First Report of Lasiodiplodia pseudotheobromae Causing Trunk Cankers in Acacia mangium in Venezuela.

In May 2010, canker and wood stain symptoms in trunks and stems of 125 Acacia mangium were observed during a survey conducted in the Uverito plantations, Monagas State, Venezuela. Cankers were 20 to 65 cm long and were brownish on the margins and dark brown in the center. Many of the cankers had swollen margins and in some cases a black exudate could be seen leaking from the most severe cankers. Small pieces (4 to 5 mm) of necrotic tissues from the cankers were surface sterilized for 1 min in 1.5% NaOCl, washed twice with sterile distilled water, and plated onto potato dextrose agar amended with 0.01% tetracycline hydrochloride (PDA-tet). Plates were incubated at 25°C under near-UV light. Colonies developed from symptomatic tissue and formed a compact mycelium, which was initially white, but became dark gray with age. Based on colony morphology, isolates were tentatively identified as a member of the Botryosphaeriaceae family. Pycnidia were produced on sterilized pine needles on 2% water agar after 5 weeks of incubation at 25°C under continuous near-UV light. Conidia were ellipsoidal, initially hyaline, unicellular, becoming dark brown, and developing a thick wall, a central septum, and longitudinal striations with age. Conidia measured 26 to 31 μm long and 11 to 16 μm wide (n = 60). The conidial morphology matched that of Lasiodiplodia, a member of the Botryosphaeriaceae family (1). Primers ITS4/ITS5, Bt2a/Bt2b, and EF1-688F/EF1-1251R (2) were used to amplify and subsequently sequence the ITS1-5.8S-ITS2 region and parts of the beta-tubulin (BT) and translation elongation factor 1-alpha (TEF1-α) gene regions, respectively. The putative Lasiodiplodia isolates had 98 to 99% homology with Lasiodiplodia pseudotheobromae isolate CBS 116459 for all three loci (EF622077, EF622057, and EU673111) (1). Based on morphological characters and DNA sequencing, the canker isolates from Venezuela (CBS129752 and UCD-A1) were then identified as L. pseudotheobromae (1) and sequences were deposited in GenBank (Accession Nos. JX545091 to JX545092, JX545111 to JX545112, and JX545131 to JX545132). Pathogenicity tests were performed by inoculating 2-year-old A. mangium tree trunks with isolates CBS129752 and UCD-A1. Twenty trees per isolate were inoculated by placing a mycelium plug from the growing margin of 8-day-old colonies upside down directly into a fresh wound made with a 5-mm cork borer. Wounds were sealed with Parafilm. Ten control trees were inoculated with non-colonized PDA plugs. After 12 weeks, all inoculated seedlings showed bark swelling around the inoculation points and a brown necrosis of the wood could be observed when removing the bark. Average length necrosis above and below the point inoculation was 27.2 cm; additionally, a black exudate was observed when the outer bark was removed from inoculation points. L. pseudotheobromae was successfully reisolated from the necrotic tissue observed in symptomatic plants. No symptoms were observed in the control plants and L. pseudotheobromae was not isolated from the controls. L. pseudotheobromae has been reported in Africa, Asia, Europe, and Latin America, where it occurs on forest and fruit trees (1). This study shows L. pseudotheobromae to be highly virulent on A. mangium and, to our knowledge, this is the first report of L. pseudotheobromae on this host in Venezuela. References: (1) A. Alves et al. Fungal Diversity 28:1, 2008. (2) J. R. Úrbez-Torres et al. Plant Dis. 90:1490, 2006.