Phytochemistry 57 (2001) 99±102

www.elsevier.com/locate/phytochem

Antifungal activity of the essential oil of ¯owerheads of

garland chrysanthemum (Chrysanthemum coronarium)
against agricultural pathogens
Pino P. Alvarez-Castellanos a, Chris D. Bishop b, Maria J. Pascual-Villalobos a,*
ConsejerõÂa de Agricultura, Agua y Medio Ambiente, Centro de InvestigacioÂn y Desarrollo Agroalimentario,
a

EstacioÂn SericõÂcola 30150 La Alberca, Murcia, Spain

b
School of Biological and Earth Sciences, Liverpool John Moores University, Byrom Street, Liverpool L33AF, UK

Received 14 June 2000; received in revised form 6 November 2000

Abstract
The antifungal activity of Chrysanthemum coronarium was evaluated against 12 agricultural pathogens. Flowerhead oil was active
both in contact and headspace in vitro assays producing hyphal growth inhibition, although there was less activity on faster
growing fungi. The main compounds identi®ed in the oil were camphor (29.2%), a-pinene (14.8%), b-pinene (9.5%) and lyratyl
acetate (9.8%). The blue color of the oil was due to the presence of chamazulene (0.5%). # 2001 Elsevier Science Ltd. All rights
reserved.
Keywords: Chrysanthemum coronarium; Asteraceae; Garland chrysanthemum; Antifungal activity; Essential oil composition; Camphor; a-Pinene; b-
Pinene; Lyratyl acetate

1. Introduction spatiosum, is used as a Chinese vegetable (chop-suey).

The Anthemidae tribe of the family Asteraceae
includes medicinal species of the genera Artemisia,
Achillea, Chamaemelum, Chamomilla and Tanacetum.
The volatile components of their essential oils exhibit
strong odours and many of them have been used as ¯a-
vourings. In addition, the oils of Tanacetum species are
reported to possess antibacterial activity (GoÈren et al.,
1990). Essential oils contain monoterpenoids, sesqui-
terpenoids and other non-terpenoid compounds. Some
of them have been reported to exhibit a wide range of
biological activities (Picman, 1986).
Many species within the genera Pyrethrum and Chry-
santhemum have been transferred to the genus Tanace-
tum, including small pyrethrum [T. cinerariifolium
(Trevir.) vis.] which is used for insecticide production.
Only two species of Chrysanthemum (C. coronarium L.
and C. segetum L.) are widely distributed in the Medi-
terranean, western Africa and Asia (Trehane, 1995).
C. coronarium var. coronarium is an ornamental and can
be found as a common weed, while C. coronarium var.
* Corresponding author. Tel.: +34-68-366768; fax: +34-68-366792.
E-mail address: mjesus.pascual@carm.es (M.J. Pascual-Villalobos).
0031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(00)00461-1
Green leaves and stems of C. segetum L. are also con-
sumed as vegetables (SaÂnchez-Monge, 1991).
Flowerheads of C. coronarium, are typically bicolored
white and yellow, or only yellow, and have been used as
chamomile adulterants (Garg and Sastry, 1996). In
Japan the leaves are used for suppression of ®shy
odours in prepared foods (Kasahara and Nishibori,
1995).
The chemical composition of C. coronarium leaves has
been studied by other authors: polyacetylenic com-
pounds have been isolated from the aerial parts and
insect antijuvenile hormone activity has been detected
for some of them (Bowers and Aregullin, 1987; Sanz et
al., 1990). From ¯owerheads of C. coronarium, sesqui-
terpene lactones (cumambrin and dihydrocumambrin)
have been isolated (El-Masry et al., 1984).
In recent years, there has been a clear tendency
towards the utilization of alternative methods for pest
and disease control in agriculture, that are less dama-
ging to the environment and human health. Research on
natural products including plant extracts which might
substitute for agrochemicals or contribute to the devel-
opment of new agents (Addor, 1995) for pest control is
extremely important. Essential oils have properties as
100 P.P. Alvarez-Castellanos et al. / Phytochemistry 57 (2001) 99±102

fungicidal agents (Knobloch et al., 1987; Bishop and In Table 2, the results indicate the growth of Alter-
Thornton, 1996). naria sp., Aspergillus ¯avus and Pythium ultimum were
In this paper, we report the results of several bioas- highly reduced (>80%) when measured at the third
says to test activity of the essential oil of ¯owerheads of day. Although no clear di€erences in activity could be
garland chrysanthemum (C. coronarium) by contact or observed for many fungi, the more resistant species were
by vapours against a panel of agricultural fungal Mycocentrospora acerina and Penicillium digitatum.
pathogens. The chemical composition of this oil was In the volatile assay (Table 3), di€erences were
also studied for the ®rst time. obtained depending on species, e.g. Fusarium mon-
iliforme was less a€ected than P. ultimum. The activity
in the vapor phase was di€erent for rapid growing spe-
2. Results and discussion cies like Botrytis cinerea or Sclerotinia sclerotiorum, in
which it changed from high inhibition (>70%) at day 3
The result of GC/MS analysis of the oil is summar-
ized in Table 1. The main compounds identi®ed were
camphor (29.2%), a- and b-pinene (14.8 and 9.5%, Table 2
respectively) and lyratyl acetate (9.8%). Signi®cant Growth inhibition of fungal species in agar di€usion plate assay by oil
(20 ml/Petri dish) of Chrysanthemum coronarium
amounts of camphene (5.2%), 2,7,7-trimethylbicyclo-
[3.1.1]hept-2-en-6-ol acetate (3.8%), p-menthatriene Speciesa Day 3 Day 6
(3.4%), germacrene D (2.7%) and a-phellandrene (2.6%) Treated Control Inhibition Treated Control Inhibition
were also detected. Chamazulene, which gave the blue (mm) (mm) (%)b (mm) (mm) (%)b
color to the oil, was present (0.5%).
As 1 7 79.4*** 2 16 84.0***
To the best of our knowledge, this is the ®rst report of
Ab 5 10 53.8*** 7 17 57.3***
the chemical composition of ¯owerheads of C. coronar- Af 2 11 82.6*** 3 12 74.4***
ium. Nevertheless, some compounds have been pre- Bc 13 32 60.1*** 24 40 40.8***
viously identi®ed (Kameoka et al., 1975) from the leaf Fm 6 12 51.2*** 10 23 54.8***
oil of C. coronarium L. var. spatiosum, although only a- Fs 6 8 25.9** 9 14 36.4***
Ma 8 8 4.7ns 13 16 20.0***
and b-pinene and camphene were found in both cases.
Pd 10 13 21.4* 17 20 13.0ns
The ¯owerhead oil of C. coronarium was active both Pu 0 11 95.5*** 4 25 83.9***
in contact and on exposure to the headspace volatiles, Rs 8 13 38.1*** 14 37 62.9***
since they both signi®cantly reduced the growth of fungi Ss 27 36 24.2*** 37 40 8.0*
in comparison with the control (although some excep- Sl 5 19 74.6*** 22 40 45.1***
tions are shown in Table 3). It should be mentioned, a
See Experimental for species tested.
though, that results from the agar di€usion plate assay b
t-Test for comparison between treatment and control (ns=non-
might also include some e€ects of the oil vapours signi®cant, *P<0.05, ** P<0.01, *** P<0.001).
besides the contact action.

Table 1 Table 3
Chemical composition of the essential oil of ¯owerheads of Chry- Growth inhibition of fungal species by volatile oil (10 ml/Petri dish) of
santhemum coronarium Chrysanthemum coronarium

Component Percentage (%) Speciesa Day 3 Day 6

b
a-Pinene 14.8 Treated Control Inhibition Treated Control Inhibitonb
Camphene 5.2 (mm) (mm) (%) (mm) (mm) (%)
a-Phellandrene 2.6 As 1 4 84.2*** 6 24 76.5***
b-Pinene 9.5 Ab 2 10 82.4** 6 40 85.5***
4 Methylenecyclohexene Trace Af 4 9 57.4** 13 34 62.2***
Limonene 0.7
Bc 12 40 69.8*** 77 77 0.0
b-Ocimene 0.3 Fm 6 9 36.4*** 27 34 21.2***
Camphor 29.2 Fs 6 6 3.2ns 24 34 30.8***
2,7,7-Trimethylbicyclo- 3.8 Ma 7 9 22.2ns 35 34 1.2ns
[3.1.1]hept-2-en-6-ol acetate Pd 5 15 69.3*** 29 43 32.4***
Bornyl acetate 0.9 Pu 1 9 88.4*** 5 38 87.8***
Lyratyl acetate 9.8 Rs 12 36 66.7*** 54 68 20.5ns
b-Caryophyllene 1.1 Ss 3 52 93.5*** 77 77 0.0
Germacrene D 2.7 Sl 0 12 98.4*** 28 70 60.5***
p-Mentha-1,3,8-triene 3.4
Chamazulene 0.5 a
See Experimental for species tested.
b
Total 84.5 t-Test for comparison between treatment and control (ns=non-
signi®cant, *P<0.05, ** P<0.01, *** P<0.001).
 P.P. Alvarez-Castellanos et al. / Phytochemistry 57 (2001) 99±102
to covering all the dish at day 6, than for others like
Fusarium sp. and M. acerina whose growth was further
reduced over time (see Table 3). In general, growth inhi-
bition percentages were high, with variations depending
on the day of observation and the test species. The
activity of C. coronarium oil is clear, Bishop and Thorn-
ton (1996) reported similar results for Monarda citrio-
dora L. essential oil when using a dose ten times higher
(100 ml).
In our experiments, the volatile activity found was
signi®cant but less persistent for fast growing species
such as S. sclerotiorum, R. solani and B. cinerea
(Table 3). An interesting observation from the bioassays
was that B. cinerea, P. digitatum and S. sclerotiorum
decreased spore production in the presence of oil (either
by contact or from the headspace volatiles). Other
authors have associated secondary metabolites with
spore production (Dionigi et al., 1992); sporulation of
Streptomyces tendae was inhibited by geosmin produc-
tion (derived from a sesquiterpene precursor).
Tanacetum species contain similar compounds in their
essential oils although a- and b-pinene are found in very
low quantities in comparison with C. coronarium. T.
parthenium (L.) Bernth is characterized by a high cam-
phor (44%) and trans-chrysanthemyl acetate (23%)
content. T. vulgare L. (Hendriks et al., 1990) contains
also lyratyl acetate, thujone and germacrene (depending
on chemotype). Mosquito repellent activity of these oils
was proved but without identi®cation of the active
principles (De Pooter et al., 1989). Camphor and bornyl
acetate (Schearer, 1984) were identi®ed as some of the
more active (repellent to potato beetles) of the mixture
of products present in the oil of tansy (T. vulgare).
Probably some of the compounds present in low
amounts also contribute to these e€ects.
The blue color of the oils was attributed to the pre-
sence of chamazulene which is known to be produced
during the steam distillation. Other members of Aster-
aceae such as Chamomilla, Stevia, Artemisia and Achil-
lea also give azulenic compounds (Kaneko et al., 1971;
Sacco et al., 1983; Acosta et al., 1989; RomaÂn et al.,
1990; Abad et al., 1995). For example, chamazulene is
being used in large quantities in the pharmaceutical and
cosmetic industries.
3. Experimental
3.1. Fungal species
The fungi were obtained from the culture collection at
Liverpool John Moores University (School of Biologi-
cal and Earth Sciences). Cultures of each of the fungi
were maintained on potato dextrose agar (PDA), slants
stored at 5 C. The fungal species used in the experi-
ments were: Alternaria sp. (As), Alternaria brassicola
101
(Ab), Aspergillus ¯avus (Af), Botrytis cinerea (Bc),
Fusarium moniliforme (Fm), Fusarium solani (Fs),
Mycocentrospora acerina (Ma), Penicillium digitatum
(Pd), Pythium ultimum (Pu), Rhizoctonia solani (Rs),
Sclerotinia sclerotiorum (Ss) and Serpula lacrymans (Sl).
3.2. Essential oils
Essential oil of bicoloured ¯owerheads of Chrysanthe-
mum coronarium L. was obtained by hydrodistillation
using a Clevenger-type apparatus. Material was obtained
from plants grown in an experimental plot (Torreblanca
Exp. Stat., Campo de Cartagena) in Murcia (Spain).
Plants were sown in autumn (26 October 1995) and
samples were harvested in April. Voucher specimens (no.
207) were deposited in the Centro de InvestigacioÂn y
Desarrollo Agroalimentario (Murcia, Spain) Herbarium
(CIDAHERB). Before distillation the ¯owerheads were
freeze dried. The yield of oil was about 0.1%(v/w) and it
had a blue color and a disagreeble smell.
3.3. Contact assay
PDA plates were prepared using 9 cm glass Petri
dishes containing Twenty microlitres of PDA. A 1 cm
disc of agar was removed from the centre of the Plates.
Twenty microlitres of water (control) or undiluted oil
was pipetted into the wells. Two 5 mm diam. discs of the
test species were cut from the periphery of less than 1
week old cultures on PDA plates and placed mycelial
surface down on opposite edges of the test plates against
the sides of the dishes. The plates were incubated in the
dark at 20 C. After 3 and 6 days of inoculation, exten-
sion of hyphae towards the central well was measured
from the inner edge of the inoculum discs to the leading
edges of colonies at a point nearest the well. Mean
growth measurements were calculated from 10 repli-
cates of each of the fungal species.
3.4. Volatile assay
PDA plates were prepared using 8.2 cm plastic Petri
dishes containing 20 ml of PDA. A 5 mm diam. disc of
the test species was cut from the periphery of the
actively growing culture of the PDA plates and placed
mycelial surface down on the centre of the dish. The
Petri dishes were placed with the lid upside down. A 10
ml aliquot of water (control) or undiluted oil was pipet-
ted on the lid without agar. The plates were incubated in
the dark at 20 C. Mean growth measurements were
calculated from 10 replicates of each fungal species.
3.5. Chemical analyses
The oil was analysed by GC/MS. Operating condi-
tions were 240 C for the injection temperature and the
102 P.P. Alvarez-Castellanos et al. / Phytochemistry 57 (2001) 99±102
oven was initially at 70 C and was then gradually
increased at a rate of 4 C/min up to 220 C and held for
120 s. The Supelcowax 10 capillary column (30 m 
0.25 mm) was used. For chamazulene identi®cation, a
SE-54 capillary column (25 m  0.25 mm) was operated
at similar conditions but the oven temperature was
increased to 220 C and held for 1 h. Mass spectra were
recorded with a 5970 HP Mass Selective detector, the
electron impact source operating at 70 eV and 240 C.
Identi®cations were made by retention indices and
direct comparison with authentic standards.
3.6. Statistics
Growth inhibition of the treatment against the con-
trol was measured by percentage, using the formula (C-
T/C)  100 where C is hyphal extension (mm) of con-
trols and T is hyphal extension (mm) of oil treated
plates. A t-test was also computed for statistical sig-
ni®cance of the results in the contact and volatile assays.
Acknowledgements
This research was funded by the National Institute for
Agricultural Research in Spain with a PhD grant and
the project SC98-022. We thank C. Armstrong and W.
Harley, for their technical support in the bioassays. An
special acknowledgement to M.V. Ruiz and M.C.
Dobarganes of Instituto de la Grasa y sus Derivados
(CSIC) in Sevilla for their help in the chemical analyses
of the oil.
References
Abad, M.J., Bermejo, P., Villar, V., 1995. An approach to the genus
Tanacetum L. (Compositae): Phytochemical and pharmacological
review. Phytotherapy Research 9, 79±92.
Acosta, L., Granda, M., SaÂnchez, E., Martin, G., Rodriguez, E., 1989.
Variation in the content of essential oil and ( )-a-bisabolol in
camomile (Matricaria recutita) harvest of head ¯owers at di€erent
stages of development. Revista Plantas Medicinales 9, 25±32.
Addor, R.W., 1995. Insecticides. In: Godfrey, C.R.A. (Ed.), Agro-
chemicals from Natural Products. Marcel Dekker, New York, pp.
1±63.
Bishop, C.D., Thornton, I.B., 1996. Evaluation of the antifungal
activity of the essential oils of Monarda citriodora var. citriodora
and Melaleuca alternifolia on post harvest pathogens. Journal of
Essential Oil Research 9, 77±82.
Bowers, W.S., Aregullin, M., 1987. Discovery and identi®cation of an
antijuvenile hormone from Chrysanthemum coronarium. In: Mem.
Inst. Oswaldo Cruz (Intern. Symp. on Insects) RõÂo de Janeiro 82
(Suppl. III), pp. 51±54.
De Pooter, H.L., Vermeesch, J., Schamp, N.M., 1989. The essential oil
of Tanacetum vulgare L. and Tanacetum parthenium (L.) Schultz-
Bip. Journal of Essential Oil Research 1, 9±13.
Dionigi, C.P., Millie, D.F., Spanier, A.M., Johnsen, P.B., 1992. Spore
and geosmin production by Streptemyces tendae on several media.
Journal of Agricultural and Food Chemistry 40, 122±125.
El-Masry, S., Abou-Dania, A.H.A., Darwish, F.A., Abou-Karum,
M.A., Grenz, M., Bohlmann, F., 1984. Sesquiterpene lactones from
Chrysanthemum coronarium. Phytochemistry 23, 2953±2954.
Garg, S., Sastry, T.C.S., 1996. Indian Compositae. foods and ¯avours,
a review. In: Caligari, P.D.S., Hinds, D.J.N. (Eds.), Compositae:
Biology and utilization, Proceedings of the International Composi-
tae Conference, Vol 2, RBG Kew, UK, pp. 381±382.
GoÈren, N., Jakupovic, J., Topal, S., 1990. Sesquiterpene lactones with
antibacterial activity from Tanacetum argyrophyllum var. argyr-
ophyllum. Phytochemistry 29, 1467±1469.
Hendriks, H., Van der Elst, D.J.D., Van Putten, F.M.S., Bos, R.,
1990. The essential oil of dutch tansy (Tanacetum vulgare L.). Jour-
nal of Essential Oil Research 2, 155±162.
Kameoka, H., Kitagawa, C., Husebe, Y., 1975. The constituents of the
steam volatile oil from Chrysanthemum coronarium L. var spatio-
sum. Journal of Agricultural Chemical Society of Japan 49, 625±
627.
Kaneko, H., Naruto, S., Takahashi, S., 1971. Sesquiterpenes of Achil-
lea subirica. Phytochemistry 10, 3305±3306.
Kasahara, K., Nishibori, K., 1995. The suppressing e€ect of garland
chrysanthemum on the odor of nishibori soup stock. Fisheries Sci-
ences 61, 672±674.
Knobloch, K., Pauli, A., Iberi, B., Weis, N., Weigand, H., 1987. Mode
of action of essential oil components on whole cells of bacteria and
fungi in plate tests. In: Schreier, P. (Ed.), Bio¯avor 87: Analysis,
Biochemistry, Biotechnology (Proceedings of the International
Conference, 29±30 September 1987), WuÈrzburg, Germany, Walter
de Gruyter, Berlin, pp. 287±299.
Picman, A.K., 1986. Biological activities of sesquiterpene lactones.
Biochemical Systematics and Ecology 14, 255±285.
RomaÂn, L.U., Mora, Y., HernaÂndez, J.D., 1990. Stevia serrata, a
source of chamazulene. Fitoterapia 61, 84.
Sacco, T., Frattini, C., Bicchi, C., 1983. Constituents of essential oil of
Artemisia arborescens. Planta Med. 47, 49±51.
SaÂnchez-Monge, E., 1991. Flora AgrõÂcola, Tomo. I. MAPA, Madrid.
Sanz, J.F., Falco, E., Marco, J.A., 1990. New acetylenes from Chry-
santhemum coronarium L. Liebigs Ann. Chem., 303±305.
Schearer, W.R., 1984. Components of oil of tansy (Tanacetum vulgare)
that repel colorado potato beetles (Leptinotarsa decemlineata).
Journal of Natural Products 47, 964±989.
Trehane, P., 1995. Proposal to conserve Chrysanthemum L. with a
conserved type (Compositae). Taxon 44, 439±441.