Genomic analysis of bacteria in the Acute Oak Decline pathobiome

Abstract

The UK’s native oak is under serious threat from Acute Oak Decline (AOD). Stem tissue necrosis is a primary symptom of AOD and several bacteria are associated with necrotic lesions. Two members of the lesion pathobiome, Brenneria goodwinii and Gibbsiella quercinecans, have been identified as causative agents of tissue necrosis. However, additional bacteria including Lonsdalea britannica and Rahnella species have been detected in the lesion microbiome, but their role in tissue degradation is unclear. Consequently, information on potential genome-encoded mechanisms for tissue necrosis is critical to understand the role and mechanisms used by bacterial members of the lesion pathobiome in the aetiology of AOD. Here, the whole genomes of bacteria isolated from AOD-affected trees were sequenced, annotated and compared against canonical bacterial phytopathogens and non-pathogenic symbionts. Using orthologous gene inference methods, shared virulence genes that retain the same function were identified. Furthermore, functional annotation of phytopathogenic virulence genes demonstrated that all studied members of the AOD lesion microbiota possessed genes associated with phytopathogens. However, the genome of B. goodwinii was the most characteristic of a necrogenic phytopathogen, corroborating previous pathological and metatranscriptomic studies that implicate it as the key causal agent of AOD lesions. Furthermore, we investigated the genome sequences of other AOD lesion microbiota to understand the potential ability of microbes to cause disease or contribute to pathogenic potential of organisms isolated from this complex pathobiome. The role of these members remains uncertain but some such as G. quercinecans may contribute to tissue necrosis through the release of necrotizing enzymes and may help more dangerous pathogens activate and realize their pathogenic potential or they may contribute as secondary/opportunistic pathogens with the potential to act as accessory species for B. goodwinii. We demonstrate that in combination with ecological data, whole genome sequencing provides key insights into the pathogenic potential of bacterial species whether they be phytopathogens, part-contributors or stimulators of the pathobiome.

Brenneria spp. and Rahnella victoriana associated with acute oak decline symptoms on oak and hornbeam in Iran

“…(e) However, the most likely explanation for lack of disease development in these pathogenicity tests is that B. goodwinii is not a primary pathogen solely responsible for the symptoms on oak and hornbeam in Iran and may work in concert with other species to form a pathobiome. This point is consistent with reports on AOD (Broberg et al, ; Denman et al, ) and other pathosystems (e.g., olive knot disease Buonaurio et al, ) and supports the idea of the combined pathogenic potential of organisms and polybacterial pathobiomes (Doonan et al, ; Gilbert et al, ). In Iran, ideally further testing of field samples using metabarcoding, metagenomics and metatranscriptomic methods would be carried out as recommended by Broberg et al (), Denman et al () and Doonan et al () to fully profile the pathobiome and compare it with the microbiome of non‐symptomatic trees to identify putative disease contributing members for further testing and to elucidate important functional genes and gene products in the pathobiome.…”

“…The other Brenneria species and R. victoriana also caused a HR but it took longer. The strong positive HR tests from B. goodwinii support the discovery that these bacteria have a functional type III secretion system (TTSS) (Doonan et al, 2019), which delivers elicitor proteins such as harpin that represents a key phytopathogenic virulence factor when introduced into host tissue (Alfano & Collmer, 1997;Wei et al, 1992), which could be one of the factors responsible for inducing the HR response in the geranium (Choi, Kim, Lee, & Oh, 2013 Denman et al, 2018) and other pathosystems (e.g., olive knot disease Buonaurio et al, 2015) and supports the idea of the combined pathogenic potential of organisms and polybacterial pathobiomes (Doonan et al, 2019;Gilbert et al, 2016). In Iran, ideally further testing of field samples using metabarcoding, metagenomics and metatranscriptomic methods would These trees exist in pure and mixed plantation stands amounting to about 6.5% of the forests (Panahi, 2011), but also occur throughout the natural reserves in the area.…”

“…The other Brenneria species and R. victoriana also caused a HR but it took longer. The strong positive HR tests from B. goodwinii support the discovery that these bacteria have a functional type III secretion system (TTSS) (Doonan et al, ), which delivers elicitor proteins such as harpin that represents a key phytopathogenic virulence factor when introduced into host tissue (Alfano & Collmer, ; Wei et al, ), which could be one of the factors responsible for inducing the HR response in the geranium (Choi, Kim, Lee, & Oh, ). In this study, despite positive HR tests on geranium, in both pathogenicity inoculation trials the isolates only induced occasional tissue necrosis under greenhouse conditions in the test periods, but they were successfully re‐isolated from the almost all the inoculated plants.…”

“…In the UK studies on AOD, B. goodwinii was emphasized as having primary importance in causing the rotting tissues in AOD, although the lesions and tissue rot in AOD have a polymicrobial cause (Broberg et al, ; Denman et al, ; Doonan, Denman, Pachebat, & McDonald, ). In our studies, B. goodwinii was isolated from all diseased trees but not the healthy trees and all the isolates of B. goodwinii obtained in our study induced a strong HR on geranium leaves within 12–14 hr indicating a host defence response to invasion, emphasizing their pathogenic potential.…”

“…Whole genome sequencing of key organisms would allow linkages to be made between their pathogenic potential and the gene products present in the lesion transcriptome and may even offer insights into mechanisms used by the pathogenic organisms. Doonan et al () discussed the concept of pathogenic potential of organisms and how polybacterial pathobiomes are becoming more widely recognized, but proper ground work must precede pathogenicity testing to obtain reliable results, and if possible, transcriptomics of material from pathogenicity trials would be carried out to complete the proof of linkages.…”