First Report of Diplodia Canker of Cypress Caused by Diplodia pinea f. sp. cupressi on Mediterranean Cypress in Tunisia.

Symptoms of decline were observed on Mediterranean cypress trees (Cupressus sempervirens L.) in Tunisia in 2003 and 2004; disease specimens were vouchered as FIAF 38649. The declining, windbreak trees in the Cap Bon Region were 25 to 30 years old. Severity of symptoms varied among trees. Thus, areas of reddish, withered foliage alternated with areas that were still green. Other trees were completely withered. The bases of withered branches and tree trunks bore slightly sunken cankers with longitudinal bark cracks that oozed drops of resin. When the outer layer of a cankered area was scraped away, dark brown inner bark tissue was seen to extend up to several centimeters around the canker. Cross sections through cankers on trunks and branches revealed extensive darkened, wedge-shaped sectors in the wood. The affected bark bore numerous black pycnidia. Conidia were mostly smooth, ovoid, hyaline, and aseptate; a few were brown with a medial septum. The mean conidial dimensions (n = 100) were 27 × 11 μm; the extreme range was 19 to 31 × 8 to 13 μm. Isolates obtained from affected host tissue and conidia developed mainly floccose colonies that were white, then greyish green, and finally dark gray black on potato dextrose agar (PDA) at 25°C. Blackish pycnidial fructifications containing typical conidia were produced after 5 weeks on autoclaved cypress seeds placed on the colonies under light. Pathogenicity was tested using five 3-year-old potted Mediterranean cypress trees. These were inoculated by placing 3-mm-diameter plugs of mycelium of isolate DF IMG86 (DAOM 234788) from the edge of a 15-day-old colony on PDA on 5-mm-diameter wounds made in the bark. The wounds were covered with cotton wool moistened with sterile water and wrapped in adhesive tape. Similar wounds on five control trees received a plug of sterile PDA. Symptoms occurred as early as the third week after inoculation. The leaves first became yellow and then turned amaranth red, after which they progressively withered. Two months after inoculation, cankers were clearly visible at the inoculation site. Isolates from these cankers were morphologically similar to those used for inoculation. The control plants did not show any disease symptoms and their wounds healed normally. Morphological, cultural, and pathological characteristics of the fungus isolated from cypress with decline symptoms were similar to those of the fungus referred to as Diplodia pinea f. sp. cupressi or Sphaeropsis sapinea f. sp. cupressi (1). Identification was confirmed by marker analysis by using intersimple sequence repeat polymerase chain reaction (2). Banding patterns for isolate DF IMG86 were produced using primers HYH(GY) and (CAG) and were identical to those for Diplodia pinea f. sp. cupressi isolates 94-3 (DAOM 229437) and 95-158 (DAOM 229439) and differed from those obtained for isolates of Diplodia pinea (S. sapinea A group), D. scrobiculata (S. sapinea B group), Botryosphaeria obtusa, and B. stevensii. To our knowledge, this is the first report of this pathogen in Tunisia. The development of D. pinea f. sp. cupressi on cypress windbreaks in the Cap Bon Region may be related to a drought that has afflicted Tunisia for the past 5 years. Reference: (1) Z. Solel et al. Can. J. Plant Pathol. 9:115, 1987. (2) S. Zhou et al. Mycol. Res. 105:919, 2001.