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First report of Fusarium meridionale causing canker in hop plants

Article in Australasian Plant Disease Notes · May 2022

DOI: 10.1007/s13314-022-00462-2

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Australasian Plant Disease Notes (2022) 17:13
https://doi.org/10.1007/s13314-022-00462-2

First report of Fusarium meridionale causing canker in hop plants

Felipe Augusto Moretti Ferreira Pinto1 · Leonardo Araujo1 · Camila Cristina Lage de Andrade2 ·
Mariana Mendes Fagherazzi3 · Alberto Fontanella Brighenti4 · Mariuccia Schlichting de Martin1 ·
Larissa Bitencourt Gomes2 · Juliane Fernandes2 · Valmir Duarte2 · Cristiano João Arioli1 · Vinícius Bizolo Sommer3

Received: 20 December 2021 / Accepted: 5 May 2022

© The Author(s) under exclusive licence to Australasian Plant Pathology Society Inc. 2022

Abstract
Fusarium canker in hop is caused by Fusarium spp. Its symptoms are wilting, cankers in the crown, foliar necrosis and
death of infected plants. Morphological and molecular observations were consistent with those previously reported for F.
meridionale. Koch’s postulates were fulfilled. To the best of our knowledge, this is the first report of F. meridionale causing
Fusarium canker in hop.

Keywords Humulus lupulus · Cannabaceae · Fusarium wilt · Hop diseases

Brazil is the third largest beer producer globally. However,
production of hops began less than five years ago. Humu-
lus lupulus is a creeping, herbaceous, perennial, and dioe-
cious 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
* Felipe Augusto Moretti Ferreira Pinto
felipepinto@epagri.sc.gov.br
1
Empresa de Pesquisa Agropecuária, Extensão Rural de Santa
Catarina (EPAGRI), Santa Catarina, Brazil
2
Agronômica Laboratório de Diagnóstico Fitossanitário e
Consultoria, Porto Alegre, RS, Brazil
3
Companhia de Bebidas das Américas (AMBEV), Lages, SC,
Brazil
4
Universidade Federal de Santa Catarina (UFSC),
Florianópolis, SC, Brazil
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.
Bines from the symptomatic H. lupulus ‘Comet’ were col-
lected 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 contain-
ing 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 sus-
pension (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 morphologi-
cal characteristics were identical to those of a Fusarium

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 13 Page 2 of 4
Fig. 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-
sambucinum species complex. Single conidia were trans-
ferred on PDA and Spezieller Nährstoffarmer agar (SNA)
media to obtain pure cultures, allowing identification to the
species level (Leslie and Summerrell 2006). Their morphol-
ogy 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 Nan-
oDrop 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 deter-
mined using polymerase chain reaction (PCR) amplifica-
tion 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
13
Australasian Plant Dis. Notes (2022) 17:13
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)
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 calcu-
lated 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 analy-
ses. 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 pro-
duced 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.
Australasian Plant Dis. Notes (2022) 17:13 Page 3 of 4 13

Fig. 2  Phylogenetic tree

constructed by the Maximum
Parsimony method showing
genetic diversity of F. meridi-
onale isolates based on the
sequence analysis of the transla-
tion 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 indi-
cate the percentages with which
the given branch was supported
by bootstrap values (1000 repli-
cates). T = ex-type strain

Similarly Ceron-Bustamante et al. (2016) observed that plants. Symptoms observed in the inoculated plants were
macroconidia were falcate, (27 to 46 × 3 to 4 μm), with foliar necrosis, and girdling of bines 45 days after inocu-
4 to 5 septae. Koch's postulates were fulfilled on hop lation. Development of fluffy white mycelia was sign of

13
 13 Page 4 of 4
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% boot-
strap (Fig. 2). The sequence was deposited in GenBank
(accession number OK669057).
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. sam-
bucinum 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 sam-
ples. 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.
Acknowledgements We are thankful to the Fundação de Amparo à
Pesquisa do Estado de Santa Catarina (FAPESC) for the financial sup-
port provided, Arthur Oliveira Souza and Iran Souza Oliveira for their
technical support and assistance.
Author contributions All authors contributed to the study conception
and design. Pathogenicity test was performed by FAMF Pinto and L
Araujo. FAMF Pinto, MM Fagherazzi, AF Brighenti, MMS Martin,
CJ Arioli and VB Sommer collected samples. Images edition were
performed by L Araujo and FAMF Pinto. Morphological characteris-
tics and molecular tests were performed by Camila Cristina Lage de
Andrade, Larissa Bitencourt Gomes, Juliane Fernandes and Valmir
Duarte. The first draft of the manuscript was written by FAMF Pinto
and all authors commented on previous versions of the manuscript. All
authors read and approved the final manuscript.
Declarations
Ethical approval This article does not contain any studies with human
participants or animals performed by any of the authors.
Informed consent Informed consent was obtained from all individual
participants included in the study.
Conflict of interest Felipe Augusto Moretti Ferreira Pinto declares that
he has no conflict of interest. Leonardo Araujo declares that he has no
conflict of interest. Camila Cristina Lage de Andrade declares that she
has no conflict of interest. Mariana Mendes Fagherazzi declares that
she has no conflict of interest. Alberto Fontanella Brighenti declares
that he has no conflict of interest. Mariuccia Schlichting de Martin
declares that she has no conflict of interest. Larissa Bitencourt Gomes
declares that she has no conflict of interest. Juliane Fernandes declares
that she has no conflict of interest. Valmir Duarte declares that he has
13
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Australasian Plant Dis. Notes (2022) 17:13
no conflict of interest. Cristiano João Arioli declares that he has no
conflict of interest. Vinícius Bizolo Sommer declares that he has no
conflict of interest.
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