RESEARCH ARTICLE          

Multilocus identification and pathogenetic characterization of Colletotrichum endophyte and pathogen species isolated from cocoa leaves and pods (Theobroma cacao) in Ecuador

Fernando Espinoza-Lozano1 * ; Mirian Villavicencio-Vasquez1 ; Lizette Serrano1 ; Daynet Sosa1 ; Jonathan Coronel-León1, 2 ; Marcos Vera-Morales1, 3

1 Escuela Superior Politécnica del Litoral, ESPOL, Centro de Investigaciones Biotecnológicas del Ecuador, Campus Gustavo Galindo km 30.5 Vía Perimetral, P.O. Box 09-01-5863, Guayaquil, Ecuador.

2 Escuela Superior Politécnica del Litoral, ESPOL, Facultad de Ingeniería Mecánica y Ciencias de la Producción, Campus Gustavo Galindo km 30.5 Vía Perimetral, P.O. Box 09-01-5863, Guayaquil, Ecuador.

3 Universidad Politécnica Salesiana, UPS, Grupo de Investigación en Aplicaciones Biotecnologicas, GIAB, Carrera de Biotecnología, Campus María Auxiliadora, km 19.5 Vía a La Costa, Guayaquil, Ecuador.

* Corresponding author: rofeespi@espol.edu.ec (F. Espinoza-Lozano).

Received: 18 July 2024. Accepted: 7 February 2025. Published: 18 February 2025.

Abstract

Cacao cultivation is one of the main agricultural products of Ecuador, known internationally for its quality and aroma. However, it is affected by fungal diseases including Moniliophthora roreri, Moniliophthora perniciosa, Phytophthora spp., and Colletotrichum spp. The genus Colletotrichum spp. is known for its characteristics that complicate traditional taxonomic identification. In cacao cultivation, it is one of the most frequently found species as an endophyte of leaves and fruits, yet it is also reported to cause the disease known as anthracnose on leaves and fruits. The objective of this work was to identify at the species level 16 Colletotrichum isolates, 13 from healthy leaf endophytes and 3 from pods with symptoms, through multilocus analysis of the ITS1, 5.8S, and ITS2 region, and partial sequences of the TUB2 and GAPDH genes. Subsequently, their pathogenicity was evaluated by inoculating healthy cacao pods and measuring the damage caused. The 16 isolates were identified as follows: from the gloeosporioides complex, C. siamense 6, C. chrysophilum 6, C. theobromicola 2 and from the boninense complex, C. karstii 2. The most frequently found species were those that caused symptoms, especially C. siamense, to which the strains were isolated from symptomatic pods belonged. This work provides relevant and accurate information about the diversity of Colletotrichum species that colonize cocoa plantations and identifies which species might cause the disease known as anthracnose. Additionally, it allows for a more precise diagnosis and consequently better treatment.

Keywords: Anthracnose; Phylogenetic Analyze; Multi-locus; Endophyte; Pathogen.

DOI: https://doi.org/10.17268/sci.agropecu.2025.014

Cite this article:

Espinoza-Lozano, F., Villavicencio-Vasquez, M., Serrano, L., Sosa, D., Coronel-León, J., & Vera-Morales, M. (2025). Multilocus identification and pathogenetic characterization of Colletotrichum endophyte and pathogen species isolated from cocoa leaves and pods (Theobroma cacao) in Ecuador. Scientia Agropecuaria, 16(2), 167-177.

1. Introduction

Cocoa has been a fundamental crop worldwide, not only because of the chocolate industry but also due to its economic and social impact in producing countries. The main global producers are in Africa, with Ivory Coast and Ghana accounting for more than 60% of the world's production. In Latin America, Brazil, Colombia, Peru, and Ecuador stand out, mainly for the qualit y of their product (Soares & Oliveira, 2022)

The cocoa cultivation in Ecuador is considered one of the greatest economic importance for the country, in 2024 was exported 362,296 MT with a total of $2,787.2 million of dollars (Ministerio de Producción, Comercio Exterior, Inversiones y Pesca, 2025) in addition, the country is the world leader in the production and export of fine aroma cocoa with 70% of the world total production and is a livelihood for around one hundred thousand families (ProEcuador, 2019). This production is affected by diseases mainly of fungal origin such as witch´s broom, frosty pod rot (de Novais et al., 2023), in smaller quantity is found the fungi Colletotrichum that in pathogenic condition is found causing the disease known as anthracnose that can affect a number of crops of economic interest (Wijaya et al., 2023).

Many morphological and molecular studies of Colletotrichum have been carried out mainly because of the various characteristics it presents (Angeli et al., 2024; Asare et al., 2021), among them its adaptability that makes it easier for it to have a life as an endophyte and has been found in a large number of hosts without causing apparent damage, however, many authors have typecast the endophytes as inactive saprophytes (Ebadi et al., 2024; Whalley, 1996), latent pathogens (Stone et al., 2000) or mutualists (Herre et al., 2007; Mejía et al., 2008).

Endophytic fungi have the ability to stimulate the development of the host plant, enhance the activity of antioxidant defense enzymes, and induce the synthesis and storage of secondary metabolites (Xu et al., 2023). In cocoa cultivation, it has been shown that Colletotrichum, as an endophyte, provides protection to the plant by reducing the incidence of diseases caused by fungi, primarily (Tao et al., 2013; Yu et al., 2022).

Previously, identification through taxonomic tech-niques was very common; however, it presented inconsistencies as it heavily relied on the specific technique used (Cai et al., 2009; Tao et al., 2013), hence the importance of phylogenetic studies in this case the multi-locus analysis by the difficulty of performing a taxonomic identification by the morphological characteristics presented by Colletotrichum, In addition, the use of a single gene or part of it is very uninformative as is the case of the ITS region of rDNA. For this reason, it is very important to supplement the use of the ITS region with other genes or parts that are preserved but provide a variability for their use. In order to differentiate the isolated endophytes previously identified as Colletotrichum spp, it was considered the objective of inferring their genetic relationship based on a multi-locus phylogenetic analysis of 13 endophytes from T. cocoa leaves and 3 isolated strains of cocoa pods with disease symptoms by sequencing three genes (Beta Tubulin 2, Internal Transcribed Spaces, Gliceraldehyde-3-phosphate dehydrogenase) and relate their pathogenic or nonpathogenic capacity by inoculating healthy cobs with their identification, thus providing a guideline for the management of this disease..

2. Methodology

Origin of the strains

For this study, the endophytic strains were isolated from healthy leaves of the National type, cocoa variety, with more than 50 years of age located in the provinces of Guayas and Azuay (Table 1). Small fragments (2x2 cm) were washed with tap water and dried with sterile paper towels. Plant tissues were rinsed with 70% ethanol and 0.5% sodium hypochlorite for 2 minutes and washed with sterile distilled water three times. Eight fragments were placed in a 90 mm diameter Petri dish containing agar with 2% malt extract (Arnold et al., 2003) and incubated at 25 °C in the dark for 10 days. Colonies with different morphology were observed every two days, they were isolated and purified on potato-dextrose-agar. The pure endophytic fungal strains were kept in the Collection of Microorganism Cultures of the Ecuadorian Center for Biotech-nological Research (CCM-CIBE).

For the isolation of the pathogenic strains, the fruits with symptoms were collected, brought to the laboratory and followed the protocol of Arnold et al. (2003) for the planting of the plant material (Table 1), later the pure isolates were obtained and deposited in the (CCM-CIBE).

Table 1

Origin of the samples

DNA extraction, PCR, sequencing and identifying

The DNA was extracted from the fungal mycelium, obtained from pure cultures in DIFCO Potato Dex­trose Agar medium (PDA), following the Cenis pro­tocol (Cenis, 1992). The ITS 1, 5.8S, ITS 2 region was amplified by polymerase chain reaction (PCR), using the universal primers ITS-1F (5'-TCCGTAGGTGAACCTGCGG-3') (Gardes & Bruns, 1993) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) (White et al., 1990). The volume of the final reaction was 25 µl; containing the following mixture at final concentration: 1X buffer solution (Invitrogen), 0.2 mM dNTPs, 1.5 mM Mg2Cl, 0.4 µM of each primer, 0.5 U Taq polymerase per reaction (Invitrogen) and 2 µl of template DNA (10-50 ng). PCR reactions were carried out with an initial denaturation of 94 °C for 1 min followed by 30 cycles consisting of de­naturation at 94 °C for 1 min, annealing at 55 °C for 1 min, and an extension at 68 °C for 1 min; and a final extension of 68 °C for 3 min for extension. The PCR products were visualized in 2% agarose gel.

The amplified products were sequenced at the Interdisciplinary Center for Biotechnology Research at the University of Florida (ICBR). The quality of the sequences was analyzed with the FinchTV program Version 1.4.0 (http://www.geospiza.com/finchtv). The sequences obtained were compared with the existing information in the database of the gene bank of the National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/), using the BLAST searches and were aligned using the MEGA 6 program (Tamura et al., 2013).

Once the isolates were preliminary identified using the ITS gene, they were further analyzed using par­tial gene sequences of another two genomic loci: the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β tubulin 2 (TUB2) genes. The primers GDF1 (5'–GCCGTCAACGACCCCTTCATTGA-3') and GDR1 (5'– GGGTGGAGTCGTACTTGAGCATGT-3') were used to amplify and sequence the GAPDH (Guerber et al., 2003), and for TUB2 the primers Btub2Fd (5'-GTBCACCTYCARACCGGYCARTG-3´) and Btub4Rd (5'-CCRGAYTGRTCCRGAYTGRT) were used (Woudenberg et al., 2009).

The PCR conditions for GAPDH were an initial de­naturation at 94 °C for 4 min followed by 34 cycles consisting of 94 °C for 45 s, 60 °C for 45 s, and 72 °C for 1 min: a final step of 72 °C for 10 min. (Prihastuti et al., 2009). TUB2 PCR consisted of an initial denaturation at 94 °C for 5 min followed by 35 cycles of 94 °C for 30 s, 52 °C for 30 s, and 72 °C for 30 s, and extension at 72 °C for 7 min (Woudenberg et al., 2009).

The purified PCR products were sequenced, in both directions, by Macrogen Inc., Korea. The quality of the nucleotide sequences and the consensus assembly was carried out using Geneious version 2020.1.2. Then, the assembled sequences were compared to the NCBI database using BAST.

Phylogenetic analysis

The phylogenetic analysis included the 16 sequences from the isolates of this study and 62 sequences belonging to 47 species from gloeosporioides and 13 boninense complex, that were downloaded from GenBank at NCBI (https://www.ncbi.nlm.nih.gov/) as model sequences (Table 2) and sequences from acutatum complex: C. acutatum (CBS112996) and C. nymphaeae (CBS_515.78) were used as outgroup. The sequences of ITS, GAPDH, and TUB2 were aligned independently with ClustalW software in MEGA X program (Kumar et al., 2018). Then, a multi-gene analyses were performed using a concatenated dataset of the three loci. The trees were visualized in FigTree v1.4.4 (https://tree.bio.ed.ac.uk/software/fgtree/). For the maximum-likelihood method (ML), the Tamura-Nei model + G nucleotide substitution model was implemented with 500 bootstrap repetitions. ML analyses were performed using Molecular Evolutionary Genetics Analysis (MEGA) 10.2 software, and the best substitution model was decided using CIPRES in jModelTest 2.1.6 (Darriba et al., 2012). Bayesian probability (BP) analysis was performed using BEAST v1.10.4 software package. The Hasegawa-Kishino-Yano (HKY) model with a Gamma distribution with an uncorrelated relaxed clock strict clock was selected as the optimal model. the Markov Chain Monte Carlo (MCMC) method was run for 10 million generations and sampled every 5000 steps in two repetitions.

Pathogenicity tests

Inoculation was performed in duplicate at the apical, middle, and terminal parts of approximately 2-month-old national cocoa fruit. For this, a 6 mm diameter portion of the bark was separated using a hole punch, and a disk of the same diameter with the fungal culture grown for seven days was placed. The fruits were individually incubated in polyethylene bags with damp cotton at 28 °C for 7 days. The variables that were evaluated were the external diameter, the internal diameter, and the depth of the damage. The external diameter was measured directly on the surface of the inoculation site, and for the evaluation of the other 2 variables, a longitudinal cut of the pod was made, and the surface of the damage was measured if it existed (Figure 1) (Montri et al., 2009).

Table 2

Strains of Colletotrichum used in multilocus analysis in this study. Details are provided about complex, species, strain and GenBank accessions of the reference sequence

Figure 1. Illustration of a cocoa pod with inoculation points. A. Whole pod showing the evaluation of external diameter. B. Longitudinal cut of the cacao pod showing the evaluation of internal diameter and depth of damage.

The results obtained were analyzed using ANOVA, and the means were compared using Tukey's test at the significance level of p ≤ 0.05, using INFOSTAT.

3. Results and discussion

Phylogenetic analysis

This study was based on the examination of Colletotrichum, which has been reported as an endophyte, pathogen, and saprobe and is distributed worldwide, colonizing various hosts (Hyde et al., 2014; Jayawardena et al., 2016; Zheng et al., 2022). In cocoa cultivation, it is one of the most commonly found foliar endophytic fungi (Villavicencio-Vásquez et al., 2018), and also causes the disease known as anthracnose in cocoa cultivation (Asare et al., 2021; Rojas et al., 2010).

To elucidate the molecular phylogenetic position of our isolate, a BLAST search was performed in the NCBI database, and phylogenetic analyses were conducted. The isolates were first classified up to the genus level by performing a BLAST of their partial nucleotide sequences of ITS, GAPDH, and TUB2 (Table 3). Their identity was further confirmed at the species level, based on the multi-locus phylogenetic analysis of those three loci using our 16 sequences of Colletotrichum isolates along with reference sequences retrieved from GenBank (Table 2). The final dataset contained 1288 bp, including gaps, comprising 519, 267, and 502 positions from ITS, GAPDH, and TUB2, respectively.

The multilocus analysis conducted was primarily based on the difficulty in morphological identification of the genus Colletotrichum (Jayawardena et al., 2016), the results obtained from the BLAST analysis with the ITS1, 5.8S, and ITS2 regions were inconclusive, as high-percentage similarity identities were found with several isolates in this study, such as: C. fructicola, C. siamense, C. theobromicola, C. crysophyllum, C. gloeosporioides, C. pandanicola, C. alienum, C. karstii, and C. phyllanthi, When analyzing the sequences of the ITS region, ITS1, 5.8S, and ITS2, the results were inconclusive due to a lack of information using only one gene (Yu et al., 2022), which also made differentiation between C. tropicale and C. siamense or C. fructicola, C. aeschynomene and C. chrysophilum (Weir et al., 2012), nearly impossible, However, it was clearly differentiated that these isolates were entirely related to the Colletotrichum gloeosporioides and boninense complexes. On the other hand, partial sequences of the TUB2 and GAPDH genes, and their combined use in phylogenetic or Bayesian inference analyses, are frequently employed in the study of these fungi, providing greater accuracy to the results; a study conducted on C. truncatum, C. dematium, and C. gloeosporioides indicated that the GAPDH locus is essential for resolving relationships between closely related Colletotrichum species (Mahmodi et al., 2014; Samarakoon et al., 2018). For this reason, the sequencing of the GAPDH and TUB2 regions was performed, obtaining similar results (Table 3), but also showing similarity with other species. Therefore, a phylogenetic analysis with Bayesian inference was carried out.

The phylogenetic analysis revealed that the 16 isolates were assigned into two species complexes (Figure 2), 14 allocated within the C. gloeosporioides complex, and the remaining two belonged to the C. boninense complex. The isolates within the gloeosporioides complex are clustered in three clades, six leaf endophytic isolates (C12, C82, C83, C107, C146, and C164) with C. chrysophilum, despite the blast indicating mostly C. fructicola. This can be explained by the close relationship between C. fructicola and C. chrysophilum; however, the use of multiple genes for phylogenetic analysis helps to separate them (Vieira et al., 2017), moreover, according to Vieira et al. (2017), studies conducted by Weir et al. (2012) using isolates from Malus in the USA and Brazil consider C. fructicola as conspecific with C. chrysophilum; two isolated were clustered with C. theobromicola, and six isolates including those obtained from pods with anthracnose symptoms (PAT1, PAT2, and PAT6) and those from healthy leaves (C75, C133, and C160) clustered with C. siamense and C. pandanicola This can be explained by their high genetic similarity, as noted by Zhang et al. (2023), who indicate that there are fewer nucleotide differences between C. pandanicola and C. siamense. However, there are no reports of C. pandanicola in cocoa cultivation since it was reported less than six years ago by Tibpromma et al. (2018) in leaves of Pandanus sp. For this reason, in this case, we will consider the information obtained in the BLAST that identifies the isolates as C. siamense. However, to comple-ment and clarify, an analysis with more genes could be performed as indicated by Chang et al. (2022) and Yu et al. (2022). The 2 isolates within the boninense complex clustered with C. karstii.

Pathogenicity tests

No significant differences were observed among all treatments; however, a marked difference was observed between the damage caused by C. siamense (greater) and C. crysophilum (lesser). Of the isolates evaluated, 8 showed external and internal damage on the pods, with 5 of the 6 isolates identified as C. siamense (3 from diseased pods and 2 leaf endophytes). The most aggressive was PAT6 (39 mm external diameter, 29.35 mm internal diameter, and 20.22 mm depth).

Table 3

Molecular identification by the three sequenced genes

Figure 2. Maximum likelihood (ML) tree of the gloeosporioides and boninense species complex based on combined data sets of ITS, GAPDH, and TUB2 sequences (1288 bp including gaps). C. acutatum (CBS 112996) and C. nymphaeae (CBS 515.78) are used as an outgroup. ML bootstrap values and Bayesian posterior probability (BP) analysis are shown at the nodes (BP/ BS). BS > 60% and BP > 0.60 are shown, Branches that are unsupported with BS or BP are denoted by “–”. Sequences obtained in the present study are indicated in blue.

Among the isolates identified as C. chrysophilum (all endophytes), 3 out of 6 showed symptoms, which were milder than those caused by the C. siamense isolates. The most aggressive in this case was C107 (18.98 mm external diameter, 16.25 mm internal diameter, and 10.74 mm depth) (Figure 3). None of the isolates identified as C. theobromicola showed symptoms, and regarding the two isolates from the boninense complex, they also did not show any damage in the evaluations.

The species found in this study, mainly those be­longing to the gloeosporioides complex, have been reported causing damage in different crops world­wide, such as C. chrysophilum in blueberry (Brazil) (Soares et al., 2021a) and cassava (Brazil) (Machado et al., 2021), C. theobromicola in wild cassava (Brazil) (Oliveira et al., 2018), cocoa (French West Indies) (Rojas et al., 2010), C. siamense in cocoa (Puerto Rico) (Serrato-Diaz et al., 2019), wild cassava (Brazil) (Oliveira et al., 2018), mango (China) (Qin et al., 2017), and chili (China) (Liu et al., 2016).

In the case of C. karstii, which belongs to the boninense complex, it has been reported causing anthracnose in soursop, passion fruit, banana, and tamarillo (Colombia) (Oliveira et al., 2018), strawberry (Brazil) (Soares et al., 2021b), Natal plum (Spain) (Garcia-Lopez et al., 2021), Mango (Brazil) (Zakaria, 2021), Dragon fruit (Brazil) (Nascimento et al., 2019), however, in this study, when pathogenicity tests were carried out, no damage was shown in the inoculated tissues, this is very common since C. karstii has been reported as endophytes in other crops such as Citrus (Europe) (Guarnaccia et al., 2017) Coffee (Colombia) (Poma-Angamarca et al., 2024).

On the other hand, it is notable that the four strains identified as C. siamense isolated from diseased pods were collected from different localities; however, they present symptoms when inoculated, as did the asymptomatic endophytes belonging to the same species. This could be explained according to (Photita et al., 2004), as endophytes can change their condition to pathogens under certain stress conditions.

Therefore, it is presumed that the endo­phytic isolates of C. siamense and C. crhysophilum changed their endophytic condition to pathogenic, even though they originated from leaves. This is well-supported, as Colletotrichum is one of the most frequently isolated endophytes from many crops (Baralt et al., 2012; Osorio et al., 2021; Vázquez Cruz et al., 2023).

Figure 3. A – D. Damage caused by inoculation of Colletotrichum isolates, entire pod, longitudinal section, and recovery of isolate in Petri dish with PDA. A. PAT6, B. C83, C. C133, D. C107. E – G. Inoculated isolates that did not cause damage to pods. E. C65, F. C6, G. CONTROL.

Figure 4. Averages of the three damage variables evaluated in mm (external diameter, internal diameter, and depth of damage) for the Colletotrichum isolates.

4. Conclusions

Through phylogenetic analysis, two complexes of the Colletotrichum group were identified in cocoa cultivation: the gloeosporioides complex (C. crysophilum, C. siamense, C. theobromicola) and the boninense complex (C. karstii). Among these, C. crysophilum has not been previously reported in cocoa cultivation. Pathogenicity tests demonstrate that isolates of C. siamense are the main cause of necrosis symptoms in cocoa pods, while isolates of C. crysophilum cause much less damage. The isolates C. theobromicola and C. karstii did not cause any damage in the inoculated pods.

This study reports that the main species causing damage in cocoa cultivation is C. siamense. However, the possibility that C. crysophilum might change its condition from endophytic to patho­genic cannot be ruled out. Future studies should conduct periodic sampling to identify possible changes in the pathogen population and its geo­graphic distribution in order to develop integrated management strategies that include cultural and biological practices to control the pathogens.

Conflicts of Interest

There are no conflicts of interest.

Authors' Contribution:

F. Espinoza-Lozano: conceptualization, data curation, research, methodology, writing the initial draft, and review. L. Serrano-Mena: Research, data curation, software, and review. M. Villavicencio-Vasquez: Conceptualization, research, formal analysis, and review. M. Vera-Morales: Formal analysis, data curation, and review. J. Coronel-León: Formal analysis, review, and supervision. D. Sosa-Castillo: Conceptualization, research, formal analysis, review, and supervision.

ORCID

F. Espinoza-Lozano  https://orcid.org/0000-0002-2051-2682

L. Serrano-Mena  https://orcid.org/0000-0001-8450-8511

M. Villavicencio-Vasquez  https://orcid.org/0000-0003-3237-0295

M. Vera-Morales  https://orcid.org/0000-0003-2342-6269

J. Coronel-León  https://orcid.org/0000-0001-6535-0261

D. Sosa-Castillo  https://orcid.org/0000-0001-5403-9072

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