Structure and Activity of Bacterial Community Inhabiting Rice Roots and the Rhizosphere

Blackwell Publishing LtdOxford, UKEMIEnvironmental Microbiology 1462-2912© 2006 The Authors; Journal compilation © 2006 Society for Applied Microbiology and Blackwell Publishing Ltd? 20068813511360Original ArticleBac- teria inhabiting rice roots and the rhizosphereY. Lu, D. Rosencrantz, W. Liesack and R. Conrad 中国科技论文在线 http://www.paper.edu.cn

Environmental Microbiology (2006) 8(8), 1351–1360 doi:10.1111/j.1462-2920.2006.01028.x

Structure and activity of bacterial community inhabiting rice roots and the rhizosphere

1,2 2 2 Yahai Lu, Dirk Rosencrantz, Werner Liesack and Plants, the primary producers, assimilate CO2 and distrib- Ralf Conrad2* ute the organic assimilates to the below-ground biota. The 1College of Resources and Environmental Sciences, below-ground biota, the degraders, transform the organic China Agricultural University, Beijing 100094, China. nutrients into inorganic compounds facilitating the recy- 2Max-Planck-Institute for Terrestrial Microbiology, Karl- cling of nutrients by the primary producers. This above- von-Frisch-Straße, 35043 Marburg, Germany. and below-ground feedback interaction constitutes the basis for ecological functioning of soil ecosystems (Wardle et al., 2004). The below-ground carbon ﬂow has been Summary investigated in various soil–plant systems (Lynch and Root-derived carbon provides a major source for Whipps, 1990). However, little is known about the soil

microbial production and emission of CH4 from rice microbiota that mediate the rhizosphere carbon dynamics. field soils. Therefore, we characterized the structure In carbon dynamics models (Paustian et al., 1997), the and activity of the bacterial community inhabiting rice below-ground biota, which consists of complex and roots and the rhizosphere. In the first experiment, diverse eukaryotic and prokaryotic life, is often considered DNA retrieved from rice roots was analysed for bac- as a ‘black box’. terial 16S rRNA genes using cloning, sequencing and In rice field soils, the below-ground carbon flow provides

in situ hybridization. In the second experiment, rice an important carbon source for methane (CH4) production 13 plants were pulse-labelled with CO2 (99% of atom and emission. Early studies speculated that increasing 13C) for 7 days, and the bacterial RNA was isolated root exudation due to accelerated plant growth caused the

from rhizosphere soil and subjected to density late season maxima of CH4 emission (Seiler et al., 1984; gradient centrifugation. RNA samples from density Schütz et al., 1989a). Isotope tracer experiments proved fractions were analysed by terminal restriction that the plant-photosynthesized carbon was rapidly (within fragment length polymorphism ﬁngerprinting, cloning 3–5 h) allocated to the below-ground biosphere, trans-

and sequencing. The experiments showed that the formed to CH4 and emitted into the atmosphere (Minoda dominant bacteria inhabiting rice roots and the and Kimura, 1994; Minoda et al., 1996; Dannenberg and rhizosphere particularly belonged to the Alphapro- Conrad, 1999). However, the microbiological mechanisms

teobacteria, Betaproteobacteria and Firmicutes. The of root exudate decomposition and CH4 production

RNA stable isotope probing revealed that the bacteria remained unclear. Owing to leakage of O2 and organic actively assimilating C derived from the pulse- substances from roots, the rice roots and the rhizosphere labelled rice plants were Azospirillum spp. (Alphapro- provide niches for diverse organisms performing various

teobacteria) and members of Burkholderiaceae biogeochemical processes. The leakage of O2 supports (Betaproteobacteria). Both anaerobic (e.g. Clostridia) the oxidation of ammonia to nitrite, sulﬁde to sulfate, fer-

and aerobic (e.g. Comamonas) degraders were rous iron to ferric iron, and CH4 to CO2. On the other hand, present at high abundance, indicating that root envi- denitriﬁcation, reduction of iron and sulfate and methano- ronments and degradation processes were highly het- genesis occur in the adjacent zone of anaerobic soil (Lie- erogeneous. The relative importance of iron and sack et al., 2000). Organic substances released from rice sulfate reducers suggested that cycling of iron and roots serve as an important carbon and energy source for sulfur is active in the rhizosphere. the microbial activities in the rhizosphere. Some of the organisms responsible for the turnover of C, N, S and Fe have been identiﬁed using either cultivation-dependent or Introduction cultivation-independent methods. For example, the meth- It is widely recognized that the above-ground and below- ane-oxidizing bacteria in the rice rhizosphere include both ground biosphere supports and codepends on each other. type I and type II methanotrophs (Bodelier et al., 2000; Eller and Frenzel, 2001; Horz et al., 2001), but rice plants Received 14 December, 2005; accepted 22 February, 2006. *For + correspondence. E-mail [email protected]; Tel. (+49) and NH4 fertilization apparently stimulate the activity of 6421 178 801; Fax (+49) 6421 178 809. the type I methanotrophs (Bodelier et al., 2000; Eller and

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1352 Y. Lu, D. Rosencrantz, W. Liesack and R. Conrad

Frenzel, 2001). Nitrosomonas is considered the major Table 1. Phylogenetic affiliation and number of bacterial 16S rRNA ammonia oxidizer on rice roots (Nicolaisen et al., 2004). gene clones retrieved from rice roots at the age of 45 days and 90 days. The structure of nitrate-reducing bacteria on rice roots is unclear, but the addition of nitrate largely increased the Clone library Day 45 Day 90 growth of Bacillus and Dechloromonas (Scheid et al., Phylogenetic group 2004), which are capable of denitrification. Both Gram- Alphaproteobacteria positive Desulfosporosinus spp. and members of Desulfo- Bradyrhizobiaceae 1 bacteraceae and Desulfovibrionaceae are possibly Caulobacteraceae 1 Rhizobiaceae 2 responsible for sulfate reduction in the Italian rice soils Hyphomicrobiaceae 11 (Scheid and Stubner, 2001; Scheid et al., 2004). Geo- Rhodospirillaceae 1 bacter, Pelobacter and, interestingly, the Anaeromyxo- Sphingomonadaceae 4 Rickettsiaceae 4 bacter group seem to be involved in ferric iron reduction Betaproteobacteria in the rhizosphere (Treude et al., 2003; Scheid et al., Comamonadaceae 215 2004). Rhodocyclaceae 26 Hydrogenophilaceae 2 An analysis of carbon flow through the different com- Deltaproteobacteria partments of the rhizospheric microbiota is essential to Geobacteraceae 2 understand the microbe-driven C and nutrient cycling in Myxococcales 66 Gammaproteobacteria the rhizosphere. Phospholipid fatty acids-based stable Pseudomonadaceae 1 isotope probing (SIP) has been used to link the rhizo- Firmicutes spheric carbon flow to the microbial community (Butler Clostridium 87 Sporomusa 25 et al., 2003; Lu et al., 2004; Treonis et al., 2004). However, Cyanobacteria 1 phospholipid fatty acids-SIP can only provide a coarse Actinobacteria 11 resolution of the microbial phyla (Singh et al., 2004). Acidobacteria 52 Bacteroidetes 27 Recently, Rangel-Castro and colleagues (2005) applied 13 RNA-SIP to a CO2-pulse-labelled grassland community and found that only a few members of the highly diverse community of bacteria, archaea and fungi became specif- daceae and Rhodocyclaceae of the Betaproteobacteria ically labelled if the grassland had been limed. each accounted for approximately 5% of total clones. We have previously identified the key archaeal groups In the 90-day library, Comamonadaceae-like clone

involved in CH4 production in the rice rhizosphere by sequences were most abundant, followed by those 13 applying RNA-SIP to a CO2-pulse-labelled archaeal assigned to Clostridia and Bacteroidetes. Myxococcales, community (Lu and Conrad, 2005). In the present study, Rhodocyclaceae, Sporomusa, Sphingomonadaceae and the bacterial community inhabiting rice roots and rhizo- Rickettsiaceae each accounted for 5–10% of total clones. sphere was determined by a combination of 16S rRNA The frequency of Deltaproteobacteria-like clone se- gene clone library analysis and rRNA-targeted fluores- quences relatively decreased while the clone frequency cence in situ hybridization (FISH). The active populations of Alpha- and Betaproteobacteria (20% and 36% of 64 were identified by RNA-SIP analysis using material from clones respectively) increased in the 90-day library as our previous pulse-labelling experiment (Lu and Conrad, compared with the 45-day library. The sequences related 2005). to Proteobacteria accounted for 44–66% of total clones sequenced in both clone libraries. Results Fluorescence in situ hybridization revealed that bacterial cells were distributed both on and inside the root Structure of bacterial community on rice roots tissues (Fig. 1A and B). Highly dense populations of Bac- (experiment I) teria were detected on root tips (Fig. 1C). Members of the Two clone libraries were constructed to characterize the Betaproteobacteria were the most abundant populations bacterial community inhabiting rice roots: one from 45- both on 27- and 85-day-old roots. Alphaproteobacteria, on day-old rice roots (34 clones) and the other from 90-day- the other hand, were barely detectable by FISH. old rice roots (64 clones). Comparative analyses of environmental 16S rRNA gene sequences showed that the Active bacteria associated with rhizosphere carbon flow root-associated bacterial community is phylogenetically (experiment II) diverse (Table 1). In the 45-day library, clostridial-like clones were most abundant, followed by those assigned Rice plants were grown for 7 days with repeated pulses 13 13 to the Myxococcales (Deltaproteobacteria) and Acidobac- of CO2 (99% of atom C). We have previously shown

teria. Members of the Bacteroidetes, and Comamona- that c. 15% of CH4 and 18% of CO2 in the porewater of

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Bacteria inhabiting rice roots and the rhizosphere 1353

Fig. 1. Fluorescence in situ hybridization A B (FISH) of indigenous bacteria on rice roots with oligonucleotide probe EUB338 (labelled with Cy5), ALF1b (Texas Red) and BET42a (fluores- cein), representing the Bacteria, Alpha- and Betaproteobacteria respectively. The pictures were created with confocal laser scanning microscopy (Grosskopf et al., 1998); the colours are not identical with those represented by the dye used. The scale bars indicate 5 µm. A. FISH detection of bacteria on the root sur- face of 27-day-old rice roots. Violet and blue dots (examples indicated by arrows) represent the Betaproteobacteria and unspecified Bacte- ria respectively. The Alphaproteobacteria were rarely detected. 5 µm 5 µm B. FISH detection of bacteria inside the tissues of 17-day-old rice roots. Violet and green dots C (examples indicated by arrows) represent the Betaproteobacteria and Alphaproteobacteria respectively. C. FISH detection of bacteria on the tip of 17- day-old rice roots. Violet, green and blue dots (examples indicated by arrows) represent the Betaproteobacteria, Alphaproteobacteria and unspecified Bacteria respectively.

5 µm

the rice rhizosphere were 13C-labelled (Lu and Conrad, A 2005). RNA-SIP showed that the Rice Cluster I lineage 1.2 was highly labelled compared with other archaeal metha- Total RNA RNA with 13C labelling RNA without labellabelling nogens. Here we examine the bacterial communities in 0.8 the same samples with a similar SIP approach. RNA 13 0.4

isolated from both the C-labelled soil and the non- m quantities) labelled rhizospheric soil was subjected to density frac- 0.0 ximu tionation, followed by reverse transcription polymerase 1.75 1.77 1.79.1.811.83 1.81 1.83 chain reaction (RT-PCR) and terminal restriction frag- of ma ment length polymorphism (T-RFLP) ﬁngerprinting of B tio tio 1.2

bacterial 16S rRNA. The average buoyant density (BD) of (ra Bacterial RNA total as well as bacterial RNA was slightly greater for the 13C-labelled soil than for the non-labelled control (Fig. 2A 0.8 and B). The T-RFLP fingerprints showed that the bacterial 0.4 community was similar in RNA fractions with BD of RNA abundance ≤1.796 g ml−1(Fig. 3A). However, a significant change 0.0 occurred at BD of ≥1.801 g ml−1 for the labelled soil. 1.75 1.771. 1.79 1.81 1.83 Specifically, the 150 bp T-RF markedly increased in the RNA buoyant density in CsTFA (g ml-1) fractions of BD between 1.801 g ml−1 and 1.816 g ml−1. ≥ Fig. 2. Caesium trifluoroacetate density gradient centrifugation of The T-RF with a length of 475 bp appeared at BD RNA extracted from rice rhizosphere soil with and without 13C label- − 1.801 g ml 1 and markedly increased with increasing of ling. The amount of RNA in gradient fractions was quantified fluoro- RNA BD. On the other hand, the 439 bp T-RF was most metrically (A) and by real-time PCR using the bacterial domain- specific primer system Ba519f/Ba907r (B). abundant in the ‘light’ fractions, but decreased in the heavy fractions. The RNA T-RFLP fingerprints from the

1354 Y. Lu, D. Rosencrantz, W. Liesack and R. Conrad

A RNA from labelled soil B RNA from non-labelled soil

439 [1772] 150 490 [1772] 160 - 164 516 - 520

LrhB [1793] [1784]

[1796] [1793]

[1801] [1801] 475

[1808] [1808]

HrhB [1816] [1816]

[1823] [1823]

100 200 300 400 500 600 700 800 T-RF length (bp) T-RF length (bp)

Fig. 3. Bacterial T-RFLP ﬁngerprints of RNA retrieved from density-resolved gradient fractions of 13C-labelled rhizospheric soil (A) and non-labelled control soil (B). The amplicons were generated by RT-PCR with bacterial 27f/907r primers and digested with MspI. CsTFA buoyant densities of the respective gradient fractions (mg/ml) are given in brackets. The numbers near the T-RF peaks denote the T-RF lengths. LrhB and HrhB indicate the RNA fractions used for construction of bacterial ‘light’ and ‘heavy’ clone libraries.

non-labelled soil did not reveal obvious change within any Within the Proteobacteria, the Alphaproteobacteria of the gradient fractions (Fig. 3B). The fingerprints of the accounted for more than 50% of the sequences in both non-labelled soil were all similar to those of the ‘light’ RNA HrhB and LrhB (Table 2). In the HrhB library, the fractions from the labelled soil. The comparison of T-RFLP sequences related to Azospirillum within the Alphaproteo- fingerprints between labelled soil and non-labelled soil bacteria were most abundant followed by those related to indicates that in particular the bacterial populations with the members of the Burkholderiaceae within the Beta- the characteristics of 150 bp and 475 bp T-RFs became proteobacteria. In contrast, the Magnetospirillum-like labelled with 13C derived from rice assimilates. sequences were most abundant in the LrhB library, while To identify organisms present in the rhizosphere and to those related to Azospirillum were less abundant. None assign the T-RFs to the individual populations, we con- of the Burkholderiaceae-like sequences were detected structed two clone libraries for bacterial 16S rRNA from in the LrhB library. The frequency and diversity of the the ‘light’ RNA (LrhB) and ‘heavy’ RNA (HrhB) fractions. sequences affiliated with the Deltaproteobacteria, Of 92 clones sequenced from the HrhB library, 77% were Clostridia and Actinobacteria decreased in the HrhB ver- affiliated to Proteobacteria, 5% clustered with Clostridium, sus LrhB library. Pseudomonadaceae and Caulobacteri- and the remaining 18% belonged to diverse phylogenetic aceae were detected in the HrhB library at low clone groups including Actinobacteria, Fusobacteria, Cyano- frequency, whereas Methylomonadaceae, Nitromona- bacteria, Acidobacteria, Chloroflexi, Bacteroidetes, Verru- daceae, Syntrophobacteraceae and Desulfovibrionaceae comicrobia and Planctomycetes (Fig. 4 and Table 2). Of were exclusively detected in the LrhB library (Table 2). 84 clones sequenced from the LrhB library, 69% were In silico prediction of T-RF sizes from sequence data affiliated to Proteobacteria, 11% belonged to Firmicutes, allows the assignment of the major T-RFs observed in the and 20% belonged to the same diverse groups listed bacterial fingerprints to defined phylogenetic lineages above. These results indicated that the Proteobacteria (Fig. 4). The comparison between T-RFLP fingerprinting were predominant, while the Firmicutes and other groups and clone library indicated that the relative increase of the were relatively less abundant in the rice rhizosphere. 150 bp and 475 bp T-RFs in the ‘heavy’ RNA fractions for

Bacteria inhabiting rice roots and the rhizosphere 1355

Azospirillum brasilense, Z29617 HRhB31, [150] Azospirillum HRhB09, [150] Azospirillum strain, AY118223 HRhB67, [150] Azospirillum lipoferum, Z29619 LRhB84, [439] HRhB90, [439] Magnetospirillum Dechlorospirillum strain, AY530551 Magnetospirillum magnetotactic, Y10110 Magnetospirillum strain, Y17390 LRhB35, [401] HRhB69, [401] Rhizobiaceae Rhizobium radiobacter, AJ389890 Sinorhizobium strain, AF357225 Methylocystis strain, AB015608 HRhB68, [150] Rasbo bacterium, AF007948 Methylocystaceae LRhB45, [152] Rice soil clone KCB90, AJ229247 HRhB08, [475] HRhB29, [475] HRhB17, [475] Burkholderiales Paucimonas lemoignei, X92554 Ralstonia solanacearum, AB024609 Burkholderia cepacia, X87275 LRhB05, [428] HRhB24, [428] Dechloromonas strain, AF170354 Rhodocyclaceae Azospira oryzae, AF011347 Soil clone Rufe9b, AY235688 Variovorax paradoxus, AB008000 HRhB02, [490] Comamonadaceae LRhB76, [457] Acidovorax strain, AF235013 HRhB10, [483] Pseudomonaceae Pseudomonas saccharophila, AF368755 Geobacter bremensis, U96917 LRhB63, [161] Geobacteraceae HRhB14, [163] Geobacter strain, Y19190 Desulfacinum hydrothermale, AF170417 LRhB79, [129] HRhB15, [129] Anaeromyxobacter dehalogenans, AF382400 Myxococcales Cystobacter violaceus, AJ233905 Myxococcales strain, AJ233948 LRhB20, [164] LRhB38, [164] Desulfovibrionaceae Desulfovibrio strain, Y17756 Desulfovibrio burkinabensis, AF053752 Clostridium acidisoli, AJ237756 LRhB59, [516] Clostridium strain, Y15985 LRhB10, [520] Clostridium aurantibutyricum, S46736 X68183 LRhB23, [528] Firmicutes Caldoanaerobacter indicus, X75788 Clostridium strain, AJ229234 LRhB25, [476] Soil clone, AF050580 HRhB27, [207] Clostridium papyrosolvens, X71852 Actinobacteria

Fusobacteria

Cyanobacteria

Acidobacteria

Chloroflexi

Bacteroidetes

Verrucomicrobia

Planctomycetes

Methanosarcina mazei, X69874 Methanomicrobium mobile, M59142 Methanopyrus kandleri, M59932

0.10

Fig. 4. Phylogenetic relationship of bacterial 16S rRNA clone sequences retrieved from density-resolved ‘heavy’ and ‘light’ RNA fractions. The T- RF sizes of clone sequences digested with MspI are shown in brackets. The scale bar represents 10% sequence divergence. GeneBank accession numbers of the reference sequences are indicated.

1356 Y. Lu, D. Rosencrantz, W. Liesack and R. Conrad

Table 2. Phylogenetic affiliation and number of bacterial 16S rRNA Methylocystaceae, Burkholderiaceae, Comamonadaceae, clones for ‘light’ (LrhB) and ‘heavy’ (HrhB) RNA retrieved from rice Rhodocyclaceae, Pseudomonadaceae, Geobacteraceae, rhizosphere soil. Myxococcales, Desulfovibrionaceae, Clostridia, Acido- RNA fractions ‘Light’ (LrhB) ‘Heavy’ (HrhB) bacteria, Verrucomicrobia and Bacteroidetes (listed according to taxonomy). Azospirillum spp. are capable of Alphaproteobacteria Azospirillum 10 23 fixing atmospheric N2 and hence could promote plant Magnetospirillum 18 9 growth (Steenhoudt and Vanderleyden, 2000). Magneto- Caulobacteraceae 1 spirillum spp. can grow anaerobically with nitrate as ter- Methylocystaceae 31 Rhizobiaceae 23minal electron acceptor or aerobically with atmospheric Betaproteobacteria O2 (Taoka et al., 2003). The strictly aerobic Burkholderi- Burkholderiaceae 13 aceae, Comamonadaceae and Pseudomonadaceae are Rhodocyclaceae 34 Gammaproteobacteria physiologically diverse and could degrade wide ranges of Pseudomonadaceae 4 organic substances. Members of the Clostridia, Acidobac- Comamonadaceae 22teria and Bacteroidetes are probably responsible for the Methylomonadaceae 1 Nitrosomonadaceae 1 anaerobic decomposition of decaying root residues. Coex- Deltaproteobacteria istence of these aerobic and anaerobic degraders strongly Geobacteraceae 67suggests that root environments and degradation Myxococcales 54 Syntrophobacteraceae 2 processes are highly heterogeneous. Anaeromyxobacter Desulfovibrionaceae 5 spp. within the order Myxococcales together with Geo- Firmicutes bacter spp. accounted for 16–17% of total clones obtained Clostridia 85 Bacillaceae 1 from rhizospheric microbial RNA (Tables 1 and 2). These Actinobacteria 51iron reducers (Lonergan et al., 1996; Treude et al., 2003) Fusobacteria 11possibly play an important role in C and Fe dynamics in Cyanobacteria 21 Acidobacteria 13the rice rhizosphere where re-oxidation of Fe(II) occurs Chloroflexi 33(Ratering and Schnell, 2001). Approximately 8% of clones Bacteroidetes 21in the LrhB library belonged to Desulfovibrioaceae. Sulfate Verrucomicrobia 25 Planctomycetes 11reduction can contribute to carbon metabolism in rice soils, especially when sulfate fertilizers are applied (Scheid et al., 2004). It appeared that the bacterial com- the 13C-labelled soil (Fig. 3A) was in agreement with the munity on rice roots changed from Clostridia as dominant higher abundance of the Azospirillum-like and the member at 45 days to Comamonadaceae at 90 days Burkholderiaceae-like sequences in the HrhB versus the (Table 1). Furthermore, the Betaproteobacteria were more LrhB library (Table 2). The predominance of the 439 bp T- abundant on rice roots (Table 1 and Fig. 1) than in the RF in the ‘light’ fractions was consistent with the domi- rhizosphere where the Alphaproteobacteria appeared to nance of Magnetospirillum-like sequences in the LrhB be dominant (Table 2). A study in Japanese rice soil library. The reduced abundance of the 516–520 bp T-RFs showed that bacterial community structure systematically in ‘heavy’ fractions was in agreement with the reduced changed with root development and growth, presumably number of Clostridium-like sequences in the HrhB versus because of dynamic variation of niche conditions sur- the LrhB. rounding roots (Ikenaga et al., 2003). The impacts of plant

growth on release of O2 and organic substances are probably the main reason for the dynamic changes of root Discussion communities. 13 Rice ﬁeld soils represent one of the most important By using pulse-labelling of rice plants with CO2 fol- sources of methane emitted into the atmosphere lowed by RNA-SIP analysis, we have previously shown (Lelieveld et al., 1998; Wang et al., 2004). Understanding that the uncultured Rice Cluster I were the highly active

organic matter dynamics in rice soils is essential for sus- archaea producing CH4 in rice rhizosphere (Lu and Con- taining soil fertility and rice production as well as mitigat- rad, 2005). Here we showed that the most active bacteria ing the emission of methane. In the present study, two in the rice rhizosphere were those closely related to experiments were conducted to characterize the structure Azospirillum spp. within the Alphaproteobacteria, and to and activity of the bacterial community involved in rhizo- members of Burkholderiaceae within the Betaproteobac- sphere carbon dynamics. The results showed that teria (Table 2). RNA-SIP revealed that these bacterial the dominant organisms (≥ 5% of sequences analysed groups incorporated carbon into their RNA that was sufﬁ- in different clone libraries) on rice roots and in the ciently enriched in 13C to allow separation by density gra- rhizosphere included Azospirillum, Magnetospirillum, dient centrifugation. The only source of such highly

Bacteria inhabiting rice roots and the rhizosphere 1357 enriched 13C was that released from roots of the pulse- rice field soil is mainly driven by acetate as carbon sub- labelled rice plants. The chemical nature of the released strate (Chidthaisong and Conrad, 2000). The fact that 13C substrates assimilated by the Azospirillum and ammonia oxidizers and methanotrophs were not labelled Burkholderiales is unknown. However, the amounts of 13C indicates that these organisms were not active in assimi- substrates excreted during the 7 days of repeated pulse- lating the root-derived carbon during the experiment labelling were apparently sufficient to result in a bacterial despite the fact that their ribosomal RNA could be community whose RNA was slightly heavier than that of retrieved. a control community, which was only exposed to unla- The previous studies using either substrate-enriched belled root exudates (Fig. 2). cultivation or phylogenetic analysis of DNA retrieved from Azospirillum spp. were often found in the rhizosphere rice bulk soil revealed that the bacterial populations in of various plants (Steenhoudt and Vanderleyden, 2000). anoxic bulk soil were dominated by Clostridia, Bacillus, Inoculation of rice roots with Azospirillum increased in rice Bacteroidetes, Verrucomicrobia and Actinobacteria (Chin fields total plant N uptake by 19–47% and rice yield by et al., 1999; Hengstmann et al., 1999). The Clostridium 22% (Choudhury and Kennedy, 2004). Thus, a positive spp., in particular, appeared to be predominant in the feedback interaction is likely to occur between rice plant anoxic zone of rice soil (Lüdemann et al., 2000) and when and Azospirillum: the plants distribute carbon to and organic matter such as rice straw was incorporated receive nitrogen from the Azospirillum spp. (Weber et al., 2001). We show in the present study that The members of Burkholderiaceae are metabolically although Clostridia were dominant, the members of Pro- highly diverse. The closest relative to our sequences is teobacteria were more active in the root environments Paucimonas lemoignei (Fig. 4), which is known to degrade (Tables 1 and 2). The plant-assimilated carbon was mainly poly(3-hydroxybutyrate) (Jendrossek, 2001). The next distributed to Alpha- and Betaproteobacteria, specifically closest relative is Ralstonia solanacearum, which is a Azospirillum spp. and members of the Burkholderiaceae. pathogen to various plants (Salanoubat et al., 2002). The The function of Clostridia and other anaerobic degraders 475 bp T-RF characteristic for these organisms increased is likely to decompose the recalcitrant root tissues and markedly in the ‘heavy’ fractions, but was barely detected residues in the rice rhizosphere that are not highly in the ‘light’ fractions of the labelled soil (Fig. 3A) and in enriched in 13C. all the fractions of the non-labelled soil (Fig. 3B). Appar- ently, this group of organisms was highly active in assimilating the 13C-labelled substrate released from the rice Conclusions roots, but had only a minor population in the rhizosphere. Rhizodeposition is one of the most important processes The reason why these organisms actively utilized the in soil ecosystems. We have shown that RNA-SIP can be plant-derived carbon but did not increase their population applied to plant–soil systems to identify the active organ- size is presently unclear. isms involved in rhizosphere carbon dynamics. Our study It is noteworthy that organisms that were apparently demonstrates that the members of Alpha- and Betapro- abundant in the rice rhizosphere did not appear to assim- teobacteria are the most active ones in assimilating the 13 ilate much of the root-released C. For example, the 16S plant-derived carbon. On the other hand, the iron and rRNA of Magnetospirillum spp., which were abundant and sulfate reducers, which were also dominant in the rhizo- potentially capable of denitrification (Taoka et al., 2003), sphere, did not assimilate much of the root-derived was mainly distributed to the ‘light’ RNA fractions (Table 2 carbon. Both anaerobic (e.g. Clostridia, Acidobacteria and Fig. 3). The sequences related to iron reducers and Bacteroidetes) and aerobic (e.g. Burkholderiaceae, (Geobacter spp. and Anaeromyxobacter dehalogenans) Comamonadaceae and Pseudomonadaceae) degraders showed no difference in abundances between HrhB and were present in the rhizosphere, indicating that root LrhB clone libraries (Table 2). Methylomonadaceae, environments and degradation processes were highly Nitromonadaceae and Desulfovibrionaceae were only heterogeneous. The effects of the rhizodepositions on detected in the LrhB library (Table 2). Possibly, the prima- biogeochemical processes and on the ecophysiology of 13 rily released and highly C-enriched compounds were not the corresponding organisms deserve further research. suitable substrates for these physiological groups that rather assimilate degradation products, which were no longer highly enriched in 13C. This interpretation is consis- Experimental procedures tent with the observation that the specific radioactivity of 14 Structure of the bacterial community on rice roots substrates detected in CO2-labelled rice microcosms decreased as they were further fermented, i.e. (experiment I) glucose > lactate > propionate > acetate (Dannenberg Rice growth and collection of root material. Rice soil was and Conrad, 1999) and that sulfate and iron reduction in obtained from wetland rice fields at the Italian Rice Research

1358 Y. Lu, D. Rosencrantz, W. Liesack and R. Conrad

13 13 Institute in Vercelli, Italy, and rice (Oryza sativa var. Roma, was placed over the plants and 35 ml of CO2 (99% of C type japonica) was grown in the greenhouse following the atom) was introduced into the chamber through a septum on procedure by Frenzel and colleagues (1992). The soil was a chamber wall every hour from 9:00 h to 16:00 h (seven times sandy loam with 1–2% organic carbon and neutral pH after a day). The labelling was performed continuously for 7 days. flooding (Schütz et al., 1989a,b). Rice roots of different ages 13 were collected for cultivation-independent retrieval of bacte- RNA extraction and CsTFA fractionation of C-RNA. At rial 16S rRNA genes (45 days and 90 days) and FISH anal- the end of labelling, the rhizosphere soil from the microcosms ysis of the bacterial community (27 days and 85 days). Root was collected by destructive sampling. Soil samples were − ° samples were carefully washed with tap water to remove frozen in liquid N2 immediately and stored at 80 C. RNA was adhering soil particles. Root material was lyophilized and extracted using the bead-beating method described before stored at −20°C until further use. (Lu and Conrad, 2005; Lu et al., 2005). The raw extracts of nucleic acids were purified by G-75 sephadex gel filtration DNA extraction, cloning and sequencing. Genomic DNA and digested with DNase I to remove DNA (Lueders et al., was extracted by the procedures described by Grosskopf and 2004a). RNA was fractionated by caesium trifluoroacetate colleagues (1998). The final DNA pellets were re-suspended (CsTFA) equilibrium density gradient centrifugation (Lueders in 200 µl of TE buffer (10 mM Tris, 1 mM EDTA; pH 8.0). The et al., 2004b; Lu and Conrad, 2005). The BD of RNA fractions amount of DNA was checked by agarose gel electrophoresis. was determined (Lueders et al., 2004b) and the amount of Clone libraries of 16S rRNA genes were constructed using RNA was quantified fluorometrically and by real-time PCR root material from 45-day-old and 90-day-old microcosms. using bacteria-specific primers Ba519f/Ba907r (Lueders PCR amplification of bacterial 16S rRNA genes was per- et al., 2004a). formed using the primer set Ba27f/Ba1492r as described by Weisburg and colleagues (1991). PCR products were purified T-RFLP fingerprinting. For T-RFLP analysis, the standard and ligated into the pGEM-T Vector (Promega) following the procedure was followed (Chin et al., 1999). RNA was ampli- manufacturer’s instruction. Plasmids were transformed into fied by RT-PCR using primer set Ba27f/Ba907r (Lueders Escherichia coli cells and clones were randomly selected for et al., 2004a). The forward primer was FAM (5-carboxyfluo- cycle sequencing reaction using a BigDye-terminator cycle rescein)-labelled. The PCR products were digested with MspI sequencing kit (Applied Biosystems). The products were (Promega). The digestion products were size-separated in an electrophoretically analysed in an ABI 377 DNA sequencer ABI 373 DNA sequencer (Applied Biosystems) (Chin et al., (Applied Biosystems). 1999).

Fluorescence in situ hybridization. FISH analysis followed Cloning, sequencing and phylogenetic analysis. RT-PCR the procedure described by Grosskopf and colleagues amplification used the same primer set as above. The ampl- (1998). The fresh root material was washed in PBS (pH 7.2) icons were ligated into pGEM-T vector and transformed into and fixed with freshly prepared 4% paraformaldehyde E. coli cells. Randomly selected clones were sequenced by (Amann et al., 1990). Samples were washed and dehydrated the ADIS DNA core facility (Max-Planck-Institute for Plant in 50%, 80% and 100% ethanol. The oligonucleotide probes Breeding Research, Cologne, Germany) using BigDye- used for in situ hybridization were: (i) EUB338 for Bacteria terminator cycle sequencing chemistry (Applied Biosystems) (Amann et al., 1990); (ii) ALF1b for Alphaproteobacteria (Lueders et al., 2004a; Lu et al., 2005). (Manz et al., 1992); and (iii) BET42a for Betaproteobacteria Phylogenetic analysis. The sequence data were analysed (Manz et al., 1992). Following hybridization, the slides were using the ARB software package (http://www.arb-home.de). stained with 4,6-diamidino-2-phenylindole (Porter and Feig, The 16S rRNA gene sequences were aligned and integrated 1980), and covered with Citifluor AF1 (Citifluor Products, into ARB database consisting of 17 000 complete or partial Citifluor, Canterbury, UK). The root samples were examined bacterial 16S rRNA sequences. The phylogenetic trees were with a confocal laser scanning microscope equipped with a calculated using neighbour-joining, maximum likelihood and krypton-argon laser (model TCS NT; Leica, Heidelberg, Ger- tree-puzzle programs. Different algorithms were used to find many) (Grosskopf et al., 1998). the topology of a consensus tree. Highly variable nucleotide positions and those with possible alignment errors were Active bacteria associated with rhizosphere carbon flow excluded from the phylogenetic analysis by using only positions present in at least 50% of all the sequences compared (experiment II) in this study. Rice growth and 13C labelling. Preparations of rice micro- Accession numbers. The sequences generated in this study cosm and 13C labelling have been previously described (Lu were deposited in the EMBL, GenBank and DDBJ nucleotide and Conrad, 2005). Briefly, rice was directly seeded into a sequence databases under the following accession numbers: root mesh bag, which was placed in a pot containing 3.5 kg AM159220–AM159499. of soil (dry weight). Root bags had a mesh size of 24 µm, which allowed the nutrients to pass freely while roots were prevented from penetrating into the bulk soil. We defined the Acknowledgements soil inside the root bag as rhizosphere soil. Soil was fertilized at rates of 0.28 g N, 0.14 g K and 0.07 g P per kilogram of We thank Peter Frenzel for the help in the preparation of rice soil and flooded throughout the growing period. The 13C label- microcosm, T. Lueders and M. Friedrich for introduction of the ling was performed at 45 days after seeding. A 10 l chamber SIP technique, and Melanie Klose and Peter Claus for excel-

Bacteria inhabiting rice roots and the rhizosphere 1359

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