A primary assessment of the endophytic bacterial community in a xerophilous moss (Grimmia montana) using molecular method and cultivated isolates

SHORT COMMUNICATION

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

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ABSTRACT

Investigating the endophytic bacterial community in special moss species is fundamental to understanding 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 estimated 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 (Proteobacteria, 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.

Introduction

In plant-endophyte interactions, plants provide nutrients and residency for the bacteria, while the bacteria in exchange directly or indirectly improve plant growth and health (Mastretta et al., 2006). Once inside the plant, endophytes either reside in specific plant tissues such as the root cortex or the xylem, or colonize the plant systematically by transport through the vascular system or the apoplast (Quadt-Hallmann et al., 1997). Of the nearly 300 000 plant species on earth, each species is host to one or more species of endophytes (Strobel et al., 2004). The complete description of endophytic species has only been enumerated and characterized for a handful of plant species, and the majority of these are common higher plants. Few studies have examined the endophytes of bryophytes, which represent the simplest extant land plants and have been classified by prominent bryologists as "living fossils" (Hornschuh et al., 2002). Consequently, the opportunity to find new and beneficial endophytic microorganisms among the diversity of plants in different ecosystems is considerable.

The mosses, one kind of bryophytes, are a diverse group of land plants that usually colonize habitats with either moist or extremely variable conditions. One of their most important features is their life cycle, which involves alteration between a diploid sporophyte and a dominant free-living haploid gametophyte generation (Opelt and Berg, 2004). Mosses are unique host plants for microorganisms in numerous ways. For example, the small size of mosses results in limited availability of the substratum. In addition, most mosses display an extraordinarily high tolerance to extreme desiccation and can resume normal metabolism very rapidly after rehydration. Hence, successful microbial colonization requires adaptation to these special conditions (DoÈbbeler, 1997). Analysis of the epiphytes on the gametophyte of Funaria hygrometrica detected numerous bacterial species on the surface of the phylloid. Among these species, two Methylobacterium strains were found to be able to simulate the well-known effect of cytokinin application on bud formation in Funaria protonema and they also promoted the growth of protonemal filaments (Hornschuh et al., 2002). Endophytic methanotrophic bacteria were also found in the hyaline cells and on the stem leaves of Sphagnum mosses; here, they provided carbon for photosynthesis via in situ oxidation of methane to carbon dioxide (Raghoebarsing et al., 2005).

Opelt and Berg (2004) isolated and identified many antagonistic bacteria associated with three moss species (Tortula ruralis, Aulacomnium palustre and Sphagnum rubellum) in the nutrient-poor habitats of the Baltic Sea Coast in Germany. These species belong to nine different genera, among which Burkholderia, Pseudomonas and Serratia were dominant, but the richness and diversity of antagonistic species were moss species-dependent, and the highest number of species with antagonistic activity was isolated from S. rubellum. Another study examined the function and diversity of bacterial species associated with two Sphagnum species (S. fallax and S. magellanicum) that grow in a temperate mire ecosystem. Species belonging to the genus Burkholderia were predominant in Sphagnum species and this genus was possibly involved in antagonism/pathogen defense and nitrogen-fixation. The authors concluded that Sphagnum is a reservoir for powerful and extraordinary antagonists and potentially facultative human pathogens (Opelt et al., 2007). Thus, thorough research on the bacteria associated with other mosses in different niches would be also useful in discovering bacterial resources and helpful in understanding the interactions 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 Liu, 2007), and can often be found growing in extreme environments. It always lives under extreme desiccation conditions and can resume normal metabolism very rapidly after rehydration. In this paper, our aim is to study the diversity and community structure of its endophytes using 16S rDNA library and culture-dependent approaches, and hope to make a well known on the interactions between endophytes and G. montana and try to find some bacterial resources with the strong tolerance to the stresses.

Material and Methods

Sampling and surface disinfection

Grimmia montana were sampled from the surface of one large stone in Beijing Songshan National Nature Reserve located at an altitude of 890 m, at N: 40º31'00.45" by E:115º49'33.20" on the 19^th of April, 2011. About 3 g of plant material, approximately more than one thousand of entire plants was collected after absorbing enough water, and then mixed together and immediately transported to the laboratory for surface disinfection as described previously (Li et al., 2010). The plants were first washed many times with tap water to remove attached substratum. Subsequently, they were immersed in 70% ethanol for 3 min, washed with 15% sodium hypochlorite solution for 10 min, rinsed three times with 70% ethanol for 30 s, and finally washed five times with sterile distilled water. To confirm that the disinfection process was successful, aliquots of the sterile distilled water in the final rinse were used to determine the results of surface disinfection. Bacteria were cultivated by setting 100 µL of the final rinse on R2A and TSA medium plates, and then examining the plates for bacterial growth after incubation at 28 ºC for 3 days. Molecular detection of bacterial species was accomplished by 16S rRNA gene PCR detection based on the primers 799f (5'-AACAGGATTAGATACCCTG-3') and 1492r (5'-GGTTACCTTGTTACGACTT-3') (Chelius and Triplett, 2001) using the final rinse as template. The 50 µL PCR reaction mixture contained 5 µL of the final rinse, 5 µL 10x Taq reaction buffer (including 1.5 mM MgCl₂), 10 pmol of each primer, 200 /M each dNTP, and 1.5 U of Taq DNA polymerase (Takara Co.). After initial denaturation at 94 ºC for five minutes, each thermal cycling was as follows: denaturation at 94 ºC for one minute, annealing at 53 ºC for one minute, and elongation at 72 ºC for one minute. At the end of 30 cycles, the final extension step was at 72 ºC for 15 min. Products of four parallel PCRs were combined and electrophoretically separated by 1% agarose. Finally, plant samples were determined to be successfully surface disinfected if no bacterium was identified via cultivation and PCR. These plants were used for the subsequent analyses.

DNA extraction and amplification of the bacterial 16S rRNA genes

About2gof surface-disinfected G. montana was frozen with liquid nitrogen and ground to a fine powder in a sterilized and precooled mortar. Next, the cetyltrimethylammonium bromide (CTAB) procedure was used to extract total DNA as previously described (Xie et al., 1999). The DNA was resuspended in 150 µL sterile Milli-Q water. The primer pair 799f and 1492r was selected to amplify the 16S rDNA of the endophytic bacteria. The PCR reaction mixture and programs are the same as described above in the section of surface disinfection. We excised the approximately 730 bp band from a 1% agarose gel, following electrophoresis of the DNA, and purified the DNA using the Gel Extraction Kit (Omega Co.), as described by the manufacturer.

Construction of the 16S rDNA clone library

The purified 730 bp PCR products were ligated into the pMD18-T vector (Takara Co.). Escherichia coli Top10 competent cells (Tiangen Co.) were transformed with the ligation products and spread onto Luria-Bertani agar plates with ampicillin (100 mg L^-1) for standard blue and white screening (Sambrook et al., 1989). Randomly selected colonies were screened directly for inserts by performing colony PCR with primers RV-M (5'-GAGCGGATAACAATTTCACACAGG-3') and M13-47 (5'-CGCCAGGGTTTTCCCAGTCACGAC-3') for the vector (Takara Co.). Two hundred fifty clones containing inserts of the correct size were sequenced using an ABI PRISM 3730 automatic sequencer (Shanghai Sangon Co., Ltd).

Phylogenetic analysis

After being trimmed by cutting the vector sequences using the Editseq program in the DNAStar package (Burland, 2000) and removing all the bad sequences as determined by the chimera sequence detection software Mallard 1.02 (www.cardiff.ac.uk/biosi/research/biosoft), all other manually verified nucleotide sequences were submitted to the NCBI GenBank database. Clones of 16S rRNA gene sequences showing 97% similarity or higher were considered to belong to the same phylotype by sequencher 4.8 (Gene Codes, Ann Arbor, MI) and assigned to an Operational Taxonomic Unit (OTU). Sequences of all phylotypes were compared to the NCBI database using BlastN or aligned by the identify analysis of EzTaxon-e (Kim et al., 2012). Clones with a 16S rDNA sequence similarity larger than 97% were assigned to the same species; those with > 95% identity were assigned to the same genus; those with < 95% were determined to be uncultured bacterial species. Next, those sequences assigned to uncultured bacteria were aligned using Clustal W (Thompson et al., 1994), and tree constructions were done with the MEGA 5 program package (Tamura et al., 2011) using the neighbor-joining method (Saitou and Nei, 1987) to infer their classification. Bootstrap analysis was performed with 1,000 replicates.

Estimation of the size of the clone library

To estimate the representation of the library, the clone coverage was calculated with the following equation based on the sequencing results: C= (1-n1/N) x 100%, where n1 represents the number of phylotypes occurring only once and N is the number of clones being examined. Diversity of the clone library was investigated using rarefaction analysis. Rarefaction curve was calculated using the Ecosim 7.0 software (Gotelli and Entsminger, 2004).

Isolation of culturable endophytes and determination of CFU

To isolate the endophytes from the plants, 1 mL of sterile 0.85% NaCl was added to 0.5 g (fresh weight) of surface disinfected G. montana and samples were homogenized in a small sterile mortar. The resultant mixture was serially diluted with sterile 0.85% NaCl and plated onto R2A and TSA media (Difco, Detroit, MI). Plates were incubated for 3 days at 28 ºC, after which Colony-Forming Units (CFU) were counted to calculate the average number of colonies per gram of moss. Isolates obtained by plating were purified and stored at -70 ºC in sterile broth containing 40% glycerol.

ARDRA analysis and identification of the isolates by sequencing

1 uL of the bacterial suspension derived from each isolate was used to amplify the 16S rDNA fragments using the primers 27f and 1492r. The PCR reaction mixture and programs are the same as described above in the section on surface disinfection. The approximately 1490 bp band was excised from a 0.8% agarose gel, and purified using the Gel Extraction Kit (Omega Co.) as described by the manufacturer. Next, the purified products were enzymatically digested with Hae III and Hha I at 37 ºC for 4 h, respectively. According to their electrophoresis pattern on a 1.0% agarose gel, these isolates were classified into different OTUs. Finally, the PCR products of isolates with different OTUs were sequenced using an ABI PRISM 3730 automatic sequencer (Shanghai Sangon Co., Ltd). After trimming the low quality nucleotides, the sequence similarities were calculated using the EzTaxon-e (Kim et al., 2012).

Results

16S rDNA library analysis of endophytic bacterial community

Bacterial 16S rDNA fragments were amplified from total DNA that was extracted from surface disinfected G. montana, using the primers 799f and 1492r. The amplified DNA displayed only one distinct and one weak band, of approximately 730 bp and 1000 bp, respectively. The sequencing result showed that the 730 bp band represented the bacterial 16S rRNA fragment, while the 1000 bp fragment was mainly derived from the mitochondria of the mosses. Thus, the purified 730 bp PCR products were used to construct a 16S rDNA clone library for the endophytic bacteria.

Of 250 clones, two-hundred and twelve individual sequences were verified. They were determined as 90 phylotypes by sequencher 4.8 and the sequences were deposited in GenBank (Accession No.: JX042330-JX042419). Of them, 48 phylotypes occurring only once, and the calculated coverage of the clone library was 77.4%.The rarefaction curve also showed that the clones detected could reflect the main information of endophytes (Figure 1).

Sequence alignment revealed that 196 individual sequences exhibited > 95% similarity with those of cultivable bacteria. Of these, 90 clones (45.9%) were affiliated with Proteobacteria, 54 clones (27.6%) with Firmicutes, 29 (14.8%) with Actinobacteria, and 23 (11.7%) with Cytophaga/Flavobacterium/Bacteroides (CFB) group. Details of all alignments in the clone library are listed in Table 1.

The sequences attributed to Proteobacteria, which includes alpha, beta and gamma classes, made up the largest fraction of the clone library. Of the 90 clones affiliated with Proteobacteria, 67 clones (or 74.4%) exhibited high similarity to Gammaproteobacteria. The proportion of clones that grouped with the alpha and beta classes was 20% and 5.6%, respectively. However, there were no sequences with > 95% similarity to genera in the delta or epsilon class. The 67 clones of Gammaproteobacteria were related to four orders of bacteria, including Pseudomonadales (34 clones), Enterobacteriales (22 clones), Aeromonadales (10 clones) and Xanthomonadales (1 clone). Of these, the dominant genera include: Acinetobacter, Aeromonas, Citrobacter, Enterobacter, Leclercia, Pseudomonas and Psychrobacter; the dominant species were Acinetobacter johnsonii, Acinetobacter junii, Leclercia adecarboxylata, Aeromonas punctata and Enterobacter cancerogenus (Table 1). Alphaproteobacteria was the second-most abundant subgroup of Proteobacteria in our survey. The 18 clones in this subgroup represented bacteria in four orders (Rhizobiales, Sphingomonadales, Rhodobacterales and Caulobacterales) (Table 1). The dominant genera were Brevundimonas, Microvirga, Rhizobium and Sphingomonas. Of the 5 clones affiliated with Betaproteobacteria, four belonged to bacterial species in Burkholderiales and only one was grouped into Methylophilales. All of them were assigned to different genera, including Bordetella, Comamonas, Methylophilus, Ramlibacter and Variovorax (Table 1).

Among the non-Proteobacteria, 54, 29 and 23 clones exhibited high similarity to bacterial species in the phyla Firmicutes, Actinobacteria and CFB respectively (Table 1). In Firmicutes, 43 clones were closely related to bacteria in Bacillales, 9 clones to Clostridiales and only 2 to Lactobacillales. The dominant genera included Paenisporosarcina, Planococcus, Planomicrobium, and the most abundant species were Paenisporosarcina macmurdoensis and Planococcus rifietoensis. Of the 29 clones grouped into Actinomycetales of phylum Actinobacteria, twelve clones were grouped with the Arthrobacter genus, while the others grouped with many other genera including Aeromicrobium and Ornithinicoccus (Table 1). Arthrobacter sulfonivorans was the most common species. In the 23 clones belonging to the CFB phylum, bacteria occurred in four orders, the Sphingobacteriales, Cytophagales, Bacteroidales and Flavobacteriales. The dominant genera were Adhaeribacter and Segetibacter, and Segetibacter koreensis was the most common species.

Finally, the 16S rDNA sequence of 16 clones, showed < 95% similarity to the previously cultivated bacteria. The phylogenetic analysis showed that these clones exhibited a close relationship with Actinobacteria (4 clones), Alphaproteobacteria (3 clones), Acidobacteria (3 clones), Bacteroidetes (2 clones), Betaproteobacteria (1 clone) and Firmicutes (3 clones) (Figure 2).

Endophytic bacteria communities detected by cultivation method

The isolation result showed that the number of colony-forming units (CFU) as determined for samples grown on R2A medium was higher than the number of CFUs grown on TSA medium. The counts (expressed as g^-1 fresh weight) were 2.0*10⁵ and 3.3*10⁴ on R2A and TSA medium, respectively. Totally 49 isolates were sequenced on the basis of 16S rDNA fragments, the ARDRA analysis resulted in the delimitation of 14 OTUs. Based on their 16S rDNA sequences (Genbank no. JX042420 -JX042433), they were assigned to 8 genera in three phyla (Proteobacteria, Actinobacteria and Firmicutes). The strains that were successfully cultivated included some genera in the Proteobacteria (Burkholderia, Massilia, Pseudomonas, Spingomonas, Yersinia), and some genera in Firmicutes and Actinobacteria such as Curtobacterium, Brevibacterium and Streptomyces. The most abundant species were Curtobacterium flaccumfaciens, Massilia brevitalea, Pseudomonas azotoformans and Pseudomonas libanensis (Table 2).

Compared the above bacterial communities with those discovered by 16S rDNA library technique, the cultivated species only involved in three phyla (Firmicutes, Proteobacteria and Actinobacteria) and no bacteria in group CFB was cultivated. The species and genera discovered by cultivation were much less than those detected by molecular method. In addition, some of genera cultivated also could not be found by molecular method, like Curtobacterium, Massilia, Burkholderia and Yersinia.

Discussion

In this study, we provide a thorough description of the endophytic bacterial community of G. montana, using a combined approach of molecular methods and cultivationdependent techniques. G. montana individuals were sampled from stone surfaces poor in nutrient availability and subject to strong stresses, such as a wide range of temperatures and extreme drought conditions. As far as we know, ours is the first description to date of the endophytic community of a xerophilous moss species in the Grimmiaceae.

Bacterial species detected by 16S rDNA library technique belong to 4 phyla and 54 genera, with a high proportion of Gammaproteobacteia, Firmicutes and Actinobacteria. Isolates from R2A and TSA media also discovered species in these groups, no bacteria in phylum CFB was cultivated. Although some of the genera discovered by these two methods were inconsistant, it reflected that using the combination of 16S rDNA library and cultivated method would be helpful to discover the bacterial information completely. Both of them proved that many different species coexisted in this small host (G. montana).

Compared to published accounts of bacterial communities associated with other moss species growing in peat bog, such as Sphagnum, our study revealed the different endophytes inhabiting the tissue of G. montana. In previous studies, Serratia and Pseudomonas of the Gammaproteobacteria, Burkholderia of the beta subgroup, Methylocella and Methylocapsa of the alpha subgroup (Raghoebarsing et al., 2005) and Staphylococcus of the Firmicutes (Opelt et al., 2007) were reported to be associated with Sphagnum species. In this survey, of the Gammaproteobacteria subgroup, Acinetobacter, Leclercia and Aeromonas were the dominant genera. Rhizobium of the Alphaproteobacteria, Massilia, Burkholderia and five of other genera of betaproteobacteria were also detected. In addition, there were also a high proportion of Gram positive bacteria detected in our library. Of them, clones assigned to Firmicutes comprised 25.5% of the total. Planococcus, Paenisporosarcina, Planomicrobium and Bacillus were the dominant genera; while Arthrobacter and Curtobacterium of Actinobacteria were also abundant. The inconsistent endophytic bacterial community in G. montana and Sphagnum species proved that plant species and niches could cooperatively shape the structure of endophytic bacterial communities (Berg and Smalla, 2009).

Analyzing the function of those bacteria dominanted in G. montana would be helpful to understand the interactions between endophytes and hosts. Of gammaproteobacteria class, the dominant species Acinetobacter johnsonii has been reported to produce alkaline and lowtemperature lipase (Wang et al., 2011a); Acinetobacter junii was considered to be a kind of cellulolytic bacterium that can produce xylanase, cellulose and pectinase (Lo et al., 2010; Zhai et al., 2010) and also could remove (via accumulation) phosphate from synthetic wastewater (Hrenovic et al., 2010); Leclercia adecarboxylata could degrade two and three benzene-ring polycyclic aromatic hydrocarbon compounds (Sarma et al., 2004; Sarma et al., 2010); Aeromonas veronii and Aeromonas punctata subsp. caviae, could produce enzymes such as the amino acid racemase, and xylanase (Cao et al., 2007; Cruz et al., 2008; Silver et al., 2011). As with the Sphagnum bacterial communities, Pseudomonas was also the dominant genus in our study. The isolated species Pseudomonas azotoformans (Komeda et al., 2004; Nie et al., 2011) could degrade Cyhalofop-butyl, while Pseudomonas libanensis could produce the biosurfactant viscosin (Dabboussi et al., 1999; Saini et al., 2008). Rhizobium pusense of the Alphaproteobacteria was first isolated from the rhizosphere of chickpea plants and considered to be a non-symbiotic rhizobium.In our survey utilizing a 16S rDNA library, five clones of Rhizobium pusense were detected, indicating that this species could be in symbiosis with G. montana.

Of bacteria assigned to Firmicutes, Planococcus rifietensis and Paenisporosarcina macmurdoensis were the dominant species, which have ever been previously isolated from algal or cyanobacterial mats in sulfurous springs (Reddy et al., 2003; Romano et al., 2003). Four Planomicrobium species were also found, which have been previously isolated from coastal sediments (Dai et al., 2005), seafood jeotgal (Yoon et al., 2001) and glaciers (Zhang et al., 2009a); they were considered as the cold tolerant bacteria (Yang et al., 2011; Zhang et al., 2009a). In addition, Bacillus simplex was isolated by cultivation, which was ever provided to have strong antioxidant activity (Wang et al., 2011b). Among the Actinobacteria, Arthrobacter sulfonivorans could produce membrane-associated dimethylsulfone-and dimethylsulfoxide-reductases (Borodina et al., 2002); Arthrobacter agilis could release N,N-dimethyl-hexadecanamine (dimethylhexadecylamine) to directly affect plant morphogenesis (Fong et al., 2001; Velazquez-Becerra et al., 2011) and could contribute to membrane stabilization in response to thermal and salt stress by increasing carotenoid accumulation (Fong et al., 2001); Curtobacterium was a dominant genus discovered in the cultures, and Curtobacterium flaccumfaciens, as the most dominant species in this group, also was known to reduce symptoms caused by Xylella fastidiosa in Catharanthus roseus (Lacava et al., 2007); the cultivable Streptomyces griseoplanus could produce anticapsin and Erythromycin-a, and might probably help to resist pathogens in the host (Boeck et al., 1971; Thompson et al., 1971).

The dominant species Segetibacter koreensis from CFB phylum was first isolated from ginseng fields in South Korea (An et al., 2007), while Adhaeribacter tereus and Adhaeribacter aquaticus were ever isolated from soil (Zhang et al., 2009b) and water biofilms (Rickard et al., 2005), respectively. This is the first time that these species have been found as endophytes, and their possible functions remain unclear.

In conclusion, the most important findings of this study were: (1) a high endophytic bacterial diversity and complex community structure were found associated with G. montana, using a combination of molecular and cultivation techniques; (2) community structure differed from that of endophytic communities of Sphagnum mosses, especially in the abundance of Actinobacteria and Firmicutes (higher in G. Montana); and (3) Some bacterial species found endophytically in G. montana are known to have possible beneficial effects on plants, but whether this is the case in G. Montana is not proven. Thus, in order to improve our understanding of the concrete mechanisms through which endophytic bacteria (such as those of G. montana) adapt to extreme environments and discover new bacterial resources, further work needs to be done in the future.

Acknowledgments

We would like to thank E.B.M. Drummond at the University of British Columbia for her assistance with English language and grammatical editing of the manuscript. We also thank professor Guisen Du at Capital Normal University for the species identification of moss. This work was funded by the Scientific Research Program of National Natural Science Foundation of China (No. 31100004).

Submitted: December 27, 2012

Approved: April 1, 2013.

All the content of the journal, except where otherwise noted, is licensed under a Creative Commons License CC BY-NC.

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Publication Dates

• Publication in this collection
  29 May 2014
• Date of issue
  2014

History

• Accepted
  01 Apr 2013
• Received
  27 Dec 2012