We found one particularly protective seed-associated microbial community that was able to significantly decrease the density of P. syringae pv tomato DC3000 growth on seedlings and reduce disease symptoms across multiple tomato types. Community profiling uncovered that Pantoea spp. dominated this seed microbiome, regardless of which seedling type it was applied to, and we were able to culture specific Pantoea strains directly from the surface of these seeds. When we applied these culturable isolates to seeds, we found that individual strains were as protective when applied in isolation as when combined. In order to understand how application density impacts protection, we varied the dose of isolates ZM1, ZM2, and ZM3, and we found a non-linear pattern of inoculation density correlation with pathogen density. The seed surface is the primary site of contact between seed and fruits, and it is known to harbor a diversity of microbes across plant species. Despite this, few studies have included seed epiphytes when investigating seed-associated microbes, focusing primarily on seed endophytes , endogenous seed-microbiota were found to suppress disease symptoms in juvenile seedlings of their natural hosts when challenged with a common tomato pathogen, Pst . When TT4 microbiota was inoculated onto two other field tomato types, it was able to significantly reduce disease symptoms and decrease the density of Pst by 10 to 100-fold . Although the tomato types themselves differed in their overall susceptibility to disease, we did not observe that any single tomato type was more protected by the TT4 microbiome than another. This may suggest that the pathogen- suppressive effects of TT4, pipp mobile storage whether attributable to microbiome members with antagonistic activities against Pst or immune system priming, are capable of acting independently of their host genotypic context.

Due to the way in which tomatoes were collected , we were not able to point to the specific differences amongst host genotypes, but this would be useful in future studies. Furthermore, as we did not sequence the microbiome of adult plants from which seeds were collected, future work should explore if differences amongst seed microbiomes are driven by differences in the microbiome composition of the adult plants themselves. These microbiome differences may be a result of field location, host genotype, or other unknown factors. Our data suggest that it may be possible to breed plants to specifically recruit or harbor beneficial seed microbiomes that may ensure a more disease resistant crop in subsequent generations. To better understand the protective effects observed, we sequenced the bacterial communities associated with seedlings inoculated with the TT4 microbiome and found the communities to be dominated by Pantoea spp . This is in line with community profiling results from the seed surface of Triticum and Brassica. We then isolated culturable bacteria from seeds, and again found primarily Pantoea spp. Inoculation of seeds with our Pantoea isolates showed that they are highly protective against Pst, both in terms of colonization and disease . Pantoea spp. is a known antagonist of many bacterial as well as fungal pathogens, and they are common biocontrol strains. Pantoea dispersa strain ZM1 appears to be novel and not previously described as a biocontrol species, but provides protection that is on par with, if not better than, currently commercially available strains. Genome sequencing will reveal if P. agglomerans strains ZM2 and ZM3 are novel bio-control strains. Our work also helps to disentangle the link between diversity and disease protection. Although there exists a speculative relationship between taxonomic diversity and the strength of a microbiome’s disease-resistance effect, little empirical evidence exists to support or disprovethis.

A recent study on the protective effect of a constructed community against Pst shows that variation in inoculum diversity affects disease-resistant effects in a significantly non-linear manner, demonstrating that increasing taxonomic diversity can have no impact, or even decrease, the protective effect of the community. In this study, seedling bacterial communities are low in richness and diversity, dominated primarily by one genus: Pantoea, although there are multiple species and strains of Pantoea. It is possible that a greater diversity of bacteria existed on the seeds, but we still find that inoculation of individual strains of Pantoea is sufficient in seedlings for protection against the pathogen used in this study. We are also aware that the fermentation step used to collect seeds might have enriched certain members of the seed microbiome that are able to survive acidic conditions. However, this may be a biologically relevant filtering step for epiphytic seed microbes, as seeds are likely to experience acidic conditions both during fermentation of fruit in the field or through the digestive track of animals. Although we only test the protective ability of isolates against a bacterial pathogen, the Pantoea isolates and the Bacillus isolate may have other growth promoting capabilities as well, as a recent paper describes various growth promotion traits of tomato seed endophytes. They may also have protective effects against fungal pathogens, as has been previously demonstrated in Pantoea species. Practically, seed associated bacteria are an excellent target for probiotic/biocontrol application, and it may even be possible to apply the protective strain to the flowers of the previous generous, as was demonstrated with a plant growth promoting endophyte. Taken together, these studies and our results suggest that the common agricultural practice of seed sterilization may be disrupting persistent mutualisms between plants and microbes across generations.

While seed sterilization is an agriculturally important procedure to purge seed-transmitted pathogens, we and other groups have shown that it may also be removing beneficial symbionts. How the simultaneous disruption of pathogenic and mutualistic symbioses would impact host health over ecological time scales, and how agricultural practices should preserve the beneficial traits conferred by the vertically transmitted microbiome while still preventing the spread of pathogens, are outstanding questions in need of future research. The focus of this work was to examine the potential protective effects of seed epiphytic communities rather than describe the mechanisms underlying protection. Previous work has demonstrated that some Pantoea spp. are protective through antibiosis activity, or it may also be mediated through competition for resources. In addition to direct interactions between microbes, application of Pantoea spp. to seeds ensures that germinating seedlings are in immediate contact with microbes, and this may prime the plant’s immune system so that it is better able to mount a response against Pst, thus indirectly protecting against disease. In our experiments, the data suggest that both direct and indirect mechanisms are mediating protection. We find that all strains of live bacteria, including a non-plant associated strain of E. coli, are capable of decreasing disease severity symptoms when compared to non-treated controls. When seeds are treated with UV-killed bacteria, we find that none of the strains are capable of decreasing disease severity. Our results suggest that all bacteria included in our experiment can protect seedlings against Pst through direct interactions, as UV-killed bacteria were unable to decrease disease severity. Additionally, we found that all live isolates except for E.coli lowered Pst densities in seedlings, suggesting that this characteristic may be unique to our Pantoea isolates. When seedlings were treated with UV-killed bacteria, we again found that ZM1, ZM2, and ZM3 were capable of lowering Pst densities, but E.coli was not. This suggests that some of the protective capability we are observing is conferred through indirect mechanisms, grow rooms perhapsthrough immune activation by UV-resistant membrane-bound antigens. The inability of UVkilled E. coli to decrease Pst density suggests that these Pantoea strains may have plant host or pathogen specific protective traits. Future work will explore the protective ability of these isolates in adult plants and will further dissect direct versus indirect mechanisms of protection.

By varying the concentration of Pst inoculated onto seedlings, we observed that increasing Pst increases AUDPC, as expected . Interestingly, we also observed a decoupling of plant disease symptoms and pathogen density. This was similarly observed when we tested for protective effects of each isolate . Here, we saw a linear increase of disease severity as Pst inoculation was increased in inoculation density, but saw a much weaker linear correlation between dose and Pst densities. We posit that this non-linear increase of Pst density might either be due to 1) a carrying capacity of Pst density that is reached on the seedling leaves, or 2) the possibility that the detection of dead or inactive Pst cells disguises a linear pattern. Furthermore, disease severity was calculated based on foliar symptoms, but it is very likely that Pst also asymptomatically colonizes the seedling root tissue. The entire seedlings, including roots, were homogenized prior to Pst quantification; this may have obscured differences in foliar Pst densities. By varying the dose of the protective strain , we were able to find that an increased dose does not necessarily correlate with decreased pathogen density, as was recently uncovered in a study investigating the protective effects of the phyllosphere community in adult tomato plants. We vary the dosage of protective Pantoea strains from less than one CFU/seed to 108 CFU/seed, the highest of which is five orders of magnitude higher than the concentration at which we originally recovered bacteria on the seeds . When analyzing Pst density seven days after inoculation, all culturable isolates’ ability to Pst suppress growth resulted in a non-linear pattern of pathogen density, whereby increasing Pantoea does did not linearly correlate with decreasing Pst density. The same is true for the two commercially available bio-control strains. Most notably, all three TT4 isolates exhibit optimal suppression of Pst at densities close to that found in naturally occurring seeds . At isolate densities above 104 CFU/seed, Pst density as detected by ddPCR, reached a similarly high level for all strains, suggesting a maximum density beyond which additional cells of the protective strains do not result in further protection. In light of our results that UV-killed Pantoea are capable of decreasing Pst density through presumed plant-immune activation, we posit that this activation, or priming, may be dependent on bacterial density on the seeds. In such a model, induction of resistance responses in the plant would be fully activated when such a threshold of signal is achieved. This is further supported by the result that UV-killed isolate C9-1 was the only Pantoea isolate unable to decrease Pst density , and its dose response curve was also the only one that did not follow the cubic pattern observed in the other Pantoea isolates. To rule out the possibility that higher densities of Pantoea resulted in the killing of Pst, a scenario that would be undistinguishable because of the use of ddPCR to quantify the pahtogen, we treated samples with a PMAxx TM and repeated the ddPCR. The data are quantitatively similar , indicating that even when only live cells are quantified, Pst densities reach an asymptote. Our results are suggestive of the possibility that plants may not only preferentially passage beneficial symbionts, . Tomato Type 1-3 were collected from non-neighboring lanes from one field, and the heirloom variety program; USDA-NIFA award # 2015-51300-24157 was collected from a neighboring field. Fruits were transported to UC Berkeley on ice and immediately stored in 4°C until processing. Intact tomato fruits from the same type were pooled in a sterile 1L beaker until they reached roughly the 500 mL line . To ensure that no additional microbes other than those found naturally were introduced to the seed surface, we surface sterilized the tomato fruits themselves before processing of seeds. Tomatoes were submerged in 75% ethanol for 20 minutes. They were then washed with sterile double-distilled H2O three times. The last wash was plated onto Kings Broth agar, and no colony forming units were detected. Sterilized tomatoes were then pooled into another sterile one-liter bottle, crushed with sterile forceps and spatula until becoming a thick fruit mixture, and allowed to ferment at room temperature for seven days. We employed this as a common seed collection method for removal of seeds from the fruit endocarp. After fermentation, seeds were then strained out from the fermented liquid with a sterilized metal strainer, minimally washed with sterile ddH2O to remove any excess fruit, and dried on filter paper within sterile petri dishes. All procedures were carried out sterilely in a Biological Safety Cabinet. Harvested seeds were stored in sterile petri dishes in darkness at 21°C, and these same seed stocks were used for all experiments.