Brazilian Journal of Microbiology 45, 1, 163-173 (2014) Copyright © 2014, Sociedade Brasileira de Microbiologia ISSN 1678-4405 www.sbmicrobiologia.org.br Research Paper A primary assessment of the endophytic bacterial community in a xerophilous moss (Grimmia montana) using molecular method and cultivated isolates Xiao Lei Liu, Su Lin Liu, Min Liu, Bi He Kong, Lei Liu, Yan Hong Li College of Life Science, Capital Normal University, Haidian District, Beijing, China. Submitted: December 27, 2012; Approved: April 1, 2013. Abstract Investigating the endophytic bacterial community in special moss species is fundamental to under- standing the microbial-plant interactions and discovering the bacteria with stresses tolerance. Thus, the community structure of endophytic bacteria in the xerophilous moss Grimmia montana were esti- mated using a 16S rDNA library and traditional cultivation methods. In total, 212 sequences derived from the 16S rDNA library were used to assess the bacterial diversity. Sequence alignment showed that the endophytes were assigned to 54 genera in 4 phyla (Proteobacteria, Firmicutes, Actinobacteria and Cytophaga/Flexibacter/Bacteroids). Of them, the dominant phyla were Proteobacteria (45.9%) and Firmicutes (27.6%), the most abundant genera included Acinetobacter, Aeromonas, Enterobacter, Leclercia, Microvirga, Pseudomonas, Rhizobium, Planococcus, Paenisporosarcina and Planomicrobium. In addition, a total of 14 species belonging to 8 genera in 3 phyla (Proteo- bacteria, Firmicutes, Actinobacteria) were isolated, Curtobacterium, Massilia, Pseudomonas and Sphingomonas were the dominant genera. Although some of the genera isolated were inconsistent with those detected by molecular method, both of two methods proved that many different endophytic bacteria coexist in G. montana. According to the potential functional analyses of these bacteria, some species are known to have possible beneficial effects on hosts, but whether this is the case in G. montana needs to be confirmed. Key words: bacterial diversity, endophytes, moss, molecular method, cultivated isolates. prominent bryologists as “living fossils” (Hornschuh et al., Introduction 2002). Consequently, the opportunity to find new and bene- In plant-endophyte interactions, plants provide nutri- ficial endophytic microorganisms among the diversity of ents and residency for the bacteria, while the bacteria in ex- plants in different ecosystems is considerable. change directly or indirectly improve plant growth and The mosses, one kind of bryophytes, are a diverse health (Mastretta et al., 2006). Once inside the plant, endo- group of land plants that usually colonize habitats with ei- phytes either reside in specific plant tissues such as the root ther moist or extremely variable conditions. One of their cortex or the xylem, or colonize the plant systematically by most important features is their life cycle, which involves transport through the vascular system or the apoplast alteration between a diploid sporophyte and a dominant (Quadt-Hallmann et al., 1997). Of the nearly 300 000 plant free-living haploid gametophyte generation (Opelt and species on earth, each species is host to one or more species Berg, 2004). Mosses are unique host plants for microorgan- of endophytes (Strobel et al., 2004). The complete descrip- isms in numerous ways. For example, the small size of tion of endophytic species has only been enumerated and mosses results in limited availability of the substratum. In characterized for a handful of plant species, and the major- addition, most mosses display an extraordinarily high toler- ity of these are common higher plants. Few studies have ex- ance to extreme desiccation and can resume normal metab- amined the endophytes of bryophytes, which represent the olism very rapidly after rehydration. Hence, successful simplest extant land plants and have been classified by microbial colonization requires adaptation to these special Send correspondence to Y.H. Li. College of Life Science, Capital Normal University, Xisanhuan North Road 105#, Haidian District, 100048 Beijing, China. E-mail: [email protected]. 164 Liu et al. conditions (DoÈbbeler, 1997). Analysis of the epiphytes on entire plants was collected after absorbing enough water, the gametophyte of Funaria hygrometrica detected numer- and then mixed together and immediately transported to the ous bacterial species on the surface of the phylloid. Among laboratory for surface disinfection as described previously these species, two Methylobacterium strains were found to (Li et al., 2010). The plants were first washed many times be able to simulate the well-known effect of cytokinin ap- with tap water to remove attached substratum. Subse- plication on bud formation in Funaria protonema and they quently, they were immersed in 70% ethanol for 3 min, also promoted the growth of protonemal filaments washed with 15% sodium hypochlorite solution for 10 min, (Hornschuh et al., 2002). Endophytic methanotrophic bac- rinsed three times with 70% ethanol for 30 s, and finally teria were also found in the hyaline cells and on the stem washed five times with sterile distilled water. To confirm leaves of Sphagnum mosses; here, they provided carbon for that the disinfection process was successful, aliquots of the photosynthesis via in situ oxidation of methane to carbon sterile distilled water in the final rinse were used to deter- dioxide (Raghoebarsing et al., 2005). mine the results of surface disinfection. Bacteria were culti- Opelt and Berg (2004) isolated and identified many vated by setting 100 mL of the final rinse on R2A and TSA antagonistic bacteria associated with three moss species medium plates, and then examining the plates for bacterial (Tortula ruralis, Aulacomnium palustre and Sphagnum growth after incubation at 28 °C for 3 days. Molecular de- rubellum) in the nutrient-poor habitats of the Baltic Sea tection of bacterial species was accomplished by 16S rRNA Coast in Germany. These species belong to nine different gene PCR detection based on the primers 799f genera, among which Burkholderia, Pseudomonas and (5’-AACAGGATTAGATACCCTG-3’) and 1492r Serratia were dominant, but the richness and diversity of (5’-GGTTACCTTGTTACGACTT-3’) (Chelius and antagonistic species were moss species-dependent, and the Triplett, 2001) using the final rinse as template. The 50 mL highest number of species with antagonistic activity was PCR reaction mixture contained 5 mL of the final rinse, isolated from S. rubellum. Another study examined the 5 mL 10x Taq reaction buffer (including 1.5 mM MgCl ), function and diversity of bacterial species associated with 2 10 pmol of each primer, 200 mM each dNTP, and 1.5 U of two Sphagnum species (S. fallax and S. magellanicum) that Taq DNA polymerase (Takara Co.). After initial denatur- grow in a temperate mire ecosystem. Species belonging to ation at 94 °C for five minutes, each thermal cycling was as the genus Burkholderia were predominant in Sphagnum follows: denaturation at 94 °C for one minute, annealing at species and this genus was possibly involved in antago- 53 °C for one minute, and elongation at 72 °C for one min- nism/pathogen defense and nitrogen-fixation. The authors ute. At the end of 30 cycles, the final extension step was at concluded that Sphagnum is a reservoir for powerful and 72 °C for 15 min. Products of four parallel PCRs were com- extraordinary antagonists and potentially facultative hu- bined and electrophoretically separated by 1% agarose. man pathogens (Opelt et al., 2007). Thus, thorough re- Finally, plant samples were determined to be successfully search on the bacteria associated with other mosses in surface disinfected if no bacterium was identified via culti- different niches would be also useful in discovering bacte- vation and PCR. These plants were used for the subsequent rial resources and helpful in understanding the interactions analyses. between mosses and their associated microbes. Grimmia montana is a xerophilous moss, and has a high tolerance to drought, cold and UV radiation (Yi and DNA extraction and amplification of the bacterial Liu, 2007), and can often be found growing in extreme en- 16S rRNA genes vironments. It always lives under extreme desiccation con- ditions and can resume normal metabolism very rapidly About2gofsurface-disinfected G. montana was after rehydration. In this paper, our aim is to study the di- frozen with liquid nitrogen and ground to a fine powder in a versity and community structure of its endophytes using sterilized and precooled mortar. Next, the cetyltrimethyl- 16S rDNA library and culture-dependent approaches, and ammonium bromide (CTAB) procedure was used to extract hope to make a well known on the interactions between total DNA as previously described (Xie et al., 1999). The endophytes and G. montana and try to find some bacterial DNA was resuspended in 150 mL sterile Milli-Q water. The resources with the strong tolerance to the stresses. primer pair 799f and 1492r was selected to amplify the 16S rDNA of the endophytic bacteria. The PCR reaction mix- Material and Methods ture and programs are the same as described above in the section of surface disinfection. We excised the approxi- Sampling and surface disinfection mately 730 bp band from a 1% agarose gel, following elec- Grimmia montana were sampled from the surface of trophoresis of the DNA, and purified the DNA using the one large stone in Beijing Songshan National Nature Re- Gel Extraction Kit (Omega Co.), as described by the manu- serve located