Establishment Limitation Constrains the Abundance of Lactic Acid Bacteria in the Napa Cabbage Phyllosphere

Abstract

Patterns of phyllosphere diversity have become increasingly clear with high-throughput sequencing surveys, but the processes that control phyllosphere diversity are still emerging. Through a combination of lab and field experiments using Napa cabbage and lactic acid bacteria (LAB), we examined how dispersal and establishment processes shape the ecological distributions of phyllosphere bacteria. We first determined the abundance and diversity of LAB on Napa cabbage grown at three sites using both culture-based approaches and 16S rRNA gene amplicon sequencing. Across all sites, LAB made up less than 0.9% of the total bacterial community abundance. To assess whether LAB were low in abundance in the Napa cabbage phyllosphere due to a limited abundance in local species pools (source limitation), we quantified LAB in leaf and soil samples across 51 vegetable farms and gardens throughout the northeastern United States. Across all sites, LAB comprised less than 3.2% of the soil bacterial communities and less than 1.6% of phyllosphere bacterial communities. To assess whether LAB are unable to grow in the phyllosphere even if they dispersed at high rates (establishment limitation), we used a gnotobiotic Napa cabbage system in the lab with experimental communities mimicking various dispersal rates of LAB. Even at high dispersal rates, LAB became rare or completely undetectable in experimental communities, suggesting that they are also establishment limited. Collectively, our data demonstrate that the low abundance of LAB in phyllosphere communities may be explained by establishment limitation.IMPORTANCE The quality and safety of vegetable fermentations are dependent on the activities of LAB naturally present in the phyllosphere. Despite their critical role in determining the success of fermentation, the processes that determine the abundance and diversity of LAB in vegetables used for fermentation are poorly characterized. Our work demonstrates that the limited ability of LAB to grow in the cabbage phyllosphere environment may constrain their abundance on cabbage leaves. These results suggest that commercial fermentation of Napa cabbage proceeds despite low and variable abundances of LAB across different growing regions. Propagule limitation may also explain ecological distributions of other rare members of phyllosphere microbes.

Keywords: 16S; cabbage; community assembly; lactic acid bacteria; microbiome; phyllosphere.

FIG 1

Conceptual overview of propagule limitation and the farm-to-ferment assembly of fermented vegetable microbiomes. (A) The three types of propagule limitation are source, dispersal, and establishment. Source limitation is where not enough propagules are present in local species pools to disperse to the phyllosphere. Dispersal limitation is where enough propagules are produced in local species pools, but they cannot reach all available habitats due to lack of dispersal opportunities. Establishment limitation is where local population sizes are constrained by the availability of habitats that can be colonized, for example, by competition with other phyllosphere bacteria. (B) Local species pools consist of bacteria that could disperse to cabbage leaves to form the phyllosphere microbiome. When chopped up and fermented, the phyllosphere microbiome is reassembled to become the fermented vegetable microbiome.

FIG 2

Microbial diversity and LAB abundance in the Napa cabbage phyllosphere. (A) Abundance of total culturable bacteria (TS agar in blue) and lactic acid bacteria (MRS agar in red) in the Napa cabbage phyllosphere at three sites in the Boston, MA, area. Site 1, n = 9; site 2, n = 10; site 3, n = 7. (B) Relative abundance of bacterial phyla and Lactobacillales families identified in the Napa cabbage phyllosphere using amplicon sequencing of the 16S rRNA gene. Each column represents an individual cabbage showing the initial phyllosphere microbiome of the lab-grown cabbage (n = 5), site 1 phyllosphere (n = 9) and fermented (n = 4), site 2 phyllosphere (n = 10) and fermented (n = 4), and site 3 (n = 7) and fermented (n = 4). Members of the Lactobacillales are shown at the family level in order to indicate the prevalence of major LAB groups. (C) Abundance of total culturable bacteria (TS agar in blue) and lactic acid bacteria (MRS agar in red) in the Napa cabbage phyllosphere from cabbages purchased at five supermarkets. For each supermarket, n = 6. (D) Relative abundance of phyla identified in the Napa cabbage phyllosphere purchased from one of the five supermarkets. Each column represents an individual cabbage. As with panel B, members of the Lactobacillales are shown at the family level in order to indicate the prevalence of major LAB groups.

FIG 3

LAB are rare in species pools. (A) Abundance of total culturable bacteria (TS agar in blue) and lactic acid bacteria (MRS agar in red) in leaf and soil species pools from 51 farms through the northeastern United States. Each point represents CFU data collected from a pool of five leaf or soil samples from each site. (B) Relative abundance of bacteria found in leaf samples and soil samples at a subset of farms from panel A, as determined with amplicon sequencing of the 16S rRNA gene (V4 region). As with Fig. 2, members of the Lactobacillales are shown at the family level in order to indicate the prevalence of major LAB groups. Each column represents sequence data collected from a pool of five leaf or soil samples from each site. Data from 14 sites are presented. (C) Relative abundance of Lactobacillales in amplicon sequence data collected as part of the Earth Microbiome Project. Points indicate means, and error bars indicate one standard deviation.

FIG 4

Gnotobiotic cabbages demonstrate that LAB are establishment limited in the Napa cabbage phyllosphere. (A) Photograph of gnotobiotic Napa cabbages used in experiments. (B) Overview of experimental design showing four treatments (LAB grown alone, LAB grown 1:1 with synthetic phyllosphere community [SPC], LAB grown 1:10 with SPC, and LAB grown 1:100 with SPC). Treatments were applied using sterile brown amber spray bottles. Four lactic acid bacteria were used in these experiments: Pediococcus pentosaceus strain B6N, Leuconostoc mesenteroides strain BN10, Lactobacillus plantarum strain MKR2, and Lactococcus lactis strain D119. (C) Abundance of the SPC (TS agar in blue) and LAB (MRS agar in red) in the input (I) inoculum and output (O) after 10 days of growth in the NCP is shown. Input and output were significantly different from one another in all tested LAB species (t test, P < 0.5, corrected for repeat sampling using a Hochberg correction; n = 8; ND, not detected).

FIG 5

LAB are establishment limited in the phyllosphere of field-grown Napa cabbages. (A) Overview of the experimental design. Napa cabbages were grown in the lab and were sprayed with either PBS as a control or an equal mix of three LAB (Pediococcus pentosaceus strain B6N, Leuconostoc mesenteroides strain BN10, and Lactobacillus plantarum strain MKR2) for the +LAB treatment. Input data were collected right after inoculation, and output data were collected after 30 days of growth in the field. After 10 days of growth, cabbages were inoculated again with the same treatments. Input data were collected at the second inoculation, and output data were collected after 17 more days of growth. (B) Abundance of LAB (MRS agar in red) and other phyllosphere bacteria (TS agar in blue) in the input (I) inoculum and output (O) leaf harvests for the first period of the experiment. An asterisk indicates a significant difference between input (I) and output (O) bacterial densities (t test, P < 0.5, corrected for repeat sampling using a Hochberg correction). The number of replicates is indicated at the bottom of the graph. ND, not detected. (C) Same as panel B, but for the second set of inputs and outputs. (D) Abundance of LAB during fermentation of cabbages from the Control and +LAB treatments in panels B and C. Points indicate mean values (n = 3). Error bars represent one standard deviation. (E) pH during fermentation of cabbages from the Control and +LAB treatments in panels B and C. Points indicate mean values (n = 3). Error bars represent one standard deviation. Control and +LAB treatments were not significantly different from one another in panel D or E (see the text for repeated-measures ANOVA statistics).