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Truffles are generally known to form a mycorrhizal relationship with plants. Kalaharituber pfeilii (Hennings) Trappe & Kagan-Zur is a species of desert truffle that is found in the southern part of Africa. The life cycle of this truffle has not been fully investigated as there are many unconfirmed plant species that have been suggested as potential hosts. Many mycorrhizal associations often involve other role players such as associated bacteria that may influence the establishment of the mycorrhizal formation and function. As part of an effort to understand the life cycle of K. pfeilii, laboratory experiments were conducted to investigate the role of ascocarp associated bacteria. Bacterial isolates obtained from the truffle ascocarps were subjected to microbiological and biochemical tests to determine their potentials as mycorrhizal helper bacteria. Tests conducted included stimulation of mycelia] growth in vitro, indole acetic add (IAA) production and phosphate solubilising. A total of 17 bacterial strains belonging to the Proteobacteria, Firmicutes and Actinobacteria were isolated from the truffle ascocarps and identified with sequence homology and phylogenetic methods. Three of these isolates showed potential to be helper bacteria in at least one of the media tested through the stimulation of mycelial growth. Furthermore, four isolates produced IAA and one was able to solubilise CaHPO3 in vitro. One isolate, identified as a relative of Paenibacillus sp. stimulated mycelial growth on all the media tested. Other bacterial isolates that showed potential stimulation of mycelia] growth were identified molecularly as a Bacillus sp. and two strains of Rhizobium sp. This study has contributed to the existing knowledge on the biotic interactions with K. pfeilii which may be useful in further symbiont and re-synthesis investigations. (C) 2013 SAAB. Published by Elsevier B.V. All rights reserved.