© The Authors, 2025, Published by the Universidad del Zulia*Corresponding author: jverar12@unemi.edu.ec
Keywords:
Barcoding
CCN-51 cocoa
EF1-α
Phytopathology
ITS
Detection of Diaporthe sp. in cacao plants (cv. CCN 51) in Guayas Province, Ecuador
Detección de Diaporthe sp. en plantas de cacao (cv. CCN 51) en la Provincia de Guayas, Ecuador
Detecção de Diaporthe sp. em plantas de cacau (cv. CCN 51) na província de Guayas, Equador
José Humberto Vera-Rodríguez
1
*
Mónica del Rocío Villamar-Aveiga
1
Robinson J. Herrera-Feijoo
2
Jaime David Sevilla-Carrasco
1
Denny Moreno
1
Cesar Stalin Gavin-Moyano
3
Oscar Mauricio Chenche-López
1
Rev. Fac. Agron. (LUZ). 2025, 42(4): e254246
ISSN 2477-9407
DOI: https://doi.org/10.47280/RevFacAgron(LUZ).v42.n4.III
Crop production
Associate editor: Dra. Evelyn Pérez Pérez
University of Zulia, Faculty of Agronomy
Bolivarian Republic of Venezuela
1
Universidad Estatal de Milagro, Facultad Ciencias e
Ingeniería, Milagro, Guayas, Ecuador, 091050.
2
Universidad Técnica Estatal de Quevedo, Quevedo,
Ecuador, 120550.
3
Universidad Agraria del Ecuador, Extensión Ciudad
Universitaria “Dr. Jacobo Bucaram Ortíz” – Milagro,
Facultad de Ciencias Agrarias
Received: 26-04-2025
Accepted: 10-09-2025
Published: 08-10-2025
Abstract
Cacao cultivation contributes signicantly to the global
economy. However, a decline in production is evident due to the
presence of pathogens, especially within the fungi kingdom, where
some remain unidentied. The objective of this study was to
identify the presence of Diaporthe sp. in cacao plants of the CCN-
51 cultivar in Ecuador. Samples of cacao branches with symptoms
of rot and necrotic tissue were collected (cankers). The samples
were disinfected and processed in the Microbiology Laboratory of
the Milagro State University, Ecuador. Cambium fragments were
cultured on potato dextrose agar (PDA) and incubated at 27 °C.
After culture purication, morphological characterization and
molecular identication were performed using ITS and EF1-α
barcoding methods. The sequences were compared with the NCBI
GenBank database for validation. A phylogenetic analysis was
performed between the strains found and those reported in Puerto
Rico and Australia. Morphological identication placed the isolates
within the genus Diaporthe, which was conrmed molecularly.
Phylogenetic analysis demonstrates marked genetic diversity
among isolates within the genus Diaporthe. These ndings suggest
that Diaporthe spp. is prevalent in Ecuadorian cacao plantations
and that molecular methods are eective for its identication.
The presence of this pathogen implies the need for management
strategies to mitigate its impact on cacao production.
This scientic publication in digital format is a continuation of the Printed Review: Legal Deposit pp 196802ZU42, ISSN 0378-7818.
Rev. Fac. Agron. (LUZ). 2025, 42(4): e254246 October-December. ISSN 2477-9409.
2-7 |
Resumen
El cultivo de cacao contribuye signicativamente a la economía
mundial. Sin embargo, es evidente la disminución de su producción
a consecuencia de la presencia de agentes patógenos, en especial
dentro del reino fungi donde algunos de ellos no se encuentran
identicados. El objetivo del estudio fue identicar la presencia
de Diaporthe sp. en plantas de cacao del cultivar CCN-51 en
Ecuador. Se recolectaron muestras de ramas de cacao con síntomas
de pudrición y tejido necrótico (cancros). Las muestras fueron
desinfectadas y procesadas en el laboratorio de Microbiología de la
Universidad Estatal de Milagro, Ecuador. Fragmentos del cámbium
se cultivaron en agar papa dextrosa (PDA) e incubaron a 27 °C. Tras
la puricación de cultivos, se realizó una caracterización morfológica
y una identicación molecular mediante métodos de barcoding ITS y
EF1-α. Las secuencias se compararon con la base de datos del NCBI
GenBank para la validación. Se efectuó un análisis logenético entre
las cepas encontradas y las reportadas en Puerto Rico y Australia.
La identicación morfológica situó a los aislamientos dentro del
género Diaporthe, lo que fue conrmado molecularmente. El análisis
logenético demostró una marcada diversidad genética entre aislados
del género Diaporthe. Estos hallazgos sugieren que Diaporthe spp.
es prevalente en plantaciones de cacao en Ecuador y que los métodos
moleculares son ecaces para su identicación. La presencia de este
patógeno implica la necesidad de estrategias de manejo para mitigar
su impacto en la producción de cacao.
Palabras clave: barcoding, cacao CCN-51, EF1-α, topatología,
ITS.
Resumo
O cultivo do cacau contribui signicativamente para a economia
global. No entanto, um declínio na produção é evidente devido à
presença de patógenos, especialmente dentro do reino dos fungos,
onde alguns permanecem não identicados. O objetivo deste estudo foi
identicar a presença de Diaporthe sp. em plantas de cacau da cultivar
CCN-51 no Equador. Amostras de ramos de cacau com sintomas de
podridão e tecido necrótico foram coletadas (cancros). As amostras
foram desinfetadas e processadas no Laboratório de Microbiologia da
Universidade Estadual de Milagro, Equador. Fragmentos de câmbio
foram cultivados em ágar batata dextrose (PDA) e incubados a 27
°C. Após a puricação da cultura, a caracterização morfológica e a
identicação molecular foram realizadas usando os métodos de código
de barras ITS e EF1-α. As sequências foram comparadas com o banco
de dados GenBank do NCBI para validação. Uma análise logenética
foi realizada entre as cepas encontradas e aquelas relatadas em Porto
Rico e Austrália. A identicação morfológica situou os isolados
dentro do gênero Diaporthe, o que foi conrmado molecularmente.
A análise logenética demonstra acentuada diversidade genética com
alta similaridade dentro do gênero Diaporthe. Esses achados sugerem
que Diaporthe spp. é prevalente nas plantações de cacau equatorianas
e que métodos moleculares são ecazes para sua identicação. A
presença desse patógeno implica a necessidade de estratégias de
manejo para mitigar seu impacto na produção de cacau.
Palavras-chave: código de barras, cacau CCN-51, EF1-α,
topatologia, ITS.
Introduction
Cocoa (Theobroma cacao L.), native to tropical America, has
played an essential role in various cultures throughout history, prized
for its many culinary, medicinal, and economic uses (Vera-Rodríguez
et al., 2021). Ecuador, in particular, has experienced remarkable
growth in its cocoa production. By 2019, 25,435 hectares of crops
were registered, with an annual production of 283,680 tonnes,
positioning the country as the third largest producer in the world (Vera
et al., 2021). This increase is partly due to the decline in production
in the main African producing countries, such as Côte d’Ivoire and
Ghana, which has made Ecuador a preferred alternative for the global
market (Colombatti Moran et al., 2024). The renowned quality and
ne aroma of its cocoa, together with a solid international reputation,
give Ecuador a signicant comparative advantage over other markets
(Córdova Durán, 2025).
Despite this, the incidence of disease in cocoa crops is a growing
concern globally, posing a signicant threat to the economy and
agricultural sustainability (de Novais et al., 2023; Cobos Mora et al.,
2024). Fungal diseases can devastate entire crops, drastically reducing
production and aecting cocoa quality, with direct repercussions on
the chocolate industry (Fernández Muñoz et al., 2022). Furthermore,
the prevalence of these diseases not only compromises the economic
viability of farmers, but also threatens the livelihoods of families who
depend on cocoa as their main source of income (Ramón Guanuche
et al., 2024). Recent research highlights the vulnerability of cocoa to
a wide range of rapidly spreading diseases with devastating eects,
underscoring the urgency of eective management measures (Rêgo
et al., 2023).
The impact of fungi and oomycetes on cocoa cultivation
is a signicant challenge faced by cocoa producers worldwide
(Perrine-Walker, 2020). Pathogens such as Moniliophthora
roreri, Moniliophthora perniciosa, Lasiodiplodia theobromae,
and Phytophthora palmivora cause devastating diseases such as
moniliasis, witches’ broom, pod rot, and plant rot, respectively, which
can drastically reduce harvests in a matter of weeks (Delgado-Ospina
et al., 2021; Chóez-Guaranda et al., 2023; Puig, 2023). These diseases
deteriorate the quality and quantity of cocoa produced, negatively
impacting the economy of producers and the chocolate industry
(Rodríguez Velázquez et al., 2024).
The spread of fungal diseases in cocoa crops is intensied
by factors such as climate change, deforestation, and the lack of
sustainable agricultural practices (Delgado-Ospina et al., 2021).
Often, the genetic resistance of cocoa varieties is insucient to
counteract the rapid evolution of pathogenic fungi (Ruiz-Chután
et al., 2024). Therefore, addressing these challenges requires a
comprehensive approach that includes the development of resistant
varieties, the implementation of sustainable agricultural practices,
and cooperation between producers, governments, and international
organisations (Ramos et al., 2024; Sousa et al., 2024).
In recent years, new fungi have been identied that pose a
signicant threat to cocoa production, intensifying concern among
producers, researchers, and the industry in general (Debnath et al.,
2023; Chávez et al., 2024). The identication in Puerto Rico of
Diaporthe tulliensis and Diaporthe pseudomangiferae as causal
agents of pod rot in cocoa underscores the importance of recognising
and understanding new pathogens. This nding is the rst report of
these species aecting cocoa, highlighting the urgent need to develop
eective control measures (Serrato-Diaz et al., 2022).
This scientic publication in digital format is a continuation of the Printed Review: Legal Deposit pp 196802ZU42, ISSN 0378-7818.
Vera-Rodríguez et al. Rev. Fac. Agron. (LUZ). 2025, 42(4): e254246
3-7 |
Four plants with these symptoms were randomly selected from
each farm, and four samples of branches showing signs of necrosis
were taken from each plant.
The samples were processed in the Microbiology Laboratory
of the Biotechnology Department of the Faculty of Science and
Engineering at the Universidad Estatal de Milagro (UNEMI) in
Ecuador. Following the suggestions of Jiménez et al. (2022), the
samples were washed with neutral soap and rinsed with distilled water,
then disinfected by immersing them in 1 % sodium hypochlorite
for 1 minute, followed by washing with distilled water. Next, with
the aid of a scalpel, the branches were cut transversely into pieces
of approximately 5 cm, placed in 70 % ethanol for 1 min and then
in the safety cabinet (BIOBASE model FH1200(X), China) until
processing. Small fragments near the necrotic tissue were then cut,
the bark was removed, and a cut of approximately 1 cm² was made
in the cambium. The plant tissue samples were individually seeded
in the centre of Petri dishes with potato dextrose agar (TM MEDIA)
in triplicate and incubated at 27 °C for eight days. The cultures were
then puried by transplanting hyphae with the microbiological loop
into four new plates with PDA (TM MEDIA) and incubated at 27 °C
for 14 days.
Subsequently, a macroscopic morphological characterisation of
the puried fungal cultures was carried out to identify characteristics
such as shape, colour, and texture. Microscopic morphological
characterisation was performed using lactophenol blue staining
and observation of the samples under a trinocular microscope (40x
objective, Motic™, model Panthera S., China). Given the marked
uniformity in the macroscopic characteristics observed in the isolated
fungal colonies, 25 % of the isolates from each farm were selected for
molecular identication.
Molecular typing of the fungal isolates was performed using
barcoding techniques based on ITS and EF1-α markers. DNA
extraction was performed using the commercial Mini Genomic DNA
FUNGUS kit (Biotium
®
), following the manufacturer’s instructions,
The genus Diaporthe is now considered paraphyletic according
to Gao et al. (2017), the asexual state formerly known as Phomopsis
(Zhu et al., 2023), includes pathogenic fungi that aect a wide variety
of plants, including agricultural crops, fruit trees, and ornamental
plants, with a considerable impact on agricultural production and
ecosystem health (Mena et al., 2023). Several species of Diaporthe
cause symptoms such as leaf spots, wilting, root rot, and branch
dieback, which can have devastating consequences for the yield and
health of host plants (Sánchez et al., 2015). These fungi play a variety
of ecological roles, acting as pathogens, endophytes, or saprophytes,
which underscores the importance of studying and understanding
them for eective management (Thompson et al., 2015; Liu et al.,
2024).
Cocoa producers face the dicult task of protecting their crops
from fungal diseases, unaware of the presence of new pathogenic
fungi that threaten them, while seeking to maintain the protability of
their business. The study aimed to identify the presence of the fungus
Diaporthe sp. in CCN-51 cocoa plants from crops in Ecuador.
Materials and methods
The research was carried out in the Hermanos Quito area
(2°11’45.468” S, 79°18’36.486” W, 105 m.a.s.l), Lorenzo de Garaicoa
Parish, Simón Bolívar Canton, northeast of the Province of Guayas,
Ecuador, during the summer season (September, 2024). Three farms
(A, B and C) with CCN-51 cocoa plantations showing symptoms of
branch canker (Figure 1) were taken as a reference. The 10-year-old
plants were planted in loamy soil at a planting distance of 3 x 3 m
between plants and rows. The farms have a subfoliar irrigation system.
According to reports from producers, the plantations have suered
a loss of fruiting of around 40 % caused by these symptoms in the
plants. The aected plants show generalised necrosis on branches
(Figure 1: A1, B1, C1), cankers on branches (Figure 1: A2, B2, C2)
and rot at the base of the trunk (Figure 1: A3, B3, C3).
Figure 1. A1, B1, C1) Generalised necrosis in branches; A2, B2, C2) Cankers in branches; A3, B3, C3) Rot at the base of the trunk.
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4-7 |
from approximately 100 mg of mycelium scrapings from each fungal
sample (Barh et al., 2023).
DNA integrity and quality were assessed using microvolume
spectrophotometry with NanoDrop (Thermo Scientic ND 2000C,
USA) and 1 % agarose gel electrophoresis. The DNA was then
diluted to a concentration of 20 ng.µL
-1
and used for polymerase
chain reaction (PCR) amplication, using primers from Macrogen
and BlasTaq™ ITS1/ITS4 and EF1-983F/EF1-2218R, respectively
(Rehner and Buckley, 2005; White et al., 1990). The PCR conditions
were as follows: initial denaturation (98 °C, 1 min), followed by 40
cycles of denaturation (94 °C, 30 s), annealing (60 °C for ITS and 65
°C for EF1-α, 15 s), and extension (72 °C, 1 min). Finally, the nal
extension (72 °C, 10 min) and maintenance at 8 °C were performed.
The nal reaction volume was (20 µL). Subsequently, the amplied
products were sequenced in the 5’ → 3’ direction using the Sanger
method, using the same primers used for the amplication of each
gene.
The resulting sequences were cleaned and assembled using
Geneious bioinformatics software version 11.1.2. Finally, the
sequences assembled and aligned by BLAST were compared with
the NCBI GenBank nucleotide database to determine the taxonomic
identication of the isolates.
Partial sequences obtained from the ITS and EF1-α genes under
study were used to design the phylogenetic tree, as well as strains
referenced by Thompson et al. (2015) and Serrato-Diaz et al. (2022)
deposited in GenBank, which were individually aligned using the
MUSCLE algorithm with the MEGA X programme, ensuring accurate
alignment. Subsequently, the aligned sequences of both regions were
concatenated in FASTA format, joining the homologous sequences by
strain and checking their correct correspondence by code.
Phylogenetic analysis was performed in this same software using
the Maximum Likelihood method under the Tamura-Nei substitution
model with 1000 bootstrap replicates, measuring evolutionary
distances as the number of substitutions per site. Finally, the Tree
Explorer tool in MEGA X was used to visualise and edit the tree,
adjusting labels, topology and distances, highlighting strains of
interest through their respective codes to facilitate their identication
in the comparative analysis.
Results and discussion
Morphological characterisation
Macroscopic morphological characterisation of randomly selected
colonies of the strains revealed irregular edges and a soft biomass
with white cottony growth (Figure 2).
Figure 3. Macroscopic morphology of Diaporthe spp. isolation
after 30 days of growth on PDA. View of the plate: A
(top) growth of the fungal colony, B (bottom) formation
of reproductive structures.
Figure 4 shows the microscopic structures of the fungus,
highlighting the presence of mycelium with its septate and hyaline
hyphae (Figure 4A). Its conidiophores (Figure 4B) and light-coloured
cylindrical conidia (Figure 4C) can also be seen, with characteristics
that conrm the genus Diaporthe sp., similar to the ndings of Gao
et al. (2017).
Figure 2. Macroscopic morphology of Diaporthe spp. isolates
after 14 days of growth on PDA. H630, H767, H768: code
of isolates corresponding to a dierent farm.
Conidiophores (small black pustules) were observed after 30
days, characteristic of the asexual reproductive phase of the fungus
for spore release (Figure 3B). Findings consistent with morphological
characteristics described by Santos et al. (2011) and Vidić et al. (2011).
Figure 4. Microscopic morphology of the H630 Diaporthe spp.
isolate after 30 days of growth. a (fungal mycelium
and hyphae), b (conidiophores), c (conidiospores).
Motic Images Plus 3.0 software. Images captured at 40x
magnication.
In this context, recent research has delved deeper into the study
of the Diaporthe genus. For example, Long et al. (2019) highlighted
the use of morphological data and molecular data using key genes for
the classication of species within this genus. Furthermore, Wang et
al. (2021) reported the identication of 52 new species of Diaporthe
that infect a variety of plants, acting as endophytes, saprophytes, or
pathogens. This nding is supported by morphological, phylogenetic,
and molecular evidence, as conrmed by Perera et al. (2018).
Molecular identication
For molecular characterisation, high-quality DNA (A260/A280
~2.05) was extracted, allowing ecient amplication, with bands of
approximately 650 bp for the ITS marker and 1,200 bp for the EF1-α
marker (Figure 5).
Table 1 shows a summary of the results of the molecular
identication of the fungal isolates. The sequences obtained from
Sanger sequencing conrmed the presence of the following species
in the samples with a high level of identity: H630, H767, and H768.
Accession No. OP753556.1; OP698111.1 under fragment EF1-α (D.
longicolla); H630 Accession No. NR_147535.1 for the ITS fragment
(D. miriciae); H767 and H768 Accession No. MF070235.1 by means
of the ITS gene (D. ueckeri).
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5-7 |
Figure 5. 1% agarose gel with PCR products for ITS and EF1-α
fragments. MW: Molecular weight marker, NC: ITS negative control,
NC*: EF1-α negative control. bp = base pairs. (A = H630; B = H767
and C = H768): Code for Diaporthe spp. isolates.
The identication of isolates as D. longicolla with the EF-α gene
indicates that this fungus could be the causal agent of the symptoms
observed in cocoa. Many fungi of the genus Diaporthe are known
phytopathogens that cause rot in a wide range of hosts. In fact, recent
studies by Sun et al. (2021) reported the rst case in China of D.
longicolla causing leaf spots on Kalanchoe pinnata, a medicinal
plant. Meanwhile, Mena et al. (2024) reported the rst case of D.
miriciae as the cause of soybean stem canker in Uruguay. Dos Santos
et al. (2024) presented the rst report of soybean seed rot caused by
D. ueckeri in Brazil, while Guo et al. (2024) reported the rst case in
China of brown spot caused by D. ueckeri in bottle palms (Hyophorbe
lagenicaulis).
Phylogenetic analysis
The phylogenetic tree shown in Figure 6 shows the evolutionary
relationships between dierent species of the genus Diaporthe
reported (*) by Thompson et al. (2015) and Serrato-Diaz et al. (2022),
and the sequences with which they showed the greatest identity
when performing the Blast of the strains under study (OBJ), using
concatenated data from the ITS and EF1-α regions, and rooted using
the species Diaporthella corylina, reported in the study by Wang et
al. (2021), as an outgroup.
The isolates from the study were grouped into three dierent
species: two sequences as D. miriciae (OBJ1 and OBJ2), one as D.
ueckeri (OBJ3) and another as D. longicolla (OBJ4). In all cases, the
OBJ sequences were positioned within the clades corresponding to
the reference species deposited in GenBank, with high support values
(≥ 97 %), conrming their correct identication.
In particular, the D. longicolla clade showed a bootstrap support
of 99 %, clearly grouping isolate OBJ4 with reference sequences
KC343004.1 and OP753556.1. Similarly, isolate OBJ3 was grouped
with D. ueckeri (MF070235.1) with 97 % support, while isolates
OBJ1 and OBJ2 were grouped with D. miriciae (KJ197283.1 and
NR_147535.1), also with 97 % support. These results demonstrate
the consistency between the sequences obtained in this study and
those reported, ensuring the reliability of species-level identication.
Table 1. Summary of molecular identication results for Diaporthe spp. isolates from CCN-51 cocoa plants showing disease symptoms
in Hermanos Quito, Lorenzo de Garaicoa Parish, Simón Bolívar Canton, northeast of Guayas Province.
Code
DNA quality (%) Organism Fragment Identity (%) Nº Accession Coverage (%) E-value
H630
100 Diaporthe longicolla EF1-α 98.41
OP753556.1 100 0.0
100 Diaporthe miriciae ITS 99.26
NR_147535.1 100 0.0
H767
89.5 Diaporthe longicolla EF1-α 96.98
OP753556.1 100 0.0
95.2 Diaporthe ueckeri ITS 99.38
MF070235.1 100 0.0
H768
100 Diaporthe longicolla EF1-α 98.45
OP698111.1 100 0.0
100 Diaporthe ueckeri ITS 99.82
MF070235.1 100 0.0
Figure 6. Phylogenetic tree based on partial sequences of the
ITS and EF1-α genes with the highest identity when
performing the Blast of the strains under study (OBJ)
and strains reported (*) by Thompson et al. (2015),
Serrato-Diaz et al. (2022) and Wang et al. (2021).
The identication of the genus Diaporthe as a pathogen allows
for a better understanding of the aetiology of diseases that could
previously be attributed to other factors or pathogen complexes (Mena
et al., 2023). This knowledge is crucial for developing more eective
and targeted management strategies, including the implementation
of preventive measures, the development of resistant cultivars, and
the timely application of specic phytosanitary treatments (Chávez
et al., 2024). Diaporthe’s ability to cause a variety of symptoms in
dierent parts of the plant, such as cankers, leaf spots, and fruit rot
(Patiño-Moscoso et al., 2023), underscores the need for rigorous
phytosanitary surveillance to prevent signicant economic losses in
agriculture.
This scientic publication in digital format is a continuation of the Printed Review: Legal Deposit pp 196802ZU42, ISSN 0378-7818.
Rev. Fac. Agron. (LUZ). 2025, 42(4): e254246 October-December. ISSN 2477-9409.
6-7 |
Conclusions
The macro and micro-morphological characteristics observed
in the colonies allowed the fungal isolates to be identied within
the genus Diaporthe, which was conrmed molecularly. Likewise,
phylogenetic analysis of the strains found versus those reported in
Australia and Puerto Rico shows marked genetic diversity; although
they are grouped separately, they maintain a high similarity within the
genus Diaporthe.
Further studies are recommended to explore the pathogenicity
of these species and their interaction with dierent cocoa cultivars,
facilitating early detection and rapid response to new outbreaks.
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