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Erythrina brucei is a leguminous tree commonly used in agro-forestry practices in southern and southwestern Ethiopia. It has desirable agro-forestry attributes that make it a good candidate for agro-forestry systems. However, there is a scarcity of information regarding species diversity and spore density of arbuscular mycorrhizal fungi (AMF) associated with E. brucei growing in different land use types. Therefore, this study was initiated to determine species diversity and richness in trap cultures of AMF associated with E. brucei growing in different geographic locations in various land use types of Ethiopia. Soil samples were collected from various E. brucei growing areas with different land use patterns. Trap culture was established using a portion of each soil sample by planting E. brucei as the host plant. AMF spore extraction was carried out in field and trap culture soils, and AMF species identification was done using spore morphology. Erythrina brucei root colonization with AMF structures was studied using trap culture, and AMF spore density in both field and trap culture soil samples was determined. A total of 11 AMF genera and 33 AMF species were recovered. The highest number of genera was recovered from forest land use type at Rebu Gebeya, followed by Adiskdam, live fence land use type. Glomus and Acaulospora were the dominant AMF genera covering 73% of the samples and contained 10 and 9 AMF species, respectively. All the soil samples contained indigenous AMF spore populations regardless of their origin and land use types. The highest mean AMF spore density in a field soil sample was recorded at a farmland in Bodite (280 spores 100 g−1 of dry soil), while the lowest was from Hossana (172 spores 100 g−1of dry soil). However, the highest mean AMF spore density in trap culture was detected from a farm land at Humbo (715 spores 100 g−1 of dry soil) and the lowest from Dhakallo (217 spores 100 g−1 of dry soil). Direct extraction of AMF from field soil samples showed lesser AMF spore density compared with trap culture in the same soil sample. All E. brucei root segments were colonized with the AMF structures, and the colonization status ranged from 34.4% to 49.1%. Both geographic location and land use type significantly affected AMF species richness (p |