Journal of Apicultural Research and Bee World 48(1): 77-78 (2009) © IBRA 2009

DOI 10.3896/IBRA.1.48.1.15

NOTES AND COMMENTS

In vitro antibacterial and antiparasitic effect of

citrus fruit essential oils on the honey bee pathogen
Paenibacillus larvae and the parasitic mite Varroa destructor.

Sandra Rosa Fuselli1,2*, Matías Maggi1,3, Susana Beatriz García de la Rosa1, Judith Principal4,
Martín Javier Eguaras1,3 and Rosalía Fritz1
1
Universidad Nacional de Mar del Plata (UNMdP), Facultad de Ciencias Exactas y Naturales, Mar del Plata, Argentina.
2
Comisión de Investigaciones Científicas (CIC), La Plata, Argentina.
3
Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina.
4
Estación de Apicultura, Decanato de Ciencias Veterinarias, Universidad Centroccidental “Lisandro Alvarado” (UCLA), Tarabana,
Estado Lara, Venezuela.

Received 29 December 2007, revised manuscript received 8 July 2008, accepted for publication 13 August 2008.

*Corresponding author: Email: sfuselli@mdp.edu.ar

Keywords: Honey bee, Apis m ellifera, pathogens, Paenibacillus larvae, American foulbrood (AFB), Varroa destructor, Citrus paradisi,
Citrus sinensis

Two of the most serious diseases or pests affecting Apis
mellifera L. colonies throughout the world are the bacterium
Paenibacillus larvae and the parasitic mite Varroa destructor. These
are currently mainly controlled using antibiotics, synthetic
pyrethroids and organophosphorus compounds, but the
excessive use of these treatments has already produced
problems with resistance in certain areas of Europe, USA, Canada, solutions were prepared diluting 1, 2, 4, 8, 10, 30 and 40 µl of each
Argentina and Asia. In addition, synthetic chemical substances leave oil in 2 ml of ethanol. Each solution obtained was poured into the
toxic residues in honey and other bee products, even when
beekeepers have used them appropriately. Some plant essential
oils have shown antibacterial, antifungal and acaricidal activity for the
in vitro control of bee pests (Eguaras et al., 2005; Fuselli et al., 2006;
2007), offering a natural alternative to synthetic chemical
substances.
This work evaluates the in vitro bioactivity of grapefruit
(Citrus paradisi Mac Fad) and sweet orange (Citrus sinensis L.
Osbeck) essential oils as a potential control of V. destructor and P.
larvae. Analysis of citrus essential oil composition and identification of
active compounds were made by GC/MS- SPME analysis, according to
the methods of Fuselli et al. (2007). Thirteen P. larvae strains were
isolated from honey combs from hives exhibiting clinical symptoms of
American foulbrood (AFB), in different regions of Argentina. Isolation
and identification of the strains were carried out using biochemical
and physiological tests and API CH50 kits. Antimicrobial activity of the
citrus oils was tested by minimal inhibitory concentration (MIC) using
the broth microdilution method (Fuselli et al., 2007). Citrus oil stock
solutions used were of 3600, 3200, 2800, 2400 and 2000 mg/l. A
twofold serial dilution was made and 100 µl of a microbial biomass
suspension of 107-108 cells/ml was added, the final concentrations
being 2.34 mg/l, 2.08 mg/l, 1.82 mg/l, 1.56 mg/l and 1.30 mg/l. From
a MIC negative microtitre plate, 100 µl were transferred on MYPGP
solid agar and incubated at 36±0.5 ˚C for 48 h, to determine minimal
bactericide concentration (MBC) values.
To test mortality of V. destructor, grapefruit and sweet orange oil
bottom of a petri dish (140 x 20 mm) and left to evaporate. Three
newly emerged adult worker honey bees and five female V. destructor
were added to each dish and incubated at 28 ºC and 60 % RH. Food
and water were provided for the bees. Ethanol and fluvalinate were
included as controls. Mite mortality was assessed after 24 h and
mortality values were corrected according to Abbott. The attractant
and repellent effects of each essential oil on the mite were
determined using petri dishes (60 x 20 mm) divided into two zones,
with a bee pupa placed in each zone. One pupa was treated with 8 µl
of essential oil, while the other remained untreated. One mite was
placed on the middle of each dish and its position was recorded after
90 min. Twenty replicates were conducted for each essential oil. A Chi
-square test was carried out to determine differences between the
expected and observed results.
GC/MS analysis identified 12 compounds in the essential oils of
grapefruit and sweet orange, representing 98.24 % and 96.29 % of the
total peak area of the headspace, respectively. Within the terpenes,
the largely predominant component in both oils is limonene (69.87 % and 74.42
%), with b-myrcene the second major constituent (9.63 % and 11.28
%). a-pinene and sabinene were present with percentages under 5 %.
 78 Fuseli, Maggi, Garcia de la Rosa, Principal, Eguaras, Fritz

Table 1. Mite (V. destructor) and honey bee (A. mellifera) mortality for grapefruit (C. paradisi) and sweet orange (C. sinensis) essential oils,
expressed as percentage of dead individuals after 24 h. Five replicates for each experimental unit were carried out (N=25).

Organism Oil concentration (µl/cage)

tested
0 1 2 4 8 10 30 40

V. destructor 15.0 33.3 32.0 53.8 76.0 - - - Grape fruit oil

A. mellifera (C. paradisi )
0.0 0.0 0.0 0.0 0.0 - - -
V. destructor 10.0 16.7 26.7 25.0 - 26.7 36.4 40.0 Sweet orange oil
(C. sinensis)
A. mellifera 0.0 0.0 0.0 0.0 - 0.0 0.0 0.0

All P. larvae strains were highly susceptible to grapefruit oil with alternative compounds for treating honey bee colonies suffering from
MIC and MBC mean values of all strains of 336.31 mg/l and 400.0 mg/ AFB and V. destructor, to be incorporated in Integrated Management
l, respectively. MIC and MBC mean values obtained for sweet orange Programmes.
were 800.0 mg/l and 933.33 mg/l, respectively. Fisher exact test
results indicated highly significant differences between the essential
oils tested (P< 0.01) and no significant differences between the
Acknowledgments
P. larvae strains tested (P> 0.05). Neither grapefruit nor sweet orange
essential oils produced mortality of honey bees at the concentrations This research was supported by Project EXA 309/05 from Mar del

tested. An increase in grapefruit oil concentration by eight times Plata National University (UNMdP) to PhD Rosalía Fritz and PICT 04-

increased mite mortality from 15.0 % to 76.0 %. Sweet orange oil 443-ANPCyT grant to Dr Martín Eguaras.

showed lower mortality values than those produced by grapefruit oil,

mortality being only slightly increased even with a forty fold increase
in concentration (Table 1). Mite mean LC50 for 24 h was 4.53 µl/cage
and 377.00 µl/cage for grapefruit and sweet orange oils, respectively.
References
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2
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