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

Authors: Tancos, Matthew; Frederick, Reid

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/19/2019
Publication Date: 2/12/2020
Citation: Tancos, M.A., Frederick, R.D. 2020. First Report of Xanthomonas campestris infecting Invasive Garlic Mustard in the United States. Plant Disease. 104:1251. https://doi.org/10.1094/PDIS-09-19-1963-PDN.
DOI: https://doi.org/10.1094/PDIS-09-19-1963-PDN

Interpretive Summary: Garlic mustard is a non-native invasive plant that is rapidly invading the eastern and midwestern United States and is displacing native flora. Recently, diseased garlic mustard populations were observed in Maryland and a bacterial pathogen (Xanthomonas campestris) was isolated and identified as the causative agent. This is the first report of an endemic Xanthomonas campestris strain found to naturally infect garlic mustard in the United States. Sequence analysis determined that this strain does not appear related to other agronomically-damaging X. campestris strains, and has the potential to be developed as an effective biological control agent of garlic mustard. Moreover, this discovery highlights how non-native invasive plants could act as reservoirs for potential agricultural plant pathogens.

Technical Abstract: Garlic mustard (Alliaria petiolata) is an aggressive non-native cruciferous (Brassica) plant from Eurasia that was introduced into North America around the 19th century. 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 2nd year mature flowering plants and 1st year immature rosettes. Disease incidence in the affected area was 75-80%. Symptomatic leaf tissue was surface sterilized, macerated, and streaked onto yeast extract dextrose calcium carbonate agar, from which bright yellow, mucoid bacterial colonies were isolated. The 16S rRNA gene was sequenced to confirm the bacterium was Xanthomonas campestris, followed by sequencing eight conserved loci (atpD, dnaK, efP, glnA, gyrB, rpoD, tpiA, and fyuA) for multilocus sequence analysis and pathovar determination. Gene sequences were aligned, concatenated, and compared to reference Brassica-associated X. campestris strains, for a total length of 4485 nucleotides, and used to generate a maximum likelihood phylogram using W-IQ-TREE. Sequence analysis revealed the garlic mustard isolate did not cluster with the agronomically-damaging X. campestris pv. campestris or pv. raphani strains, but instead clustered with non-pathogenic X. campestris strains, the weakly pathogenic strain CFBP 5814, and strains of X. campestris pv. incanae with bootstrap support of 99%. To fulfill Koch’s postulates, true leaves of a garlic mustard rosette were wound-inoculated with a sterile pin dipped into a 24-hour 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. A third leaf was mock inoculated with sterile water. Plants were grown in a growth chamber with 14 hours of light at 26ºC. Symptoms appeared between 10-14 days post inoculation, with chlorotic V-shaped leaf lesions extending from the sites of inoculation, while 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 plants extensive distribution and potential to serve as a reservoir for X. campestris.