Bacterial communities in floral nectar

Environmental Microbiology Reports (2012) 4(1), 97–104

doi:10.1111/j.1758-2229.2011.00309.x

Bacterial communities in floral nectar Svetlana Fridman,1 Ido Izhaki,1 Yoram Gerchman2 and Malka Halpern1,2* 1 Department of Evolutionary and Environmental Biology, Faculty of Natural Sciences, University of Haifa, Mount Carmel, 31905 Haifa, Israel. 2 Department of Biology and Environment, Faculty of Natural Sciences, University of Haifa, Oranim, 36006 Tivon, Israel. Summary Floral nectar is regarded as the most important reward available to animal-pollinated plants to attract pollinators. Despite the vast amount of publications on nectar properties, the role of nectar as a natural bacterial habitat is yet unexplored. To gain a better understanding of bacterial communities inhabiting floral nectar, culture-dependent and -independent (454-pyrosequencing) methods were used. Our findings demonstrate that bacterial communities in nectar are abundant and diverse. Using culturedependent method we showed that bacterial communities of nectar displayed significant variation among three plant species: Amygdalus communis, Citrus paradisi and Nicotiana glauca. The dominant class in the nectar bacterial communities was Gammaproteobacteria. About half of the isolates were novel species (< 97% similarities of the 16S rRNA gene with known species). Using 454-pyrosequencing we demonstrated that nectar microbial community are distinct for each of the plant species while there are no significant differences between nectar microbial communities within nectars taken from different plants of the same species. Primary selection of the nectar bacteria is unclear; it may be affected by variations in the chemical composition of the nectar in each plant. The role of the rich and diverse nectar microflora in the attraction–repulsion relationships between the plant and its nectar consumers has yet to be explored. Introduction Floral nectar is considered the most important reward animal-pollinated plants furnish to attract pollinators (Forcone et al., 1997; Bernardello et al., 1999). Despite Received 22 July, 2011; accepted 27 October, 2011. *For correspondence. E-mail [email protected]; Tel. (+972) 4 9838727; Fax (+972) 4 9838911.

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the dominance of sugar (> 90% dry weight) in nectar, non-sugar compounds play an important role. These compounds (< 10% dry weight) include amino acids, organic acids, lipids, essential oils, polysaccharides, vitamins, antioxidants, minerals and secondary metabolites (Baker and Baker, 1983; Dafni, 1992; Carter et al., 2006). Nectar consumers such as insects, birds and bats were suggested to transfer microflora among flowers, and between flowers and other plant organs (Sandhu and Waraich, 1985). However, floral nectar was suggested to be not suitable as bacterial habitat and was even demonstrated to have antimicrobial properties (Sasu et al., 2010). These properties could be due to several chemical components that were suggested to limit growths of microflora in the nectar: (i) high sugar concentration in nectars may impose high osmotic pressure that constrains microbial growth (Pusey, 1999; Brysch-Herzberg, 2004); (ii) nectar-associated proteins were suggested to function as a defensive mechanism against microorganism infections by producing reactive oxygen molecules (Carter and Thornburg, 2004a–c; González-Teuber et al., 2009; Harper et al., 2010); and (iii) secondary metabolites such as phenolics have also been suggested to play an antimicrobial role in nectar (Hagler and Buchmann, 1993). Based on these constraints (e.g. Minorsky, 2007), one may expect that floral nectar is populated by only a few microbiota groups that are adapted to live in such extreme environment. Although some publications have indicated the presence of microorganisms in floral nectar, most of them described fungi and yeasts (Sandhu and Waraich, 1985; Lachance et al., 2001; Brysch-Herzberg, 2004; Manson et al., 2007; Herrera et al., 2008; 2009; Pozo et al., 2009; 2011) and only one addressed the presence of bacteria (Gilliam et al., 1983). A significant negative correlation was found between yeast density and sugar content, as well as yeast density and nectar concentration in a Watsonia species (de Vega et al., 2009). Herrera and Pozo (2010) described a phenomenon whereby the sugar catabolism of yeast populations inhabiting floral nectar can increase its temperature and thus modify the thermal microenvironment within the flower. However, despite the vast amount of publications on nectar properties, the role of nectar as a natural habitat for microorganisms and specifically for bacteria is yet unexplored. Microbial communities in nectar may affect the nectar’s chemical profile, thus directly controlling nectar consumption by flower visitors such as pollinators and nectar thieves, and

98 S. Fridman, I. Izhaki, Y. Gerchman and M. Halpern consequently indirectly governing plant fitness (Herrera et al., 2008; 2009; Herrera and Pozo, 2010). The aim of the current study was to gain a better understanding of floral nectar bacterial communities. To that end, we studied the bacterial communities in flower nectar of three plant species: Nicotiana glauca, Amygdalus communis and Citrus paradisi in Northern Israel. Here we show that (i) bacteria are common inhabitants of floral nectar and (ii) each plant species floral nectar support a unique bacterial community. Results and discussion The presence of bacteria in floral nectar Yeasts have been shown to inhabit flower nectar (Manson et al., 2007; Herrera et al., 2008; 2009; Manson, 2009). However, as far as we know, nectar has not been considered a bacterial habitat. On the contrary, Minorsky (2007) raised the question why microbes do not grow in nectar, and quoted Carter and colleagues (2007) who found that nectarins, proteins which accumulate in the nectar of ornamental tobacco plants, produce very high levels of hydrogen peroxide (up to 4 mM). This might be the case for some bacterial strains, but as we show here, many others thrive in floral nectar. Nectar sampling and bacterial counts Flower nectar from three plant species: N. glauca (Tree Tobacco), A. communis (Almond) and C. paradisi (Grapefruit) were collected from flowers of each sampled plant (five different plants for each plant species) between March and June 2009. All the sampled plants were located within a radius distance of up to 10 kilometres in Northern Israel. These three plant species were chosen because their floral nectar is known to contain secondary metabolites which are considered as an antimicrobial agent: N. glauca contains nicotine and anabasine, A. communis contains amygdalin and C. paradisi contains caffeine (Detzel and Wink, 1993; Kretschmar and Baumann, 1999; LondonShafir et al., 2003; Tadmor-Melamed et al., 2004). Using DAPI, we found that bacterial counts per millilitre in the nectar samples from N. glauca, A. communis and C. paradisi were 1.4 ¥ 107 (⫾ 1.6 ¥ 106), 1.7 ¥ 107 (⫾ 4.8 ¥ 106) and 3.1 ¥ 107 (⫾ 2.0 ¥ 106) respectively. Bacterial cfu ml-1 in the nectar samples from the different plants were approximately 50%, 25% and 10% from the DAPI counts respectively. These results demonstrate that bacteria thrive in floral nectar. These high abundances of bacteria (> 106 cfu per millilitre nectar) were about two magnitude higher compared with what was observed for yeasts in the floral nectar of Helleborus foetidus, Aquilegia vulgaris and Aquilegia pyrenaica cazorlensis (Herrera et al., 2008).

Culturable microbial communities structure in nectar of different plant species Nectar samples were collected aseptically from flowers of A. communis, C. paradisi and N. glauca and were spread onto R2A agar (Himedia) and R2A agar supplemented with 20% sucrose. One hundred representative isolates were identified by amplifying and sequencing the 16S rRNA gene (Appendix S1). About 33%, 75% and 42% of A. communis, C. paradisi and N. glauca nectar isolates, respectively, were found to be novel species (< 97% similarities in the 16S rRNA gene sequences to known species) (Table 1). This demonstrates that indeed, the nectar is an unexplored bacterial niche. Representatives of the Gammaproteobacteria class dominated all nectar samples, accounting for 59%, 82% and 45% of the nectar isolates in A. communis, C. paradisi and N. glauca respectively (Table 1). Isolates belonging to the Bacilli class also occurred in the nectar from all plant species. Actinobacteria was identified only from A. communis and N. glauca. Representatives of the Alphaproteobacteria and Flavobacteria classes were found only in the A. communis nectar. Interestingly, the most abundant species in the nectar were a novel unidentified Enterobateriaceae species in A. communis and C. paradisi and Acinetobacter sp. in C. paradisi and N. glauca (Table 1). Significant differences were found between nectar bacterial communities from different plant species (Table 1 and Fig. 1). Figure 1 displays the results from the canonical correspondence analysis using the CANOCO computer program. The distribution of the bacterial species along the ordinates was not random according to the Monte Carlo test (F = 1.14, P < 0.05) and thus can be explained by their different plant species source. Plant species explained 42% of the variation in the bacterial community composition whereas the horizontal and the vertical axes explained 24% and 18% of the variation respectively (Fig. 1).

454-pyrosequencing of 16S rRNA genes Bacterial diversity in all nectar samples was surveyed by 454-pyrosequencing of 16S rRNA genes (five samples per plant species, 15 samples in total). A total of approximately 10 000 sequences per sample were obtained. Nevertheless, after chloroplasts and Archaea sequences were removed from the analysis, about 3200–7000 sequences per sample were analysed (77 077 sequences, in total) (see also Appendix S1). Sequences were assigned to species-level operational taxonomic units (OTUs) using a 97% pairwise-identity cut-off. In sum, 2197 OTU’s were obtained for all 15 samples with an average of 401, 379 and 207 OTU’s

© 2011 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Reports, 4, 97–104

Bacterial communities in floral nectar

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Table 1. List of bacterial isolates from nectar of Amygdalus communis, Citrus paradisi and Nicotiana glauca. Class

Closest relative in GenBank database

Amygdalus communis

Citrus paradisi

Nicotiana glauca

Alphaproteobacteria Asaia astilbes Bartonella rattaustraliani

1 (100) 1 (97.2)

Gammaproteobacteria Acinetobacter baumannii Acinetobacter baylyi Acinetobacter bouvetii Acinetobacter calcoaceticus Acinetobacter gerneri Acinetobacter johnsonii Acinetobacter radioresistens Acinetobacter schindleri Erwinia amylovora Erwinia persicina Pantoea agglomerans Pantoea septica Pseudomonas flectensa Pseudomonas lutea Pseudomonas rhizosphaerae Pseudomonas trivialis Pseudomonas viridiflava Pseudomonas synxantha

2 (94.7–96.0) 6 (95.5–96.1) 1 (95.1) 1 (96.2) 6 (95.8–96.3) 2 (95.2–95.5) 1 (96.1) 2 (95.1–96.2)

1 (96.1) 1 (96.6) 3 (95.9–96.1) 5 (96.9–97.4)

2 (98.5–99.6) 2 (99.3) 1 (98.7) 14 (96.2–97.1) 2 (98.8–99.9) 1 (99.0) 1 (100) 2 (99.3–100)

17 (96.5–97.1)

3 (94.5–96.7)

1 (99.7)

Actinobacteria Arthrobacter tumbae Arthrobacter pascens Curtobacterium flaccumfaciens Kocuria kristinae

1 (99.7)

1 (99.8) 1 (99.9)

1 (98.5) 1 (98.0)

Bacilli

Flavobacteria

Bacillus megaterium Bacillus safensis Paenibacillus illinoisensis Paenibacillus validus Staphylococcus epidermidis Staphylococcus warneri Chryseobacterium indoltheticum

3 (97.9–99.6) 1 (99.5) 1 (94.8) 2 (99.1) 1 (100) 1 (98.5)

4 (98.9–100) 1 (99.9)

a. Isolates that were identified as most closely related to Pseudomonas flectens do not belong to the Pseudomonas genus and are in fact novel species in a novel genus in the Enterobacteriaceae family (M. Halpern, S. Fridman and I. Izhaki, unpubl. data). See also Fig. 5. The number before the parentheses indicates the number of isolates, the number within the parentheses indicates the percentage of the 16S rRNA gene similarities to the closest known species. Isolates with less than 97.5% 16S rRNA gene similarities to known species are most likely novel species and the name of their closest relative species is marked in bold. The isolates were identified by comparing their 16S rRNA gene sequences to that of the GenBank database (EZtaxon version 2.1. http://www.eztaxon.org). Sequences lengths were at least 850 bp. Sequences length obtained for most Acinetobacter species and for all the isolates that were identified as closely related to Pseudomonas flectens were 1300– 1500 bp. Accession numbers of the 16S rRNA gene sequences are HQ284799–HQ284831, HQ284869–HQ284906 and HQ284948–HQ284970.

per sample and coverage of 95.8%, 97.2% and 91.4% for A. communis, C. paradisi and N. glauca nectar bacterial communities, respectively. Chao1 richness estimator for A. communis, C. paradisi and N. glauca nectar bacterial communities was, 689, 629 and 418, respectively (see also Appendix S1). This analysis revealed clustering of samples by their plant origin, except for one nectar sample from N. glauca that overlapped with C. paradici samples (Fig. 2). However, when AMOVA analysis was applied, significant differences were found between the bacterial communities from the nectar samples that originated from the three different plant species (F7,14 = 3.14; P < 0.01). AMOVA post hoc analysis revealed significant differences between the bacterial communities from

the different plant species nectar (A. communis vs. C. paradisi, F1,9 = 3.97, P < 0.01; A. communis vs. N. glauca, F1,9 = 3.06, P < 0.01; C. paradisi vs. N. glauca F1,9 = 3.97, P < 0.05). This indicates that nectar from each plant species has a distinct microbial community (Fig. 2). The majority of the sequences from all the nectar samples were classified as Proteobacteria (> 83.0%). Gammaproteobacteria was the dominant class and comprised 79.5%, 92.8% and 72.9% of the sequences in C. paradisi, N. glauca and A. communis respectively (Fig. 3, upper graph). The most prevalent families were Moraxellaceae and Enterobacteriaceae. Acinetobacter was the dominant genus with the frequency of 49%, 90% and 78% of the bacterial species in A. communis,

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100 S. Fridman, I. Izhaki, Y. Gerchman and M. Halpern Novel bacterial species in floral nectar

Fig. 1. Ordination diagram (calculated with CANOCO software) showing variation in the abundance of bacterial species isolates among the three plant species. This joint plot data analyse the relationship between bacterial species and plant species. The environmental variables are displayed as arrows radiating from the centre of the diagram. The length of the arrows represents the contribution of each plant species to the variation of the sample. The angle in between two arrows is a measure for the correlation between the two variables (small angle means high correlation), and the projection of a taxa point on an arrow is a measure for the relative value of that point; in other words, for the position of that point on the gradient described by the arrow. The green triangles represent different bacterial species. The identity of the species is as follows: group 1: Erwinia amylovora, Arthrobacter tumbae, Kocuria kristinae, Bacillus megaterium, Staphylococcus epidermidis; group 2: Acinetobacter radioresistens; group 3: Acinetobacter johnsonii; group 4: Arthrobacter tumbae, Staphylococcus warneri; group 5: Acinetobacter gerneri; group 6: Bacillus safensis, Pseudomonas synxantha, Acinetobacter schindleri, Acinetobacter bouvetii, Acinetobacter baumannii, Acinetobacter baylyi, Paenibacillus illinoisensis; group 7: Enterobacteriaceae nov. genus (the former Pseudomonas flectens); group 8: Acinetobacter calcoaceticus, Paenibacillus validus, Curtobacterium flaccumfaciens, Erwinia persicina, Asaia siamensis, Pantoea agglomerans, Pantoea septica, Bartonella rattaustraliani, Chryseobacterium indoltheticum, Pseudomonas rhizosphaerae, Pseudomonas trivialis, Pseudomonas viridiflava, Pseudomonas lutea.

C. paradisi and N. glauca nectar samples respectively (Fig. 3). AMOVA analysis revealed significant differences between Acinetobacter species from the nectar bacterial communities that originated from the different plant species (F7,13 = 3.03; P < 0.01), demonstrating that although Acinetobacter was the main genus in all nectar samples, different plant nectar inhabits different Acinetobacter species. The other bacterial classes that were found in much lower abundances in the bacterial communities from the different plants are specified in Fig. 3 (lower part).

Acinetobacter. Acinetobacter species (Gammaproteobacteria) seemed to play an important role in the bacterial communities of flower nectar, as 25% of the cultivated nectar species were identified as novel Acinetobacter species (Table 1). Acinetobacter was also the dominant genus in the 454-pyrosequencing results (Fig. 3, upper part). To assure the novelty of the isolated Acinetobacter species, the Z1-Z2 region of the rpoB gene (coding for RNA polymerase B) of the isolates was amplified, sequenced and compared to known Acinetobacter species (Fig. 4). The different isolates shared 82–85% similarities with the following species: Acinetobacter grimontii (10 isolates), Acinetobacter tjernbergiae (seven isolates), Acinetobacter gerneri (three isolates), Acinetobacter baumannii and Acinetobacter ursingii (one isolate), demonstrating that they belong not only to novel species but most likely to novel genera (Fig. 4). The phylogenetic analyses of the rpoB gene sequences demonstrated that Acinetobacter isolates belong to at least two different groups, both forming an out-group to all known Acinetobacter type strains (Fig. 4).

Fig. 2. Nectar bacterial diversity clustering by plant species. Bacterial diversities of all nectar samples were surveyed by 454-pyrosequencing of 16S rRNA genes. The first two principal coordinates (PC1 and PC2) from the principal coordinate analysis of unweighted UniFrac are plotted for each sample. Each symbol represents a sample, coloured by plant species (N. glauca, green; C. paradisi, red; A. communis, blue). The variance explained by the PCs is indicated on the axes. AMOVA analysis showed significant differences between the bacterial communities from the nectar samples that originated from the different plant species (F7,14 = 3.14; P < 0.01). The 454-pyrosequencing technique demonstrates that nectar from each of the three plant species has a distinct microbial community, while there are no significant differences between nectar microbial communities within nectar samples of the same plant species.

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Fig. 3. Mean class abundances of bacterial communities from nectar samples of the different plant species A. communis, C. paradisi and N. glauca. Most of the sequences belonged to the Gammaproteobacteria (upper part of the figure). The prevalence of the genus Acinetobacter out of the Gammaproteobacteria sequences is indicated. All the rest of the classes are specified in the lower part of the figure.

It seems that a significant fraction of the bacteria in nectar (at least for C. paradisi) are culturable species as seen in the food industry [e.g. raw milk, in which culturable bacteria are considered more than 50% of the total bacteria (Hantsis-Zacharov and Halpern, 2007)]. Interestingly, Acinetobacter isolates grew well on LB or R2A agar plates with the supplement of 20% sucrose but very poorly without the addition of sucrose. Furthermore, isolates could not be transferred more than 10 times from their first isolation (data not shown), suggesting that they are lacking some nutrition from the flower’s nectar. Another interesting phenomenon was that most of the Acinetobacter isolates seemed to produce a mucus matrix. The floral nectar contains high sucrose concentrations – the mean value of 64.4% sucrose, for example, was found in the nectars of 278 plant species pollinated by hummingbirds (Nicolson and Fleming, 2003). It is possible that the nectar’s sucrose is used by the bacteria to produce polysaccharides. However, it is unclear what the chemical composition of these polysaccharides is, and how the bacteria or the plant may benefit from them. Enterobateriaceae gen. nov. sp. nov. Another bacterial species that showed high prevalence in the nectar with 34 isolates from all three plant species were novel Enterobateriaceae species. The novel species showed the highest similarity (but less than 97%) to Pseudomonas flectens which is misclassified as a member of the genus Pseudomonas (Table 1, Fig. 5). Pseudomonas flectens was included in the family Enterobacteriaceae, but an extensive study comparing this species with others from

that family is required for definite taxonomic conclusion (Anzai et al., 2000). Chanbusarakum and Ullman (2008) isolated an unidentified bacterial strain from a western flower thrip. This unidentified species was closely related to our isolates (Fig. 5), thus, possibly indicating that thrips, which are tiny, slender insects, feeding on pollen, might be the vectors of transmission of this species in the flower’s nectar. Secondary metabolites and nectar bacterial isolates Given that the tested nectar is known to contain secondary metabolites, antagonistic interactions of these metabolites with the nectar isolates were tested. The secondary metabolites concentrations in the different plant species are: N. glauca nicotine (0.56 ⫾ 0.12 ppm) and anabasine (5.4 ⫾ 0.90 ppm), A. communis amygdalin (4–10 ppm) and C. paradisi caffeine (94.26 ⫾ 2.90 ppm) (Detzel and Wink, 1993; Kretschmar and Baumann, 1999; London-Shafir et al., 2003; Tadmor-Melamed et al., 2004). Representative isolates from different plant species were spread onto R2A supplemented with 10% sucrose. Secondary metabolites in different concentrations [amygdalin (5, 50 and 1000 ppm), caffeine (95, 200 and 1000 ppm), nicotine (0.6, 5 and 1000 ppm) and anabasine (5, 10 and 1000 ppm)] were added onto paper disks which were placed in the middle of the agar plates. No antagonistic interactions were found between the secondary metabolites and the bacterial isolates. Plant secondary metabolites in floral nectar are mainly recognized as deterrents and toxins for a variety of

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102 S. Fridman, I. Izhaki, Y. Gerchman and M. Halpern 11NS3 10NS2 10N4 13N3 17N2 76 10NS6 10N2 97 12NS2 97 14N2 86 14N1 87 9N6 12N1 18N3 97 66 11NS4 13NS1 11N2 68 11N1 54 9N1 Acinetobacter ursingii NIPH 137T 58 Acinetobacter lwoffii CCM 5581T Acinetobacter schindleri NIPH 1034T 51 Acinetobacter bouvetii CCM 7196T Acinetobacter berezinae LMG 1003T 81 Acinetobacter baylyi CCM 7195T Acinetobacter gerneri CCM 7197T Acinetobacter towneri CCM 7201T Acinetobacter grimontii DSM14968 100 Acinetobacter baumannii ATCC 19606T Genomic sp. 13TU LMG 993 54 Acinetobacter johnsonii LMG 999T Acinetobacter beijerinckii NIPH 838T Acinetobacter parvus NIPH 384T 78 Acinetobacter tjernbergiae CCM 7200T 81 Genomic sp. 16 ATCC 17988 100 Genomic sp. 15BJ LUH 1729 Acinetobacter haemolyticus CCM 2358T Pseudomonas aeruginosa LMG 1242T

Fig. 4. Neighbour-joining phylogenetic tree of bacterial partial rpoB gene sequences. The tree presents isolates that are affiliated to the genus Acinetobacter (Gammaproteobacteria) and type strains in this genus. All Acinetobacter isolates represent novel species. The phylogenetic tree was constructed in MEGA 4.1 software. Bootstrap values greater than 50% are shown at the branch points. Pseudomonas aeruginosa LMG 1242T was used as an out-group. The bar indicates 10% sequence divergence. The letter N in the isolate’s name indicates its nectar origin. Isolate names with no special mark: C. paradisi nectar isolates; bold, underlined letters: N. glauca nectar isolates.

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organisms (Wink, 1999). Hagler and Buchmann (1993) suggested that secondary metabolites in nectar could have an antimicrobial function although currently there is no published data evaluating the antimicrobial effect of nectar secondary metabolites (Adler, 2000). In our experiments we did not find any antagonistic effect of amygdalin, caffeine, nicotine or anabasine (all found in the nectar of the tested plant species) on growth of the bacterial isolates from the different plant species. We tested the effect of the secondary metabolites in vitro. However, it might be the case that these secondary compounds affect bacteria differently in the presence of the other chemical constituents of the nectar (in vivo). Our study indicates that floral nectar is a rich medium for microbial growth despite the notion that several constrains such as osmotic

pressure, nectarins and secondary metabolites may limit bacterial growth. Conclusions Here we demonstrate, for the first time, that floral nectar is a unique, specialized and diverse bacterial habitat. Using culturable and molecular methods we showed that different plant species have distinctive bacterial community composition. Significant differences were found between nectar bacterial communities from different plant species but not between different plants of the same plant species (Figs 1 and 2). Primary selection of the nectar bacteria is unclear; it may be affected by the different chemical compositions of the nectar in

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Bacterial communities in floral nectar 8N6 10NS3 10 4N3 13NS 13 NS3 8N4 9N5 9N 8NS6 5NS5 10N3 13N2 13 9NS3 9NS Enterobacteriaceae Ente obacteriaceae bacterium BFo-2 (EU029106) 9NS5 9NS 5NS1 19NS3 4N1 4NS3 62 5NS2 8N2 Enterobacteriaceae Ente obacteriaceae bacterium M515 (AB461801) 11NS5 8N3 9NS6 9NS 8NS1 12N3 91 9N2 4NS5 19NS2 64 9NS1 9NS 10NS 10 NS5 96 12N2 10NS 10 NS1 13NS 13 NS4 88 19NS1 8NS7 64 10N1 Unidentified thrip gut bacteriu bacterium (AF024609) Pseudomonas flectens ATCC TCC 12775T Tatu tumella ella cit citrea LMG 22049T 80 Yers rsinia ia mass massilie iliens nsis CCUG 5344 53443T Budvicia aquatica DSM 5075T Cedece davisae DS Cedecea DSM 4568T Pantoea eucalypti LMG 24198T Escheric ichia coli KCTC CTC 2441T 57 Cronobacter onobacter turicensis Z3032T Hafnia alvei ATCC TCC 13337T Raoultell ltella plan antic ticola DSM 3069T Kluyvera cryoc cryocrescens escens ATCC TCC 33435T Serr Se rratia atia ureilytic eilytica Ni NiVa 51T 58 Leclerci Lecle eclerci cia ad adec adecarboxylata ecarbo arboxyl xylata GT GTC 1267T 86 99 Ente Enterobacter obacter ludwigii DSM 16688T Moellerella Moelle ella wisconsensi wisconsensis DSM 5676T Pseudomonas aeruginosa LMG 1242T

103

Fig. 5. Neighbour-joining phylogenetic tree of bacterial partial 16S rRNA gene sequences. The tree presents isolates that are affiliated to the family Enterobacteriaceae and type strains in this family. All isolates represent a novel genus. The phylogenetic tree was constructed in MEGA 4.1 software. Bootstrap values greater than 50% are shown at the branch points. The bar indicates 1% sequence divergence. Pseudomonas aeruginosa LMG 1242T was used as an out-group. The letter N in the isolate’s name indicates its nectar origin. Isolate names with no special mark: C. paradisi nectar isolates; bold, underlined letters: N. glauca nectar isolates; white letters on grey background: A. communis nectar isolates.

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the different plant species. The effect of the bacteria on the nectar is also unknown; these bacteria may have a crucial influence on the chemical profile of the nectar. For instance, they may produce volatile compounds which can impact visitation rate by nectar consumers such as pollinators and nectar thieves, thus affecting plant pollination and fitness. Further study on the effect of pollinators or specific vectors that consume the nectar, on bacterial community composition in nectar, distribution and transfer between flowers is needed. Acknowledgements This study was supported by a grant from the Israel Science Foundation (ISF, grant no. 189/08).

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© 2011 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Reports, 4, 97–104