Comparative Analyses of Phyllosphere Bacterial Communities and Metabolomes in Newly Developed Needles of Cunninghamia lanceolata (Lamb.) Hook. at Four Stages of Stand Growth

Abstract

Host-plant-associated bacteria affect the growth, vigor, and nutrient availability of the host plant. However, phyllosphere bacteria have received less research attention and their functions remain elusive, especially in forest ecosystems. In this study, we collected newly developed needles from sapling (age 5 years), juvenile (15 years), mature (25 years), and overmature (35 years) stands of Chinese fir [Cunninghamia lanceolata (Lamb.) Hook]. We analyzed changes in phyllosphere bacterial communities, their functional genes, and metabolic activity among different stand ages. The results showed that phyllosphere bacterial communities changed, both in relative abundance and in composition, with an increase in stand age. Community abundance predominantly changed in the orders Campylobacterales, Pseudonocardiales, Deinococcales, Gemmatimonadales, Betaproteobacteriales, Chthoniobacterales, and Propionibacteriales. Functional predictions indicated the genes of microbial communities for carbon metabolism, nitrogen metabolism, antibiotic biosynthesis, flavonoids biosynthesis, and steroid hormone biosynthesis varied; some bacteria were strongly correlated with some metabolites. A total of 112 differential metabolites, including lipids, benzenoids, and flavonoids, were identified. Trigonelline, proline, leucine, and phenylalanine concentrations increased with stand age. Flavonoids concentrations were higher in sapling stands than in other stands, but the transcript levels of genes associated with flavonoids biosynthesis in the newly developed needles of saplings were lower than those of other stands. The nutritional requirements and competition between individual trees at different growth stages shaped the phyllosphere bacterial community and host-bacteria interaction. Gene expression related to the secondary metabolism of shikimate, mevalonate, terpenoids, tocopherol, phenylpropanoids, phenols, alkaloids, carotenoids, betains, wax, and flavonoids pathways were clearly different in Chinese fir at different ages. This study provides an overview of phyllosphere bacteria, metabolism, and transcriptome in Chinese fir of different stand ages and highlights the value of an integrated approach to understand the molecular mechanisms associated with biosynthesis.

Keywords: Cunninghamia lanceolata; functional genes; host-bacteria interaction; metabolic profile; phyllosphere bacterial community.

Figure 1

Structure of phyllosphere bacterial communities of Chinese fir in stands of different ages. (A) Venn diagram of phyllosphere bacterial operational taxonomic units (OTUs); (B) ACE index for each stand age; (C) Chaos index for each stand age; (D) Rarefaction curve of number of OTUs with increasing sequencing depth. SM5, SM15, SM25, and SM35 represent stands of age 5, 15, 25, and 35 years, respectively (n = 18).

Figure 2

Beta diversity in the phyllosphere bacterial community of Chinese fir. (A) Scatterplot of axes 1 and 2 from a PCoA based on unweighted UniFrac dissimilarity matrix; (B) unweighted ANOSIM; (C) UPGMA dendrogram. SM5, SM15, SM25, and SM35 represent stand ages of 5, 15, 25, and 35 years, respectively.

Figure 3

Abundance of phyllosphere bacterial communities with relative abundance > 0.8% as classified to (A) family; (B) genus; and (C) species taxonomic levels. (D) The top 30 bacterial orders identified by the predictive Random Forest model using the mean decreasing Gini score. SM5, SM15, SM25, and SM35 represent stand ages of 5, 15, 25, and 35 years, respectively.

Figure 4

The linear discriminant analysis (LDA) effect size (LEfSe) analysis of bacterial taxa showing significant differences in relative abundance among Chinese fir stands of different ages. (A) Cladogram using LEfSe method indicating the phylogenetic distribution of phyllosphere bacterial communities among different ages; (B) LDA scores showed the significant bacterial difference among four ages; (C–G) the relative abundance of Massilia, Pantoea, Burkholderiaceae, Betaproteobacteriales, and Gammaproteobacteria in each sample from the four groups. SM5, SM15, SM25, and SM35 represent stand ages of 5, 15, 25, and 35 years, respectively.

Figure 5

Changes in bacterial functional genes predicted by PICRUSt based on KEGG pathways (A–C,F–K) and Clusters of Orthologous Groups (COG) analysis (D,E) that differed significantly in pairwise comparisons of Chinese fir stands of different ages. (F–K) involved in (F) carbon metabolism; (G) nitrogen metabolism; (H) fatty acid elongation; (I) flavonoids biosynthesis; (J) antibiotic biosynthesis; and (K) steroid hormone biosynthesis, based on third-tier KEGG pathways. SM5, SM15, SM25, and SM35 represent stand ages of 5, 15, 25, and 35 years, respectively.

Figure 6

Leaf secondary metabolite profile of the phyllosphere of Chinese fir. (A) Metabolite classification based on HMDB data; (B) number of candidate metabolites among the 20 highest ranked KEGG pathways.

Figure 7

Metabolites that showed a significant change between any two stands of Chinese fir as indicated by partial least-squares discriminant analysis. (A) Principal component analysis (PCA) of the foliar metabolites of 28 independent samples collected from Chinese fir aged 5, 15, 25, and 35 years; (B) heatmap for all significantly changed metabolites. Metabolites satisfied the following conditions in pairwise comparisons: (1) ratio ≥ 2 or ratio ≤ 0.5; (2) q value < 0.05; (3) VIP ≥ 1; (4) annotation with a secondary metabolite; (C) level of metabolites that significantly changed at least four times in six pairwise comparisons.

Figure 8

Relationship between metabolites and phyllosphere bacterial communities of Chinese fir. (A) SparCC correlations between metabolite classes and genera; (B) network constructed for strongly correlated genera and metabolites (|R| > 0.6). *p < 0.05, **p < 0.01. Red lines indicate positive relationships; green lines indicate negative relationships.

Figure 9

Expression of unigenes in Chinese fir at four stages. (A) Numbers of DEGs in each comparison; (B) Venn diagram of analyses of differentially and stage-specific expression genes per comparison. SM5, SM15, SM25, and SM35 represent stand ages of 5, 15, 25, and 35 years, respectively.

Figure 10

Schematic diagram of the transcriptional changes of genes involved in secondary metabolism in leaves of Chinese fir at different stand ages. The log₂FC of DEGs in SM15, SM25, and SM35 compared with SM5, SM15, and SM35 is presented using the MapMan visualization platform. SM5, SM15, SM25, and SM35 represent stand ages of 5, 15, 25, and 35 years, respectively.