Journal of Essential Oil Research

ISSN: 1041-2905 (Print) 2163-8152 (Online) Journal homepage: http://www.tandfonline.com/loi/tjeo20

In Vitro Activity of Essential Oils from San Luis-

Argentina Against Ascosphaera apis

Alejandro D. Dellacasa , Pedro N. Bailac , Marta I. Ponzi , Sergio R. Ruffinengo

& Martín J. Eguaras

To cite this article: Alejandro D. Dellacasa , Pedro N. Bailac , Marta I. Ponzi , Sergio R.
Ruffinengo & Martín J. Eguaras (2003) In Vitro Activity of Essential Oils from San Luis-
Argentina Against Ascosphaera apis , Journal of Essential Oil Research, 15:4, 282-285, DOI:
10.1080/10412905.2003.9712143

To link to this article: http://dx.doi.org/10.1080/10412905.2003.9712143

Published online: 09 Dec 2011.

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Download by: [Southern Illinois University] Date: 14 January 2016, At: 11:42
 J. Esscnt. Oil Res., 15,282-285 (July/August 2003)

In Vitro Activity of Essential Oils from San Luis-

Argentina Against Ascosphaera apis

Alejandro D. Dellacasa, Pedro N. Bailac"and Marta 1. Ponzi

Facultad de Ingeniena y Ciencias Econdnzico Sociales, Universidad Nacional de San Luis, INTEQUI-CONICET. Av. 25
de Mayo 384. C P 5730, Villa MercecEes, San Luis, Argentina
Sergio R. Ruffinengo
Cbtedra d e Apicultura, FCA, UNMdP-PROAPI, Ruta 226, K n z 73.5, (7620) Balcarce, Argentina
Martin J. Eguaras
Lab. de Artrdpodos, FCEyN, UNMdE CONZCET, PROAPI, Funes 3350. (7600) Mar cEel Plata, Argentina
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Abstract
Chalkbrood is an invasive mycosis produced by Ascosphaera apis affecting exclusively the larvae growth of Apis
mellifera L.There exists no pharmacological treatment and the chemical products used are not able to control the
disease generating resistances and residues in the apicultural production. An ecological alternative is the use of
essentialoils as natural products to controlthismycosis. Eight oils were screenedagainst A. apis for fungicidalactivity.
The oils of Baccharis coridi$olia and Eupatorium patens did not possess any activity while the oils of Tessaria
absinthioides,Aloysia gratissima, Heterotheca latifolia, Lippia juneliana, L. integrifolia and L. turbinata exhibited
varying levels of fungicidal activity.

Key Word Index

Apis mell+v-a,hcosphaera apis, Tessaria absinthioides, Aloysia gratissima, Heterotheca latifolia, Lippia
juneliana, Lippia integrifolia,Lippia turbinata, Baccharis coridifolia, Eupatoriumpatens, Asteraceae,Verbenaceae,
chalkbrood, honey bee diseases, antifungal activity.

Introduction this mycosis disease; chemical products used were also found
to be ineffective, primarily due to the fact that they do not kill
Chalkbrood is an invasive mycosis affecting exclusively the spores that are persistent in the beehive (4).An ecological
Apis mellifcra L. (honeybee) larvae growth. It is a mycotic alternative is the use of essential oils to control chalkbrood.
disease-most frequently found in honeybees-produced by In the present work, the in vitro fungicidal activityagainst
the ascomycetefungusAscosphaera apis (Mausenex Claussen) A. apis of some oils of wild species of San Luis province
Oliver et Spiltoir, a dioic species. (Argentina) was evaluated.
In Argentina, the appearance and expansion process of
chalkbrood in honeybees was similar to that of other coun- Experimental
tries. By the end of 1978, it was observed in different apiaries
of Buenos Aires province; it was considered as a minor In order to determine the fungicidal activity,oils obtained
problem up to 1988. From that time, a constant increase has by hydrodistilation from the following species were used:
occurred, reaching at the present time high apiary infection Tessaria absinthioides (Hook et Am.) D.C. (fresh plants),
levels spread throughout a large number of provinces. This Bacchariscoridifolia D. C. (femaleplants), Heterotheca latifolia
fungal disease occurs widely in temperate regions; its spread Buck., Aloysia gratissinta (Gill. et Hook)Tronc., Eupatorium
in the apiary is made easier by Varroa (1). patens Don ex Hooker et Amott, Lippia juneliana (Mold.)
The occurrence of different circumstances is necessary Tronc., L. integrifolia (Gris.) Hier. and L. turbinata Gris.
for the chalkbrood development. Different causes are men- Eua2uation ofactioity: Agar glucose 4% SABOURAUD
tioned that are able to predispose the appearance of this was used as the culture medium (reagent for Merck diagno-
mycosis, although it has only been demonstrated that brood sis); 25 mL of this molten culture medium was placed in each
cooling is the most important aspect in this process (2-3). Petri dish, and was cooled up to 45°C. The medium was
Pharmacological treatment has not been effective against inoculated with the fungus. The agar diffusion method was

'Address for correspondence Received: May 2001

Revised: December 2001
1041-2905/03/0004-0282$6.00/~2003 Allured Publishing Corp. Accepted:February 2002

2WJoumal of Essential Oil Research Vol. 15, July/August 2003

 Ascosphaera apis

Table 1. Number of replicates of oils screened against Ascosphaera apis at 24 h and 72 h exhibiting growth and halo of growth
inhibition

24 h 72 h
Essential Oil G NG Halo G NG Halo
~
~ ~ ~

E. patens 3 0 0.0 3 0.0

B. coridifolia 3 0 0.0 3 0.0
A. gratissima 0 3 2.0 0 2.0
H. latifolia 0 3 2.5 0 2.5
T. absintioides 0 3 2.5 0 2.5
L. juneliana 0 3 3.0 0 3.0
L. integrifolia 0 3 3.0 0 3.0
L. turbinata 0 3 3.0 0 3.0
Ketoconazole 0 3 4.0 0 4.0

G = growth detected; NG = no growth detected; Halo = diameter of growth inhibition in mm

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used with filter paper discs (5-7);5 pL of each oil was used to gicidal activity against A. apis. On the other hand, the oils of
impregnate filter paper discs of 6 mm diameter under sterile B. coridifolia from female plants and of E. patens did not
conditions. They were placed on the agar to which the fungus exhibit any fungicidal activity.
had been previouslyinoculated at such a distance so as five or Table 11shows the principal compounds (> 5%)of the oils
six discs in equidistant way were placed in each plate with screened. The 2 absinthioides oil contained caryophyllene
control discs with ketoconazole (concentration: 1 mg/mL). oxide as its principal component (9), B. coridifolia
The culture plates were incubated for 72 h at 28°C. Finally, isocaryophylleneand P-caryophyllene(lo),H. latifolia borneol
the reading and comprehension of results was performed, and and camphor ( l l ) , A. gratissinta epi-a-cadinol and
the fungicidal activity of the oils was determined. caryophyllene oxide (12), E. patens P-caryophyllene and y-
Medium preparation: The MY20 medium was used for muurolene (13),L.junelianapiperitenone oxide andlimonene,
the maintenance ofA. apis (8).It was prepared by mixing 200 L. integrifolia P-caryophylleneand L. turbinata limoneneand
g of glucose, 5 g of peptone, 3 g of malt extract, 3 g of yeast piperitenone oxide (14).
extract and 20 g of agar in 1000 mL of distilled water using Colin et al. (15) studied other species for the in vitro
moderate agitation until no evidence of separation of the control of A. apis: the oils of two varieties of Origanum
components from the medium agar was observed. The mix- vulgare, Saturqa nwntana and 16 clones of Thymus vulgaris.
ture was heated slowly, autoclaved for 15 min at 121°C and The most effective oils were the 0. vulgare variety from
then cooled at 30°C. Culture plates were prepared using this Drome, S. montana and the clones 756,133,077 and 557 of Z
medium and under aseptic conditions. A. apis cultures on vulgaris.The S . montana oilconsists offour main compounds:
MY20were kept at 30"C, in aerobiosisand dark conditions for carvacrol (31.5%), p-cymene (20.8%),y-terpinene (11.8%)
15 days, time when some signs of ageing could be observed. and thymol(4.2%).The 2 vulgarisin the clone 756: p-cymene
C u b a t i o n andscodng: White mummiesofA. mellifera, (25.3%),thymol(9.7%)and carvacrol(39.9%),the clone 133:
asepticallyextracted from growth frames of honeybee heaves, p-cymene (22.2%),thymol (10.1%) and carvacrol (38.6%),
were used. Diseased heaves were located on the country of the clone 077: p-cyniene (28.6%),thymol(8.7%)and carvac-
Balcarce, on the southeast of Buenos Aires province, Argen- rol (32.0%) and the clone 557: p-cymene (31.3 B), thymol
tina. Mummies kept in glass vials were stored in a refrigerator (5.7%),carvacrol (27.3%) and linalool (8.4%). The oils that
until used. Isolateswere maintained on MY20 by subculturing showed more antifungalactivity had carvacrol concentrations
every 10 to 15 days. of more than 30%.
Bazzoni et al. (16)have studied the oils from the following
Results and Discussion species: 2 capitatus, 2 herba-barona,Rosmarinus oficinalis
(threesamples),Myrtuscommunis,Eucalyptusglobulus,Salvia
Bio-tests reported in Table I show that the biggest halo of desoleana, S . officinalis, Helichrysum italicum and
growth inhibitionwas 4 mm diameter using control discs with Cinnarnomum species. The oils that had an effect against A.
ketoconazole.The L.juneliana, L. integrifolia and L. turbinata apis were: Z capitatus that contained carvacrol(68.01%),y-
oils were the most effective for the A. apis control. They terpinene (6.33%)and p-cyniene (6.17%)as its main compo-
produced a halo of growth inhibition of 3 mm diameter at 24 nents; 2 herba-barona contained carvacrol (60.04%) and
h. The Z absinthioidesand H.latifolia oils produced a halo of p-cymene (6.16%), Cinnamomum species contained
growth inhibition of 2.5 mm diameter. The A. gratissima oil cinnmicaldehyde(79.3%)andeugenol(ll.9%)andH. italicum
produced a halo of growth inhibition of 2 mm diameter. contained neryl acetate (51.59%)and nerol(8.22%).
From the eight o h evaluatedin the vegetativecycle of the Larrh et al. (17) used oils from species cultivated in La
fungus,sixofthem--l: absinthioides,H . latifolia,A. gratissimu, Platazone:coriander (CoriandmmsatiuztmL.),laurel (Laurus
L.juneliana, L. integrifolia and L. turbinuta-xhibited fun- nobilis L.), false camphor (Cinnamomumglandulifera Nees),

Vol. 15, July/August 2003 Journal of Essential Oil Researchn83

 Dellacasa et al.

Table II. Principal compounds (> 5%) of oils screened against Ascosphaera apis

Species Compounds Percentage' Reference

T. absinthiodes caryophyllene oxide 12.2

(E)-P-damascenone 8.6
y-eudesmol 8.5 9
y-gurjunene 5.8
terpinen-4-01 5.4
B. coridifolia isocaryophyllene 34.3
P-caryophyllene 10.8 10
caryophyllene oxide 9.8
p-selinene 8.2
H. latifolia borneol 31.5
camphor 27.2 11
limonene 7.2
camphene 6.4
A. gratissirna epi-a-cadinol 33.0 12
caryophyllene oxide 11.0
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E. patens P-catyophyllene 14.2

y-muurolene 13.5
a-pinene 11.1 13
bicyclogermacrene 7.6
a-santalene 5.7
(Z)-p-ocimene 5.5
L. juneliana piperitenone oxide 36.5
limonene 23.1 14
camphor 7.9
spathulenol 6.5
L. integrifolia P-caryophyllene 18.4
limonene 8.2 14
spathulenol 6.6
bomeol 5.7
L. turbinata limonene 43.3
piperitenone oxide 24.8 14
1,8-cineole 14.7

*relative percentage obtained from GC peak area

Table 111. Percentages of monoterpenes and sesauitermnes of oils screened

Monoterpenes' Sesquiterpened
Species Hydrocarbon Oxygenated Hydrocarbon Oxygenated Total

E. patens 28.3 0.7 54.7 10.0 93.7

B. coridifolia 1.o N 66.0 22.2 89.2
A. gratissima N 8.2 13.7 60.3 82.2
H. latifolia 22.4 62.0 6.3 N 90.7
T. absintioides N 33.2 16.6 34.9 84.7
L. juneliana 25.3 52.1 4.8 6.5 88.7
L. integrifolia 10.8 8.0 33.5 23.9 76.2
L. turbinata 45.4 43.1 0.8 2.7 92.0

'percentage of relative area; N = not detected

basil (Ocimum basilicum L.), tagetes (Tagetes minuta L.), as their main compound (68.4% and42.3%, respectively),and
rosemary (A.officinalis L.), eucalyptus (Eucalyptus globulus tagetesoilcontained(Z)-p-ocimene(44.3%)and (Z)-ocimenone
Labill), and lavandin (Lavandula xintermedia Emeric ex (12.2%).
Loiseleur).All were assayed to prove their fungistaticactivity It can also be found in the literature that Calderone et al.
at different concentrations: 700, 800 and 900 pUL. At all (18) studied eight plant extracts. They were selected for
concentrations tested, coriander oil was the most effective evaluation of their potential to inhibit the growth of A. apis.
fungistatic control while basil and tagetes oils were effective Fungicidal activity of bay oil [Pimentu rucenwsu (Mill.) J.W.
only at 800 p n . Coriander and basil oils contained linalool Moore], camphor,clove oil [Syzygiumaromaticurn (L.) Men:

284/Joumal of Essential Oil Research Vol. 15, July/August 2003

 Ascosphaera apis

Aspectosgenerales y causaspredisponentes. Gestibn Apicola, 3 (16).

et L.M.Perry], cinnamon oil (Cinnaniomum species), cit- 7-10 (1999).
ronellal, Spanish origanum oil (?: capitatus),a-terpinene, and 3. J. M. Flores, M. A. Palacio, F.Puerta, J. M. Ruiz, J. M. Ruz and S.
thyniol was assayed at four concentrations in agar media to Ruffinengo, Crla yesificada ( Ascosferiosis) en la abeja mellfera. I/:
Diagndstico y fomas de lucha. Gestidn Apicola, 4 (17), 24-26 (2000).
determine their activity thresholds. Cinnamon oil (ex C.
4. L. Bailey and 8. Ball. Honey Bee Pathology. Academic Press Inc., Sari
aqlanicurn Blume)completelyinhibited the growth ofA. apis Diego, CA92101, (1991).
at 100 ppni for 168 h. Bay oil, citronellal, clove oil, origanum 5. A.N. Bauer, W. M. M. Kirby, J. C. Sterns and M. Truck, The antimicrobic
oil and thymol completely inhibited the growth at 1,000 ppm susceptibilify test: principles and practices.Ann. J. Clin. Microbiol., 45,
439-442 (1966).
for 168h. Camphor inhibited all growth at 10,000ppm for 168
6. P. W. Waterworth, Antimicrobial properties of plant essential oils:
h, and a-terpinene inhibited all growth for i.2h at 10,000ppni. practices. Churchil Livingstone, Edinburgh, 37-40 (1978).
A previous study of the same H. latifolia oil used in our 7. J. L. Rios. M. C. Recio and A. Villar, Antimicrobial activify of aroma
study showed that it had a high acaricidal effect against the chemicals and essential oils. J. Ethnopharmacol., 23, 127-132 (1988).
destructive Varroa mite (11)a carrier of A. apis (19). 8. S. Ruffinengo, N. PeAa, G. Clemente, M. Palacio and A. Escande,
Suitability of culture media for the production of ascospores and
The monoterpenesand sesquiterpenespercentages of the maintenance of Ascosphaera apis. J. Apicultural Res., 39, 143-148
oils from the species studied in this work are reported in Table (2000).
111. The percentageswere established taking into account the 9. P. Bailac, C. Duschatzky, A. Carrascull, N. Firpo and M. Ponzi.
total of the identified conipounds grouped in hydrocarbons Composition of the essential oils of Tessaria absinthioides (Hook et
Am.) D. Candole. J. Essent. Oil Res.,lO. 89-91 (1998).
and oxygenatedcompounds.The H. latifolia, L.jundiana and
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10. P. N. Bailac, A. D. Dellacasa, H. 0. Bernasconi, N. Firpo and M. 1. Ponzi,

L. turbinata oils possessed a higher percentage of oxygenated Essential oil of female plants of Baccharis coridifolia De Candole. J.
compounds.The L. integrifolia oil contained a lower percent- Essent. Oil Res., 13,23-24 (2001).
age of oxygenatedcompounds than the more effective species 11. S. R. Ruffinengo, M. J. Eguaras, D. Cora, E. Rodriguez, E.
Bedascarrasbure, P. N. Bailac and M. 1. Ponzi. Biological activity Of
and a lower percentage of identifed compounds: (76.2%), Heterotheca latifolia essential oil against Varroajacobsoni. J. Essent.
while the L.jundiana and L. turbinata oils contained a larger Oil Res., 14,462-464 (2002).
quantity of identified compounds (88.7%and 92.0%,respec- 12. P. N. Bailac, A. D. Dellacasa,C. B. Duschatzky, N. Firpo and M. I. Ponzi.
Composici6n del aceite esencial y actividad antimicrobiana de Aloysia
tively). The A. gratissima and L. integrifolia oils contained a gratissima de San Luis-Argentina.Anales de la Asociacidn Quimica
higher percentage of oxygenated sesquiterpenes. The six Argentina, 87, (3/4), 149-153 (1999).
studied oils that proved to have antifungal activity showed 13. P. N. Bailac. A. D. Dellacasa, H. 0.Bernasconi, N. Firpo and M. I. Ponzi,
concentrationsabove the 30% of oxygenated terpenes. Composici6ndelaceiteesencialyacfividadantimicrobianade Eupatorium
patens. Bol. SOC.Chil. Qulm., 45,207-21 1 (2000).
Experiments carried out in vitro show that six of the oils
14. C. Duschatzky, P. Bailac, N. Firpo y M. Ponzi. Composicidn de los
tested possessed good fungicidal activity. As a result, we aceites esencialesde Lippiajuneliana,Lippiaintegrifoliay Lippia turbinata
believe that they deserve to be tested in bee hives. However, de la provincia de San Luis (Argentina). Revista Colomb. de Qulm., 27,
an adequate way of application must be found to pennit the (2), 9-16 (1998).
15. M.E. Colin, J. Ducos de Lahitte, E. Larribau and T. BouB, Activitd des
successful reduction of fungal infection in bee colonies. huiles essentielles de Labi6es sur Ascosphaera apis et traitement d' un
rucher. Apidolcgie, 20,221-228, (1989).
Acknowledgments
16. E. Bazzoni, I. FlorisandM. D.L. Moretti, In vitroactivityofsomeessenfial
We would like to thank CONICET, ANPCyT forfinuncial sup- oils against Ascosphaera apis. J. Essent. Oil Res., in press (2001).
port on this research, as well as the Universidad Nacional de Sun Luis 17. S. Larrdn, J. a. Ringuelet, M. R. Carranza, C. P. Henning, M. S. RB, E.
and Universidad Nacional de Mar del Plata. L. Cerimele and M. 1. Urrutia, In vitro fungistatic effect of essential oils
against Ascosphaera apis. J. Essent. Oil Res., 13, 122-124 (2001).
18. N. W. Calderone, H. Shimanuki and G. Allen-Wardell, An in vitro
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