Tropical Plant Pathology (2023) 48:97–103

https://doi.org/10.1007/s40858-022-00543-8

SHORT COMMUNICATION

Nucleic acid extraction and multiplex analysis for simultaneous

detection of the corn stunt complex pathogens in plant and insect
tissues
Eduardo Silva Gorayeb1 · Matheus Rodrigues Magalhães Albuquerque1 · Jacson Ferreira2 ·
Samara Campos do Nascimento1 · Thiago da Silva da Silva1 · Daian Marcos Savaris2 · Leandro Prado Ribeiro2 ·
Maria Cristina Canale2 · Fábio Nascimento da Silva1

Received: 2 June 2022 / Accepted: 10 November 2022 / Published online: 28 November 2022
© The Author(s), under exclusive license to Sociedade Brasileira de Fitopatologia 2022

Abstract
The corn stunting complex (CSC), composed of two Mollicutes (phytoplasma and spiroplasma) and one marafivirus (maize
rayado fino virus, MRFV), is the most important phytosanitary problem of maize crops in Brazil. Currently, MRFV detection
is performed separately from mollicutes, which contributes to the omission of virus detection in CSC monitoring activities
and basic studies. Here, a complete diagnostic method for simultaneous detection of CSC pathogens is presented, comprising
the optimization of an RNA/DNA extraction protocol and a triplex PCR, in which a newly designed MRFV-specific primer
pair was included along with the widely used R16F2n/R2 and CSSF2/CSSR6 primers for the mollicutes. As a result, a newly
designed MRFV-specific primer targeting the 5′ end of the polyprotein gene was developed and efficiently amplified MRFV
from different geographical locations and tissues (plant and insect) in the multiplex reaction. The designed MRFV primer
pair was species-specific, sensitive, and did not form secondary structures or dimerize with each other, nor with the other
primers used to detect the mollicutes. This method proved to be effective, cheaper, faster, and easier to perform than other
methods previously reported. Thus, it is a helpful tool for both basic and applied studies as well as monitoring programs
aiming to understand the MRFV presence in the CSC and maize crops.

Keywords Corn stunt spiroplasma · Maize bushy stunt phytoplasma · Molecular virus-detection · Plant disease
monitoring · Triplex PCR

The corn stunt is one of the most important diseases affect-
ing maize in the American Continent. It is associated with
a complex of three pathogens: the maize bushy stunt phyto-
plasma (MBSP) (‘Candidatus Phytoplasma asteris’), corn
stunt spiroplasma (CSS) (Spiroplasma kunkelii), and maize
rayado fino virus (MRFV) (Maize rayado fino virus) (Nault
* Maria Cristina Canale
cristinacanale@epagri.sc.gov.br
* Fábio Nascimento da Silva
fabio.silva@udesc.br
1
Departament of Agronomy, Universidade do Estado de
Santa Catarina, Luís de Camões 2090, Conta Dinheiro,
88.520‑000, Lages, SC, Brazil
2
Research Center for Family Agriculture, Agricultural
Research and Rural Extension Company of Santa Catarina
(CEPAF/EPAGRI), Ferdinando Tusset – São Cristóvão,
89.801‑970, Chapecó, SC, Brazil
et al. 1980; Hammond and Bedendo 2005; Orlovskis et al.
2017).
Maize bushy stunt phytoplasma is a wall-less pleo-
morphic prokaryote, classified in the the16S rRNA gene
group 16SrI, subgroup I-B (Lee et al. 2004). It has a cir-
cular genome of 576,118 base pairs (bp), containing 531
genes, similar to that of endosymbiotic bacteria (Orlovskis
et al. 2017). The corn stunt spiroplasma is a characteristi-
cally helical motile bacterium. It is a wall-less prokaryote
with an internal cytoskeleton, which is composed of fibrils
located under the cell membrane. Its genome has been fully
sequenced and consists of a 1,463,926 bp circular chromo-
some and four plasmids (Davis et al. 2015).
Maize rayado fino virus is the type species of the Marafi-
virus genus (Family Tymoviridae), along with ten other spe-
cies (ICTV 2020). It is a phloem-restricted virus, possessing
an icosahedral particle of approximately 30 nm in diam-
eter, containing a single 6336 bp long positive-sense RNA

13
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98
genome (Edwards and Weiland 2011; ICTV 2020; Mlotshwa
et al. 2020).
All CSC pathogens are obligate parasites, being, in the
plant, restricted to the phloem tissues, and transmitted by
corn leafhopper [Dalbulus maidis (Delong and Wolcott
1923) (Hemiptera: Cicadellidae)] in a circulative-propaga-
tive manner (Özbek et al. 2003; Massola Junior 2004).
Infected plants develop symptoms of shortening of the
internodes, over-production of ears, sterility of tassels, low
grain formation, short-grain cobs, root tipping, chlorotic
streaks between leaf veins, and leaf reddening (Henríquez
et al. 1999).
In Brazil, the adoption of a second crop season during the
1980s reduced the interval between cultivations favoring the
permanence of the corn leafhopper and, consequently, the
corn stunt complex (CSC) occurrence in the field (Oliveira
et al. 2002). Since then, several outbreaks have been
observed in different Brazilian states, such as Goiás, Minas
Gerais, Rio Grande do Sul, Santa Catarina, and Bahia, gen-
erating up to 100% losses in some areas (Sabato 2017).
Currently, CSC is mainly managed through insecticide
usage, targeting the leafhopper vector through seed treat-
ment and post-emergence spraying (Sabato 2017). However,
since the 2017/2018 season, the constant presence of the
corn leafhopper in Brazilian maize cultivations has resulted
in a significant increase in the usage of insecticides (around
75%), which has increased production costs and can contrib-
ute to reduce pesticide effectiveness, as well as, the contami-
nation of maize kernels (Jones and Medina 2020).
One possible solution for this problem is in the associa-
tion between insecticide applications and CSC monitoring
programs, which involve the molecular detection of patho-
gens in plants and/or insects, and consequently, help grow-
ers plan their actions in the field (Carloni et al. 2013). In
addition, pest monitoring can reveal the distribution of D.
maidis and CSC pathogens among regions, which helps to
focus management strategies in the most affected regions,
combining different control methods, such as the elimination
of voluntary plants, sanitary empty (a period without maize
cultivation), the usage of tolerant cultivars, and chemical or
biological insect control.
However, monitoring activities are mainly focused on
mollicute detection since different nucleic acid extraction
and PCR methods are used to detect MRFV, given its genetic
material’s RNA genome (Prince et al. 1993; Hammond et al.
1997; Marzachi et al. 1998; Barros et al. 2001; Gomes et al.
2004; Gonçalves et al. 2007; Galvão et al. 2021).
Although studies showed the presence of MRFV along
with the mollicutes causing the corn stunt (Hammond and
Bedendo 2001), the full contribution of the virus to CSC is
still not completely elucidated. However, this virus infec-
tion can generate symptoms of long chlorotic dotted stripes
along the leaf veins generally between 7 and 14 days after
13
Tropical Plant Pathology (2023) 48:97–103
inoculation, and yield losses ranging from 10 to 100%,
depending on genotype susceptibility, insect vector inci-
dence, and climate conditions (Vásquez and Mora 2007).
While the mollicutes are widespread in Brazil, MRFV
was reported in the states of São Paulo, Minas Gerais, Par-
aná, and more recently in Santa Catarina (CABI and EPPO
1997; Hammond and Bedendo 2005; Gonçalves et al. 2007;
Albuquerque et al. 2022). As the three pathogens are trans-
mitted by the same vector, it is expected that MRFV is pre-
sent in other CSC-affected maize production regions.
In the present study, a faster and easier diagnostic method
than those previously used was developed, requiring only
one nucleic acid extraction procedure and one triplex PCR to
detect the mollicutes, unlike what was being used, in which
two nucleic acid extractions and two PCRs were necessary to
detect the same three pathogens. For detecting the three CSC
pathogens in a single end-point multiplex PCR, an DNA/
RNA extraction protocol was adapted, and new primer pair
targeting MRFV was designed and tested in combination
with the already widely used R16F2n/R2 (Gundersen and
Lee 1996) and CSSF2/CSSR6 (Barros et al. 2001) primer
pairs.
The simultaneous extraction of DNA and RNA of all
samples was performed using a CTAB protocol adapted
from Marzachi et al. (1998) (detailed in supplementary File
1). The extracted nucleic acid's concentration and quality
were assessed through spectrophotometry (NanoDrop®
2000, Thermo Fisher Scientific) and agarose gel electro-
phoresis (1%).
In order to design a new pair of MRFV-specific prim-
ers, the two MRFV complete sequences (AF2655566 and
KM523134) available in GenBank (https://​www.​ncbi.​nlm.​
nih.​gov/​genba​nk/) were downloaded and aligned using the
muscle tool available in MEGA X software (Kumar et al.
2018). A unique consensus sequence was generated and
used as a template for designing primer pairs with Primer3
software (v. 0.4.0) (Koressaar and Remm 2007; Untergasser
et al. 2012) following the recommendations proposed by
Hyndman and Mitsuhashi (2003). The occurrence of second-
ary structures and primer dimerization (also including het-
erodimer analyses with R16F2n/R16R2 and CSSF2/CSSR6
primer pairs), as well as primer specificity, were evaluated in
silico with the IDT oligo analyzer (https://​www.​idtdna.​com/​
pages/t​ ools/o​ ligoa​ nalyz​ er) and the blastn tools (https://b​ last.​
ncbi.​nlm.​nih.​gov), respectively.
The best pair of primers obtained from in silico analy-
ses were denominated MRFVAF (Forward, 5′ TCG​CAC​
TCT​TCT​TCG​TTG​ 3′) and MRFVAR (Reverse, 5′ GTC​
TCG​ATG​GTC​TTG​TGG​A 3′). The primers aligned at the
5′ end of the ORF1 and the 5′ end of ORF2, resulting in
a fragment of ~ 274 bp of a region that encodes methyl-
transferase. Both primers presented similar melting tem-
peratures and acceptable GC content (50 and 52.6%, for
Tropical Plant Pathology (2023) 48:97–103 99

MRFVAF and MRFVAR, respectively). Secondary struc-
ture analysis revealed that primers are not likely to form
hairpins at temperatures above 25.8 °C. The potential for
homodimer formation was low, with changes of Gibbs free
energy of bonding (ΔG) of − 3.61 and − 6.76 for MRFVAF
and MRFVAR, respectively. The potential for heterodimer
formation with ΔG below − 9 was not found among the six
primers used in the multiplex analysis, and blastn analysis
showed that except for the complete MRFV sequences cur-
rently available, the primers designed did not hybridize to
any distinct type of pathogen or any sequence of the maize
genome, confirming in silico tests.
The MRFV-specific primer pair was tested in vitro
using an MRFV-positive sample [detected using MRFV-
09/MRFV-10 primers (Hammond et al. 1997)] from Pin-
hão, Paraná, Brazil (25°41′54.2″S, 51°39′30.6″W). First,
complementary DNA (cDNA) was synthesized using
the MMLV reverse transcriptase enzyme (Promega),
with approximately 0.5–2 ug of nucleic acid from each
extracted sample as a template, following the manufac-
turer’s instructions and the newly designed MRFV reverse
primer (MRFVAR). The obtained cDNA was used as a
template for PCR with the enzyme GoTaq® Flexi DNA
Polymerase (Promega), following the manufacturer’s
recommendations.
For testing primer analytical sensitivity, the cDNA of the
MRFV-positive sample was subjected to serial dilutions,
decreasing by one order of magnitude from 10 to 1­ 0−6 ng/
µL, and used as a template for PCR. Clear bands were
observed in all dilutions, detecting the presence of MRFV
cDNA down to a concentration of 13 pg/µL (Fig. 1A).
The best annealing temperature was assessed with a PCR
using the following thermocycler (Veriti™, Thermo Fisher)
conditions: 94 °C for 30 s; 35 cycles of 94 °C for 15 s, 50,
52, 54, 56, 58, or 60 °C for 15 s, and 72 °C for 20 s, followed
by a final extension at 72 °C for 5 min. The PCR products
were analyzed on 2% agarose gels and stained with SYBR
Safe™ (Invitrogen).
Clear bands were observed in all temperatures, but
slightly unspecific amplification was seen in reactions using
higher annealing temperatures (54–60 °C) (Fig. 1B). Given
this result, the temperature of 52 °C was chosen for primer
validation and multiplex analyses, which is also closest to
the optimal annealing temperature calculated for each primer
(51 and 51.3 for MRFVAF and MRFVAR, respectively)
(Rychlik et al. 1990).

Fig. 1  In vitro sensitivity test of

MRFVAF/MRFVAR primers
(A) and temperature gradient
PCR testing (B). Lanes 1 and
9: 1 kb DNA ladder (Promega).
Lanes 8 and 16: no template
control. Lanes 2–7: sensitivity
test of MRFV-specific primers
using an MRFV-infected sample
(extracted from a single maize
plant), with a cDNA (produced
with MRFVAR primer) concen-
tration gradient of 1300 ng/µL,
130 ng/µL, 13 ng/µL, 1.3 ng/
µL, 130 pg/µL, and 13 pg/µL,
respectively. Lanes 10–15: PCR
testing using an MRFV-infected
sample (extracted from a single
maize plant) with a temperature
gradient ranging from 50, 52,
54, 56, 58, to 60 °C, respec-
tively

13
100
All multiplex PCR testing were performed with 25 µL
reactions, using 7.5 units of the enzyme the GoTaq® Flexi
DNA Polymerase (Promega), 7.5 mM ­MgCl2 (Promega),
1.2 mM dNTP mix (Promega), R16F2n/R16R2, CSSF2/
CSSR6, and MRFVAF/MRFVAR primers (0.1 µM each), 1
µL of cDNA, and 2 µL of DNA of each sample.
An initial test was performed with three separate posi-
tive controls (previously tested using R16F2n/R2, CSSF6/
CSSR2, and MRFV09/MRFV10 primers). Two insect sam-
ples (pool of three insects), infected with CSS and MBSP/
MRFV, respectively, obtained in experimental colonies
from the Superior School of Agriculture Luiz de Quei-
roz, Piracicaba, SP, Brazil, and one MRFV-infected sam-
ple, from corn plants, obtained from Pinhão, PR, Brazil
(25°41′54.2″S, 51°39′30.6″W). Bands of approximately 274,
500, and 1250 bp were clearly observed for MRFV, CSS, and
MBSP, respectively (Fig. 2A). In order to confirm primer
functionality, positive samples were purified (Wizard®
SV Gel and PCR Clean-Up System, Promega) and sent for
Sanger sequencing (ACTGene, Alvorada, RS, Brazil). The
sequences obtained for MRFV (215 bp), MSBP (935 bp),
and CSS (466 bp) were compared with those in GenBank
using the blastn tool (https://​blast.​ncbi.​nlm.​nih.​gov), show-
ing 88.8%, 99.3%, and 100% of identity with MRFV (Gen-
Bank Accession N° AF2655566 and KM523134), Aster
yellows phytoplasma (GenBank Accession N° EU416200),
and CSS (GenBank Accession N° KX925443) isolates,
respectively. In addition, a triple-infected plant (previously
indexed using R16F2n/R2, CSSF6/CSSR2, and MRFV09/
MRFV10 primers), collected in a commercial area from
Campos Novos, SC, Brazil (27°22′00.1″S, 51°15′28.5″ W),
was subjected to a PCR using a temperature gradient ranging
from 50 to 60 °C in the annealing step, to verify the optimal
Fig. 2  In vitro separate testing of primers (A) and temperature gradi-
ent multiplex PCR testing (B). Lanes 1 and 6: 1 kb DNA ladder (Pro-
mega). Lanes 2 and 13: no template control. Lane 3: corn stunt spiro-
plasma-infected sample (extracted from a single maize plant). Lane 4:
maize bushy stunt phytoplasma and maize rayado fino virus (MRFV)-
13
Tropical Plant Pathology (2023) 48:97–103
PCR conditions for the reaction. Clear bands were observed
in all temperatures, but unspecific amplification was seen in
reactions using higher annealing temperatures (56–60 °C)
(Fig. 2B), which confirmed 52 °C as the annealing tempera-
ture for the multiplex reaction.
The standardized multiplex-PCR was validated with nine
plant and nine insect samples collected from commercial and
experimental areas. All plant samples were symptomatic,
exhibiting at least one typical symptom of CSC, whereas
insects were collected using yellow sticky traps installed
at the border of commercial maize plantations. In addition,
CSC-free samples (two originated from maize plants and two
from insects) were analyzed using the triplex PCR, in order
to test the possible formation of nonspecific amplifications.
Plants were obtained in the Brazilian municipalities
of Piracicaba, SP, (22°42′47.1″S, 47°37′37.6″W); Fran-
cisco Beltrão, PR, (26°05′57.7″S, 52°57′35.7″W); Pinhão,
PR, (25°44′28″S, 51°50′14″W); Caiapônia, GO, Brazil
(16°55′25.9″S 51°47′46.7″W); Mineiros, GO (17°33′24.5″S
52°35′59.8″W); Rio Verde, GO (two samples, 17°50′41.5″S
50°55′04.4″W); and Chapadão do Sul, MS (two samples,
18°49′08.8″S, 52°39′32.8″W).
Insect samples were obtained in the municipali-
ties of Piracicaba, SP, (three samples, 22°42′47.1″S,
47°37′37.6″W); Chapecó, SC (two samples, 27°04′24.3″S,
52°44′52.1″W); Morro da Fumaça, SC (two samples,
28°38′25.7″S, 49°14′50.6″W); Videira, SC (27°00′58.9″S,
51°05′00.3″W); and São Miguel do Oeste, SC (26°46′34.3″S,
53°30′33.8″W). The previously used triple-infected sam-
ple from Campos Novos, SC, Brazil (27°22′00.1″S,
51°15′28.5″W) was used as a positive control. Nucleic acid
extraction, cDNA synthesis, and PCR conditions were per-
formed as described earlier (using 52 °C for annealing).
infected sample (extracted from a sample composed of three Dalbu-
lus maidis adults). Lane 5: MRFV-infected sample (extracted from a
single maize plant). Lanes 7–12: PCR testing with the triple-infected
sample (extracted from a single maize plant), with temperature gradi-
ent ranging from 50 to 60 °C, changing 2 °C respectively
Tropical Plant Pathology (2023) 48:97–103
All tested samples resulted in clear positive bands with
the expected size for at least one CSC pathogen, giving
conclusive results for both insect and plant-borne samples.
MRFV was the most prevalent CSC pathogen, present in 16
out of 18 samples, followed by CSS in 12 out of the 18 sam-
ples tested, and MBSP, found in three samples (Fig. 3). The
differences in the size of the amplified bands correspond-
ing to the spiroplasma observed in samples 5 and 6 (Fig. 3)
may be related to a shift in the mobility of the amplified
DNA, already observed for SYBr safe, other SYBr products,
GelRed, and other gel staining dyes (Huang and Fu 2005;
Truman et al. 2022). In addition, no unspecific bands were
observed when the triplex PCR was performed with CSC-
free samples, regardless of whether they originated from
insects or plants (Supplementary file 2).
This study presents for the first time an innovative
protocol of a fast and sensitive technique for molecular
diagnosis of the three pathogens involved in the CSC
simultaneously. Herein, a complete method (from nucleic
acid extraction to pathogen detection) was developed
and adjusted, allowing researchers to extract RNA/DNA
from plant and insect samples rapidly, as well as to detect
MRFV, MBSP, and CSS in a single end-point multiplex
PCR (Fig. 3). The extraction protocol was efficient for
gathering enough nucleic acids from both D. maidis and
maize tissue samples, containing the parameters required
for performing cDNA synthesis and conventional PCR,
Fig. 3  Results of the primer validation test (geographic location
of each sample is in parenthesis). Lanes 1 and 13: 1 kb DNA lad-
der (Promega). Lanes 2 and 14: positive control (extracted from
maize plants). Lanes 3 and 15: no template control. Samples in
lanes 4, 5, 6 [Piracicaba, SP, Brazil (22°42′47.1″S, 47°37′37.6″W)],
7, 8 [Chapecó, SC, Brazil (27°04′24.3″S, 52°44′52.1″W)], 9, 10
[Morro da Fumaça, SC, Brazil (28°38′25.7″S, 49°14′50.6″W)],
11 [Videira, SC, Brazil (27°00′58.9″S, 51°05′00.3″W)], and 12
[São Miguel do Oeste, SC, Brazil (26°46′34.3"S, 53°30′33.8"W)]
101
which became evident with the results of the analyses pre-
sented in Figs. 1, 2, and 3. In addition, this protocol aims
to reduce extraction costs by avoiding virus-RNA extrac-
tion separately.
The insertion of a primer pair for MRFV detection in
the multiplex technique broadens the diagnostic process of
the CSC pathogens since the MBSP and CSS are already
detected simultaneously (Gomes et al. 2004; Albuquerque
et al. 2022). Therefore, it would make more sense to design
a third pair of primers corresponding to MRFV to be used
in combination with those already utilized than to design
new pairs for all pathogens. In addition, in silico analyses
of other MRFV-specific primers available in the literature
(Hammond et al. 1997; Mlotshwa et al. 2020) revealed high
probabilities of self-dimerization and heterodimer forma-
tion with the mollicutes-specific primers (data not shown).
Avoiding primer dimerization is crucial for performing good
PCR because when two primers interact, the efficiency of
hybridizing the target is significantly reduced (Hyndman and
Mitsuhashi 2003).
In this study, we successfully obtained a single primer
pair in which those undesirable features were not happen-
ing, and, in addition to the standardization of the method,
another factor of great importance was the absence of
primer-dimers and unspecific amplification in the multiplex
reaction (Figs. 2 and 3), thus evidencing the high precision
and efficiency of the presented multiplex technique.
were originated from insects. Samples in lanes 16 [Pinhão, PR,
Brazil (25°44′28″S, 51°50′14″W)], 17 [Piracicaba, SP, Brazil
(22°42′47.1″S, 47°37′37.6″W)], 18 [Francisco Beltrão, PR, Bra-
zil (26°05′57.7″S, 52°57′35.7″W)], 19 [Caiapônia, GO, Bra-
zil (16°55′25.9″S 51°47′46.7″W)], 20, 21 [Rio Verde, GO, Bra-
zil (17°50′41.5″S 50°55′04.4″W)], 22 [Mineiros, GO, Brazil
(17°33′24.5″S 52°35′59.8″W)], 23, and 24 [Chapadão do Sul, MS,
Brazil (18°49′08.8″S, 52°39′32.8″W)] were originated from maize
plants
13
102
The target region amplified by the primer corre-
sponds to the 5′ end of the viral polyprotein, in which
few sequences are available in GenBank, and no variabil-
ity data has been published yet. Because of this, samples
from different locations in Brazil were included to validate
primer robustness in broader surveys. Surprisingly, MRFV
was detected in 16 of 18 samples, showing the efficiency
of the primers designed in this study for MRFV isolates
from different regions (Fig. 3).
The implementation of a monitoring system of CSC in
corn plants and Dalbulus maidis will result in a more effi-
cient and durable control strategy since the most affected
areas will be revealed. Therefore, management strategies
can be strengthened in a given region, whereas areas with
lower incidences can reduce management activities and
costs, such as post-emergence insecticide application.
In addition, D. maidis monitoring during the off-season
can help guide cultural practices, such as removing vol-
untary plants, which can harbor CSC pathogens and the
leafhopper vector. Carloni et al. (2013) highlighted the
importance of monitoring D. maidis in Argentinian tem-
perate regions, principally in years with mild winters, in
order to assess the presence of leafhoppers and develop a
quick management response before the start of the season.
In summary, the present study showed a complete pro-
tocol for simultaneously detecting the three pathogens of
the corn stunting complex. Given its sensitivity, specific-
ity, and efficiency for both plant and insect samples, this
technique will be helpful in diagnosis, epidemiological
studies, and monitoring activities of the corn stunting
complex, improving CSC management in the field.
Supplementary Information The online version contains supplemen-
tary material available at https://d​ oi.o​ rg/1​ 0.1​ 007/s​ 40858-0​ 22-0​ 0543-8.
Author contribution Conceptualization: ESG, JF, TSS, DMS, LPR,
MCC, and FNS; methodology: ESG, JF, LPR, MCC, and FNS; formal
analysis and investigation: ESG, MRMA, JF, SCN, TSS, and DMS;
writing—original draft preparation: ESG and JF; writing—review and
editing: LPR, MCC, and FNS; funding acquisition: LPR, FNS, and
MCC; resources: FNS and MCC; supervision: FNS.
Funding This study was financed in part by the Coordenação de Aper-
feiçoamento de pessoal de Nível Superior, Brazil, (CAPES) [Finance
Code 001]. Financial support was received from Fundação de Amparo
à Pesquisa e inovação de Santa Catarina (FAPESC) [Programa moni-
tora Milho Santa Catarina (Project nº 2021TR1246)]. F.N.S. and L.P.R.
both receive CNPq (Conselho Nacional de Desenvolvimento Científico
e Tecnológico) fellowships.
Data availability All data generated or analyzed during this study are
included in this published article [and its supplementary information
files].
Declarations
Conflict of interest The authors declare no competing interests.
13
Tropical Plant Pathology (2023) 48:97–103
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