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. 2013 Feb 26;4(2):e00602-12.
doi: 10.1128/mBio.00602-12.

Unexpected diversity during community succession in the apple flower microbiome

Affiliations

Unexpected diversity during community succession in the apple flower microbiome

Ashley Shade et al. mBio. .

Abstract

Despite its importance to the host, the flower microbiome is poorly understood. We report a culture-independent, community-level assessment of apple flower microbial diversity and dynamics. We collected flowers from six apple trees at five time points, starting before flowers opened and ending at petal fall. We applied streptomycin to half of the trees when flowers opened. Assessment of microbial diversity using tag pyrosequencing of 16S rRNA genes revealed that the apple flower communities were rich and diverse and dominated by members of TM7 and Deinococcus-Thermus, phyla about which relatively little is known. From thousands of taxa, we identified six successional groups with coherent dynamics whose abundances peaked at different times before and after bud opening. We designated the groups Pioneer, Early, Mid, Late, Climax, and Generalist communities. The successional pattern was attributed to a set of prevalent taxa that were persistent and gradually changing in abundance. These taxa had significant associations with other community members, as demonstrated with a cooccurrence network based on local similarity analysis. We also detected a set of less-abundant, transient taxa that contributed to general tree-to-tree variability but not to the successional pattern. Communities on trees sprayed with streptomycin had slightly lower phylogenetic diversity than those on unsprayed trees but did not differ in structure or succession. Our results suggest that changes in apple flower microbial community structure are predictable over the life of the flower, providing a basis for ecological understanding and disease management.

Importance: Flowering plants (angiosperms) represent a diverse group of an estimated 400,000 species, and their successful cultivation is essential to agriculture. Yet fundamental knowledge of flower-associated microbiotas remains largely unknown. Even less well understood are the changes that flower microbial communities experience through time. Flowers are particularly conducive to comprehensive temporal studies because they are, by nature, ephemeral organs. Here, we present the first culture-independent time series of bacterial and archaeal communities associated with the flowers of apple, an economically important crop. We found unexpected diversity on apple flowers, including a preponderance of taxa affiliated with Deinococcus-Thermus and TM7, phyla that are understudied but thought to be tolerant to an array of environmental stresses. Our results also suggest that changes in microbial community structure on the apple flower may be predictable over the life of the flower, providing the basis for ecological understanding and disease management.

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Figures

FIG 1
FIG 1
Study design and flower anatomy. (A) Time course for sampling the apple microbiome. Fifteen pooled flowers from each of six Gala apple trees were collected at five time points over the life span of the flowers, for a total of 30 samples. Collection of phenologically matched flowers began before they opened and ended when petals fell. Arrows indicate streptomycin application. A precipitation event (1.30 cm) occurred on 30 April 2010. (B) Anatomy of a flower. All flower parts, including sepals, pistil, petals, and stamens, were included in the sampling. The current study provides a picture of the microbial communities that includes all of these flower compartments. (Reprinted from reference with permission of the publisher.)
FIG 2
FIG 2
Distribution of taxon abundances among OTUs (singletons omitted) detected on apple flowers.
FIG 3
FIG 3
Temporal trends in the apple flower microbiome. For each time point, n = 6 (1 sample of DNA extracted in bulk from 15 flowers from each of six trees). The inner-quartile ranges are shown by the box boundaries, nonoutlier extremes are shown by the whiskers, the median is shown by the thick middle line, and outliers are shown by the outliers’ black points. Statistics were summarized across each of six trees sampled at each time point, and the communities were analyzed at the 97% OTU level. Trees 4, 5, and 6 were sprayed with streptomycin on 29 and 30 April 2010. (a) Rarefied Faith’s phylogenetic diversity of microorganisms. There were 10 resamples at a depth of 1,531 sequences for each tree at each time point. Letters indicate significant differences in phylogenetic diversity across days, assessed by analysis of variance (F = 101.56, 4 degrees of freedom [df], P < 0.001) and post hoc testing with Tukey’s HSD test (P < 0.05). (b) Variability in community structure (assessed by analysis of beta dispersion, a metric of variability). Though modest differences were detected (multivariate homogeneity of group dispersions; F = 2.34, 4 df, P = 0.08), a post hoc test revealed that only 27 and 29 April were different (Tukey’s HSD test; P = 0.09).
FIG 4
FIG 4
Discovery, dynamics, and characteristics of apple flower successional groups. (a) Hierarchical clustering (complete linkage-based Bray-Curtis similarities among OTUs defined at 97% sequence identity) to determine OTUs having coherent dynamics. The analysis was conducted on 1,677 OTUs, each represented as a branch tip of the dendrogram. The y axis height represents within-cluster Bray-Curtis similarity. (b) Successional group dynamics indicating the mean relative abundance of members belonging to each group over the lifetime of apple flowers. Error bars indicate the standard deviation around the mean of 6 trees.
FIG 5
FIG 5
Dynamics of the five most prevalent OTUs detected for each successional group. OTU IDs correspond to the taxonomic assignments in Table 1. Error bars are standard errors around the mean OTU’s relative abundance at one time point across six trees. (a) Pioneer taxa; (b) Early succession taxa; (c) Mid succession taxa (secondary axis is for OTU 6932); (d) Late succession taxa; (e) Climax taxa; (f) Generalist taxa.
FIG 6
FIG 6
Distribution and representation of apple flower microorganisms. The tree includes OTUs identified at least to the class level. The branch shading corresponds to the different phyla detected (phylum names indicated in boxes). The branch tip colors correspond to the successional groups of each taxon (OTUs with 97% tag sequence identity). The height and label of the colored bar indicate the relative abundance of each OTU in the data set, and unlabeled OTUs have relative abundances below 0.01. Note that Fig. 6 is a tool for visualizing the representation of OTUs among phyla and is not intended to depict evolutionary relationships.
FIG 7
FIG 7
Characteristics of community structure that contribute to changes in beta diversity though time. (a) Prevalent members of the community are detected often (persistent through time and prevalent across trees), while rare members are detected infrequently (transient). Each OTU is a point, the blue line is the log-linear model (adjusted r2 = 0.68, slope = 6.49, P < 0.001), and gray shading represents the standard error. Percent occurrence is out of 30 total observations (six trees and five sampling times). (b) Partition of temporal beta diversity (measured as Sørenson’s similarity [Sor]; blue) into components that represent taxa replacement (Simpson’s similarity [Sim]; green) and nestedness (Nes; red). Each point is multivariate community similarity calculated for a time series from one tree, and the analysis was repeated for each of six trees and at various levels of cutoff to remove less prevalent OTUs. The line is an average across the trees; gray shading represents the standard error. (c) Prevalent members of the community are detected often (persistent), while rare members are detected infrequently (transient). Each OTU is a point, the color shows the tree in which the OTU was detected, and the lines are the log-linear models for each tree (all adjusted r2 values are >0.45, slopes range between 3.99 and 5.01, all P values are <0.001). Percent occurrence is out of five time points per tree. (d) Nestedness metric based on overlap and decreasing fill (NODF). Each point is calculated for a time series from one tree, and the analysis was repeated for each of six trees and at various levels of cutoff to remove less-prevalent members. The line is an average across the trees; gray shading represents the standard error.
FIG 8
FIG 8
Association network of prevalent taxa (top 20% most abundant), color coded by their successional group. OTUs are nodes, and node size is relative to its number of sequences, with larger shapes indicating more-abundant OTUs. Node shape indicates the phylum-level affiliation of the OTU. Edges (lines) are significant associations assessed by local similarity analysis (P < 0.001, q < 0.05). Light-gray edges are time-lagged associations, while dark edges are unilateral associations (see the supplemental results in Text S1 in the supplemental material). The distance between two OTUs (length of the edge) was optimized to distinguish associations between successional groups.

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