First Report of Xanthomonas campestris Infecting Invasive Garlic Mustard in the United States

Garlic mustard (Alliaria petiolata) is an aggressive nonnative cruciferous (Brassica) plant from Eurasia that was introduced into North America around the 19th century (Durka et al. 2005). The biennial Brassica herb has since rapidly invaded and displaced native flora in much of the eastern and midwestern United States and Canada, thriving in woodlands, hedgerows, lawns, and other shady environments. In May of 2019, foliar leaf symptoms developed on garlic mustard along the edge of a forested property in Frederick County, Maryland. Initial leaf symptoms were chlorotic angular V-shaped lesions originating from the leaf margins, characteristic of a bacterial infection. As the disease progressed, leaves wilted and lesions became necrotic, covering entire leaf margins. The disease was patchy along the property and nearby forest edge, but the disease was observed on both second-year mature flowering plants and first-year immature rosettes. Disease incidence in the affected area was 75 to 80%. Symptomatic leaf tissue was surface sterilized, macerated, and streaked onto yeast extract dextrose calcium carbonate agar (Wilson et al. 1967), from which bright yellow, mucoid bacterial colonies were isolated. The 16S rRNA gene was sequenced to confirm the bacterium was a Xanthomonas sp. (accession MN823168), followed by sequencing eight conserved loci (atpD, dnaK, efP, glnA, gyrB, rpoD, tpiA, and fyuA) for multilocus sequence analysis and pathovar determination (accessions MN444860 to MN444867) (Fargier et al. 2011). Gene sequences were aligned, concatenated, and compared with reference Brassica-associated X. campestris strains, for a total length of 4,485 nucleotides, and used to generate a maximum likelihood phylogram using W-IQ-TREE (Fargier et al. 2011; Lange et al. 2016; Trifinopoulos et al. 2016). Sequence analysis revealed the garlic mustard isolate (strain FDWSRU 18048) did not cluster with the agronomically damaging X. campestris pv. campestris or pv. raphani strains but instead clustered with nonpathogenic X. campestris strains, the weakly pathogenic strain CFBP 5814, and strains of X. campestris pv. incanae with bootstrap support of 99% (Fargier et al. 2011; Lange et al. 2016). To fulfill Koch’s postulates, true leaves of a healthy transplanted garlic mustard rosette were wound-inoculated with a sterile pin dipped into a 24-h-old culture and inoculated into the outer margin of a single leaf, and the petiole and midrib of a second leaf on the same plant (Vicente et al. 1998). A third leaf was mock inoculated with sterile water. Plants were grown in a growth chamber with 14 h of light at 26°C. Symptoms appeared between 10 and 14 days postinoculation, with chlorotic V-shaped leaf lesions extending from the sites of inoculation, whereas the petiole inoculation led to vascular blackening and leaf wilting. No symptoms were observed on the mock-inoculated leaves. Symptoms were identical to diseased field samples. Inoculations were repeated with the same results. Bacteria reisolated from symptomatic plants were sequence confirmed to be X. campestris. To our knowledge, this is the first confirmed report of X. campestris naturally infecting garlic mustard in the United States. Future studies will investigate the Brassica host range for this isolate due to the plant’s extensive distribution and potential to serve as a reservoir for X. campestris.