First report of aster yellows phytoplasma (‘<i>Candidatus</i> Phytoplasma asteris’) in <i>Thlaspi arvense</i> (pennycress) in the United States

Thlaspi arvense, commonly known as pennycress, field pennycress, stinkweed, fanweed, or French weed is a winter or summer annual flowering plant, belonging to the Brassicaceae family (Esmailbegi et al., 2018). It was introduced to North America from Europe. Thlaspi arvense has been domesticated and cultivated as a novel winter annual oilseed cover crop in the Midwest of USA for the sustainable biofuel feed stock market. In May 2022, adult pennycress plants in breeding trials grown in Minnesota, USA, exhibited symptoms typical of aster yellows phytoplasma (AYP) infection. Since then, AYP symptoms have been observed in all six environments where pennycress breeding trials were grown from 2022 to 2024 (Table 1). Symptoms include yellowing of leaves, phyllody, stunting, shoot proliferation and axillary shoots, a reduced number or absence of pods, and witches’ broom (Figs. 1, 2). The symptoms were most prevalent after the anthesis stage, but were only observed at the vegetative stage in early October 2024 in St. Paul, MN. To characterise the disease, we collected 17 diseased single-plant samples from pennycress breeding trials grown in 2022–2024 in Minnesota and North Dakota, USA (Table 1). Symptomless plants were also collected in 2024. For phytoplasma testing, leaf and petiole tissues were ground in liquid nitrogen with a mortar and pestle, then DNA was extracted using a DNeasy Plant Mini Kit according to the manufacturer's instructions (Qiagen Sciences Inc., USA). Extracted DNAs were PCR tested using primer pair P1/P7 (Deng & Hiruki, 1991; Schneider et al, 1995) followed by nested PCR with R16F2n/R16R2 (Gundersen & Lee, 1996). To further identify the pathogen, primers fTuf1 and rTuf1 that target the phytoplasma elongation factor TU (tuf) gene were used in nested PCR (Schneider & Gibb, 1997). PCR amplicons were sequenced in both directions, and the sequences were deposited in GenBank with Accession Nos. PV225529, and PV231216, respectively, for the 16S rRNA sequence and the TU gene sequence. BLAST analysis showed 100% sequence identity between sequences from pennycress phytoplasma and several strains of ‘Candidatus Phytoplasma asteris’, for example sequence CP128397 (‘Candidatus Phytoplasma asteris’, strain M33) and CP000061 (Aster yellows witches’-broom phytoplasma, strain AYWB). The high nucleic acid identity among the 16S rRNA gene fragment and the tuf fragment for the ‘Ca. P. asteris’ strain from pennycress indicates a high genetic homology. Based on the high levels of sequence identities the phytoplasma infecting pennycress in Minnesota is affiliated to the aster yellows phytoplasma group (group 16SrI). Though 100% of plants with AYP symptoms tested positive, 25% of asymptomatic plants also tested positive for phytoplasma (Table 1). These were collected in the general proximity of diseased plants and possibly represent more recent infections that had not yet developed clear symptoms. Phytoplasmas have a wide range of hosts and many weeds can act as a reservoir for the transmission of the pathogen to susceptible crops. To our knowledge, this is the first report of a phytoplasma disease on pennycress in the United States and globally.