In vitro cultivation of arbuscular mycorrhizal fungi (AMF) is challenging due to their biotrophic symbiosis. The principal aim of this study was to demonstrate the effect of establishing in vitro dual cultures of arbuscular mycorrhizal fungi (AMF) inoculated on Swietenia macrophylla (mahogany) roots on plant growth. Furthermore, it was sought to demonstrate that plant colonization by Glomeromycota can be achieved with a replicable protocol. This study established monoxenic cultures of carrot (Daucus carota) Ri T-DNA ROC inoculated with Glomus sp. on two-compartment plates. At 75 days, hyphal growth reached 223.93 mm in the root compartment and 103.71 mm in the hyphal compartment. Spores produced in vitro measured 26.14 ± 1.70 µm, smaller than ex vitro spores (101.2 ± 4.22 µm). Rhodotorula mucilaginosa was isolated from cultures and appeared to stimulate hyphal growth and root–fungal contact. From these cultures, a dual culture of mahogany inoculated with Glomus sp. was established. No significant differences were observed between inoculated and non-inoculated plants in stem length, root length, root number, or leaf number at 30 days. Spore production ranged from 10,166 to 27,696 per plate, averaging 14,795 ± 3301, with hyphal lengths of 3655.46 ± 308.75 mm. Hyphal development included running and branching patterns, with solitary and clustered spores. Spore diameter averaged 27.68 ± 3.85 µm. Arbuscular colonization reached 41.49% at 30 days and 52.13% at 75 days, exceeding rates reported for other culture systems. Monoxenic cultures are a reliable, aseptic source of high-quality inoculum, supporting biofertilizer production and biotechnological applications. These methods provide valuable tools for studies involving AMF, such as those demonstrated with mahogany.

Four rhizobial strains isolated from root nodules of tropical leguminous plants in Costa Rica were characterized. Strain LEGMi-135b was isolated from Lonchocarpus felipei root nodules, LEGMi-12c from Cojoba arborea , LEGMi-198b from Calliandra haematocephala and Rhizobium altiplani LEGMi-166a from Mimosa pigra . Phylogenetic analyses based on the 16S rRNA gene and four concatenated housekeeping genes ( recA, atpD, GATase and gyrB ) placed these strains within the genera Martinezella (LEGMi-12c, LEGMi-135b, and LEGMi-198b) and Rhizobium (LEGMi-166a). Genome-based analyses showed that strain LEGMi-135b exhibited an average nucleotide identity (ANI) value of 94% and a digital DNA-DNA hybridization (dDDH) value of 53.2% compared with its closest related species, Martinezella hainanensis CCBAU 57015 ^T , supporting its classification as a distinct species. LEGMI-12c exhibited ANI and dDDH values of 98% and 87%, respectively, compared with Martinezella hainanensis CCBAU 57015 ^T . LEGMi-198b showed the highest similarity to Rhizobium cerradonense CNPSo 3464, with ANI and dDDH values of 98% and 81.4%, respectively; however, this taxon has not been validly published. LEGMi-166a exhibited ANI and dDDH values of 100% and 99.3%, respectively, compared with Rhizobium altiplani BR 10423ᵀ, confirming its assignment to this species. Additionally, differentiation of strains LEGMi-135b and LEGMi-198b from their closest phylogenetic relatives was supported by phenotypic, physiological, and biochemical analyses. Based on the combined genomic, phenotypic, and biochemical evidence, we propose the establishment of the novel rhizobial species Martinezella legmii sp. nov., with strain LEGMi-135b designated as the type strain (LEGMi-135bᵀ), and Martinezella ivanii sp. nov., with strain LEGMi-198b designated as the type strain (LEGMi-198bᵀ). This study expands our understanding of rhizobial diversity associated with nitrogen-fixing symbioses, particularly among tropical leguminous plants from Costa Rica. Data summary: The raw reads of the three Martinezella spp. and one Rhizobium sp. isolates have been deposited in GenBank under BioProject PRJNA1084876 and SRA accession SRP493729. The corresponding BioSample and genome accessions are: Martinezella sp. LEGMi-12c (SAMN44800730; GCF_054114145.1), LEGMi-135b (SAMN44800728; GCF_054114165.1), Rhizobium sp. LEGMi-166a (SAMN46966679; GCF_054114185.1), and Martinezella sp. LEGMi-198b (SAMN44800729; GCF_054114205.1). The 16S rRNA sequences are available under accessions PP439182 , PP439210 , PP439229 , and PP439245 , respectively.