First Report of Bacterial Leaf Spot of a Hardy Pink (Dianthus gratianopolitanus hybrid) Caused by Burkholderia andropogonis in Virginia

In December 2015, the Virginia Tech Plant Disease Clinic received hardy pinks (Dianthus gratianopolitanus hybrid Scent First ‘Passion’) with severe leaf spotting from a commercial grower in Virginia, who reported that 20% of the crop was affected. Leaf spots were tan, circular, and 1 to 2 mm in diameter with purple margins and water-soaking. Bacterial streaming from lesions was observed. Symptomatic leaf tissue was surface-sterilized for 2 min in 40 ml of 10% NaClO with one drop of Tween-20, then rinsed in sterile, distilled water, and blotted dry. Small (1 mm2) sections were excised from leaf spot margins and placed in 1 ml of sterile, distilled water for 15 min. The suspension was transferred onto King’s medium B. Culture plates were incubated for 3 days at 28°C (Duan et al. 2009). Small, nonfluorescent, white colonies were consistently recovered. The bacterium was identified as Burkholderia andropogonis (recently proposed to be renamed Paraburkholderia andropogonis; Sawana et al. 2014) with the Gen III Microstation System (Biolog, Hayward, CA), following manufacturer instructions. PCR was used to amplify the 16s rRNA region. The product was cleaned enzymatically, then sequenced bidirectionally by Eurofins (Louisville, KY). A consensus sequence was assembled using DNASTAR Lasergene 10 (DNASTAR Inc.). The sequence (accession no. KY464993) was compared with sequences in GenBank using BLAST and had 99% identity (893/905 bp) to the B. andropogonis type strain (X67037). To complete Koch’s postulates, inoculum was prepared from a single B. andropogonis isolate by transferring one loopful of bacteria from a pure, 3-day-old culture on King’s medium B into 50 ml of nutrient yeast extract-broth (NYEB), which was shaken at 150 rpm, 28°C. After 15 h, 5 ml of the culture was transferred to 45 ml of NYEB and shaken as described above until an absorbance of 0.1 at 600 nm was reached. Serial dilutions were prepared in phosphate-buffered saline (0.1 M, pH 7.2), and the number of colony forming units was determined by dilution-plating. Prior to inoculating Dianthus Scent First ‘Passion’ plants, each plant was wounded by penetrating leaves with a 25G 5/8 needle at 10 locations on eight leaves per shoot, two shoots per plant. Plants were sprayed to runoff with inoculum or sterile phosphate-buffered saline immediately after wounding as follows: three plants with 8.8 × 109cfu/ml; five plants with 8.8 × 108cfu/ml, and two plants with sterile buffer. Plants were covered with plastic bags for 48 h and maintained in a greenhouse set to a minimum temperature of 84°F and a maximum of 95°F. Small, water-soaked lesions developed around wounds on inoculated plants 10 days post inoculation. After 2 weeks, lesion development on nonwounded tissue of inoculated plants was also apparent, and plants inoculated with 8.8 × 109cfu/ml and 8.8 × 108cfu/ml had an average total of 86 and 15 lesions on all plant tissue per plant, respectively. After 3 weeks, all inoculated plants had a high incidence of lesions on both wounded and nonwounded tissue. No symptoms were observed on control plants throughout this period. Bacteria were isolated from lesions on both wounded and nonwounded tissue and identified as B. andropogonis with the Gen III Microstation System. To our knowledge, this is the first report of bacterial leaf spot of hardy pinks caused by B. andropogonis in Virginia. © 2017, American Phytopathological Society. All rights reserved.