Bacteria Associated with Acute Oak Decline: Where Did They Come From? We Know Where They Go

Abstract

Acute oak decline is a high-impact disease causing necrotic lesions on the trunk, crown thinning and the eventual death of oak. Four bacterial species are associated with the lesions-Brenneria goodwinii, Gibbsiella quercinecans, Rahnella victoriana and Lonsdalea Britannica-although an epi-/endophytic lifestyle has also been suggested for these bacteria. However, little is known about their environmental reservoirs or their pathway to endophytic colonisation. This work aimed to investigate the ability of the four AOD-associated bacterial species to survive for prolonged periods within rhizosphere soil, leaves and acorns in vitro, and to design an appropriate method for their recovery. This method was trialled on field samples related to healthy and symptomatic oaks. The in vitro study showed that the majority of these species could survive for at least six weeks within each sample type. Results from the field samples demonstrated that R. victoriana and G. quercinecans appear environmentally widespread, indicating multiple routes of endophytic colonisation might be plausible. B. goodwinii and L. britannica were only identified from acorns from healthy and symptomatic trees, indicating they may be inherited members of the endophytic seed microbiome and, despite their ability to survive outside of the host, their environmental occurrence is limited. Future research should focus on preventative measures targeting the abiotic factors of AOD, how endophytic bacteria shift to a pathogenic cycle and the identification of resilient seed stock that is less susceptible to AOD.

Keywords: AOD; acorns; acute oak decline; enrichment; leaves; niches; recovery; rhizosphere.

Figure 1

The combined methods workflow used for the spiking of bacteria into oak-related microcosms and the Enterobacteriaceae enrichment method used for their recovery.

Figure 2

Map detailing the location of samples taken from Hatchlands Park site. Healthy (blue) and AOD-symptomatic (red) oak were selected in a paired model, with odds and evens being paired from 1 and 2, through to 19 and 20. Map made in ArcGIS.

Figure 3

The identities of AOD-associated bacteria recovered from the three inoculated niches, (A) indicates the identifications made in soil, (B) acorns and (C) leaves. The inoculum of each species was adjusted to an OD₆₀₀ of 0.5, which was added to each substrate sample except the controls, to which 1 mL of sterile ¼ strength Ringers was added. Isolation from each sample type took place at weekly intervals over the course of six weeks before identification by HRM in triplicate. Re-isolations are separated by week and the initial inoculated bacteria. Identifications that do not match the original species that were inoculated into spiked substrates can be seen in a number of the columns and the control samples, indicating that AOD bacteria were already present in the majority of samples.

Figure 4

The HRM curves generated for each microcosm at week 1 and week 6 of the experiment. From left to right, the coloured peaks indicate the presence of B. goodwinii (Bg), R. victoriana (Rv), L. britannica (Lb) and G. quercinecans (Gq). Solid colour lines represent positive controls, while dashed lines represent samples.

Figure 5

The number of AOD bacterial species recovered from different sample types taken from 20 oak trees at Hatchlands Park, Guildford, UK. The recovery count is the number of trees each bacterium was identified from per sample type; each single identification was confirmed by high-resolution melt analysis in triplicate.