Chalkbrood is one of the most dangerous disease of the honeybees, Apis mellifera. It causes a significant reduction in brood and honey production thus, leading to an economic loss in apiculture. In present study, the pathogenic fungus was isolated from infected larvae in Northern Governorate- Palestine and identified based on morphological and cultural characters as Ascosphaera apis. Crude aqueous extracts of ten different plant species including Punica granatum, Artemisia monosperma, A. absinthium, A. herba-alba, Cyperus rotundus, Callistemon viminalis, Cinnamomum zeylanicum, Tagetes patula, Annona squamosa and Psidium guajava were evaluated in vitro for their antifungal activities against A. apis. Results showed that extract of P. granatum was the most active one among all. Other extracts vis., P. guajava, C. zeylanicum, and C. viminalis demonstrated a moderate activity with MICs of 1.25, 2.5 and 5% respectively. The remaining extracts of A. monosperma, A. absinthium, A. herba-alba, T. patula, C. rotundus, and A. squamosa were totally inactive against A. apis. For further evaluation of the antifungal properties of the different plant extracts, the radial mycelial growth inhibition test was also performed on agar media supplemented by plant extracts to achieve different concentrations of 5%, 10% and 20%. The results showed that the average radial mycelia growth of A. apis was significantly reduced by P. granatum, P. guajava, C. viminalis and C. zeylanicum (P < 0.05). Contrary to this, The remaining extracts of A. monosperma, A. absinthium, A. herba-alba, C. rotundus, T. patula and, A. squamosa were found to have weak antifungal activities at all concentrations tested. In order to assess the antifungal effect of the P. granatum extract on A. apis, the growth of profile for the fungus was followed at extract free agar media after incubation at different concentrations of P. granatum extract. The results found that the extract of P. granatum flower exhibits a fungistatic effect because it causes changes on the normal growth profile of A. apis at the different concentrations tested (5%, 10% and 20%). Since the extract of P. granatum flower was proved to be the most effective in vitro against A. apis, its toxicity to worker bees was evaluated. The results of the toxicity test showed that, the flower extract of P. granatum was not lethal to adult workers at low concentrations tested since the a cumulative mortality percentages were 4.4%, 1.1 % and 3.6% at 0% 12.5% and 25% concentrations respectively. In conclusion, results from these findings suggest that the aqueous extract of P. granatum flower may be used as natural antifungal agents to inhibit growth of A. apis. These findings however need to be progressed to field applications to evaluate the efficacy of the most active antifungal plant extract identified in this study against the causative agent of chalkbrood disease in an apiary system
