First report of Brenneria goodwinii, Gibbsiella quercinecans and Rahnella victoriana in declining oaks in France

Acute Oak Decline (AOD) is mediated by abiotic factors (temperature and precipitation) and triggered by insects (mainly the bark-boring beetle Agrilus biguttatus) and a complex of bacterial species (mainly Brenneria goodwinii, Gibbsiella quercinecans and Rahnella victoriana) (Denman et al., 2017; Doonan et al., 2020). Given the extent of oak dieback and the prevalence of A. biguttatus in France (Saintonge & Goudet, 2020; Sallé et al., 2022), a preliminary study was done in five French forests to assess the prevalence of AOD symptoms (bleeding cortical lesions on the trunk associated with insect emergence holes and dieback) and to determine the bacteria associated with those symptoms. The mean prevalence of AOD symptoms was estimated at 37% arround 20 trees assessed in each of the five forests. Bark samples and, when possible, exudates were taken from lesions associated with insect emergence holes and/or cracks (Fig. 1). Bacterial isolations were made from 43 bark samples and 11 exudate samples by plating on three agar media (Luria, Gifu Anaerobic and Eosin Methylene Blue) and incubated at 22°C for one to five days. Bacterial strains were identified by high-resolution melting (Brady et al., 2016) or 16S rRNA sequencing (Denman et al., 2016). The sequenced strains had 100% identity with sequences of reference strains (GenBank Accession Nos. CP014137.1, CP014136.1 and NR_146847.1). The percentage of trees infected with G. quercinecans, B. goodwinii and R. victoriana was 21, 16 and 12, respectively. These AOD-associated bacteria were detected with a higher success rate in exudates (81%) than in bark (25%). Gibbsiella quercinecans and B. goodwinii were isolated in four forests, R. victoriana in three forests. All three species were detected on both Q. robur and Q. petraea. The co-occurrence of two species in one lesion was observed in five trees. Sequences have been deposited in GenBank (OR554234, OR554237 and OR554232). For pathogenicity tests, excised branches (3 cm in diameter) from three actively growing Q. robur trees were incubated (21°C with a 16-hour photoperiod) in a jar containing water, with the upper part of the section sealed with plastic film. Brenneria goodwinii, G. quercinecans and R. victoriana (strains FRB141T, FRB97T, FRB225T) were applied to wounds (three replicates per stem and per tree) (Denman et al., 2017) using a sterile loop charged with growth from 24-hour cultures on Luria agar medium. For control inoculations, only a wound was made. After eight weeks, cortical necrosis was apparent around all inoculation points, while only brownish discolouration was observed for control wounds (Fig. 2). The inoculated bacteria were isolated from the necrotic tissue of the inoculated stems but not from the controls. This is the first report of B. goodwinii, G. quercinecans and R. victoriana in France. Further research into the role of these bacteria in the observed oak decline and their interactions with pathogenic or endophytic fungi and other factors involved in AOD is underway. The acknowledgements are expressed to the Ministère de l'Agriculture et de la Souveraineté Alimentaire and INRAE which funded the research activities and to INRAE ECODIV for a grant.