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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
  Bacterial communities in floral nectar emi4_309 97..104 Svetlana Fridman, 1 Ido Izhaki, 1 Yoram Gerchman 2 and Malka Halpern 1,2 * 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. SummaryFloral nectar is regarded as the most importantreward available to animal-pollinated plants to attractpollinators. Despite the vast amount of publicationson nectar properties, the role of nectar as a naturalbacterial habitat is yet unexplored. To gain a betterunderstanding of bacterial communities inhabitingfloral nectar, culture-dependent and -independent(454-pyrosequencing) methods were used. Our find-ings demonstrate that bacterial communities innectar are abundant and diverse. Using culture-dependent method we showed that bacterial commu-nities of nectar displayed significant variation amongthree plant species: Amygdalus communis  , Citrus paradisi  and Nicotiana glauca  . The dominant class inthe nectar bacterial communities was Gammaproteo- bacteria  .About half of the isolates were novel species( < 97% similarities of the 16S rRNA gene with knownspecies). Using 454-pyrosequencing we demon-strated that nectar microbial community are distinctfor each of the plant species while there are no sig-nificantdifferencesbetweennectarmicrobialcommu-nities within nectars taken from different plants of thesame species. Primary selection of the nectar bacte-ria is unclear; it may be affected by variations in thechemical composition of the nectar in each plant. Therole of the rich and diverse nectar microflora in theattraction–repulsion relationships between the plantand its nectar consumers has yet to be explored.Introduction Floral nectar is considered the most important rewardanimal-pollinated plants furnish to attract pollinators(Forcone et al  ., 1997; Bernardello et al  ., 1999). Despitethe dominance of sugar ( > 90% dry weight) in nectar,non-sugar compounds play an important role.These com-pounds ( < 10% dry weight) include amino acids, organicacids, lipids, essential oils, polysaccharides, vitamins,antioxidants, minerals and secondary metabolites (Bakerand Baker, 1983; Dafni, 1992; Carter et al  ., 2006).Nectar consumers such as insects, birds and bats weresuggested to transfer microflora among flowers, andbetween flowers and other plant organs (Sandhu andWaraich, 1985). However, floral nectar was suggested tobe not suitable as bacterial habitat and was even demon-strated to have antimicrobial properties (Sasu et al  .,2010). These properties could be due to several chemicalcomponents that were suggested to limit growths ofmicroflora in the nectar: (i) high sugar concentration innectars may impose high osmotic pressure that con-strains microbial growth (Pusey, 1999; Brysch-Herzberg,2004); (ii) nectar-associated proteins were suggested tofunction as a defensive mechanism against microorgan-ism infections by producing reactive oxygen molecules(Carter and Thornburg, 2004a–c; González-Teuber et al  .,2009; Harper et al  ., 2010); and (iii) secondary metabolitessuch as phenolics have also been suggested to play anantimicrobial role in nectar (Hagler and Buchmann, 1993).Based on these constraints (e.g. Minorsky, 2007), onemay expect that floral nectar is populated by only a fewmicrobiota groups that are adapted to live in such extremeenvironment.Although some publications have indicated the pres-ence of microorganisms in floral nectar, most of themdescribed 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 correlationwas found between yeast density and sugar content, aswell as yeast density and nectar concentration in a Wat- sonia  species (de Vega et al  ., 2009). Herrera and Pozo(2010) described a phenomenon whereby the sugarcatabolism of yeast populations inhabiting floral nectarcan increase its temperature and thus modify the thermalmicroenvironment within the flower. However, despitethe vast amount of publications on nectar properties, therole of nectar as a natural habitat for microorganismsand specifically for bacteria is yet unexplored. Microbialcommunities in nectar may affect the nectar’s chemicalprofile, thus directly controlling nectar consumption byflower visitors such as pollinators and nectar thieves, and Received 22 July, 2011; accepted 27 October, 2011. *For correspon-dence. E-mail; Tel. ( + 972) 4 9838727;Fax ( + 972) 4 9838911. Environmental Microbiology Reports (2012) 4 (1), 97–104 doi:10.1111/j.1758-2229.2011.00309.x  © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd  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 under-standing of floral nectar bacterial communities. To thatend, we studied the bacterial communities in flower nectarof three plant species: Nicotiana glauca, Amygdalus com- munis  and Citrus paradisi  in Northern Israel. Here weshow that (i) bacteria are common inhabitants of floralnectar and (ii) each plant species floral nectar support aunique 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 consid-ered 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 thatnectarins, proteins which accumulate in the nectar ofornamental tobacco plants, produce very high levels ofhydrogen peroxide (up to 4 mM). This might be the casefor some bacterial strains, but as we show here, manyothers thrive in floral nectar. Nectar sampling and bacterial counts  Flower nectar from three plant species: N. glauca  (TreeTobacco) , A. communis  (Almond) and C. paradisi  (Grape-fruit) were collected from flowers of each sampled plant(fivedifferentplantsforeachplantspecies)betweenMarchand June 2009.All the sampled plants were located withina radius distance of up to 10 kilometres in Northern Israel.Thesethreeplantspecieswerechosenbecausetheirfloralnectar is known to contain secondary metabolites whichare considered as an antimicrobial agent: N. glauca  con-tains nicotine and anabasine, A. communis  containsamygdalin and C. paradisi  contains caffeine (Detzel andWink, 1993; Kretschmar and Baumann, 1999; London-Shafir et al  ., 2003; Tadmor-Melamed et al  ., 2004).Using DAPI, we found that bacterial counts per millilitrein the nectar samples from N. glauca, A. communis  and C. paradisi  were 1.4 ¥ 10 7 ( Ϯ 1.6 ¥ 10 6 ), 1.7 ¥ 10 7 ( Ϯ 4.8 ¥ 10 6 ) and 3.1 ¥ 10 7 ( Ϯ 2.0 ¥ 10 6 ) respectively.Bacterial cfu ml - 1 in the nectar samples from the differentplants were approximately 50%, 25% and 10% from theDAPI counts respectively. These results demonstrate thatbacteria thrive in floral nectar. These high abundances ofbacteria ( > 10 6 cfu per millilitre nectar) were about twomagnitude higher compared with what was observed foryeasts 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 spreadonto R2A agar (Himedia) and R2A agar supplementedwith 20% sucrose. One hundred representative isolateswere identified by amplifying and sequencing the 16SrRNA gene (Appendix S1).About 33%, 75% and 42% of A. communis  , C. paradisi  and N. glauca  nectar isolates, respectively, were found tobe novel species ( < 97% similarities in the 16S rRNAgenesequences to known species) (Table 1). This demon-strates that indeed, the nectar is an unexplored bacterialniche. 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). Isolatesbelonging to the Bacilli  class also occurred in the nectarfrom all plant species. Actinobacteria  was identified onlyfrom A. communis  and N. glauca  . Representatives of the Alphaproteobacteria  and Flavobacteria  classes werefound only in the A. communis  nectar. Interestingly, themost abundant species in the nectar were a novel uniden-tified 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 bac-terial communities from different plant species (Table 1and Fig. 1). Figure 1 displays the results from the canoni-cal correspondence analysis using the CANOCO computerprogram. The distribution of the bacterial species alongthe ordinates was not random according to the MonteCarlo test ( F  = 1.14, P  < 0.05) and thus can be explainedby their different plant species source. Plant speciesexplained 42% of the variation in the bacterial communitycomposition whereas the horizontal and the vertical axesexplained 24% and 18% of the variation respectively(Fig. 1). 454-pyrosequencing of 16S rRNA genes  Bacterial diversity in all nectar samples was surveyed by454-pyrosequencing of 16S rRNA genes (five samplesper plant species, 15 samples in total). A total of approxi-mately 10 000 sequences per sample were obtained.Nevertheless, after chloroplasts and Archaea  sequenceswere removed from the analysis, about 3200–7000sequences per sample were analysed (77 077sequences, in total) (see also Appendix S1).Sequences were assigned to species-level operationaltaxonomic units (OTUs) using a 97% pairwise-identitycut-off. In sum, 2197 OTU’s were obtained for all 15samples with an average of 401, 379 and 207 OTU’s98 S. Fridman, I. Izhaki, Y. Gerchman and M. Halpern   © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Reports  , 4 , 97–104  per sample and coverage of 95.8%, 97.2% and 91.4% for A. communis  , C  . paradisi  and N. glauca  nectar bacterialcommunities, respectively. Chao1 richness estimator for A. communis  , C  . paradisi  and N. glauca  nectar bacterialcommunities was, 689, 629 and 418, respectively (seealso Appendix S1).This analysis revealed clustering of samples by theirplant srcin, except for one nectar sample from N. glauca  that overlapped with C. paradici  samples (Fig. 2).However, when AMOVA analysis was applied, significantdifferences were found between the bacterial com-munities from the nectar samples that originatedfrom the three different plant species ( F  7,14 = 3.14; P  < 0.01). AMOVA post hoc analysis revealed significantdifferences between the bacterial communities fromthe different plant species nectar ( A. communis  vs. C  . paradisi, F  1,9 = 3.97, P  < 0.01; A. communis  vs. N. glauca, F  1,9 = 3.06, P  < 0.01; C  . paradisi  vs. N. glauca F  1,9 = 3.97, P  < 0.05). This indicates that nectar fromeach plant species has a distinct microbial community(Fig. 2).The majority of the sequences from all the nectarsamples were classified as Proteobacteria  ( > 83.0%). Gammaproteobacteria  was the dominant class and com-prised 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, 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  1 (100) Bartonella rattaustraliani  1 (97.2) Gammaproteobacteria  Acinetobacter baumannii  2 (94.7–96.0) Acinetobacter baylyi  6 (95.5–96.1) Acinetobacter bouvetii  1 (95.1) Acinetobacter  1 (96.2) calcoaceticus Acinetobacter gerneri  6 (95.8–96.3) 1 (96.1) Acinetobacter johnsonii  2 (95.2–95.5) 1 (96.6) Acinetobacter radioresistens  1 (96.1) 3 (95.9–96.1) Acinetobacter schindleri  2 (95.1–96.2) Erwinia amylovora  5 (96.9–97.4) Erwinia persicina  2 (98.5–99.6) Pantoea agglomerans  2 (99.3) Pantoea septica  1 (98.7) Pseudomonas flectens  a 14 (96.2–97.1) 17 (96.5–97.1) 3 (94.5–96.7) Pseudomonas lutea  2 (98.8–99.9) Pseudomonas rhizosphaerae  1 (99.0) Pseudomonas trivialis  1 (100) Pseudomonas viridiflava  2 (99.3–100) Pseudomonas synxantha  1 (99.7) Actinobacteria Arthrobacter tumbae  1 (99.7) 1 (99.8) Arthrobacter pascens  1 (99.9) Curtobacterium flaccumfaciens  1 (98.5) Kocuria kristinae  1 (98.0) Bacilli Bacillus megaterium  3 (97.9–99.6) Bacillus safensis  1 (99.5) Paenibacillus illinoisensis  1 (94.8) Paenibacillus validus  2 (99.1) Staphylococcus epidermidis  4 (98.9–100) Staphylococcus warneri  1 (100) 1 (99.9) Flavobacteria Chryseobacterium indoltheticum  1 (98.5) a. Isolates that were identified as most closely related to Pseudomonas flectens  do not belong to the Pseudomonas  genus and are in fact novelspecies 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 rRNAgene similarities to the closest known species. Isolates with less than 97.5% 16S rRNA gene similarities to known species are most likely novelspecies and the name of their closest relative species is marked in bold.The isolates were identified by comparing their 16S rRNAgene sequencesto that of the GenBank database (EZtaxon version 2.1. Sequences lengths were at least 850 bp. Sequences lengthobtained 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. Bacterial communities in floral nectar  99  © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Reports  , 4 , 97–104  C  . paradisi  and N. glauca  nectar samples respectively(Fig. 3). AMOVA analysis revealed significant differencesbetween Acinetobacter  species from the nectar bacterialcommunities that originated from the different plantspecies ( F  7,13 = 3.03; P  < 0.01), demonstrating thatalthough Acinetobacter  was the main genus in all nectarsamples, different plant nectar inhabits different Acineto- bacter  species. The other bacterial classes that werefound in much lower abundances in the bacterial commu-nities from the different plants are specified in Fig. 3(lower part). Novel bacterial species in floral nectar Acinetobacter  . Acinetobacter  species ( Gammaproteo- bacteria  ) seemed to play an important role in the bacterialcommunities of flower nectar, as 25% of the cultivatednectar species were identified as novel Acinetobacter  species (Table 1). Acinetobacter  was also the dominantgenus in the 454-pyrosequencing results (Fig. 3, upperpart). To assure the novelty of the isolated Acinetobacter  species, the Z1-Z2 region of the rpoB  gene (coding forRNA 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 gri- montii  (10 isolates), Acinetobacter tjernbergiae  (sevenisolates), Acinetobacter gerneri  (three isolates), Acineto- bacter baumannii  and Acinetobacter ursingii  (one isolate),demonstrating that they belong not only to novel speciesbut most likely to novel genera (Fig. 4). The phylogeneticanalyses of the rpoB  gene sequences demonstrated that Acinetobacter  isolates belong to at least two differentgroups, both forming an out-group to all known Acineto- bacter  type strains (Fig. 4). Fig. 1. Ordination diagram (calculated with CANOCO software)showing variation in the abundance of bacterial species isolatesamong the three plant species. This joint plot data analyse therelationship between bacterial species and plant species. Theenvironmental variables are displayed as arrows radiating from thecentre of the diagram. The length of the arrows represents thecontribution of each plant species to the variation of the sample.The angle in between two arrows is a measure for the correlationbetween the two variables (small angle means high correlation),and the projection of a taxa point on an arrow is a measure for therelative value of that point; in other words, for the position of thatpoint on the gradient described by the arrow.The green triangles represent different bacterial species. Theidentity 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  . Fig. 2. Nectar bacterial diversity clustering by plant species.Bacterial diversities of all nectar samples were surveyed by454-pyrosequencing of 16S rRNA genes. The first two principalcoordinates (PC1 and PC2) from the principal coordinate analysisof unweighted UniFrac are plotted for each sample. Each symbolrepresents a sample, coloured by plant species ( N. glauca  , green; C  . paradisi, red; A. communis  , blue). The variance explained by thePCs is indicated on the axes. AMOVA analysis showed significantdifferences between the bacterial communities from the nectarsamples that srcinated from the different plant species( F  7,14 = 3.14; P  < 0.01). The 454-pyrosequencing techniquedemonstrates that nectar from each of the three plant species hasa distinct microbial community, while there are no significantdifferences between nectar microbial communities within nectarsamples of the same plant species. 100 S. Fridman, I. Izhaki, Y. Gerchman and M. Halpern   © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Reports  , 4 , 97–104  It seems that a significant fraction of the bacteria innectar (at least for C. paradisi  ) are culturable species asseen in the food industry [e.g. raw milk, in which culturablebacteria are considered more than 50% of the total bac-teria (Hantsis-Zacharov and Halpern, 2007)]. Interest-ingly, Acinetobacter  isolates grew well on LB or R2A agarplates with the supplement of 20% sucrose but verypoorly without the addition of sucrose. Furthermore, iso-lates could not be transferred more than 10 times fromtheir first isolation (data not shown), suggesting that theyare lacking some nutrition from the flower’s nectar.Another interesting phenomenon was that most of the Acinetobacter  isolates seemed to produce a mucusmatrix. The floral nectar contains high sucrose concentra-tions – the mean value of 64.4% sucrose, for example,was found in the nectars of 278 plant species pollinatedby hummingbirds (Nicolson and Fleming, 2003). It is pos-sible that the nectar’s sucrose is used by the bacteria toproduce polysaccharides. However, it is unclear what thechemical composition of these polysaccharides is, andhow the bacteria or the plant may benefit from them. Enterobateriaceae  gen. nov. sp. nov . Another bacterialspecies that showed high prevalence in the nectar with 34isolates from all three plant species were novel Enteroba- teriaceae  species. The novel species showed the highestsimilarity (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 anextensive study comparing this species with others fromthat family is required for definite taxonomic conclusion(Anzai et al  ., 2000). Chanbusarakum and Ullman (2008)isolated an unidentified bacterial strain from a westernflower thrip. This unidentified species was closely relatedto our isolates (Fig. 5), thus, possibly indicating that thrips,which are tiny, slender insects, feeding on pollen, might bethe vectors of transmission of this species in the flower’snectar. Secondary metabolites and nectar bacterial isolates  Given that the tested nectar is known to contain second-ary metabolites, antagonistic interactions of thesemetabolites with the nectar isolates were tested. The sec-ondary metabolites concentrations in the different plantspecies are: N. glauca  nicotine (0.56 Ϯ 0.12 ppm) andanabasine (5.4 Ϯ 0.90 ppm), A. communis  amygdalin(4–10 ppm) and C. paradisi  caffeine (94.26 Ϯ 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 plantspecies were spread onto R2A supplemented with 10%sucrose. Secondary metabolites in different concentra-tions [amygdalin (5, 50 and 1000 ppm), caffeine (95, 200and 1000 ppm), nicotine (0.6, 5 and 1000 ppm) and ana-basine (5, 10 and 1000 ppm)] were added onto paperdisks which were placed in the middle of the agar plates.No antagonistic interactions were found between the sec-ondary metabolites and the bacterial isolates.Plant secondary metabolites in floral nectar are mainlyrecognized as deterrents and toxins for a variety of Fig. 3. Mean class abundances of bacterialcommunities from nectar samples of thedifferent plant species A. communis  , C  . paradisi  and N. glauca  . Most of thesequences belonged to the Gammaproteobacteria  (upper part of thefigure). The prevalence of the genus Acinetobacter  out of the Gammaproteobacteria  sequences is indicated.All the rest of the classes are specified in thelower part of the figure. Bacterial communities in floral nectar  101  © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Reports  , 4 , 97–104
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