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.

Bacterial etiolation and decline (BED), caused by Acidovorax avenae, is an emerging disease of creeping bentgrass on golf courses in the United States. We performed the first comprehensive analysis of A. avenae on a nationwide collection of turfgrass- and maize-pathogenic A. avenae. Surprisingly, our results reveal that the turfgrass-pathogenic A. avenae in North America are not only highly divergent but also belong to two distinct phylogroups. Both phylogroups specifically infect turfgrass but are more closely related to maize pathogens than to each other. This suggests that, although the disease is only recently reported, it has likely been infecting turfgrass for a long time. To identify a genetic basis for the host specificity, we searched for genes closely related among turfgrass strains but distantly related to their homologs from maize strains. We found a cluster of 11 such genes generated by three ancient recombination events within the type III secretion system (T3SS) pathogenicity island. Ever since the recombination, the cluster has been conserved by strong purifying selection, hinting at its selective importance. Together our analyses suggest that BED is an ancient disease that may owe its host specificity to a highly conserved cluster of 11 T3SS genes.

Dickeya dadantii is a bacterial plant pathogen that causes soft rot disease on a wide range of host plants. The type III secretion system (T3SS) is an important virulence factor in D. dadantii. Expression of the T3SS is induced in the plant apoplast or in hrp-inducing minimal medium (hrp-MM), and is repressed in nutrient-rich media. Despite the understanding of induction conditions, how individual cells in a clonal bacterial population respond to these conditions and modulate T3SS expression is not well understood. In our previous study, we reported that in a clonal population, only a small proportion of bacteria highly expressed T3SS genes while the majority of the population did not express T3SS genes under hrp-MM condition. In this study, we developed a method that enabled in situ observation and quantification of gene expression in single bacterial cells in planta. Using this technique, we observed that the expression of the T3SS genes hrpA and hrpN is restricted to a small proportion of D. dadantii cells during the infection of potato. We also report that the expression of T3SS genes is higher at early stages of infection compared to later stages. This expression modulation is achieved through adjusting the ratio of T3SS ON and T3SS OFF cells and the expression intensity of T3SS ON cells. Our findings not only shed light into how bacteria use a bi-stable gene expression manner to modulate an important virulence factor, but also provide a useful tool to study gene expression in individual bacterial cells in planta.

Erwinia amylovora is a Gram-negative bacterial plant pathogen in the family Enterobacteriaceae and is the causal agent of fire blight, a devastating disease of apple and pear. Fire blight is traditionally managed by the application of the antibiotic streptomycin during bloom, but this strategy has been challenged by the development and spread of streptomycin resistance. Thus, there is an urgent need for effective, specific, and sustainable control alternatives for fire blight. Antisense antimicrobials are oligomers of nucleic acid homologs with antisense sequence of essential genes in bacteria. The binding of these molecules to the mRNA of essential genes can result in translational repression and antimicrobial effect. Here, we explored the possibility of developing antisense antimicrobials against E. amylovora and using these compounds in fire blight control. We determined that a 10-nucleotide oligomer of peptide nucleic acid (PNA) targeting the start codon region of an essential gene acpP is able to cause complete growth inhibition of E. amylovora. We found that conjugation of cell penetrating peptide (CPP) to PNA is essential for the antimicrobial effect, with CPP1 [(KFF)3K] being the most effective against E. amylovora. The minimal inhibitory concentration (MIC) of anti-acpP-CPP1 (2.5 μM) is comparable to the MIC of streptomycin (2 μM). Examination of the antimicrobial mechanisms demonstrated that anti-acpP-CPP1 caused dose-dependent reduction of acpP mRNA in E. amylovora upon treatment and resulted in cell death (bactericidal effect). Anti-acpP-CPP1 (100 μM) is able to effectively limit the pathogen growth on stigmas of apple flowers, although less effective than streptomycin. Finally, unlike streptomycin that does not display any specificity in inhibiting pathogen growth, anti-acpP-CPP1 has more specific antimicrobial effect against E. amylovora. In summary, we demonstrated that PNA–CPP can cause an effective, specific antimicrobial effect against E. amylovora and may provide the basis for a novel approach for fire blight control.