Brazil nut (Bertholletia excelsa) is an Amazonian native species whose seeds are valuable non-timber forest products in Brazil (IBGE 2021a). The seeds are harvested by local people, and their commercialization has high social and economic values for many Amazonian indigenous and riverine communities (Ribeiro et al. 2014). In addition, Brazil nuts are a selenium-rich food resource with high contents of proteins, bioactive compounds, and other micronutrients (Cardoso et al. 2017).

Brazil and Bolivia are the largest producers of Brazil nut and have been competing for leading production every year. However, in 2020, Bolivia exported $162 M, whereas Brazil exported only $21.8 M (OEC 2021). In 2020, Brazil produced 33.118 ton of Brazil nut valued at $18 million, and it was the third major non-timber forest product. The states of Amazonas, Acre, and Pará accounted for 86% of the total production in Brazil (IBGE 2021b). In 2016, canker symptoms were observed on stems and branches of 1.5-year-old Brazil nut trees in an experimental field at Embrapa Amazônia Oriental in Tomé-Açu, Pará. These symptoms were observed 30 days after the trees were grafted by budding (Fig. 1a and b).

A cross section of the cankers revealed reddish vascular lesions. Samples from the margin of dead tissue were extracted for indirect pathogen isolation. The samples were disinfected with 70% alcohol for 30 s and 2% sodium hypochlorite for 30 s, washed with distilled water, and then placed on potato dextrose agar (PDA) in Petri dishes. The plates were maintained at 25 °C under a 12-h fluorescent light-and-dark cycle.

After 3 days, a light-gray culture was observed, which grew darker with age. Colonies produced black pycnidia with elliptical conidia. Immature conidia were initially hyaline and unicellular. Mature conidia became dark brown with central septa, thick walls, and longitudinal streaks. The conidia measured 20–25 μm in length and 10–15 μm in width (Fig. 1E), similar to the characteristics of Lasiodiplodia theobromae described by Lima et al. (2013), Alves et al. (2008), and Netto et al. (2014).

Fig. 1
figure 1

External symptoms of canker in the branch (A) and internal with the darkening of the vessels (B) in a Brazil nut seedling in field. Symptoms in Brazil Nut seedling 10 (C) and 17 days (D) after inoculation with Lasiodiplodia theobromae CMAA 1775. Young (hyaline and unicellular) and mature conidia with dark coloration and centralized septum of L. theobromae CMAA 1775 (E).

For molecular identification, DNA was extracted from the mycelia of a colony grown on PDA for 7 days. The ribosomal DNA internal transcribed spacer (ITS) regions and partial translation elongation factor 1 alpha loci (EF1) were PCR-amplified using the primer sets ITS4/ITS5 (White et al. 1990) and EF1-688 F/EF1-1251R (Phillips et al. 2005), respectively.

The PCR products were purified and sequenced. The nucleotide sequences of ITS and EF1 were deposited in GenBank (MN210328 and MN217399) and compared with the sequences in GenBank using BLASTN. The nucleotide sequences were then subjected to multiple sequence alignment using the ClustalW program. A phylogenetic tree was obtained by applying Maximum Parsimony analysis of taxa using MEGA software.

The phylogenetic analysis indicated that the isolate clustered in the L. theobromae clade (GenBank accession numbers CBS1649 and CBS111530) with an 85% bootstrap value (Fig. 2). The L. theobromae isolate from Brazil nut was deposited in the Culture Collection of Microorganisms of Agricultural and Environmental Importance, Jaguariúna, Brazil (strain designation CMAA 1757).

Fig. 2
figure 2

Phylogenetic tree generated from Maximum Parsimony Inference based on the alignments of ITS and EF1 sequence data of isolates of Lasiodiplodia spp. The isolate of Lasiodiplodia theobromae CMAA 1775 associated with canker of Brazil nut is highlighted in bold type font. Node labels indicate posterior support probability. The tree was rooted with Macrophomina phaseolina PD112 as the outgroup taxon.

The isolate was then tested for pathogenicity on Brazil nut seedlings. It was cultured on PDA and maintained at 25 °C for 7 days. A mycelium disc of CMAA 1775 was superimposed on the seedling stems previously injured with a scalpel, and sealed with parafilm. A disc of only PDA was used on control plants. The plants were maintained in a greenhouse set to a temperature range from 26° C to 30° C. After 15 days, all the inoculated plants displayed the same symptoms initially observed in the field (Fig. 1c). The fungus was re-isolated, and its morphological characteristics were identical with those of L. theobromae CMAA 1757, fulfilling Koch’s postulates.

In Brazil, L. theobromae causes canker symptoms in several important economic crops, such as rubber (Gasparotto et al. 1984), grapevine (Ribeiro et al. 1992; Correia et al. 2016), paricá (Tremacoldi et al. 2009), African mahogany (Tremacoldi et al. 2013), teak (Borges et al. 2015), cashew, pitomba, and seriguela (Coutinho et al. 2017). This paper is the first to report L. theobromae causing canker in Brazil nut trees in the world as well as in Brazil.

Brazil nut is an important non-timber forest resource in Brazil and a promising species for reforestation of degraded or deforested areas (Melo et al. 2018). Canker caused by L. theobromae negatively impacts the production of Brazil nut seedlings. Further studies on epidemiology and control measures are required to develop management strategies for canker caused by L. theobromae in Brazil nut.