau.edu.ng;
mensahuche@yahoo.com.
Azubike-izah Faith Omonigho: +2347062250578.
Email: faithoyemonlan@yahoo.com
Ofoegbulam, Amarachi Helen: Email:
helysilky@yahoo.com
World Journal of Microbiology
Vol. 3(1), pp. 038-042, July, 2016. © www.premierpublishers.org, ISSN: 2141-5032x
Research Article

2. Characterization and In vitro antifungal potential of Rosmarinus officinalis and Eucalyptus globulus essential oils on phytopathogen
Colletotrichum sp.
Osaro-Matthew et al. 038
The search for natural substance that can mitigate fruit
spoilage in recent years and concern about the
numerous side effects of synthetic compounds have
channeled research interest to plant resources.
Essential oils which are volatile secondary metabolites
of plant, have industrial application especially in food
and pharmaceutical industries (Adorjan and Buchbauer,
2010; Burt, 2004). Many essential oils and their
chemical composition have been evaluated for their
antimicrobial activities against some fungi and bacteria
in many research reports (Kalemba and Kunicka,
2003). Due to the variation in the component of the
essential oils, the antimicrobial activities may be
attributed to several mechanisms of actions (Bakkali et
al., 2008; Buchbauer, 1993; Di Pasqua et al., 2007;
Hammer et al., 2008).
To face the challenge of food security, research on bio-
preservatives has become a multidisciplinary task (Jing,
2007). This study evaluated the chemical profile and
antifungal activities of essential oils of Rosmarinus
officinalis (Rosemary), and Eucalyptus globules
(Eucalyptus) on the fungal isolate from tomato
anthracnose.
MATERIALS AND METHOD
Sample collection
Diseased tomato fruits were purchased randomly from
markets in Umuahia, Abia state and allowed to develop
anthracnose, the Eucalyptus, Rosemary and Lemon
essential oils were purchased from the market..
Gas chromatography- Mass spectometry (GC-MS)
The essential oils were analyzed by GC-MS to identify
the constituents of the essential oils and also to confirm
the quality and authenticity of the essential oils. The
analysis was performed using a GC-MS-QP2010 PLUS
SHIMADZU JAPAN model. The carrier gas used was
Helium, with column flow rate rate of 0.99ml/min.
Injection temperature was 250
O
C, Oven temperature
programmed from 60
O
C (3min) to 280
O
C (12min) at
3min/
O
C. Ion source temperature 200
O
C. MS start time
3.50min end time 24.00min. The constituents of
essential oil was identified based on the retention
indices and spectrum comparison with NIST MS
libraries (NIST, USA). The GC-MS analysis was carried
out at NARICT (National Research Institute for
Chemical Technology) Zaria Nigeria.
Isolation and Identification of Test Phytopathogen
Sabouraud Dextrose Agar (SDA) was prepared
according to manufactures instruction, a sterilized bled
was used to cut little portion from the affected area of
the tomato and inoculated onto the SDA then incubated
for 7-14 days at 25
O
C. After which the morphological
characteristics was observed both the obverse and
reverse of the colony were observed, the conidia and
appresoria were identified using lactophenol cotton blue
stain (Lubbe et al., 2004; Leck, 1999).
Assays for Antimicrobial Activity
The agar well and agar incorporation methods were
used for the evaluation of the essential oils antifungal
activities.
Agar well diffusion assay
2mls of the test fungal suspension of 10
5
spores per ml
with a Haemocytometer determination, prepared from
agar plate of actively growing fungi was inoculated onto
an SDA agar. Into three wells (6mm) which were made
on the agar plate equidistant to each other using sterile
cork borer, 0.1ml (100µl) of the essential oil at 75% and
100% concentrations were added into the wells while
the third well contained acetone as negative control.
The tests were carried out for each of the three
essential oils. The plates were then left at room
temperature (25
o
C) for 72hours. The antimicrobial
activities of the essential oils were measured as the
diameter (mm) of clear zone of growth inhibition
(Smania et al., 1995).
Agar incorporation assay
The antifungal activity was also carried out by the
incorporation method 1ml 100% concentration of the
essential oils were mixed with molten Sabouraud
Dextrose Agar (45-50
0
C) and allowed to solidify, 1ml of
acetone was used as the negative control. After cooling
and solidification, the medium was inoculated with 1ml
suspension of the actively growing fungus and
incubated at 25
0
C for 3 days. The antifungal activity of
the essential oils was calculated by the percentage of
inhibition (%I) from the diameter of colonies in the
control (CD) and the diameter colonies in the test plates
(ED) both measured on the 3
rd
day. %I= CD-ED/CD X
100 (Suleiman and Abdallah, 2014).
RESULTS
The colonial characteristic of the isolated fungus is
shown in Table (1) while Table (2) describes the
microscopic features of the fungus (Colletotrichum sp).
Tables (3) and (4) show the antifungal activities of the
essential oils using Agar well diffusion method and
Agar incorporation methods respectively. Eucalyptus
essential oil had the highest zones of inhibition, while
Lemon essential oil had the least zones of inhibition at
75% and 100% concentrations respectively. Using the
Agar incorporation method, Eucalyptus essential oil
showed a high percentage inhibition of 100%.
GC-MS analysis qualitative and quantitative analysis
result of essential oils of Rosmarinus officinalis and
Eucalyptus globulus Tables (5 and 6) and Figure 1
and 2.

3. Characterization and In vitro antifungal potential of Rosmarinus officinalis and Eucalyptus globulus essential oils on phytopathogen
Colletotrichum sp.
World J. Microbiol. 039
Table 1. Colonial features of isolates
Isolate Age of culture Pigment of colony Texture of
colony
Size of colony
Colletotrichum
Species
7-14days White surface
White reverse
Wooly/cottony Covered the agar
surface
Table 2. Microscopic observation of isolate
Isolate Hyphae Appressorium Conidia
Colletotrichum species Hyaline with defined
septa
Long and unbranched Straight unicellular
fusiform conidia
Table 3. Zone of inhibition obtained on ColletotrichumSp using Agar well diffusion method
Essential oil Concentration
75% 100%
Rosemary No inhibition No inhibition
Eucalyptus 0.3mm 0.8mm
Control No inhibition No inhibition
Table 4. Percentage of inhibition obtained against Colletotrichum species using Agar incorporation method
Essential oil % of inhibition
Rosemary 95.24
Eucalyptus 100
Table 5. Chemical constituents of Rosmarinus officinalis
S/N Name of compound Percentage (%)
1 alpha-pinene 0.31
2 beta-pinene 0.87
3 Eucalyptol 32.83
4 1,6-Octadien-3-ol,3,
7-dimethyl (β-Linalool)
15.12
5 Bicyclo[2.2.1] heptan-
2-one,1,7,7-trimethyl (Camphor)
24.17
6 Isoborneol 5.22
7 p-Menth-1-en-8-ol 7.80
8 Z,Z,Z-4,6,9-Nonad-Ecatriene 0.85
9 Oleic acid 12.82
Figure 1. Chromatogram of Rosmarinus officinalis essential oil

4. Characterization and In vitro antifungal potential of Rosmarinus officinalis and Eucalyptus globulus essential oils on phytopathogen
Colletotrichum sp.
Osaro-Matthew et al. 040
Table 6. Chemical constituents of Eucalyptus globulus essential oil
N0 Name Of Comound Percentage
1 IR-alpha-Pinene 5.37
2 beta-Pinene 0.71
3 beta-Myrcene 0.37
4 alpha-Phellandrene 0.27
5 Cyclohexene,4-methyl-3-(1-methylethylidene) 2.96
6 Benzene,1-methyl-2-(1-methylethyl) 6.02
7 Eucalyptol 26.67
8 1,4-Cyclohexadiene,1-methyl-4-(1-methylethyl) 7.66
9 Terpinolen 2.37
10 Terpinen-4-ol 25.08
11 p-menth-1-en-8-ol 7.53
12 Zingiberene 0.55
13 Caryophyllene 1.03
14 Aromadendrene 2.82
15 α –Farnesene 2.37
16 Naphthalene,1,2,3,5,6,8a-hexahydro-4,7-
dimethyl-1-(1-methylethyl)
1.23
17 Ledol 0.49
18 2-Naphthalenemethanol,decahydro-
alpha,alpha,4a-trimethyl-8-methylene,[2R-(2-
alpha4a-alpha,8a-beta)]
2.00
19 n-Hexadecanoic acid 1.10
20 Oleic acid 2.96
21 Octadecanoic acid 0.44
Figure 2. Chromatogram of Eucalyptus globulus essential oil
DISCUSSION
The antifungal effect of essential oils particularly
against Colletotrichum is interesting owing to the role of
the fungus as a phytopathogen. From this research
work, Eucalyptus and Rosemary essential oils had
antifungal activity against the fungus Colletotrichum
which causes the post-harvest anthracnose disease in
tomato fruits compared to the control acetones.
Using the Agar incorporation method, The Eucalyptus
globulus essential oil at 100% concentration inhibited
the mycelial growth of the experimental fungus
(Colletotrichum sp) this is in line with the result obtained
by Katooli et al.(2012) which Eucalyptus essential oil
had fungistatic activity against Colletotrichum
gloeosporioides.
The essential oil of Rosmarinus officinalis also showed
antifungal activity against the test fungus and the
findings of the present study are in concordance with
the result obtained by Idris et al. (2015) who revealed
that Rosmarinus officinalis essential oil significantly
inhibited mycelia growth of Colletotrichum musae in
vitro and delayed the development of anthracnose
disease on Banana in vivo.
Other researchers have independently shown that
essential oil has fungistatic and fungicidal activities,

5. Characterization and In vitro antifungal potential of Rosmarinus officinalis and Eucalyptus globulus essential oils on phytopathogen
Colletotrichum sp.
World J. Microbiol. 041
Elhoussine et al. (2013) reported the effect of
Eucalyptus on Penicillium citrinum, Lee et al. (2007),
also demonstrated that Tymus vulgaris oil inhibited
mycelial growth of three phytopathogenic fungi such as
C. gloeosporioides , Rhizoctonia solani and Fusarium
oxysporum. Barrera-Necha et al. (2008) revealed the
efficacy of nine essential oil on C. gloeosporioides. The
antifungal effect of numerous essential oil can be linked
to the chemical constituents of the oil which include:
monoterpenes, aldehydes, alcohols, ketones , oxides
and esters.
With the Agar well diffusion method the result obtained
is negligible this might explain the high volatility of the
essential oil, may be the oil evaporated before it
properly diffuse into the medium.
In this study the chemical profile of Eucalyptus
essential oil shows 21 compounds in which the most
abundant compounds are Eucalyptol (26.67%), 1,4-
Cyclohexadiene,1-methyl-4-(1-methylethyl) (7.66%),
Terpinen-4-ol (25.08%) and p-Menth-1-en-8-ol (7.53%)
which are synonymous to 1,8-Cineole, γ-Terpinen, 4-
Carvomenthenol and α-Terpineol respectively, which is
in accordance with the report of Elhoussine et al.
(2013) 1.8-cineole (22.35%), terpinen-4-ol (3.10%) and
α-terpinéol (1.10%). Also the result of Boukhatem et al.
(2014) shows 1,8-Cineole(51.08 %) and α-pinene
(24.6 %). Result obtained from phytochemical analysis
of Eucalyptus globulus from different region shows that
1,8-Cineole (Eucalyptol) is the major compound, with
percentages of: 64.5 % in Uruguay, 77 % in Cuba,
86.7 % in California, 58 % to 82 % in Morocco, 48.7
% in Africa, and 50 % to 65 % in Argentina as reported
by Viturro et al. (2003), the different percentage of
abundance could be attributed to variation in climatic
conditions.
The qualitative analysis of Rosmarinus officinalis
essential oil shows nine compound in which Eucalyptol
(32.83%), β-Linalool (15.12%), Camphor (24.17%) and
Oliec acid (12.82%)were quantitatively most abundant
in concordance with the result of this research work
presence of Camphor, Cineol, Borneol were reported
by Mahmoud, (2010) on Rosmarinus officinalis L.
essential oil, also Kesatebrhan and Tesema, (2014)
report on comparison of chemical constituent of
Ethiopian Rosemary essential oil with other countries
shows presence of compounds such as 1,8 Cineole,
Camphor, Isoborneol, β-Mercene, Linalool, α-Pinene
which are in agreement with the result of this study.
However, the qualitative analysis of Eucalptus and
Rosemary essential oil in this study shows that they
both contain Eucalyptol, β- Pinene, Oleic acid although
they quantitatively differ, which might explain the close
result obtained with the Agar incorporation method
Eucalyptus (100%) and Rosemary (95.24%).
CONCLUSION
From this research work it has been shown that
essential oils of Rosemary and Eucalyptus educed in
vitro the mycelia growth of Colletotrichum. Previous
studies have also indicated that constituents of these
essential oils are generally regarded as safe
compounds, since their toxicity to the human being is
very low. Base on these evidence we can conclude that
Rosemary, Eucalyptus and Lemon essential oils could
be used as possible bio-fungicides as an alternative to
synthetic fungicides against tomato fungal rot.
However, in vivo experiments need to be conducted
before making recommendations with regard to the
efficacy of these essential oils putative antifungal
agents to increase the post-harvest storage life of
tomato.
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Accepted 02 May, 2016.
Citation: Osaro-Matthew RC, Azubike-Izah FO,
Ofoegbulam AH (2016). Characterization and In vitro
antifungal potential of Rosmarinus officinalis and
Eucalyptus globulus essential oils on phytopathogen
Colletotrichum sp.. World Journal of Microbiology, 3(1):
038-042.
Copyright: © 2016 Osaro-Matthew et al. This is an
open-access article distributed under the terms of the
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unrestricted use, distribution, and reproduction in any
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