First report of causing bacteria wilt on eucalyptus in Brazil.

The Ralstonia solanacearum Species Complex (RSSC) is the most significant plant pathogen group with a wide host range. It is genetically related but displays distinct biological features, such as restrictive geography occurrence. The RSSC comprises three species: Ralstonia pseudosolanacearum (phylotype I and III), Ralstonia solanacearum (phylotype IIA and IIB), and Ralstonia syzygii (phylotype IV) (Fegan and Prior 2005). In Brazil, eucalyptus production has been growing as one of the leading industries to produce cellulose, energy, and other products from renewable sources, covering approximately 7.8 million hectares, representing 76% of all planted forests in 2023 (IBÁ, 2024). The eucalyptus bacterial wilt (BW), caused by Ralstonia solanacearum and Ralstonia pseudosolanacearum, members of RSSC (Lopes and Rossato 2018), is one of the most important eucalyptus diseases, causing damage to nurseries and eucalyptus plantations (Alfenas et al., 2009). We collected Eucalyptus urophylla clone plants showing typical wilt symptoms in a commercial nursery with an average size of 400 m2 harboring 300k cuttings plant in rooting, showing 5% symptom incidence in February 2024 in the Bahia State, Brazil. Affected plants showed wilting of leaves and branches, reduced growth, and death at a higher level of disease severity. The vascular tissues showed darkening and intense bacterial exudation. Next, we aimed to determine the presence of RSSC bacteria in these plants by using the isolation method. For this, the stem macerate of symptomatic plants was plated on a semi-selective SMSA medium (ELPHINSTONE et al. 1996) and incubated for 72h at 28oC. It was possible to isolate bacteria with typical morphology of RSSC in all samples. Using multiplex PCR (Fegan & Prior, 2005), 96.77% of isolates were classified as phylotype II and 2.58% as phylotype I. Notably, we identified one isolate (0,65%), BR19, which exhibited an amplification pattern characteristic of phylotype IV. This isolate demonstrated typical RSSC growth, forming a slimy colony with a red internal region on CPG medium supplemented with tetrazolium. To fulfill Koch's postulates, eight plants of Eucalyptus urophylla were inoculated with bacterial suspension (20 µL adjusted to 108 CFU/mL of saline solution), and eight plants injected with distilled water were used as controls. The bacterial inoculum or the distilled water was injected at the base of the stem, and the plants were kept in a greenhouse (28 ± 2 °C), according to the methodology described by Fonseca et al. (2016). Then, after 30 days of post-inoculation with the bacterium, all plants showed wilt symptoms, and it was re-isolated from plating stem tissue macerates on SMSA medium. The identity of the bacteria was confirmed using RSSC species phylotype multiplex PCR (Fegan & Prior, 2005). Using Long-read Nanopore technology, we were able to sequence the draft complete genome composed of a chromosome and a megaplasmid, which were deposited at GenBank with assembly accession number GCF_042494895.1. The completeness of the BR19 genome was found to be 92.1%, according to the BUSCO program. Phylogenomic analysis was performed according to Subedi et al. (2024), revealing that BR19 is genetically closer to two other isolates of Ralstonia syzygy subsp. syzygii (GenBank assemblies: ASM1591059v1 and ASM2921996v1) isolated from clove (Syzygium aromaticum) in Indonesia. It is the first report of the detection of R. syzygii subsp. syzygii phylotype IV away from Southeast Asia and the first report of R. syzygii subsp. syzygii infecting eucalyptus. This result expands our knowledge about the geographic distribution and host range of RSSC.