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Activity of Tea Tree (Melaleuca alternifolia, Cheel)

and thyme (Thymus vulgaris, Linnaeus.) Essential
Oils against Some Pathogenic Seed Borne Fungi
a b
Luca Riccioni & Laura Orzali
a
CRA-PAV, Plant Pathology Research Centre , Rome , Italy
b
CRA-PAV, Plant Pathology Research Centre , Rome , Italy
Published online: 07 Feb 2012.

To cite this article: Luca Riccioni & Laura Orzali (2011) Activity of Tea Tree (Melaleuca alternifolia, Cheel) and thyme
(Thymus vulgaris, Linnaeus.) Essential Oils against Some Pathogenic Seed Borne Fungi, Journal of Essential Oil Research,
23:6, 43-47, DOI: 10.1080/10412905.2011.9712280

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 M. alternifolia

Activity of Tea Tree (Melaleuca alternifolia, Cheel)

and thyme (Thymus vulgaris, Linnaeus.) Essential
Oils against Some Pathogenic Seed Borne Fungi

Luca Riccioni*
CRA-PAV, Plant Pathology Research Centre, Rome, Italy
Laura Orzali
CRA-PAV, Plant Pathology Research Centre, Rome, Italy

Abstract
Downloaded by [University Of Pittsburgh] at 10:38 12 November 2014

Melaleuca alternifolia (tea tree) and Thymus vulgaris (thyme) essential oils were tested for their antifungal activ-
ity to investigate the possibility of their use for seed treatment. The ability of the two oils to inhibit mycelial growth
was studied by in vitro assay on agar medium containing different concentrations of the essential oils (0%, 0.05%,
0.1%, 0.25%, 0.5%, 1% v/v). Seven seed-borne pathogen fungi of large interest Ascochyta rabiei, Colletotrichum
lindemuthianum, Fusarium graminearum, F. culmorum, Drechslera avenae, Alternaria radicina and A. dauci
were transferred on the modified medium in order to test the oils antifungal activity, by calculating the Percentage
of Mycelial Growth Inhibition (%MGI) and the Minimum Inhibitory Concentration (MIC). Results showed that
both the oils had a clear reducing effect on fungal growth, that was dose-dependent and it differed depending on
the fungal species, confirming what is already reported in literature, with T. vulgaris oil to be one of the most potent
agent against fungi. M. alternifolia and T. vulgaris oils can be considered potential alternative natural fungicides to
the synthetic chemicals that are currently used to prevent and control seed-borne diseases, and could be used in
agriculture for safe and eco-friendly seed-treatments.

Key Word Index

Ascochyta rabiei, Colletotrichum lindemuthianum, Fusarium spp, Drechslera avenae, Alternaria spp, mycelial
growth, Minimum Inhibitory Concentration.

Introduction Most crops, mainly wheat and vegetable cultures, can be

exposed to different seed borne diseases which results in con-
In recent years, interest has grown in developing alternative
siderable losses in yields: it is well known that seed can hide
measures to chemical treatments for crop and seed protection,
pathogen organisms which represent an important threat to
including physical methods, biocontrol agents and plant extracts
crop yield and quality, causing germinability reduction, seed-
(1). Concerning plant extracts, many studies have been focused
ling damages, primary infection onset and species spreading.
on the pharmacological actions of essential oils derived from
Therefore, seed borne diseases represent a critical problem for
aromatic and medicinal plants: these are metabolic products
successful production at both qualitative and quantitative level,
accumulating by plants and extracted from leaves, flowers,
especially in organic farming systems, where less efficient plant
roots and barks, and their active principles (alkaloids, phenols,
protection agents are available for managing plant diseases. In
flavonods, monoterpenes and sesquiterpenes isoprenoids)
this work, some seed-borne pathogen fungi of large interest
have been broadly studied due to their many antimicrobial
on two legumes (chick-pea and bean), wheat and carrot are
and antioxidant properties (2) (3). In particular, tea tree oil
studied: Ascochyta rabiei (Pass.) Labrousse, Colletotrichum
(Melaleuca alternifolia Cheel, TTO) and thyme oil (Thymus
lindemuthianum (Sacc. & Magn.) Briosi & Cavara, Fusarium
vulgaris Linnaeus, TO) have been reported to possess anti-
graminearum Schwabe, F. Culmorum (W.G. Smith) Saccardo,
fungal activity (4) (5) (6) and are considered to be potentially
Drechslera avenae (Eidam) Scharif, Alternaria radicina (Meier)
very important for agriculture, thanks to their effectiveness,
Drechsler & Eddy, A. Dauci (Kühn) Groves & Skolko.
low cost and easy availability.
*Address for correspondence: [email protected]

1041-2905/11/0001-06$14.00/0­—© 2011 Allured Business Media

Vol. 23, November/December 2011 Journal of Essential Oil Research/43

 Riccioni et al.

The aim of this study is to test the efficacy of the antifungal isolated from durum wheat seeds; the strains of A. dauci, A.
activity of TTO and TO against these pathogenic seed-borne rabiei and C. lindemuthianum were isolated from carrot, chick-
fungi by in vitro assay, evaluating the potential use of the two pea and bean seeds, respectively; the strain of A. radicina was
oils for safe and eco-friendly seed-treatments. provided from Centraalbureau voor Schimmelcultures (CBS)
Fungal Biodiversity Centre (Utrecht, The Netherlands).
Experimental Oils: Stocks of medicinal TTO and TO were purchased
Fungi: A total of 7 fungi (F. graminearum, F. culmorum, A. from Sovimpex (Marseille, France) and from Esperis s.p.a.
radicina, A. dauci, A. rabiei, C. lindemuthianum, D. avenae), (Milan, Italy) respectively. The main active components of
maintained on solid PDA medium (potato dextrose agar), were the two oils used for in vitro trials are: terpinen-4-ol (40.7%)
used for antifungal activity evaluation of TTO and TO. The and g-terpinene (20.3%) for TTO, thymol (41%) for TO, as
strains of F. graminearum, F. culmorum and D. avenae were reported by respective producer Companies.
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Figure 1. Micelial growth means of F. graminearum, F. culmorum, A. radicina, A. dauci, A. rabiei, C. lindemuthianum, D.
avenae in presence of TO at different concentrations. Data of mycelial growth are expressed as radius length measured at
the 3rd, 4th, 5th, 6th and 7th days after inoculation. The error bar show the Standard Deviation.

44/Journal of Essential Oil Research Vol. 23, November/December 2011

 M. alternifolia

In vitro activity: Mycelial growth was determined on solid values in control and treatments, respectively, measured for
PDA medium amended with TTO and TO at the following each fungus at the seventh day after inoculation, obtained in
concentrations: 0% (control), 0.05%, 0.1%, 0.25%, 0.5%, 1% both experiments. Minimum inhibitory concentration (MIC),
(v/v) and in presence of Tween20 (0.5% v/v) as emulsifying the minimum dose that produced a 50% reduction of growth
agent. Experiments were conducted twice in duplicate in 90 mm compared with the growth of the oil-free control, was computed
Petri dishes containing the medium modified and inoculated for each combination of fungus/compound at the seventh day
after inoculation using regression equation and FORCAST tool
with 6 mm plugs of PDA from actively growing cultures; after
available with Microsoft Excel. In F. graminearum experiments,
inoculations dishes were kept in the dark at 24°C.
since mycelium covered the whole dish area on the sixth day
Radial growth of colonies was measured every day (from
after inoculation, the fifth day was taken as the last useable
the third to the seventh after inoculation) at two perpendicular day to calculate MGI and MIC.
points along the diameter of the plate, and the mean of these Analysis of variance (ANOVA) was performed on the data
two readings was used to calculate the mean daily radius of obtained using CoStat-Statistics Software version 6.4.
the fungal colony. Percentage inhibition of mycelial growth The significance of the differences among treated and control
(%MGI) by the oils was calculated using the formula: (C-T) samples was evaluated using the Student-Newman-Keuls test
x 100/C where C and T are the average of the colonies radius for multiple comparison (significance level 0.05).
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Figure 2. Micelial growth means of F. graminearum, F. culmorum, A. radicina, A. dauci, A. rabiei, C. lindemuthianum, D.
avenae in presence of TTO at different concentrations. Data of mycelial growth are expressed as radius length measured
at the 3rd, 4th, 5th, 6th and 7th days after inoculation. The error bar show the Standard Deviation.

Vol. 23, November/December 2011 Journal of Essential Oil Research/45

 Riccioni et al.

Results and Discussion inhibition. The growth of the seven fungi on TO and TTO
amended medium, compared to the growth of the oil-free
Figures 1 and 2 show the mean radial growth of F.
control, was significantly reduced (p=0.00) for all the TO and
graminearum, F. culmorum, A. radicina, A. dauci, A. rabiei,
TTO concentrations, excepting two cases, were the growth of
C. lindemuthianum and D. avenae colony on solid medium
F. culmorum and A. radicina was enhanced (p=0.00) at the
containing TO and TTO, respectively. The standard deviations
lowest experimental concentration levels of TTO: 0.05% TTO
are also reported. The ANOVA analysis showed not significant
for F. culmorum and 0.05% and 0.1% TTO for A. radicina. At
differences between the two experiments (p>0.05). Data on
these concentrations, they showed in both repetitions/tests
the percentage inhibition of mycelial growth (%MGI) are
mycelial growth stimulation, which results in minus sign in
reported in Tables I and II. In general, the fungi showed
Table II data, supporting data previously reported by Angelini
different mycelial growth depending on the strain and the
and colleagues (7) for Pleurotus species.
oil concentration, proving a dose-dependent way of growth
Although essential oils are known to be good antimicrobial
agents, some microorganism are stimulated by them and use
them as a carbon energy source (8); this may have been the case
for the A. radicina and F. culmorum examined in this study.
Table 1. Thyme oil (TO) in vitro activity: the percentage The MICs of TTO were evaluated for all the tested fungi,
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inhibition of mycelial growth (%MGI) is calculated for each and data are reported in Table III. The highest MIC value
fungus at different oil concentration as mean radius at the 7th was 0.40% for A. radicina, while the lowest one was 0.08%
(*) and the 5th (**) day after inoculation of both experiments. TTO for D. avenae. About TO, all fungi showed no mycelial
100 value refers to complete inhibition with no mycelial growth even at the lowest TO concentration tested, so the TO
growth. Significance differences (p ≤ 0.05) found using the MIC value is surely <0.05% for each fungus. However, the
Student-Newman-Keuls are indicated as letters a-e, where comparison of MIC values of the two oils showed that TO is
the letter a (not reported in the table) refers to the respective
more effective than TTO in mycelial growth inhibition, and
control range. Value followed by the same letter within the
this occurred with all the fungi tested.
same row do not differ significantly.
Results obtained from this work are consistent with previ-
%MGI ous studies (4) (5) (6), which demonstrate that TTO and TO
ORGANISM TO TO TO TO TO posses antifungal activities, and these activities depend on oil
0.05% 0.1% 0.25% 0.5% 1% concentration and on the fungal specie. Actually, first stud-
ies on essential oils were mainly focused on pharmacological
F. graminearum ** 93b 100c 100c 100c 100c
F. culmorum * 91b 100c 100c 100c 100c
and cosmetical potential of the oils against bacteria (9) (10),
C. lindemuthianum* 100b 100b 100b 100b 100b dermatophytes fungi and other filamentous fungal species like
A. rabiei * 96b 100c 100c 100c 100c members of the genus Candida (11) (5) (12). Only in more
A. dauci * 92b 100c 100c 100c 100c recent years interest has grown in investigating essential oil
A. radicina* 56b 82c 100d 100d 100d potentialities as alternative measures to chemical treatments
D. avenae * 99b 100b 100b 100b 100b for crop and seed protection, and many authors have begun to
study essential oil activities against plant pathogenic fungi of
agricultural interest, and valuable results have been achieved.
Zaker and Mosallanejad (13) studied the antifungal ability of
Table II. Tea tree oil (TTO) in vitro activity : the percentage five plant extracts against Alternaria alternata, the causal agent
inhibition of mycelial growth (%MGI) is calculated for each of Alternaria leaf spot of potato; Marin et al. (14) tested cin-
fungus at different oil concentration as mean radius at the 7th namon, clove, oregano, palmarosa and lemongrass oils against
(*) and the 5th (**) day after inoculation of both experiments. Fusarium graminearum, one of the main causal agents of the
100 value refers to complete inhibition with no mycelial root and foot rot and fusarium ear blight in wheat; Faria et al.
growth. Significance differences (p ≤ 0.05) found using the (15) investigated the activity of Ocimum gratissimum essential
Student-Newman-Keuls are indicated as letters a-e, where
the letter a (not reported in the table) refers to the respective
control range. Value followed by the same letter within the
same row do not differ significantly. Table III. Minimum inhibitory concentration (MIC) of thyme oil
%MGI (TO) and tea tree oil (TTO) at the 7th (*) and the 5th (**) day
after inoculation
ORGANISM TO TO TO TO TO
0.05% 0.1% 0.25% 0.5% 1% ORGANISM TO MIC TTO MIC
F. graminearum ** 16b 28c 91d 100e 100e F. graminearum ** <0.05% 0.19%
F. culmorum * -5b 29c 80d 99e 100e F. culmorum * <0.05% 0.31%
C. lindemuthianum* 17b 69c 100d 100d 100d C. lindemuthianum* <0.05% 0.11%
A. rabiei * 26b 47c 96d 100d 100d A. rabiei * <0.05% 0.14%
A. dauci * 9b 28c 83d 100e 100e A. dauci * <0.05% 0.20%
A. radicina* -30b -10b 74c 98d 100d A. radicina* <0.05% 0.40%
D. avenae * 34b 63c 100d 100d 100d D. avenae * <0.05% 0.08%

46/Journal of Essential Oil Research Vol. 23, November/December 2011

 M. alternifolia

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