Microbes Environ. Vol. 32, No. 3, 288-292, 2017 https://www.jstage.jst.go.jp/browse/jsme2 doi:10.1264/jsme2.ME17027 Short Communication Isolation of Novel Bacteria Including Rarely Cultivated Phyla, Acidobacteria and Verrucomicrobia, from the Roots of Emergent Plants by Simple Culturing Method Yasuhiro Tanaka 1 *, hiroaki MaTsuzawa 2 , hideYuki TaMaki 3 , Masahiro Tagawa 3 , Tadashi ToYaMa 4 , Yoichi kaMagaTa 3 , and kazuhiro Mori 4 1 Department of Environmental Sciences, Faculty of Life and Environmental Sciences, University of Yamanashi, 4–4–37 Takeda, Kofu, Yamanashi 400–8510, Japan; 2 International Research Centre for River Basin Environment, University of Yamanashi, 4–3–11 Takeda, Kofu, Yamanashi 400–8511, Japan; 3 Bioproduction Research Institute, AIST, 1–1–1 Higashi, Tsukuba, Ibaraki 305–8566, Japan; and 4 Department of Civil and Environmental Engineering, Faculty of Engineering, University of Yamanashi, 4–3–11 Takeda, Kofu, Yamanashi 400–8511, Japan (Received February 16, 2017—Accepted May 31, 2017—Published online July 22, 2017) A number of novel bacteria including members of rarely cultivated phyla, Acidobacteria and Verrucomicrobia, were successfully isolated from the roots of two emergent plants, Iris pseudacorus and Scirpus juncoides, by a simple culturing method. A total of 47.1% (66 strains) for I. pseudacorus and 42.1% (59 strains) for S. juncoides of all isolates (140 strains from each sample) were phylogenetically novel. Furthermore, Acidobacteria and Verrucomicrobia occupied 10.7% (15 strains) and 2.9% (4 strains) of I. pseudacorus isolates, and 2.1% (3 strains) and 3.6% (5 strains) of S. juncoides isolates, respectively, indicating that plant roots are attractive sources for isolating rarely cultivated microbes. Key words: emergent plant, Acidobacteria, Verrucomicrobia, microbial community Molecular ecological techniques based on 16S rRNA gene sequences have revealed that most prokaryotes in nature have yet to be cultured. The number of bacterial phyla was previously presumed to be 100, only 30 of which contain cultivated representatives according to the Greengenes and SILVA databases as of August 2014 (26). Hence, limited information is available on the entity of organisms in more than 70 phyla (candidate bacterial phyla). Furthermore, most of the cultivated microbes are affliated with four bacterial phyla: Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria (8), and another 26 bacterial phyla have a small number of axenic cultures and already reported species. The phyla Acidobacteria and Verrucomicrobia are widely distributed and abundantly detected in natural ecosystems (3–7, 11–13, 15, 16, 19, 25), but harbor only 37 and 43 authentic species, respectively, indicating that these groups mainly comprise rarely cultivated microbes. Many researchers have attempted to cultivate members of Acidobacteria and Verrucomicrobia, and, thus, novel microbes within these phyla were, to some extent, successfully isolated. However, extensive efforts and unique laborious strategies for cultiva- tion are still needed, as follows: (i) in situ cultivation using diffusion chambers (1); and the combined use of (ii) a tradi- tional plate culturing method or (iii) advanced cultivation techniques and screening methods with specifc PCR primers and hybridization probes (10, 17, 18, 21). Since there are very few studies on microbes associated with the roots of aquatic plants, we recently analyzed micro- bial communities in the roots of three aquatic plants: Spirodela polyrhiza (a foating aquatic plant), Phragmites australis (an emergent plant), and Lythrum anceps (an emergent plant), and succeeded in isolating a number of novel microbes including rarely cultivated organisms within the phyla Verrucomicrobia and Armatimonadetes (formerly called candidate phylum OP10) (14, 23). These fndings prompted a tentative theory that aquatic plant roots may be an effective isolation source for a number of yet-to-be cultured microbes. In order to verify this hypothesis, further studies on microbial isolation targeting a greater variety of aquatic plant species are needed. In the present study, we conducted the isolation and phylogenetic characterization of microbes inhabiting the roots of two emergent plants, Iris pseudacorus and Scirpus juncoides, which are taxonomically far from (at the level of order) each other and the three already-targeted aquatic plants described above. We eventually found that root samples of the two aquatic plants were effective sources for isolating varieties of novel microbes representing members of rarely cultured bacterial phyla Acidobacteria and Verrucomicrobia by using a simple culturing method without an extensive effort. Two plants, I. pseudacorus and S. juncoides, were har- vested from a pond in Yamanashi prefectural wood park “Kanegawa-no-mori” (Fuefuki, Yamanashi, Japan; 35°38ʹ23ʺ N, 138°40ʹ36ʺ E). In order to remove free-living microbes in pond water, which are unlikely to interact with the roots of plants, root samples (0.3 g wet weight) were gently rinsed twice with 30 mL of sterilized modifed Hoagland solution (24), and applied to total DNA extraction using the ISOIL kit for bead beating (Nippon gene, Tokyo, Japan). The Ultraclean water DNA kit (MO BIO Laboratories, Carlsbad, CA, USA) was used for DNA extraction from a pond water sample (100 mL) collected near the plants. The PCR amplifcation of 16S rRNA gene fragments was conducted using a previously described method (14) with two bacterial universal primers, Eub8F and Eub1512R, except for the number of cycles; these numbers were adjusted to 19, 20, and 21 cycles for I. * Corresponding author. E-mail: yasuhiro@yamanashi.ac.jp; Tel: +81–55–220–8771; Fax: +81–55–220–8666.