The diversity of Rhizobia , Sinorhizobia and novel non-Rhizobial Paenibacillus nodulating wild herbaceous legumes
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Original Paper
The diversity of Rhizobia, Sinorhizobia and novel non‑Rhizobial Paenibacillus nodulating wild herbaceous legumes Sadia Latif · SamiUllah Khan · Muhammad Naveed · Ghulam Mustafa · Tasmia Bashir · Abdul Samad Mumtaz
Received: 1 November 2012 / Revised: 27 March 2013 / Accepted: 17 July 2013 © Springer-Verlag Berlin Heidelberg 2013
Abstract The objective of the present study was to isolate and characterize nodulating bacteria associated with wild legumes. For this purpose, we recovered twenty isolates from root nodules of five wild legume species: Melilotus alles, Melilotus officinalis, Trifolium pratense, Trifolium repens and Medicago sp. Most of the isolates were morphologically analogous with only few exceptions in colony shape, appearance and incubation time. All isolates were Gram negative except T.P2-4. Random amplification of polymorphic DNA showed genetic variation among isolates. The 16S rRNA sequence analysis revealed these isolates as Rhizobium, Sinorhizobium and Paenibacillus. Each of these was also screened for nod D and nod F genes with marked variation at these loci; however, the nucleotide sequence analysis confirmed the presence of nod genes. The assignment of strains to their hosts revealed a unique symbiotic association of Paenibacillus sp. nodulating T .pratense which is being reported here for the first time. Keywords Proteobacteria · Symbiosis · Nod D · Nod F · 16S rRNA · Trifolii
Introduction The ability to fix atmospheric nitrogen is restricted only to the root nodulating microbes associated with legumes called Rhizobia. The nitrogen so fixed and available to plants, meets approximately half of the total nitrogen
S. Latif · S. Khan · M. Naveed · G. Mustafa · T. Bashir · A. S. Mumtaz (*) Department of Plant Sciences, Quaid-i-Azam University, Islamabad 45320, Pakistan e-mail: [email protected]; [email protected]
required to agriculture (Handley et al. 1998). Rhizobia are rod-shaped, Gram-negative soil bacteria that exhibit the capability to infect plant roots, establish a symbiotic relationship and fix nitrogen. The symbiosis is manifested through the formation of nodules, where atmospheric nitrogen is converted into ammonia for plants to assimilate (Riely et al. 2004). Rhizobia are extensively associated with crops and wild legumes alike. As these being described, their classification is progressing too. Hitherto, forty species and seven genera have been described in α-subclass of proteobacteria, which included Allorhizobium, Azorhizobium, Bradyrhizobium, Mesorhizobium, Rhizobium, Sinorhizobium (Wei et al. 2002) and a species in the genus Methylobacterium (Sy et al. 2001). The species within the β-subclass of proteobacteria have been defined by two genera: Burkholderia and Cupriavidus (Wei et al. 2002). Similarly, members of γ-proteobacteria have also been found to be involved in nodulation (Benhizia et al. 2004). Bacteria are found as structures called bacteriods, which are involved in nitrogen fixation (Stacey et al. 2006). Interestingly, all members of the family F
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