In September 2010, live oak (Quercus virginiana Mill.) trees in an Alachua County, FL, shopping center parking lot were observed with shoot dieback and cankers on small branches. Isolations were made from canker margins by surface sterilizing tissue in 2.5% sodium hypochlorite and plating on potato dextrose agar (PDA) and incubating at 23°C. Fungi morphologically similar to Diplodia quercivora Linaldeddu & A.J.L. Phillips (mycelium initially velvety and white and later turning pale to dark olivaceous and grayish in reverse) were consistently isolated from symptomatic tissue (2). The two loci used by Linaldeddu et al. (2) in the description of D. quercivora were sequenced to identify a representative isolate (PL1345) as D. quercivora. The internal transcribed spacer (ITS) (GenBank Accession No. KF386635) and translation factor 1-alpha (EF-1α) (KF386636) regions were amplified and sequenced using primers ITS1F/ITS4 (3) and EF1-728F/EF1-986R (1). BLASTn searches of the two sequences resulted in 99% (467 of 469 and 257 of 259, respectively) homology with D. quercivora CBS 133852, confirming the fungal isolates' identity as D. quercivora. In October 2011, Koch's postulates were verified by inoculating, repeated twice, three Q. virginiana saplings (stem diameters, 12 to 14 mm; at inoculation sites approximately 50 mm above soil line) with isolate PL1345. Agar plugs (3 × 3 mm) taken from the margin of a 12-day-old culture on PDA were inserted into flaps in the stems made by a sterile blade with the mycelia facing the cambial tissue. One negative control tree was mock inoculated with a sterile PDA plug. All inoculation sites were sealed with Parafilm and maintained in a greenhouse (19 to 29°C). Trees were assessed for symptoms 90 days after inoculation. External bleeding was noted on all but one tree, and all flaps became necrotic. Pycnidia were observed on the outer surface of the flap on one inoculated tree. Negative controls showed no bleeding and their tissue flaps remained alive. Vertical length of phloem necrosis and percent of stem girdling were measured after removing the bark. Mean necrotic length and percent girdling for inoculated saplings were 48 mm (standard error [SE] = 10.6) and 26.6% (SE = 5.7) for the first inoculation and 46 mm (SE = 17) and 25% (SE = 5) for the second, respectively. Controls showed no internal necrosis and all produced healthy callus tissue at inoculation sites. Two of the pathogen-inoculated trees per inoculation were sampled and the pathogen was re-isolated from each. Recovered fungal isolates were confirmed as D. quercivora based on morphology and 100% ITS sequence homology to PL1345. D. quercivora was first described as causing shoot dieback and cankers on Q. canariensis in Tunisia and was found to be pathogenic to three additional Mediterranean oak species, Q. ilex, Q. pubescens, and Q. suber (2). To our knowledge, this is the first report of D. quercivora causing cankers on Q. virginiana and the first report of the fungus outside of Tunisia. Given the damage this pathogen has caused there, efforts to monitor the spread of this disease would seem warranted. More research is needed to assess the risk this pathogen poses to North American oaks, however.
References: (1) I. Carbone et al. Mycologia 91:553, 1999. (2) B. T. Linaldeddu et al. Mycologia 105:1266, 2013. (3) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.
