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Morphological and Genome Characterization of Alternaria alternata Causing Blueberry (Vaccinium corymbosum L.) Leaf Spot in Peru

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Morphological and Genome Characterization of Alternaria alternata Causing Blueberry (Vaccinium corymbosum L.) Leaf Spot in Peru

Ricardo Velasquez 1,† Valentina Osorio 2,† Ana Maria Leiva 2 Juan Manuel Pardo 2 Alejandra Gil-Ordóñez 2 Ida Bartolini 3 Wilmer J. Cuellar 2,*

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1
Laboratorio de Entomología y Manejo Integrado de Plagas, Instituto Nacional de Innovación Agraria (INIA), Av. Chancay—Huaral KM 5.500, Lima 15026, Peru
2
Crop Protection Research Area, Cassava Program, Crops for Nutrition and Health, International Center for Tropical Agriculture (CIAT), the Americas Hub, Km 17, Recta Cali-Palmira, Palmira 763537, Colombia
3
Laboratorio de Biología Molecular del Servicio Nacional de Sanidad Agraria (SENASA), La Molina Nº 1915, La Molina Lima 12, Lima 15026, Peru
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.

Received: 26 January 2026 Revised: 10 April 2026 Accepted: 09 May 2026 Published: 20 May 2026

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© 2026 The authors. This is an open access article under the Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/).

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Fungal Res. 2026, 1(1), 10002; DOI: 10.70322/fungalres.2026.10002
ABSTRACT: Blueberries (Vaccinium corymbosum L.), valued for their nutritional benefits and economic significance, have become Peru’s leading agro-export crop. However, intensive cultivation can lead to phytosanitary problems if not addressed promptly, posing a serious threat to blueberry production. This study aimed to isolate and identify the causal agent of leaf spot symptoms initially observed in blueberries cultivated in Peru, marking the first formal documentation of its presence in the country. In 2022, leaf spot symptoms were recorded on V. corymbosum cv. Biloxi, in the north of Lima, Peru. Field observations revealed necrotic, sunken spots on leaves and fruits, with 4.84% of leaves diseased and 1.28% of fruits affected. Pathogen isolation and microscopic studies identified Alternaria alternata as the primary causal agent, which was confirmed by genome sequencing using Oxford Nanopore Technology. Pathogenicity tests demonstrated the fungus’ ability to reproduce symptoms identical to those observed in the field, fulfilling Koch’s postulates. Under experimental conditions, disease severity increased over time, with the affected leaf area ranging from 9.35% to 25.61% between 7 and 14 days post-inoculation. This study establishes A. alternata as a pathogen of blueberries in Peru and provides essential insights for future research and strategies to mitigate its impact on the industry.
Keywords: Fungal identification; Conidiophores; Conidia; Nanopore; Pathogenicity; Crop protection

1. Introduction

Blueberries (Vaccinium corymbosum L.), a perennial crop native to North America and part of the Ericaceae family, are highly valued for their nutritional properties and global economic importance. The increasing global demand for blueberries has driven expansion in Peru’s production. Although commercial production began in 2007, the industry experienced remarkable growth following a national campaign launched in 2012, expanding from just 50 hectares to over 18,000 hectares in 2022. That year, blueberries had become Peru’s top agro-export crop, generating $1.435 billion in revenue and accounting for approximately 14% of the nation’s total agro-export earnings [1]. Today, Peru ranks as the world’s third-largest blueberry producer, following the United States (U.S.) and Canada, and is the leading producer in Latin America. The country exports to major markets, including the U.S., Europe, and China [2,3].

Nevertheless, this crop is highly susceptible to fungal pathogens that cause severe economic losses. Among them, Alternaria spp. are a broad-host-range pathogen responsible for yield reductions of 20% to 80% widely [4], and postharvest losses of up to 46% in China. Economic losses in the blueberry industry due to Alternaria spp. infections are estimated to be between $230 million and $1 billion globally [5]. These substantial financial impacts underscore the critical importance of early detection and timely implementation of management to contain the spread of the disease and lower the associated costs of disease management at such scales.

Globally, the most frequently reported Alternaria species affecting blueberries are A. alternata and A. tenuissima, which have been associated with leaf spot in major producing countries such as the U.S., China, South Korea, and Australia [6,7,8,9]. Additionally, these species have been linked to fruit rot in blueberries in the U.S. and Argentina [10,11]. These are polycyclic pathogens that persist in soil and plant debris through various survival structures. Under favorable environmental conditions, conidia initiate infections through natural openings and wounds [12,13]. However, despite this extensive global documentation of Alternaria species affecting blueberries, no formal reports have documented these pathogens in blueberry crops in Peru. Although Alternaria spp. have been reported affecting other crops in Peru, such as citrus and tomato [14,15], this knowledge gap represents a phytosanitary concern that limits effective disease management.

Therefore, the objective of this study was to isolate, identify, and characterize the causal agent of leaf spot symptoms observed in blueberry plants in Peru, integrating morphological, molecular, and biological approaches (including fulfillment of Koch’s postulates). By scientifically documenting its identity, this work provides a robust foundation for future epidemiological studies and targeted disease management strategies.

2. Materials and Methods

2.1. Sample Collection and Pathogen Isolation

In 2022, leaf spots were first observed on V. corymbosum cv. Biloxi in a field in northern Lima (–11.51478, –77.23869). Plants were evaluated for symptoms by examining lesion characteristics, including color, shape, pattern distribution, and presence of fungal signs. Disease incidence, calculated as the percentage of affected leaves per plant, was assessed using a stratified random sampling of 30 plants across three homogeneous field strata (10 plants per stratum) [16], covering a total area of 1800 m2. Fruit rot incidence was evaluated by collecting 9250 fruits from the same 30 plants across three harvest periods and expressing it as the percentage of symptomatic fruits. For pathogen isolation, five plants showing moderate symptom severity were selected to avoid advanced lesions potentially colonized by secondary microorganisms. From these plants, ten symptomatic leaves were collected from the middle canopy, as this location represents the active disease front, ensuring that tissues retain cellular integrity and nutrients necessary for pathogen metabolism, unlike lower leaves already compromised by advanced senescence or upper leaves that may not yet have been colonized [17]. Small tissue fragments (5 mm diameter) were excised from the margins of necrotic lesions using a sterile scalpel.

Samples were surface disinfected by sequential immersion in 1% NaClO for 1 min, followed immediately by 70% ethanol for 1 min, and then rinsed three times with sterile distilled water. All procedures were conducted under aseptic conditions without interruption. Disinfected tissues were placed on potato dextrose agar (PDA) and incubated at 25 °C under continuous darkness. Fungal growth was observed after three days, and single-spore purification was subsequently performed to obtain pure cultures.

2.2. Morphological Characterization

A total of five fungal isolates were obtained from symptomatic tissues, all showing consistent macroscopic and microscopic characteristics typical of Alternaria alternata. Due to this uniformity, one representative isolate was selected for detailed characterization. Macroscopic characteristics were evaluated on PDA, including colony color (surface and reverse), texture, margin type, and mycelial appearance. For microscopic characterization, cultures were grown on potato carrot agar (PCA), a medium known to promote sporulation in Alternaria spp., and incubated at 25 °C under an 8 h light/16 h dark photoperiod for 7 days. Conidia were examined using a light microscope (BA210, Motic, Xiamen, China) at 40× magnification, and measurements were obtained from 50 randomly selected conidia following the criteria described by Simmons (2007) [18].

2.3. Genome Characterization

A single representative isolate obtained by single-spore purification was selected for genomic analysis and designated as Alternaria alternata isolate Per1. For DNA extraction, isolate Per1 was grown on PDA medium at 25 °C under continuous darkness for 2–4 weeks. Approximately 0.03 g of fresh mycelium was scraped from agar plates and processed according to the protocol described by Gil-Ordóñez et al. (2024) [19]. DNA integrity was confirmed by gel electrophoresis, and concentration was determined using Qubit™ dsDNA HS Assay Kit (Invitrogen, Life Technologies, Carlsbad, CA, USA). Whole-genome sequencing was performed using Oxford Nanopore Technology (FLO-MIN114 R10 chemistry, LSK114 kit). Genome assembly was conducted using Flye v2.9 [20] with default parameters, specifying only the input read (--nano-raw) and computational settings. Assembly and sequencing coverage metrics were calculated using BBMap v38.18 [21] and Qualimap v2.2.2 [22], respectively. Genome completeness and quality were assessed with BUSCO v5.2.2 using the eukaryota_odb10 (8 January 2024) and pleosporales_odb10 (8 January 2024) datasets asreferences [23]. Subsequently, in silico prediction of coding regions was performed using AUGUSTUS, previously trained with the genome of A. alternata (GenBank: GCA_036971565). Orthologous gene clusters were inferred using OrthoVenn3 (https://orthovenn3.bioinfotoolkits.net/home, accessed on 20 January 2025) and compared with the reference genomes of A. alternata (GenBank: GCA_036971565), A. arborescens (GenBank: GCA_004154835), and A. tenuissima (GenBank: GCA_041430775) using the OrthoMCL algorithm (e-value = 1 × 10−10), with all other parameters set to default. Phylogenetic relationships were inferred based on conserved single-copy orthologs using a maximum likelihood approach implemented within the OrthoVenn3 pipeline [24].

2.4. Pathogenicity Test

Pathogenicity tests were conducted on whole blueberry plants grown in pots under screen-house conditions to fulfill Koch’s postulates [8,9,25]. The experiment followed a completely randomized design using five healthy six-month-old blueberry plants (biological replicates), with six leaves per plant (technical replicates), totaling 30 leaves per treatment. The experiment was conducted twice. Inoculations were done using a conidial suspension (3.0 × 105 conidia mL−1) of A. alternata obtained from a 10-day-old single-spore culture grown on PDA, suspended in sterile distilled water containing 0.025% Tween 20. Before inoculation, four wounds per leaf were made using a sterile needle to ensure consistent infection. The conidial suspension was applied using sterile brushes to cover the entire leaf surface, while control leaves received sterile water containing 0.025% Tween 20. All leaves remained attached to living plants throughout the experiment. Plants were maintained at >70% relative humidity and 26 ± 2 °C for 14 days. Disease severity was evaluated at 0, 7, and 14 days post-inoculation (DPI) using paired measurements from five leaves per plant, with one additional leaf per plant reserved for re-isolation and documentation. Necrotic leaf area was quantified through manual analysis using the R package pliman [26]. Data were analyzed descriptively using means and standard deviations.

2.5. Re-Isolation and Validation

Following pathogenicity testing, re-isolation was attempted from both inoculated symptomatic tissues and mock-inoculated leaves using the same isolation procedure described in Section 2.1. Re-isolated organisms were subjected to morphological characterization following the same methodology described in Section 2.2, to confirm identity with the original isolate. Rapid molecular validation was conducted by amplification of the internal transcribed spacer (ITS) regions of nuclear ribosomal DNA using the primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) [27]. PCR reactions were performed in 25 μL volumes containing GoTaq® Green Master Mix (12.5 μL, 1× final concentration), upstream and downstream primers (0.5 μL each, 10 μM), DNA template (3 μL, 20 ng), and nuclease-free water to a final volume. Thermal cycling conditions included initial denaturation at 94 °C for 4 min, followed by 35 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min, with final extension at 72 °C for 10 min. PCR products were sequenced using the Sanger method at Macrogen Co., Ltd. (Daejeon, Korea). Additionally, we carried out whole-genome sequencing and assembly of the re-isolated pathogen as previously described for multilocus validation (including regions gapdh, rpb2, tef1-α, and Alt-a1). Sequences were compared with those from the original isolate using BLASTn analysis (e-value < 1 × 10−25).

3. Results and Discussion

3.1. Field Symptoms and Disease Incidence

Leaf spot symptoms were characterized by brown, circular or irregular necrotic lesions with sunken, dark-brown centers and concentric rings, often surrounded by yellow halos and small black dots (Figure 1a). Lesions initially appeared as small spots (approximately 3–7 mm in diameter), predominantly on the abaxial leaf surface, and became evident on both surfaces as the disease progressed. Fruit rot symptoms were also observed in the field, characterized by flattening of mature fruits with a characteristic dry appearance, later covered by a mass of mycelium. Damage assessment at the field level revealed that 4.80% of fruits were affected during the first production cycle and 1.28% in the last production cycle of the year. Additionally, leaf spot incidence was recorded at 4.8%, a level comparable to previous reports of leaf spot incidence in South Korea [8].

3.2. Morphological Characterization

Five isolates were obtained that showed consistent morphology, with simple conidiophore architecture and short conidia chains (up to seven conidia) characteristic of A. alternata. Fungal colonies exhibited a dark olive color on the surface and a dark gray color on the reverse. The colonies had white filamentous margins, a circular form, and a velvety texture. They produced loose, aerial hyphae on the culture surfaces, with a cottony mycelial appearance (Figure 1b), consistent with the characteristics reported in other studies [28,29]. Single suberect or erect pale brown conidiophores were observed, ranging in size from 23.7 to 66.6 μm (mean: 36.5 ± 1.7 μm) and producing up to seven chained conidia (Figure 1c). Each conidium measured from 20.2 to 43.7 μm in length, with a mean of 28.6 μm ± 4.7 μm (n = 50), and 8.2 to 16.9 μm in width, with a mean of 11.7 μm ± 1.9 μm (n = 50). They contained three to six transverse septa and up to two longitudinal septa. Conidia were olivaceous or dull green-brown in color, ellipsoid to ovoid, often featuring a short conical or cylindrical beak (Figure 1d). The first conidium produced on a conidiophore was also narrowly elliptical, with three transverse septa.

Figure_1_1

Figure 1. Disease symptoms on V. corymbosum caused by A. alternata and its cultural and morphological characteristics. (a) Necrotic lesions on blueberry leaves taken from the field; (b) Colony morphology after 10 days of growth on PDA medium; (c) Conidiophores and conidia after 7 days of growth on PCA medium; (d) Microscopic view showing the diverse shapes of macroconidia (40×).

Based on these key features, the isolate was identified as A. alternata, distinct from closely related species in the Alternaria complex, according to Simmons’ taxonomic key [18]. Key distinguishing features included conidial dimensions consistent with the ellipsoid form, simple conidiophore architecture with short chains (up to 7 conidia) typical of Group J species rather than the longer unbranched chains (10–25+ conidia) characteristic of A. tenuissima (Group H), and typical beak morphology and septation patterns. The observed morphological traits are consistent with previous reports describing conidia with 3 to 6 transverse septa and dimensions ranging from 16 to 42 × 7 to 16 μm [7]. Likewise, other authors have described conidia as obclavate to elliptical [30]. However, differences were observed when comparing conidial dimensions with isolates from California, which showed average measurements of 22.04 μm in length and 8.7 μm in width [11]. Such variation may be influenced by geographic factors or local adaptation, emphasizing the importance of regional studies for understanding pathogen variability and its possible implications in disease management.

A. alternata has been confirmed as the causal agent of leaf spots on blueberries in South Korea and southern Poland [8,31]. Similarly, in California, 61.5% of Alternaria isolates from decayed blueberries were identified as A. alternata [11]. However, other studies have reported A. tenuissima as responsible for leaf spots in Western Australia and Argentina [9,32]. These results underscore the complexity of Alternaria spp. in different regions and highlight the need for accurate identification to develop precise, pathogen-targeted disease management strategies.

In other crops, Alternaria alternata has been reported causing brown spots on citrus crops across several Peruvian provinces [14]. Given that citrus crops are well-established in Peru, particularly in regions such as Lima [33], they could serve as a potential reservoir for A. alternata populations affecting blueberries. The widespread occurrence of A. alternata across Latin America, including citrus in Argentina, alfalfa in Brazil, quinoa in Colombia, hazelnut in Chile, broccoli in Ecuador, and weeds in Uruguay, underscores its adaptability to different environmental conditions and hosts [34,35,36,37,38,39]. The presence of many of these crops in Peru may have facilitated the expansion and adaptation of A. alternata to the more recently introduced blueberries.

3.3. Genome Characterization

DNA had high integrity and yielded 90.4 ng/µL before sequencing. Whole-genome sequencing yielded approximately 6 Gb of estimated bases (506,720 reads, average read length: 11.8 kb, N50 raw reads: 20.04 kb, mean quality score: Q10), and resulted in an assembled genome of 34.2 Mb with a coverage of ~166×, comprising 17 contigs (N50: 435,246 bp, L50: 18, largest contig: 6,834,098 bp) and a GC content of 49.93%. BUSCO analysis revealed 98.8% completeness (98.8% complete, 0.8% fragmented) and 95.3% completeness (94.8% complete, 0.5% fragmented) based on eukaryota_odb10 and pleosporales_odb10 databases, respectively. The genome size of A. alternata Per1 (34.2 Mb) and GC content fall within the expected range for this species, which has been reported to vary between 30–35 Mb and ~50%, respectively, across isolates from different host plants using various sequencing technologies, including Nanopore [6,40]. Protein-based gene cluster analysis was performed using the entire predicted proteome (all protein-coding genes) derived from the assembled genome of isolate Per1 (12,169 protein-coding genes). Orthologous gene cluster analysis identified 11,720 clusters and showed strong clustering with reference genomes of A. alternata (Figure 2).

Figure_2_1

Figure 2. Phylogenetic relationships and orthologous cluster analysis of an Alternaria isolate from Peruvian blueberries. (a) Diagram showing distinct and shared orthologous clusters among A. alternata Per1 (orange dots) and the reference genomes of A. alternata, A. tenuissima, and A. arborescens available in the OrthoVenn3 web service. The bar graph on the left displays the count of orthologous clusters. (b) Maximum likelihood phylogenetic tree illustrating evolutionary relationships and genetic distances based on single-copy conserved genes. The tree was constructed using the JTT+CAT evolutionary model. This analysis provides insights into the genetic relationships between the Peruvian Alternaria isolate and other reference genomes.

3.4. Pathogenicity Test

Symptoms began to appear on inoculated leaves seven days post-inoculation, and by day 14, all inoculated leaves had developed necrotic spots identical to those observed in the field. In contrast, control plants remained asymptomatic (Figure 3). These results are consistent with previous studies reporting symptom onset between 7 and 8 days after inoculation [27,41]. Similarly, leaf spot symptoms observed after 15 days were comparable to those occurring under natural conditions [10]. Disease severity increased over time, with the affected leaf area ranging from 9.35 ± 0.71% at 7 days to 25.61 ± 6.2% at 14 days post-inoculation (Figure 3).

Figure_3_1

Figure 3. Results of the pathogenicity test showing A. alternata causing leaf spots 14 days post-inoculation. (a) No symptoms on a control leaf at 14 days post-inoculation; (b) Necrotic symptoms on a leaf 7 days post-inoculation; (c) Necrotic symptoms on a leaf 14 days post-inoculation. All leaves shown were attached to living, whole plants at the time of inoculation and symptom development.

The pathogen was successfully re-isolated from 86.7% of symptomatic tissues, confirming pathogenicity and fulfilling Koch’s postulates. Fusarium spp. were occasionally recovered as contaminants; however, contamination was controlled through colony purification and morphological differentiation, as Fusarium spp. colonies exhibited white coloration distinct from the characteristic olive-green appearance of A. alternata isolates.

Morphological and cultural comparisons confirmed the re-isolated organism as A. alternata, consistent with the inoculated pathogen (Figure 4). Comprehensive molecular validation included multilocus sequence analysis (extracted from whole-genome data) of ITS, gapdh, rpb2, tef1-α, Alt-a 1 locus, which confirmed the identity of the re-isolated fungus with that of the inoculated isolate (sequences for all loci shared 100% identity; GenBank nros: PV069777, PZ316321-PZ316324). These results fully satisfy Koch’s postulates through both morphological and robust molecular confirmation.

Figure_4_1

Figure 4. Morphological confirmation of re-isolated A. alternata. (a) Colony morphology after 10 days of growth on PDA medium; (b) Conidiophores and conidia after 7 days of growth on PCA medium; (c) Microscopic view of conidia (40×).

4. Conclusions

Alternaria alternata has been identified as the causal agent of leaf spot symptoms in blueberries in Peru. The combined use of morphological, pathogenicity, and whole-genome analyses enabled precise identification of the pathogen and confirmed its pathogenicity. These results establish a foundation for targeted disease management and inform the selection of specific control strategies.

Statement of the Use of Generative AI and AI-Assisted Technologies in the Writing Process

During the preparation of this manuscript, the authors used Grammarly in order to improve the English language and writing clarity. After using this tool, the authors reviewed and edited the content as needed and take full responsibility for the content of the published article.

Acknowledgments

We extend our gratitude to David Pavel Casanova Núñez Melgar of INIA–Peru; Jesús Iván Chumpitaz Fernández at the Peruvian Institute of Agroexporters; and Jacobo Trujillo, Technical Support at INIA EEA Donoso–Huaral and the National Agrarian Health Service (SENASA), for their valuable assistance in molecular laboratory work, with special thanks to Arturo Olortegui Livia. We also acknowledge the technical support provided by Viviana Domínguez and Ronal Montes from CIAT.

Author Contributions

Conceptualization, R.V., J.M.P. and W.J.C.; Biological test, R.V., V.O. and J.M.P.; Formal Analysis, R.V., V.O., A.M.L. and A.G.-O.; Validation, R.V. and V.O.; Data Curation, V.O., A.M.L. and A.G.-O.; Writing—Original Draft Preparation, All authors; Writing—Review & Editing, W.J.C.; Supervision, J.M.P. and W.J.C.; Project Administration, R.V., I.B. and W.J.C.

Ethics Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The genome assembly and associated data of isolate Per1 and re-isolate have been deposited in NCBI under accession JBMHEI000000000, BioProject PRJNA1189477, and BioSample SAMN44971619 and SAMN57370065 and SAMN57370065.

Funding

This research was funded by the Ministry of Agriculture of Peru (MINAGRI) through the Secretaría Técnica de Cooperación del CGIAR (STC-CGIAR).

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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