Inoculation of Stigma-Colonizing Microbes to Apple Stigmas Alters Microbiome Structure and Reduces the Occurrence of Fire Blight Disease

Abstract

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.

Complete Genome Sequences of Curtobacterium , Pantoea , Erwinia , and Two Pseudomonas sp. Strains, Isolated from Apple Flower Stigmas from Connecticut, USA

“…The genus Curtobacterium , belonging to the phylum Actinobacteria , was much less abundant in the microbiome and was investigated as a representative of the apple stigma “rare biosphere” ( 3 ). These four strains were identified in our previous study by 16S rRNA gene comparisons ( 1 ). Acquisition of the full-genome sequence information of these strains will advance future investigations concerning the metabolic and functional requirements of the flower microbiome.…”

“…15A4, and Pseudomonas sp. 1079, were isolated from apple flower stigma samples collected on the apple cultivar ‘Early Macoun’ ( Malus x domestica NY75414-1) planted at Lockwood Farm in Hamden, CT (41.406°N, 72.906°W) ( 1 ). Our previous data showed that Enterobacteriaceae and Pseudomonadaceae are the two predominant families of bacteria on apple stigma ( 2 , 3 ).…”

“…The stigma portion of an apple flower was dissected and immersed into 200 μl of 0.5× phosphate-buffered saline (PBS) in a sterile 1.5-ml microcentrifuge tube. The stigma sample was then sonicated for 5 min, followed by vortexing for 30 s. Then, 5 ml of PBS was spread onto a lysogeny broth (LB) agar plate to collect stigma resident bacteria ( 1 ). A single colony of each strain was inoculated into LB broth and incubated at 28°C overnight with shaking.…”

The Floral Microbiome and Its Management in Agroecosystems: A Perspective

“…Moreover, there is growing interest in taking an expanded, community-level perspective on microbe–microbe interactions within the anthosphere, and calls have been made for development of mBCA consortia, which could include “helper strains” to encourage mBCA establishment and performance . For instance, the floral microbiome naturally plays a role in mediating pathogen establishment, and there is some evidence to suggest that more complex microbial communities can enhance disease resistance. , Given variable performance of solitary mBCAs, it is likely that complex interactions among microbiome constituents can contribute to such observed outcomes; however, this requires more thorough investigation. As next-generation sequencing continues to be increasingly embraced as a tool for identifying members of the floral microbiome, combining it with culture-based approaches and chemical analyses could allow researchers to identify novel mBCAs or microbial consortia for improved control, as well as the mechanisms involved.…”

“…49 For instance, the floral microbiome naturally plays a role in mediating pathogen establishment, and there is some evidence to suggest that more complex microbial communities can enhance disease resistance. 50,51 Given variable performance of solitary mBCAs, it is likely that complex interactions among microbiome constituents can contribute to such observed outcomes; however, this requires more thorough investigation.…”