Ruffinengo

J. Essent. Oil Res., 18, et al.

704-707 (November/December 2006)

Laboratory Evaluation of Heterothalamus

alienus Essential Oil Against Different Pests of
Apis mellifera

Sergio R. Ruffinengo, Matías Maggi and Sandra Fuselli

Lab. de Artrópodos, FCEyN, Cátedra de Apicultura, FCA UNMdP, CIC Funes 3350, (7600) Mar del Plata, Argentina
Ignazio Floris
Dipartamento di Protezione delle Piante, Universitá degli Studi di Sassari Via E De Nicola - 07100 Sassari, Italy
Gladys Clemente
FCA, UNMdP, Ruta 226, Km 73.5, (7620) Balcarce, Argentina
Norberto H. Firpo
CINDECA-CONICET. Calle 47 N°257, La Plata, Bs. As., Argentina
Pedro N. Bailac* and Marta I. Ponzi
Facultad de Ingeniería y Ciencias Económico Sociales, Universidad Nacional de San Luis, INTEQUI-CONICET. Av. 25
de Mayo 384. CP 5730, Villa Mercedes, San Luis, Argentina

Abstract
The Heterothalamus alienus oil was investigated in laboratory for control of different pests that affect the colonies
of bees, Apis mellifera, against Varroa destructor mite, the bacterium that causes the American Foulbrood, Paenibacil-
lus larvae and the fungus that produces the chalkbrood, Ascosphaera apis. The oil composition was analyzed by GC
and GC/MS, the main components of the oil were β-pinene (44.4%) and trans-muurola-4(14),5-diene (9.2%). The
concentration to kill 50% of the mites in 24 h (LC50) was 0.59 mg/cage. Inferior and superior limits were the following
ones: 0.34 mg/cage and 1.01 mg/cage, LC50 was estimated for 48 h, and 72 h showed a slight increase with respect to
the record of the 24 h. Paenibacillus larvae strains were Gram positive and catalase negative, the oil presented MIC
values of 800–900 mg/L and MBC of 1000–1200 mg/L. Disks of impregnated filter paper with H. alienus oil around
colonies of A. apis in growth inhibited the micelial growth significantly by 51% in the first experiment (seven days)
and by 31% in the second experiment (eight days).

Key Word Index

Heterothalamus alienus, Asteraceae, essential oil composition, β-pinene, trans-muurola-4(14),5-diene, honeybee
pathogens, Ascosphaera apis, Paenibacillus larvae, parasitic bee mite, Varroa destructor.

Introduction
The main plagues that affect the colonies of bees, Apis
mellifera L., in general have been controlled using pyrethroids
and antibiotics. The excessive use of different active principles
(fluvalinate, coumaphos, oxytetracycline) have generated re-
sistance phenomena to these plagues (1,2), and at the same
time they cause contamination of the wax and honey produced
by the bees (3).
Natural pesticides based on essential oils may represent
an alternative, in particular to those pests related to the food
provision. In this sense, the control of pathogens and parasites of
honeybees with natural substances is fundamentally important
since honey, the principal product of bee keeping, is a natural
product that must be free of any contaminants.
The organic acids, such as formic and oxalic (4,5) are the
most investigated substances applied under field conditions.
Essential oils have also been studied as potential controllers of
plagues of the bees with diverse results. Some oils may been
effective in the control of certain bee pests (6-7). However, in
general these oils have not been incorporated in commercial
formulations, except in some cases (8).

*Address for correspondence Received: June 2005

Revised: November 2005
1041-2905/06/0006-0704$14.00/0­—© 2006 Allured Publishing Corp. Accepted: February 2006

704/Journal of Essential Oil Research Vol. 18, November/December 2006

 H. alienus
This work, evaluates the activity of the Heterothalamus
alienus oil in different experiments of laboratory on the Var-
roa destructor mite, the bacterium (Paenibacillus larvae) that
causes the American Foulbrood, and the fungus (Ascosphaera
apis) that produces the chalkbrood.
Experimental
Plant material and essential oil isolation: Heterothala-
mus alienus (Sprengel) O K, Asteraceae, was collected from
Merlo, province San Luis, Argentina (April 2001) as a wild
species. A voucher specimen number 2950 was deposited in the
Herbarium of the Facultad de Ingeniería y Ciencias Económico-
Sociales, Universidad Nacional de San Luis. Air-dried aerial
parts in the vegetative fructification stage were distilled for 4
h in stainless equipment to obtain the oil using the method of
distillation with steam according to Aldicara (9). The oil was
separated from water by decantation, dried with anhydrous
sodium sulfate and then kept under refrigeration to avoid its
deterioration. The oil yield (v/w) was 0.7%.
Essential oil analysis: The oil composition was analyzed
by GC and GC/MS, using a Shimadzu GC-17A chromatograph
equipped with a DB-1 fused silica capillary column (60 m
x 0.248 mm, film thickness 0.25 µm). The temperature was
programmed from 60ºC (5 min) to 220ºC at 3ºC/min and the
final temperature was held for 22 min, injector and detector
temperatures: 230ºC and 250ºC, respectively, detector FID;
carrier gas nitrogen at a flow of 0.9 mL/min. The GC/MS
analysis was performed on a Perkin-Elmer, Q-Mass 910 GC,
separation was accomplished on a 30 m x 0.25 mm fused silica
capillary column (DB-5), film thickness 1.0 µm. The injector and
detector temperatures 250ºC, oven temperature programmed
from 60ºC (5 min), 60°–220ºC (3ºC/min) and 220ºC (8 min),
carrier gas He at a flow of 1 mL/min, operating at 70 eV. The
identification of components was based on comparison of
their mass spectra with those reported in literature (10) and
by computer search of their 70 eV mass spectra with those
stored in the library of the GC/MS data system, as well as by
retention indices.
Mite lethality test: The treatment was assayed in unmodi-
fied Petri dishes (60 x 20 mm). The oil (0.5 µL, 1 µL, 2,5 µL,
4 µL, 6 µL, 8 µL, 12 µL, 15 µL 20 µL and 30 µL) was diluted
in 1 mL of ethanol and the solution was applied to the bot-
tom of the Petri dish. In each dish, five newly emerged adult
worker bees (between zero and three days) and five Varroa
mite females obtained from brood cells were used. The mites
used were adults Varroa females removed from the brood cells,
since according to Milani (1) they present less variation than
the ones originating from adult bees. A candy was placed inside
for food. Complete exposure was tested for each oil concentra-
tion. Solvent, untreated controls and fluvalinate were included
as controls. Before and during experiments Varroa females
were placed in an incubator at 33º–34ºC and 70% RH. Five
replicates for each experimental unit were done. Mite mortal-
ity was controlled at 24, 48 and 72 h. LC50 values and inverse
95% fiducial confidence limits of V. destructor were estimated
according to Lindberg et al. (11). Variables were transformed
with the probit function (12,13) and for the calculation, the
process PROC LOGISTIC of the SASV8 was used (14).
Vol. 18, November/December 2006 Journal of Essential Oil Research/705
Adult bee toxicity test and ratio selection: LC50 values
and inverse 95% fiducial confidence limits of A. mellifera were
estimated from total exposure test, according to Lindberg et
al. (11). Variables were transformed with the probit function
(12,13) and for the calculation the process PROC LOGISTIC
of the SASV8 was used (14). Ratio selection was obtained as:
LC50 of A. mellifera/LC50 of V. destructor. This ratio will only
be considered selectivity indicator if overlap does not exist
among the intervals of confidence of the compared LC.
Paenibacillus larvae bioassay: Three strains of P. larvae
ssp. larvae were used in this study, being isolated from honey
combs of hives exhibiting clinical symptoms of American Foul-
brood Disease (AFB). The strains came from combs placed
at the south of Buenos Aires Province mainly from Miramar
(38°15’S-57°50’W), Balcarce (37°52’S-58°15’W) and Vidal
(37°27’S-57°44’W) localities. Isolation and identification of
the strains were done using techniques previously described
(15,16). Then, biochemical and physiological tests to confirm
P. larvae ssp. larvae were made (17) and to API CH50 kits (Api
System, BioMérieux S.A., Marcy l’Étoile, FR). The cultures of
P. larvae ssp. larvae were stored at -20ºC on MYPGP agar with
20% v/v of glycerol, up to their use. Vegetative cells of P. larvae
ssp. larvae were grown on MYPGP agar (Mueller-Hinton-yeast
extract–glucose-sodium piruvate) for 48 h at 35ºC ± 0.5ºC and
then they were suspended in double distilled sterile water. The
standardization was made by turbidimetry method carrying
the turbidity level about 107–108 cells/mL. This concentration
corresponding to an absorbance of 0.258 at a wavelength of
620 nm measured with a spectrophotometer Bausch & Lomb
(Spectronic 20, USA).
Bacterial serial suspensions were cultivated in Mueller-
Hinton broth (extract of meat with 2.0 g/L; hydrolyzed casein
17.5 g/L and starch 1.5 g/L) plus thiamine 0.1 g/L for every
bacterial strain. The oil was emulsified with a 5.9% solution
of propylene glycol (1,2-propanediol), and was diluted up to
concentrations from 50–1500 mg/L. These emulsions were
prepared at room temperature using a Vortex dispersing tool
for tubes (Fbr by Decalab SRL) with agitation. All the tubes as
well as positive and negative controls were incubated at 35ºC
± 0.5ºC for 48 h in order to determine the minimal inhibitory
concentrations (MIC) values.
From MIC negative tubes, known suspension volumes
were spread on MYPGP agar (Mueller-Hinton-yeast extract-
glucose-sodium piruvate) with nalidixic acid addition (18).
After the bacterial suspensions were absorbed into the agar,
the plates were incubated in inverted position at 35ºC ± 0.5ºC
for 48 h in order to determine minimal bactericide concentra-
tions (MBC) values.
Positive and negative control test of Mueller-Hinton broth,
MYPGP agar and propylene glycol were made. MIC and MBC
tests were performed by triplicate.
Statistical analysis: MIC and MBC data obtained from
essential oil were comparative analyzed by a double classifica-
tion ANOVA software, which was used to measure differences
between bacterial strains. Results statistical significance between
essays were assessed using a comparative student’s T-test.
Ascosphaera apis assay: An A. apis strain was grown on
agar MY20 (19,20). In the center of each Petri dish a disk of
agar of 0.8 cm diameter from the border of colonies of A. apis
 Ruffinengo et al.

of seven days of growth was sowed. Around the agar disk with
A. apis four disks of paper of filter of 0.8 cm diameter were
placed symmetrically, each one impregnated with 1 µL of oil of
H. alienus. The cultivations were incubated at 30ºC, in darkness
and aerobiosis and the diameter from the colonies to the seven
days in a first experiment and to the eight days in the second
experiment were determined. The experiment was conducted
using a random block design with four replications, with each
Petri dish acting as an experimental unit. The variance of the
data was analyzed and the averages were compared by means
of a test of Waller-Duncan (14).

Results and Discussion

The composition of the H. alienus oil can be seen in Table
I. Twenty-three components were identified, with β-pinene
Figure 1. Percent mite mortality to Heterothalamus
(44.4%) and trans-muurola-4(14),5-diene (9.2%) as the major alienus after 24 h, by logarithm of dose
ones.
Results obtained in tests of total exposition as percentage
of dead mites with relation to the dose logarithm are shown in Table II. Lethal concentration 50 (LC50) estimated for Varroa
Figure 1. LC50 mites in 24 h was 0.59 mg/cage. The confidence destructor and Apis mellifera, according to exposition time to
interval for LC50 was estimated, inferior and superior limits Heterothalamus alienus oil
were 0.34 mg/cage and 1.01 mg/cage. LC50 estimated for 48
Organism Time (h) LC50a Lia Lsa
h and 72 h showed a slight increase with respect to the record
of the 24 h (Table II). These values of LC50 are intermediate Varroa destructor 24 0.59 0.34 1.01
when they are compared with the oils of other Argentine wild 48 1.37 1.03 1.73
72 1.29 0.91 1.58
species (21). For A. mellifera the LC50 after 24 h was 7.88 µL
Apis mellifera 24 7.88 6.27 9.17
(Li 6.27 µL – Ls 9.17 µL). The ratio selection was larger at
48 5.51 3.55 9.62
13 for 24 h and it decreased to 4 after 48 h. Analysis of results 72 NC
revealed that no damages to the bees were observed not even
Li = inferior limit; Ls = superior limit; a = mg/cage; NC = not considerable
at values higher than the LC50 reached for Varroa. This is a
very important point since many oils with acaricidal activity

possess adverse effects on honey bees and cause damage to

Table I. Principal compounds of Heterothalamus alienus oil
Compounds RIa Percentage
α-pinene 941 2.6
sabinene 974 0.5
β-pinene 979 44.4
limonene 1029 3.2
(E)-β-ocimene 1046 3.7
perillene 1118 0.2
α-terpineol 1183 0.3
α-cubebene 1361 0.2
α-copaene 1387 0.3
β-elemene 1396 0.9
β-caryophyllene 1433 4.6
α-humulene 1468 3.3
γ-muurolene 1486 1.6
trans-muurola-4(14),5-diene 1495 9.2
valencene 1505 0.7
cis-calamene 1541 0.3
α-calacorene 1546 0.5
bicyclogermacrene 1510 5.9
δ-cadinene 1530 2.8
spathulenol 1581 3.3
caryophyllene oxide 1587 0.9
α-muurolol 1636 1.3
α-cadinol 1652 0.9
retention indices referred to n-alkanes, determined on DB-1 column
a
706/Journal of Essential Oil Research Vol. 18, November/December 2006
the colonies (8).
Paenibacillus larvae strains were Gram positive and catalase
negative. These microorganisms did not hydrolyze starch and
produce indole; characteristics that are common to the species
(15). All strains corresponded to the biochemical type II; with
reduction of nitrate to nitrite and the lack of acid production
from D(+)-mannitol or D(+)-salicin (17). For the different
bacterial strains analyzed, the H. alienus oil presented MIC
values of 800–900 mg/L and MBC values of 1000–1200 mg/L.
No MIC significant differences for bacterial strains (Balcarce,
Miramar and Vidal) were found (P< 0.01). The same results
were obtained analyzing MBC values when bacterial strains
(P< 0.01). According to Alippi et al. (22), other oils such as
lemongrass (Cymbopogon citratus [DC] Stapf) and thyme
(Thymus vulgaris L.) showed MIC values of 50–100 mg/L and
100–150 mg/L, respectively. These oils had the greatest inhibi-
tory effect on the growth of P. larvae ssp. larvae. Floris et al. (6)
obtained significant results with the oils of Citrus sinensis (L.)
Osbeck, Cinnamomum zeylanicum Nees, Cuminum cyminum
L., Eugenia ssp., Thymus vulgaris and a reconstituted oil of
Verbena. Cinnamomun zeylanicum oil was found to be the most
effective against P. larvae ssp. larvae, with minimal bactericidal
and sporicidal concentrations of 50 and 100 mg/kg, respectively.
In comparison, the oil of H. alienus with 800-900 mg/L MIC
values would be considered as an antibacterial substance with
a little activity against P. larvae ssp. larvae.
 H. alienus
Figure 2. Diameter of Ascosphaera apis colonies growing
at 30°C in agar MY20 where were prepared disks of
filter paper impregnated with the oil of Heterothalamus
alienus; results are averages of four replications and
different letters indicate significant differences among
treatments (p = 0.0288 and p = 0.0007, respectively)
Disks of impregnated filter paper with H. alienus oil around
colonies of A. apis in growth inhibited the micelial growth
significantly in each one of the experiments (p = 0.0288 and
p = 0.0007, respectively), as shown in Figure 2. The presence
of these disks with oil inhibited 51% of A. apis growth in the
first experiment and 31% in the second experiment.
The oil of H. alienus shows in vitro miticidal, germicidal
and fungicidal activity. This oil also revealed a high selection
index that gives it a margin of security for its use under field
conditions. It is possible that this oil can be used in combination
with others in a handling strategy integrated for the plagues
of bee colonies. It would not be advisable to use it alone be-
cause it only possessed moderate action against some of the
investigated plagues.
Acknowledgments
We thank the financial support to the ANPCyT, CONICET PEI
6229, in addition to Martín Eguaras.
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