First Report of Stem Canker Caused by Lasiodiplodia subglobosa on Carya illinoinensis in Brazil

Pecan [Carya illinoinensis(Wangenh.) K. Koch], a member of the Juglandaceae, is important to the economy of southern Brazil. Stem canker symptoms were observed on 3-year-old trees of cultivars Desirable, Shawnee, Success, Jackson, and Barton in orchards located in Santa Maria (29[degrees]43'06" S; 53[degrees]43'00" W), Rio Grande do Sul State, Brazil. The disease resulted in 100% loss of symptomatic plants. Approximately half of the 2-ha orchard studied was infected. This disease can be a serious problem if disseminated. Initial symptoms appeared as small, elliptically-shaped, protruding lesions on the trunk bark and branches, progressing to cankers ranging from 6 to 60 cm and eventually leading to plant death. Necrosis and dark streaks were observed in longitudinal sections of the trunk, near the vascular tissue. Portions of symptomatic trunks and branches were surface disinfected with 70% ethanol for 3 min and 1% sodium hypochlorite for 5 min. Portions were rinsed 3x in sterile distilled water, placed in gearboxes, and incubated in a moist chamber at 25 + or - 2[degrees]C with a 12-h photoperiod. After 3 days, fungal structures were observed. Fungal material was transferred first to potato dextrose agar (PDA) medium and subsequently to leaf-pine-agar (LPA) at 25 + or - 2[degrees]C for 12 days to induce the production of reproductive structures. On PDA, the colony color ranged from dark gray to black. On LPA, the pathogen produced pycnidia covered with aerial mycelium. Conidia when young ranged from ovoid to subglobose, were hyaline, and had no septa. The conidia became dark brown with a median septum and presented longitudinal grooves, and were 17 to 25 x 12 to 16 [mu]m (avg. 50 conidia). DNA extraction was performed employing the reagent CTAB as described by Doyle and Doyle (1991). A region of the elongation factor 1-[alpha] gene was amplified (PCR conditions: initial denaturation at 94[degrees]C/2 min; 40 cycles of 94[degrees]C/30s, 54[degrees]C/30s, 72[degrees]C/40s, final extension at 72[degrees]C/4 min) with primers EF1 and EF2 (O'Donnell and Cigelnik, 1997) and showed 100% similarity to Lasiodiplodia subglobosa(GenBank Accession No. KF226721) Accession No. KT895370. Based on morphological and molecular criteria the fungus was identified as L. subglobosa(Machado et al. 2014). A pathogenicity test to fulfill Koch's postulate was performed on 1-year-old and 6-month-old pecan seedlings under greenhouse conditions. The inoculation of the fungus was perf