First Report of <i>Pseudomonas amygdali</i> Causing Leaf Spot on <i>Hibiscus rosa-sinensis</i> in a Greenhouse in New York State, U.S.A.

<i>Hibiscus rosa-sinensis</i>, commonly known as the "Chinese hibiscus", is a widely cultivated shrub with ornamental and medicinal applications (Jadhav et al., 2009). However, it is known to be susceptible to a range of pathogens including bacteria (Chase, 1986). In March 2019, <i>Hibiscus rosa-sinensis</i> plants in production at a greenhouse in New York, but originally from Florida, developed widespread, conspicuous leaf spots and associated chlorosis for a month after arrival. "Hibiscus 35-1" was isolated from a suspension of symptomatic plant tissue pieces soaked in sterile deionized water and streaked on potato dextrose agar (PDA) (González-Tobón et al., 2023), resulting in numerous white bacterial colonies with a uniform appearance. Previously we reported the complete species identification for this bacterial isolate using whole genome sequencing via Oxford Nanopore and Illumina platforms (BioProject: PRJNA765326) as a <i>Pseudomonas amygdali</i> with an ANI ≥ 95% ± 0.5%. (González-Tobón et al., 2023). The hypersensitive response (HR) to <i>P. amygdali</i> 35-1 was first evaluated in three tobacco (<i>Nicotiana tabacum</i>) and three tomato (<i>Solanum lycopersicum</i>) plants. An overnight culture was washed, resuspended, adjusted to an OD600 of 0.2-0.3, and used to inoculate two leaves per plant via syringe infiltration (Chakravarthy et al., 2017). HR was observed on all leaves at the inoculation site as early as 48hpi for tobacco and 24hpi for tomato. No symptoms were observed for mock inoculations. The pathogenicity of <i>P. amygdali</i> 35-1 in hibiscus was then tested using six 'Social Butterfly' plants in 4.0 in pots inoculated by hand spraying. Inoculum was made from a 72 h culture on solid King's B medium resuspended in sterile deionized water with 2 drops/L of Tween 80 and sprayed at a concentration of 3.6 x 108 bacteria/mL, as determined by dilution plating. Inoculated plants and six matching controls were sealed inside plastic bags and placed in a randomized complete block in a growth chamber at 30 ºC with a 12h photoperiod for 40 days. No changes were observed on any of the control plants but spotting and chlorosis similar to the original symptoms gradually developed on five of the inoculated plants. After 40 days, seven 2-mm tissue squares were excised from the lesion margins or from the asymptomatic tissue of controls, surface-sterilized with 0.75% NaCl, and plated on King's B. No bacteria were recovered from the control plants but white colonies resembling those of the original inoculation were recovered from several diseased tissue pieces. Genomic DNA was extracted from this bacterium using the New England Biolabs Monarch Genomic DNA Extraction Kit. Polymerase chain reaction (PCR) tests were performed to amplify five genes (<i>16S</i>, <i>cts</i>, <i>gapA</i>, <i>gyrB</i>, and <i>rpoD</i>) typically used for multilocus sequence typing (MLST) (Sarkar & Guttman, 2004). The amplicons were Sanger sequenced and BLAST analyses confirmed 100% match to <i>P. amygdali</i> 35-1 (GenBank: CP084212). To our knowledge, our lab is the first to report P. amygdali causing disease on hibiscus in New York. Similar symptoms on hibiscus plants in Florida were reported in the past as caused by other <i>Pseudomonas</i> species of the <i>P. syringae</i> G group, mainly <i>P. syringae</i> and <i>P. cichorii (</i>Chase, 1986; Gardan et al., 1999: Girard et al., 2021; Jones et al., 1986). Given the lack of consistent reporting on bacterial leaf spot pathogens of hibiscus, as well as the economic importance of this ornamental, further research on the disease is needed.