A bacterial gall disease of Myrica rubra caused by Pseudomonas amygdali pv. myricae in Taiwan.

Chinese bayberry (Myrica rubra), or yamamomo, is cultivated for fruit production and ornamental purposes. In 2023, an unknown disease affecting M. rubra was reported in the Huisun Experimental Forest, National Chung Hsing University, Renai, Nantou County, Taiwan. The disease is expressed as galls on petioles, young shoots, twigs, and trunks, particularly on twig branches. These galls appeared cracked, dark brown, irregular, and rough on surfaces. The disease incidence was 100% among approximately 80 trees. Samples collected in March 2023 exhibited internal vascular discoloration and bacterial streaming was observed. Diseased tissues were immersed in sterile water, and the bacterial suspensions were streaked on nutrient agar for isolation. Purified strains underwent hypersensitive response (HR) assays on Nicotiana benthamiana leaves, identifying six bacterial strains (MrBG-1 to MrBG-6) that triggered a HR reaction. Pathogenicity assays were performed on lower woody stems and upper non-woody stems of six 15-month-old M. rubra seedlings in a greenhouse (22 to 25℃) under natural daylight. The six bacterial strains were inoculated on three seedlings while the other three seedlings were treated with sterile water. A suspension of each strain (20 ul; 108 CFU / mL) was dropped on an inoculation site which was wounded with needles in an area of 5 mm2. Inoculation sites swelled with cracked surfaces at one month post-inoculation (mpi) and formed galls on woody tissue and cankers on non-woody tissue at three mpi, while wounds in the controls healed. The pathogens were re-isolated and confirmed identical to the original inoculated bacteria via 16S rDNA sequencing (PV082408 to PV0824013). Since isolates MrBG-1 to MrBG-6 produced fluorescent pigment on King's B medium, LOPAT tests were performed (Schaad et al. 2001). Results indicated that these isolates produced levan, induced HR in tobacco, lacked oxidase and arginine dihydrolase activity, and failed to cause soft rot in potato tuber slices. These characteristics were consistent with those of P. amygdali pv. myricae (formerly P. syringae pv. myricae) strains causing gall symptoms on M. rubra in Japan (Ogimi and Higuchi 1981). Phylogenetic identification utilized concatenated gene sequences of DNA gyrase subunit B (gyrB, PV089501 to PV089506), RNA polymerase beta subunit (rpoB, PV089507 to PV089512), and RNA polymerase sigma factor (rpoD, PV089513 to PV089518) by using the maximum likelihood method. The primers used for amplifying the three genes and the reference strains included in the phylogenetic analysis were referred from a previous study (Maleki-Zadeh et al. 2022). The phylogenetic results clustered MrBG-1 to MrBG-6 in the clade of Pseudomonas amygdali pv. myricae Ogimi and Higuchi with a 86% bootstrap value. Based on the pathogenicity assay, phenotypic tests, and phylogenetic analysis, we concluded that the disease is caused by P. amygdali pv. myricae. Although the disease in Taiwan has only been observed in an experimental forest, it has also been reported in Japan (Okinawa) (Ogimi and Higuchi, 1981) and China (Zhang and He, 1991). Since the pathogen affects all economic cultivars of Myrica rubra (Ogimi and Higuchi, 1981), it poses a significant threat to Chinese bayberry utilization.