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Contrasting Response Patterns of Rice Phyllosphere Bacterial Taxa to Elevated CO2
Pedosphere 24(4): 544–552, 2014 ISSN 1002-0160/CN 32-1315/P c 2014 Soil Science Society of China Published by Elsevier B.V. and Science Press
Contrasting Response Patterns of Rice Phyllosphere Bacterial Taxa to Elevated CO2 ∗1 REN Gai-Di1,2 , ZHU Jian-Guo1 and JIA Zhong-Jun1,∗2 1 Institute
of Soil Science, Chinese Academy of Sciences, Nanjing 210008 (China) of Chinese Academy of Sciences, Beijing 100049 (China)
2 University
(Received June 4, 2013; revised March 19, 2014)
ABSTRACT A vast number of microorganisms colonize the leaf surface of terrestrial plants, known as the phyllosphere, and these microorganisms are thought to be of critical importance in plant growth and health. However, the taxonomic identities and ecological functions of the microorganisms inhabiting the rice phyllosphere remain poorly understood. Using a massive, parallel pyrosequencing technique, we identified the phyllosphere bacterial taxa of four different rice varieties and investigated the microbial response to elevated CO 2 (eCO2 ) in a rice field of a free-air CO2 enrichment (FACE) facility located in Jiangsu Province, China. The results showed that the dominant phylotype, the Enterobacteriaceae family of Gammaproteobacteria, accounted for 70.6%–93.8% of the total bacterial communities in the rice phyllosphere. The dominant phylotype was stimulated by eCO2 , with its relative abundance increasing from 70.6%–75.2% at ambient CO2 (aCO2 ) to 86.5%–93.8% at eCO2 in the phyllosphere of rice varieties IIYou084 (TY-084), YangLiangYou6 (YLY-6), and ZhenXian96 (ZX-96). The rare phylotypes, including the bacterial taxa of Sphingobacteriaceae, Xanthomonadaceae, Oxalobacteraceae, Clostridiaceae, and Pseudomonadaceae, were suppressed and their relative abundance decreased from 13.4%–23.0% at aCO 2 to 1.47%– 6.11% at eCO2 . Furthermore, the bacterial diversity indices decreased at eCO2 in the phyllosphere of the rice varieties TY-084, YLY-6, and ZX-96. In contrast, an opposite response pattern was observed for the rice variety of YangDao8 (YD-8). In the phyllosphere of this variety, the relative abundance of the dominant phylotype, Enterobacteriaceae, decreased from 94.1% at aCO 2 to 81.4% at eCO2 , while that of the rare phylotypes increased from 3.37% to 6.59%. In addition, eCO2 appeared to stimulate bacterial diversity in the rice variety YD-8. Our results suggest that the phyllosphere microbial response to eCO2 might be relative abundance-dependent in paddy fields. Key Words:
bacterial diversity, free-air CO2 enrichment, microbial response, paddy field, pyrosequencing, rice variety
Citation: Ren, G. D., Zhu, J. G. and Jia, Z. J. 2014. Contrasting response patterns of rice phyllosphere bacterial taxa to elevated CO2 . Pedosphere. 24(4): 544–552.
INTRODUCTION The leaf surface of terrestrial plants, the phyllosphere, provides an extensive habitat for microorganisms. The planetary phyllosphere bacterial population is estimated to be as large as 1026 cells on a global leaf surface area of approximately 6.4 × 108 km2 , and bacteria are often found in numbers of 106 to 107 cells cm−2 (up to 108 cells g−1 ) of leaf (Lindow and Brandl, 2003). A large fraction of the phyllosphere bacteria are thought to significantly contribute to the health and growth of higher plants. For example, intensive studies have demonstrated the importance of phyllosphere bacterial taxa for promoting plant growth (Chinnadurai et al., 2009) and plant pathogen resistance (Costa et al., 2008; Balint-Kurti et al., 2010). Nitrogen-fixing bacteria have also been ∗1 Supported
found in the phyllosphere (Freiberg, 1998; F¨ urnkranz et al., 2008) of various higher plants, the growth of which may be significantly stimulated by these phyllosphere nitrogen fixers (Freiberg, 1998; Papen et al., 2002; F¨ urnkranz et al., 2008). In addition, the phyllosphere of higher plants represents a unique environment for testing ecological concepts and theories, as illustrated by a recent work (Redford and Fierer, 2009) showing that phyllosphere bacterial communities follow predictable succession patterns at different temporal scales. The global climate changes associated with rising atmospheric CO2 are projected to profoundly impact microbial processes that sustain ecosystem function (Singh et al., 2010). However, most researches have been focused on microbially mediated processes in soil (Lipson et al., 2006; Austin et al., 2009; Zhou et al.,
by the International S&T Cooperation Project of the Ministry of Science and Technology of China (No. 2010DFA22770) and the National Natural Science Foundation of China (No. 41090281). ∗2 Corresponding author. E-mail: [email protected].
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2012) rather than the phyllosphere of higher plants despite the potential importance of phyllosphere bacteria. Rice, a C3 crop, is one of the most important staple foods for human nutrition, and it nourishes approximately 50% of world’s population. Understanding the composition and diversity of bacteria colonizing the rice phyllosphere is essential to elucidating the microbial mechanisms underlying sustainable rice cultivation. Culture-dependent approaches have often been employed to illustrate the structure of the rice phyllosphere bacterial taxa (Mano et al., 2006; Costa et al., 2008; Chinnadurai et al., 2009). However, the versatile lifestyles of microorganisms and environmental heterogeneity make it difficult to reach consistent conclusions using traditional techniques, particularly with respect to fundamental questions such as which microorganisms are present and what they are doing. Recently, metagenome- and proteome-based analyses have proved to be powerful tools to illustrate the taxonomic identities and functional importance of rice phyllosphere bacterial communities. For example, plant genotype has been shown to be an important factor shaping the structure of phyllosphere microbial communities (Whipps et al., 2008), the growth of which were affected to different extents by differences in the availability of water and nutrients. The rising atmospheric CO2 levels are expected to have profound consequences on agricultural ecosystems (Ainsworth and Long, 2005; Ainsworth et al., 2008). Previous work has shown that elevated CO2 (eCO2 ) alters the overall taxonomic composition and structure of soil bacterial communities (He et al., 2012). However, little is known about the response of rice phyllosphere bacteria to eCO2 and whether there are consistent response patterns among different rice varieties. This study was performed in an attempt to delineate the taxonomic identity of the phyllosphere bacterial communities of different rice varieties and to explore the microbial response patterns of phyllosphere bacterial taxa to eCO2 . MATERIALS AND METHODS Study site description and sample collection The study was conducted at a free-air CO2 enrichment (FACE) facility located in the town of Xiaoji, Yangzhou City, Jiangsu Province, China (119◦ 42 0 E, 32◦ 35 5 N), a typical region for rice-wheat rotation in China. The mean annual temperature at this site was 15 ◦ C, and the annual precipitation was 900–1 000 mm. The average annual sunshine was approximately 2 132
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h, with an annual frost-free period of > 220 d. The FACE site has been described in greater detail in a previous report (Ren et al., 2007). The FACE field experiment was established in 2004. The field plots were subjected to two levels of CO2 as treatments, including ambient CO2 (aCO2 ) and eCO2 . The CO2 concentration at eCO2 was 200 ± 40 μmol mol−1 higher than that at aCO2 (355 ± 15 μmol mol−1 ). All field plots were randomly arranged for each CO2 treatment with three replicates. The soil of the study site was classified as a Shajiang-Aquic Cambiosol (Cooperative Research Group on Chinese Soil Taxonomy, 2001), and it contained 57.8% sand (2–0.02 mm), 28.5% silt (0.02–0.002 mm), and 13.7% clay (< 0.002 mm). The soil bulk density was 1.16 g cm−3 . Four rice varieties were tested, including two hybrid varieties, IIYou084 (TY-084) and YangLiangYou6 (YLY-6), and two indica varieties, ZhenXian96 (ZX96) and YangDao8 (YD-8). Rice leaves were sampled on October 10, 2010 at the maturation stage of rice plants and pathogen-infected leaves were removed before subsequent molecular analysis. Each rice variety was processed with multiple replicates. The rice varieties TY-084, YLY-6, ZX-96, and YD-8 had 2, 3, 3, and 2 replicated leaf samples for the aCO2 treatment and 2, 3, 2, and 2 replicated leaf samples for the eCO2 treatment, respectively, totaling 19 leaf samples. Phyllosphere bacterial cell harvest and DNA extraction The leaf samples were placed into sterile TrisEDTA (TE) buffer (10 mmol L−1 Tris-HCl, 1 mmol L−1 EDTA, pH 8.0), and the microbial cells were disassociated from the leaf surface by vigorous shaking. The resulting cell suspension was filtered by sterile glass wool to remove visible large plant particles before centrifugation. The steps of disassociation, filtration and centrifugation of the phyllosphere bacterial cells were repeated three times, and the resulting cell suspensions were pooled to maximize the recovery efficiency for subsequent DNA extraction. Three cycles of freezing in liquid nitrogen for 10 min and thawing at 65 ◦ C for 30 min were conducted, and cell lysis was further enhanced by addition of lysozyme. Sodium dodecyl sulfate (SDS) and proteinase K were added, and the resulting solution was incubated at 37 ◦ C overnight. Sodium chloride (NaCl, 5 mol L−1 ) was added before centrifugation. The supernatant was then purified with an equal volume of chloroform/isoamyl alcohol solution (24:1). The DNA pellets were precipitated by adding isopropanol to the resulting aqueous phase, incubation for 30 min at 4 ◦ C, and centrifugation at
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12 000 r min−1 . The purified DNA was washed in 70% (v/v) pre-cooled ethanol, air-dried, and dissolved in TE buffer.
gan package of R software version 2.15.0 (R Development Core Team, USA).
Pyrosequencing and sequence analysis
The data were analyzed using analysis of variance (ANOVA) with Duncan’s multiple range test to assess the difference in the relative abundances of bacterial taxa at P < 0.05. All statistical analysis was performed with the statistical software package SPSS (version 16.0).
Bidirectional pyrosequencing was performed on a Roche 454 GS FLX titanium sequencer (Roche Diagnostics Corporation, USA) by analyzing the V4 region of the 16S rRNA genes at the whole microbial community level. Fusion primers made up of adapter A or B, key sequence, and tag sequences were used to amplify the 16S rRNA genes. In brief, the primers were designed as 5 -adapter A or B + key sequence + (tag) + (template-specific sequence)-3 . For example, the 515F forward primer was made as follows: 5 -CGTATCGCCTCCCTCGCGCCA + TCAG + (6 bp tag) + (GTGCCAGCMGCCGCGG)-3 , and the 907R reverse primer was 5 -CTATGCGCCTTGCCAGCCCGC + TCAG + (6 bp tag) + (CCGTCAATTCMTTTRAGTTT)-3 . The polymerase chain reaction (PCR) amplicons were purified and visualized on 2.0% (w/v) agarose gels. The concentration of the purified PCR amplicons was determined, and the purified PCR amplicons were then combined in equimolar ratios into a single tube in preparation for pyrosequencing analysis. The 454 high-throughput pyrosequencing data were processed and analyzed following a previously described procedure (Fierer et al., 2008) using the Quantitative Insights Into Microbial Ecology (QIIME) pipeline (http://qiime.sourceforge.net). In brief, quality control of the pyrosequencing data was performed to remove low quality sequence reads by excluding reads shorter than 200 bp, with an average quality score of < 25, ambiguous nucleotides of > 0, homopolymes of > 6, or primer mismatches. In addition, the 6 bp barcode tag sequences were manually examined and used to assign sequence reads to different samples. The clustering method was then used to assign similar sequences to operational taxonomic units (OTUs) at a 97% sequence similarity level (Edgar, 2010). The most abundant sequence within an OTU was chosen by the PyNAST pipeline (Caporaso et al., 2010) using a relaxed neighbor-joining tree building algorithm (Price et al., 2009) for subsequent analysis. The taxonomic identity of each OTU was determined using the RDP Classifier (Wang et al., 2007) with a confidence cutoff of 0.80. Principal component analysis (PCA) of the pyrosequencing data was performed to examine whether eCO2 affects the community structure of phyllosphere bacterial taxa. The PCA was performed using the Ve-
Statistical analysis
RESULTS Bacterial phylotype distribution on the rice phyllosphere We obtained 120 085 high-quality sequences across all 19 samples (Table I) after removal of low-quality sequences, and the sequences had an average read length of 402 bp. An average read number of 6 320 sequences was observed for each sample. No archaeal sequences were detected, and all of the 16S rRNA gene sequences were affiliated with the domain bacteria. A total of 6 869 OTUs were obtained across all samples (Table II), and 850 OTUs per sample could be identified on the basis of 97% sequence similarity. The 16S rRNA gene sequences were classified into groups at different taxonomic resolutions for 6 phyla, 10 classes, 22 orders, 46 families, and 85 genera. At the phylum level, 5 765 out of 6 869 OTUs were affiliated to Proteobacteria, followed by Bacteroidetes with 422 OTUs, Firmicutes with 345 OTUs, Actinobacteria with 88 OTUs, and Planctomycetes with 2 OTUs (Table II). Of the 5 765 OTUs affiliated with Proteobacteria, 5 471 OTUs could be assigned to Gammaproteobacteria, accounting for 79.29% of Proteobacteria (Table II). The proportions of 16S rRNA gene sequences affiliated with the classes Alphaproteobacteria and Betaproteobacteria were 6.14% and 8.57%, respectively (Table II). Dominant bacterial phylotype in rice phyllosphere To understand the dominant phylotype in the phyllosphere of different rice varieties, we analyzed the microbial communities at the phylum level or lower. On average, ≥ 95.6% of the 16S rRNA gene sequences could be classified at the above-family levels, but only 34.8% of the sequences were classified at the genus level (Table I). This result suggests that subsequent detailed analysis could be performed with great confidence at the family level. Pyrosequencing of the total microbial communities in the rice phyllosphere revealed that Enterobacteriaceae-like sequences within the class Gammaproteobacteria held the highest rela-
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TABLE I Pyrosequencing reads and percentages identified at different taxonomic levels for bacterial species from the phyllosphere of the four rice varieties tested Rice varietya)
CO2 treatmentb)
No. of high quality reads
Percentage identified at different taxonomic levels Phylum
Class
Order
Family
Genus
% Hybrid variety TY-084 YLY-6 Indica variety ZX-96 YD-8
7 791±1 201c) 6 958±522 4 954±747 5 481±510
aCO2 eCO2 aCO2 eCO2
7 037±641 6 023±1 316 6 452±1 062 6 613±1 156 120 085
aCO2 eCO2 aCO2 eCO2
Total
99.6±0.4 99.8±0.3 99.7±0.2 98.8±1.1
97.6±2.2 99.4±0.6 99.2±0.6 98.6±1.0
95.3±3.1 97.6±0.9 95.6±3.8 97.8±1.1
93.9±4.6 97.4±1.3 94.7±3.9 95.8±4.5
34.6±4.9 29.5±12.9 49.5±15.3 22.4±25.2
99.2±1.0 99.5±0.7 99.9±0.0 99.5±0.6 99.5
97.8±1.8 98.4±2.0 99.5±0.4 98.5±1.5 98.6
95.4±2.5 97.2±2.9 98.0±0.7 96.9±1.9 96.7
94.0±3.8 95.9±4.3 97.9±0.7 96.1±3.0 95.6
38.7±8.6 31.4±7.9 25.0±18.9 44.8±8.5 34.8
a) TY-084
= IIYou084; YLY-6 = YangLiangYou6; ZX-96 = ZhenXian96; YD-8 = YangDao8. = ambient CO2 ; eCO2 = elevated CO2 . c) Mean±standard error. b) aCO
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TABLE II Number of operational taxonomic units (OTUs)a) detected in all bacterial phyla and the classes of Proteobacteria in the phyllosphere of the four rice varieties tested Phylum
Class
No. of OTUs
Percentage in total No. of OTUs
5 765 4 571 494 354 346 422 345 88 2 1 246 6 869
83.93 66.55 7.19 5.15 5.04 6.14 5.02 1.28 0.03 0.01 3.58
Percentage in total No. of Proteobacteria OTUs %
Proteobacteria Gammaproteobacteria Betaproteobacteria Alphaproteobacteria Unclassified-Proteobacteria Bacteroidetes Firmicutes Actinobacteria Planctomycetes Deinococcus-Thermus Unclassified bacteria Total a) OTUs
79.29 8.57 6.14 6.00
were categorized on the basis of 97% sequence similarity.
tive abundance at the family level across all samples, ranging from 70.6% to 94.1% of total 16S rRNA sequence reads (Fig. 1). The rare phylotypes with low relative abundance consisted of 5 families, including Xanthomonadaceae, Pseudomonadaceae, Sphingobacteriaceae, Oxalobacteraceae, and Clostridiaceae (Fig. 1). It is interesting to note that there were slight differences in phyllosphere bacterial taxa among rice varieties. For example, the 16S rRNA sequences of Clostridiaceae were absent from rice variety YLY-6, but they were observed with high relative abundance for the rice varieties TY-084, ZX-96, and YZ-8 (Fig. 1). Response patterns of rice phyllosphere bacterial phylotypes to eCO2 The response patterns of phyllosphere bacterial
taxa to eCO2 were analyzed in great detail at the family level (Fig. 2). Exposure to eCO2 stimulated the dominant phylotype but suppressed the rare phylotypes in rice varieties TY-084 (Fig. 2a), YLY-6 (Fig. 2b), and ZX-96 (Fig. 2c), although no statistically significant differences were observed in some cases. The most abundant phylotype in rice variety YLY-6 was found to be Enterobacteriaceae-like bacteria, accounting for 70.6% of the total communities, at aCO2 (Fig. 1), and it was stimulated, accounting for 93.8% at eCO2 (Figs. 1 and 2b). The rare phylotypes with low relative abundance were found to be Sphingobacteriaceae, Xanthomonadaceae, Oxalobacteraceae, Clostridiaceae, and Pseudomonadaceae (Fig. 1). The overall relative abundance of these bacterial communities decreased significantly (P < 0.05),
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Fig. 1 Relative abundance of the bacterial taxa at the family level in the phyllosphere of four rice varieties tested, including two hybrid varieties, TY-084 (a) and YLY-6 (b), and two indica varieties, ZX-96 (c), and YD-8 (d), under ambient atmospheric CO 2 (aCO2 ) and elevated CO2 (eCO2 ). TY-084, YLY-6, ZX-96, and YD-8 denote the rice varieties IIYou084, YangLiangYou6, ZhenXian96, and YangDao8, respectively.
Fig. 2 Relative abundance of the dominant and rare phylotypes of bacterial taxa at the family level in the phyllosphere of four rice varieties tested, including two hybrid varieties, TY-084 (a) and YLY-6 (b), and two indica varieties, ZX-96 (c) and YD-8 (d), under ambient atmospheric CO2 (aCO2 ) and elevated CO2 (eCO2 ). Enterobacteriaceae is the dominant phylotype with high relative abundance and Sphingobacteriaceae, Xanthomonadaceae, Oxalobacteraceae, Clostridiaceae, and Pseudomonadaceae are the rare phylotypes with low relative abundance. The error bars represent the standard error of the mean. Bars with the same letter are not significantly different at P < 0.05 as determined by analysis of variance (ANOVA). TY084, YLY-6, ZX-96, and YD-8 denote the rice varieties IIYou084, YangLiangYou6, ZhenXian96, and YangDao8, respectively.
from 23.0% at aCO2 to 1.47% at eCO2 (Fig. 2b). Similar results were obtained for phyllosphere bacterial taxa in rice varieties TY-084 (Fig. 2a) and ZX-96 (Fig. 2c), where the bacterial communities were dominated by phylotypes closely related to Enterobacteriaceae, with exceptionally high relative abundance of 72.2% and 75.2% at aCO2 (Fig. 1), respectively. Notably, contrasting response patterns were observed in the rice variety YD-8: eCO2 suppressed the dominant phylotype and stimulated the rare phylotypes, although no significant differences (P > 0.05) were observed between aCO2 and eCO2 (Fig. 2d). The overall relative abundance of the dominant phylotype decreased from 94.1% at aCO2 to 81.4% at eCO2 (Fig. 2d) and the overall relative abundance of the rare phylotypes increased from 3.37% at aCO2 to 6.59% at eCO2 (Fig. 2d). It should be emphasized that the response patterns were delineated by using the total abundance of individual dominant or rare phylotypes in the phyllosphere. However, a similar response pattern could be observed at the individual phylotype level (Fig. 1). Effect of eCO2 on bacterial diversity in rice phyllosphere For the rice varieties TY-084, YLY-6, and ZX96, eCO2 appeared to suppress the phyllosphere bacterial richness, measured as the number of observed OTUs (Fig. 3a–c), and the diversity, measured as the Shannon index (Fig. 3e–g) and phylogenetic diversity (Fig. 3i–k). However, the phyllosphere bacterial richness (Fig. 3d) and diversity (Fig. 3h, l) were enhanced
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Fig. 3 Bacterial richness and diversity in the phyllosphere of four rice varieties tested, including two hybrid varieties, TY-084 (a, e, and i) and YLY-6 (b, f, and j), and two indica varieties, ZX-96 (c, g, and k) and YD-8 (d, h, and l), under ambient atmospheric CO 2 (aCO2 ) and elevated CO2 (eCO2 ). The operational taxonomic units (OTUs) were categorized on the basis of 97% sequence similarity. TY-084, YLY-6, ZX-96, and YD-8 denote the rice varieties IIYou084, YangLiangYou6, ZhenXian96, and YangDao8, respectively.
by eCO2 in rice variety YD-8, although the differences were not statistically significant. To examine whether eCO2 affects the bacterial community structure of the phyllosphere, PCA was performed on the pyrosequencing data. Overall, the phyllosphere bacterial communities at aCO2 were largely separated from those at eCO2 across the four rice varieties tested (Fig. 4). These indicate that eCO2 altered the overall bacterial community structure of the phyllosphere of the four rice varieties tested. DISCUSSION One of the fundamental challenges in microbial ecology is to decipher the mechanisms driving the emergence and maintenance of microbial diversity. The phyllosphere of higher plants thus provides an ideal model to investigate the shifts in microbial communities in response to environmental disturbances because it is generally believed that microbial communities on leaves are simple and dominated by a few genera. Traditional culture-dependent techniques have
demonstrated that the phyllosphere of rice plants is dominated by a few taxa, such as Bacillus megatarium (Costa et al., 2008), Methylobacterium sp. (Chinnadurai et al., 2009), Agrobacterium larrymoorei, Aurantimonas altamirensis, Bacillus pumilus, B. subtilis, Curtobacterium citreum, C. flaccumfaciens, C. pusillum, and Hyphomicrobium facilis (Mano et al., 2006). These bacterial taxa could be classified into taxonomic groups containing only 7 families: Bacillaceae (Costa et al., 2008), Methylobacteriaceae (Chinnadurai et al., 2009), Rhizobiaceae, Aurantimonadaceae, Bacillaceae, Microbacteriaceae, and Hyphomicrobiaceae (Mano et al., 2006). Our high-throughput sequencing results provided a comprehensive survey of the microbial diversity in the rice phyllosphere, revealing 6 869 distinctly different OTUs that can be grouped into 6 phyla, 10 classes, 22 orders, 46 families, and 85 genera (Table II). The proteobacteria, including three subphyla, Alpha-, Beta-, and Gammaproteobacteria, was the most represented phylum. 66.55% of total 16S rRNA gene sequences were assigned to Gammaproteobacteria. In contrast, previous reports show that Alphaproteoba-
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Fig. 4 Principal component (PC) analysis of bacterial communities in the phyllosphere of four rice varieties tested, including two hybrid varieties (TY-084 and YLY-6) and two indica varieties (ZX-96 and YD-8), under ambient atmospheric CO 2 (aCO2 ) and elevated CO2 (eCO2 ). The percentages in parentheses indicate the proportions of variation explained by each ordination axis. TY-084, YLY-6, ZX-96, and YD-8 denote the rice varieties IIYou084, YangLiangYou6, ZhenXian96, and YangDao8, respectively.
cteria comprised up to 70% of the total rice phyllosphere bacterial communities (Vorholt, 2012). This discrepancy may be explained by the differences in rice genotypes (Whipps et al., 2008), environmental heterogeneity, and microbial interactions (Hunter et al., 2010). In addition, the considerable diversity of the Proteobacteria in morphology, physiology, and metabolism (Zavarzin et al., 1991) may significantly impact the community structure of phyllosphere bacterial taxa. The groups of Proteobacteria have been shown to play important roles in supplying nutrients such as carbon (Badger and Bek, 2008), nitrogen (Galloway, 1998), sulfur (Sievert et al., 2007), and phosphate (Longnecker et al., 2010). To the best of our knowledge, these results represent the first study to catalogue the taxonomic identities of phyllosphere bacteria of rice by using pyrosequencing technique. Enterobacteriaceae-like sequences within the Gammaproteobacteria class were found to be the most abundant group in the phyllosphere across all four rice varieties, in agreement with previous reports (Redford and Fierer, 2009; Hunter et al., 2010). At the genus level, Pantoea-like bacteria dominated the sequences affiliated with the Enterobacteriaceae family and showed significant shifts at eCO2 . This group has long been recognized as containing plant growthpromoting bacteria. Pantoea agglomerans strains have been demonstrated to possess many plant-promoting characteristics, such as nitrogen fixation (Feng et al., 2003), 1-aminocyclopropane-1-carboxylate (ACC) deaminase, siderophore, and phytohormone produc-
tion (such as indole-3-acetic acid), and phosphate solubilization (Teng et al., 2010). In addition, this genus of Pantoea (such as Pantoea stewartii subsp. stewartii) has also been reported to be involved in control of plant pathogenic virulence by producing quorumsensing regulatory proteins (Schu et al., 2009). These versatile traits may be an important factor that enables them to readily adapt to the changing environment induced by eCO2 . The link between the metabolic activity of phyllosphere bacterial taxa and rice physiology remains poorly understood and needs more detailed study. In this study, eCO2 showed a stimulating effect on the dominant phylotype but appeared to suppress the rare phylotypes in the rice varieties TY-084 (Fig. 2a), YLY-6 (Fig. 2b), and ZX-96 (Fig. 2c); however, a reverse trend was observed in the rice variety YD-8 (Fig. 2d). These contrasting response patterns may be due to the difference in the relative abundance of each phylotype in the aCO2 control. The relative abundance of the dominant phylotype at aCO2 was similar among the rice varieties TY-084, YLY-6, and ZX-96, ranging from 70.6% to 75.2%. In the rice variety YD-8, the dominant phylotype comprised up to 94.1%, a measurement dramatically higher than the observations for the other three varieties. Therefore, the response of the phyllosphere bacterial taxa to eCO2 may depend on their relative abundance at aCO2 . This finding is consistent with a previous study indicating the critical role of relative abundance in driving the seasonal changes of the microbial communities (Ellis et
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al., 1999). There may be a threshold value of relative abundance at which the response pattern of rice phyllosphere bacteria to eCO2 would behave differently. This was supported by the diversity index results to some extent, as the rice varieties TY-084, YLY-6, and ZX96 showed decreases in the bacterial richness and diversity indices at eCO2 , while YD-8 showed increasing trends. In addition, the microbial communities may respond in a rice-variety specific manner. The composition and diversity of phyllosphere communities have been reported to reflect the immigration survival and growth of microbial colonists (Vorholt, 2012). This process may be influenced by numerous environmental factors in addition to the plant genotype. Plant genotypes can lead to variations in physiological states, including water transport, photosynthesis, respiration, and stomatal conductance, which could affect the microbial response patterns to environmental stress (Whipps et al., 2008). The PCA results further demonstrated that the phyllosphere bacterial taxa at eCO2 were distinct from those at aCO2 , although the response patterns to eCO2 diverged across all rice varieties tested. Additionally, the phyllosphere bacterial community structure was altered at eCO2 for all rice varieties, and this observation is consistent with the response of soil bacterial community to eCO2 (He et al., 2012). It appears that the environmental disturbance by eCO2 plays an important role in influencing the bacterial diversity as well as the structure of microbial communities in the phyllosphere of rice. CONCLUSIONS The results of this study demonstrated that the most dominant phylotype on the four rice varieties tested was the Enterobacteriaceae-like sequences within the class Gammaproteobacteria. The phyllosphere bacteria of rice showed contrasting response patterns to eCO2 . The rice varieties TY-084, YLY-6, and ZX96 had an increasing trend in the relative abundance of the dominant phylotype (Enterobacteriaceae), from 70.6%–75.2% at aCO2 to 86.5–93.8% at eCO2 ; the rare phylotypes including Xanthomonadaceae, Pseudomonadaceae, Sphingobacteriaceae, Oxalobacteraceae, and Clostridiaceae showed a tendency to decrease in relative abundance. A reverse response pattern to eCO2 was observed for the rice variety YD-8. On this variety, the dominant phylotype, Enterobacteriaceae, was suppressed, with its relative abundance decreasing from 94.1% at aCO2 to 81.4% at eCO2 , while the relative abundance of the rare phylotypes increased from 3.37% to 6.59%. This revealed a relative abundancedependent response pattern. The contrasting response
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patterns of these phyllosphere bacterial taxa may lead to a divergence in bacterial diversity at eCO2 . The results of this study provided a mechanistic basis for moving toward a predictive understanding of microbially mediated ecosystem feedback resulting from rising atmospheric CO2 and global warming. ACKNOWLEDGEMENTS The authors would like to extend their gratitude to Drs. ZHOU Juan and LAI Shang-Kun and Prof. YANG Lian-Xin from Yangzhou University, China for their help in sample collection. The authors would also like to thank Dr. ZHANG Hua-Yong from the Institute of Soil Science, Chinese Academy of Sciences for bioinformatics assistance. REFERENCES Ainsworth, E. A., Leakey, A. D. B., Ort, D. R. and Long, S. P. 2008. FACE-ing the facts: inconsistencies and interdependence among field, chamber and modeling studies of elevated [CO2 ] impacts on crop yield and food supply. New Phytol. 179: 5–9. Ainsworth, E. A. and Long, S. P. 2005. What have we learned from 15 years of free-air CO2 enrichment (FACE)? A metaanalytic review of the responses of photosynthesis, canopy properties and plant production to rising CO2 . New Phytol. 165: 351–372. Austin, E. E., Castro, H. F., Sides, K. E., Schadt, C. W. and Classen, A. T. 2009. Assessment of 10 years of CO2 fumigation on soil microbial communities and function in a sweetgum plantation. Soil Biol. Biochem. 41: 514–520. Badger, M. R. and Bek, E. J. 2008. Multiple Rubisco forms in proteobacteria: their functional significance in relation to CO2 acquisition by the CBB cycle. J. Exp. Bot. 59: 1525– 1541. Balint-Kurti, P., Simmons, S. J., Blum, J. E., Ballar, C. L. and Stapleton, A. E. 2010. Maize leaf epiphytic bacteria diversity patterns are genetically correlated with resistance to fungal pathogen infection. Mol. Plant Microbe Interact. 23: 473– 484. Caporaso, J. G., Bittinger, K., Bushman, F. D., DeSantis, T. Z., Andersen, G. L. and Knight, R. 2010. PyNAST: a flexible tool for aligning sequences to a template alignment. Bioinformatics. 26: 266–267. Chinnadurai, C., Balachandar, D. and Sundaram, S. P. 2009. Characterization of 1-aminocyclopropane-1-carboxylate deaminase producing methylobacteria from phyllosphere of rice and their role in ethylene regulation. World J. Microbiol. Biotechnol. 25: 1403–1411. Cooperative Research Group on Chinese Soil Taxonomy. 2001. Chinese Soil Taxonomy. China Science and Technology Press, Beijing. De Costa, D. M., Samarasinghe, S. S. T., Dias, H. R. D. and Dissanayake, D. M. N. 2008. Control of rice sheath blight by phyllosphere epiphytic microbial antagonists. Phytoparasitica. 36: 52–65. Edgar, R. C. 2010. Search and clustering orders of magnitude faster than BLAST. Bioinformatics. 26: 2460–2461. Ellis, R. J., Thompson, I. P. and Bailey, M. J. 1999. Temporal fluctuations in the pseudomonad population associated with
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Contrasting Response Patterns of Rice Phyllosphere Bacterial Taxa to Elevated CO2 ∗1 REN Gai-Di1,2 , ZHU Jian-Guo1 and JIA Zhong-Jun1,∗2 1 Institute
of Soil Science, Chinese Academy of Sciences, Nanjing 210008 (China) of Chinese Academy of Sciences, Beijing 100049 (China)
2 University
(Received June 4, 2013; revised March 19, 2014)
ABSTRACT A vast number of microorganisms colonize the leaf surface of terrestrial plants, known as the phyllosphere, and these microorganisms are thought to be of critical importance in plant growth and health. However, the taxonomic identities and ecological functions of the microorganisms inhabiting the rice phyllosphere remain poorly understood. Using a massive, parallel pyrosequencing technique, we identified the phyllosphere bacterial taxa of four different rice varieties and investigated the microbial response to elevated CO 2 (eCO2 ) in a rice field of a free-air CO2 enrichment (FACE) facility located in Jiangsu Province, China. The results showed that the dominant phylotype, the Enterobacteriaceae family of Gammaproteobacteria, accounted for 70.6%–93.8% of the total bacterial communities in the rice phyllosphere. The dominant phylotype was stimulated by eCO2 , with its relative abundance increasing from 70.6%–75.2% at ambient CO2 (aCO2 ) to 86.5%–93.8% at eCO2 in the phyllosphere of rice varieties IIYou084 (TY-084), YangLiangYou6 (YLY-6), and ZhenXian96 (ZX-96). The rare phylotypes, including the bacterial taxa of Sphingobacteriaceae, Xanthomonadaceae, Oxalobacteraceae, Clostridiaceae, and Pseudomonadaceae, were suppressed and their relative abundance decreased from 13.4%–23.0% at aCO 2 to 1.47%– 6.11% at eCO2 . Furthermore, the bacterial diversity indices decreased at eCO2 in the phyllosphere of the rice varieties TY-084, YLY-6, and ZX-96. In contrast, an opposite response pattern was observed for the rice variety of YangDao8 (YD-8). In the phyllosphere of this variety, the relative abundance of the dominant phylotype, Enterobacteriaceae, decreased from 94.1% at aCO 2 to 81.4% at eCO2 , while that of the rare phylotypes increased from 3.37% to 6.59%. In addition, eCO2 appeared to stimulate bacterial diversity in the rice variety YD-8. Our results suggest that the phyllosphere microbial response to eCO2 might be relative abundance-dependent in paddy fields. Key Words:
bacterial diversity, free-air CO2 enrichment, microbial response, paddy field, pyrosequencing, rice variety
Citation: Ren, G. D., Zhu, J. G. and Jia, Z. J. 2014. Contrasting response patterns of rice phyllosphere bacterial taxa to elevated CO2 . Pedosphere. 24(4): 544–552.
INTRODUCTION The leaf surface of terrestrial plants, the phyllosphere, provides an extensive habitat for microorganisms. The planetary phyllosphere bacterial population is estimated to be as large as 1026 cells on a global leaf surface area of approximately 6.4 × 108 km2 , and bacteria are often found in numbers of 106 to 107 cells cm−2 (up to 108 cells g−1 ) of leaf (Lindow and Brandl, 2003). A large fraction of the phyllosphere bacteria are thought to significantly contribute to the health and growth of higher plants. For example, intensive studies have demonstrated the importance of phyllosphere bacterial taxa for promoting plant growth (Chinnadurai et al., 2009) and plant pathogen resistance (Costa et al., 2008; Balint-Kurti et al., 2010). Nitrogen-fixing bacteria have also been ∗1 Supported
found in the phyllosphere (Freiberg, 1998; F¨ urnkranz et al., 2008) of various higher plants, the growth of which may be significantly stimulated by these phyllosphere nitrogen fixers (Freiberg, 1998; Papen et al., 2002; F¨ urnkranz et al., 2008). In addition, the phyllosphere of higher plants represents a unique environment for testing ecological concepts and theories, as illustrated by a recent work (Redford and Fierer, 2009) showing that phyllosphere bacterial communities follow predictable succession patterns at different temporal scales. The global climate changes associated with rising atmospheric CO2 are projected to profoundly impact microbial processes that sustain ecosystem function (Singh et al., 2010). However, most researches have been focused on microbially mediated processes in soil (Lipson et al., 2006; Austin et al., 2009; Zhou et al.,
by the International S&T Cooperation Project of the Ministry of Science and Technology of China (No. 2010DFA22770) and the National Natural Science Foundation of China (No. 41090281). ∗2 Corresponding author. E-mail: [email protected].
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2012) rather than the phyllosphere of higher plants despite the potential importance of phyllosphere bacteria. Rice, a C3 crop, is one of the most important staple foods for human nutrition, and it nourishes approximately 50% of world’s population. Understanding the composition and diversity of bacteria colonizing the rice phyllosphere is essential to elucidating the microbial mechanisms underlying sustainable rice cultivation. Culture-dependent approaches have often been employed to illustrate the structure of the rice phyllosphere bacterial taxa (Mano et al., 2006; Costa et al., 2008; Chinnadurai et al., 2009). However, the versatile lifestyles of microorganisms and environmental heterogeneity make it difficult to reach consistent conclusions using traditional techniques, particularly with respect to fundamental questions such as which microorganisms are present and what they are doing. Recently, metagenome- and proteome-based analyses have proved to be powerful tools to illustrate the taxonomic identities and functional importance of rice phyllosphere bacterial communities. For example, plant genotype has been shown to be an important factor shaping the structure of phyllosphere microbial communities (Whipps et al., 2008), the growth of which were affected to different extents by differences in the availability of water and nutrients. The rising atmospheric CO2 levels are expected to have profound consequences on agricultural ecosystems (Ainsworth and Long, 2005; Ainsworth et al., 2008). Previous work has shown that elevated CO2 (eCO2 ) alters the overall taxonomic composition and structure of soil bacterial communities (He et al., 2012). However, little is known about the response of rice phyllosphere bacteria to eCO2 and whether there are consistent response patterns among different rice varieties. This study was performed in an attempt to delineate the taxonomic identity of the phyllosphere bacterial communities of different rice varieties and to explore the microbial response patterns of phyllosphere bacterial taxa to eCO2 . MATERIALS AND METHODS Study site description and sample collection The study was conducted at a free-air CO2 enrichment (FACE) facility located in the town of Xiaoji, Yangzhou City, Jiangsu Province, China (119◦ 42 0 E, 32◦ 35 5 N), a typical region for rice-wheat rotation in China. The mean annual temperature at this site was 15 ◦ C, and the annual precipitation was 900–1 000 mm. The average annual sunshine was approximately 2 132
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h, with an annual frost-free period of > 220 d. The FACE site has been described in greater detail in a previous report (Ren et al., 2007). The FACE field experiment was established in 2004. The field plots were subjected to two levels of CO2 as treatments, including ambient CO2 (aCO2 ) and eCO2 . The CO2 concentration at eCO2 was 200 ± 40 μmol mol−1 higher than that at aCO2 (355 ± 15 μmol mol−1 ). All field plots were randomly arranged for each CO2 treatment with three replicates. The soil of the study site was classified as a Shajiang-Aquic Cambiosol (Cooperative Research Group on Chinese Soil Taxonomy, 2001), and it contained 57.8% sand (2–0.02 mm), 28.5% silt (0.02–0.002 mm), and 13.7% clay (< 0.002 mm). The soil bulk density was 1.16 g cm−3 . Four rice varieties were tested, including two hybrid varieties, IIYou084 (TY-084) and YangLiangYou6 (YLY-6), and two indica varieties, ZhenXian96 (ZX96) and YangDao8 (YD-8). Rice leaves were sampled on October 10, 2010 at the maturation stage of rice plants and pathogen-infected leaves were removed before subsequent molecular analysis. Each rice variety was processed with multiple replicates. The rice varieties TY-084, YLY-6, ZX-96, and YD-8 had 2, 3, 3, and 2 replicated leaf samples for the aCO2 treatment and 2, 3, 2, and 2 replicated leaf samples for the eCO2 treatment, respectively, totaling 19 leaf samples. Phyllosphere bacterial cell harvest and DNA extraction The leaf samples were placed into sterile TrisEDTA (TE) buffer (10 mmol L−1 Tris-HCl, 1 mmol L−1 EDTA, pH 8.0), and the microbial cells were disassociated from the leaf surface by vigorous shaking. The resulting cell suspension was filtered by sterile glass wool to remove visible large plant particles before centrifugation. The steps of disassociation, filtration and centrifugation of the phyllosphere bacterial cells were repeated three times, and the resulting cell suspensions were pooled to maximize the recovery efficiency for subsequent DNA extraction. Three cycles of freezing in liquid nitrogen for 10 min and thawing at 65 ◦ C for 30 min were conducted, and cell lysis was further enhanced by addition of lysozyme. Sodium dodecyl sulfate (SDS) and proteinase K were added, and the resulting solution was incubated at 37 ◦ C overnight. Sodium chloride (NaCl, 5 mol L−1 ) was added before centrifugation. The supernatant was then purified with an equal volume of chloroform/isoamyl alcohol solution (24:1). The DNA pellets were precipitated by adding isopropanol to the resulting aqueous phase, incubation for 30 min at 4 ◦ C, and centrifugation at
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12 000 r min−1 . The purified DNA was washed in 70% (v/v) pre-cooled ethanol, air-dried, and dissolved in TE buffer.
gan package of R software version 2.15.0 (R Development Core Team, USA).
Pyrosequencing and sequence analysis
The data were analyzed using analysis of variance (ANOVA) with Duncan’s multiple range test to assess the difference in the relative abundances of bacterial taxa at P < 0.05. All statistical analysis was performed with the statistical software package SPSS (version 16.0).
Bidirectional pyrosequencing was performed on a Roche 454 GS FLX titanium sequencer (Roche Diagnostics Corporation, USA) by analyzing the V4 region of the 16S rRNA genes at the whole microbial community level. Fusion primers made up of adapter A or B, key sequence, and tag sequences were used to amplify the 16S rRNA genes. In brief, the primers were designed as 5 -adapter A or B + key sequence + (tag) + (template-specific sequence)-3 . For example, the 515F forward primer was made as follows: 5 -CGTATCGCCTCCCTCGCGCCA + TCAG + (6 bp tag) + (GTGCCAGCMGCCGCGG)-3 , and the 907R reverse primer was 5 -CTATGCGCCTTGCCAGCCCGC + TCAG + (6 bp tag) + (CCGTCAATTCMTTTRAGTTT)-3 . The polymerase chain reaction (PCR) amplicons were purified and visualized on 2.0% (w/v) agarose gels. The concentration of the purified PCR amplicons was determined, and the purified PCR amplicons were then combined in equimolar ratios into a single tube in preparation for pyrosequencing analysis. The 454 high-throughput pyrosequencing data were processed and analyzed following a previously described procedure (Fierer et al., 2008) using the Quantitative Insights Into Microbial Ecology (QIIME) pipeline (http://qiime.sourceforge.net). In brief, quality control of the pyrosequencing data was performed to remove low quality sequence reads by excluding reads shorter than 200 bp, with an average quality score of < 25, ambiguous nucleotides of > 0, homopolymes of > 6, or primer mismatches. In addition, the 6 bp barcode tag sequences were manually examined and used to assign sequence reads to different samples. The clustering method was then used to assign similar sequences to operational taxonomic units (OTUs) at a 97% sequence similarity level (Edgar, 2010). The most abundant sequence within an OTU was chosen by the PyNAST pipeline (Caporaso et al., 2010) using a relaxed neighbor-joining tree building algorithm (Price et al., 2009) for subsequent analysis. The taxonomic identity of each OTU was determined using the RDP Classifier (Wang et al., 2007) with a confidence cutoff of 0.80. Principal component analysis (PCA) of the pyrosequencing data was performed to examine whether eCO2 affects the community structure of phyllosphere bacterial taxa. The PCA was performed using the Ve-
Statistical analysis
RESULTS Bacterial phylotype distribution on the rice phyllosphere We obtained 120 085 high-quality sequences across all 19 samples (Table I) after removal of low-quality sequences, and the sequences had an average read length of 402 bp. An average read number of 6 320 sequences was observed for each sample. No archaeal sequences were detected, and all of the 16S rRNA gene sequences were affiliated with the domain bacteria. A total of 6 869 OTUs were obtained across all samples (Table II), and 850 OTUs per sample could be identified on the basis of 97% sequence similarity. The 16S rRNA gene sequences were classified into groups at different taxonomic resolutions for 6 phyla, 10 classes, 22 orders, 46 families, and 85 genera. At the phylum level, 5 765 out of 6 869 OTUs were affiliated to Proteobacteria, followed by Bacteroidetes with 422 OTUs, Firmicutes with 345 OTUs, Actinobacteria with 88 OTUs, and Planctomycetes with 2 OTUs (Table II). Of the 5 765 OTUs affiliated with Proteobacteria, 5 471 OTUs could be assigned to Gammaproteobacteria, accounting for 79.29% of Proteobacteria (Table II). The proportions of 16S rRNA gene sequences affiliated with the classes Alphaproteobacteria and Betaproteobacteria were 6.14% and 8.57%, respectively (Table II). Dominant bacterial phylotype in rice phyllosphere To understand the dominant phylotype in the phyllosphere of different rice varieties, we analyzed the microbial communities at the phylum level or lower. On average, ≥ 95.6% of the 16S rRNA gene sequences could be classified at the above-family levels, but only 34.8% of the sequences were classified at the genus level (Table I). This result suggests that subsequent detailed analysis could be performed with great confidence at the family level. Pyrosequencing of the total microbial communities in the rice phyllosphere revealed that Enterobacteriaceae-like sequences within the class Gammaproteobacteria held the highest rela-
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TABLE I Pyrosequencing reads and percentages identified at different taxonomic levels for bacterial species from the phyllosphere of the four rice varieties tested Rice varietya)
CO2 treatmentb)
No. of high quality reads
Percentage identified at different taxonomic levels Phylum
Class
Order
Family
Genus
% Hybrid variety TY-084 YLY-6 Indica variety ZX-96 YD-8
7 791±1 201c) 6 958±522 4 954±747 5 481±510
aCO2 eCO2 aCO2 eCO2
7 037±641 6 023±1 316 6 452±1 062 6 613±1 156 120 085
aCO2 eCO2 aCO2 eCO2
Total
99.6±0.4 99.8±0.3 99.7±0.2 98.8±1.1
97.6±2.2 99.4±0.6 99.2±0.6 98.6±1.0
95.3±3.1 97.6±0.9 95.6±3.8 97.8±1.1
93.9±4.6 97.4±1.3 94.7±3.9 95.8±4.5
34.6±4.9 29.5±12.9 49.5±15.3 22.4±25.2
99.2±1.0 99.5±0.7 99.9±0.0 99.5±0.6 99.5
97.8±1.8 98.4±2.0 99.5±0.4 98.5±1.5 98.6
95.4±2.5 97.2±2.9 98.0±0.7 96.9±1.9 96.7
94.0±3.8 95.9±4.3 97.9±0.7 96.1±3.0 95.6
38.7±8.6 31.4±7.9 25.0±18.9 44.8±8.5 34.8
a) TY-084
= IIYou084; YLY-6 = YangLiangYou6; ZX-96 = ZhenXian96; YD-8 = YangDao8. = ambient CO2 ; eCO2 = elevated CO2 . c) Mean±standard error. b) aCO
2
TABLE II Number of operational taxonomic units (OTUs)a) detected in all bacterial phyla and the classes of Proteobacteria in the phyllosphere of the four rice varieties tested Phylum
Class
No. of OTUs
Percentage in total No. of OTUs
5 765 4 571 494 354 346 422 345 88 2 1 246 6 869
83.93 66.55 7.19 5.15 5.04 6.14 5.02 1.28 0.03 0.01 3.58
Percentage in total No. of Proteobacteria OTUs %
Proteobacteria Gammaproteobacteria Betaproteobacteria Alphaproteobacteria Unclassified-Proteobacteria Bacteroidetes Firmicutes Actinobacteria Planctomycetes Deinococcus-Thermus Unclassified bacteria Total a) OTUs
79.29 8.57 6.14 6.00
were categorized on the basis of 97% sequence similarity.
tive abundance at the family level across all samples, ranging from 70.6% to 94.1% of total 16S rRNA sequence reads (Fig. 1). The rare phylotypes with low relative abundance consisted of 5 families, including Xanthomonadaceae, Pseudomonadaceae, Sphingobacteriaceae, Oxalobacteraceae, and Clostridiaceae (Fig. 1). It is interesting to note that there were slight differences in phyllosphere bacterial taxa among rice varieties. For example, the 16S rRNA sequences of Clostridiaceae were absent from rice variety YLY-6, but they were observed with high relative abundance for the rice varieties TY-084, ZX-96, and YZ-8 (Fig. 1). Response patterns of rice phyllosphere bacterial phylotypes to eCO2 The response patterns of phyllosphere bacterial
taxa to eCO2 were analyzed in great detail at the family level (Fig. 2). Exposure to eCO2 stimulated the dominant phylotype but suppressed the rare phylotypes in rice varieties TY-084 (Fig. 2a), YLY-6 (Fig. 2b), and ZX-96 (Fig. 2c), although no statistically significant differences were observed in some cases. The most abundant phylotype in rice variety YLY-6 was found to be Enterobacteriaceae-like bacteria, accounting for 70.6% of the total communities, at aCO2 (Fig. 1), and it was stimulated, accounting for 93.8% at eCO2 (Figs. 1 and 2b). The rare phylotypes with low relative abundance were found to be Sphingobacteriaceae, Xanthomonadaceae, Oxalobacteraceae, Clostridiaceae, and Pseudomonadaceae (Fig. 1). The overall relative abundance of these bacterial communities decreased significantly (P < 0.05),
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Fig. 1 Relative abundance of the bacterial taxa at the family level in the phyllosphere of four rice varieties tested, including two hybrid varieties, TY-084 (a) and YLY-6 (b), and two indica varieties, ZX-96 (c), and YD-8 (d), under ambient atmospheric CO 2 (aCO2 ) and elevated CO2 (eCO2 ). TY-084, YLY-6, ZX-96, and YD-8 denote the rice varieties IIYou084, YangLiangYou6, ZhenXian96, and YangDao8, respectively.
Fig. 2 Relative abundance of the dominant and rare phylotypes of bacterial taxa at the family level in the phyllosphere of four rice varieties tested, including two hybrid varieties, TY-084 (a) and YLY-6 (b), and two indica varieties, ZX-96 (c) and YD-8 (d), under ambient atmospheric CO2 (aCO2 ) and elevated CO2 (eCO2 ). Enterobacteriaceae is the dominant phylotype with high relative abundance and Sphingobacteriaceae, Xanthomonadaceae, Oxalobacteraceae, Clostridiaceae, and Pseudomonadaceae are the rare phylotypes with low relative abundance. The error bars represent the standard error of the mean. Bars with the same letter are not significantly different at P < 0.05 as determined by analysis of variance (ANOVA). TY084, YLY-6, ZX-96, and YD-8 denote the rice varieties IIYou084, YangLiangYou6, ZhenXian96, and YangDao8, respectively.
from 23.0% at aCO2 to 1.47% at eCO2 (Fig. 2b). Similar results were obtained for phyllosphere bacterial taxa in rice varieties TY-084 (Fig. 2a) and ZX-96 (Fig. 2c), where the bacterial communities were dominated by phylotypes closely related to Enterobacteriaceae, with exceptionally high relative abundance of 72.2% and 75.2% at aCO2 (Fig. 1), respectively. Notably, contrasting response patterns were observed in the rice variety YD-8: eCO2 suppressed the dominant phylotype and stimulated the rare phylotypes, although no significant differences (P > 0.05) were observed between aCO2 and eCO2 (Fig. 2d). The overall relative abundance of the dominant phylotype decreased from 94.1% at aCO2 to 81.4% at eCO2 (Fig. 2d) and the overall relative abundance of the rare phylotypes increased from 3.37% at aCO2 to 6.59% at eCO2 (Fig. 2d). It should be emphasized that the response patterns were delineated by using the total abundance of individual dominant or rare phylotypes in the phyllosphere. However, a similar response pattern could be observed at the individual phylotype level (Fig. 1). Effect of eCO2 on bacterial diversity in rice phyllosphere For the rice varieties TY-084, YLY-6, and ZX96, eCO2 appeared to suppress the phyllosphere bacterial richness, measured as the number of observed OTUs (Fig. 3a–c), and the diversity, measured as the Shannon index (Fig. 3e–g) and phylogenetic diversity (Fig. 3i–k). However, the phyllosphere bacterial richness (Fig. 3d) and diversity (Fig. 3h, l) were enhanced
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Fig. 3 Bacterial richness and diversity in the phyllosphere of four rice varieties tested, including two hybrid varieties, TY-084 (a, e, and i) and YLY-6 (b, f, and j), and two indica varieties, ZX-96 (c, g, and k) and YD-8 (d, h, and l), under ambient atmospheric CO 2 (aCO2 ) and elevated CO2 (eCO2 ). The operational taxonomic units (OTUs) were categorized on the basis of 97% sequence similarity. TY-084, YLY-6, ZX-96, and YD-8 denote the rice varieties IIYou084, YangLiangYou6, ZhenXian96, and YangDao8, respectively.
by eCO2 in rice variety YD-8, although the differences were not statistically significant. To examine whether eCO2 affects the bacterial community structure of the phyllosphere, PCA was performed on the pyrosequencing data. Overall, the phyllosphere bacterial communities at aCO2 were largely separated from those at eCO2 across the four rice varieties tested (Fig. 4). These indicate that eCO2 altered the overall bacterial community structure of the phyllosphere of the four rice varieties tested. DISCUSSION One of the fundamental challenges in microbial ecology is to decipher the mechanisms driving the emergence and maintenance of microbial diversity. The phyllosphere of higher plants thus provides an ideal model to investigate the shifts in microbial communities in response to environmental disturbances because it is generally believed that microbial communities on leaves are simple and dominated by a few genera. Traditional culture-dependent techniques have
demonstrated that the phyllosphere of rice plants is dominated by a few taxa, such as Bacillus megatarium (Costa et al., 2008), Methylobacterium sp. (Chinnadurai et al., 2009), Agrobacterium larrymoorei, Aurantimonas altamirensis, Bacillus pumilus, B. subtilis, Curtobacterium citreum, C. flaccumfaciens, C. pusillum, and Hyphomicrobium facilis (Mano et al., 2006). These bacterial taxa could be classified into taxonomic groups containing only 7 families: Bacillaceae (Costa et al., 2008), Methylobacteriaceae (Chinnadurai et al., 2009), Rhizobiaceae, Aurantimonadaceae, Bacillaceae, Microbacteriaceae, and Hyphomicrobiaceae (Mano et al., 2006). Our high-throughput sequencing results provided a comprehensive survey of the microbial diversity in the rice phyllosphere, revealing 6 869 distinctly different OTUs that can be grouped into 6 phyla, 10 classes, 22 orders, 46 families, and 85 genera (Table II). The proteobacteria, including three subphyla, Alpha-, Beta-, and Gammaproteobacteria, was the most represented phylum. 66.55% of total 16S rRNA gene sequences were assigned to Gammaproteobacteria. In contrast, previous reports show that Alphaproteoba-
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Fig. 4 Principal component (PC) analysis of bacterial communities in the phyllosphere of four rice varieties tested, including two hybrid varieties (TY-084 and YLY-6) and two indica varieties (ZX-96 and YD-8), under ambient atmospheric CO 2 (aCO2 ) and elevated CO2 (eCO2 ). The percentages in parentheses indicate the proportions of variation explained by each ordination axis. TY-084, YLY-6, ZX-96, and YD-8 denote the rice varieties IIYou084, YangLiangYou6, ZhenXian96, and YangDao8, respectively.
cteria comprised up to 70% of the total rice phyllosphere bacterial communities (Vorholt, 2012). This discrepancy may be explained by the differences in rice genotypes (Whipps et al., 2008), environmental heterogeneity, and microbial interactions (Hunter et al., 2010). In addition, the considerable diversity of the Proteobacteria in morphology, physiology, and metabolism (Zavarzin et al., 1991) may significantly impact the community structure of phyllosphere bacterial taxa. The groups of Proteobacteria have been shown to play important roles in supplying nutrients such as carbon (Badger and Bek, 2008), nitrogen (Galloway, 1998), sulfur (Sievert et al., 2007), and phosphate (Longnecker et al., 2010). To the best of our knowledge, these results represent the first study to catalogue the taxonomic identities of phyllosphere bacteria of rice by using pyrosequencing technique. Enterobacteriaceae-like sequences within the Gammaproteobacteria class were found to be the most abundant group in the phyllosphere across all four rice varieties, in agreement with previous reports (Redford and Fierer, 2009; Hunter et al., 2010). At the genus level, Pantoea-like bacteria dominated the sequences affiliated with the Enterobacteriaceae family and showed significant shifts at eCO2 . This group has long been recognized as containing plant growthpromoting bacteria. Pantoea agglomerans strains have been demonstrated to possess many plant-promoting characteristics, such as nitrogen fixation (Feng et al., 2003), 1-aminocyclopropane-1-carboxylate (ACC) deaminase, siderophore, and phytohormone produc-
tion (such as indole-3-acetic acid), and phosphate solubilization (Teng et al., 2010). In addition, this genus of Pantoea (such as Pantoea stewartii subsp. stewartii) has also been reported to be involved in control of plant pathogenic virulence by producing quorumsensing regulatory proteins (Schu et al., 2009). These versatile traits may be an important factor that enables them to readily adapt to the changing environment induced by eCO2 . The link between the metabolic activity of phyllosphere bacterial taxa and rice physiology remains poorly understood and needs more detailed study. In this study, eCO2 showed a stimulating effect on the dominant phylotype but appeared to suppress the rare phylotypes in the rice varieties TY-084 (Fig. 2a), YLY-6 (Fig. 2b), and ZX-96 (Fig. 2c); however, a reverse trend was observed in the rice variety YD-8 (Fig. 2d). These contrasting response patterns may be due to the difference in the relative abundance of each phylotype in the aCO2 control. The relative abundance of the dominant phylotype at aCO2 was similar among the rice varieties TY-084, YLY-6, and ZX-96, ranging from 70.6% to 75.2%. In the rice variety YD-8, the dominant phylotype comprised up to 94.1%, a measurement dramatically higher than the observations for the other three varieties. Therefore, the response of the phyllosphere bacterial taxa to eCO2 may depend on their relative abundance at aCO2 . This finding is consistent with a previous study indicating the critical role of relative abundance in driving the seasonal changes of the microbial communities (Ellis et
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al., 1999). There may be a threshold value of relative abundance at which the response pattern of rice phyllosphere bacteria to eCO2 would behave differently. This was supported by the diversity index results to some extent, as the rice varieties TY-084, YLY-6, and ZX96 showed decreases in the bacterial richness and diversity indices at eCO2 , while YD-8 showed increasing trends. In addition, the microbial communities may respond in a rice-variety specific manner. The composition and diversity of phyllosphere communities have been reported to reflect the immigration survival and growth of microbial colonists (Vorholt, 2012). This process may be influenced by numerous environmental factors in addition to the plant genotype. Plant genotypes can lead to variations in physiological states, including water transport, photosynthesis, respiration, and stomatal conductance, which could affect the microbial response patterns to environmental stress (Whipps et al., 2008). The PCA results further demonstrated that the phyllosphere bacterial taxa at eCO2 were distinct from those at aCO2 , although the response patterns to eCO2 diverged across all rice varieties tested. Additionally, the phyllosphere bacterial community structure was altered at eCO2 for all rice varieties, and this observation is consistent with the response of soil bacterial community to eCO2 (He et al., 2012). It appears that the environmental disturbance by eCO2 plays an important role in influencing the bacterial diversity as well as the structure of microbial communities in the phyllosphere of rice. CONCLUSIONS The results of this study demonstrated that the most dominant phylotype on the four rice varieties tested was the Enterobacteriaceae-like sequences within the class Gammaproteobacteria. The phyllosphere bacteria of rice showed contrasting response patterns to eCO2 . The rice varieties TY-084, YLY-6, and ZX96 had an increasing trend in the relative abundance of the dominant phylotype (Enterobacteriaceae), from 70.6%–75.2% at aCO2 to 86.5–93.8% at eCO2 ; the rare phylotypes including Xanthomonadaceae, Pseudomonadaceae, Sphingobacteriaceae, Oxalobacteraceae, and Clostridiaceae showed a tendency to decrease in relative abundance. A reverse response pattern to eCO2 was observed for the rice variety YD-8. On this variety, the dominant phylotype, Enterobacteriaceae, was suppressed, with its relative abundance decreasing from 94.1% at aCO2 to 81.4% at eCO2 , while the relative abundance of the rare phylotypes increased from 3.37% to 6.59%. This revealed a relative abundancedependent response pattern. The contrasting response
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patterns of these phyllosphere bacterial taxa may lead to a divergence in bacterial diversity at eCO2 . The results of this study provided a mechanistic basis for moving toward a predictive understanding of microbially mediated ecosystem feedback resulting from rising atmospheric CO2 and global warming. ACKNOWLEDGEMENTS The authors would like to extend their gratitude to Drs. ZHOU Juan and LAI Shang-Kun and Prof. YANG Lian-Xin from Yangzhou University, China for their help in sample collection. The authors would also like to thank Dr. ZHANG Hua-Yong from the Institute of Soil Science, Chinese Academy of Sciences for bioinformatics assistance. REFERENCES Ainsworth, E. A., Leakey, A. D. B., Ort, D. R. and Long, S. P. 2008. FACE-ing the facts: inconsistencies and interdependence among field, chamber and modeling studies of elevated [CO2 ] impacts on crop yield and food supply. New Phytol. 179: 5–9. Ainsworth, E. A. and Long, S. P. 2005. What have we learned from 15 years of free-air CO2 enrichment (FACE)? A metaanalytic review of the responses of photosynthesis, canopy properties and plant production to rising CO2 . New Phytol. 165: 351–372. Austin, E. E., Castro, H. F., Sides, K. E., Schadt, C. W. and Classen, A. T. 2009. Assessment of 10 years of CO2 fumigation on soil microbial communities and function in a sweetgum plantation. Soil Biol. Biochem. 41: 514–520. Badger, M. R. and Bek, E. J. 2008. Multiple Rubisco forms in proteobacteria: their functional significance in relation to CO2 acquisition by the CBB cycle. J. Exp. Bot. 59: 1525– 1541. Balint-Kurti, P., Simmons, S. J., Blum, J. E., Ballar, C. L. and Stapleton, A. E. 2010. Maize leaf epiphytic bacteria diversity patterns are genetically correlated with resistance to fungal pathogen infection. Mol. Plant Microbe Interact. 23: 473– 484. Caporaso, J. G., Bittinger, K., Bushman, F. D., DeSantis, T. Z., Andersen, G. L. and Knight, R. 2010. PyNAST: a flexible tool for aligning sequences to a template alignment. Bioinformatics. 26: 266–267. Chinnadurai, C., Balachandar, D. and Sundaram, S. P. 2009. Characterization of 1-aminocyclopropane-1-carboxylate deaminase producing methylobacteria from phyllosphere of rice and their role in ethylene regulation. World J. Microbiol. Biotechnol. 25: 1403–1411. Cooperative Research Group on Chinese Soil Taxonomy. 2001. Chinese Soil Taxonomy. China Science and Technology Press, Beijing. De Costa, D. M., Samarasinghe, S. S. T., Dias, H. R. D. and Dissanayake, D. M. N. 2008. Control of rice sheath blight by phyllosphere epiphytic microbial antagonists. Phytoparasitica. 36: 52–65. Edgar, R. C. 2010. Search and clustering orders of magnitude faster than BLAST. Bioinformatics. 26: 2460–2461. Ellis, R. J., Thompson, I. P. and Bailey, M. J. 1999. Temporal fluctuations in the pseudomonad population associated with
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