Brazil is the third largest beer producer globally. However, production of hops began less than five years ago. Humulus lupulus is a cree**, herbaceous, perennial, and dioecious plant of the Cannabaceae family mainly used as a raw material in the beer brewing industry (Gonsaga et al. 2021). Fusarium spp. have been associated with canker and cone tip blight on hop plants (Pethybridge et al. 2001; O’Neal et al. 2015). The disease is associated with wet winters, areas with poor drainage or damage of bines or rhizomes by wind (Mahaffee et al. 2009). The former causes wilting, girdling and cankers in the crown, foliar necrosis, and death of infected plants (O’Neal et al. 2015).

During spring 2020, approximately 40 hops plants (Humulus lupulus) of variety ‘Comet’ showing Fusarium canker symptoms were observed in a commercial hop yard (established at 2019), in Brazil (Fig. 1A). The plants were poorly developed with chlorotic leaves. (Fig. 1A, B). Bines wilted, plants swelled, and frequently girdling was observed near the base of bines at the soil line (Fig. 1C, D). Symptoms were not observed on cones until the harvest. The objective of this study was to identify the Fusarium species associated with canker symptoms in hop in Brazil.

Fig. 1
figure 1

Symptoms of canker caused by Fusarium meridionale in leaves and bines of the Humulus lupulus variety ‘Comet’. Poorly developed plants with chlorotic leaves (A, B). Disease symptoms on the bines with canker and girdling near the base (C, D). Seven-days-old colonies of F. meridionale growing on a potato dextrose agar dishes (E). Conidia of F. meridionale (F). Bar: 50 μm (F). Photos: F. A. M. F. Pinto (A, C, E), M. M. Fagherazzi (B and D) and A. O. Souza (F)

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Bines from the symptomatic H. lupulus ‘Comet’ were collected from a commercial hopyard in Lages, Santa Catarina (27º49′53"S, 50º15′59"W), Brazil. To obtain a pure fungal isolate, the bines were examined under a stereomicroscope (Leica EZ4E, Germany) equipped with a digital camera (5.0 Megapixel resolution). Pieces of the infected hop bines were surface-treated with 2% sodium hypochlorite for 1 min, placed on 2% water agar, and incubated at 22 ± 2 °C. After three days, colonies were transferred to Petri dishes containing potato dextrose agar (PDA). The plates were incubated at 22 °C with a photoperiod of 12 h for 7 days to develop colonies (Pethybridge et al. 2001). The fungal isolate was preserved and deposited in the Micological Collection of the "Santa Verônica Giuliani" in Porto Alegre, RS (registration number SVG00115-F). Pathogenicity tests (for fulfillment of Koch’s postulates) were performed by inoculating hop plants ‘Comet’ and mature hop cones (N = 20) with a conidial suspension (1.0 × 106 spores per milliliter) until runoff. Four leaf petioles (per plant) from a stem (two leaf scars/shoot) were detached using a sterilized scalpel. A 30 μl drop of ascospore suspension was placed on the wounds on the stem, and the inoculated wounds were covered with parafilm and incubated at 22 ± 2 °C in a sealed box on plastic mesh over tissue wetted with sterile distilled water (Pethybridge et al. 2001).

Colonies grew rapidly on PDA and their morphological characteristics were identical to those of a Fusarium sambucinum species complex. Single conidia were transferred on PDA and Spezieller Nährstoffarmer agar (SNA) media to obtain pure cultures, allowing identification to the species level (Leslie and Summerrell 2006). Their morphology was observed in BX41 microscope (Olympus, Japan); images of conidia were captured digitally (Q-Color™, 5.0 Megapixel resolution) and processed further using Olympus cellSens Dimension software.

For DNA extraction, the fungal tissue harvested from PDA plates was ground to a fine powder in liquid N2 using a frozen pestle and mortar. Total DNA was extracted with 100 mg of homogenized ground material using the Wizard Genomic DNA Purification Kit (Promega, Madison, USA). DNA integrity and concentration were assessed using NanoDrop spectrophotometer (ND-1000; Thermo Scientific, New Hampshire, USA). The DNA was stored at -20 °C until further analysis.

The molecular identity of the fungal isolate was determined using polymerase chain reaction (PCR) amplification and sequencing of the translation elongation factor (EF) 1-α coding region according to O´Donnell et al. (1998). The EF primers used were EF-1 and EF-2. The PCR comprised denaturation at 94 °C for 2 min, followed by 35 cycles of annealing at 94 °C for 30 s, 54 °C for 30 s and 72 °C for 1 min, and extension at 72 °C for 10 min. The sequences obtained were edited using BioEdit 7.0.5.3 software, and consensus sequences were analyzed using Molecular Evolutionary Genetics Analysis (MEGA X) software (Kumar et al. 2018). The similarity of the isolate nucleotide sequences was calculated using the BLAST algorithm (Basic Local Alignment Search Tool). Sequences from reference strains of Fusarium meridionale (NRRL28436) (Yli-Matilla et al. 2009; Laraba et al. 2021) available in GenBank were added to the analyses. Fusarium musarum (NRRL 28,507) was selected as the outgroup taxon. Phylogenetic analysis was performed using the maximum parsimony method with 1000 repetitions using MEGA X software (Kumar et al. 2018). A phylogenetic tree was constructed using the EF data. Sequences generated in this study were deposited in GenBank.

After seven days of incubation, fungal colonies produced large amounts of dense mycelia and red pigments (Fig. 1E). Macroconidia were abundant, falcate, 28.4–59.7 (x̅ = 45.3) × 3.2–5.7 (x̅ = 4.0) μm, and 4–5 septae (x̅ = 6.8) (n = 30) (Fig. 1F). Microconidia were not observed. Similarly Ceron-Bustamante et al. (2016) observed that macroconidia were falcate, (27 to 46 × 3 to 4 μm), with 4 to 5 septae. Koch's postulates were fulfilled on hop plants. Symptoms observed in the inoculated plants were foliar necrosis, and girdling of bines 45 days after inoculation. Development of fluffy white mycelia was sign of the pathogen. Symptoms were not observed in inoculated cones. The Fusarium isolate SVG00115-F was identical to F. meridionale (accession number NRRL28436), which was confirmed by phylogenetic analysis, with 95% bootstrap (Fig. 2). The sequence was deposited in GenBank (accession number OK669057).

Fig. 2
figure 2

Phylogenetic tree constructed by the Maximum Parsimony method showing genetic diversity of F. meridionale isolates based on the sequence analysis of the translation elongation factor (EF) 1 alpha sequence data. Scale bar represents genetic distance. The tree is rooted to Fusarium musarum (NRRL 28,507). Numbers below branches indicate the percentages with which the given branch was supported by bootstrap values (1000 replicates). T = ex-type strain

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Thus, morphological and molecular observations of this study indicate that F. meridionale is the causal agent of the symptoms observed in hop plants in Brazil. While F. sambucinum is the predominant Fusarium species associated with hop diseases, many Fusarium species have been found in hop worldwide (Pethybridge et al. 2001; Bienapfl et al. 2005; O’Neal et al., 2015). F. sambucinum has been reported in Brazil (Iwase et al. 2020), but was not identified in our samples. In Southern Brazil, F. meridionale has been reported to cause head blight in barley and wheat (Astolfi et al. 2011; Arruda et al. 2021), ear rot in maize (Machado et al. 2021), and is also associated with rice seeds (Gomes et al. 2015). However, this is the first report of it causing disease in hop. To the best of our knowledge, this is the first report of F. meridionale causing Fusarium canker in hop in Brazil. More studies are required to understand the potential economic impacts of this disease in Brazilian climatic conditions.