First Report of Bipolaris secalis Causing Disease on Jangada Brava (Heliocarpus americanus) Seeds in Brazil

Abstract

Jangada Brava (a local name) (Heliocarpus americanus L.) is a dioecious plant, native to South America, that can reach 12 m in height and 50 cm in diameter. It can be found in the semideciduous broadleaved forest of the Parana Basin (Lorenzi 1992). Its wood is used in the manufacture of pencils, toys, and cross laminated timber (CLT). The tree is planted as a quick replenishment for degraded areas due to its good growth. In January 2017, seeds collected directly from native trees around a neighborhood of the city of Engenheiro Beltrao, Parana, Brazil (23°47′49″S, 52°16′08″W), had a fungal infection in 60% of the samples sent for diagnostic testing. Contaminated seeds were surface-disinfested in a 0.5% NaOCl solution for 1 min and placed onto potato dextrose agar (PDA). A fungus was recovered after 5 days of incubation at 25°C showing a grayish black colony with an irregular shape, elevated surface, and a border ranging from white to light gray. On the seventh day of incubation, the colony produced free spindle conidia, fusiform, pale to dark-brown color. The conidia presented straight to slightly curved ranging from 49 to 95 μm in length and 11 to 23 μm in width, presenting an average of 10 septa. Conidiophores were mostly simple, producing conidia at their apices. Morphological characteristics of the isolates were similar to those of Bipolaris secalis as described by Sisterna (1989). To obtain a DNA based identification, amplification and sequencing of the internal transcribed spacer (ITS) region using ITS1 and ITS4 primers was performed (White et al. 1990). A sequence of ∼400 bp of the ITS region was deposited in GenBank (accession no. MF770710), and BLAST analysis revealed that the isolate was 99% identical to the B. secalis isolate on GenBank (KU554556.1) differing from B. shoemakeri by base substitutions in the ITS positions 103 (G) and 339 (insertion or deletion of nucleotide) (Tan et al. 2016). To fulfill Koch’s postulates, a sample of 200 healthy seeds was placed in direct contact with the fungal mycelia, grown in Petri dishes containing PDA, and then incubated at 25°C for 72 h. A sample of 200 of the same seeds, placed on PDA only, was used as a negative control. The seeds were sowed in a commercial substrate and incubated for 25 days at 25°C with a 12-h day/night cycle. The fungus inhibited 80% of the seed’s germination when compared with the control group. The remaining seeds that germinated produced leaves with necrotic, sporulating lesions with dark brown stains, characteristic of the fungus as described above. In total, about 89% of the seedlings (with 75% of the leaves on each seedling) exhibited symptoms whereas the control group remained asymptomatic. The fungus was reisolated from symptomatic areas and reidentified with morphological bases. Koch’s postulates were repeated once and showed similar results. This is the first report of an incidence of B. secalis causing necrosis in seedlings and reducing germination in Jangada Brava seeds in Brazil.