INHIBITORY ACTIVITY OF CERTAIN NATURAL PRODUCTS ON THE GROWTH OF ASCOSPHAERA APIS النشاط التثبيطى لبعض المنتجات الطبيعية على نمو فطر اسکوسفيرا آبيس

INHIBITORY ACTIVITY OF CERTAIN NATURAL PRODUCTS ON THE GROWTH OF ASCOSPHAERA APIS

Abou El-Enain, H.T.^*; M.F.Abdel-Rahman^* and K.A.M. Abo-Elyousr ^**

*Beekeeping Research Department, Plant Protection Research Institute (PPRI),
 Agricultural Research Center, Dokki, Giza, Egypt,

**Plant Pathology Dept. Faculty of Agriculture, Assiut University

Corresponded author E-mail: m_fathalla70@yahoo.com

INTRODUCTION:

                Ascosphaera apis (Olive and Spiltoir) is a fungal pathogen causing chalkbrood disease in honeybee, Apis mellifera L., larvae. It is common in most beekeeping countries (Bailey and Ball, 1991). This disease rarely kills colonies but results in persistent loss of brood, which weakens colonies, leading to a reduction in honey surplus.

                Several histological studies have been made on larvae infected with chalkbrood (Carrera et al., 1987; Bamford and Heath, 1982 and Puerta et al., 1994). Nevertheless, there is still controversy about the route of invasion of the fungus into larvae.

Infection seems to be initiated by ascospores (Heath, 1982), although some authors suggest that infection is directly produced by invading hypha (Gilliam et al., 1978). Larvae can ingest the fungus at an early stage, but only stretched larvae, inside capped cells, present symptoms of the disease. Gilliam (1978) and Gilliam et al. (1978) demonstrated that eggs and pupae are not susceptible to laboratory infection.

                For the control of bee pests and diseases chemicals used, which must be constrained and legally recommended otherwise bee products will be contaminated enough to be dangerous for humans (Delaplane, 1997). Currently, there are no products available for the management of chalkbrood, despite reports that the disease has become more prevalent recently (Gilliam and Vandenburg, 1990). Possibly the increasing interest in the use of alternative therapies in the result of the development of antibiotic resistance in some microorganisms becoming a major problem. Some of these alternative therapies are essential oils; propolis and honey. Several studies have shown essential oils to be effective in controlling bee diseases such as chalkbrood (Higes et al., 1998).

                Essential oils are the result of a vapour hydrodistillation plant species, which are thus separated because of being immiscible in water. They are complex mixtures in whose composition there are mainly terpenic compounds, and phenols, which are being continuously studied, e.g., as natural biocide agents (Pedro et al., 2006). Colin et al., 1989 using in vitro tests, demonstrated fungicidal activity of essential oils of Thymus vulgaris; Satureja Montana and Origanum vulgare against chalkbrood. In similar work, Davis and Ward, 2003 studied the antifungal efficacy of over 50 natural products and they found that, a number of essential oils were particularly efficacious at controlling, in vitro, the growth of Ascosphaera apis.

Honey is the natural sweet substance produced by honeybees from the nectar of plants. Honeybee products and some of its therapeutic values were mentioned in the Holy Quran. The ancient Egyptians used honey in combination with other herbs and on its own, to treat wounds and diseases of the gut (Zumla and Lulata, 1989).

                Propolis or “bee glue” is a well-known substance that beekeepers find in their hives. It is one of the natural materials being used in human medicine and veterinary (Caillas, 1978) with a large spectrum of biological action.

Several authors have reported on the antimicrobial activity of propolis on fungi (Lindenfelser, 1967; Brumfit et al., 1990 and Tosi et al., 1996). Pepeljnjak et al., (1982) found that, for pure propolis extracts, a concentration of 15-30 mg/ml was needed to inhibit the growth of Candida albicans; Aspergillus flavus; A. ochraceus; Penicillium viridicatum and P. notatum.

Obaseiki-Ebor et al., (1983) found that, at 0.5% v/v of distillated honey was fungicidal to Candida albicans and fungistatic to Penicillium spp. and Aspergillus niger. Radwan et al., (1984) stated that, growth of colonies from 30-60% of the fungi from sewage; soil; air; and trap water was found to be prevented by 25% honey.

The aim of the present work was to conduct a laboratory study on the efficacy of some natural products (e.g. propolis; honey and essential oils) against the chalkbrood fungus, Ascosphaera apis.

MATERIALS AND METHODS:

The present work was carried out in Plant Pathology Dept., Faculty of Agriculture, Assiut University.

1- Isolations and identification of the causal pathogen:

Larvae mummies were collected from different localities of Assiut Governorate in 2008 season. They were washed with tap water, surface sterilized for three minutes with 2% sodium hypocholorite solution, then rinsed several times in sterilized distilled water and dried between folds of sterilized filter papers. The surface sterilized mummies were plated on to Potato Dextrose Agar (PDA) medium and incubated at 27+1ºC. After 4-5 days incubation period, the developed fungal colonies were purified by hyphal tip and single spore isolation techniques. Identification of the fungal isolates was carried out by using the morphological characteristics of mycelia and spores (Bailey & Ball, 1991), and confirmed by Assiut University Mycological Center. Assiut, Egypt.

2- Tested agents:

                Ethanol propolis extract (EPE) prepared by ten grams of crude propolis, collected from Sahel Seleim, Assiut Governorate, were dissolved in 90 ml ethanol 70% (v/v). The mixture was shaken for 1/2 hour and left at room temperature for 24 h. This procedure was repeated daily for 5 successive days. The extraction was kept in a screw-capped tube and refrigerated until use. Fennel honey extracted from honeybee colonies from apiary at Assiut region. In addition to propolis and fennel honey, eighteen essential oils were obtained from El-Captain Company (Cap Farm) for extracting Natural oils; Herbs and Cosmetics El-Obour City, Cairo, were tested to controlling chalkbrood disease (Table 1).

Table (1): Identification of the tested essential oils

3-Effect of certain natural products on Ascosphaera apis under laboratory conditions:

                Laboratory works were directed to study the effect of some natural products on linear growth of Ascosphaera apis. Natural products essential oils; fennel honey and EPE were added singly to PDA medium at 1000 ppm in order to test their direct effect on the tested fungi. Tested products were filtrate sterilized by Seitz filter before added to the medium. Petri dishes (9 cm in diameter) containing PDA medium were inoculated in the center with disks (5 mm) of the isolate fungi growing 7 days old culture. Four replicates were used for each treatment. Plates containing PDA medium without tested materials were used as control. Plates were incubated at 27+1°C. Linear growth of fungi was measured in mm. when fungal growth filled up control plates. Percentage of reduction in linear growth of the tested oils was determined using the following formula:

R = [C – (T/C)] × 100

Where: R=Percentage of growth reduction,
C=Diameter of the control hyphal growth,
T= Diameter of the treated hyphal growth.

4- Statistical analysis:

Inhibition percentages of A. pis were transformed using arcsin method, then, analysis of variance (ANOVA) was carried out to determine if the treatments differed from control according to the method of Waller and Duncan (Waller and Duncan, 1969).

RESULTS AND DISCUSSION:

Pathogenicity capability of this isolate for Larvae mummies were carried out by spry 200 larvae with 30 ml of spore suspension and the larvae diseases were recorded after 7 days from spry (data not shown).

Data in Table (2), showed that cinnamon; cloves; ginger; thyme, celery; chamomile; garlic; jojoba; pepper, peppermint, fennel, henna, onion, rose, worm-wood oils and propolis caused antifungal activities against Ascosphaera apis. Cinnamon; cloves; rose; thyme oils and propolis, showed the strongest activity against A. apis. They produced inhibition rate 74.44, 71.11, 66.11, 71.44 and 68.11%, respectively.

Essential oils are complex mixture in whose composition, there are mainly terpenic compounds, and phenols, which are being continuously studied, e.g., as natural biocide agents. Oils that contained oxygenated terpenes as major constituents showed the highest inhibition percentage (Pedro et al., 2006). In vitro (Stranks, 1977) reported that, citral and geraniol inhibited the fungus Ascosphaera apis which causes chalkbrood disease in the honeybee, Apis mellifera. Cinnamon oil exhibits activity against mycotoxigenic moulds, Penicillium spp. and Clostridium botulinum. Thymol, a major component of thyme oil, is highly active against Aspergillus parasiticus (Buchanan and Shepherd,1981) and Clostridium botulinum (Karapinar and Aktug, 1987).

Pepeljnjak et al., (1982) found that concentrations of 1500–3000 mg/ml. from pure propolis extract were needed to inhibit the growth of Candida albicans and Aspergillus flavus. The flavonoids in propolis (mainly piocembrin) have been considered to responsible for its inhibitory effect on Candida (Metzner et al., 1979).

Celery; chamomile; garlic; jojoba; pepper and peppermint oils, were exhibited the moderate inhibition against Ascosphaera apis where as the growth reduction to 50.0, 46.78, 48.11, 56.33, 55.89 and 40.78%, respectively. While, the lowest inhibitory reaction against A. Apis 20.33, 25.89, 27.44, 29.67 and 18.11% were recorded in case of the tested materials, fennel, ginger, henna, onion and worm-wood oils. The remaining essential oils and fennel honey were shown to be ineffectual against Ascosphaera apis in vitro test system. Our results are in agreed with several report, Efem et al., (1992) found that, growth inhibition of fungi causing surgical infections or wound contaminations, was complete in the media containing 100% unprocessed honey, partial in media containing 50% and no inhibition was produced by 20% honey.

Molan, 1992a stated that Fungi are generally much more tolerant than bacteria to the high osmotic effect. The same author (1992b) also stated that, no fungi can grow in fully ripened honey, but the more diluted honey becomes, the more species can grow in it.

Our results showed that, cinnamon; cloves; rose; thyme oils and propolis were efficacious in inhibition growth Ascosphaera apis in vitro.

From our results we can conclude that, some of natural products such as, cinnamon; cloves; rose; thyme oils and propolis may be particularly useful against Ascosphaera apis, pathogenic fungus documented to cause chalkbrood disease in honeybee. These materials, however, need to be researched more fully in the mode of actions of tested materials as well as the interaction between such materials and the pathogens before they may be commercially acceptable. We are continuing with this investigation to assess the practical value of the therapeutic application of these products.

Table (2): Antifungal activity of the tested natural products against the growth of  Ascosphaera apis

Means followed by different letters within the same column are significantly different (P < 0.05, ANOVA, LSD).

REFERENCES:

Bailey, L. and B.V. Ball (1991): Honeybee pathology (second edition). Academic Press, London, pp. 53-63, 154-158.

Bamford, S. and L.A.F. Heath (1989): The effects of temperature and pH on the germination of spores of the Chalkbrood fungus, Ascosphaera apis. J. apic. Res., 28: 36-40.

Brumfit, W.; J. M. T. Hamilton and I. Franklin (1990): Antibiotic activity of natural products:1-Propolis. Microbios, 62:19-22.

Buchanan, R. L. and A.J. Shepherd (1981): Inhibition of Aspergillus parasiticus by thymol. Journal of Food Science, 46:976 - 977.

Caillas, A. (1978): Propolis. A remarkable hive product. Propolis Apimondia Puplishing House, Bucharest: 5-8.

Carrera, P.; A. Sommaruga and G. Vailati (1987): The development of Ascosphaera apis within larvae of Apis mellifera ligustica. J.  apic. Res., 26: 59-63.

Colin, M. E.; J. Ducos de Lahitte; E. Larribau E. and T. Boue (1989): Activity of essential oils of Lamiaceae on Ascosphaera apis and treatment of an apiary. Apidologie, 20: 221-228.

Davis, C. and W. Ward (2003): Control of chalkbrood disease with natural products. A report for the Rural Industries Research and Development Corporation, www.rirdc.gov.au .

Delaplane, K. (1997): Practical science-research helping beekeepers: 3-Varroa. Bee World, 78 (4): 155-164.

Efem, S.E.E.; K.T. Udoh and C. I. Iwara (1992): The antimicrobial spectrum of honey and its clinical significance. Infection, 20 (4): 227-229.

Gilliam, M. (1978): Chalkbrood status today and hopes for control. Amer. Bee J., 118: 468-471.

 Gilliam, M. and J.D. Vandenberg (1990): "Fungi" Honeybee Pests, Predators and Diseases. Second edition, R.A. Morse and R. Nowogrodzki, eds. Cornell Univ. Press, Ithaca, NY, pp. 64-90.

Gilliam, M.; S. Taber and J.B. Rose (1978): Chalkbrood disease of honey bees,
Apis mellifera L.: A progress report. Apidologie, 9: 75-89.

Heath, L.A.F. (1982): Development of Chalkbrood in a honeybee colony: A review. Bee World., 63: 119-130.

Higes, P. M.; R. M. Saurez; M. J. Llorente; V. M. J. Paya and M. A. Vincente (1998): The efficiency of essential oil (Saturega montana) in controlling the ascospherosis in honeybee (Apis mellifera) under field conditions. Revista Iberoamericana de Micrologia, 15 (3): 151-154.

Karapinar, M. and S.E. Aktug (1987): Inhibition of foodborne pathogens by thymol, eugenol, menthol and anethone. International Journal of Food Micro-biology, 4: 161-166.

Lindenfelser,L.A. (1967): Antimicrobial activity of propolis. American Bee Journal, 107: 90-92.

Metzner, J.; H. Bekemeier; M. Paintz and E. Shneidewind (1979): On the anti-microbial activity of propolis and propolis constituents. Pharmazie, 34 (2): 97-102.

Molan, P. C. (1992a): The antimicrobial activity of honey: 1-The nature of antibacterial activity. Bee World, 73 (1): 5-28.

Molan, P. C. (1992b): The antimicrobial activity of honey: 2-Variation in the poteny of the antibacterial activity. Bee World, 73 (2): 59-76.

Obaseiki-Ebor, E. E.; T. C. Afonya and A. O. Onyekweli (1983): Preliminary report on the antimicrobial activity of honey distillate. J. Pharm. Pharmacol., 35 (11): 748-749.

Pedro, N.; G. Liesel; G. Alejandro; F. Rosalia; I. P. Marta and E. Martin (2006): Control of Ascosphaera apis and Paenibacillus larvae subsp. Larvae by the use of essential oils for obtaining beehive products without toxic residues. Molecular Medicinal Chemistry, 11: 1– 2.

Pepeljnjak, S.; D. Maysinger and I. Jalsenjak (1982): Effect of propolis extract on some fungi. Scientia Pharmacentica, 50 (2): 165-167.

Puerta, F.; J.M. Flores; M. Bustos; F. Padilla and F. Campano (1994): Chalkbrood development in honeybee brood under controlled conditions Apidologie, 25: 540-546.

Radwan, S. S.; A. A. El-Essawy and M. M. Sarhan (1984): Experimental evidence for the occurrence in honey of
specific substances active against microorganisms. Zentralblatt für Mikrobiologie, 139 (4): 249-255.

Stranks, W. (1977): Essential oils as sapstain fungicides. Bimon. Res. Notes Fish. Environ. Canada, 33:44-55.

Tosi, B.; A. Donini; C. Romagnoli and A. Bruni (1996): Antimicrobial activity of some commercial extracts of propolis prepared with different solvents. Phytotherapy Research 10: 335-336.

Waller, R.A. and D.P. Duncan (1969): A bays rule for symmetric multiple comparison problem. Amer. Stat. Assoc. J. December, 1485-1503.

Zumla, A. A. and A. Lulata (1989): Honey – a remedy rediscovered. Journal of the Royal Society of Medicine, 82: 384-385.