A bacterial disease of hellebore caused by Pseudomonas viridiflava in New Zealand Robert K. Taylor & Megan K. Romberg & Brett J. R. Alexander Received: 3 February 2011 /Accepted: 27 April 2011 /Published online: 6 May 2011 # Australasian Plant Pathology Society Inc. 2011 Abstract Pseudomonas viridiflava is reported causing a disease of hellebore (Helleborus × hybridus) in New Zealand. The bacterium was isolated from diseased plants exhibiting distinct symptoms consisting of black leaf spots, necrotic petal and stem lesions. P. viridiflava was identified on the basis of biochemical and molecular tests and confirmed as the causal agent by fulfilling Koch’ s postulates. Keywords Pseudomonas viridiflava . Hellebore Hellebores are grown in New Zealand as an ornamental plant and as a commercial crop to supply cut flowers to local and export markets. The most popular hellebores are Helleborus orientalis and its hybrids (H. × hybridus). Hellebores are mainly propagated in shade houses and watered by drip irrigation. In August 2010, diseased hellebore plants (H. × hybridus Double White) were observed in a nursery in Tauranga, New Zealand. Symptoms developed after several days of moderate rainfall and more than 90% of the plants were affected, resulting in unsuitability for sale. Symptoms consisted of circular, black leaf spots which were 1.5– 2 mm in diameter, black stem lesions and dry, grey to brown lesions with distinct margins on the flower petals (Fig. 1). The symptoms were different from those caused by other leaf-spotting pathogens of hellebore such as the fungus Coniothyrium sp. and the bacterium Xanthomonas sp. (Gleason et al. 2009). Saline suspensions of macerated diseased leaf, petal and stem tissues were streaked onto King’ s B medium (King et al. 1954) and pale yellow, fluorescent bacterial colonies were consistently growing following 48 h incubation at 25°C. All isolates were Gram negative and were levan, oxidase and arginine dehydrolase negative. They all produced a hypersensitive reaction on tobacco leaves after 24–36 h and showed pectolytic activity on potato tuber slices. The bacterium was identified as P. viridiflava on the basis of these biochemical tests and is in group II of the LOPAT determinative scheme of Lelliott et al. (1966). The house- keeping gene gyrB (Sarkar and Guttman 2004) was amplified from two isolates (ICMP 18563, 18564), produc- ing a 600 bp amplicon. BLASTn analysis of the gyrB amplicons showed that the region was 99.0–99.8% homol- ogous to P. viridiflava sequences in GenBank, with highest homology to accessions AY606756 and AY606738. Pseu- domonas viridiflava was isolated consistently from leaf, petal and stem tissue adjacent to leaf spots, petal and stem lesions. Pathogenicity tests with two selected isolates of P. viridiflava (ICMP 18563, 18564) were performed on hellebore (H. × hybridus) cut flowers, young detached leaves and petals. Each isolate was inoculated onto three detached petals and leaves by placing 20 μl of bacterial suspension (10 7 cfu/mL) on the surface and pricking through the drops using sterile needles. After inoculation, the detached petals and leaves were kept in Petri dishes lined with moist filter paper, incubated at 25°C and checked daily for symptoms. Three healthy cut flowers with stems submerged in flasks of water were inoculated per isolate in a similar manner, enclosed in clear polyethylene bags and incubated at 25°C. Controls were inoculated in the same way using sterile physiological saline (0.85% NaCl). Necrosis developed on the cut flowers, leaf and petal tissues 3 days after inoculation. The symptoms induced on cut flowers, R. K. Taylor (*) : M. K. Romberg : B. J. R. Alexander Plant Health and Environment Laboratory, Ministry of Agriculture and Forestry, PO Box 2095, Auckland 1140, New Zealand e-mail: robert.taylor@maf.govt.nz Australasian Plant Dis. Notes (2011) 6:28–29 DOI 10.1007/s13314-011-0010-1