Antimicrobial Activity

of Some Essential Oils

Against Paenibacillus larvae,

the Causal Agent

of American Foulbrood Disease

Adriana M. Alippi

Jorge A. Ringuelet

Elsa L. Cerimete

Maria S. Re

Cynthia P. Henning

 

ABSTRACT. Essential oils from savory (Satureja hortensis), lavandin (Lavandula hybrids), eucalyptus globulus), lemon grass (Cymbopogon citratus), peppermint (Mentha x piperita), oregano (Origanum vulgare), rosemary (Rosmarinus offlcinalis), and thyme (Thymus vulgaris) were tested for antimicrobial activities against Paenibacillus larvae, the causal agent of American Foulbrood Disease (AFB) of honeybees. Trials for determining the minimum inhibitory


Adriana M. Alippi is Research Scientist from C.I.C. and is affiliated with the Laboratorio de Fitopatologia, Facultad de Ciencias Agrarias y Forestales, Universidad Nacional de La Plata, La Plata, Argentina.

Jorge A. Ringuelet, Elsa L. Cerimete, Maria S. Re, and Cynthia P. Henning are affiliated with the Curso de Fitoquimica, Facultad de Ciencias Agrarias y Forestales, Universidad Nacional de La Plata, La Plata, Argentina.

A financial grant (B- 1 859/2) of the International Foundation for Science (IFS), Sweden, to Andriana M. Alippi is gratefully acknowledged. This work was also supported by the U.N.L.P. and by the C.I.C. The authors thank Dr. Bandoni for his collaboration in the analytical determinations by gas chromatography and to Dr. M. Colin for his valuable suggestions. Received July 24, 1995.

Concentration (MIC) of these oils revealed that lemon grass and thyme were the most effective with MIC values between 50 to 100 ml/liter and 100 to 150 ml/liter, respectively, in tests with 8 Paenibacillus larvae strains from different Argentinean geographical regions. The findings indicate that lemon grass and thyme oils could be used as effective inhibitors of AFB in honeybee colonies. [Article copies available for a fee from 7he Haworth Document Delivery Service: 1-800-342-9678. E-mail address: getinfo@haworth.coml

 

KEYWORDS. Apis mellifera, herbs, honeybees, medicinal plants, veterinary medicine

 

INTRODUCTION

American Foulbrood (AFB), caused by the spore-forming bacterium Paenibacillus larvae (White) (3), is the most serious disease of bacterial origin affecting the larvae and pupae stages of honeybees (Apis mellifera L.) (4,2 1). Currently, the alternative to burning the infected hives for control of this disease is treatment with antibiotics. Treating hives with an antibiotic, however, can contaminate the honey with antibiotic residue, reducing the quality of the product.

Essential oils extracted from aromatic plants have been demonstrated through in vitro tests to possess antimicrobial activity (6,9,13,17,18,23, 25,26). Essential oils have been used in bee colonies for the control of chalkbrood, a fungal disease caused by the pathogen Ascosphaera apis (8), and varroasis, an affliction caused by the parasitic mite Varroa jacobsoni (16,24). In addition, Calderone et al. (6) evaluated botanical compounds for in vitro control of Paenibacillus larvae, Ascosphaera apis, and Paenibacillus alei. Floris and Carta (10) also demonstrated that cinnamon oil (Cinnamomum zeylanicum) was effective on colonies of honeybees infected with American Foulbrood disease.

The purpose of the present work was to evaluate, in vitro, the antibacterial activity of 8 essential oils, not previously tested, against 8 strains of Paenibacillus larvae isolated from different areas of Argentina.

 

MATERIALS AND METHODS

Plant material. Essential oils of proven antimicrobial effects from 8 botanical species (6,8,23,26) and cultivated in Argentina were selected for testing: savory (Satureja horiensis L., family Labiatae); lavandin (Lavandula x intermedia Emeric ex Loiseleur, family Labiatae), eucalyptus (Eucalyptus globulus Labill., family Myrtaceae); lemon grass (Cymbopogon citratus (D.C.) Stapf, family Poaceae), peppermint (Mentha piperita L., family Labiatae); oregano (Origanum vulgare L., family Labiatae); rosein . (Rosmarinus officinalis L., family Labiatae); and thyme (7hymus vulgaris L., family Labiatae).

Preparation of extracts. The essential oils were extracted by steam distillation with a Clevenger apparatus (12). In the case of lavandin, lemon grass, thyme, oregano, savory and peppermint, the analysis of principal components was done by gas chromatography on methylsilicone and Carbowax 20 M columns (5). For rosemary oil, infrared spectroscopy was used for analysis (19) and for eucalyptus oil, a chemical analysis using resorcinol was employed (20). The formulations of essential oils used in testing were made with sterile, double distilled water and propylene glycol at 5 percent as emulsifier.

Bacterial strains. The 8 strains of Paenibacillus larvae used in these studies were isolated from honeycombs of hives exhibiting clinical symptoms of American Foulbrood Disease and located at the following Argentinean localities: La Plata, ZArate, Punta Alta, Lobos, Lincoln, Pigue, Lujdn and Concordia. Isolation and identification of the strains were done using previously described techniques (1,2,1 1) and Paenibacillus larvae cultures were characterized according to their biochemical type (14).

Determination of minimal inhibitory concentrations. J-agar was employed as basal medium (I 1) for growth of the bacterial strains during testing for effects of essential oils. The culture medium (9 ml) was poured into Petri dishes (9 cm in diameter) and maintained at 45'C until the essential oils were incorporated into the agar. The oils were added as I ml of formulation using an automatic micropipette while constantly stirring to assure a uniform distribution. Each essential oil was tested at a concentration of 20, 50,100,150,200,250,300, 350,400,450,500,550,600, 650, and 700,ulA. For controls, J-agar with the addition of propylene glycol in aqueous solution (but no essential oil), was used.

Vegetative cells of Paenibacillus larvae previously grown on J-agar for 48 h of incubation at 36 I 'C were suspended in sterile, double distilled water and standardized to a turbidity level approximating 107 CCIIS/n-d (absorbance of 0.258 at a wavelength of 620 nm measured with a photocolorimeter (Crudo Camafio, Model Nan 11). The different bacterial strains were layered by using an automatic micropipette to place 8 drops (30,ul each) over the surface of the solidified culture medium containing an essential oil. This procedure was repeated twice for each bacterial strain and each oil.

After the bacterial suspensions were absorbed into the agar, the plates were incubated in inverted position at 36 I 'C for 48 h. Bacterial growth was monitored visually and the minimum inhibitory concentration (MIC) of essential oil for each strain and essential oil combination was determined (complete inhibition of bacterial growth on the test plates, disregarding a single colony or faint haze caused by inoculum, according to the NCCLS standards) (22).

RESULTS

Analysis of main essential oil constituents indicated the rosemary oil corresponded to that from a myrcene chemotype (according to infrared -spectroscopy) and that for the eucalyptus oil the principal component was cineole (averaging 65%) (data not shown). Constituents of oils in other tested plant material were within expected ranges (Table 1).

All Paenibacillus larvae strains were gram positive and catalase negative. These micro-organisms did not hydrolyze starch, produce indole, or withstand serial transfer in nutrient broth, common characteristics of the species (I 1). All strains corresponded to the biochemical type ll with reduction of nitrate to nitrite and no acid production from d(+)mannitol or d(+)salicin (14).

Of all tested essential oils, those which had the greatest antibacterial action against Paenibacillus larvae were lemon grass with MIC values ranging from 50 to 100,ulll (depending upon the tested strain) and thyme with MIC values ranging form 100 to 150, m1/1 (Table 2). MIC values for Rosemary were 700 m1/1 and more than 700 m1/1 for eucalyptus, indicating little antibacterial activity against Paenibacillus larvae. Oils of savory and oregano had intermediate MIC values from 250m1/1 to 450 m1/1 (depending upon the tested strain).

 

DISCUSSION

Antimicrobial activity of citral, the principal component of lemon grass essential oil, against bacterial species such as Clostridium sporogenes, S'almonella pullorum, Staphylococcus aureus, and Klebsiella pneumoniae @as been previously demonstrated (9). Thymol, the principal oil component of the thyme clones used in this study, has also been reported to be an

TABLE 1. Principal components of essential oils.

Component

Cymbopogon citratus

Lavandula x intermedia

Mentha x piperita

Origanum vulgare

Satureja horlensis

Thymus vulgaris

(---------------------------------------------- %-----------------------------------------------)

a-Pinene

ND

ND

ND

0.5

1.1

ND

a-Terpinene

ND

ND

ND

ND

3.6

1.8

a-Terpineol

ND

ND

ND

3.7

ND

ND

b -Caryophyllene

ND

2.5

ND

1.5

1.9

1.5

b -Pinene

ND

ND

ND

1.1

2.2

2.0

Bormeol

ND

2.5

ND

14.1

0.3

ND

Camphor

ND

10.0

ND

ND

ND

ND

Carvacrol

ND

ND

ND

0.4

35.5

2.4

1,8-Cineole

ND

7.6

ND

ND

ND

ND

Citral

66.7

ND

ND

ND

ND

ND

Free menthol

ND

ND

44.6

ND

ND

ND

r-Pinene

ND

ND

ND

ND

ND

0.8

r-Terpinene

ND

ND

ND

7.0

40.4

13.1

Limonene

ND

1.3

ND

ND

ND

ND

Linalool

ND

44.6

ND

1.6

ND

2.4

Linalyl acetate

ND

3.6

ND

ND

ND

ND

Mentho-furan

ND

ND

10.0

ND

ND

ND

Menthone

ND

ND

20.0

ND

ND

ND

Menthyl acetate

ND

ND

7.4

ND

ND

ND

Myrcene

0.8

ND

ND

ND

ND

ND

Ocimene

ND

5.0

ND

ND

ND

ND

p-Cymene

ND

ND

ND

3.0

4.8

18.1

Thymol

ND

ND

ND

25.1

0.3

39.9

Terpinolene

ND

ND

ND

1.5

ND

ND

Tricyclene

ND

ND

ND

0.7

1.4

1.3

ND = not detected.

antibacterial agent against Salmonella typhimuritim and Staphylococcus aureus (I 5). Calderone et al. (6) have observed that cinnamon oil (Cinnamomum spp.) completely inhibits the growth of Paenibacillus larvae in vitro tests at concentrations as low as 10 ppm and that camphor and citronellal (3,7-dimethyl-6-octenal) were effective at 100 ppm. With bay (Pimenta racemosa) oil, clove (Sysygium aromaticum) oil, Spanish origanum (Thymus capitatus) oil and thymol (5-methyl-2-[ 1 -methylethyl] phenol), inhibition was obtained with concentrations of 1000 ppm. In beehives, thyme oil has been reported to inhibit the development of the fungus Ascosphaera apis (8) and thymol is also employed in combin ' ation with other essence compounds in commercial preparations (Apilife VAR) for the control of the mite Varroajacobsoni (16,24).

TABLE 2. Minimal inhibitory concentrations of essential oils against Paenibacillus larvae.

Paenibacillus larvae strain

Essential oil

Zarate

Ralta

LaPlata

Lobos

Lincoln

Pigu6

Lujiln

E.Rios

(-----------------------------m1/1 essential oil/liter media ------------------------------)

Cymbopogon citralus

50

50

50

50

100

50

50

100

Eucalyptus globulus

>700

>700

>700

>700

>700

>700

>700

>700

Lavandula x intermedia

450

550

600

350

600

450

450

550

Mentha x piperita

600

600

600

600

650

600

600

600

Origanum vulgare

300

250

300

300

350

450

250

350

Rosmarinus officinalis

700

700

700

700

700

700

700

700

Satureja hortensis

300

250

300

200

300

200

300

300

Thymus vulgaris

150

150

150

150

150

150

100

100

 

Our results with savory and oregano coincide with those obtained by Colin (M. Colin, 1993, Pers. Com.) (7). In the present study, the essential oil of lemon grass had the greatest inhibitory effect on the growth of Paenibacillus larvae. The principal component of this oil is citral which is also the main constituent of Lippia citriodora H.B. cultivars tested by Colin (M. Colin, 1993, unpublished data) (7).

The 8 strains of Paenibacillus larvae isolated in Argentina exhibited similar MIC values for the 8 essential oils tested. The essential oils from lemon grass and thyme could be used as effective inhibitors of American Foulbrood Disease in honey bee colonies if the toxicity to bees, residual effects in honey, and efficiency of use present no obstacles. Use of the essential oils would represent a natural alternative to antibiotics in control of American Foulbrood Disease.

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