The similarity of changes in community structure both across replicates and genotypes over the course of the passaging experiment led us to predict that these microbiomes were adapting to the local plant and greenhouse environment. To further determine if the community changes we observed from P1 to P4 were due to habitat selection rather than neutral processes, we employed a community coalescence competition experiment. In this experiment , phyllosphere communities from the end of P1 and the end of P4 were inoculated onto a new cohort of plants, either on their own or in an approximately 50:50 mixture of live cells . To ensure that our method for the mixed inoculum was effective, we sequenced multiple replicates of the P1, P4, and Mix inoculums and found that source of inoculum explains 88% of dissimilarity amongst samples . To ensure that the Mix inoculum was significantly different than both P1 and P4 separately, we compared P1 and Mix inocula directly and found that 75% of difference between samples can be explained by this variable . Similarly, when P4 and Mix are compared directly, 74% of variation in the community is explained . This consistent difference among the three inocula allowed us to compare the communities colonizing plants from each treatment. We first measured final bacterial abundance and found that colonization was lower on these plants than in previous experiments, pipp mobile storage systems but does not significantly differ among treatments , apart from control plants, where bacterial colonization was greatly reduced .
We then compared bacterial communities again using 16S amplicon sequencing and ordinated samples on a PCoA based on Bray-Curtis distances. Plants that received P1 inoculum had distinctly different communities than those that received either P4 or the Mixed inoculum. Plants that received the Mixed inoculum clustered together with those receiving P4 and were relatively indistinguishable. Using ADONIS tests, we determined that inoculum source can explain 45% of Bray-Curtis dissimilarity amongst samples , and there was no effect of plant genotype . In a pairwise analysis between P1 and Mixed, inoculum source explains 31% of the community dissimilarity . In contrast, inoculum source does not explain any significant variation in dissimilarity amongst P4 and Mixed inoculum plants . Together, these results suggest that the plants receiving the 50:50 mixed inoculum were indistinguishable in community composition from those receiving the pooled, P4 adapted microbiomes, and that these selected communities were not invadable by the microbial communities from the start of the experiment. Consistent with our results from the passaging experiment itself, alpha diversity was highest in P1 plants compared to both P4 and Mixed plants . Alpha diversity did not differ amongst communities colonizing plants from the P4 and Mixed inoculums, despite being different between the two inocula themselves. We also examined compositional makeup of the communities , and consistent with P1 to P4 passaging results, we see differentially abundant taxa between groups .
Again, two Pseudomonas OTUs are more abundant in P1 plants as compared to P4 and Mix, in which there was an unclassified Pseudomonaceae that was higher in relative abundance.The impact of a microbiome on host health and fitness depends not only on which microbial organisms are present in the community, but also on how they interact with one another within the microbiome. Unlocking the great potential of microbiome manipulation and pre/probiotic treatment in reshaping host health will therefore depend on our ability to understand and predict these interactions. We took a microbiome passaging approach, inspired by classic experimental evolution, to test how selection for growth in the tomato phyllosphere under greenhouse conditions would impact microbiome diversity and adaptation across genotypes that differ in disease resistance genes. Across independently selected lines passaged on five tomato genotypes, we observed a dramatic shift in community structure and composition, accompanied by a loss of alpha diversity . We also found that host genotype shapes community composition early in passaging , explaining over 24% of variation amongst samples, but diminishes over time. The relative importance of host genotype and environment in shaping microbiome composition remains highly debated. Our results suggest that the relative importance of genotype versus other factors, such as the growth environment or strength of within-microbiome interactions, changes over the course of passaging on a constant host background. We did observe that even in the absence of a strong genotype effect, there remains a legacy of genotype effect, in that OTUs found to be significantly associated with particular genotypes early on are more likely to be present at the end of passaging than those that did not exhibit any host preference.
In order to test if the phyllosphere microbiome undergoes habitat filtering, we chose to begin the experiment with a diverse inoculum. This starting community generated from field grown tomato plants likely contained microbes from other surrounding plant species, dust, soil, and other sources. In particular, neighboring plants have been shown to contribute to both the density and composition of local airborne microbes. We found that although the total number of these field inoculum OTUs decreased over the course of the experiment, the taxa that remained consistently made up 78-95% of the community. This provides evidence that the original spray inoculum underwent strong niche selection over the course of the experiment. To test the alternative hypothesis that community changes were due to neutral processes such as bottle necking or random dispersal, we first fit our data to neutral and null models, finding a poorer fit over time. We then tested this experimentally by conducting a community coalescence experiment to measure fitness of passaged microbiomes as compared to those from the start of the experiment. The results of this experiment strongly support the idea that these phyllosphere microbiomes adapted to the plant host environment over the course of four passages . Independent of overall bacterial abundance, P4 microbiomes were able to dramatically outcompete the less-adapted P1 microbiomes. This community coalescence approach allowed us to demonstrate non-neutral adaption of a bacterial community that is independent of host genotype and resistant to invasion by a more diverse, less-adapted community. This community coalescence approach was used by others in a study conducted on methanogenic bacterial communities. The authors found that when multiple methanogenic communities were combined, a single dominant community emerged from the mix. This emergent dominant community resembles the single community with the highest methane production that went into the combination, suggesting that the most-fit community is capable of reassembly, even in the presence of other bacteria.The impact of a microbiome on host health and fitness depends not only on which microbial organisms are present in the community, but also on how they interact with one another within the microbiome. Unlocking the great potential of microbiome manipulation and pre/probiotic treatment in reshaping host health will therefore depend on our ability to understand and predict these interactions. We took a microbiome passaging approach, cannabis growing systems inspired by classic experimental evolution, to test how selection for growth in the tomato phyllosphere under greenhouse conditions would impact microbiome diversity and adaptation across genotypes that differ in disease resistance genes. Across independently selected lines passaged on five tomato genotypes, we observed a dramatic shift in community structure and composition, accompanied by a loss of alpha diversity . We also found that host genotype shapes community composition early in passaging , explaining over 24% of variation amongst samples, but diminishes over time. The relative importance of host genotype and environment in shaping microbiome composition remains highly debated. Our results suggest that the relative importance of genotype versus other factors, such as the growth environment or strength of within-microbiome interactions, changes over the course of passaging on a constant host background. We did observe that even in the absence of a strong genotype effect, there remains a legacy of genotype effect, in that OTUs found to be significantly associated with particular genotypes early on are more likely to be present at the end of passaging than those that did not exhibit any host preference. In order to test if the phyllosphere microbiome undergoes habitat filtering, we chose to begin the experiment with a diverse inoculum. This starting community generated from field grown tomato plants likely contained microbes from other surrounding plant species, dust, soil, and other sources. In particular, neighboring plants have been shown to contribute to both the density and composition of local airborne microbes.
We found that although the total number of these field inoculum OTUs decreased over the course of the experiment, the taxa that remained consistently made up 78-95% of the community. This provides evidence that the original spray inoculum underwent strong niche selection over the course of the experiment. To test the alternative hypothesis that community changes were due to neutral processes such as bottle necking or random dispersal, we first fit our data to neutral and null models, finding a poorer fit over time. We then tested this experimentally by conducting a community coalescence experiment to measure fitness of passaged microbiomes as compared to those from the start of the experiment. The results of this experiment strongly support the idea that these phyllosphere microbiomes adapted to the plant host environment over the course of four passages . Independent of overall bacterial abundance, P4 microbiomes were able to dramatically outcompete the less-adapted P1 microbiomes. This community coalescence approach allowed us to demonstrate non-neutral adaption of a bacterial community that is independent of host genotype and resistant to invasion by a more diverse, less-adapted community. This community coalescence approach was used by others in a study conducted on methanogenic bacterial communities. The authors found that when multiple methanogenic communities were combined, a single dominant community emerged from the mix. This emergent dominant community resembles the single community with the highest methane production that went into the combination, suggesting that the most-fit community is capable of reassembly, even in the presence of other bacteria.Seeds were surface sterilized using TGRC recommendations as follows: seeds were soaked in 2.7% bleach solution for 20 minutes. Sterilized seeds were then transferred onto 1% water agar plates and placed in the dark at 21°C until emergence of the hypocotyl. At that point, seedling plates were moved into a growth chamber and allowed to continue germination for 1 week. Growth chamber conditions were 25°C, 65% humidity and 16 h daylight per day. After approximately one week, seedlings were transferred planted in sunshine mix #1 soil in seedling trays. After approximately one more week of growth, seedlings were transplanted into 8” diameter pots, making the plants approximately 2.5-3 weeks old at the first time of microbial inoculation. Age of inoculation varied slightly from experiment to experiment but was kept identical amongst genotypes within an experiment.Microbial inoculum for the first passage of the experiment was generated from field-grown tomato plants from the UC Davis Student Organic Farm collected in September and October of 2016. One-gallon Ziploc bags were filled with leaf, stem, and some flower material from tomato plants. One bag was collected from each of nine different sites, spread through four different fields. Plant material was collected from various genotypes of tomatoes. Other plant types, such as lettuce, eggplant, corn, and oak trees, surrounded the tomato fields. During the October collection, soil was also collected at each site. The top ~2cm of soil was brushed away, and a 50mL conical was pushed directly into the soil at the base of a plant which was in the middle of each collection site. Plant material and soil were transferred to the lab on ice and stored at 4°C briefly until processing. Sterile phosphate freezing buffer was added to the bags of leaves, and the entire bags were placed in a Branson M5800 sonicating water bath. Material was sonicated for 10 minutes. This gentle sonication washes microbes from the surfaces of the leaves but does not damage cells. The resulting leaf wash from each site was pooled. From the September collection, leaf wash was pelleted for 10 mins at 4000 x G, resuspended in glycerol freezing buffer, and stored at -80 for approximately one month. This was then thawed, re-spun to remove the freezing buffer, and combined with the October leaf wash.