Disease symptoms in blueberry associated with species of Pestalotiopsis include leaf spot, fruit rot, stem canker and twig dieback (Espinoza et al. 2008; Maharachchikumbura et al. 2014). Leaf spot caused by Pestalotiopsis microspora is one of the major fungal diseases in blueberry production and if not treated promptly can lead to plant death (Yi-Lan et al. 2021). In Brazil, one blueberry plant (Vaccinium sp.) of the cultivar Climax showing leaf spot symptoms was observed in a commercial orchard (established in 2007), during November of 2020 (spring) (Fig. 1A to C). Necrotic leaf spot symptoms gradually appeared from leaf edges to middle tissues on the adaxial surface (Fig. 1A to C). Later, these necrotic leaf spots expanded and coalesced resulting in severe defoliation of leaves (Fig. 1A). Symptoms were not observed on the fruit of affected plant, but aborted fruit (decay) with signs of Pestalotiopsis spp were detected in soil under the affected plant (Fig. 1D). Fungal acervuli were observed in abaxial leaf surface (Fig. 1C) and around all fruit (Fig. 1D). Thus, the objective of the present study was to confirm the causal agent of the symptoms observed in blueberry plant in Brazil.

Fig. 1
figure 1

Symptoms of leaf spot in leaves (A to C) and decay in fruit (D) caused by Pestalotiopsis trachycarpicola (ex-type Pestalotiopsis trachycarpicola) in blueberry tree cv. Climax. Necrotic leaf spot symptoms gradually appeared from leaf edges to middle tissues (A to C). Infected tissues generally resulted in severe defoliation of leaves (A) and fruit abortion (D). Asterisks (*) indicate the abaxial leaf surface (C) and fruit (D) where the acervuli occurred. Front (E) view of ten-days-old colonies of P. trachycarpicola (ex-type P. trachycarpicola) growing on a potato dextrose agar dishes. Conidia of P. trachycarpicola (ex-type P. trachycarpicola) (F). Bars: 5 (C) and 20 (D) mm; 20 μm (F)

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Leaves from symptomatic blueberry plants cv. Climax were collected from a commercial orchard in São Joaquim, Santa Catarina (28°14’33.62’’S, 50°09’07.59’’W, Altitude 1,142 m), Brazil. Infected tissues (3 cm2) were surface sterilized for 30 s in 75% ethanol and 60 s in 0.5% sodium hypochlorite (NaOCl), followed by rinsing three times in sterile water before placing on potato dextrose agar (PDA). Plates were incubated in the dark at 22 °C ± 2 °C for 10 days to develop colonies. The Pestalotiopsis spp. isolate was preserved and deposited in the Mycological Collection of the “Santa Veronica Giuliani” in Porto Alegre, RS, under registration number SVG00116-F. Leaf spots, fruit and plates containing fungal structures were examined under a stereomicroscope (Leica EZ4E, Germany) fitted with a digital camera (5.0 Megapixel resolution). Morphological observations were made under a BX41 37 microscope (Olympus, Japan), and images were acquired digitally (Q-Color™, 5.0 38 Megapixel resolution).

Detached terminal shoots (20–30 cm long) and leaves, immature and mature fruit of blueberry trees cv. Climax (orchard was established in 2004) were used to satisfy Koch’s postulates according to Luan et al. (2008) and Araujo et al. (2022). Blueberry tissues were inoculated with the fungus by spraying a suspension of 2 × 105 conidia mL− 1 until runoff. Blueberry tissues were placed inside plastic boxes (10 L – 16 × 40 × 29 cm) and incubated at 26 °C and 12 h photoperiod. Control blueberry tissues were sprayed with sterile water.

For DNA extraction, the harvested fungal material obtained from the agar cultures was ground to a fine powder, in liquid N2, using a frozen pestle and mortar. Total DNA extraction using 100 mg of homogenized ground material was performed using the Wizard Genomic DNA Purification Kit (Promega, Madison, USA). DNA integrity and concentration were assessed using a NanoDrop™ spectrophotometer (ND-1000; Thermo Scientific, New Hampshire, USA). The DNA was stored at -20 °C until the analysis was performed. Polymerase chain reaction amplifications of the internal transcribed spacer (ITS) region, translation elongation factor 1 a (tef1) and b-tubulin (tub2) partial gene were carried out with ITS1 + ITS4 (White et al. 1990) primers the EF-1 + EF-2 (O’Donnell et al. 1998), Tb2a + Tb2b (Glass and Donaldson 1995) primers, respectively. Amplicons were purified using ReliaPrep™ DNA Clean-Up and Concentration System (Madison, U.S.A.) and sequenced in both directions at the ACTGene Company, Porto Alegre, RS. The sequences obtained from sequencer were edited using the BioEdit 7.0.5.3 software, and consensus sequences were analyzed using the Molecular Evolutionary Genetics Analysis (MEGA X) software (Kumar et al. 2018). The similarity of the nucleotide sequences of the isolate was calculated using the BLAST algorithm (Basic Local Alignment Search Tool). Sequences from reference strains of Pestalotiopsis spp. (Maharachchikumbura et al. 2014) available in GenBank were added to the analyses and the sequence obtained in this work was aligned with these reference sequence Truncatella angustata (CBS 356,33) as an outgroup taxon. The Bayesian phylogenetic tree was reconstructed using database from ITS, TEF and TUB. The best nucleotide substitution models (GTR + I) for ITS, (SYM + G) for TEF and (HKY + G) for TUB was determined using the MrModeltest software (Posada and Buckley 2004). The CIPRES web portal (Miller et al. 2010) was used to run MrBayes v3.2.1 (Ronquist and Huelsenbeck 2003). The Markov Chain Monte Carlo (MCMC) analysis was run with a total of 10 million generations, sampling every 1,000 generations. The first 25% of the sampled were discarded as burn-in, and the posterior probability values were calculated using the remaining trees (Rannala and Yang 1996). The phylogenetic tree (Fig. 2) was visualized using the FigTree software (http://tree.bio.ed.ac.uk/software/figtree/). Sequences generated in this study were deposited in GenBank.

Fig. 2
figure 2

The Bayesian phylogenetic tree based on ITS, TEF and TUB sequences of Pestalotiopsis spp. (four ex-types). The Bayesian posterior probabilities values are indicated above the nodes, and the scale bar represents the number of expected changes per site. The reference specimen CBS 356,33 of Truncatella angustata was used as outgroup

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After 10 days of incubation, fungal colonies reached 90 mm in diameter with a full edge. Colony colour was white to pale yellow, with dense aerial mycelium on surface, fruiting bodies black (colony conidia), developing in concentric circles, the back of culture was yellow (Fig. 1E). Black acervuli conidiomata (approximately 100 to 500 μm in diameter) were formed superficially on the PDA medium, leaves and fruit (Fig. 1C–E). Conidiophores were not observed in fungal colonies developed on PDA plates. Conidia contained five cells (4-septate) with apical and basal appendages (n = 30) (Fig. 1F). Basal cell was conic to acute, hyaline, thin-walled, 3.9–6.3 (x̅ = 5.0) µm long × 1.8–3.2 (x̅ = 2.5) µm wide. The apical cell was conic to subcylindrical, hyaline, thin-walled, 3.7–5.5 (x̅ = 4.2) µm long × 1.9–4.0 (x̅ = 2.8) µm wide. The middle three cells were all olive to brown color, thick-verruculose-walled, from the apical to basal cells, the second cell was 3.4–5.5 (x̅ = 4.4) µm long × 2.9–5.1 (x̅ = 4.3) µm wide, the third was 3.9–5.5 (x̅ = 4.5) µm long × 3.8–5.4 (x̅ = 4.7) µm wide, the fourth was 4.0–6.2 (x̅ = 4.5) µm long × 3.5–5.2 (x̅ = 3.9) µm wide. Two to three apical appendages varied from 5.0 to 18.5 (x̅ = 11,8) µm long, while the basal appendage (only) varied from 2.1 to 5.8 (x̅ = 3,4) µm long. The Pestalotiopsis spp. isolate SVG00116-F was identical (morphological observations) to Pestalotiopsis trachycarpicola (four ex-types Pestalotiopsis trachycarpicola), which was confirmed by phylogenetic analysis, with 99% bootstrap (Fig. 2) using phylogenetic analysis. The sequences were deposited in GenBank under accession number ON238108 (ITS), OP895025 (TEF) and OP895026 (TUB). The classification of Pestalotiopsis species is based on a combination of morphological characteristics and molecular analyses (Espinoza et al. 2008; Gonzales et al. 2012; Maharachchikumbura et al. 2014). Fulfilling Koch’s postulates showed that Pestalotiopsis spp. isolate SVG00116-F was a pathogen of leaves and fruit blueberry, causing the same symptoms as observed in the field (Supplementary Information). The pathogen was reisolated from symptomatic leaves and identified by morphological characteristics and molecular method (Figs. 1 and 2).

In a study about the occurrence and prevalence of fungal species causing foliar diseases on blueberry in the Georgia, the Pestalotia leaf spot was the third most common foliar disease, occurring on about 25% of the samples in two years of the survey (Scherm et al. 2008). In South America the pathogen has been observed in blueberry orchards in Argentina (Wright et al. 1998), Chile (Espinoza et al. 2008) and Uruguay (Gonzales et al. 2012). To our knowledge, this is the first report of Pestalotiopsis trachycarpicola (ex-type P. trachycarpicola) causing spot leaf disease in blueberry in Brazil. Little is known about the behavior of P. trachycarpicola in the Brazilian climate and additional studies are required to understand the potential economic impacts of this pathogen on blueberry orchards.