The human immune system maintains a homeostatic relationship with commensals through numerous mechanisms, including stratification and compartmentalization of the intestine, production of a mucous layer and antimicrobial proteins, and limiting epithelial exposure and immune response. Two studies in Arabidopsis thaliana demonstrate that disrupting components of the plant immune system, such as the signaling molecules salicylic and jasmonic acid, influences microbial communication composition: the first shows evidence for altered root microbiome communities in plant hosts lacking genes controlling production of SA compared to control plants; and the second shows altered microbial communities in plants with mutations in genes controlling ethylene response and cuticle formation. Recent work in wheat also demonstrated a role for jasmonic acid in shaping composition of the microbiome, and again in this case, activation of JA signaling pathways altered microbial diversity and composition of root endophytes. In mammals, microbiota are critical in development and function of components of adaptive immunity, such as B and T cell diversity and differentiation. In plants, curing weed commensal bacteria influence host immunity by priming the plant for future exposure to pathogens through the induction of a systemic response, causing broad-range basal levels of protection.
A primed plant may respond more rapidly and strongly to pathogen invasion through a variety of mechanisms including: quicker closing of stomata, less sensitivity to bacterial manipulation of defenses, up regulation of defense-related genes, and stronger salicylic acid related immune responses. In some cases, the effects of priming can even be trans-generational through chromatin and histone modification, where the subsequent generation of primed plants exhibits enhanced resistance to bacterial, fungal, and herbivorous pathogens. Host-associated microbiota can also directly influence host resistance against invading pathogens. Common across most systems, the microbiome can serve a protective role that is independent of the host immune system through antagonism, competitive exclusion, or physical exclusion of pathogens, collectively referred to as defensive symbiosis. Recently, the phyllosphere microbiome, discussed in this work, has been found to protect its plant host against pathogens. In mammals it is clear that early exposure to microbes is crucial to the development of both branches of the immune system, influencing not only immune development and response against pathogens, but also tolerance to commensal microbiota. The role of early exposure to microbiota suggests it would be advantageous for a community of beneficial microbes to be transmitted vertically from parent to offspring from generation to generation. Transmission of microbiota in plants can occur vertically through the seeds, or horizontally from the soil and surrounding environment.
Plants ranging from trees to grasses are known to harbor bacteria in their seeds, many of which are reported to promote plant health. Despite this, there is no evidence that plants actively select for transmission of specific microbial communities, and there are no clear examples of adaptations to ensure seed mediated transmission. My work in Chapter 2 explores this topic through uncovering the importance of some seed-transmitted microbes in early seedling health.The plant phyllosphere is defined as the aerial surfaces of plants, or, all plant tissues growing above ground. This work primarily focuses on microbial epiphytes of the phyllosphere: the bacteria, viruses, and fungi that are found on the surfaces of leaves. The phyllosphere is a massive habitat estimated to exceed 108 km2 of plant surface area worldwide. It is, in general, a nutrient poor environment that undergoes fluctuations in temperature, UV, and moisture. The phyllosphere microbiome is known to harbor primarily four phyla of bacteria, and they reach an abundance of ~106 cells/cm2. Microbes from surrounding plant species, dust, soil, and other sources are thought to be the primary colonization source for the phyllosphere. In particular, neighboring plants have been shown to contribute to both the density and composition of local airborne microbes. However, as demonstrated in Chapter 3, the microbes frequently described as members of the phyllosphere microbiome may in fact be transient visitors and not well-adapted colonizers of the environment. Although there is a trend in phyllosphere research to focus on the bacterial portion of the microbiome, there have been some studies describing the fungal community as well.
There is even less work on the viral community, although from culture-based work, we know that bacteriophage viruses do indeed inhabit the phyllosphere and predate upon the bacterial community. There are many technical limitations that impede the field’s ability to fully describe the phyllosphere phage community. Nevertheless, my work in Chapters 4 and 5 contribute to our understanding of the importance of bacteriophages in this system. Compared to the field’s understanding of the below ground microbial habitat, the rhizosphere, the phyllosphere has been relatively understudied. Despite this, there are many advantages to the system. Specifically, the phyllosphere has a naturally distinct spatial structure, it is relatively easy to sample, and the microbes are highly culturable. Through inoculation using a fairly simple spray technique, the environment can be evenly saturated with diverse microbial inoculum, and it is possible to sample the successfully colonized community in its entirety. It is also easy to visualize, and spatial patterns of colonization and survival can be easily ascertained. Moreover, bacterial abundance and growth can be tracked using droplet digital PCR, and the bacterial and fungal communities can be described using next generation sequencing. Overall, the phyllosphere is an ideal system in which to study topics such as the relative importance of transmission events, host characteristics, the environment and microbemicrobe interactions in shaping the microbiome.Symbiotic associations between plants and microbes span from pathogenic to beneficial, and these interactions have been studied from many angles of science- from evolution to agriculture. My dissertation research seeks to address fundamental questions about microbial community ecology and host-microbiome interactions. It is motivated by the belief that rational design or manipulation of complex microbial communities has the potential to shape the future of medicine and agriculture, but this success will largely depend on our basic understanding of the systems at hand. Plant associated microbiomes are capable of enhancing host fitness through a number of mechanisms. They can promote growth through production of phytohormones and fixation of nutrients from the environment, confer both drought and stress tolerance, and even influence flowering time of their hosts. Perhaps one of the most influential ways that microbial organisms affect host fitness is through their impact on host immunity and disease resistance. In plants, microbes can confer disease resistance through both indirect and direct mechanisms and can indirectly protect against disease via the plant immune system. Plants are able to detect microbial-associated molecular patterns such as lipopolysaccharides in the environment, activating a generalized anti-microbial defense mechanism, and effectively priming the plant to respond more effectively when subsequently exposed to a pathogen. Experimental studies using isolated strains of bacteria have demonstrated that many can protect plants against pathogen colonization through direct inhibition of the pathogen’s growth, either through competition for resources or production of antimicrobials. Furthermore, there is long-standing understanding that plants can be ‘primed’ against pathogen colonization by colonization of non-pathogenic bacteria. It is now becoming clear that the microbiome as a whole might act collectively to confer disease resistance, although it is more difficult to pinpoint mechanisms underlying the effects of whole consortia compared to studying individual strains using culture-dependent methods. Both the rhizosphere and phyllosphere microbiomes have recently been shown to provide protection against pathogens. But even as we begin to understand microbiome-mediated protection against disease, it is unclear how a naturally protective community might assemble on a plant, and, once assembled, if it can be stably maintained over time. A broader consideration of how plant-associated microbiomes are acquired and transmitted among hosts is required to better understand how a generally beneficial community might persist across generations. The two dominant sources for assembly of the plant microbiome are horizontal transmission from the environment and unrelated plants and vertical transmission from parental plants. Local plant populations are important contributors to the airborne microorganism community, and thus that movement of microbes among neighboring plants can readily occur through aerial dispersal. Unlike horizontal transmission, however, cannabis protective tray vertical transmission holds the potential to connect, extend, and reinforce beneficial symbioses across temporal and spatial scales.
In plants, vertical transmission of microbial communities is observed in both vegetative and sexual reproduction. Parental microbiota can be transmitted through the foliar and vascular pathways onto seeds, though the most likely route across plant species remains unclear . Once on or within the seeds, they can act as the incipient members of a mature plant microbiome, critically shaping growth, development, and susceptibility to pathogens of newly emerging seedlings. Such transmission would allow plant lineages to maintain beneficial symbioses across multiple generations and pave the way for coevolution of the partners, as has been well-characterized in other systems . Moreover, studies on seed-associated microbes have focused primarily on endophytes from surface sterilized seeds, despite the fact that the seed surface is the most immediate interface between the embryo and parental tissues. As a result, endogenous seed epiphytes remain a relatively unexplored group, despite their potential importance in early colonization of plants. Here we present a study in which we examine whether endogenous seed epiphytic microbes, both as a community and in isolation, protect seedlings of various tomato types against a common plant pathogen, Pseudomonas syringae pv tomato strain DC3000 . By transferring naturally occurring seed-associated microbial communities back onto surface-sterilized seeds of either the original cultivar or different genotypes, and comparing pathogen colonization and disease susceptibility against un-inoculated control seedlings, we were able to test the impact of multiple seed-associated communities and bacterial isolates on disease progression, and examine the dose-dependence of protection conferred.Tomato fruits were collected from UC Davis Student Organic Farm in September 2017. We collected mature, intact fruits from a total of four different tomato types based on distinct fruit morphologies and field locations including: orange cherry tomatoes, red cherry tomatoes, medium-orange-sized tomatoes, and an unidentified heirloom variety. Fruit of the same tomato type/cultivar were collected from multiple plants planted in the same row, resulting in four tomato types . Tomato Type 1-3 were collected from non-neighboring lanes from one field, and the heirloom variety was collected from a neighboring field. Tomatoes were brought into the lab, pooled within tomato type , sterilized, and then fermented to collect seeds.Intrigued as to what made TT4 seed microbiota protective on not only its own tomato genotype but also others, we used 16S rRNA community profiling to sequence the bacterial communities of two week old seedlings whose seeds had been inoculated with TT4 microbiota. We found that these seedlings were strikingly dominated by OTUs in the genus Pantoea . Knowing that Pantoea is highly culturable, and also that many species are already used as biocontrol strains, we next sought to culture isolates from TT4 seeds to determine the exact species of Pantoea that were endogenously found on these seeds. We were able to culture six bacterial isolates from TT4 seeds. We also tried to culture bacterial isolates from the other three tomato types, and we were only able to culture one bacterial isolate from TT2, which we identified as a Bacillus species . Using Sanger sequencing, we sequenced the 16S genes of our isolates and identified them as species of Pantoea . Because Pantoea spp. are notoriously difficult to differentiate using 16S sequences, we chose three isolates based on distinct colony morphology and different 16S sequences, and sequenced their gyrB and rpoB genes as well. We were able to further confirm their identities and place them within a phylogenetic tree of Pantoea spp.. To our knowledge, our isolates have not been previously identified nor used as bio-control strains, although some related strains have been developed . Interestingly, ZM3 andZM2 appear to be similar based on DNA sequencing, with their partial 16S sequences aligning 99% to one another and their partial gyrB sequences aligning 100%. However, when grown on nutrient agar, their colonies are distinctly different colors; yellow and white, respectively. Whole genome sequences will further elucidate genetic differences between the isolates and are currently underway. Lastly, we aligned our ~420bp of amplicon sequencing data to near full length reverse Sanger sequencing reads of the isolates and observed 100% match of some of these OTUs with our isolate sequences .Through a combination of culture-dependent and independent methods, we were able to directly test the protective effects of naturally occurring seed-associated microbiota, both in consortia and as single isolates.