The apple flower is a transient organ that can shed light on how plant-associated phytobiomes are established and structured. Stigmas, stamens, receptacles, and petals were dissected from flowers, and the microbiome of each structure was characterized. Each flower part harbored a largely overlapping set of bacterial taxa, predominantly within the groups Pseudomonas and Enterobacteriaceae. However, the structure of the communities differed. The stigmas showed a high dominance of a small number of operational taxonomic units (OTUs; 97% sequence identity) whereas OTUs on petals were more even in relative abundance. Results from the OTU analysis of phytobiomes from stigmas from three apple cultivars, Braeburn, McIntosh, and Sunrise, indicated cultivar did not significantly influence community structure. Correlation analysis of bacterial taxa in the apple phytobiome found a negative correlation between Pseudomonas and Enterobacteriaceae, suggesting a potential niche exclusion relationship between these taxa. In this respect, the phytobiome of the apple flower is relatively simple but different flower parts, particularly the stigma, enrich the relative abundance of specific bacterial populations. Correlations in the community structure point to potential antagonistic relationships, which could be used to manipulate the structure of the microbiome for biological control of pathogens or other orchard management strategies.

Plant microbiomes have important roles in plant health and productivity. However, despite flowers being directly linked to reproductive outcomes, little is known about the microbiomes of flowers and their potential interaction with pathogen infection. Here, we investigated the temporal spatial dynamics of the apple stigma microbiome when challenged with a phytopathogen Erwinia amylovora, the causal agent of fire blight disease. We profiled the microbiome from the stigmas of individual flowers, greatly increasing the resolution at which we can characterize shifts in the composition of the microbiome. Individual flowers harbored unique microbiomes at the operational taxonomic unit level. However, taxonomic analysis of community succession showed a population gradually dominated by bacteria within the families Enterobacteriaceae and Pseudomonadaceae. Flowers inoculated with E. amylovora established large populations of the phytopathogen, with pathogen-specific gene counts of >3.0 × 107 in 90% of the flowers. Yet, only 42% of inoculated flowers later developed fire blight symptoms. This reveals that pathogen abundance on the stigma is not sufficient to predict disease outcome. Our data demonstrate that apple flowers represent an excellent model in which to characterize how plant microbiomes establish, develop, and correlate with biological processes such as disease progression in an experimentally tractable plant organ.

Summary Erwinia amylovora is the causal agent of fire blight, one of the most devastating diseases of apple and pear. Erwinia amylovora is thought to have originated in North America and has now spread to at least 50 countries worldwide. An understanding of the diversity of the pathogen population and the transmission to different geographical regions is important for the future mitigation of this disease. In this research, we performed an expanded comparative genomic study of the Spiraeoideae‐infecting (SI) E. amylovora population in North America and Europe. We discovered that, although still highly homogeneous, the genetic diversity of 30 E. amylovora genomes examined was about 30 times higher than previously determined. These isolates belong to four distinct clades, three of which display geographical clustering and one of which contains strains from various geographical locations (‘Widely Prevalent’ clade). Furthermore, we revealed that strains from the Widely Prevalent clade displayed a higher level of recombination with strains from a clade strictly from the eastern USA, which suggests that the Widely Prevalent clade probably originated from the eastern USA before it spread to other locations. Finally, we detected variations in virulence in the SI E. amylovora strains on immature pear, and identified the genetic basis of one of the low‐virulence strains as being caused by a single nucleotide polymorphism in hfq, a gene encoding an important virulence regulator. Our results provide insights into the population structure, distribution and evolution of SI E. amylovora in North America and Europe.

17Plant microbiomes have important roles in plant health and productivity. However, 18 despite flowers being directly linked to reproductive outcomes, little is known about the 19 microbiomes of flowers and their potential interaction with pathogen infection. Here, we 20 investigated the temporal dynamics and spatial traits of the apple stigma microbiome 21 when challenged with a phytopathogen Erwinia amylovora, the causal agent of fire blight 22 disease. We profiled the microbiome from the stigmas of a single flower, greatly 23 increasing the resolution at which we can characterize shifts in the composition of the 24 microbiome. Individual flowers harbored unique microbiomes at the OTU level. 25However, taxonomic analysis of community succession showed a population gradually 26 dominated by bacteria within the families Enterobacteriaceae and Pseudomonadaceae. 27 Flowers inoculated E. amylovora established large populations of the phytopathogen, 28 with pathogen specific gene counts of >3.0 x 10 7 in 90% of the flowers. Yet, only 42% of 29 inoculated flowers later developed fire blight symptoms. This reveals pathogen amount 30 on the stigma is not sufficient to predict disease outcome. Our data demonstrate that 31 apple flowers represent an excellent model in which to characterize how plant 32 microbiomes establish, develop, and interact with biological processes such as disease 33 progression in an experimentally tractable plant organ.34 35 36 37 38 39 40 Flowers, the reproductive organs of angiosperms, play a critical role in the plant's 41 lifecycle. The most important function of flowers is to provide a mechanism for 42 pollination, the union of sperm contained within pollen, to the ovules contained in the 43 ovary. The fertilized ovules produce seeds that will later germinate to become the next 44 generation of plants. Yet, unlike other vegetative organs such as the roots, stems, and 45 leaves that are present through a large part of the plant's lifecycle, flowers develop on 46 mature plants and are typically present for the limited period during bloom. As such, 47 research characterizing the microbiome of the flower is generally less developed than for 48 other plant organs. 49 Flowers of apple (Malus domestica) have been subject to considerable research 50 attention as they are the direct precursors of apple fruits, one of the most consumed fruits 51 worldwide (1). The ephemeral nature of apple flowers, with mature flowers from petal 52 open to petal fall only lasting for 5-10 days in spring, offers a unique environment in 53 which to study community succession (1, 2). During bloom, petals open up in a relatively 54 short period of time, typically within one day, which exposes the internal flower parts to 55 the environment and microorganisms. Several of these internal flower parts exude various 56 types of nutrient-rich secretions including nectar, stigmatic exudate, and pollen exudate, 57 for the purpose of attracting pollinators, and inducing the germination of pollen grains (1, 58 3). These secretio...

Flowers secrete nutrient rich exudates that support the growth of an assemblage of microorganisms, including both beneficial and pathogenic members, most of which belong to the phylum Proteobacteria. Given the potential role of the microbiome in plant health, manipulating the microbiome to promote growth of beneficial members holds promise in controlling plant diseases. In this study we inoculated four different bacterial strains that were originally isolated from apple stigmas, alone or in mixtures of increasing complexity, onto apple flowers during bloom. We tested if such treatments would influence fire blight occurrence, a disease caused by Erwinia amylovora, and if we could detect a shift in the structure of the microbiome due to the treatments. We show that various inoculations did influence the occurrence of fire blight, although the level of disease suppression was dependent upon specific bacterial strains. Furthermore, treatments using different strains or strain mixtures predominantly resulted in increased representation of the inoculated strains, suggesting that disease suppression was due to an alteration of the stigma microbiome structure. Compared to treatments with single strains, a Pantoea-Pseudomonas strain mixture produced a homogeneous microbiome structure with less inter-flower variability. Findings from this study suggest the microbiome on the flower stigma can be manipulated through microbial inoculation. Due to flowers’ short life span yet important role in plant disease infection, even a shot-term influence on microbiome composition may result in significant decreases in disease susceptibility.