Inhibitory Effects of Some Medicinal Plant Extracts on the Growth of Ascosphaera apis

Transcription

1 Inhibitory Effects of Some Medicinal Plant Extracts on the Growth of Ascosphaera apis P. Chantawannakul and T. Puchanichanthranon Department of Biology Faculty of Science Chiang Mai University Chiang Mai, Thailand S. Wongsiri Department of Biology Faculty of Science Chulalongkorn University Bangkok, Thailand Keywords: antifungal property, honey bee, Chalkbrood disease, Cinnamomum cassia, Piper betel Abstract Ascosphaera apis is a fungal pathogen causing Chalkbrood disease in honey bee larvae. Chalkbrood is most frequent during damp conditions. Infected larvae turn chalky white color, become hard, and then turn black. It can be regarded as the most widespread infectious disease in Thailand and this has led to economic loss in apiculture. A. apis strains were isolated on Potato Dextrose Agar (PDA) from the dead honey bee larvae, collected from infected hives in Northern Thailand. The fungal strains were identified as A. apis by their morphology in comparison with the A. apis IFO9831. We aim to find an alternative approach by using medicinal plants in combating and controlling the disease. Studies on the effect of crude medicinal plant extracts, therefore, have been carried out. Dried powdered plants (Allium sativum Linn, Eugenia caryophyllum Bullock&Harrison, Piper betel, Curcuma longa Linn, Illicium verum Hook, Cinnamomum cassia, Rhinacanthus nasutus Kurz, Azadirachta siamensis, Acorus calamus Linn., and Stemona tuberosa Lour) were extracted in seven different solvents by incubating for 48 hours. The aqueous extracts in suitable solvent of E. caryophyllum, P. betel, I. verum, C. cassia, A. calamus, and gave inhibitory effect when tested with Thai isolates of Ascosphaera apis. Various concentrations ( %) of the extract of the five chosen plants were experimented. Cinnamomum cassia and Piper betel ( % (w/v)) gave best inhibitory effects on the fungal growth. These results suggest that either cinnamon or betel piper extract, alone or in combination, could well be used for Chalkbrood control in the future. INTRODUCTION The honey bee is one of the most beneficial insects known to man. Besides being the producer of valuable natural products like honey and wax, they play a significant role in the ecosystem and the survival of many flowering-plant species. In Thailand, honey imports of Thailand rose from 1,843 Mt in 1999 to 3,000 Mt in 2000 due to the introduction of the European honey bee (Apis mellifera). However, as mellifera beekeeping being conducted increased, honey production is gravitated by microbial diseases which are found to infect the honey bee larvae (Bailey and Ball, 1991). Chalkbrood disease, caused by the fungus Ascosphaera apis Massen ex Claussen (Spiltoir and Olive, 1955), is a serious threat causing up to bee mortality in Thailand. The disease causes economic damage in apiaries worldwide. The initial report of chalkbrood in the U.S. came in 1968; Canada first reported it in 1972; Thailand reported in 1987 (Caron, 1994). The disease is initiated at the larval stage when bee larvae are infected with spores carried in pollens or spread by the nurse worker bees. Larvae mummified by Chalkbrood often look like tiny sticks of chalk. Mummies are white, black or grayish and mottled. Research on control is focused on three areas: management practices, chemicals (e.g. antimycotic drug) and genetics of bees. Management practices are very laborious and carried out with great care including strict inspection and quarantine when disease incidence occurs. Efficacious chemotherapeutic agent is not universally acceptable or Proc. WOCMAP III. Vol. 4: Targeted Screening of MAPs, Economics & Law Eds. C. Franz, Á. Máthé, L.E. Craker and Z.E. Gardner Acta Hort. 678, ISHS

2 available for use against the Chalkbrood fungus in honey bees. Adding to that, many chemicals used by beekeepers may not be beneficial in anyway other than stimulating the cleaning instinct of the bees (Gilliam, 1993). In some countries, the application of antibiotics has been an option to combat the diseases, however, evolution of resistance in pathogens and parasites to nowadays antibiotics and strict regulations on the chemical residues permitted in the human food chain are being greatly concerned. This means that new antifungal substances are continually being sought and alternative approaches of controlling diseases are needed. Plant oils and extracts have been long used for a wide variety of purposes (Jones, 1996). Antimicrobial activity of essential oils and other plant extracts has been examined (e.g. Thymus essential oils) (Cosentino et al., 1999; Hammer et al., 1999). It has been confirmed the possibility of using some plant oils or some of their components in human food chain because these products and compounds are of GRAS status, the safety concern of using them to prevent the microbial growth is minimal. Thus, the possibility of controlling Chalkbrood disease by medicinal plants has been recently investigated. One of the effective substance is azadirachtin extracted from Neem (Azadirachta indica A. Juss). However, azadirachtin is sensitive to UV light. Also, after application to the bee hives, azadirachtin was easily degraded by ph and temperature changes (Liu, 1995). Screening for other new substances from medicinal plant is being examined. The purpose of this work is to evaluate the possible control of Chalkbrood by using medicinal plants in vitro. MATERIAL AND METHODS Samples Collection and Identification Forty six samples of infected honey bee larvae were collected from six apiaries (five apiaries in Chiang Mai and one apiary in Payao Province, Thailand). The samples were cultured in Potato Dextrose Agar (PDA) at 30 C for 5 days. The morphology was investigated under compound microscopy and Scanning Electron Microscopy (SEM). The fungal species were identified by their morphology of their fruiting bodies in comparison with a type strain, A. apis IFO9831 (purchased from NITE Biological Resource Center, Japan) (Bissett et al., 1996). Preparation of Medicinal Plant Extracts Commercial available supplies of plant materials used through this study are presented in Table 1. Each ground plant was soaked in various kinds of solvents (hexane, dichloromethane, ethyl acetate, buthanol, ethanol, methanol and water). Each kind of solvent was used in the ratio of 1:3 (50 mg of ground plant/150 ml of solvent) and for 48 hours in extraction. The extracts were then evaporated under reduced pressure by a rotary evaporator and stored in a sterilized vial at 4 C for further analysis. Determination of the Inhibitory Effect of the Plant Extracts on A. apis 1. Screening for Antifungal Activity. The medicinal extracts were tested for antifungal activity by using agar disk diffusion method (Barry and Thronsberry, 1991). A. apis MR (isolated from Thai apiary) was grown on Potato Dextrose Agar (PDA) at 30 C for 3-4 days. A 6-mm diameter antibiotic test disk was saturated with each medicinal plant extracts (20 µl) and placed on the surface of the agar at 0.5 cm apart from the fungal hyphae. Plates were incubated for 3 days at 30 C. The growth zone of fungus was measured and compared to controls every 24 h. 2. Minimum Inhibitory Concentration Assay. Medicinal plant exhibiting inhibitory effect on the growth of A. apis were examined for Minimum Inhibitory Concentration (MIC). Each of the tested plant extracts was used at different concentrations: % (w/v). Each extract was mixed with the agar. A. apis MR was inoculated by placing agar plugs (5 mm in diameter) containing 3-day-old fungal hyphae (triplicates were used for each treatment). Two perpendicular diameters of the growth zone were measured from which the average growth area was calculated. Negative control was solvent used for each 184

3 extraction. Nystatin in DMSO (1 mg/ml) was used for positive control. 3. Thin Layer Chromatography (TLC). The plant extract (1 µl) was spotted on silica gel thin layer glass and separated with suitable mobile phase. The TLC plate was then placed on PDA containing A. apis and incubated for 3-4 days at 30 C. The antimycotic activity was visualized by clearing zones. RESULT AND DISCUSSION In the survey of Chalkbrood disease in Apis mellifera in Northern Thailand from September 2001 to March 2002, forty-six samples of infected honeybee larvae were collected from six apiaries (five apiaries in Chiang Mai and an apiary in Payao province). Chalkbrood disease was the most widespread disease in honey bee (Apis mellifera) larvae. It may be caused by humidity and temperature which are suitable to the pathogen germination and growth. Optimum temperature for germination of the fungus is similar to that required for sporulation and is within the range for vegetative stage. Therefore, an optimum of C was appropriate for all stages (Bamford and Heath, 1989). Fungal strains from mummified larvae were isolated and cultured on potato dextrose agar incubated at 30 C for five days. All fungal strains were identified as Ascosphaera apis, based on their morphology under a compound microscope and a scanning electron microscope (Fig. 1). Some spices are already known to contain essential oils that posses antimicrobial activity (e.g. eugenol in cloves, allicin in garlic, and cinnamic aldehyde and eugenol in cinnamon (Bullerman et al., 1977; Holt et al., 1995)). Thyme, cinnamon, anise and spearmint were also found to exhibit complete inhibition of fungal growth. The use of the essential oil in inhibiting of mycotoxin production of stored grains is suggested accordingly (Soliman and Badeaa, 2002). In addition, antifungal effects of eugenol and thymol on Penicillium citrinum in spanish cheese is reported (Vazquez et al., 2001). Extracts with various solvents from ten medicinal plants were screened for inhibitory effects on A. apis MR (Thai isolate). Our experiments showed that Eugenia caryophyllum, Piper betel, Illicium verum, Cinnamomum cassia, and Acorus calamus highly inhibited the growth of the fungal pathogen. In contrast, the pathogenic fungus was not at all sensitive to extracts from Azadirachta siamensis and Allium sativum as in Table 2. To determine their activity thresholds, the five medicinal plants (E. caryophyllum, P. betel, I. verum, C. cassia, and A. calamus) at various concentrations ( %) were assayed. Ethyl acetate Piper betel extract and hexane Cinnamomum cassia extract showed greater antifungal activity than that of the others (Table 3). MIC values of ethyl acetate extract of Piper betel and hexane extract of cinnamon are 3.5% and 1.5% (w/v) as shown in Table 4 and 5. No differences in inhibitory activity of the plant extracts between strains were observed. It is previously found that pure cinnamon oil has a fungicidal effect on the Chalkbrood pathogen (Calderone and Shimanuki, 1994). However, in this study, ethyl acetate Piper betel extract exhibited efficiently antifungal effect similar to that of cinnamon. When the crude extract of Piper betel was separated by using thin layer chromatography with various solvents, the suitable mobile phase was toluene: ethylacetate (93:7 (v/v)). The chromatogram gave visible six bands with the Rf values of 0.03, 0.13, 0.2, 0.39, 0.42, 0.86 respectively. After overlaid with PDA containing A. apis culture, the fungal growing plate gave the clearing zone observed at Rf after 3-4 days at 30 C. The further analysis of these compounds is in process to compare with that of cinnamon extracts. Therefore, the application of these plant extracts, alone or in combination, could be safely used as a substitute for chemical fungicides since they are natural, and non-toxic to humans - they are commonly used as ingredient in Asian cooking for thousand years. Further investigation is being conducted to evaluate the stability of bioactive compounds, delivery mechanism of these compounds to bee hives, their effects on bees and also economic of the use in pilot and commerical scales. 185

4 ACKNOWLEDGEMENTS P.C. gratefully acknowledges Thailand Research Fund (PDF ) for financial support. Thanks also go to Department of Biology, Faculty of Science, Chiang Mai University, Thailand for providing all laboratory facilities. Literature Cited Bailey, L. and Ball, B.V Honey bee pathology. 2 nd ed., Academic Press, London. Bamford, S. and Heath, L.A.F The effects of temperature and ph on the germination of spores of the Chalkbrood fungus, Ascoshaera apis. J. Apicultural Res. 28(1): Barry, A.L. and Thornsberry, C Susceptibility test: Diffusion test procedures. In: A. Balows, W.J. Hausler, K.L. Herrmann, H.D. Isenberg and H. Jean Shadomy (eds.), Manual of Clinical Microbiology. 5 th ed., American Society for Microbiology, Washington DC. Bissett, J., Duke, G. and Goettel, M Ascosphaera acerosa sp. nov. isolated from the alfalfa leafcutting bee, with a key to the species of Ascosphaera. Mycologia 88(5): Bullerman, C.U., Lieu, F.Y. and Seier, S Inhibition of growth an alflatoxin production by cinnamon and cloves oils, cinnamic aldehyde and eugenol. J. Food Sci. 42: Calderone, N.W. and Shimanuki, H An in vitro evaluation of botanical compounds for the control of the honeybee pathogens Bacillus larvae and Ascosphaera apis, and the secondary invader B. alvei. J. Ess. Oil Res. 6: Caron, D Chalkbrood: No known cures, but lots of help. Bee culture 122(7): Cosentino, S., Tuberosa, C.I.G., Pisano, B., Satta, M., Mascia, V., Arzedi, E. and Palmus, F In vitro antimicrobial activity and chemical composition of Sardinian Thymus essential oils. Letters in Appl. Microbiol. 29: Gilliam, M Chalkbrood control. p In: L. Connor, T. Rinderer, H.A. Sylvester and S. Wongsiri (eds.), Asian Apiculture, Wicwas Press, Connecticut, USA. Hammer, K.A., Carson, C.F. and Riley, T.V Antimicrobial activity of essential oils and other plant extracts. J. Appl. Microbiol. 86: Holt, D.L. and Gomez-Almonte, N.A A reseach note: antimycotic activity of garlic extracts and extract fractions in vitro and plant. J. Food Prot. 58: Jones, F.A Herbs-useful plants. Their role in history and today. Eur. J. Gastroenterology and Hepatology 8: Liu, T.P A possible control of Chalkbrood and Nosema disease of the honey bee with Neem. Canadian Beekeeping 18(5): Vazquez, B.I., Fente, C., Franco, C.M., Vazquez, M.J. and Cepeda, A Inhibitory effects of eugenol and thymol on Penicillium citrinum strains in culture media and cheese. Intl. J. Food Microbiol. 67: Spliltoir, C.F. and Olive, L.S A reclassification of the genus Pericystis. Mycologia 47: Soliman, K.M. and Badeaa, R.I Effect of oil extracted from some medicinal plants on different mycotoxigenic fungi. Food and Chem. Toxicol. 40:

5 Tables Table 1. List of test plant species, their common names and medicinal properties. Scientific name Acorus calamus Allium sativum Common name (English and Thai) Sweet flag Wan-Nam Garlic Kra-Team Parts used Rhizome Medicinal use* Alleviate stomach acidity Clove Inhibit formation of cholesterol, suppress blood clotting, ease asthma, prevent heart attacks and strokes, and inhibit the growth of cancerous tumors Leaf Leprocy, eye problems, and skin ulcer Azadirachta siamensis Neem Sa-Dao Cinnamomum cassia Cinnamon Bark Stomach upset, relieve flatulence and Ob-Cheuy bloating, and restore appetite Curcuma longa Tumeric Rhizome Anti-gastric ulcers, anti inflammatory, Ka-Min dyspepsia, antipruitic and cholagogic properties Eugenia caryophyllum Clove Flower buds Antiseptic and local anesthetic Kan-Plu properties used for curing toothache Illicium verum Star anise Fruit Soothing, stimulating or carminative (relieving gas) Piper betel Betel piper Leaf Use as mouth freshener, help digestive Plu tract in functioning, and relieve pain Rhinacanthus nasutus - Leaf Thong-Pan-Chung Good for colds, relieve fevers, refresh the lungs, relieve early stage of tuberculosis, headache from hypertention, reduce blood pressure, sore throat, and constipation Stemona tuberosa - Root Lung tuberculosis, chronic bronchitis, Nohn-Tai-Yak and chronic dry cough * These plants had been used in local folk medicinal remedies in different forms of various afflictions. However, no mention of their antimicrobial use has been reported in systematic manner. Table 2. Antifungal activity of aqueous extract of plant species. Scientific name Allium sativum Eugenia caryophyllum Piper betel Curcuma longa Illicium verum Cinnamomum cassia Rhinacanthus nasutus Azadirachta siamensis Acorus calamus Stemona tuberosa Common name Garlic Clove Betel pepper Turmeric Star anise Cinnamon - Neem tree Sweet flag - Fungal growth inhibition Hex. Dic. Eth. But. Alc. Met. Wat Hex, Hexanes; Dic, Dichloromethane; Eth, Ethylacetate; But, Buthanol; Alc, Ethanol; Met, Methanol; Wat, Water = cm, ++++ = cm, +++ = cm, ++ = cm and, - = > 1 cm (Measurement was done by the growth of fungus incubated for three days after laying the impregnated paper disks with tested substances) 187

6 Table 3. MIC values of some plant species which exhibited high antimycotic activities on A. apis MR. Plant species Solvent used Minimal Inhibition Concentration* (% (w/v)) Cinnamomum cassia hexane 1.5 Cinnamomum cassia dichlormethane 2 Cinnamomum cassia ethylacetate 10 Piper betel ethylacetate 3.5 Piper betel hexane 7.5 Piper betel ethyl alcohol 7.5 Piper betel dichloromethane 7.5 Eugenia caryophyllum hexane 7.5 Illicium verum dichloromethane 10 * Other plant extracts, which had MIC values more than 10% (w/v), are not shown here. Table 4. Concentrations of hexane Cinnamomum cassia extracts and percentage of inhibition when tested with Ascoshaera apis MR on potato dextrose agar. Concentration of crude extract (w/v) (Control) Diameter of mycelium (cm) Average growth inhibition (%) Table 5 Concentrations of ethyl acetate Piper betel extract and percentage of inhibition when tested with Ascoshaera apis MR on potato dextrose agar. Concentration of crude extract (w/v) (Control) Diameter of mycelium (cm) Average growth inhibition (%)

7 Figures Fig. 1. Ascosphaera apis MR (left, fruiting body or spore cyst; right, spore balls released from a spore cyst) under SEM. 189