Endophytic bacterial diversity in banana ‘Prata Anã’ (Musa spp.) roots

The genetic diversity of endophytic bacteria in banana ‘Prata Anã’ roots was characterized. Two hundred and one endophytic bacteria were isolated, 151 of which were classified as Gram-positive and 50 as Gram-negative. No hypersensitivity response was observed in any of the isolates. The rep-PCR technique generated different molecular profiles for each primer set (REP, ERIC and BOX). Fifty readable loci were obtained and all of the fragments were polymorphic. Amplified ribosomal DNA restriction analysis (ARDRA) of the isolates based on cleavage with four restriction enzymes yielded 45 polymorphic bands and no monomorphic bands. PCR amplified the nifH gene in 24 isolates. 16S rDNA sequencing of the 201 bacterial isolates yielded 102 high-quality sequences. Sequence analyses revealed that the isolates were distributed among ten bacterial genera (Agrobacterium, Aneurinibacillus, Bacillus, Enterobacter, Klebsiella, Lysinibacillus, Micrococcus, Paenibacillus, Rhizobium and Sporolactobacillus) and included 15 species. The greatest number of isolates belonged to the genus Bacillus. The bacteria identified in this study may be involved in promoting growth, phosphate solubilization, biological control and nitrogen fixation in bananas.


Introduction
Bananas are important agricultural products in most tropical countries, with the world production estimated to be 90.7 million tons. Brazil is ranked fourth among banana producers, with a production of 7,116,808 tons and a harvested area of 510,825,000 hectares (FAO, 2010). Plants, including banana trees, are complex micro-ecosystems in which different niches are filled by a wide variety of microorganisms, including endophytes (Mia et al., 2010). In recent years, there has been considerable interest in the study of endophytic microorganisms and the determination of their role in plants. Endosymbionts act as biological control agents in numerous diseases (Jie et al., 2009), in the promotion of plant growth (Ryan et al., 2008) and in the bioremediation of polluted areas (Germaine et al., 2009). The use of these microorganisms is preferred compared to chemical fertilizers and pesticides because of their lower cost and their contribution to sustainable agriculture (Aung et al., 2011).
Various molecular techniques have been used to characterize endophytic bacteria, including repetitive extragenic palindromic sequence PCR (rep-PCR), which uses the conserved sequences of ERIC, REP and BOX, 16S rDNA amplification and restriction, and the cloning and sequencing of the amplified genes (Ryan et al., 2008). Additionally, primers specific for the amplification of important bacterial genes, such as nifH (necessary for nitrogen fixation), or genes involved in the degradation of organic pollutants, have been used to study the potential participation of endophytic bacteria in important processes of the host plant (Ryan et al., 2008).
For more than 60 years, bacteria have been known to co-exist with plants without causing any damage. The first reports of endophytic bacteria in banana trees were published in the 1990s and increased from 2000 onwards. However, few advances have been made in isolating and characterizing these endophytic bacteria and in understanding their diversity and functions in bananas. Some genera have been described as banana colonizers, including Azospirillum amazonense, Azospirillum brasilense, Bacillus, Burkholderia cepacia, Burkholderia spp., Citrobacter sp., Enterobacter spp., Klebsiella spp., Klebsiella variicola, Ochrobactrum, Pantoea, Serratia and Staphylo-coccus epidermidis (Rosenblueth et al., 2004;Thomas et al., 2008;Ting et al., 2008;Jie et al., 2009).
The objective of this study was to isolate and identify banana endophytic bacteria and to assess their genetic diversity based on rep-PCR, ARDRA and partial 16S rDNA sequencing.

Plant sampling and bacterial isolation
Roots from juvenile 'Prata Anã' plants were collected in four counties in Minas Gerais State and in one county in Bahia State, Brazil. The banana root fragments were immersed in 70% ethanol for 1 min and 4% sodium hypochlorite (NaClO) for 3 min and then washed three times in sterile, distilled water. The fragments were subsequently exposed to ultraviolet light in a flow chamber for 10 min and subjected to an ultrasound bath for 10 min. After two baths, the fragments were macerated and the suspensions were diluted 10 fold. A 0.1 mL aliquot was plated onto each of the following media: nutrient yeast dextrose agar (NYDA; 10 g dextrose, 5 g yeast extract, 3 g beef extract, 5 g peptone and 18 g agar), potato dextrose agar (PDA; 200 g potato starch, 20 g dextrose and 20 g agar) and tryptic soy agar (30 g TSA). The plates were incubated for 48 h at 25 ± 1°C on a 12 h photoperiod. The different media described above were used for bacterial isolation and further culturing.
The bacterial isolates were characterized by Gram staining and, to assess pathogenicity, the isolates were inoculated into non-host plants, including pepper (Capsicum annuum Mill), tobacco (Nicotiana tabacum L.) and common bean (Phaseolus vulgaris L.) seedlings grown in a greenhouse. The infected plants were monitored for hypersensitivity responses as described by Romeiro (2001).

Total DNA extraction and 16S rDNA sequencing
For DNA extraction, each isolate was grown in liquid tryptic soy broth (TSB) for 24 h at 37°C under constant mixing at 180 rpm. Bacterial genomic DNA was extracted with a DNA extraction kit (Qiagen, Valencia, CA, USA), according to the manufacturer's recommendations.
The isolates were identified by partial sequencing of the 16S region. Initially, the 16S region was amplified using primers 27 (5'-AGAGTTTGATC(AC)TGGCTCAG--3') and 1492R (5'-ACGG(CT)TACCTTGTTACGAC TT-3'). The reactions consisted of 2 mL of dNTPs (2.0 mM each), 2.5 mL of 10X buffer, 0.75 mL of 50 mM MgCl 2 , 2.5 mL of each primer (5 mM), 0.3 mL of Taq polymerase (5 U/mL), 50 ng of template DNA and sterile Milli-Q water in a final volume of 25 mL. The amplification conditions consisted of an initial denaturation at 94°C for 3 min, followed by 30 cycles of 94°C for 30 s, 50°C for 30 s and 72°C for 1 min and a final extension at 72°C for 7 min.
DNA was purified using a QIAquick gel extraction kit (Qiagen). Sample sequencing was done using an automated sequencer (ABI-PRISM 3100 Genetic Analyzer). The resulting sequences were compared with those present in the National Center for Biotechnology Information (NCBI) database (www.ncbi.nlm.nih.gov) by using the basic local alignment search tool (BLAST) software for nucleotides (Altschul et al., 1997). The bacterial isolates were identified to the species level when similarity values varied between 98% and 100% and to the genus level when similarity values were < 98%.

Nucleotide sequence accession numbers
The nucleotide sequence data reported in this paper have been deposited in the GenBank nucleotide sequence database under accession numbers JQ979307-JQ979408.

ARDRA amplification
Endophytic bacterial DNA was amplified with the following primers: FGPS1490 5'-TGCGGCTGGATCAC CTCCTT-3' and FGPS132 5'-CCGGGTTTCCCCATTC GG-3'. The amplification reactions contained 0.8 mL of dNTPs (2.0 mM each), 2.5 mL of 10X buffer, 0.75 mL of 50 mM MgCl 2 , 0.25 mL of each primer (5 mM), 0.2 mL of Taq polymerase (5 U/mL) and 50 ng of DNA in a final volume of 25 mL. The amplification conditions consisted of an initial denaturation step (95°C for 3 min) followed by 35 cycles of 94°C for 1 min, 60°C for 1 min and 72°C for 2 min, with a final extension at 72°C for 3 min.
The amplification products were digested with the restriction enzymes HinfI, MspI, NdeI and RsaI. Each enzy-matic reaction contained 10 mL of PCR product, 2 mL of 10X enzyme-specific buffer, 2 mL of enzyme (5 U/reaction) and 18 mL of sterile Milli-Q water. The reactions were incubated for 16 h in a water bath at 37°C. The fragments were analyzed by electrophoresis on 1.2% agarose gels in 1X TBE at 100 V for 3 h. The size of the amplified fragments was estimated with a 100-bp molecular weight DNA ladder.
nifH gene PCR amplification DNA from root isolates was analyzed for the presence of the nifH gene using the universal primers 19f F (5'-GGAATTCTGTGACCTAAAGCTGA-3') and 407 R (5'-AGCATACATTGCCATCATTTCACC-3'). The amplification reaction mixtures contained 2.0 mL of dNTPs (2.0 mM each), 2.5 mL of 10X buffer, 1 mL of 50 mM MgCl 2 , 0.5 mL of each primer (5 mM), 0.6 mL of Taq polymerase (5 U/mL) and 50 ng of DNA in a final volume of 25 mL. The amplification conditions consisted of denaturation at 94°C for 2 min, followed by 30 cycles of 94°C for 30 s, 52°C for 30 s and 72°C for 30 s, with a final extension at 72°C for 7 min. The amplification products were analyzed by electrophoresis on 1.2% agarose gels and fragments of~270 bp were expected for the nifH gene. The size of the amplified fragments was estimated with a 100-bp molecular weight DNA ladder.

Statistical analyses
The ARDRA and rep-PCR results were analyzed cumulatively with R 2.13 software based on the coefficient of simple matching. Cluster analysis was done by the unweighted pair group method with arithmetic mean (UPGMA) using MEGA 5 software (Tamura et al., 2011).

Results
Bacterial isolation and 16S rDNA sequencing 201 isolates of endophytic bacteria were obtained from the roots of 'Prata Anã' banana plants; 150 of these isolates were classified as Gram-positive and 51 as Gramnegative. There was no hypersensitivity response in tobacco (N. tabacum L.), bell pepper (C. annuum Mill) and common beans (P. vulgaris L.) five days after inoculation of the bacterial isolates.
The genus Bacillus was identified most frequently (87.3% of isolates), followed by the genus Lysinibacillus 254 Endophytic bacteria in Musa spp. subtilis predominated among the species identified in this study and represented 20.6% and 9.8% of the isolates identified, respectively (Table 1).

REP-PCR and ARDRA analysis
The rep-PCR technique generated different molecular profiles for each primer (REP, ERIC and BOX) separately. 50 readable loci were obtained and all of the fragments were polymorphic.
Nine major clusters were identified (Figure 1). Cluster I contained 34 isolates from three genera (Paenibacillus, Bacillus and Lysinibacillus), all belonging to the phylum Firmicutes. Despite the low dissimilarity among the isolates, this group showed high genetic diversity, with repre- Organism with the partial 16S rDNA sequence most homologous to that of the banana isolate.  sentatives from eight species of Bacillus. In general, the groups were formed based on phylogenetic criteria. Groups I, II, III and V were formed exclusively by bacteria of the phylum Firmicutes while group IV consisted of a single representative of the genus Lysinibacillus. Group VI contained different bacterial genera, two of which (Klebsiella and Rhizobium) belonged to the phylum Proteobacteria. 75% of the isolates in group VIII belonged to the phylum Firmicutes, the exception being Micrococcus luteus, which belonged to the phylum Actinobacteria. Group IX contained three genera (Agrobacterium tumefasciens, Enterobacter sp. and Bacillus subtilis) that belonged to three classes (Alphaproteobacteria, Gammaproteobacteria and Bacilli, respectively). ARDRA diversity analysis of the bacterial isolates, which compared the cleavage products from four restriction enzymes, yielded 45 polymorphic and non-monomorphic bands. The restriction enzyme RsaI provided the most resolution by generating 13 bands, followed by MspI with 12, HinfI with 11 and NdeI with nine bands.
Phylogenetic analysis based on the ARDRA results showed that most (96%) of the isolates were affiliated with Firmicutes, including the dominant genus Bacillus. Figure 2 shows the phylogeny of Bacillus and Lysinibacillus, with the formation of eight groups. Groups III, V, VI, VII and VIII consisted exclusively of representatives belonging to Bacillus. The four representatives of Lysinibacillus formed three groups, with groups I and II containing one isolate each while group IV contained two isolates.
The phylum Proteobacteria accounted for 3.9% of all isolates. The only member of Actinobacteria identified was Micrococcus luteus (Figure 3). Figure 3 shows six groups in which groups II, V and VI were represented by a single bacterium each. There was no common criterion by which the bacteria were grouped. Group III contained two bacteria belonging to the phylum Firmicutes (Paenibacillus sp. and Aneurinibacillus sp.).

Analysis of the nifH gene
PCR amplification of the nifH gene was detected in 24 of the 102 bacterial isolates; 79% of the isolates belonged to the genus Bacillus. Seven species of Bacillus were nifH-positive, including: B. amyloliquefaciens, B. cereus, B. flexus, B. licheniformis, B. pumilus, B. subtilis

DISCUSSION
Studies of plants and endophytic bacteria have demonstrated the importance of such interactions for plant adaptation to diverse ecosystems and for enhancing soil health and quality. However, little is known about the colonizing species, the relationship between these bacteria and banana trees, and the possible benefits of this interaction.
Of the 201 bacterial isolates collected in this study, 75% were classified as Gram-positive. In contrast, Thomas et al. (2008), in a study of endophytic bacteria isolated from banana shoot tip cultures during the first passage in vitro encountered more Gram-negative organisms (75%). Together, these findings agree with other reports regarding the marked diversity of Gram-negative and Gram-positive endophytic bacteria in banana (Habiba et al., 2002;Ganen et al., 2009).
The lack of a hypersensitivity reaction for the 201 bacterial isolates suggested the absence of phytopathogenic bacteria. The species A. tumefaciens was identified here but did not elicit a hypersensitivity response in non-host plants, although it behaved as an endophytic bacterium in banana trees. According to Kobayashi and Palumbo (2000), some  phytobacteria genera are typical endophytic bacteria without causing any symptoms of illness in the associated plant. The host and pathogen developmental stage, environmental changes and host defense responses (Schulz and Boyle, 2005), as well as genetic, structural and physiological factors can determine whether a microorganism is endophytic or phytopathogenic (Andreote et al., 2008).
Sequence analyses using BLASTn revealed high bacterial diversity in the roots of 'Prata Anã' banana plants.
Many bacterial genera, such as Bacillus and other species identified here have also been identified in other studies of endophytic bacteria. The genera Bacillus, Enterobacter and Pantoea have been identified as endophytes in several plants, such as citrus, sugar cane and soybean (Medrano and Bell, 2007;Magnani et al., 2010).
Bacillus was the most frequently encountered genus in this study. The Bacillus species identified here (B. cereus, B. subtilis, B. megaterium and B. pumilus) have been described as endophytic species (Elvira-Recuenco and Van Vuurde, 2000;Araújo et al., 2002) and have also been isolated from contaminated banana explants in tissue culture (Odutayo et al., 2007;Jie et al., 2009). Bacillus bacteria are classified as growth promoters (characterized by the production of auxins and gibberellins by B. pumilus), nitrogen fixers and phosphate solubilizers (Forchetti et al., 2007).
Lysinibacillus was the second most frequently identified genus. Species belonging to this genus have been associated with ginseng plants and the isolates evaluated were positive for most of the plant growth promoters (Vendan et al., 2010). Despite the predominance of Bacillus isolates, species of Paenibacillus were also identified and are of considerable biotechnological potential. Paenibacillus species not only have the capacity to produce plant growth hormones (auxins and cytokinins), enzymes (chitinases, amylases and proteases) and antibiotics but also can solubilize organic phosphates (Coelho et al., 2009).
Only one representative each of Enterobacter and Rhizobium was found to be associated with banana roots. Enterobacter species are endophytes in Citrus spp., soybean and banana (Araujo et al., 2002;Kuklinsky-Sobral et al., 2004;Thomas et al., 2008;Jie et al., 2009). Enterobacter species can fix nitrogen and solubilize phosphate (Asis Jr and Adachi, 2003;Dalton et al., 2004), thereby increasing plant growth and productivity.
Rhizobium species have been widely used as biofertilizers for various legume species. In banana, a few reports have described Rhizobium spp. associated with Musa spp. A study by Martinez et al. (2003) reported significant increases in stem and leave fresh weight in bananas after the inoculation of some Rhizobium isolates.
Although A. tumefaciens is described as phytopathogenic this species has been reported as an endophytic bacterium that is asymptomatically associated with the roots of Triticum aestivum L. (Sharma et al., 2005), the stems of Rosa grandiflora (Martí et al., 1999) and the roots and stems of Crotalaria pudica, Crotalaria pallida and Mimosa pudica (Wang et al., 2008). Wang et al. (2008) also observed that A. tumefaciens was able to thrive in nodules formed by Sinorhizobium meliloti in Melilotus dentatus. According to Llop et al. (2009), the asymptomatic association of A. tumefaciens with host plants may reflect a loss of pathogenicity. To our knowledge, this is the first report on identify A. tumefaciens living as an endophyte in Musa spp. This association suggests that these isolates may be present in the soil and enter the plant through secondary roots.
The BOX primer yielded the greatest number of polymorphic bands and had the best resolving power among the bacterial isolates. According to Van Berkum (1999), BOX regions are associated with a high degree of polymorphisms and consequently participate in adaptive evolution by mediating the interactions of microorganisms with harsh or adverse environments.
The ARDRA technique was efficient in identifying variation among bacterial isolates evaluated by polymorphisms and estimates of genetic distance. The variations observed among bacteria are based on the generation of different band profiles that reveal the diversity among and within bacterial groups and group individuals of similar genotypes (Kuklinsky-Sobral et al., 2004). Assumpção et al. (2009) stated that the diversity and structure of microbial 262 Endophytic bacteria in Musa spp. communities varies with the sample size because the probability of finding rare species increases with increasing sample size. The detection of the nifH gene indicated that 23.5% of the endophytic bacteria analyzed could act as nitrogen fixers. Most of these nitrogen fixers belonged to the genus Bacillus, which has already been described as potential nitrogen fixers (Raymond et al., 2004). The nifH gene was also present in the genus Rhizobium and K. pneumoniae. The presence of this gene is strong evidence of the nitrogen-fixing ability in bacteria because this gene encodes the Fe-nitrogenase subunit of the nitrogenase complex. The nifH gene has therefore become a useful marker for studying the diversity of endophytic bacteria with the potential to fix N 2 in independent culture studies (Izquierdo and Nüsslein, 2006).
The distribution of phylogenetic groups showed a predominance of members of Firmicutes (96%). In contrast, Jie et al. (2009), in a study of the re-introduction of naturally-occurring endophytes into tissue culture banana plantlets, noted a high proportion of members of the phylum Proteobacteria (87.7%). Both studies demonstrated the potential effect of cultivars, climatic conditions and soil on the genetic diversity of endophytic bacteria in bananas.
The isolation, identification and screening of endophytic bacteria as plant growth regulators, as well as their ability to increase plant nutrient absorption and stimulate the development of resistance towards abiotic and biotic stress, are essential leads for establishing their applications in agriculture, especially the cultivation of bananas (Cao et al., 2004;Jaizme-Vega et al., 2004;Jie et al., 2009).
The marked genetic diversity observed here and in other studies of endophytic bacteria in banana represents an emerging trend in biotechnology. However, further studies on the molecular and biochemical mechanisms of growth promotion and on the usefulness of artificial inoculation must be done in order to meet the expectations of largescale banana producers.