WJM

World Journal of Microbiology

Vol. 3(1), pp. 038-042, July, 2016. www.premierpublishers.org, ISSN: 2141-5032x

Research Article

Characterization and In vitro antifungal potential of

Rosmarinus officinalis and Eucalyptus globulus
essential oils on phytopathogen Colletotrichum sp.
1*

Osaro-Matthew Ruth Chiamaka, 2Azubike-Izah Faith Omonigho, 1Ofoegbulam Amarachi

Helen
1*

Department of Microbiology, Michael Okpara University of Agriculture Umudike, Nigeria.

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

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 biopreservatives 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.

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
5

2mls of the test fungal suspension of 10 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 (100l) 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
o
temperature (25 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).

MATERIALS AND METHOD

Agar incorporation assay
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.
O
Injection temperature was 250 C, Oven temperature
O
O
programmed from 60 C (3min) to 280 C (12min) at
O
O
3min/ C. Ion source temperature 200 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
O
for 7-14 days at 25 C. After which the morphological
characteristics was observed both the obverse and

The antifungal activity was also carried out by the

incorporation method 1ml 100% concentration of the
essential oils were mixed with molten Sabouraud
0
Dextrose Agar (45-50 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
0
incubated at 25 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
rd
(ED) both measured on the 3 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.

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

Colletotrichum
Species

7-14days

White surface
White reverse

Texture
of
colony
Wooly/cottony

Size of colony
Covered
surface

the

agar

Table 2. Microscopic observation of isolate

Isolate
Colletotrichum species

Hyphae
Hyaline
septa

with

defined

Appressorium
Long and unbranched

Conidia
Straight
unicellular
fusiform conidia

Table 3. Zone of inhibition obtained on ColletotrichumSp using Agar well diffusion method

Essential oil

Concentration
75%
No inhibition
0.3mm
No inhibition

Rosemary
Eucalyptus
Control

100%
No inhibition
0.8mm
No inhibition

Table 4. Percentage of inhibition obtained against Colletotrichum species using Agar incorporation method

Essential oil
Rosemary
Eucalyptus

% of inhibition
95.24
100

Table 5. Chemical constituents of Rosmarinus officinalis

S/N
1
2
3
4

Percentage (%)
0.31
0.87
32.83
15.12

6
7
8

Name of compound
alpha-pinene
beta-pinene
Eucalyptol
1,6-Octadien-3-ol,3,
7-dimethyl (-Linalool)
Bicyclo[2.2.1]
heptan2-one,1,7,7-trimethyl (Camphor)
Isoborneol
p-Menth-1-en-8-ol
Z,Z,Z-4,6,9-Nonad-Ecatriene

Oleic acid

12.82

24.17
5.22
7.80
0.85

Figure 1. Chromatogram of Rosmarinus officinalis essential oil

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
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18

19
20
21

Name Of Comound
IR-alpha-Pinene
beta-Pinene
beta-Myrcene
alpha-Phellandrene
Cyclohexene,4-methyl-3-(1-methylethylidene)
Benzene,1-methyl-2-(1-methylethyl)
Eucalyptol
1,4-Cyclohexadiene,1-methyl-4-(1-methylethyl)
Terpinolen
Terpinen-4-ol
p-menth-1-en-8-ol
Zingiberene
Caryophyllene
Aromadendrene
Farnesene
Naphthalene,1,2,3,5,6,8a-hexahydro-4,7dimethyl-1-(1-methylethyl)
Ledol
2-Naphthalenemethanol,decahydroalpha,alpha,4a-trimethyl-8-methylene,[2R-(2alpha4a-alpha,8a-beta)]
n-Hexadecanoic acid
Oleic acid
Octadecanoic acid

Percentage
5.37
0.71
0.37
0.27
2.96
6.02
26.67
7.66
2.37
25.08
7.53
0.55
1.03
2.82
2.37
1.23
0.49
2.00

1.10
2.96
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,

Characterization and In vitro antifungal potential of Rosmarinus officinalis and Eucalyptus globulus essential oils on phytopathogen
Colletotrichum sp.

World J. Microbiol.

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%), 1,4Cyclohexadiene,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, -Terpinen, 4Carvomenthenol and -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
-terpinol (1.10%). Also the result of Boukhatem et al.
(2014) shows 1,8-Cineole(51.08 %) and -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%), -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, -Mercene, Linalool, -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, - 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

041

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.

REFERENCE
Adorjan B, Buchbauer G (2010). Biological properties of
essential oils: an updated review. Flavour Frag. J. 25:
407-426.
th
Agrios GN (2005). Plant Pathology. (5 Edition). San
Diego: Academic Press, USA. 385-614.
Bakkali F, Averbeck S, Averbeck D, Idaomar M (2008).
Biological
effects
of
essential
oils.
Food
Chem.Toxicol 4:446-475.
Barrera-Necha
LL, Bautista-Banos S, FloresMoctezuma EH, Estudillo RA (2008). Efficacy of
Essential Oils on the Conidial Germination, Growth of
Colletotrichum gloeosporioides and Control of
Postharvest Diseases in Papaya (Caricapapaya L.). J
Plant Pathol. 7: 174-178.
Barth M, Hankinson RT, Zhuang H, Breidt F (2009).
Compendium of the microbiological spoilage of foods
and beverages: Microbiological Spoilage of Fruits and
Vegetables. Food Microbiology and food safety,
Speber and Doyle (eds.), Springer Science and
Business media. pp135-183.
Boukhatem MN, Amine FM, Kameli A, Saidi F, Walid K,
Mohamed SB (2014). Quality assessment of the
essential oil from Eucalyptus globulus Labill of Blida
(Algeria) origin. Int. Lett. Chem. Phys. Astron. 36
:303-315.
Buchbauer G (1993). Molecular interaction: biological
effects and modes of action of Essential oils. Int. J.
Aromather. 5: 11-14.
Burt S (2004). Essential oils: their antibacterial
properties and potential applications in Foods. Int J
Food Microbiol. 94: 223-253.
Cannon PF, Damm U, Johnston PR, Weir BS (2012).
Colletotrichum- Current Status and Future Directions.
Stud Mycol. 73: 181 213.
Di Pasqua R, Betts G, Hoskins N, Edwards M, Ercolini
D, Mauriello G (2007).Membrane toxicity of
antimicrobial compounds from essential oils. J Agric.
Food Chem. 55: 4863-4870.
Elhoussine D, Benziane Z, Rachida C, Amina Bouseta.
(2013). Antifungal activity and gas chromatography
coupled with mass spectrometry (GC-MS) leaf oil
analysis of essential oils extracted from Eucalpitus
globulus
(Myrtaceae) of north centre region of
Morocco, Afr. J Pharm. Pharmacol. 7(19):1157-1162.
Eni AO, Ibokunoluwa O, Oranusi U (2010). Microbial

Characterization and In vitro antifungal potential of Rosmarinus officinalis and Eucalyptus globulus essential oils on phytopathogen
Colletotrichum sp.

Osaro-Matthew et al.

042

Quality of Fruits and Vegetables. Afri J Food Sci. 4

(5): 291 296.
Hammer KA, Carson CF, Dunstan JA, Hale J, Lehmann
H, Robinson CJ, Prescott SL, Riley TV (2008).
Antimicrobial and anti-inflammatory activity of five
Taxandria fragrans oils in vitro. Microbiol. Immun.
52:522-530.
Hyde KD, Cai L, Mckenzie EHC, Yan YL, Zhag JZ,
Prihastuti H (2009). Colletotrichum: A Catalogue of
Confusion. Fungal Division, 39: 1 17.
Idris MF, Ibrahim MA, Forsido FS. (2015.) Essential
Oils to Control Colletotrichum musae in vitro and in
vivo on Banana Fruits. Am Eurasian J Agric. Environ.
Sci. 15 (3): 291-302,
Jing H. (2007). Identification of variation in adaptively
important traits and genome-wide analysis of trait
markers association. J. Exp. Bot. 58(13):3749-3764.
Kalemba D, Kunicka A. (2003). Antibacterial and
antifungal properties of essential oils.Curr. Med.
Chem., 10(10): 0929-8673.
Katooli N, Maghsodlo R, Hosein H, Razavi ES. (2012).
Fungistatic activity of Essential oil of Thyme and
Eucalyptus against Postharvest and soilborne Plant
Pathogenic fungi, Glob. J. Med. Plant Res. 1(1): 1-4.
Leck, A. (1999). Preparation of lactophenol cotton blue
slide mounts. Community Eye Health Journal,
12(30):24.
Lee SO, Choi GJ, Jang KS, Kim JC. (2007). Antifungal
Activity of Five Plant Essential Oils as Fumigant
Against Postharvest and Soilborne Plant Pathogenic
Fungi. Plant Pathol., J. 23(2): 97-102.
Lubbe CM, Denman S, Cannon PF, Groenewald JK,
Lamprecht SC, Crous PW (2004) Characterization of
Colletotrichum species associated with disease of
Proteaceae. Mycologia 96:1268-1279.
Mahmoud KS (2010). Gas Chromatography-Mass
Spectrometry
(GC-MS)
Analysis
of
Active
Compounds Produced from Rosemary (Rosmarinus
officinalis L.) Callus and Leaf Extracts, AlMustansiriya J. Sci. 21(4):111-118.
Nashwa SMA,
Abo-Elyousr KA (2012). Effect of
aqueous extract against the early blight disease of
tomato plants under greenhouse and field conditions.
Plant Prot. Sci. 48:74-79.
Smania A, Monache DF, Gil ML, Benchetrit LC, Cruz
FS (1995). Antibacterial activity of substance
produced by the fungus Pycnoporus sanguineus (Fr)
merr. J. Ethnopharmacol, 45: 177-181.
Suleimam AE, Abdallah BH (2014). In vitro activity of
Garlic (Alliumsativum) on some pathogenic fungi.
European J. Med. Plants. 4(10): 1240-1250
Thangavelu, R., Sundararaju, P, Sathiamoorthy S
(2004). Management of anthracnose disease of
banana caused by Colletotrichum musae using plant
extracts. J. Hortic. Sci. Biotechnol. 48:74-79.
Viturro IC, Molina AC, Heit CI (2003). Volatile
components of Eucalyptusglobulus, J. Essent. Oil
Res. 15(3):206-208.
Wani AH (2011) An overview of the fungal rot of
tomato. Mycopath 9(1): 33-38.

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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Characterization and In vitro antifungal potential of Rosmarinus officinalis and Eucalyptus globulus essential oils on phytopathogen
Colletotrichum sp.