First Report of Bacterial Wilt on Southern Highbush Blueberries (Vaccinium corymbosum), Caused by Ralstonia solanacearum, in North Carolina.

Abstract

North Carolina is the seventh-largest producer of blueberries in the United States, with an estimated value of $104.6 million (USDA-NASS). In the Spring of 2024, a patch of approximately 50 contiguous southern highbush blueberries (SHB) (Vaccinium corymbosum cv. Rebel) exhibited dieback, leaf scorch, and wilt in one Bladen County, NC farm. Eleven woody stems were excised from different SHB bushes, surface sterilized, and cut into 5 cm pieces. Cut stems were then immersed in sterile deionized water (SDW) to test for bacterial streaming. A stream of fine, slimy strands of bacterial ooze exuded from all the stems. A plastic loop was used to streak 10 µl of the bacterial streaming supernatant onto Casamino Acid-Peptone-Glucose agar amended with Triphenyl Tetrazolium Chloride (Kelman 1954). After 48 hours of incubation at 28ºC, multiple white mucoid single colonies with a soft pink center, characteristic of Ralstonia solanacearum, were observed. Eleven isolates were randomly selected and stored in 30% glycerol at -80ºC. The 11 isolates were molecularly characterized as R. solanacearum phylotype II using Multiplex PCR (Fegan and Prior, 2005), where GMI1000, K60, CMR15, and PSI07 genomic DNA represent phylotype I, II, III, IV, respectively. None of the strains amplified the 357 bp band characteristic of the R. solanacearum Select Agent strains using the 630/631 primer pair (Fegan et al. 1998). Sequevar was determined by amplifying and sequencing the 750 bp region of the egl gene using ENDO-F/ENDO-R primers (Ji et al. 2007). The egl sequences were deposited to NCBI (GenBank accessions: PQ417932 to PQ417942) and compared to publicly available sequences. The egl sequence of the 11 isolates shared 100% (704/704 bp) and 99.7% (702/704 bp) identity with Sequevar 7 strains of R. solanacearum RF75 and K60, respectively (Cellier et al. 2023). This confirms the 11 isolates recovered from SHB bushes in NC are R. solanacearum, phylotype IIA, Sequevar 7 strains. Two isolates, AHR105 and AHR109, were randomly selected to perform Koch's postulates. Bacterial inoculum was prepared from 24 h cultures grown with shaking at 225 rpm at 28°C. Bacteria were pelleted and washed three times with SDW before resuspending in SDW to an O.D.600 of 0.2 (~ 2x108 CFU/ml). The roots of five six-month-old SHB ('cv. Rebel') were submerged in 500 ml of inoculum or water as a negative control, for 10 min at transplanting. Each experiment consisted of five plants per strain or water control and was repeated twice. All plants were incubated at 28°C, 80% relative humidity, and 8 h:16 h light-dark cycle in a growth chamber. The first symptoms appeared at four weeks post-inoculation, and by the sixth week, all AHR105 and AHR109 treated plants showed defoliation, dieback, and wilting. No symptoms were observed in the water-treated plants. Six weeks post-inoculation, all the plants were destructively sampled, and white mucoid single colonies with a soft pink center, similar in morphology to those initially inoculated, were recovered and confirmed to be AHR105 and AHR109 via PCR and sequencing; no bacteria were recovered from water-treated plants. R. solanacearum is known to cause wilting in tomato and tobacco-producing counties in NC but has not been previously reported on SHB in NC. The disease, however, was reported on blueberries in Florida and Georgia (Norman et al. 2017; Oliver et al. 2022). Accurate and timely detection of R. sol in SHB in NC is paramount for deploying specific and localized management tactics to safeguard NC SHB production.