In vitro antibacterial and antiparasitic effect of citrus fruit essential oils on the honey bee pathogen Paenibacillus larvae and the parasitic mite Varroa destructor.

FUSELLI SANDRA; MAGGI MATIAS; GARCIA DE LA ROSA BEATRIZ; PRINCIPAL JUDITH; EGUARAS MARTÍN; FRITZ ROSALIA

JOURNAL OF APICULTURAL RESEARCH

INTERNATIONAL BEE RESEARCH ASS

Año: 2009 vol. 48 p. 78 - 79

0021-8839

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. Theseare 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, Argentina and Asia. In addition, synthetic chemical substances leave toxic residues in honey and other bee products, even whenbeekeepers 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 ofAmerican 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) usingthe 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 solutions were prepared diluting 1, 2, 4, 8, 10, 30 and 40 μl of eachoil in 2 ml of ethanol. Each solution obtained was poured into the 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 attractantand 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 %.All P. larvae strains were highly susceptible to grapefruit oil with MIC and MBC mean values of all strains of 336.31 mg/l and 400.0 mg/l, respectively. MIC and MBC mean values obtained for sweet orange 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 P. larvae strains tested (P> 0.05). Neither grapefruit nor sweet orange essential oils produced mortality of honey bees at the concentrations tested. An increase in grapefruit oil concentration by eight timesincreased mite mortality from 15.0 % to 76.0 %. Sweet orange oil 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. LC50 for the fluvalinate control was 0.28 μl/cage.The acaricidal effect of grapefruit oil is promising, with lethal effects greater than other oils previously tested using similar methodologies (Ruffinengo et al., 2005). Differences of the attractant and repellent effects of the essential oils on V. destructor mites were statisticallysignificant (χ2= 16.925; df= 1; χ2 (1; 0.01)= 6.635). Mite numbers in petri dishes with grapefruit oil were significantly lower than those with sweet orange oil (χ2= 19.500; df= 1; χ2 (1; 0.01)=6.635), concluding that grapefruit oil has a significant repellent effect. No literature isavailable on the use of citrus fruit essential oils to potentially control AFB and V. destructor. The inhibition of P. larvae could be attributed to the volatile specific component limonene, the principal component of grapefruit and sweet orange oils. Sweet orange oil, however, showed lower antibacterial activity than grapefruit oil, probably due to different monoterpenes composition. grapefruit oil had a moderate inhibitory effect against P. larvae, compared with other essential oils(Alippi et al., 1996; Fuselli et al., 2006). No honey bee mortality was observed with any citrus oils concentration. This is important, since many oils with acaricidal activity also have adverse effects on honey bees, causing damage to the colonies. Grapefruit oil had a repellent effect on mites, but no similar results were obtained using sweet orange oil. Grapefruit oil showed the best results against V. destructor regarding the LC50 and the repellent effect.The oil of grapefruit thus showed significant in vitro antibacterial and antiparasitic activity, providing a contribution to the screening of alternative compounds for treating honey bee colonies suffering from AFB and V. destructor, to be incorporated in Integrated ManagementProgrammes.