First Report of Xylella fastidiosa Associated with Oleander Leaf Scorch in Louisiana.

Oleander (Nerium oleander L.) is an evergreen shrub native to the Mediterranean Region and Southeast Asia. Despite being poisonous, it is a popular ornamental plant for use in landscapes, gardens, parks, roadsides, and highway medians. During the fall of 2008, several oleander plants with leaf scorch symptoms were observed at Arsenal Park in Baton Rouge, LA. Symptomatic oleander samples were also received from a commercial nursery in Baton Rouge, LA and a homeowner in Thibodeaux, LA. Symptoms resembled leaf scorch caused by Xylella fastidiosa Wells et al. and included chlorotic mottling of the leaves that started from the tips and margins and progressed toward the midribs. As disease developed, leaf tips and margins became necrotic. Severely infected plants defoliated and died. Leaf petioles from 13 samples (8 from Arsenal Park, 3 from the commercial nursery, and 2 from the homeowner) from symptomatic plants gave positive reactions for X. fastidiosa by ELISA (Agdia, Inc., Elkhart, IN). Leaf petioles from six healthy oleander plants gave a negative reaction for X. fastidiosa by ELISA. Isolation of X. fastidiosa was attempted from eight ELISA-positive and six ELISA-negative oleander samples. Leaf petioles weighing 0.05 g from each sample were used for isolation. The petioles were surface sterilized in 70% ethanol for 1.5 min and then in 2% sodium hypochlorite for 1.5 min, followed by three 1-min washes in sterile water. The petioles were chopped into small pieces under aseptic conditions and soaked in 500 μl sterile water for 30 min. One hundred microliters of the suspension were spread onto periwinkle wilt (PW) plates and incubated in the dark at 28°C. After incubation for 7 days, bacterial colonies typical of X. fastidiosa appeared on five of eight ELISA-positive sample plates. No colonies were observed on six ELISA-negative sample plates. Single colonies were transferred to fresh PW plates to obtain pure cultures. Bacterial colonies from five pure cultures were suspended in nuclease-free water and boiled for 10 min to obtain DNA. DNA from eight symptomatic and six healthy oleander plants was extracted with a DNeasy Plant Mini kit (Qiagen Inc., Valencia, CA) according to the manufacturer's guidelines. Primers (QHOLS-08 and QHOLS-05) (1) specific to the oleander strain of X. fastidiosa amplified a 274-bp portion of DNA from both symptomatic oleander tissues and pure culture of X. fastidiosa isolated from symptomatic tissue. No such amplification was observed in healthy tissue. These primers amplify a portion of DNA encoding a hypothetical protein of unknown function that has been shown to be unique to oleander strains of X. fastidiosa. The PCR product was sequenced and compared with the whole genome shotgun sequence of the oleander strain Ann-1 of X. fastidiosa (GenBank Accession No. AAAM03000099), which resulted in 100% identity with nucleotides 11343 to 11616 in contig 228. X. fastidiosa has been previously reported to cause oleander leaf scorch in California (3), Florida (4), and Texas (2). To our knowledge, this is the first report of X. fastidiosa associated with oleander leaf scorch in Louisiana, extending the geographic range of this important bacterial disease. References: (1) Q. Huang. Curr. Microbiol. 58:393, 2009. (2) Q. Huang et al. Plant Dis. 88:1049, 2004. (3) A. H. Purcell et al. Phytopathology 89:53, 1999. (4) R. L. Wichman et al. Plant Dis. 84:198, 2000.