Diversity and phylogeny of the bacterial strains isolated from nodules of fenugreek (Trigonella foenum-graecum L.) in Iran

The fenugreek is used in many parts of the world due to its nutritional and therapeutical properties. It is nodulated in Iran by strains of Ensifer in the symbiovar meliloti that are phylogenetically related to Ensifer meliloti and E. kummerowiae. The diversity of fenugreek (Trigonella foenum-graecum L.) microsymbionts has been barely studied even though it is of great interest for being a spice and a medicinal plant. Here, we analyzed 59 bacterial strains isolated from fenugreek nodules originating from different geographic and climatic areas of Iran. Most of these strains exhibit phenotypic characteristics compatible with rhizobia and they nodulate fenugreek. Analysis of the recA and atpD genes shows that representative strains of ERIC-BOX-PCR groups cluster with the type strains of Ensifer meliloti and E. kummerowiae as well as with strains capable of nodulating different Trigonella species found in other countries. The closeness of E. meliloti and E. kummerowiae suggests there is a need to revise the taxonomic status of the latter species. The nodC gene analysis shows that most Trigonella-nodulating strains belong to the symbiovar meliloti except those nodulating Trigonella arcuata in China, which belong to the symbiovar rigiduloides. This analysis shows that the type strains of E. kummerowiae, E. meliloti, and E. medicae belonged to three well-defined groups within the symbiovar meliloti, with the Iranian strains belonging to the E. kummerowiae subgroup. The small group of strains unable to nodulate fenugreek isolated in this study belong to Enterobacter cloacae, reported for the first time as being a possible endophyte of fenugreek nodules.