Polyphasic taxonomic description of Frankia abietis sp. nov., Frankia limosa sp. nov., Frankia palustris sp. nov. and Frankia luti sp. nov. isolated from Alnus spp.

Frankia strain AiPa1T was isolated from root nodules of Alnus incana induced from soil under Picea abies in Finland. Strains AgKG'84/4T, AgB32T and AgB1.8T were isolated from Alnus glutinosa nodules from Germany. AiPa1T, AgB32T and AgKG'84/4T are infective on A. glutinosa and fix nitrogen in symbiotic nodules and pure culture, and strain AgB1.8T is infective on A. glutinosa but does not fix nitrogen and does not have the nif genes. Colonies of the four strains are white, and they form hyphae and sporangia. N2-fixing vesicles are formed by AiPa1T, AgB32T and AgKG'84/4T in NH4+-free medium but not by AgB1.8T. Comparative 16S rRNA gene sequence analyses showed AiPa1T, AgB32T and AgKG'84/4T were in cluster 1 (Fig. S1), while strain AgB1.8T was in cluster 4. A whole-genome phylogeny confirmed this positioning. Digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values between the four strains and all validly named Frankia species were below the defined threshold for prokaryotic species demarcation, with dDDH and ANI values at or below 64 and 71%, respectively. Strains AiPa1T, AgB32T and AgKG'84/4T have genome sizes of 7.00, 6.40 and 6.71 Mb, respectively, while the genome size of AgB1.8T is 9.82 Mb. Phenotypic, physiological and phylogenomic analyses confirmed the assignment of strain AiPa1T (=DSM 114742T=LMG 32596T) to a novel species, for which the name Frankia abietis sp. nov. is proposed. Strain AgKG'84/4T (=DSM 114736T=LMG 32597T) is assigned to a novel species, for which the name Frankia limosa sp. nov. is proposed. Strain AgB32T (=DSM 114735T=LMG 32598T) is assigned to a novel species, with AgB32T as the type strain, for which the name Frankia palustris sp. nov. is proposed. Strain AgB1.8T (=DSM 114738T=LMG 33088T) is assigned to a novel species, for which the name Frankia luti sp. nov. is proposed.