In vitro activity of essential oils from San Luis-Argentina against Ascosphaera apis

Dellacasa, Alejandro D

Received: May 2001

Revised: December 2001

Accepted: February 2002

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 essential oils as natural products to control this mycosis. Eight oils were screened against A. apis for fungicidal activity. The oils of Baccharis coridifolia 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 mellifera, Ascosphaera apis, Tessaria absinthioides, Aloysia gratissima, Heterotheca latifolia, Lippia juneliana, Lippia integrifolia, Lippia turbinata, Baccharis coridifolia, Eupatorium patens, Asteraceae, Verbenaceae, chalkbrood, honey bee diseases, antifungal activity.

Introduction

Chalkbrood is an invasive mycosis affecting exclusively Apis mellifera L. (honeybee) larvae growth. It is a mycotic disease-most frequently found in honeybees-produced by the ascomycete fungus Ascosphacra apis (Maasen ex Claussen) Oliver et Spiltoir, a dioic species.

In Argentina, the appearance and expansion process of chalkbrood in honeybees was similar to that of other countries. By the end of 1978, it was observed in different apiaries of Buenos Aires province; it was considered as a minor problem up to 1988. From that time, a constant increase has occurred, reaching at the present time high apiary infection levels spread throughout a large number of provinces. This fungal disease occurs widely in temperate regions; its spread in the apiary is made easier by Varroa (1).

The occurrence of different circumstances is necessary for the chalkbrood development. Different causes are mentioned that are able to predispose the appearance of this mycosis, although it has only been demonstrated that brood cooling is the most important aspect in this process (2-3).

Pharmacological treatment has not been effective against this mycosis disease; chemical products used were also found to be ineffective, primarily due to the fact that they do not kill the spores that are persistent in the beehive (4). An ecological alternative is the use of essential oils to control chalkbrood.

In the present work, the in vitro fungicidal activity against A. apis of some oils of wild species of San Luis province (Argentina) was evaluated.

Experimental

In order to determine the fungicidal activity, oils obtained by hydrodistillation from the following species were used: Tessaria absinthioides (Hook et Arn.) D.C. (fresh plants), Bacchariscoridifolia D. C. (female plants), Heterotheca latifolia Buck., Aloysia gratissima (Gill, et Hook) Tronc., Eupatorium patens Don ex Hooker et Arnott, Lippia juneliana (Mold.) Tronc., L. integrifolia (Gris.) Hier, and L. turbinata Gris.

Evaluation of activity: Agar glucose 4% SABOURAUD was used as the culture medium (reagent for Merck diagnosis); 25 mL of this molten culture medium was placed in each Petri dish, and was cooled up to 45[degrees]C. The medium was inoculated with the fungus. The agar diffusion method was used with filter paper discs (5-7); 5 [mu]L of each oil was used to impregnate filter paper discs of 6 mm diameter under sterile conditions. They were placed on the agar to which the fungus had been previously inoculated at such a distance so as five or six discs in equidistant way were placed in each plate with control discs with ketoconazole (concentration: 1 mg/mL). The culture plates were incubated for 72 h at 28[degrees]C. Finally, the reading and comprehension of results was performed, and the fungicidal activity of the oils was determined.

Medium preparation: The MY20 medium was used for the maintenance of A. apis (8). It was prepared by mixing 200 g of glucose, 5 g of peptone, 3 g of malt extract, 3 g of yeast extract and 20 g of agar in 1000 mL of distilled water using moderate agitation until no evidence of separation of the components from the medium agar was observed. The mixture was heated slowly, autoclaved for 15 min at 121[degrees]C and then cooled at 30[degrees]C. Culture plates were prepared using this medium and under aseptic conditions. A. apis cultures on MY20 were kept at 30[degrees]C, in aerobiosis and dark conditions for 15 days, time when some signs of ageing could be observed.

Cultivation and scoring: White mummies of A. mellifera, aseptically extracted from growth frames of honeybee heaves, were used. Diseased heaves were located on the country of Balcarce, on the southeast of Buenos Aires province, Argentina. Mummies kept in glass vials were stored in a refrigerator until used. Isolates were maintained on MY20 by subculturing every 10 to 15 days.

Results and Discussion

Bio-tests reported in Table I show that the biggest halo of growth inhibition was 4 mm diameter using control discs with ketoconazole. The L. juneliana, L. integrifolia and L. turbinata oils were the most effective for the A. apis control. They produced a halo of growth inhibition of 3 mm diameter at 24 h. The T. absinthioides and H. latifolia oils produced a halo of growth inhibition of 2.5 mm diameter. The A. gratissima oil produced a halo of growth inhibition of 2 mm diameter.

From the eight oils evaluated in the vegetative cycle of the fungus, six of them-T. absinthioides, H. latifolia, A. gratissima, L. juneliana, L. integrifolia and L. turbinata-exhibited fungicidal activity against A. apis. On the other hand, the oils of B. coridifolia from female plants and of E. patens did not exhibit any fungicidal activity.

Table II shows the principal compounds (> 5%) of the oils screened. The T. absinthioides oil contained caryophyllene oxide as its principal component (9), B. coridifolia isocaryophyllene and [beta]-caryophyllene (10), H. latifolia borneol and camphor (11), A. gratissima epi-[alpha]-cadinol and caryophyllene oxide (12), E. patens [beta]-caryophyllene and [gamma]muurolene (13), L. juneliana piperitenone oxide and limonene, L. integrifolia [beta]-caryophyllene and L. turbinata limonene and piperitenone oxide (14).

Colin et al. (15) studied other species for the in vitro control of A. apis: the oils of two varieties of Origanum vulgare, Satureja montana and 16 clones of Thymus vulgaris. The most effective oils were the O. vulgare variety from Drome, S. montana and the clones 756, 133, 077 and 557 of T. vulgaris. The S. montana oil consists of four main compounds: carvacrol (31.5%), p-cymene (20.8%), [gamma]-terpinene (11.8%) and thymol (4.2%). The T. vulgaris in the clone 756: p-cymene (25.3%), thymol (9.7%) and carvacrol (39.9%), the clone 133: p-cymene (22.2%), thymol (10.1%) and carvacrol (38.6%), the clone 077: p-cymene (28.6%), thymol (8.7%) and carvacrol (32.0%) and the clone 557: p-cymene (31.3 %), thymol (5.7%), carvacrol (27.3%) and linalool (8.4%). The oils that showed more antifungal activity had carvacrol concentrations of more than 30%.

Bazzoni et al. (16) have studied the oils from the following species: T. capitatus, T. herba-barona, Rosmarinus officinalis (three samples), Myrtus communis, Eucalyptus globulus, Salvia desoleana, S. officinalis, Helichrysum italicum and Cinnamomum species. The oils that had an effect against A. apis were: T. capitatus that contained carvacrol (68.01%), [gamma]-terpinene (6.33%) and p-cymene (6.17%) as its main components T. herba-barona contained carvacrol (60.04%) and p-cymene (6.16%), Cinnamomum species contained cinnamicaldehyde (79.3%) and eugenol (11.9%) and H. italicum contained neryl acetate (51.59%) and nerol (8.22%).

Larran et al. (17) used oils from species cultivated in La Platazone: coriander (Coriandrumsativum L.), laurel (Laurus nobilis L.), false camphor (Cinnamomum glandulifera Nees), basil (Ocimum basilicum L.), tagetes (Tagetes minuta L.), rosemary (R. officinalis L.), eucalyptus (Eucalyptus globulus Labill), and lavandin (Lavandula xintermedia Emeric ex Loiseleur). All were assayed to prove their fungistatic activity at different concentrations: 700, 800 and 900 [mu]L/L. At all concentrations tested, coriander oil was the most effective fungistatic control while basil and tagetes oils were effective only at 800 [mu]L/L. Coriander and basil oils contained linalool as their main compound (68,4% and 42.3%, respectively), and tagetes oil contained (Z)-[beta]-ocimene (44.3%) and (Z)-ocimenone (12.2%).

It can also be found in the literature that Calderone et al. (18) studied eight plant extracts. They were selected for evaluation of their potential to inhibit the growth of A. apis. Fungicidal activity of bay oil [Pimenta racemosa (Mill.) J.W. Moore], camphor, clove oil [Syzygium aromaticum (L.) Merr. et L.M. Perry], cinnamon oil (Cinnamomum species), citironellal, Spanish origanum oil (T. capitatus), [alpha]-terpinene, and thymol was assayed at four concentrations in agar media to determine their activity thresholds. Cinnamon oil (ex C. zeylanicum Blume) completely inhibited the growth of A. apis at 100 ppm for 168 h. Bay oil, citronellal, clove oil, origanum oil and thymol completely inhibited the growth at 1,000 ppm for 168 h. Camphor inhibited all growth at 10,000 ppm for 168 h, and [alpha]-terpinene inhibited all growth for 72 h at 10,000 ppm.

A previous study of the same H. latifolia oil used in our study showed that it had a high acaricidal effect against the destructive Varroa mite (11) a carrier of A. apis (19).

The monoterpenes and sesquiterpenes percentages of the oils from the species studied in this work are reported in Table III. The percentages were established taking into account the total of the identified compounds grouped in hydrocarbons and oxygenated compounds. The H. latifolia, L. juneliana and L. turbinata oils possessed a higher percentage of oxygenated compounds. The L. integrifolia oil contained a lower percentage of oxygenated compounds than the more effective species and a lower percentage of identified compounds: (76.2%), while the L. juneliana and L. turbinata oils contained a larger quantity of identified compounds (88.7% and 92.0%, respectively). The A. gratissima and L. integrifolia oils contained a higher percentage of oxygenated sesquiterpenes. The six studied oils that proved to have antifungal activity showed concentrations above the 30% of oxygenated terpenes.

Experiments carried out in vitro show that six of the oils tested possessed good fungicidal activity. As a result, we believe that they deserve to be tested in bee hives. However, an adequate way of application must be found to permit the successful reduction of fungal infection in bee colonies.

Acknowledgments

We would like to thank CONICET, ANPCyT for financial support on this research, as well as the Universidad Nacional de San Lius and Universidad Nacional de Mar del Plata.

References

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Alejandro D. Dellacasa, Pedro N. Bailac* and Marta I. Ponzi

Facultad de Ingenieria y Ciencias Economico Sociales, Universidad Nacional de San Luis, INTEQUI-CONICET. Av. 25 de Mayo 384. CP 5730, Villa Mercedes, San Luis, Argentina

Sergio R. Ruffinengo

Catedra de Apicultura, FCA, UNMdP-PROAPI, Ruta 226, Km 73.5, (7620) Balcarce, Argentina

Martin J. Eguaras

Lab. de Artropodos, FCEyN, UNMdP, CONICET, PROAPI, Funes 3350. (7600) Mar del Plata, Argentina

*Address for correspondence

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