Molecular and biological characterization of Peruvian isolates of zucchini yellow mosaic virus reveals differential severity and partial resistance breakdown in zucchini cultivars

Caracterização molecular e biológica de isolados peruanos do vírus do mosaico amarelo da abobrinha revela gravidade diferencial e quebra parcial de resistência em cultivares de abobrinha

Abstract

Zucchini yellow mosaic virus (ZYMV) is one of the most important potyviruses affecting cucurbit crops worldwide and has recently been reported in Peru. However, information regarding the biological variability and host response of Peruvian isolates remains limited. This study aimed to characterize two Peruvian ZYMV isolates obtained from conventional and organic zucchini production systems, as well as to evaluate their aggressiveness in four commercial zucchini cultivars under greenhouse and field conditions. Symptom severity was assessed using a proposed diagrammatic scale, while genomic characterization was performed through high-throughput sequencing, followed by phylogenetic analysis based on the complete sequences of the polyprotein-coding region. Both isolates, ZYMV_Lim and ZYMV_Chos, induced typical ZYMV symptoms; however, ZYMV_Chos caused more severe symptoms, particularly in the cultivar ‘Hurakan F1’, indicating greater aggressiveness. Comparative genomic analysis revealed amino acid differences mainly in the P1 and HC-Pro regions, which may contribute to differences in pathogenicity and host interaction in potyviruses. Phylogenetic analysis grouped the Peruvian isolates with sequences from the United Kingdom, the United States, and Australia, suggesting independent introduction events. Under field conditions, the cultivar ‘Rocío F1’ exhibited lower symptom incidence, reduced disease progression, and superior agronomic performance. The biological and molecular differences identified among the Peruvian isolates were associated with contrasting responses in the evaluated cultivars. The use of tolerant cultivars such as ‘Rocío F1’ may represent a practical alternative for reducing ZYMV damage under field conditions.

Keywords:
ZYMV; Cucurbita pepo; viral resistance; management of viral disease

Resumo

O vírus do mosaico amarelo da abobrinha (ZYMV) é um dos potyvírus mais importantes que afetam cucurbitáceas em todo o mundo e foi recentemente relatado no Peru. Contudo, as informações sobre a variabilidade biológica e a resposta do hospedeiro frente a isolados peruanos ainda são limitadas. Este estudo teve como objetivo caracterizar dois isolados peruanos de ZYMV, obtidos de sistemas de produção convencional e orgânica de abobrinha, e avaliar sua agressividade em quatro cultivares comerciais de abobrinha sob condições de casa de vegetação e campo. A severidade dos sintomas foi avaliada por meio de uma escala diagramática proposta, enquanto a caracterização genômica foi realizada por sequenciamento de alto rendimento, seguida de análise filogenética baseada nas sequências completas da região codificadora da poliproteína. Ambos os isolados, ZYMV_Lim e ZYMV_Chos, induziram sintomas típicos do ZYMV; no entanto, ZYMV_Chos causou sintomas mais severos, particularmente na cultivar ‘Hurakan F1’, indicando maior agressividade. A análise genômica comparativa revelou diferenças de aminoácidos, principalmente nas regiões P1 e HC-Pro, as quais estão associadas à patogenicidade em potyvírus. A análise filogenética agrupou os isolados peruanos com sequências do Reino Unido, Estados Unidos e Austrália, sugerindo eventos independentes de introdução. Em condições de campo, a cultivar ‘Rocío F1’ apresentou menor incidência de sintomas, menor progresso da doença e desempenho agronômico superior. As diferenças biológicas e moleculares identificadas entre os isolados peruanos foram associadas a respostas contrastantes nas cultivares avaliadas. O uso de cultivares tolerantes, como ‘Rocío F1’, pode representar uma alternativa prática para reduzir os danos causados pelo ZYMV em condições de campo.

Palavras-chave:
ZYMV; Cucurbita pepo; resistência viral; manejo de doenças virais

1. Introduction

Zucchini yellow mosaic virus (ZYMV) is one of the most economically important viruses affecting cucurbit crops worldwide (Desbiez, 2020). It belongs to the genus Potyvirus and possesses a positive-sense single-stranded RNA genome of approximately 10 kb (Maghamnia et al., 2018; Khanal and Ali, 2019). The virus is primarily transmitted by aphids in a non-persistent manner (Lecoq and Katis, 2014; Fereres et al., 1992), although seed transmission has also been reported in Cucurbita pepo at low frequencies, ranging from 1 to 5% (Riedle-Bauer et al., 2002; Simmons et al., 2011, 2013).

ZYMV was first identified in southern Europe in 1973 (Topkaya et al., 2019; Abdel Aleem et al., 2021). Major epidemics were subsequently recorded during 1979–1980, and by 1996 the virus had spread to most cucurbit-growing regions worldwide (Caciagli, 2008). In Peru, ZYMV was recently confirmed infecting Cucurbita maxima in several coastal production areas (Valencia et al., 2025), and it has also been detected in Cucurbita moschata, C. pepo, and Citrullus lanatus (Risco, 2024, unpublished data).

Symptoms induced by ZYMV vary according to host species and cultivar. In zucchini, infection typically causes severe yellow mosaic, leaf deformation, and fruit distortion. In melon (Cucumis melo), symptoms range from chlorosis to leaf necrosis, whereas in squash, infected fruits are generally smaller and may exhibit yellow rings, mottling, and surface protuberances (Abdel Aleem et al., 2021; Seal et al., 2017; Simmons et al., 2013). In all susceptible hosts, infection commonly results in stunted growth and reduced yield (Lisa and Lecoq, 1984; Sharma, 2023).

Previous phylogenetic studies have shown that ZYMV isolates frequently cluster according to geographic origin. In some cases, host association has also been reported, suggesting ongoing adaptation of viral populations to local agroecosystems (Ahsan et al., 2023; Rabadán and Gómez, 2023; Morelli et al., 2024). This genetic variability may also be associated with differences in biological aggressiveness and adaptation to local hosts.

Management of ZYMV relies mainly on the use of resistant or tolerant cultivars (Desbiez and Lecoq, 1997; Fidan et al., 2023; Li et al., 2024; Hao et al., 2024), together with the use of healthy seeds and seedlings during crop establishment (Lecoq and Katis, 2014). Several resistance genes have been identified in Cucurbita spp. (Paris et al., 1988; Gilbert-Albertini et al., 1993; Pachner et al., 2011; Brown et al., 2003; Yaşar et al., 2025), particularly dominant genes in C. moschata (Brown et al., 2003; Shrestha et al., 2021; Li et al., 2024). However, their incorporation into commercial cultivars remains limited (Lecoq and Desbiez, 2008). Furthermore, the high genetic variability of ZYMV populations may promote the emergence of more aggressive variants capable of overcoming host resistance mechanisms (Farag et al., 2025; Sharma, 2023; Bibiano et al., 2025). For this reason, characterization of local ZYMV populations may provide useful information for resistance breeding and cultivar selection under Peruvian conditions.

In this context, the present study aimed to molecularly and biologically characterize two Peruvian ZYMV isolates obtained from conventional and organic zucchini production systems. Symptom expression and genetic variability were compared between isolates. In addition, the differential response of four commercial zucchini cultivars to both isolates was evaluated under controlled conditions, together with the agronomic impact of ZYMV infection under open-field conditions.

Understanding the biological diversity of local ZYMV populations is essential because resistance sources identified under one epidemiological context may fail when exposed to genetically distinct viral populations.

2. Material and Methods

2.1. Study site and experimental conditions

The experiment was conducted under two experimental conditions: (i) greenhouse and insect-proof screenhouse facilities, protected against aphid infestation, located at the Department of Phytopathology, Faculty of Agronomy, Universidad Nacional Agraria La Molina, Peru; and (ii) open-field conditions in a commercial production plot located in Huachinga (km 44.5), district of Santa Eulalia, in the Chosica area, where previous growing seasons had reported viral disease incidence in zucchini crops.

2.2. Virus isolates

Under greenhouse and insect-proof screenhouse conditions, two isolates of zucchini yellow mosaic virus were evaluated. The first isolate, designated ZYMV_Lim, was previously identified and isolated from C. maxima plants grown under organic management; its complete genome sequence is available in GenBank under accession number OQ181225 (Valencia et al., 2025). The ZYMV_Lim isolate was not re-sequenced in the present study; all comparative genomic analyses were performed using the previously generated genome sequence (OQ181225). The second isolate, ZYMV_Chos, was collected in October 2023 from naturally infected plants of C. pepo cultivated in the Chosica area, in the same field where the open-field experiment was established (Section 2.1), under conventional crop management. Infected leaf tissue from both isolates (Lim and Chos) was preserved by lyophilization in silica gel and stored at 4 °C until use. Under open-field conditions, ZYMV infection occurred through natural infection.

2.3. Virus identification

2.3.1. High-throughput sequencing

Total RNA was extracted from the “Chos” leaf sample collected from C. pepo cv. ‘Rocio F1’ using the RNeasy Plant Mini Kit from QIAGEN, following the manufacturer’s instructions. High-throughput sequencing (HTS) was performed using the Illumina platform NextSeq 200, with the TruSeq Stranded Total RNA Sample Preparation kit and rRNA depletion using Ribo-Zero Plant. Sequencing generated 150-bp reads, with a total output of approximately 13 million reads.

2.3.2. HTS data analysis

Bioinformatic analysis of the HTS data was performed following the procedures previously described by Valencia et al. (2025) and Mendoza et al. (2022). Read quality was assessed using FastQC v0.12.0 (Andrews, 2010) on the Galaxy platform. Subsequently, Trimmomatic v0.32 (Bolger et al., 2014) was used to remove short and low-quality reads. Contigs were assembled using SPAdes v3.12.0 (Bankevich et al., 2012). In Geneious R11 (Biomatters Ltd., Auckland, New Zealand), contigs were subjected to BLASTn analysis against a viral reference sequence database (RefSeq 2025) downloaded from NCBI. Finally, the complete viral genome sequence was reconstructed using a de novo assembly strategy.

2.3.3. Phylogenetic analysis

A total of 159 complete genome nucleotide sequences of ZYMV were retrieved from National Center for Biotechnology Information GenBank. Multiple sequence alignment was performed using the MAFFT v7.450 algorithm (Katoh and Standley, 2013), implemented in Geneious R11 (Geneious, 2017), under default parameters. The dataset was constructed using the polyprotein-coding region, resulting in a final alignment of 9,269 nucleotides (nt). Recombination analysis was conducted using RDP4 (Martin et al., 2015), applying the statistical methods RDP, GENECONV (Sawyer, 1989), BootScan (Martin et al., 2005; Salminen et al., 1995), MaxChi (Maynard Smith, 1992), Chimaera (Posada and Crandall, 2001), SiScan (Gibbs et al., 2000), and 3Seq (Boni et al., 2007). Recombinant sequences identified by these analyses were excluded from the dataset prior to phylogenetic reconstruction. The nucleotide substitution model General Time Reversible with invariant sites and gamma distribution (GTR+I+G) (Tavaré, 1986; Yang, 1994; Gu et al., 1995) was selected using jModelTest v2.1.10 (Darriba et al., 2012). Bayesian inference (BI) analysis was subsequently performed in MrBayes v3.2.7 (Ronquist et al., 2012) using the Markov Chain Monte Carlo (MCMC) algorithm, with 2,000,000 generations (ngen = 2,000,000) and a sampling frequency of 1,000 (samplefreq = 1000). The resulting phylogenetic tree was visualized and edited using FigTree v1.4 (Rambaut, 2014).

2.4. Greenhouse bioassay under controlled conditions

From October 2023 to February 2024, a bioassay was conducted under controlled conditions through mechanical inoculation of the two zucchini yellow mosaic virus isolates, ZYMV_Lim and ZYMV_Chos, in three commercial zucchini cultivars.

2.4.1. Plant material

For the greenhouse experiment, three commercial cultivars of C. pepo were evaluated, obtained from different seed suppliers. The plant material included the open-pollinated cultivar ‘Gray Zucchini’ and the hybrids ‘Hurakan F1’ and ‘Rocio F1’.

2.4.2. Mechanical inoculation and symptom assessment

Seeds of susceptible C. pepo ‘Gray Zucchini’, previously reported as susceptible to ZYMV (Valencia et al., 2025), were sown in REKYVA Peat Substrate Professional in 100-cell trays. Five days after emergence, seedlings were transplanted into pots containing a sterile substrate composed of sand, compost, and soil (1:1:1).

Reactivation of the ZYMV_Lim and ZYMV_Chos isolates from the lyophilized tissues “Lim” and “Chos” was performed by independent mechanical inoculation onto cotyledon leaves. Infected leaf tissue was homogenized in distilled water (pH 6.5) and mechanically inoculated using carborundum (600 mesh) as an abrasive. At 15 days post-inoculation (dpi), newly developed leaves showing clear viral symptoms, characterized by severe mosaic, were used as inoculum sources for subsequent inoculations of the three commercial cultivars of C. pepo.

Seeds of ‘Gray Zucchini’, ‘Hurakan F1’, and ‘Rocio F1’ were sown and transplanted following the same procedure described above. Cotyledon leaves were then mechanically inoculated with the reactivated ZYMV_Lim and ZYMV_Chos isolates, with three replicates per treatment. Negative controls consisted of plants inoculated only with distilled water. Following inoculation, plants were maintained in a greenhouse under natural environmental conditions, with a mean temperature of approximately 18.5 °C and a natural photoperiod of approximately 12 h 22 min until symptom evaluation at 15 dpi. Symptom presence or absence was evaluated and compared among treatments and their respective controls. Symptoms were recorded using evaluation sheets and documented photographically against a dark background.

2.4.3. Back-inoculation assay

The assay was performed by mechanically inoculating susceptible ‘Gray Zucchini’ plants with sap extracted from leaf tissue of plants that showed no visible symptoms. This assay was conducted to determine the presence of latent or asymptomatic infection by ZYMV.

2.4.4. ZYMV severity scale

A severity scale ranging from 0 to 4 was established based on foliar symptoms of ZYMV: 0 (no symptoms), 1 (mild mosaic), 2 (mosaic), 3 (mosaic with blistering and leaf deformation), and 4 (severe leaf reduction with deformation). Severity was comparatively evaluated among viral isolates and cultivars, using three replicates per treatment. Symptoms were recorded at 15 dpi through written descriptions and photographic documentation against a dark background. The proposed diagrammatic severity scale was developed to standardize symptom assessment in this study. As it was not formally validated for inter- or intra-rater reliability, it should be considered a descriptive assessment tool.

2.4.5. Symptom severity induced by ZYMV_Lim and ZYMV_Chos isolates

After determining the symptom severity caused by ZYMV independently in each zucchini cultivar (‘Gray Zucchini’, ‘Hurakan F1’, and ‘Rocio F1’), a simultaneous comparative assay was conducted using the three cultivars inoculated with the two isolates, ZYMV_Lim and ZYMV_Chos. In this experiment, all plants were inoculated during the same period under homogeneous experimental conditions. Each treatment included three replicates, along with the corresponding mock-inoculated control, in which plants were treated only with the extraction buffer used for leaf tissue maceration.

2.5. Field evaluation under natural ZYMV infection

The same three cultivars evaluated under greenhouse conditions were included in the field experiment, with the additional incorporation of the hybrid ‘Modena F1’. On 17 April 2024, the field trial was established in a commercial plot with a documented history of viral disease incidence in zucchini cultivation. Conventional agronomic practices were applied throughout the crop cycle, using a drip irrigation system. Prior to trial establishment, soil characterization was conducted to determine its physicochemical properties and to ensure suitable conditions of fertility management for an ideal crop. The agronomic management and microclimatic conditions were identical across all randomized blocks. The experiment was conducted under natural infection in a commercial horticultural area surrounded by cucurbit fields at different crop growth stages. Because ZYMV is transmitted in a non-persistent manner by transient aphids, all plots were exposed to the same natural infection environment.

2.5.1. Experimental design and statistical analysis

A randomized complete block design was used with three replicates per treatment. Each experimental plot consisted of 30 plants arranged in three rows, with 1.4 m spacing between rows and 0.9 m between plants within rows. Each plot covered an area of 37.8 m², resulting in a total block area of 151.2 m² that included the four treatments (‘Gray Zucchini’, ‘Hurakan F1’, ‘Rocío F1’, and ‘Modena F1’). The variables evaluated under field conditions included weekly disease incidence (%), yield (kg ha⁻¹), and the number and weight of fruits with and without ZYMV symptoms. Data were analyzed by analysis of variance (ANOVA), and mean comparisons were performed using Tukey’s honestly significant difference (HSD) test at a significance level of α = 0.05.

2.5.2. Field detection of ZYMV by DAS-ELISA and RT-PCR

ZYMV detection under field conditions was performed at 65 days after transplanting (DAT) (21 June 2024) using the DAS-ELISA technique with specific antibodies (QC-SOP-0087) obtained from DSMZ (Braunschweig, Germany), following the manufacturer’s instructions. The results were subsequently confirmed by RT-PCR using the specific primers designed by Valencia et al. (2025): ZYMV4274_F (5′-CGGTGAAAGTCAAAACAGAGGAC-3′) and ZYMV4663_R (5′-CTTCCTAGTCGCTGAATCCTCTC-3′), which amplify a 412-bp fragment corresponding to a partial region of the CI protein-coding gene of ZYMV.

3. Results

3.1. Detection and identification of ZYMV

During 2023, plants of C. pepo cv. ‘Rocío F1’ showing mosaic and leaf deformation symptoms were observed in commercial production fields located in Chosica, Peru (Figure 1). Serological analysis by DAS-ELISA, complemented with high-throughput sequencing of total RNA from the sample designated “Chos”, revealed that the observed symptoms were exclusively associated with ZYMV infection. Furthermore, no evidence of mixed infections with other viruses affecting cucurbits was detected.

Figure 1
Symptoms such as mosaic and leaf deformation are associated with zucchini yellow mosaic virus infection in Cucurbita pepo cv. ‘Rocio F1’, collected in Chosica (sample “Chos”) on October 29, 2023.

3.2. Severity Scale

Based on the symptoms observed under controlled conditions, five severity grades (0, 1, 2, 3, and 4) were established for ZYMV-induced disease in C. pepo, as graphically illustrated in Figure 2. Grade 0 corresponded to symptomless plants; grade 1 to plants showing mild mosaic symptoms; grade 2 to plants with mosaic and leaf deformation; grade 3 to plants exhibiting mosaic, blisters, and deformation; and grade 4 to plants with reduced leaf blades, deformation, and blisters.

Figure 2
Severity scale of zucchini yellow mosaic virus symptoms in zucchini leaves: (A) asymptomatic (G0: Grade 0), (B) mild mosaic (G:1 Grade 1), (C) mosaic (G2: Grade 2), (D) mosaic, blisters, and leaf deformation (G3: Grade 3), and (E) reduced leaf blade, blisters, and deformation (G4: Grade 4).

3.3. Differential severity of ZYMV_Lim and ZYMV_Chos isolates

Based on the proposed severity scale, the ZYMV_Chos isolate exhibited greater aggressiveness than ZYMV_Lim in the evaluated cultivars. In the cultivar ‘Hurakan F1’, ZYMV_Chos reached severity grade 4, whereas ZYMV_Lim induced symptoms corresponding only to grade 3. Comparative analysis of the three evaluated cultivars revealed that ‘Gray Zucchini’ was susceptible to both isolates, exhibiting severity grade 3. In contrast, ‘Hurakan F1’ showed tolerance to ZYMV_Lim (grade 1) but high susceptibility to ZYMV_Chos (grade 4). The cultivar ‘Rocío F1’ remained symptomless following inoculation with ZYMV_Lim (grade 0); however, it exhibited a tolerant response to ZYMV_Chos (grade 2), suggesting a potential resistance-breaking capacity associated with this viral isolate (Figure 3). Back-inoculation assays confirmed that all symptomless ‘Rocío F1’ plants (3/3) were latently infected with ZYMV_Lim, as sap extracted from these plants induced severe mosaic symptoms in the susceptible cultivar ‘Gray Zucchini’ at 15 days post-inoculation.

Figure 3
Symptoms induced by zucchini yellow mosaic virus isolates ZYMV_Lim and ZYMV_Chos in three zucchini cultivars: ‘Gray Zucchini’, ‘Hurakan F1’, and ‘Rocio F1’. Mock controls were included for each assay. Leaf symptoms were recorded 14 days post-inoculation (dpi). Severity grades are indicated as G2 (grade 2), G3 (grade 3), and G4 (grade 4). Scale bar = 2 cm. Photographs were taken in La Molina on February 8, 2024.

3.4. HTS and sequence data analysis

Based on the HTS data, the genomic sequence of the ZYMV_Chos isolate (GenBank PX442250) was assembled, revealing a genome length of 9,586 nt, a GC content of 42.7%, and a coding capacity for a polyprotein of 3,080 amino acids. Comparison with the ZYMV_Lim isolate sequence (GenBank OQ181225) showed a nucleotide identity of 99.0%. However, twenty-two amino acid differences were identified within the protein 1 (P1) and helper component-proteinase (HC-Pro) regions (Figure 4), both of which are associated with viral transmission and virulence. In contrast, no amino acid differences were detected in the coat protein (CP) region.

Figure 4
Comparison of the amino acid sequences of the Peruvian zucchini yellow mosaic virus isolates ZYMV_Lim and ZYMV_Chos. Red vertical bars in the upper panel indicate amino acid substitution sites (n = 22) across the viral polyprotein. The lower panel shows a magnified view of the 460–550 aa region, where variable amino acid residues are highlighted in blue. Sequence analysis was performed using Geneious Prime.

3.5. Phylogenetic and recombination analyses

Recombination analysis of 153 nucleotide sequences corresponding to the polyprotein-coding region of ZYMV identified 43 recombination events involving four recombinant sequences (PP256252.1, AJ307036.2, OK558795.1, and KY225545.1). These events were consistently detected using seven methods implemented in the RDP software package (RDP, GENECONV, BootScan, MaxChi, Chimaera, SiScan, and 3Seq), with statistical significance values (p) ranging from 1.69 × 10⁻⁶⁵ to 5.09 × 10⁻³.

Bayesian inference, performed using 149 sequences after excluding recombinant isolates, grouped the three Peruvian isolates (GenBank: PX442250, P443465, and OQ181225), obtained from C. pepo, C. maxima, and C. moschata, into a single clade with high statistical support. This Peruvian clade showed a close phylogenetic relationship with sequences from the United Kingdom (OM471983), the United States (PQ162451), and Australia (MN598576 and MY225547), isolated from Cucumis sativus, C. melo, and C. lanatus, respectively (Figure 5 and 6).

Figure 5
Phylogenetic tree inferred from 149 nucleotide sequences corresponding to the polyprotein region of zucchini yellow mosaic virus. The analysis was performed using Bayesian inference with MrBayes under the GTR+I+G nucleotide substitution model, with 2 million generations. Sequence labels in each clade follow the format: GenBank accession number_country_host. Values shown at the nodes represent posterior probabilities. Sequences corresponding to Peruvian isolates are highlighted in blue. Argentina (Arg), Australia (Aus), Brazil (Bras), Canada (Can), China (Chin), Côte d'Ivoire (Cot), Czech Republic (Cze), Egypt (Egy), France (Fran), Germany (Ger), Hungary (Hung), India (Ind), Iran (Iran), Israel (Isr), Italy (Ita), Japan (Jap), South Korea (Kor/SK/Sko), Papua New Guinea (Papu), Peru (Per), Portugal (Port), Réunion (RU), Singapore (Sing), Slovakia (Slov), Spain (Spa), Sudan (Sud), Taiwan (Tai), Timor-Leste (EastT), Trinidad and Tobago (Trin), Turkey (Turk), United Kingdom (UK) and United States (USA).

Figure 6
Summary of the phylogenetic tree shown in Figure 5. Blue clades correspond to Peruvian isolates of zucchini yellow mosaic virus. ZYMV_Chos: PX442250_Per_Cpepo (Genbank_Peru_Cucurbita pepo), ZYMV_Lim: OQ181225_Per_Cmax (Genbank_Peru_Cucurbita maxima), whereas red clades correspond to sequences from Brazil and Argentina.

In contrast, other available South American sequences from Argentina (KT598222) and Brazil (MN364667) clustered within a distinct clade together with Asian sequences from China and Japan, showing clear separation from the clade containing the Peruvian isolates and sequences from Iran, Canada, France, the United States, the United Kingdom, and Australia. These findings indicate a marked phylogenetic divergence between the Brazilian and Argentine isolates compared with the Peruvian isolates.

Overall, the phylogenetic analysis revealed a tendency for ZYMV sequences to cluster according to their geographic origin. This phylogenetic pattern suggests that Peruvian isolates may be associated with independent introduction routes, possibly linked to the international movement of seeds from regions with which they share greater genetic affinity.

3.6. ZYMV under field conditions

Zucchini cultivars were cultivated between April 17 and July 7, 2024, during which an average temperature of 18.52 °C was recorded, with mean minimum and maximum temperatures of 13.77 °C and 26.40 °C, respectively (SENAMHI, 2024). The experimental field soil exhibited a sandy loam texture, consisting of 55% sand, 26–28% silt, and 17–19% clay. In addition, the soil showed a slightly acidic pH (6.65), moderate electrical conductivity (2.47 dS/m), low carbonate content, and high levels of phosphorus (109.8 ppm) and potassium (640 ppm), conditions considered favorable for optimal zucchini development.

DAS-ELISA analysis of 50 plants at 65 DAT revealed that 38% (19/50) were infected with ZYMV. Viral infection was subsequently confirmed by RT-PCR, indicating high infection pressure under field conditions. Infection rates were highest in the cultivars ‘Gray Zucchini’ (10/20), ‘Hurakan F1’ (4/10), and ‘Rocío F1’ (4/10), while only one plant tested positive in the cultivar ‘Modena F1’ (1/10).

3.6.1. Differential symptom severity among Cucurbita pepo cultivars

Under field conditions with natural ZYMV infection, evaluated at 66 DAT, the open-pollinated cultivar ‘Gray Zucchini’ (Figure 7A) exhibited severe foliar symptoms characterized by yellow mosaic, blisters, leaf deformation, and reduced leaf blade development, as well as pronounced fruit deformation. In contrast, the hybrid ‘Hurakan F1’ (Figure 7B) showed moderate mosaic symptoms accompanied by leaf deformation and, similarly, deformed fruits. The cultivar ‘Rocío F1’ (Figure 7C) displayed very mild mosaic symptoms and slight fruit deformation, behaving as a tolerant cultivar under the evaluated conditions. Likewise, Modena F1’ (Figure 7D) exhibited only mild symptoms, including slight mosaic and a low incidence of fruit deformation, also confirming its tolerant profile. A healthy zucchini leaf and fruit, free of viral symptoms, are presented in Figure 7E as a reference for the normal phenotype of non-infected plants.

Figure 7
Symptoms associated with natural zucchini yellow mosaic virus infection under field conditions in Chosica at 66 days after transplanting (Jun 22, 2024). Cultivar ‘Gray Zucchini’ exhibited yellow mosaic, blisters, leaf deformation, reduced leaf blade development, and severe fruit deformation (A); ‘Hurakan F1’ showed moderate mosaic, leaf deformation, and fruits deformation (B); ‘Rocio F1’ displayed mild mosaic and slight fruit deformation (C); ‘Modena F1’ exhibited slight mosaic and fruit deformation (D); and healthy zucchini leaf and fruit free of viral symptoms are shown as a reference (E). Scale bar = 2 cm.

3.6.2. Spatial and temporal dynamics of ZYMV incidence

The spatial analysis of disease incidence revealed marked differences in the response of the evaluated cultivars. The open-pollinated cultivar ‘Gray Zucchini’ exhibited high susceptibility, reaching the highest incidence of ZYMV-infected plants (87.78%) at 81 DAT (Figure 8). In contrast, the hybrid cultivars showed lower incidence levels: ‘Hurakan F1’ recorded 16.67%, ‘Modena F1’ 13.33%, and ‘Rocío F1’ the lowest incidence, with 12.22%. Analysis of variance (ANOVA) of the temporal data, based on the Area Under the Disease Progress Curve (AUDPC) calculated from disease incidence, revealed statistically significant differences among zucchini cultivars in response to ZYMV infection. Tukey’s multiple comparison test (α = 0.05) showed that ‘Gray Zucchini’ had the highest AUDPC value (2453.5), indicating a more rapid disease progression. In contrast, the hybrids ‘Hurakan F1’, ‘Modena F1’, and ‘Rocío F1’ did not differ significantly from each other and exhibited significantly lower AUDPC values (672.11, 560.01, and 470.28, respectively) (Figure 9), suggesting a tolerant response to the pathogen.

Figure 8
Field incidence of zucchini yellow mosaic virus in zucchini cultivars grown under drip irrigation conditions, evaluated at 81 days after transplanting (July 7, 2024). Green dots indicate asymptomatic plants, whereas red dots indicate symptomatic plants. Cultivars are abbreviated as follows: Gz, ‘Gray Zucchini’; Hura, ‘Hurakan F1’; Roci, ‘Rocio F1’; and Mode, ‘Modena F1’.

Figure 9
Area under the disease progress curve (AUDPC) of zucchini yellow mosaic virus severity in four zucchini cultivars evaluated under field conditions. Boxplots represent disease progression values for ‘Gray Zucchini’, ‘Hurakan F1’, ‘Modena F1’, and ‘Rocio F1’. Different lowercase letters indicate significant differences among cultivars according to Tukey’s HSD test (p < 0.05). Mean AUDPC values are shown within each box. ANOVA revealed significant differences among treatments (F (3,6) = 45.92, p = 0.0002, η² = 0.918).

Overall, both spatial and temporal analyses confirmed that the open-pollinated cultivar was considerably more susceptible to ZYMV than the evaluated commercial hybrids.

3.6.3. Yield performance and symptom expression

The results revealed significant differences (p < 0.05) among the evaluated zucchini cultivars for all analyzed variables. Regarding the number of asymptomatic fruits, ‘Gray Zucchini’ showed intermediate values, whereas the hybrids ‘Rocío F1’ and ‘Modena F1’ recorded the highest values (71,340 and 73,280 fruits ha⁻¹, respectively), without significant differences between them but significantly higher than ‘Hurakan F1’. In contrast, the number of symptomatic fruits was significantly greater in ‘Gray Zucchini’, while ‘Rocío F1’ and ‘Modena F1’ exhibited the lowest values (Table 1).

Table 1
Agronomic performance of four commercial zucchini cultivars evaluated under field conditions naturally infected with zucchini yellow mosaic virus (ZYMV).

Variable	Cultivar z				α	C.V. (%)
	Gray Zucchini	Hurakan F1	Rocío F1	Modena F1
Number of asymptomatic fruits (fruits·ha−1)	59,877 B	57,848 B	71,340 A	73,280 A	<0.05	1.43
Number of symptomatic fruits (fruits·ha−1)	2,733.7 A	881.8 AB	88.2 B	264.6 B	<0.05	2.63
Weight of asymptomatic fruits (kg·ha−1)	47,896 C	50,821 BC	64,195 A	31,696 D	<0.05	28.15
Weight of symptomatic fruits (kg·ha−1)	1,358.6 A	440.5 AB	83.3 B	98.8 B	<0.05	23.17
Total yield (kg·ha−1)	57,455 B	56,210 B	65,715 A	34,313 C	<0.05	2.54

• ^z
  Means followed by the same letter within each row are not significantly different among cultivars according to the multiple comparison test (P < 0.05).
• C.V.: coefficient of variation.

In terms of asymptomatic fruit weight, ‘Rocío F1’ achieved the highest yield (64,195 kg ha⁻¹), followed by ‘Hurakan F1’, whereas ‘Modena F1’ showed the lowest value. Conversely, the weight of symptomatic fruits was significantly higher in ‘Gray Zucchini’ (1,358.6 kg ha⁻¹) compared with the evaluated hybrids.

Finally, total yield analysis showed that ‘Rocío F1’ achieved the highest value (65,715 kg ha⁻¹), differing statistically from the other genotypes. ‘Gray Zucchini’ and ‘Hurakan F1’ exhibited intermediate yields with no significant differences between them, whereas ‘Modena F1’ recorded the lowest yield (34,313 kg ha⁻¹). Coefficients of variation ranged from 1.43% to 28.15%, indicating adequate experimental precision for most of the evaluated variables.

4. Discussion

The recent detection of ZYMV in Peruvian cucurbit production systems has raised questions regarding the biological behavior of local viral populations. Although the virus was previously reported infecting C. maxima in coastal production areas (Valencia et al., 2025), the present study confirms a new isolate (ZYMV_Chos) affecting zucchini and demonstrates biological differences among Peruvian isolates. The symptoms observed in infected plants were like those previously described for ZYMV-infected cucurbits, particularly severe mosaic, leaf deformation, and fruit distortion (Ali et al., 2024; Mahmoud et al., 2025). In contrast, the absence of visible symptoms in some infected plants agrees with previous reports in partially resistant cultivars, indicating that symptom expression depends on the interaction between host genotype and viral isolate (Shrestha et al., 2021; Ahsan et al., 2023).

The greater aggressiveness of ZYMV_Chos compared with ZYMV_Lim, particularly in ‘Hurakan F1’, confirms that closely related isolates may differ substantially in pathogenic behavior. Similar findings have been reported for ZYMV populations infecting C. pepo, where symptom severity varies according to both isolate virulence and cultivar susceptibility (Clarke et al., 2020; Nováková et al., 2015). The partial resistance breakdown observed in ‘Rocío F1’ suggests the presence of more virulent variants within local populations, a phenomenon previously associated with intensive cultivation systems and the continuous exposure of resistant cultivars to viral selection pressure (Fereres et al., 1992; Shrestha et al., 2021). These findings reinforce that resistance to ZYMV is isolate-dependent and should be evaluated using local viral populations.

Genomic sequencing revealed amino acid differences between ZYMV_Chos and ZYMV_Lim, mainly in the P1 and HC-Pro proteins. P1 and HC-Pro are commonly associated with virulence-related functions in potyviruses, including replication efficiency and suppression of antiviral RNA silencing (Kaldis et al., 2018; Pollari et al., 2020; Goh et al., 2023). Previous studies demonstrated that mutations in HC-Pro may alter symptom expression by affecting suppression of siRNA-mediated defense pathways (Fuellgrabe et al., 2011), while changes in P1 can influence viral accumulation and infectivity (Hýsková et al., 2024). Although functional analyses were not performed, the amino acid substitutions identified in P1 and HC-Pro may be associated with the stronger symptoms induced by ZYMV_Chos; however, their direct contribution to virulence and resistance-breaking remains to be experimentally validated. In contrast, the high conservation observed in the CP region suggests stronger functional constraints on this structural protein, as previously described for ZYMV populations (Damayanti et al., 2022).

Phylogenetic analysis based on the complete polyprotein-coding region showed that the Peruvian isolates grouped with sequences from the United Kingdom, the United States, and Australia, but remained separated from South American isolates from Argentina and Brazil. A similar phylogenetic pattern has been reported for other cucurbit potyviruses, where isolate grouping appears to be more related to introduction pathways than to geographic distance (Ahsan et al., 2023; Rabadán and Gómez, 2023; Morelli et al., 2024). The observed phylogenetic relationships suggest independent introduction events into Peru, potentially associated with international seed exchange, which has been recognized as an important long-distance dissemination pathway for cucurbit viruses.

Under field conditions, cultivars differed markedly in disease incidence and yield performance. ‘Gray zucchini’ showed the highest incidence and disease progress, confirming its high susceptibility, whereas ‘Rocío F1’ exhibited lower incidence, reduced AUDPC, and higher marketable yield. Similar responses have been reported in commercial zucchini hybrids carrying partial resistance to ZYMV (Desbiez et al., 2003; Liu et al., 2022; Farinati et al., 2023). Previous studies also identified resistance-associated loci in C. moschata and differential transcriptional responses linked to tolerance mechanisms (Shrestha et al., 2021; Amoroso et al., 2022). Therefore, the superior field performance of ‘Rocío F1’ suggests that tolerant cultivars remain a valuable strategy for disease management, although their effectiveness may vary depending on the aggressiveness of local strains or isolates. While visual phenotyping clearly separates the isolates, future quantitative molecular studies will precisely determine if the mechanism involves restricted systemic movement or lower viral replication. Although ‘Modena F1’ exhibited low disease incidence and produced the highest number of asymptomatic fruits, its total yield was lower because this cultivar naturally produces smaller and thinner fruits than the other cultivars evaluated. The average fruit weight of ‘Modena F1’ was approximately 433 g, whereas the other cultivars produced fruits weighing 800–900 g on average. Therefore, the lower yield of ‘Modena F1’ reflects its inherent horticultural characteristics rather than an adverse effect of ZYMV infection.

Overall, the results demonstrate that Peruvian ZYMV isolates exhibit biological and molecular variability that influences symptom expression, cultivar response, and resistance stability. These findings highlight the importance of characterizing local viral populations and integrating durable host resistance into breeding programs to improve zucchini production under high viral pressure.

5. Conclusion

This study demonstrated that Peruvian isolates of zucchini yellow mosaic virus differ in biological aggressiveness and molecular composition, resulting in distinct symptom expression and cultivar response. The higher virulence of ZYMV_Chos was associated with amino acid variation in the P1 and HC-Pro regions; however, the biological significance of these mutations remains to be determined through functional studies. Although ‘Modena F1’ exhibited the lowest disease incidence, its lower total yield reflected its naturally smaller fruit size, an inherent characteristic of the cultivar. Among the evaluated cultivars, ‘Rocío F1’ showed the most balanced performance, combining a tolerant phytosanitary response with the highest marketable yield under high natural ZYMV pressure. Our results support the need to evaluate local ZYMV populations when selecting resistant materials for zucchini production in Peru.

Acknowledgements

The authors of this research project gratefully acknowledge Universidad Nacional Agraria La Molina for financial support through the Faculty of Agronomy Research Project Funding Program (Resolution: 493-FA-2024). The authors also thank the Virology Laboratory of the Department of Plant Pathology for the technical and scientific support provided throughout the development of this study.

Data Availability Statement

The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

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Edited by

• Editor:
  Takako Matsumura Tundisi

Publication Dates

• Publication in this collection
  11 Sept 2026
• Date of issue
  2026

History

• Received
  18 May 2026
• Accepted
  14 July 2026