SUPPLEMENTARY MATERIAL

Stonebrood and chalkbrood in Apis mellifera causing fungi: in vitro sensitivity to some

essential oils

Simona Nardoni,1 Carlo D’Ascenzi,

1 Guido Rocchigiani,

1 Roberto Amerigo Papini,

1 Luisa Pistelli,

2

Giovanni Formato, 3 Basma Najar,

2 and Francesca Mancianti

1.

1Dipartimento di Scienze Veterinarie, Università degli Studi di Pisa, Viale delle Piagge, 2 – I 56124

Pisa, Italy

2Dipartimento di Scienze Farmaceutiche, Università degli Studi di Pisa, Via Bonanno, 33 - I 56126

Pisa, Italy

3Beekeeping Laboratory Istituto Zooprofilattico Sperimentale del Lazio e della Toscana “Mariano

Aleandri" Via Appia Nuova 1411 - 00178 Roma, Italy

Correspondence author

Simona Nardoni

Tel: +39 050 2216952

E-mail: simona.nardoni@unipi.it

Abstract: Aim of the present paper was to evaluate the in vitro antimycotic activity of 17

chemically defined essential oils (EOs) both alone and as a mixture, against agents responsible for

stonebrood and chalkbrood in honeybees. Cinnamomum zeylanicum yielded the lower MIC against

Aspergillus flavus, but was not effective against Ascosphaera apis, while Litsea cubeba and

Pelargonium graveolens appeared to be effective against all checked fungi. Aspergillus niger

yielded the lower sensitivity. Mixtures were composed by L. cubeba, C. zeylanicum, and

Cymbopogon flexuosus (M1) and by L. cubeba, C. zeylanicum, P. graveolens and C. flexuosus

(M2) respectively, and resulted effective. The components of M1 showed a synergistic effect. The

use of mixtures allowed decreasing the total amount of EOs, minimizing possible adverse effects

for bees. The use of these products could be of interest for an alternative natural approach in

honeybee management.

Keywords: Aspergillus flavus, Aspergillus niger, Ascosphaera apis, essential oils mixture,

antimycotic activity

Experimental

Gas Chromatography – Mass Spectrometry Analysis

Citrus limon, Illicium verum, Origanum vulgare, Thymus vulgaris, Litsea cubeba, Citrus bergamia,

Foeniculum vulgare, Pelargonium graveolens, Salvia sclarea, Lavandula hybrida, Cymbopogon

flexuosus, Sygizium aromaticum, Origanum majorana, Cinnamomum zeylanicum, Citrus paradisi,

Citrus aurantium and Citrus sinensis EOs were used. EOs were produced by Flora SRL, Lorenzana

(Pisa, Italy) and purchased for test execution.

Volatile constituents of each EO were analyzed by GC-MS as previously reported (Nardoni et al.

2016).

Mycology

Isolates of A. flavus, 1 of A. niger, and 1 of A. apis were cultured each from larvae of A. mellifera

found spontaneously mummified in combs of hives showing clinical symptoms of stonebrood or

chalkbrood. The hives were located in the province of Pisa (Tuscany, Central Italy). The isolates

identified on the basis of their macro and microscopical morphology, were assayed in vitro by

microdilution test against 17 EOs using 96-well plates (Sarstedt, Milano, Italy). Serial dilutions of

EOs in Malt Extract Broth (MEB) were made to achieve final concentrations ranging from 0.5%. to

0.005%. Such dilutions were chosen to completely avoid toxic effects of monoterpenoids present in

EOs with regard to honeybees (Mondet et al. 2011, Alayrangues et al. 2016). Before testing, each

isolate of Aspergillus was subcultured at least twice on Potato Dextrose Agar to ensure viability and

purity. EUCAST (2008) broth microdilution method testing was performed modified by using MEB

instead of the recommended liquid medium. Inocula of 5 × 105 phialospores/ml, were incubated at

25°C until a complete growth in control wells was observed. The same inocula were achieved using

ascospores of A. apis. The MIC values were visually determined as the lowest concentration of EO

that caused complete inhibition of growth.

Mixtures

After preliminary results about the effectiveness of each single EO, the most effective were selected

to compose mixtures for a further in vitro trial.

Therefore inocula, obtained as above described, were also tested against one mixture composed by

0.02% each of L. cubeba, C. zeylanicum, and C. flexuosus (M1) and another mixture composed by

0.015% each of L. cubeba, C. zeylanicum, P. graveolens and C. flexuosus (M2). Both mixtures were

checked as undiluted and at 75% and 50% dilution, respectively.

To better understand the measurement of the effect of the combinations of EOs in the 2 mixtures,

FIC Index (FICI) was calculated as reported by Rosato et al. (2007). The FICI was interpreted as:

(i) a synergistic effect when ⩽0.5; (ii) an additive or indifferent effect when >0.5 and <1 and

(iii) an antagonistic effect when>1.

References

Alayrangues, J, Hotier, L, Massou I, Bertrand Y, Armengaud C. 2016. Prolonged effects of in-hive

monoterpenoids on the honeybee Apis mellifera. Ecotoxicol. DOI 10.1007/s10646-016-1642-x

EUCAST. 2008. Technical note on the method for the determination of broth dilution minimum

inhibitory concentrations of antifungal agents for conidia-forming moulds

http://www.eucast.org/documents/technical notes

Mondet F, Goodwin M, Mercer A. 2011. Age-related changes in the behavioural response of

honeybees to Apiguard(R), a thymol based treatment used to control the mite Varroa destructor. J.

Comp. Physiol. 197: 1055–1010.

Nardoni S, Pistelli L, Baronti I, Najar B, Pisseri F, Bandeira Reidel RV, Papini R, Perrucci S,

Mancianti F. 2016. Traditional Mediterranean plants: characterization and use of an essential oils

mixture to treat Malassezia otitis externa in atopic dogs. Nat Prod Res. 5:1-4.

Rosato A, Vitali C, De Laurentis N, Armenise D, Milillo MA. 2007. Antibacterial effect of some

essential oils administered alone or in combination with Norfloxacin. Phytomed. 14: 727-732.

Table S1. Chemical composition of essayed essential oils.

Component LRI C.a C. b C.l C. p C.s C. z C.f. F.v I.v L.h L.c O. m O.v P.g S.s S.a T.v

1 Tricyclene 926

1.1 1.9

1.4

8.6 1,4

1.5

2 Camphene 953

1.1

3 Sabinene 976

1.1 2.3

1.0 3.2

4 β-pinene 980

5.4 11.9

1.0

1.2

5

6-methyl-5-hepten-2-

one 985

1.8

1.5

6 Myrcene 991 1.9 1.0 1.8 2.2 2.3

1.6 2.2

7 α-phellondrene 1005

2.1

2.2

8 α-terpinene 1018

1.0

4.7 2.1

9 p-cymene 1026

3.0

1.9

4.2 9.3

15.3

10 limonene 1031 94.7 33.2 65.7 92.2 95.5

2.0 6.5 3,9

16.3 2.1

11 β-phellandrene 1031

5.9

12 1,8-cineole 1033

7.7 2.3

13 (Z) β-ocimene 1040

1.2

14 (E) β-ocimène 1050

2.1

15 γ-terpinene 1062

6.4 9.3

7.9 5.3

2.9

16 cis-sabinene hydrate 1068

3.2

17

cis-linalool oxide

(furanoid) 1074

2.2

18 fenchone 1087

20.1

19 terpinolene 1088

1.5

20

trans-linalool oxide

(furanoid) 1088

1.8

21 trans-sabinene idrato 1097

12.8 1.8

3.8

22 linalool 1098

14.2

6.3 1.5

31.5 1.5

3.9 8.1

23 cis-rose oxide 1111

1.0

24 camphor 1143

7.3

25 menthone 1154

1.1

26 isomenthone 1164

3.5

27 borneol 1165

2.1

1.6

28 4-terpineol 1177

4.0

17.6

2.4

29 α-terpineol 1189

2.1

2.7

30

menthyl

chavicol(=estagol) 1195

8.6

31 citronellol 1228

44.5

32 thymol methyl ether 1232

1.7

33 neral 1240

35.2

32.5

34 geraniol 1255

4.4

2.7

13.7

35 linalool acetate 1257 1.4 31.6

26.8

3.2

54.7

36 (E) cinnamaldehyde 1266

56.4

37 geranial 1270

1.2

38.4

36.4

38 citronellyl formate 1280

7.3

39 (E) anethol 1283

46.9 89.8

40 bornyl acetate 1285

2.4

41 thymol 1290

52.6

42 carvacrol 1298

20.8 65.9

43 geranyl formate 1298

1.9

44 α-limonene diepoxide 1347

8.6

45 eugenol 1356

3.0

77.9

46 α-copaene 1376

1.5

47 geranyl acetate 1383

4.2

48 β-caryophyllene 1418

10.3 2.3

2.2

1.7 3.7

8.9 6.8

49 aromadendrene 1439

2.9

50 α-humulene 1454

2.7

51 (E)-β-farnesene 1458

1.4

52 α-muurolene 1499

1.4

53 trans-γ-cadinene 1513

1.2

54 eugenol acetate 1524

12.2

55 δ-cadinene 1524

1.0

56 citronellyl butyrate 1532

1.2

57 geranyl butyrate 1564

1.1

58 caryophyllene oxide 1581

4.8

59

(E)-2-phenyl ethyl

tiglate 1583

1.2

60 (Z)-citronellyl tiglate 1658

1.2

61 geranyl tiglate 1696

1.2

62 benzyl benzoate 1762

1.0

63 scareol 2223

1.3

unknown

12.8

1.7

Total of identified compounds 100 100 100 99.2 100 99.7 97.6 99.7 100 100 99.4 98.0 98.5 99.3 86.1 100 95.6

Monoterpene hydrocarbons (mh) 97.4 49.0 94.3 96.2 98.7 15.5 3.9 22.1 7.3 6.4 21.3 27.7 22.5 21.5

Oxygenated monoterpene (om) 1.9 48.4 3.6 0.5 0.6 7.4 86.3 21.1 0.7 85.0 75.7 66.6 71.2 83.4 78.2

64.1

Sesquiterpene hydrocarbons (sh) 0.2 2.5 2.0 1.9

14.7 4.5

1.1 5.4 0.9 3.2 4.2 7.8 0.9 9.5 9.2

Oxugenated sesquiterpene (os)

0.8 0.9

0.1 1.3

0.4 0.4 6.9 4.9 0.4

Oxygenated Diterpene (od)

1.3

Non-terpene derivatives (nt) 0.5 0.1 0.1 0.6 0.7 58.3 2.0 0.9 0.1 1.9 1.5 0.1 0.1 1.2 0.8

0.8

Phenylpropanoids (pp) 3.0 55.6 90.7 0.1 90.1

Legend: LRI- on DB-5 column.

C.a (Citrus aurantium), C.b (Citrus bergamia), C.l (Citrus limon), C.p (Citrus paradisi), C.s (Citrus sinensis), C.z (Cinnamomum zeylanicum), C.f

(Cymbopogon flexuosus), F.v (Foeniculum vulgare), I.v (Illicium verum), L.h (Lavandula hybrida), L.c (Litsea cubeba), O.m (Origanum

majorana), O.v (Origanum vulgare), P.g (Pelargonium graveolens), S.s (Salvia sclarea), S.a (Syzygium aromaticum), T.v (Thymus vulgaris)

Table S2. Minimum Inhibitory Concentration of examined essential oils versus A. flavus A. niger and

Ascosphaera apis.

Essential oils Minimum Inhibitory Concentration

(%)

Aspergillus flavus Aspergillus niger Ascosphaera apis Citrus limon >0.5 >0.5 >0.5 Illicium verum 0.1 0.1 >0.5 Origanum vulgare 0.1 0.1 >0.5 Thymus vulgaris 0.1 0.1 >0.5 Litsea cubeba 0.1 0.1 0.025 Citrus bergamia >0.5 >0.5 >0.5 Foeniculum vulgare >0.5 >0.5 >0.5 Pelargonium graveolens 0.1 0.1 0.025 Salvia sclarea >0.5 >0.5 >0.5 Lavandula hybrida >0.5 >0.5 >0.5 Cymbopogon flexuosus 0.1 0.1 >0.5 Sygizium aromaticum 0.5 0.5 >0.5 Origanum majorana >0.5 >0.5 >0.5 Cinnamomum zeylanicum 0.075 0.1 >0.5 Citrus paradisi >0.5 >0.5 >0.5 Citrus aurantium >0.5 >0.5 >0.5 Citrus sinensis >0.5 >0.5 >0.5

Mixtures M1 0.06 0.06 0.06 M2 0.06 0.06 0.06

M1 (mixture of C. zeylanicum, C. flexuosus and L. cubeba, 0.02% each) M2 (mixture of C. zeylanicum, C. flexuosus, P. graveolens and L. cubeba, 0.015% each)