essential oil composition of agastache anethiodora britton (lamiaceae) infected by cucumber mosaic...

66 R. BRUNI, A. BIANCHI AND M. G. BELLARDI

Copyright © 2006 John Wiley & Sons, Ltd. Flavour Fragr. J. 2007; 22: 66–70

DOI: 10.1002/ffj

FLAVOUR AND FRAGRANCE JOURNALFlavour Fragr. J. 2007; 22: 66–70Published online 2 November 2006 in Wiley InterScience(www.interscience.wiley.com) DOI: 10.1002/ffj.1760

Essential oil composition of Agastache anethiodoraBritton (Lamiaceae) infected by cucumber mosaicvirus (CMV)

Renato Bruni,1* Alberto Bianchi1 and Maria Grazia Bellardi2

1 Dip. di Biologia Evolutiva e Funzionale, Sez. Biologia Vegetale e Orto Botanico, Università degli Studi di Parma, Parco Areadelle Scienze 11A, 43100 Parma, Italy

2 Dip. di Scienze e Tecnologie Agroambientali (DiSTA)—Patologia Vegetale, Università degli Studi di Bologna, Viale G.Fanin 42,40127 Bologna, Italy

Received 20 January 2006; Revised 8 May 2006; Accepted 20 June 2006

ABSTRACT: Giant hyssop, Agastache anethiodora Britton, cultivated at the Herb Garden of Casola Valsenio, Italy, has

been found for the first time naturally infected by cucumber mosaic virus (CMV). Characteristic symptoms on the leaves

were chlorotic or yellow mosaic, ring and line patterns and malformation, followed by yellowing and stunting of the entire

plant. CMV was mechanically transmitted to species of the families Chenopodiaceae and Solanaceae and identified by

applying PAS–ELISA and RT–PCR techniques. The essential oil of both healthy and CMV-infected plants has been evalu-

ated by means of GC–FID and GC–MS, with the object of identifying composition differences caused by the disease. The

infection of A. anethiodora by CMV was found to induce significant reduction in the yield of essential oil and several

changes in the relative composition of the main components: pulegone, menthone, iso-menthone, methyl chavicole and

limonene. Methyl chavicole content, in particular, was drastically reduced. The importance of the phytopathological status

of essential oil-bearing plants is outlined. Copyright © 2006 John Wiley & Sons, Ltd.

KEY WORDS: Agastache anethiodora; CMV disease infection; essential oil; methyl chavicole

Introduction

During recent decades in Italy, trends in herbal crops and

within the herbal market resulted in the increased culti-

vation of medicinal and essential oil-bearing plants, herbs

and spices. With these new specialty crops have come

unique diseases and pest problems, some of which were

previously rare or unknown in the wild and have instead

been promoted by cultivation.1 On the productive side,

the fact that plant pathologies may lead to considerable

losses in gross yield is well known.1 It is also note-

worthy that modifications in the abundance and quality of

secondary metabolites, included in essential oils, have

been reported.2 The measurement of the impact of fungal

diseases and mycoplasma, have in fact been the object of

some experimental studies and were found to be respon-

sible for significant variations in the composition of

essential oils.3–5 However, knowledge regarding the influ-

ence of viral diseases on the chemical composition of

essential oils is quite limited, since epidemiological

studies of virus spreading inside medicinal crops have

become more frequent only during recent years.6–8 In

Italy, cucumber mosaic virus (CMV), transmitted by

aphids in non-persistent manner, is spreading very widely

in medicinal and aromatic crops. It has been found infect-

ing almost 20 cultivated species, including: Galega

officinalis L., Hesperis matronalis L., Asclepias tuberosa

L., Echinacea purpurea L., Hyssopus officinalis L.,

Nepeta cataria L., Inula viscosa L., Origanum vulgare

L., Melilotus albus Desr., M. officinalis (L.) Pallas,

Valeriana officinalis L. and V. phu L.9 This phenomenon

is of particular relevance whenever biennial and perennial

plants are infected, as they may allow viruses to persist

from one year to another in fields and help the spread to

nearby crops. Moreover, being nuclear protein parasites

that alter the host’s cell metabolism to their own advan-

tage, viruses become an integral part of cell’s biochem-

istry. Hence, the possible influence of viral diseases on

the secondary metabolism of the host plants cannot then

be excluded and may lead to a loss in quality of essen-

tial oils,10,11 affecting fragrance and pharmacological or

functional properties. Such an influence could thus be-

come an issue in the market value definition of the final

cultivation product.

During an epidemiological survey carried out in Emila-

Romagna region (northern Italy, 2003) to identify virus

infections most frequently occurring in medicinal and

aromatic plants, some plants of Agastache anethiodora

were found showing a severe virus-like disease.

* Correspondence to: R. Bruni, Dip. di Biologia Evolutiva e Funzionale-

Sezione di Biologia Vegetale, Viale delle Scienze 11A, 43100, Università

degli Studi di Parma, Italy.

E-mail: [email protected]

ESSENTIAL OIL OF AGASTACHE ANETHIODORA 67


DOI: 10.1002/ffj

Agastache anethiodora Britton (= Agastache foeniculum

Kuntze, ex Lophanthus anisatum Benth.; family

Lamiaceae) is a large, late-flowering perennial herb with

purple flowers in terminal spikes, native to the great

plains of the northern Americas and also known as giant

hyssop or anise hyssop. Its volatile oil has been sug-

gested as potentially useful for treating colds and as

an antioxidant.12–14 Like other Agastache species, A.

anethiodora is reputed for its anis-like scented essential

oil, due to the abundance of methyl-chavicole,12 and thus

is used for flavouring foods, teas and other beverages.

The closely related A. rugosa essential oil showed good

antifungal properties and some capacity to inhibit the

proliferation of human cancer cells.15,16 Non-volatile

diterpenoids and lignans from this species also showed

various biological activities, ranging from HIV integrase

and apoptosis inhibition to cytotoxic activity.17,18 More-

over, giant hyssop is also reputed as a source of nectar

for honey bees,19 a process in which the floral scent is

deeply involved.

To evaluate the effects of infection by viral pathogens

on the quality of herbal products, the composition of

healthy and CMV-infected A. anethiodora plants were

evaluated by means of GC and GC–MS.

Experimental

Plant Material

The virus-like disease was observed on 80% of A. anethiodora

plants obtained by seed and grown in the open field at the Herb

Garden of Casola Valsenio (Ravenna, Italy); a voucher speci-

men of the species (Code no. OPR2) was deposited at the

Herbarium of Officinal Plants, Botanical Garden of Parma

University, Italy. Both healthy and infected plants were obt-

ained from a homogeneous genetic pool and grown in the same

field, under the same conditions in terms of irrigation, fertiliza-

tion, light and climate. The most characteristic symptoms on the

leaves were chlorotic or yellow mosaic, ring and line patterns

and malformation, followed by yellowing and stunting of the

entire plant. Under the Italian climate, these symptoms were

best visible during the summer. Before effecting the collection

procedure, asymptomatic and symptomatic plants were selected

and labelled by visual inspections of their aerial parts (no fungi

or bacteria infections were present). Twenty-five samples from

both the two plant batches (with and without symptoms) were

then collected and used exclusively in virological tests.

Virological Testing

Identification of the virus was performed by mechanical

inoculations on herbaceous hosts belonging to species of the

families Chenopodiaceae (Chenopodium amaranticolor Coste

et Reyn, C. album L., C. murale L. and C. quinoa Willd.) and

Solanaceae (Nicotiana glutinosa L., N. benthamiana L., N.

occidentalis L. and N. tabacum L. ‘Samsun’). The leaf material

of A. anethiodora collected in the field and of inoculated test

plants was submitted to further investigations, such as electron

microscopy, serology and biotechnology, with the aim of iden-

tifying the isolated virus. Leaf sap extracts were negatively

stained with 2% (w/v) aqueous uranyl acetate (UA) or 1%

phosphotungstic acid (PTA) neutralized at pH 7.0 and observed

using a Philips CM10 electron microscope. The serological

technique applied was protein A–double antibody sandwich–

enzyme-linked immunosorbent assay (PAS–ELISA).20 The

plates were first coated with 1 µg/ml protein-A (Sigma-Aldrich,

Milan, Italy) in carbonate buffer, pH 9.6. All the 50 samples

collected (symptomatic and asymptomatic) were homogenized

in phosphate buffer saline (PBS), pH 7.2, containing 0.05%

Tween-20, 2% polyvinyl pyrrolidone (PVP, MW 24 000) and

0.2% powdered chicken albumin. Polyclonal antisera were

added at a 1/500 dilution in PBS-Tween. Protein A-alkaline

phosphatase conjugate (Sigma-Aldrich, Milan, Italy) was diluted

(1 µg/ml) in PBS-Tween, pH 7.4. The serological reaction was

considered positive when (in Photometer Dynathech MR 7000)

the A405 value exceeded that of the healthy control by three

standard deviations. The sera to the following isometric or

bacilliform viruses were tested: cucumber mosaic virus (CMV);

arabis mosaic virus (ArMV); alfalfa mosaic virus (AMV);

broad bean wilt virus (BBWV, serotypes I and II); and tobacco

ring spot virus (TRSV). The sera to CMV (PVAS-30, from

Commelina diffusa Burm.), ArMV (PVAS-192) and TRSV

(PVAS-157) were obtained from American Type Culture

Collections (ATCC), Manassas, VA, USA; the Istituto di

Fitovirologia Vegetale, CNR, Turin, Italy provided the sera to

BBWV-I and II; the anti-AMV was available at our laboratory

(DiSTA-Patologia Vegetale, Bologna).

To confirm the results of PAS–ELISA tests, reverse

transcriptase-polymerase chain reaction (RT–PCR) was applied

to 20 A. anethiodora leaf samples (100–200 mg; healthy and

CMV-infected) according to Logemann et al.21 (for RNA isola-

tion) and by using (for the amplification) two primers specific

for the RNA-3 of CMV, CMV3R and CMV3F1, 16 and 18

bases long, respectively: 5′-AGT GAC TTC AGG CAG T-3′(upstream; genome position 1986–2001; GenBank Accession

No. Y16926); 5′-GCT TGT TTC GCG CAT TCA-3′ (down-

stream; genome position 1566–1583; GenBank Accession No.

Y16926). The first strand cDNA was synthesized (1 h at 37°C)

from 0.5 µl total RNA: 5 µl solution contained RNAs (0.5 µl),

primer CMV3F1 (5 pmol) and reverse transcriptase-RNase

(50 U; M-MLV, Promega). The PCR reaction volume was

25 µl. The reaction mix contained primers CMV3R and

CMV3F1 (5 pmol), MgCl2 (1.5 mM), Taq polymerase (1.25 U;

Promega) and 5 µl cDNA as template. The following conditions

and parameters in a Biometria T3 Thermocycler were applied:

denaturation for 5 min at 94 °C, 30 cycles of (94 °C 1 min,

56 °C 1 min and 72 °C 1 min); in the last cycle, the extension

time was 72 °C for 10 min. In RT and PCR analysis, 0.2 mM of

each dNTP was utilized. The PCR products were recovered

after electrophoresis in 1.5% ultra-pure agarose gel stained with

ethidium bromide.

Plant Material and Isolation of Essential Oil

In September, about 1 kg of leaves from virus-infected and

healthy A. anethiodora plants were collected and steam-distilled



DOI: 10.1002/ffj

immediately thereafter. The essential oil content was determined

on a volume to dry weight basis. The essential oil samples were

dried over anhydrous sodium sulphate and stored in glass vials

with Teflon-sealed caps at −18 ± 0.5 °C in the absence of light.

Gas Chromatography (GC)

GC analysis was performed on a Fisons (Rodano, Milano, Italy)

9130-9000 Series gas chromatograph equipped with a Fisons

EL980 processor, a FID detector and a MEGA SE52 (Mega,

Legnano, Italy) 5% poly diphenyl 95% dimethylsiloxane

bonded phase column (30 m × 0.32 mm i.d, film thickness

0.15 µm). Operating conditions were as follows: injector tem-

perature, 280 °C; FID temperature, 280 °C; carrier gas, helium

at a flow rate of 2 ml/min; split injection with split ratio 1:40.

Oven temperature was initially 45 °C and then raised to 100 °C

at a rate of 1 °C/min, then raised to 250 °C at a rate of 5 °C/min

and finally held at that temperature for 10 min. 1 µl of each

sample dissolved in CH2Cl2 was injected.

Gas Chromatography–Mass Spectrometry(GC–MS)

Essential oil constituents were analysed by a Hewlett-Packard

HP5890 Series II Plus gas chromatograph equipped with a

HPMS 5989b mass spectrometer operating in EI mode. The GC

conditions were as reported for GC analysis and the same

column was used. The MS conditions were as follows: ioniza-

tion voltage, 70 eV; emission current, 40 µA; scan rate, 1 scan/s;

mass range, 35–300 amu; ion source temperature, 200 °C.

Identification of Compounds

The mass spectrometry (MS) fragmentation patterns were checked

with those of other essential oils of known composition,

with pure compounds and by matching the MS fragmenta-

tion patterns with NBS75K mass spectra libraries and with

those from the literature.22,23 The relative amounts of the indi-

vidual components were obtained from GC analysis based on

peak areas without FID factor correction. The constituents of

the volatile oils were also identified by comparing their gas

chromatography (GC) retention indices. A mixture of aliphatic

hydrocarbons (C8–C24) in hexane (Sigma) was injected under

the above-described temperature programme to calculate the

retention indices, using the generalized equation by Van del

Dool and Kartz.24

Results and Discussion

Virological Study

Mechanical inoculations made it possible to infect several

herbaceous plants, including Chenopodium amaranticolor,

C. album, C. murale and C. quinoa, which showed local

symptoms (necrotic lesions) in 3–4 days. All Solanaceae

tested were infected: Nicotiana tabacum L. ‘Samsun’

and N. glutinosa L. showed systemic leaf mosaic and

malformation, N. benthamiana and N. occidentalis

showed crinkled and narrowed leaves.

In leaf-dip preparations from field-collected A.

anethiodora plants (symptomatic and asymptomatic) and

inoculated herbaceous plants, no elongated virus particles

were observed. PAS–ELISA identified the virus infecting

all the symptomatic samples as CMV, which was also

detected in inoculated host plants. RT–PCR confirmed

the serological results, highlighting that the two primers

CMV3R and CMV3F1 amplified fragments of 436 bp of

the expected size with cDNA from the CMV control only

(belonging to DiSTA collection) and from symptomatic

samples of A. anethiodora; no amplification was obtained

with asymptomatic samples.

The virological investigation carried out in the field

shows that CMV naturally infects A. anethiodora (Figure 1)

leading to severe symptoms (yellowing and stunting)

which can severely reduce the yield of the crop and of

Figure 1. Agastache anethiodora infected by CMV, showing stunting (A) and chlorotic mosaic on the malformedleaves (A, B)

ESSENTIAL OIL OF AGASTACHE ANETHIODORA 69


DOI: 10.1002/ffj

its commercial by-products. CMV has been probably

transmitted to those plants by aphids from weeds or

some medicinal species growing in the same area. The

finding of CMV described here is the first report of

its natural infection in A. anethiodora and, due to their

taxonomic proximity, other Agastache species could

suffer similar effects. It is possible that other species

belonging to the family Lamiaceae infected by the same

viral pathology could exhibit similarities to those found

in A. anethiodora, although this should be confirmed

by experimental evidence.

Essential Oil Analysis

The aerial parts of healthy and CMV-infected A.

anethiodora plants, after having undergone steam distilla-

tion, yielded a pale oil in a yield of 3.5 ml/kg and 0.4 ml/

kg, respectively; a quantitative decrease of 88% was

induced in virus-infected plants. A simple sensory evalua-

tion of the oil evidenced a different profile, confirmed by

GC and GC–MS analysis. In fact, although the chemical

compounds detected were the same (Table 1), their rela-

tive abundance was quite different (Figure 2). Table 1

reports the composition of A. anethiodora essential oil in

terms of components; 29 components of the 51 recorded

were identified, constituting >95% of the entire volatile

fraction. Of the 22 unidentified compounds, only two

exceeded 0.5%. The main constituents identified were

Table 1. Chemical composition of the essential oils of healthy and CMV-infected A. anethiodora plants

No. Compounda KI A. anethiodora (RA%)b

Healthy CMV-infected

1 α-Pinene 937 Trc Tr

2 Sabinene 976 Tr 0.2

3 1-Octen-3-ol 979 0.6 1.2

4 3-Octanone 985 0.2 0.4

5 Myrcene 992 0.2 1.1

6 p-Cymene 1024 Tr Tr

7 Limonene 1029 2.8 12.0

8 p-Cymenene 1092 Tr 0.1

9 Linalool 1096 0.3 0.4

10 Oct-1-en-3-yl, acetate 1114 0.5 0.8

11 cis-p-Mentha-2,8-dien-1-ol 1136 0.4 0.1

12 Menthone 1155 4.1 8.2

13 iso-Menthone 1167 27.0 43.9

14 α-Terpineol 1188 Tr 0.1

15 Methyl chavicole 1198 16.2 3.2

16 Pulegone 1239 31.2 18.7

17 Piperitone 1252 0.7 0.4

18 trans-Sabinyl acetate 1291 0.1 0.1

19 Piperitenone 1343 2.4 2.1

20 β-Bourbonene 1388 0.2 0.3

21 β-Elemene 1391 0.1 0.1

22 Caryophyllene 1418 3.3 2.7

23 β-Humulene 1439 0.3 0.2

24 γ-Amorphene 1496 0.7 0.5

25 Bicyclogermacrene 1501 0.8 0.6

26 α-Farnesene 1508 0.5 0.3

27 δ-Amorphene 1512 0.3 0.1

28 Germacrene D-4-ol 1577 2.2 0.6

29 Caryophyllene oxyde 1582 1.4 0.3

Total 96.5 98.7

a Compounds are listed in order of elution from a SE-52 column.b RA%, relative area percentage (peak area relative to total peak area%).c Tr, <0.05%.

Figure 2. Quantitative differences between healthyand CMV-infected A. anethiodora essential oils withregard to the main components detected



DOI: 10.1002/ffj

limonene, menthone, iso-menthone, methyl chavicole and

pulegone. From these results, CMV infection appeared

to be responsible for significant variations in the rela-

tive abundance of the major constituents of the essential

oil. In particular, the amount of methyl chavicole was

drastically reduced (80.5%) in the infected samples,

along with pulegone (40.2%). In contrast, a higher abun-

dance of limonene (76.3%), menthone (50.8%) and iso-

menthone (38.5%) was detected. The overall amount of

both monoterpene hydrocarbons and oxygenated mono-

terpenes increased from 3.07% to 13.35% and from

66.13% to 73.95%, respectively, while the sesquiterpene

content decreased frpm 9.7% to 5.49% (Figure 2).

Conclusions

Infection of A. anethiodora by CMV was found to be

associated with considerable variations in the qualitative

composition of essential oil and linked to a detection of

a 88% loss in distillation yield. The inverted relative

amounts of pulegone, limonene, methyl chavicole,

menthone and iso-menthone was the most evident vari-

ation. Methyl chavicole abundance was drastically de-

creased in the infected sample, along with pulegone,

while limonene, menthone and iso-menthone in parti-

cular increased their relative abundance. Those results,

although limited to a single species, show the possible

effects of viral diseases on medicinal and aromatic plant

crops, suggesting that the phytopathological status of

essential oil-bearing plants should be a further variable

to be considered whenever a fluctuation in essential oil

composition is reported. The damage caused by CMV

appears serious enough to suggest control measures, such

as removal of infected plants and elimination of weeds

and aphids, their main vectors. In fact, CMV (genus

Cucumovirus, family Bromoviridae) is one of the most

economically important viruses, associated with hundreds

of diseases distributed worldwide in more than 1000

botanical species belonging to 100 families. Regarding

‘herbs’, recent surveys demonstrated that CMV is the

most prevalent virus affecting (alone or mixed to other

viruses) crops located in geographically distinct areas,

the Emilia-Romagna and Trentino-Alto Adige regions.9

In addition, CMV is known to infect the seed of

several plants, some of which are herbs (such as

Hyssopus officinalis). Considering the constant presence

of aphid infestations in horticultural and ornamental

crops, medicinal and aromatic CMV-infected plants, as

well as their seed, represent a good source of CMV

inoculum for other cultivated species. Moreover, as the

biological activities and commercial value of an essential

oil are strictly correlated with its chemical composition

and relative abundance of its components, routine con-

trols of the virological status of essential oil crops should

be considered.

Acknowledgements—Special thanks are due to Sauro Biffi and to theHerb Garden of Casola Valsenio, Italy, for their help in plant collectionand cultivation.

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essential oil composition of agastache anethiodora britton (lamiaceae) infected by cucumber mosaic...

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