Etiology of Chickpea Yellowing Syndrome (CYS) in Argentina: The Role of Fungal and Viral Pathogens

Etiology of Chickpea Yellowing Syndrome (CYS) in Argentina: The Role of Fungal and Viral Pathogens | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Etiology of Chickpea Yellowing Syndrome (CYS) in Argentina: The Role of Fungal and Viral Pathogens Bruno Daniel Pugliese, Patricia Rodriguez Pardina, Juan Pablo Edwards Molina, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8534319/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 22 Mar, 2026 Read the published version in European Journal of Plant Pathology → Version 1 posted 5 You are reading this latest preprint version Abstract Chickpea Yellowing Syndrome (CYS) is a major disease complex affecting chickpea ( Cicer arietinum L.) in Argentina, yet its etiology remains poorly understood. This study aimed to identify primary fungal and viral agents associated with CYS. A comprehensive survey (2020–2022) collected symptomatic and asymptomatic plants from 22 fields in the provinces of Córdoba, Santiago del Estero, Catamarca, and Salta. Fungal agents were identified by morphological and molecular methods. Pathogenicity of 55 Fusarium spp. isolates was evaluated on susceptible cultivar 'Kiara', while aggressiveness of pathogenic isolates was characterized by the Area Under the Disease Progress Curve calculated from disease incidence. Viral presence was screened using ELISA and RT-PCR. Fusarium was the dominant fungal genus (74%), with F. oxysporum being the most prevalent species. Pathogenicity tests confirmed 82% of isolates as pathogenic. Notably, pathogenic isolates were also recovered from asymptomatic plants. Aggressiveness analysis classified isolates into distinct clusters, identifying a highly aggressive F. proliferatum isolate. Crucially, this work provides the first report of bean leafroll virus (BLRV; Luteovirus phaseoli ) and alfalfa mosaic virus (AMV; Alfamovirus AMV ) infecting chickpea in Argentina. BLRV was detected in 36% of fields and AMV in 9%. These findings demonstrate that CYS etiology is complex, resulting from the interaction of Fusarium species and viruses, providing the essential knowledge required to design effective integrated management strategies. Cicer arietinum Fusarium oxysporum Fusarium proliferatum bean leafroll virus alfalfa mosaic virus yellowing Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Chickpea ( Cicer arietinum L.) is a valuable and nutritious crop, ranking third in global pulse production after common bean and pea, with an annual output exceeding 10 million tons. Most of this production is concentrated in India, which contributes over 70% of the world's total supply. Other significant producers include Pakistan, Iran, Turkey, Australia, and Ethiopia (Muehlbauer & Sarker, 2018 ). In South America, Argentina has become a significant producer and exporter of chickpeas, ranking among the world's leading suppliers along with Canada and Australia (Merga & Haji, 2019 ). The 2024 growing season recorded the largest cultivated area and production in the past five years, with 151,000 hectares planted and a yield of 193,000 tons. The main production areas are concentrated in the provinces of Salta, Córdoba, and Santiago del Estero, with 59,600 ha (65,000 Tn), 46,800 ha (85,000 Tn), and 23,000 ha (19,000 Tn) respectively (MAGyP, 2025). Chickpea productivity is limited by a range of biotic stresses. Globally, several pathogens, including fungi, viruses, bacteria, and nematodes, are known to cause diseases that impact yield (Manjunatha et al., 2022 ). Soil-borne fungal pathogens are particularly significant, infecting the chickpea root system and causing symptoms such as wilting, yellowing, and root necrosis (Chilakala et al., 2023 ). Among these, the genus Fusarium is one of the most frequently reported and extensively studied, particularly F. oxysporum f. sp. ciceris (FOC), the causal agent of Fusarium wilt (Trapero-Casas & Jiménez-Díaz, 1985; Dubey et al., 2010 ; Živanov et al., 2022 ). However, multiple other species within this genus have also been identified affecting chickpea roots, including F. redolens (Zaim & Bekkar, 2022 ; Zhou et al., 2021 ; Saeedi & Jamali, 2021 ), F. equiseti (Zhou et al., 2021 ; Moparthi et al., 2024 ), F. solani (Saeedi & Jamali, 2021 ), and F. proliferatum (Duarte-Leal et al., 2020 ; Moparthi et al., 2024 ). In addition to Fusarium , other fungal genera such as Macrophomina (Živanov et al., 2019 ; Dell’Olmo et al., 2022 ; Cota-Barreras et al., 2022 ) and Rhizoctonia (Ram & Singh, 2018 ; Basbagci & Dolar, 2022 ; Akber et al., 2023 ), have been reported associated with this symptomatology. Regarding FOC, two different pathotypes are recognized based on the symptoms they induce in susceptible cultivars: a "yellowing" pathotype, which causes gradual foliar yellowing and eventual plant death, and a "wilting" pathotype, characterized by chlorosis, flaccidity, and premature death (Jiménez-Díaz et al., 2015 ). Furthermore, eight pathogenic races of FOC (0, 1A, 1B/C, 2, 3, 4, 5, and 6) have been described based on their differential virulence on a set of host cultivars (Haware & Nene, 1982 ). Races 0 and 1B/C are associated with the yellowing symptoms, while the remaining races cause wilt (Jiménez-Gasco et al., 2004 ). The economic impact of Fusarium wilt depends on the specific cultivar-race interaction, with yield losses reaching as high as 75–100% in cases where a virulent race infects a highly susceptible cultivar (Navas-Cortés et al., 2000 ). Viral diseases, alongside fungal pathogens, constitute another major constraint to chickpea production. At least 40 viruses from 19 genera have been documented infecting chickpeas worldwide. Viruses associated with chickpea stunt and chlorotic dwarf diseases, such as bean leafroll virus (BLRV; Luteovirus phaseoli ), beet western yellows virus ( Polerovirus BWYV ), and chickpea chlorotic stunt virus ( Polerovirus CPCSV ), are of major economic importance. Other viruses such as alfalfa mosaic virus (AMV; Alfamovirus AMV ) and cucumber mosaic virus (CMV; Cucumovirus CMV ), while perhaps of lesser economic impact, are epidemiologically significant due to their ability to be transmitted by seed (Chatzivassiliou, 2021 ). The primary mode of transmission for most chickpea viruses is via aphids, either in a persistent (e.g., BLRV) or non-persistent (e.g., AMV) manner (Kaiser et al., 1990 ). In recent years, viral diseases have become a primary factor in crop failure, with yield losses frequently ranging from 30% to 50% (Cun, 2022 ). In recent growing seasons, a high incidence of chickpea plants exhibiting a symptom complex of chlorosis, yellowing, necrosis, and plant death has been observed across Argentina. This condition has been broadly termed the "Chickpea Yellowing Syndrome" (CYS). This symptomatology is non-specific and can be caused by a variety of pathogens, leading to potential misdiagnosis, particularly with Fusarium wilt (Westerlund et al., 1974 ; Jiménez-Fernández et al., 2011 ). The widespread and severe nature of CYS in Argentina led to the hypothesis that multiple biotic agents are likely involved in its etiology. Therefore, the aim of this study was to identify the primary biotic agents associated with the occurrence of CYS in the major production regions of Argentina with a focus on fungal and viral pathogens. Materials and methods Samples collection Sample collection was conducted over three years (2020–2022) across a total of 22 fields in the main chickpea-producing provinces of Argentina: Córdoba, Santiago del Estero, Catamarca, and Salta. The specific number of fields sampled per province and year, along with their corresponding IDs used in this study, is detailed in Table 1 . The geographical distribution of these sampled fields is illustrated in Fig. 1 . Table 1 Summary of chickpea fields sampled for Chickpea Yellowing Syndrome (CYS) diagnosis between 2020 and 2022 Province Sampling year Number of fields Field IDs Córdoba 2020 6 CBA-1, CBA-2, CBA-3, CBA-4, CBA-5, CBA-6 2021 3 CBA-7, CBA-8, CBA-9 Santiago del Estero 2020 1 SDE-1 2021 3 SDE-2, SDE-3, SDE-4 2022 2 SDE-5, SDE-6 Catamarca 2021 2 CAT-1, CAT-2 2022 1 CAT-3 Salta 2021 3 SAL-1, SAL-2, SAL-3 2022 1 SAL-4 In each field, five symptomatic plants exhibiting characteristic yellowing (Fig. 2 ) were collected along with five nearby asymptomatic control plants, totaling ten plants per field. Plants were carefully extracted using a shovel, retaining their root system. Each sample was placed in an individual polyethylene bag and stored at 4°C until processing. Fungal diagnosis Fungal diagnosis was performed via in vitro culture and morphological identification of asexual reproductive and vegetative structures. Stem segments (approximately 5 cm above and 5 cm below the soil level) were excised from each sample. These segments were then sectioned longitudinally and subsequently transversely into 0.5 cm pieces. Within a laminar air flow cabinet, the resulting tissue pieces were superficially disinfected with 70% ethanol for 30 seconds, followed by a 0.5 g/L active chlorine sodium hypochlorite solution for 2 minutes. After disinfection, the tissue was washed three times with sterile distilled water and dried on sterile filter paper. Between 15 and 20 disinfected stem pieces per sample were placed in Petri dishes containing potato dextrose agar (PDA; Britania, Buenos Aires, Argentina) with streptomycin sulfate (150 mg/L) and incubated at 24°C under 12 hours light/12 hours dark cycle. The plates were inspected by optical microscopy (Nikon Eclipse E100, Tokyo, Japan) using the 10x and 40x objectives after 5 to 7 days to register any fungus growing from the vegetal tissue (Azevedo et al., 2017 ). Monosporic isolates Fungal isolates obtained from the stem pieces were first characterized based on colony morphology and microscopic features. Representative isolates of each distinct morphotype were subcultured onto fresh PDA medium and incubated at 24° C for 7 days and 12 hours photoperiod of white light. After this incubation period, monosporic cultures were generated to ensure genetic homogeneity. A conidial suspension was prepared from each isolate and adjusted to a concentration of 1×10 6 conidia/mL using a hemocytometer. A 100µl aliquot of this suspension was then spread onto water agar (WA) medium using a sterile inoculation loop to achieve single spore separation. These plates were incubated at 21° C for 24 hours with a 12-hour light/12-hour dark cycle. Following incubation, individual germinated conidia were carefully selected under a stereo microscope (Model XTD-217T, BioTraza, Hangzhou, China) and aseptically transferred to new PDA plates (Smith & Onions, 1983 ). The resulting single-colony cultures, which originated from a single spore, were considered monosporic and used for subsequent studies. Molecular identification of Fusarium oxysporum A species-specific PCR was carried out to identify the monosporic Fusarium isolates belonging to F. oxysporum . Initially, genomic DNA was extracted from each isolate using the EasyPure® Genomic DNA Kit (TransGen Biotech, Beijing, China), following the manufacturer's instructions. PCR amplification was subsequently carried out using the F. oxysporum –specific primers FOF1 and FOR1 (Table 2 ), as designed by Mishra et al. ( 2003 ). PCR was performed in a Labnet MultiGene™ OptiMax Thermal Cycler (Labnet International, Edison, NJ, USA) under the following cycling conditions: an initial denaturation step at 95°C for 1 minute, followed by 25 cycles of denaturation at 94°C for 1 minute, annealing at 58°C for 30 seconds, and extension at 72°C for 1 minute. A final extension step was conducted at 72°C for 7 minutes. The resulting amplified products were analyzed by electrophoresis on a 1.5% agarose gel, stained with GelRed™ 2.5X (Biotium, Fremont, CA, USA). The presence of F. oxysporum was confirmed by the visualization of a fragment corresponding to the expected product size of 340 bp. A subset of the 340 bp amplicons was sequenced to validate the species-specific identifications. As further molecular confirmation, two conserved gene regions, translation elongation factor 1-alpha (EF − 1α) and beta-tubulin (β-tubulin), were amplified and sequenced from ten randomly selected isolates. The EF − 1α region was amplified using primers EF1 and EF2 (O’Donnell et al., 1998 ) following the thermal cycling conditions described by Karlsson et al. ( 2016 ). The β-tubulin region was amplified using primers T1 and T22 as per the protocol of O’Donnell and Cigelnik (1997) (Table 2 ). Amplified products were visualized on a 1.5% agarose gel to confirm the expected fragment sizes of approximately 500–600 bp for EF − 1α and 1400 bp for β-tubulin. Fragments were purified using the EasyPure® PCR Purification Kit (TransGen Biotech, Beijing, China) and sent for sequencing at Macrogen (Seoul, South Korea). The resulting sequences were analyzed with the Basic Local Alignment Search Tool for nucleotides (BLASTn) against the National Center for Biotechnology Information (NCBI) database to confirm their identity as F. oxysporum . Table 2 List of primers used in this study for the molecular identification and characterization of Fusarium isolates. The table details the target gene, primer names, nucleotide sequences (5'–3'), the expected size of the amplified product in base pairs (bp), and the corresponding literature reference for each primer set Target Primer name Sequence (5' − 3') Expected size (bp) Reference F. oxysporum species identification FOF1 ACA TAC CAC TTG TTG CCT CG 340 Mishra et al. ( 2003 ) FOR1 CGC CAA TCA ATT TGA GGA ACG Translation Elongation Factor 1-alpha (EF-1α) EF1 ATG GGT AAG GAR GAC AAG AC ~ 500–600 O’Donnell et al. ( 1998 ) EF2 GGA RGT ACC AGT SAT CAT GTT Beta-tubulin (β-tubulin) T1 AAC AAC TGG GCC AAG GGT CAC ~ 1400 O’Donnell & Cigelnik (1997) T22 TCT GGA TGT TGT TGG GAA TCC Fungal pathogenicity test Inoculum A total of 55 Fusarium spp. isolates were selected from the monosporic collection established during field surveys in 2020 and 2021. These isolates were evaluated in pathogenicity tests conducted over two growing seasons in 2022 (Year 1) and 2023 (Year 2). The selection was designed to represent the diversity of the full collection, comprising isolates from different geographical regions (Córdoba, Catamarca, Santiago del Estero, and Salta) and host origins (symptomatic and asymptomatic plants). Given the high prevalence of F. oxysporum in the initial survey, the majority of selected isolates belonged to this species. However, to assess the pathogenic potential of other Fusarium species, one isolate that tested negative in the F. oxysporum -specific PCR assay was also included. The species identity of this isolate was confirmed by amplifying and sequencing the EF − 1α and β-tubulin gene regions, followed by BLASTn analysis using the protocols described above. A complete list of the isolates and their characteristics is provided in Appendix Table 1. Each isolate was cultured in Petri dishes containing PDA and incubated for 15 days under a photoperiod of 12 hours of white light and 12 hours of near-UV light (380–400 nm). The conidial suspensions were prepared by harvesting the conidia and adjusting their concentration to 1x10⁵ conidia/mL using a hemocytometer (Cachinero et al., 2002 ). Plants Chickpea seeds of the cultivar Kiara, reported as susceptible to CYS, were surface disinfected by immersion in 70% ethanol for 30 seconds, followed by a 0.5 g/L active chlorine sodium hypochlorite solution for 4 minutes. The seeds were dried under a laminar flow hood and placed on water agar in Petri dishes to germinate. After 5 days at 21°C with a 12-hour photoperiod, healthy, fungus-free seedlings were transplanted into plug trays containing a sterile 1:1 (v/v) mixture of soil and perlite. Plants were grown under greenhouse conditions for 15 days until they reached approximately 15 cm in height with three to four developed leaves. Inoculation procedure The developed plants were removed from their cells, and their roots were washed with sterile distilled water. To create infection points, small lesions were made on the apical root tissues. Roots of nine plants per isolate were submerged in the corresponding conidial suspension for 16 hours (Cachinero et al., 2002 ). Following fungal inoculation, three plants were transplanted into each pot containing a 3:1 mixture of tyndallized soil and perlite. The experiment was arranged in a randomized complete block design (RCBD) with three replications. To promote nodulation, Mesorhizobium ciceri (Rhizoliq Top®, Rhizobacter, Pergamino, Argentina) was applied directly to the root system of each plant using a micropipette, following the manufacturer's recommended dose. The pots were maintained in an experimental plot equipped with anti-aphid netting and an overhead sprinkler irrigation system to ensure the substrate remained at field capacity. Environmental Data Acquisition To characterize the environmental conditions during each experimental year, daily temperature data were acquired using the Google Earth Engine platform. Data were extracted from the ERA5-Land global reanalysis dataset. Disease Assessment and Pathogen Re-isolation Disease assessment was conducted at 7-, 14-, and 21-days post-inoculation (DPI). Plants exhibiting characteristic CYS symptoms upon visual inspection were recorded as diseased. Isolates were classified as 'pathogenic' if they induced characteristic symptoms in at least one plant during the assessment period; otherwise, they were considered non-pathogenic. Disease incidence was then calculated for each pot at each observation time point as the percentage of plants showing symptoms. To fulfill Koch's postulates, a subset of diseased plants was sampled at the end of the disease assessment period. Fusarium sp. pathogens were re-isolated from the symptomatic tissue. Data Processing and Statistical Analysis Data collected from the disease assessments were processed using the R software environment (version 4.5.0; R Core Team, 2024 ). The Area Under the Disease Progress Curve (AUDPC) was calculated for each experimental unit based on disease incidence recorded at 7, 14, and 21 days post-inoculation utilizing the agricolae package (version 1.3-7; de Mendiburu, 2023 ). Statistical analyses were performed exclusively on data from isolates confirmed as pathogenic. To characterize the aggressiveness of the isolates, AUDPC data were modeled using a linear mixed-effects model fitted with the lme4 package (version 1.1.35.5; Bates et al., 2015 ). In this analysis, the fungal isolate was defined as a fixed effect, while the experimental year and blocks nested within years were treated as random effects. Model assumptions, including normality of residuals and homogeneity of variance, were verified using the diagnostic tools available in the DHARMa package (version 0.4.7; Hartig, 2024 ). Subsequently, isolates were categorized into distinct aggressiveness groups via a post-hoc cluster analysis using the Scott-Knott algorithm ( P < 0.05 ; Scott & Knott, 1974 ) implemented in the ScottKnott package (version 1.3.3; Jelihovschi et al., 2014 ), and data visualization was performed using ggplot2 (version 3.5.2; Wickham, 2016 ). Viral diagnosis Leaf and apical shoots tissue samples from each plant originally collected from the 22 surveyed fields were analyzed by Enzyme-Linked Immunosorbent Assay (ELISA), using protocols such as DAS-ELISA, PTA-ELISA, or TAS-ELISA (Clark & Adams, 1977 ; Mowat et al., 1987; Sukhacheva et al., 1996 ) with specific antibodies for A. AMV (self-production); L. phaseoli (DSMZ, Braunschweig, Germany); the Potyvirus genus (Bioreba, Reinach, Switzerland); C. CMV , soybean dwarf virus (SbDV; Luteovirus glycinis ), tobacco ringspot virus (TRSV; Nepovirus nicotianae ), tobacco streak virus (TSV; Ilarvirus TSV ), and tomato spotted wilt virus (TSWV; Orthotospovirus tomatomaculae ) (all from Agdia, Elkhart, IN, USA). In all assays, each plate included six healthy samples and one positive sample, as controls. The reactions were quantified using a Thermo Labsystem MultisKan MS spectrophotometer (Thermo Electron Corporation, Vantaa, Finland). Samples were classified as positive when the Optical Density at 405 nm (OD405) value exceeded either 0.100 or the mean absorbance of the healthy controls plus three standard deviations (cut-off), whichever was higher. The presence of viruses was confirmed by RT-PCR (Choi et al., 2013 ) using custom-designed oligonucleotides for each identified species. The amplified products were analyzed by electrophoresis on agarose gels (1.5%), stained with GelRed™ Biotium (2.5X), to verify the presence of a fragment of the expected size. For the detected viruses, the relative incidence was calculated by field, defined as the proportion of virus-infected plants considering only symptomatic samples (Madden et al., 2007 ). Results Fungal diagnosis Three distinct fungal genera were identified through morphological diagnosis by optical microscopy: Fusarium , Macrophomina , and Rhizoctonia . Fusarium emerged as the most prevalent genus, accounting for 74.09% of the samples. Following in frequency was Macrophomina (4.09%), while Rhizoctonia (2.27%) was the least frequent. All but one of the Macrophomina detections occurred as a co-infection with Fusarium . Additionally, 25% of the samples showed no evidence of fungal growth. Fusarium revealed its presence in both symptomatic and asymptomatic plants. Among the Fusarium -isolates, 55.8% were derived from plants exhibiting symptoms, while the remaining 44.2% originated from asymptomatic plants. Fusarium was detected in all sampled regions and years (Fig. 3 ), with five fields (CBA-3, CBA-4, CBA-6, CAT-3, and SAL-2) showing 100% sample positivity. Fusarium -positive asymptomatic samples were found in all fields except for SDE-2. Regarding plants with CYS symptoms, two conditions were observed equally: in 11 of 22 fields, all symptomatic plants tested positive for Fusarium , while in the remaining 11 fields, symptomatic plants were found that tested negative for this fungal genus. A total of 169 monosporic fungal isolates were recovered in this study. The collection was predominantly composed of Fusarium spp. (n = 163), with a smaller number of Macrophomina sp. (n = 3) and Rhizoctonia sp. (n = 3) isolates also identified. These isolates are now part of the pathogen collection at the Instituto de Patología Vegetal (IPAVE-CIAP-INTA). The molecular identification by PCR using F. oxysporum -specific primers confirmed that 152 of the 163 Fusarium isolates initially identified by optical microscopy were F. oxysporum . Furthermore, sequence analysis of the EF-1α and β-tubulin gene regions from ten selected isolates confirmed their identity validating the accuracy of the species-specific PCR assay. The list of these isolates used for validation, along with their GenBank accession numbers, is provided in the Appendix Table 2. Fungal pathogenicity test The post-inoculation temperature conditions differed between the two trial years. As illustrated in Fig. 4 , the 2023 trial (Year 2) was conducted under a generally warmer thermal regime compared to the 2022 trial (Year 1). The mean daily temperature from June 15 to July 15 was 10.47°C in 2023, which was higher than the 9.2°C recorded for the same period in 2022. Among the 55 isolates evaluated, 10 (18%) failed to induce disease symptoms in either experimental year and were therefore designated as non-pathogenic. These non-pathogenic isolates were recovered from all four sampled provinces (Córdoba, Catamarca, Salta, and Santiago del Estero) and included specimens obtained from both symptomatic and asymptomatic plant tissues (Appendix Table 1). The remaining 45 isolates (82%) were confirmed as pathogenic, successfully inducing disease symptoms. This group included the single non- F. oxysporum isolate evaluated (5.1.1.2), which was identified as F. proliferatum by sequencing its EF − 1α and β-tubulin gene regions (Appendix Table 2). Notably, 14 of these pathogenic isolates were recovered from asymptomatic plants. Finally, the identity of the inoculated pathogens was verified by successful re-isolation from symptomatic tissue, fulfilling Koch's postulates. The assessment of disease incidence, modeled as the overall AUDPC across the two experimental years, revealed significant variability in aggressiveness among the isolates. The Scott-Knott cluster analysis differentiated the tested pathogens into three distinct groups (Fig. 5 ). Group A, exhibiting the highest aggressiveness, comprised only the F. proliferatum isolate 5.1.1.2 (Córdoba, 2020), which recorded the highest overall mean AUDPC, and the F. oxysporum isolate 15.2.1.2 (Córdoba, 2021). The remaining isolates were distributed between Group B (n = 26) and Group C (n = 17), exhibiting moderate and low aggressiveness, respectively. Both groups included isolates from all four sampled provinces, as well as specimens recovered from both symptomatic and asymptomatic plants. Viral diagnosis Out of all the viruses tested across both symptomatic and asymptomatic samples, only BLRV and AMV were detected. Notably, both were exclusively identified in symptomatic samples. The presence of these viruses was confirmed by PCR, using the specific primers BLRV-F/BLRV-R for BLRV (Trucco et al., 2016 ) and AMV-F/AMV-R for AMV (Trucco et al., 2022 ). In both cases, a fragment of the expected size was obtained: 955bp for BLRV and 884bp for AMV. BLRV exhibited a wider distribution and higher prevalence compared to AMV. It was detected in 8 of the 22 fields sampled, resulting in a prevalence of 36% and a highest relative incidence of 60%. In contrast, AMV was found in only 2 of the sampled fields, corresponding to a prevalence of 9%, with the highest recorded relative incidence of 40% (Figs. 6 and 7 ). The geographical distribution of the detected viruses varied. BLRV was identified across multiple provinces, including Córdoba, Catamarca, Santiago del Estero, and Salta. AMV, however, was only detected in samples from Córdoba. Furthermore, while BLRV was consistently detected in samples collected throughout all years of the study period, AMV was identified exclusively in samples from 2020. Discussion According to Agrios ( 2005 ), a syndrome is the complete set of effects exhibited by a host in response to a particular disease. This study of CYS in Argentina's main production regions reveals that yellowing symptoms frequently accompanied by vascular necrosis, stunting, or internode shortening may be caused by multiple pathogens. Our results indicate that although pathogenic fungi are predominant, CYS cannot be attributed to a single specific agent, as viruses causing chickpea yellowing were also detected. These findings underscore the complex etiology of the syndrome. Three fungal genera were detected from the collected chickpea samples: Fusarium , Macrophomina , and Rhizoctonia . Fusarium was the most prevalent genus, which is consistent with reports on similar chickpea disease complexes worldwide. Trapero-Casas and Jiménez-Díaz (1985) reported similar fungal genera, including Fusarium and Macrophomina , associated with yellowing, wilt, and root rot in chickpeas in Southern Spain. Considering the Fusarium isolates collected in our study, F. oxysporum was the most frequently identified species. This observation aligns with surveys conducted in Montana, USA (Moparthi et al., 2024 ), western Iran (Younesi et al., 2021 ), and Algeria (Sekkal et al., 2025 ), where F. oxysporum was also the main Fusarium species identified. In contrast, recent studies from Alberta, Canada (Zhou et al., 2021 ), and other regions of Iran (Saeedi & Jamali, 2021 ) reported F. redolens as the most prevalent species. This variation highlights that the composition of the Fusarium complex associated with chickpea is likely region-specific, influenced by local environmental conditions and agronomic practices. The high frequency of the genus Fusarium recovered from asymptomatic plants has been documented in other pathosystems (Kuldau & Yates, 2000 ) and might suggest a latent endophytic phase for some species of the genus within the host plant. An endophytic organism is one that lives inside a plant without causing any visible symptoms of disease (Petrini, 1991 ). Malcolm et al. ( 2013 ) proposed that some fungal species could be capable of acting as either an endophyte or a pathogen, with the outcome depending on the specific plant species, the fungal genotype, and the set of environmental conditions in a particular location. Such endophytic potential could explain the widespread presence of Fusarium across the surveyed fields of this study, even in the absence of visible disease. The results of the pathogenicity test revealed significant variability in both pathogenicity and aggressiveness among the Fusarium spp. isolates associated with chickpea in Argentina. The recovery of non-pathogenic isolates, some even from symptomatic samples, concurs with previous studies (Edel et al., 1997 ; Jiménez-Fernández et al., 2011 ) reporting that the soil and plant microbiome comprises a complex consortium of pathogenic and non-pathogenic fungi. Regarding the pathogenic isolates, the Scott-Knott analysis classified them into three distinct clusters according to the AUDPC that they generated across the two experimental years. Group A, composed of only two isolates that caused the highest disease levels, included the sole F. proliferatum isolate (5.1.1.2) evaluated. Although this species was less frequent in our survey compared to F. oxysporum , its classification suggests it possesses a high pathogenic potential. Consistent with reports of this pathogen causing chickpea yellowing, vascular wilt, and root rot in other regions (Duarte-Leal et al., 2020 ; Moparthi et al., 2024 ), our findings indicate that F. proliferatum could pose an emerging threat to chickpea production in Argentina if favorable conditions promote its spread. The remaining F. oxysporum isolates were distributed between Groups B and C, exhibiting moderate to low disease levels in the pathogenicity test. This intraspecific variability in aggressiveness is consistent with previous characterizations of F. oxysporum populations (Dubey et al., 2010 ; Bayraktar et al., 2012; Moparthi et al., 2024 ). Furthermore, the lack of a clear association between geographical origin and these pathogenic groups suggests that diverse variants are widely distributed across Argentina's production regions, rather than being restricted to specific areas. Moreover, the identification of isolates recovered from asymptomatic plants within these pathogenic groups could support the hypothesis of a latent or endophytic phase in the CYS pathosystem introduced earlier in this discussion (Malcolm et al., 2013 ). Our investigation into viral pathogens revealed that bean leafroll virus and alfalfa mosaic virus were present in symptomatic plants, highlighting their involvement in the CYS etiology. This is the first report of both viruses affecting chickpea crops in Argentina. The broader distribution and higher incidence of BLRV relative to AMV suggests its role as a more significant viral contributor to CYS in Argentina. However, the detection of AMV is epidemiologically relevant due to its ability to be transmitted by seed, with reported transmission rates ranging from 0.1% to 1% (Jones & Coutts, 1996 ; Chatzivassiliou, 2021 ). This mechanism poses a risk of introducing the virus into new production areas through infected germplasm. BLRV and AMV have been previously studied in Argentina affecting alfalfa crops (Trucco et al., 2016 , 2018 ). This context is significant as both crops coexist during the same growing season and share common aphid species that act as vectors. Alfalfa crops located near chickpea fields may therefore serve as a perennial reservoir for both viruses and their vectors such as Acyrthosiphon pisum and Myzus persicae (Ávalos et al., 2010 ; Davis et al., 2017 ; Johnstone et al., 1984 ), which could facilitate virus transmission. The geographical distribution of the detected viruses, with AMV exclusively found in Córdoba and BLRV also showing a notable presence in this province, points to regional factors influencing viral epidemiology. This pattern could be linked to the distribution and population density of their aphid vectors. Finally, the presence of CYS symptomatic plants that tested negative for the targeted pathogens could suggest that the syndrome's development involves either abiotic stressors, such as nutrient deficiencies or water stress (Chen et al., 2011 ), or other biological agents like nematodes or phytoplasmas that were not the focus of this study (Briar et al., 2023 ; Balol et al., 2021 ). Conclusion This study represents the first integral diagnosis of the biotic agents associated with CYS in Argentina, applying a consistent methodology across the country's primary production regions over three consecutive growing seasons. Our findings confirm the complex etiology of this syndrome, which cannot be attributed to a single pathogen and includes, at a minimum, fungi and viruses. The fungal component of CYS is dominated by Fusarium species, with F. oxysporum being the most prevalent agent identified. However, there is variability in aggressiveness within this population that appears independent of geographical origin, since variants exhibiting moderate to low aggressiveness were identified across all sampled regions. We also identified numerous non-pathogenic isolates, even from symptomatic plants, as well as pathogenic isolates recovered from asymptomatic plants. This suggests complex interactions between these fungi and their hosts, including potential endophytic roles. Finally, the identification of a highly aggressive F. proliferatum isolate, while less frequent, highlights its potential as an emerging threat to the crop. This work provides the first report of BLRV and AMV infecting chickpea crops in Argentina. The higher prevalence and wider distribution of BLRV indicate it is a key viral component of the CYS complex. AMV, due to its potential for seed transmission, represents a specific risk for pathogen introduction into new production areas. In conclusion, CYS in Argentina is a disease resulting from the interaction of multiple pathogens, including a dominant Fusarium group and at least two viruses. A thorough understanding of this etiology is the first necessary step for designing efficient and sustainable management strategies. Future efforts should therefore prioritize a deeper characterization of all causal agents, alongside comprehensive epidemiological studies to quantify the prevalence and impact of each causal agent. This research must also investigate how environmental factors and crop management practices modulate disease expression. Declarations Competing Interests: The authors have no relevant financial or non-financial interests to disclose. Funding: This work was supported by the Instituto Nacional de Tecnología Agropecuaria (INTA) and the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET). References Agrios, G. N. (2005). Plant pathology . Elsevier. Akber, M. A., Mubeen, M., Sohail, M. 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10:51:31","extension":"xml","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":208952,"visible":true,"origin":"","legend":"","description":"","filename":"EJPPD26000180structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8534319/v1/fe8b5ee1436f15dbe88dec57.xml"},{"id":100136848,"identity":"64ab293f-8d4c-4ec6-8d19-5a13364322d5","added_by":"auto","created_at":"2026-01-13 10:51:31","extension":"html","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":226723,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8534319/v1/7f0d98a772e4cedc1d357565.html"},{"id":100367095,"identity":"a4863d97-d22e-4de6-b01a-7c254515325e","added_by":"auto","created_at":"2026-01-16 07:56:46","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":71977,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eGeographical distribution of the 22 chickpea fields sampled in Argentina between 2020 and 2022. (A) Map of Argentina, with the main chickpea-producing region included in the study highlighted by an oval. (B) Detailed map of the sampled regions within the provinces of Salta, Catamarca, Santiago del Estero, and Córdoba. Each point represents a sampled field, with the color indicating the year of collection as specified in the legend (brown: 2020; green: 2021; yellow: 2022)\u003c/em\u003e\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8534319/v1/35cbd661c237325b83b3e268.jpg"},{"id":100366843,"identity":"f1540321-5da6-4410-87ab-817cbc6a9a09","added_by":"auto","created_at":"2026-01-16 07:56:35","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":321236,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eCharacteristic symptoms of Chickpea Yellowing Syndrome (CYS) observed in commercial chickpea fields in Argentina\u003c/em\u003e\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8534319/v1/223fd7e833488286143cdd56.jpg"},{"id":100366881,"identity":"b4aca02f-335a-42a6-bbba-57b3a287d584","added_by":"auto","created_at":"2026-01-16 07:56:36","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":144181,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eDiagnosis of genus Fusarium in symptomatic and asymptomatic chickpea plants from 22 fields. Each stacked bar represents a single field, showing the percentage of plants in each diagnostic category. Field IDs on the x-axis specify the province (CBA: Córdoba, CAT: Catamarca, SDE: Santiago del Estero, SAL: Salta), a field number, and the sampling year (2020-2022)\u003c/em\u003e\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8534319/v1/019110fc681ac3e554cea1a0.jpg"},{"id":100136823,"identity":"12d4ddee-34b0-4a1b-b8e3-705e4535c149","added_by":"auto","created_at":"2026-01-13 10:51:30","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":86570,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eDaily mean temperature (°C) for Year 1 (2022, yellow) and Year 2 (2023, grey). Shaded areas represent the daily minimum and maximum temperature range. The vertical dashed red line indicates the date of infection (June 15) for both years\u003c/em\u003e\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8534319/v1/54d2cf78d0926ae2e1b619e1.jpg"},{"id":100366348,"identity":"f312265b-6c7f-4d93-a644-6ce0080dec59","added_by":"auto","created_at":"2026-01-16 07:56:14","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":89331,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eAggressiveness of 45 pathogenic Fusarium spp. isolates, expressed as the Area Under the Disease Progress Curve (AUDPC). Yellow dots represent the estimated mean AUDPC for each isolate derived from a linear mixed model. Horizontal yellow bars indicate the 95% confidence intervals. Background grey points display the raw observed data. Distinct letters indicate significant differences in aggressiveness groups according to the Scott-Knott cluster analysis (p \u0026lt; 0.05). Isolates are ordered vertically from highest to lowest aggressiveness\u003c/em\u003e\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8534319/v1/94d83f9f429e632afe8e6a3d.jpg"},{"id":100366857,"identity":"bfe1f0c5-f4ff-47df-9a07-a88f67b63304","added_by":"auto","created_at":"2026-01-16 07:56:35","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":60480,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eRelative incidence of bean leafroll virus in the 22 sampled fields. Relative incidence was calculated as the percentage of symptomatic plants that tested positive for the virus in each field.\u003c/em\u003e \u003cem\u003eField IDs on the x-axis specify the province (CBA: Córdoba, CAT: Catamarca, SDE: Santiago del Estero, SAL: Salta), field number, and the sampling year (2020-2022)\u003c/em\u003e\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8534319/v1/7c076a5ddeeb97020fc6e849.jpg"},{"id":100136831,"identity":"ebed1bd7-22ce-4689-b223-18142dff2e4a","added_by":"auto","created_at":"2026-01-13 10:51:30","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":52861,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eRelative incidence of alfalfa mosaic virus in the 22 sampled fields. Relative incidence was calculated as the percentage of symptomatic plants that tested positive for the virus in each field. Field IDs on the x-axis specify the province (CBA: Córdoba, CAT: Catamarca, SDE: Santiago del Estero, SAL: Salta), field number, and the sampling year (2020-2022)\u003c/em\u003e\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8534319/v1/591d11417e3fdf1b0b7139c3.jpg"},{"id":105223292,"identity":"7a58a03b-7e93-4656-9c18-9b36a1debdea","added_by":"auto","created_at":"2026-03-23 16:02:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1593382,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8534319/v1/8f0f5e09-f041-4646-82dc-dc0740cd3412.pdf"},{"id":100368252,"identity":"4d06f1b8-369e-4f56-a069-aa667019b317","added_by":"auto","created_at":"2026-01-16 07:57:46","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":26005,"visible":true,"origin":"","legend":"","description":"","filename":"Appendix.docx","url":"https://assets-eu.researchsquare.com/files/rs-8534319/v1/9deaab2f93161e6eb55ff2b7.docx"}],"financialInterests":"","formattedTitle":"Etiology of Chickpea Yellowing Syndrome (CYS) in Argentina: The Role of Fungal and Viral Pathogens","fulltext":[{"header":"Introduction","content":"\u003cp\u003eChickpea (\u003cem\u003eCicer arietinum\u003c/em\u003e L.) is a valuable and nutritious crop, ranking third in global pulse production after common bean and pea, with an annual output exceeding 10\u0026nbsp;million tons. Most of this production is concentrated in India, which contributes over 70% of the world's total supply. Other significant producers include Pakistan, Iran, Turkey, Australia, and Ethiopia (Muehlbauer \u0026amp; Sarker, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn South America, Argentina has become a significant producer and exporter of chickpeas, ranking among the world's leading suppliers along with Canada and Australia (Merga \u0026amp; Haji, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The 2024 growing season recorded the largest cultivated area and production in the past five years, with 151,000 hectares planted and a yield of 193,000 tons. The main production areas are concentrated in the provinces of Salta, C\u0026oacute;rdoba, and Santiago del Estero, with 59,600 ha (65,000 Tn), 46,800 ha (85,000 Tn), and 23,000 ha (19,000 Tn) respectively (MAGyP, 2025).\u003c/p\u003e \u003cp\u003eChickpea productivity is limited by a range of biotic stresses. Globally, several pathogens, including fungi, viruses, bacteria, and nematodes, are known to cause diseases that impact yield (Manjunatha et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSoil-borne fungal pathogens are particularly significant, infecting the chickpea root system and causing symptoms such as wilting, yellowing, and root necrosis (Chilakala et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Among these, the genus \u003cem\u003eFusarium\u003c/em\u003e is one of the most frequently reported and extensively studied, particularly \u003cem\u003eF. oxysporum\u003c/em\u003e f. sp. \u003cem\u003eciceris\u003c/em\u003e (FOC), the causal agent of Fusarium wilt (Trapero-Casas \u0026amp; Jim\u0026eacute;nez-D\u0026iacute;az, 1985; Dubey et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Živanov et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, multiple other species within this genus have also been identified affecting chickpea roots, including \u003cem\u003eF. redolens\u003c/em\u003e (Zaim \u0026amp; Bekkar, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Zhou et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Saeedi \u0026amp; Jamali, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), \u003cem\u003eF. equiseti\u003c/em\u003e (Zhou et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Moparthi et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), F. \u003cem\u003esolani\u003c/em\u003e (Saeedi \u0026amp; Jamali, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and \u003cem\u003eF. proliferatum\u003c/em\u003e (Duarte-Leal et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Moparthi et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In addition to \u003cem\u003eFusarium\u003c/em\u003e, other fungal genera such as \u003cem\u003eMacrophomina\u003c/em\u003e (Živanov et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Dell\u0026rsquo;Olmo et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Cota-Barreras et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and \u003cem\u003eRhizoctonia\u003c/em\u003e (Ram \u0026amp; Singh, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Basbagci \u0026amp; Dolar, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Akber et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), have been reported associated with this symptomatology.\u003c/p\u003e \u003cp\u003eRegarding FOC, two different pathotypes are recognized based on the symptoms they induce in susceptible cultivars: a \"yellowing\" pathotype, which causes gradual foliar yellowing and eventual plant death, and a \"wilting\" pathotype, characterized by chlorosis, flaccidity, and premature death (Jim\u0026eacute;nez-D\u0026iacute;az et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Furthermore, eight pathogenic races of FOC (0, 1A, 1B/C, 2, 3, 4, 5, and 6) have been described based on their differential virulence on a set of host cultivars (Haware \u0026amp; Nene, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1982\u003c/span\u003e). Races 0 and 1B/C are associated with the yellowing symptoms, while the remaining races cause wilt (Jim\u0026eacute;nez-Gasco et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). The economic impact of Fusarium wilt depends on the specific cultivar-race interaction, with yield losses reaching as high as 75\u0026ndash;100% in cases where a virulent race infects a highly susceptible cultivar (Navas-Cort\u0026eacute;s et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2000\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eViral diseases, alongside fungal pathogens, constitute another major constraint to chickpea production. At least 40 viruses from 19 genera have been documented infecting chickpeas worldwide. Viruses associated with chickpea stunt and chlorotic dwarf diseases, such as bean leafroll virus (BLRV; \u003cem\u003eLuteovirus phaseoli\u003c/em\u003e), beet western yellows virus (\u003cem\u003ePolerovirus BWYV\u003c/em\u003e), and chickpea chlorotic stunt virus (\u003cem\u003ePolerovirus CPCSV\u003c/em\u003e), are of major economic importance. Other viruses such as alfalfa mosaic virus (AMV; \u003cem\u003eAlfamovirus AMV\u003c/em\u003e) and cucumber mosaic virus (CMV; \u003cem\u003eCucumovirus CMV\u003c/em\u003e), while perhaps of lesser economic impact, are epidemiologically significant due to their ability to be transmitted by seed (Chatzivassiliou, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The primary mode of transmission for most chickpea viruses is via aphids, either in a persistent (e.g., BLRV) or non-persistent (e.g., AMV) manner (Kaiser et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1990\u003c/span\u003e). In recent years, viral diseases have become a primary factor in crop failure, with yield losses frequently ranging from 30% to 50% (Cun, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn recent growing seasons, a high incidence of chickpea plants exhibiting a symptom complex of chlorosis, yellowing, necrosis, and plant death has been observed across Argentina. This condition has been broadly termed the \"Chickpea Yellowing Syndrome\" (CYS). This symptomatology is non-specific and can be caused by a variety of pathogens, leading to potential misdiagnosis, particularly with Fusarium wilt (Westerlund et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e1974\u003c/span\u003e; Jim\u0026eacute;nez-Fern\u0026aacute;ndez et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The widespread and severe nature of CYS in Argentina led to the hypothesis that multiple biotic agents are likely involved in its etiology. Therefore, the aim of this study was to identify the primary biotic agents associated with the occurrence of CYS in the major production regions of Argentina with a focus on fungal and viral pathogens.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eSamples collection\u003c/p\u003e \u003cp\u003eSample collection was conducted over three years (2020\u0026ndash;2022) across a total of 22 fields in the main chickpea-producing provinces of Argentina: C\u0026oacute;rdoba, Santiago del Estero, Catamarca, and Salta. The specific number of fields sampled per province and year, along with their corresponding IDs used in this study, is detailed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The geographical distribution of these sampled fields is illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cem\u003eSummary of chickpea fields sampled for Chickpea Yellowing Syndrome (CYS) diagnosis between 2020 and 2022\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProvince\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSampling year\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNumber of fields\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eField IDs\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eC\u0026oacute;rdoba\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2020\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCBA-1, CBA-2, CBA-3, CBA-4, CBA-5, CBA-6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2021\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCBA-7, CBA-8, CBA-9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eSantiago del Estero\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2020\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSDE-1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2021\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSDE-2, SDE-3, SDE-4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSDE-5, SDE-6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCatamarca\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2021\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCAT-1, CAT-2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCAT-3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSalta\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2021\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSAL-1, SAL-2, SAL-3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSAL-4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn each field, five symptomatic plants exhibiting characteristic yellowing (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) were collected along with five nearby asymptomatic control plants, totaling ten plants per field. Plants were carefully extracted using a shovel, retaining their root system. Each sample was placed in an individual polyethylene bag and stored at 4\u0026deg;C until processing.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFungal diagnosis\u003c/p\u003e \u003cp\u003eFungal diagnosis was performed via \u003cem\u003ein vitro\u003c/em\u003e culture and morphological identification of asexual reproductive and vegetative structures. Stem segments (approximately 5 cm above and 5 cm below the soil level) were excised from each sample. These segments were then sectioned longitudinally and subsequently transversely into 0.5 cm pieces. Within a laminar air flow cabinet, the resulting tissue pieces were superficially disinfected with 70% ethanol for 30 seconds, followed by a 0.5 g/L active chlorine sodium hypochlorite solution for 2 minutes. After disinfection, the tissue was washed three times with sterile distilled water and dried on sterile filter paper.\u003c/p\u003e \u003cp\u003eBetween 15 and 20 disinfected stem pieces per sample were placed in Petri dishes containing potato dextrose agar (PDA; Britania, Buenos Aires, Argentina) with streptomycin sulfate (150 mg/L) and incubated at 24\u0026deg;C under 12 hours light/12 hours dark cycle. The plates were inspected by optical microscopy (Nikon Eclipse E100, Tokyo, Japan) using the 10x and 40x objectives after 5 to 7 days to register any fungus growing from the vegetal tissue (Azevedo et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMonosporic isolates\u003c/h2\u003e \u003cp\u003eFungal isolates obtained from the stem pieces were first characterized based on colony morphology and microscopic features. Representative isolates of each distinct morphotype were subcultured onto fresh PDA medium and incubated at 24\u0026deg; C for 7 days and 12 hours photoperiod of white light. After this incubation period, monosporic cultures were generated to ensure genetic homogeneity. A conidial suspension was prepared from each isolate and adjusted to a concentration of 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e conidia/mL using a hemocytometer. A 100\u0026micro;l aliquot of this suspension was then spread onto water agar (WA) medium using a sterile inoculation loop to achieve single spore separation. These plates were incubated at 21\u0026deg; C for 24 hours with a 12-hour light/12-hour dark cycle. Following incubation, individual germinated conidia were carefully selected under a stereo microscope (Model XTD-217T, BioTraza, Hangzhou, China) and aseptically transferred to new PDA plates (Smith \u0026amp; Onions, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e1983\u003c/span\u003e). The resulting single-colony cultures, which originated from a single spore, were considered monosporic and used for subsequent studies.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMolecular identification of\u003c/b\u003e \u003cb\u003eFusarium oxysporum\u003c/b\u003e\u003c/p\u003e \u003cp\u003eA species-specific PCR was carried out to identify the monosporic \u003cem\u003eFusarium\u003c/em\u003e isolates belonging to \u003cem\u003eF. oxysporum\u003c/em\u003e. Initially, genomic DNA was extracted from each isolate using the EasyPure\u0026reg; Genomic DNA Kit (TransGen Biotech, Beijing, China), following the manufacturer's instructions. PCR amplification was subsequently carried out using the \u003cem\u003eF. oxysporum\u003c/em\u003e\u0026ndash;specific primers FOF1 and FOR1 (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), as designed by Mishra et al. (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). PCR was performed in a Labnet MultiGene\u0026trade; OptiMax Thermal Cycler (Labnet International, Edison, NJ, USA) under the following cycling conditions: an initial denaturation step at 95\u0026deg;C for 1 minute, followed by 25 cycles of denaturation at 94\u0026deg;C for 1 minute, annealing at 58\u0026deg;C for 30 seconds, and extension at 72\u0026deg;C for 1 minute. A final extension step was conducted at 72\u0026deg;C for 7 minutes. The resulting amplified products were analyzed by electrophoresis on a 1.5% agarose gel, stained with GelRed\u0026trade; 2.5X (Biotium, Fremont, CA, USA). The presence of \u003cem\u003eF. oxysporum\u003c/em\u003e was confirmed by the visualization of a fragment corresponding to the expected product size of 340 bp.\u003c/p\u003e \u003cp\u003eA subset of the 340 bp amplicons was sequenced to validate the species-specific identifications. As further molecular confirmation, two conserved gene regions, translation elongation factor 1-alpha (EF\u0026thinsp;\u0026minus;\u0026thinsp;1α) and beta-tubulin (β-tubulin), were amplified and sequenced from ten randomly selected isolates. The EF\u0026thinsp;\u0026minus;\u0026thinsp;1α region was amplified using primers EF1 and EF2 (O\u0026rsquo;Donnell et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e1998\u003c/span\u003e) following the thermal cycling conditions described by Karlsson et al. (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The β-tubulin region was amplified using primers T1 and T22 as per the protocol of O\u0026rsquo;Donnell and Cigelnik (1997) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Amplified products were visualized on a 1.5% agarose gel to confirm the expected fragment sizes of approximately 500\u0026ndash;600 bp for EF\u0026thinsp;\u0026minus;\u0026thinsp;1α and 1400 bp for β-tubulin. Fragments were purified using the EasyPure\u0026reg; PCR Purification Kit (TransGen Biotech, Beijing, China) and sent for sequencing at Macrogen (Seoul, South Korea). The resulting sequences were analyzed with the Basic Local Alignment Search Tool for nucleotides (BLASTn) against the National Center for Biotechnology Information (NCBI) database to confirm their identity as \u003cem\u003eF. oxysporum\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cem\u003eList of primers used in this study for the molecular identification and characterization of Fusarium isolates. The table details the target gene, primer names, nucleotide sequences (5'\u0026ndash;3'), the expected size of the amplified product in base pairs (bp), and the corresponding literature reference for each primer set\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTarget\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePrimer name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSequence (5' \u0026minus;\u0026thinsp;3')\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eExpected size (bp)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eReference\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eF. oxysporum\u003c/em\u003e species identification\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFOF1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eACA TAC CAC TTG TTG CCT CG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e340\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eMishra et al. (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2003\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFOR1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCGC CAA TCA ATT TGA GGA ACG\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTranslation Elongation Factor 1-alpha (EF-1α)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEF1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eATG GGT AAG GAR GAC AAG AC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e~\u0026thinsp;500\u0026ndash;600\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eO\u0026rsquo;Donnell et al. (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e1998\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEF2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGGA RGT ACC AGT SAT CAT GTT\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eBeta-tubulin (β-tubulin)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eT1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAAC AAC TGG GCC AAG GGT CAC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e~\u0026thinsp;1400\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eO\u0026rsquo;Donnell \u0026amp; Cigelnik (1997)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eT22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTCT GGA TGT TGT TGG GAA TCC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eFungal pathogenicity test\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eInoculum\u003c/h3\u003e\n\u003cp\u003eA total of 55 \u003cem\u003eFusarium\u003c/em\u003e spp. isolates were selected from the monosporic collection established during field surveys in 2020 and 2021. These isolates were evaluated in pathogenicity tests conducted over two growing seasons in 2022 (Year 1) and 2023 (Year 2). The selection was designed to represent the diversity of the full collection, comprising isolates from different geographical regions (C\u0026oacute;rdoba, Catamarca, Santiago del Estero, and Salta) and host origins (symptomatic and asymptomatic plants). Given the high prevalence of \u003cem\u003eF. oxysporum\u003c/em\u003e in the initial survey, the majority of selected isolates belonged to this species. However, to assess the pathogenic potential of other \u003cem\u003eFusarium\u003c/em\u003e species, one isolate that tested negative in the \u003cem\u003eF. oxysporum\u003c/em\u003e-specific PCR assay was also included. The species identity of this isolate was confirmed by amplifying and sequencing the EF\u0026thinsp;\u0026minus;\u0026thinsp;1α and β-tubulin gene regions, followed by BLASTn analysis using the protocols described above. A complete list of the isolates and their characteristics is provided in Appendix Table\u0026nbsp;1.\u003c/p\u003e \u003cp\u003eEach isolate was cultured in Petri dishes containing PDA and incubated for 15 days under a photoperiod of 12 hours of white light and 12 hours of near-UV light (380\u0026ndash;400 nm). The conidial suspensions were prepared by harvesting the conidia and adjusting their concentration to 1x10⁵ conidia/mL using a hemocytometer (Cachinero et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2002\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003ePlants\u003c/h3\u003e\n\u003cp\u003eChickpea seeds of the cultivar Kiara, reported as susceptible to CYS, were surface disinfected by immersion in 70% ethanol for 30 seconds, followed by a 0.5 g/L active chlorine sodium hypochlorite solution for 4 minutes. The seeds were dried under a laminar flow hood and placed on water agar in Petri dishes to germinate. After 5 days at 21\u0026deg;C with a 12-hour photoperiod, healthy, fungus-free seedlings were transplanted into plug trays containing a sterile 1:1 (v/v) mixture of soil and perlite. Plants were grown under greenhouse conditions for 15 days until they reached approximately 15 cm in height with three to four developed leaves.\u003c/p\u003e\n\u003ch3\u003eInoculation procedure\u003c/h3\u003e\n\u003cp\u003eThe developed plants were removed from their cells, and their roots were washed with sterile distilled water. To create infection points, small lesions were made on the apical root tissues. Roots of nine plants per isolate were submerged in the corresponding conidial suspension for 16 hours (Cachinero et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2002\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFollowing fungal inoculation, three plants were transplanted into each pot containing a 3:1 mixture of tyndallized soil and perlite. The experiment was arranged in a randomized complete block design (RCBD) with three replications. To promote nodulation, \u003cem\u003eMesorhizobium ciceri\u003c/em\u003e (Rhizoliq Top\u0026reg;, Rhizobacter, Pergamino, Argentina) was applied directly to the root system of each plant using a micropipette, following the manufacturer's recommended dose. The pots were maintained in an experimental plot equipped with anti-aphid netting and an overhead sprinkler irrigation system to ensure the substrate remained at field capacity.\u003c/p\u003e\n\u003ch3\u003eEnvironmental Data Acquisition\u003c/h3\u003e\n\u003cp\u003eTo characterize the environmental conditions during each experimental year, daily temperature data were acquired using the Google Earth Engine platform. Data were extracted from the ERA5-Land global reanalysis dataset.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eDisease Assessment and Pathogen Re-isolation\u003c/h2\u003e \u003cp\u003eDisease assessment was conducted at 7-, 14-, and 21-days post-inoculation (DPI). Plants exhibiting characteristic CYS symptoms upon visual inspection were recorded as diseased. Isolates were classified as 'pathogenic' if they induced characteristic symptoms in at least one plant during the assessment period; otherwise, they were considered non-pathogenic. Disease incidence was then calculated for each pot at each observation time point as the percentage of plants showing symptoms.\u003c/p\u003e \u003cp\u003eTo fulfill Koch's postulates, a subset of diseased plants was sampled at the end of the disease assessment period. \u003cem\u003eFusarium\u003c/em\u003e sp. pathogens were re-isolated from the symptomatic tissue.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eData Processing and Statistical Analysis\u003c/h3\u003e\n\u003cp\u003eData collected from the disease assessments were processed using the R software environment (version 4.5.0; R Core Team, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The Area Under the Disease Progress Curve (AUDPC) was calculated for each experimental unit based on disease incidence recorded at 7, 14, and 21 days post-inoculation utilizing the agricolae package (version 1.3-7; de Mendiburu, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Statistical analyses were performed exclusively on data from isolates confirmed as pathogenic. To characterize the aggressiveness of the isolates, AUDPC data were modeled using a linear mixed-effects model fitted with the lme4 package (version 1.1.35.5; Bates et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In this analysis, the fungal isolate was defined as a fixed effect, while the experimental year and blocks nested within years were treated as random effects. Model assumptions, including normality of residuals and homogeneity of variance, were verified using the diagnostic tools available in the DHARMa package (version 0.4.7; Hartig, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Subsequently, isolates were categorized into distinct aggressiveness groups via a post-hoc cluster analysis using the Scott-Knott algorithm (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e; Scott \u0026amp; Knott, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e1974\u003c/span\u003e) implemented in the ScottKnott package (version 1.3.3; Jelihovschi et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), and data visualization was performed using ggplot2 (version 3.5.2; Wickham, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eViral diagnosis\u003c/p\u003e \u003cp\u003eLeaf and apical shoots tissue samples from each plant originally collected from the 22 surveyed fields were analyzed by Enzyme-Linked Immunosorbent Assay (ELISA), using protocols such as DAS-ELISA, PTA-ELISA, or TAS-ELISA (Clark \u0026amp; Adams, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1977\u003c/span\u003e; Mowat et al., 1987; Sukhacheva et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e1996\u003c/span\u003e) with specific antibodies for \u003cem\u003eA. AMV\u003c/em\u003e (self-production); \u003cem\u003eL. phaseoli\u003c/em\u003e (DSMZ, Braunschweig, Germany); the Potyvirus genus (Bioreba, Reinach, Switzerland); \u003cem\u003eC. CMV\u003c/em\u003e, soybean dwarf virus (SbDV; \u003cem\u003eLuteovirus glycinis\u003c/em\u003e), tobacco ringspot virus (TRSV; \u003cem\u003eNepovirus nicotianae\u003c/em\u003e), tobacco streak virus (TSV; \u003cem\u003eIlarvirus TSV\u003c/em\u003e), and tomato spotted wilt virus (TSWV; \u003cem\u003eOrthotospovirus tomatomaculae\u003c/em\u003e) (all from Agdia, Elkhart, IN, USA). In all assays, each plate included six healthy samples and one positive sample, as controls. The reactions were quantified using a Thermo Labsystem MultisKan MS spectrophotometer (Thermo Electron Corporation, Vantaa, Finland). Samples were classified as positive when the Optical Density at 405 nm (OD405) value exceeded either 0.100 or the mean absorbance of the healthy controls plus three standard deviations (cut-off), whichever was higher. The presence of viruses was confirmed by RT-PCR (Choi et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) using custom-designed oligonucleotides for each identified species. The amplified products were analyzed by electrophoresis on agarose gels (1.5%), stained with GelRed\u0026trade; Biotium (2.5X), to verify the presence of a fragment of the expected size.\u003c/p\u003e \u003cp\u003eFor the detected viruses, the relative incidence was calculated by field, defined as the proportion of virus-infected plants considering only symptomatic samples (Madden et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eFungal diagnosis\u003c/p\u003e \u003cp\u003eThree distinct fungal genera were identified through morphological diagnosis by optical microscopy: \u003cem\u003eFusarium\u003c/em\u003e, \u003cem\u003eMacrophomina\u003c/em\u003e, and \u003cem\u003eRhizoctonia\u003c/em\u003e. \u003cem\u003eFusarium\u003c/em\u003e emerged as the most prevalent genus, accounting for 74.09% of the samples. Following in frequency was \u003cem\u003eMacrophomina\u003c/em\u003e (4.09%), while \u003cem\u003eRhizoctonia\u003c/em\u003e (2.27%) was the least frequent. All but one of the \u003cem\u003eMacrophomina\u003c/em\u003e detections occurred as a co-infection with \u003cem\u003eFusarium\u003c/em\u003e. Additionally, 25% of the samples showed no evidence of fungal growth.\u003c/p\u003e \u003cp\u003e \u003cem\u003eFusarium\u003c/em\u003e revealed its presence in both symptomatic and asymptomatic plants. Among the \u003cem\u003eFusarium\u003c/em\u003e-isolates, 55.8% were derived from plants exhibiting symptoms, while the remaining 44.2% originated from asymptomatic plants.\u003c/p\u003e \u003cp\u003e \u003cem\u003eFusarium\u003c/em\u003e was detected in all sampled regions and years (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), with five fields (CBA-3, CBA-4, CBA-6, CAT-3, and SAL-2) showing 100% sample positivity. \u003cem\u003eFusarium\u003c/em\u003e-positive asymptomatic samples were found in all fields except for SDE-2. Regarding plants with CYS symptoms, two conditions were observed equally: in 11 of 22 fields, all symptomatic plants tested positive for \u003cem\u003eFusarium\u003c/em\u003e, while in the remaining 11 fields, symptomatic plants were found that tested negative for this fungal genus.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA total of 169 monosporic fungal isolates were recovered in this study. The collection was predominantly composed of \u003cem\u003eFusarium\u003c/em\u003e spp. (n\u0026thinsp;=\u0026thinsp;163), with a smaller number of \u003cem\u003eMacrophomina\u003c/em\u003e sp. (n\u0026thinsp;=\u0026thinsp;3) and \u003cem\u003eRhizoctonia\u003c/em\u003e sp. (n\u0026thinsp;=\u0026thinsp;3) isolates also identified. These isolates are now part of the pathogen collection at the Instituto de Patolog\u0026iacute;a Vegetal (IPAVE-CIAP-INTA).\u003c/p\u003e \u003cp\u003eThe molecular identification by PCR using \u003cem\u003eF. oxysporum\u003c/em\u003e-specific primers confirmed that 152 of the 163 \u003cem\u003eFusarium\u003c/em\u003e isolates initially identified by optical microscopy were \u003cem\u003eF. oxysporum\u003c/em\u003e. Furthermore, sequence analysis of the EF-1α and β-tubulin gene regions from ten selected isolates confirmed their identity validating the accuracy of the species-specific PCR assay. The list of these isolates used for validation, along with their GenBank accession numbers, is provided in the Appendix Table\u0026nbsp;2.\u003c/p\u003e \u003cp\u003eFungal pathogenicity test\u003c/p\u003e \u003cp\u003eThe post-inoculation temperature conditions differed between the two trial years. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, the 2023 trial (Year 2) was conducted under a generally warmer thermal regime compared to the 2022 trial (Year 1). The mean daily temperature from June 15 to July 15 was 10.47\u0026deg;C in 2023, which was higher than the 9.2\u0026deg;C recorded for the same period in 2022.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAmong the 55 isolates evaluated, 10 (18%) failed to induce disease symptoms in either experimental year and were therefore designated as non-pathogenic. These non-pathogenic isolates were recovered from all four sampled provinces (C\u0026oacute;rdoba, Catamarca, Salta, and Santiago del Estero) and included specimens obtained from both symptomatic and asymptomatic plant tissues (Appendix Table\u0026nbsp;1).\u003c/p\u003e \u003cp\u003eThe remaining 45 isolates (82%) were confirmed as pathogenic, successfully inducing disease symptoms. This group included the single non-\u003cem\u003eF. oxysporum\u003c/em\u003e isolate evaluated (5.1.1.2), which was identified as \u003cem\u003eF. proliferatum\u003c/em\u003e by sequencing its EF\u0026thinsp;\u0026minus;\u0026thinsp;1α and β-tubulin gene regions (Appendix Table\u0026nbsp;2). Notably, 14 of these pathogenic isolates were recovered from asymptomatic plants. Finally, the identity of the inoculated pathogens was verified by successful re-isolation from symptomatic tissue, fulfilling Koch's postulates.\u003c/p\u003e \u003cp\u003eThe assessment of disease incidence, modeled as the overall AUDPC across the two experimental years, revealed significant variability in aggressiveness among the isolates. The Scott-Knott cluster analysis differentiated the tested pathogens into three distinct groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Group A, exhibiting the highest aggressiveness, comprised only the \u003cem\u003eF. proliferatum\u003c/em\u003e isolate 5.1.1.2 (C\u0026oacute;rdoba, 2020), which recorded the highest overall mean AUDPC, and the \u003cem\u003eF. oxysporum\u003c/em\u003e isolate 15.2.1.2 (C\u0026oacute;rdoba, 2021). The remaining isolates were distributed between Group B (n\u0026thinsp;=\u0026thinsp;26) and Group C (n\u0026thinsp;=\u0026thinsp;17), exhibiting moderate and low aggressiveness, respectively. Both groups included isolates from all four sampled provinces, as well as specimens recovered from both symptomatic and asymptomatic plants.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eViral diagnosis\u003c/p\u003e \u003cp\u003eOut of all the viruses tested across both symptomatic and asymptomatic samples, only BLRV and AMV were detected. Notably, both were exclusively identified in symptomatic samples. The presence of these viruses was confirmed by PCR, using the specific primers BLRV-F/BLRV-R for BLRV (Trucco et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) and AMV-F/AMV-R for AMV (Trucco et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In both cases, a fragment of the expected size was obtained: 955bp for BLRV and 884bp for AMV.\u003c/p\u003e \u003cp\u003eBLRV exhibited a wider distribution and higher prevalence compared to AMV. It was detected in 8 of the 22 fields sampled, resulting in a prevalence of 36% and a highest relative incidence of 60%. In contrast, AMV was found in only 2 of the sampled fields, corresponding to a prevalence of 9%, with the highest recorded relative incidence of 40% (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe geographical distribution of the detected viruses varied. BLRV was identified across multiple provinces, including C\u0026oacute;rdoba, Catamarca, Santiago del Estero, and Salta. AMV, however, was only detected in samples from C\u0026oacute;rdoba. Furthermore, while BLRV was consistently detected in samples collected throughout all years of the study period, AMV was identified exclusively in samples from 2020.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAccording to Agrios (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), a syndrome is the complete set of effects exhibited by a host in response to a particular disease. This study of CYS in Argentina's main production regions reveals that yellowing symptoms frequently accompanied by vascular necrosis, stunting, or internode shortening may be caused by multiple pathogens. Our results indicate that although pathogenic fungi are predominant, CYS cannot be attributed to a single specific agent, as viruses causing chickpea yellowing were also detected. These findings underscore the complex etiology of the syndrome.\u003c/p\u003e \u003cp\u003eThree fungal genera were detected from the collected chickpea samples: \u003cem\u003eFusarium\u003c/em\u003e, \u003cem\u003eMacrophomina\u003c/em\u003e, and \u003cem\u003eRhizoctonia\u003c/em\u003e. \u003cem\u003eFusarium\u003c/em\u003e was the most prevalent genus, which is consistent with reports on similar chickpea disease complexes worldwide. Trapero-Casas and Jim\u0026eacute;nez-D\u0026iacute;az (1985) reported similar fungal genera, including \u003cem\u003eFusarium\u003c/em\u003e and \u003cem\u003eMacrophomina\u003c/em\u003e, associated with yellowing, wilt, and root rot in chickpeas in Southern Spain. Considering the \u003cem\u003eFusarium\u003c/em\u003e isolates collected in our study, \u003cem\u003eF. oxysporum\u003c/em\u003e was the most frequently identified species. This observation aligns with surveys conducted in Montana, USA (Moparthi et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), western Iran (Younesi et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and Algeria (Sekkal et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), where \u003cem\u003eF. oxysporum\u003c/em\u003e was also the main \u003cem\u003eFusarium\u003c/em\u003e species identified. In contrast, recent studies from Alberta, Canada (Zhou et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and other regions of Iran (Saeedi \u0026amp; Jamali, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) reported \u003cem\u003eF. redolens\u003c/em\u003e as the most prevalent species. This variation highlights that the composition of the \u003cem\u003eFusarium\u003c/em\u003e complex associated with chickpea is likely region-specific, influenced by local environmental conditions and agronomic practices.\u003c/p\u003e \u003cp\u003eThe high frequency of the genus \u003cem\u003eFusarium\u003c/em\u003e recovered from asymptomatic plants has been documented in other pathosystems (Kuldau \u0026amp; Yates, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2000\u003c/span\u003e) and might suggest a latent endophytic phase for some species of the genus within the host plant. An endophytic organism is one that lives inside a plant without causing any visible symptoms of disease (Petrini, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e1991\u003c/span\u003e). Malcolm et al. (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) proposed that some fungal species could be capable of acting as either an endophyte or a pathogen, with the outcome depending on the specific plant species, the fungal genotype, and the set of environmental conditions in a particular location. Such endophytic potential could explain the widespread presence of \u003cem\u003eFusarium\u003c/em\u003e across the surveyed fields of this study, even in the absence of visible disease.\u003c/p\u003e \u003cp\u003eThe results of the pathogenicity test revealed significant variability in both pathogenicity and aggressiveness among the \u003cem\u003eFusarium\u003c/em\u003e spp. isolates associated with chickpea in Argentina. The recovery of non-pathogenic isolates, some even from symptomatic samples, concurs with previous studies (Edel et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Jim\u0026eacute;nez-Fern\u0026aacute;ndez et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) reporting that the soil and plant microbiome comprises a complex consortium of pathogenic and non-pathogenic fungi.\u003c/p\u003e \u003cp\u003eRegarding the pathogenic isolates, the Scott-Knott analysis classified them into three distinct clusters according to the AUDPC that they generated across the two experimental years. Group A, composed of only two isolates that caused the highest disease levels, included the sole \u003cem\u003eF. proliferatum\u003c/em\u003e isolate (5.1.1.2) evaluated. Although this species was less frequent in our survey compared to \u003cem\u003eF. oxysporum\u003c/em\u003e, its classification suggests it possesses a high pathogenic potential. Consistent with reports of this pathogen causing chickpea yellowing, vascular wilt, and root rot in other regions (Duarte-Leal et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Moparthi et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), our findings indicate that \u003cem\u003eF. proliferatum\u003c/em\u003e could pose an emerging threat to chickpea production in Argentina if favorable conditions promote its spread.\u003c/p\u003e \u003cp\u003eThe remaining \u003cem\u003eF. oxysporum\u003c/em\u003e isolates were distributed between Groups B and C, exhibiting moderate to low disease levels in the pathogenicity test. This intraspecific variability in aggressiveness is consistent with previous characterizations of \u003cem\u003eF. oxysporum\u003c/em\u003e populations (Dubey et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Bayraktar et al., 2012; Moparthi et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Furthermore, the lack of a clear association between geographical origin and these pathogenic groups suggests that diverse variants are widely distributed across Argentina's production regions, rather than being restricted to specific areas. Moreover, the identification of isolates recovered from asymptomatic plants within these pathogenic groups could support the hypothesis of a latent or endophytic phase in the CYS pathosystem introduced earlier in this discussion (Malcolm et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOur investigation into viral pathogens revealed that bean leafroll virus and alfalfa mosaic virus were present in symptomatic plants, highlighting their involvement in the CYS etiology. This is the first report of both viruses affecting chickpea crops in Argentina. The broader distribution and higher incidence of BLRV relative to AMV suggests its role as a more significant viral contributor to CYS in Argentina. However, the detection of AMV is epidemiologically relevant due to its ability to be transmitted by seed, with reported transmission rates ranging from 0.1% to 1% (Jones \u0026amp; Coutts, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Chatzivassiliou, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). This mechanism poses a risk of introducing the virus into new production areas through infected germplasm.\u003c/p\u003e \u003cp\u003eBLRV and AMV have been previously studied in Argentina affecting alfalfa crops (Trucco et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). This context is significant as both crops coexist during the same growing season and share common aphid species that act as vectors. Alfalfa crops located near chickpea fields may therefore serve as a perennial reservoir for both viruses and their vectors such as \u003cem\u003eAcyrthosiphon pisum\u003c/em\u003e and \u003cem\u003eMyzus persicae\u003c/em\u003e (\u0026Aacute;valos et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Davis et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Johnstone et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1984\u003c/span\u003e), which could facilitate virus transmission.\u003c/p\u003e \u003cp\u003eThe geographical distribution of the detected viruses, with AMV exclusively found in C\u0026oacute;rdoba and BLRV also showing a notable presence in this province, points to regional factors influencing viral epidemiology. This pattern could be linked to the distribution and population density of their aphid vectors.\u003c/p\u003e \u003cp\u003eFinally, the presence of CYS symptomatic plants that tested negative for the targeted pathogens could suggest that the syndrome's development involves either abiotic stressors, such as nutrient deficiencies or water stress (Chen et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), or other biological agents like nematodes or phytoplasmas that were not the focus of this study (Briar et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Balol et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study represents the first integral diagnosis of the biotic agents associated with CYS in Argentina, applying a consistent methodology across the country's primary production regions over three consecutive growing seasons. Our findings confirm the complex etiology of this syndrome, which cannot be attributed to a single pathogen and includes, at a minimum, fungi and viruses.\u003c/p\u003e \u003cp\u003eThe fungal component of CYS is dominated by \u003cem\u003eFusarium\u003c/em\u003e species, with \u003cem\u003eF. oxysporum\u003c/em\u003e being the most prevalent agent identified. However, there is variability in aggressiveness within this population that appears independent of geographical origin, since variants exhibiting moderate to low aggressiveness were identified across all sampled regions. We also identified numerous non-pathogenic isolates, even from symptomatic plants, as well as pathogenic isolates recovered from asymptomatic plants. This suggests complex interactions between these fungi and their hosts, including potential endophytic roles. Finally, the identification of a highly aggressive \u003cem\u003eF. proliferatum\u003c/em\u003e isolate, while less frequent, highlights its potential as an emerging threat to the crop.\u003c/p\u003e \u003cp\u003eThis work provides the first report of BLRV and AMV infecting chickpea crops in Argentina. The higher prevalence and wider distribution of BLRV indicate it is a key viral component of the CYS complex. AMV, due to its potential for seed transmission, represents a specific risk for pathogen introduction into new production areas.\u003c/p\u003e \u003cp\u003eIn conclusion, CYS in Argentina is a disease resulting from the interaction of multiple pathogens, including a dominant \u003cem\u003eFusarium\u003c/em\u003e group and at least two viruses. A thorough understanding of this etiology is the first necessary step for designing efficient and sustainable management strategies. Future efforts should therefore prioritize a deeper characterization of all causal agents, alongside comprehensive epidemiological studies to quantify the prevalence and impact of each causal agent. This research must also investigate how environmental factors and crop management practices modulate disease expression.\u003c/p\u003e"},{"header":"Declarations","content":" \u003ch2\u003eCompeting Interests:\u003c/h2\u003e \u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e \u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eThis work was supported by the Instituto Nacional de Tecnolog\u0026iacute;a Agropecuaria (INTA) and the Consejo Nacional de Investigaciones Cient\u0026iacute;ficas y T\u0026eacute;cnicas (CONICET).\u003c/p\u003e"},{"header":"References","content":"\u003cul\u003e\n\u003cli\u003eAgrios, G. N. (2005). \u003cem\u003ePlant pathology\u003c/em\u003e. Elsevier.\u003c/li\u003e\n\u003cli\u003eAkber, M. 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Cham: Springer international publishing. https://doi.org/10.1007/978-3-319-24277-4_9 \u003c/li\u003e\n\u003cli\u003eYounesi, H., Darvishnia, M., Bazgir, E., \u0026amp; Chehri, K. (2021). Morphological, molecular and pathogenic characterization of \u003cem\u003eFusarium\u003c/em\u003e spp. associated with chickpea wilt in western Iran. \u003cem\u003eJournal of Plant Protection Research\u003c/em\u003e, 402-413. https://doi.org/10.24425/jppr.2021.139250 \u003c/li\u003e\n\u003cli\u003eZaim, S., \u0026amp; Bekkar, A. A. (2022). First report of \u003cem\u003eFusarium redolens\u003c/em\u003e causing Fusarium yellows on chickpea in Algeria. \u003cem\u003eJournal of Plant Pathology, 104\u003c/em\u003e(2), 835-835. https://doi.org/10.1007/s42161-022-01044-y \u003c/li\u003e\n\u003cli\u003eZhou, Q., Yang, Y., Wang, Y., Jones, C., Feindel, D., Harding, M., \u0026amp; Feng, J. (2021). Phylogenetic, phenotypic and host range characterization of five \u003cem\u003eFusarium\u003c/em\u003e species isolated from chickpea in Alberta, Canada. \u003cem\u003eCanadian Journal of Plant Pathology, 43\u003c/em\u003e(5), 651-657. https://doi.org/10.1080/07060661.2020.1869830 \u003c/li\u003e\n\u003cli\u003eŽivanov, D., Tančić Živanov, S., Savić, A., Uhlarik, A., Miladinov, Z., Medic Pap, S., \u0026amp; Nagl, N. (2022). First Report of \u003cem\u003eFusarium oxysporum\u003c/em\u003e f. sp. \u003cem\u003eciceris\u003c/em\u003e on Chickpea (\u003cem\u003eCicer arietinum\u003c/em\u003e) in Serbia. \u003cem\u003ePlant Disease, 106\u003c/em\u003e(5), 1530. https://doi.org/10.1094/PDIS-09-21-1998-PDN \u003c/li\u003e\n\u003cli\u003eŽivanov, D., Živanov, S. T., Nagl, N., Savić, A., Katanski, S., \u0026amp; Milić, D. (2019). First report of \u003cem\u003eMacrophomina phaseolina\u003c/em\u003e causing dry root rot of chickpea (\u003cem\u003eCicer arietinum\u003c/em\u003e) in Serbia. \u003cem\u003ePlant Disease, 103\u003c/em\u003e(10), 2685. https://doi.org/10.1094/PDIS-03-19-0652-PDN \u003c/li\u003e\n\u003c/ul\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"european-journal-of-plant-pathology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ejpp","sideBox":"Learn more about [European Journal of Plant Pathology](http://link.springer.com/journal/10658)","snPcode":"10658","submissionUrl":"https://www.editorialmanager.com/ejpp/default2.aspx","title":"European Journal of Plant Pathology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Cicer arietinum, Fusarium oxysporum, Fusarium proliferatum, bean leafroll virus, alfalfa mosaic virus, yellowing","lastPublishedDoi":"10.21203/rs.3.rs-8534319/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8534319/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eChickpea Yellowing Syndrome (CYS) is a major disease complex affecting chickpea (\u003cem\u003eCicer arietinum\u003c/em\u003e L.) in Argentina, yet its etiology remains poorly understood. This study aimed to identify primary fungal and viral agents associated with CYS. A comprehensive survey (2020\u0026ndash;2022) collected symptomatic and asymptomatic plants from 22 fields in the provinces of C\u0026oacute;rdoba, Santiago del Estero, Catamarca, and Salta. Fungal agents were identified by morphological and molecular methods. Pathogenicity of 55 \u003cem\u003eFusarium\u003c/em\u003e spp. isolates was evaluated on susceptible cultivar 'Kiara', while aggressiveness of pathogenic isolates was characterized by the Area Under the Disease Progress Curve calculated from disease incidence. Viral presence was screened using ELISA and RT-PCR. \u003cem\u003eFusarium\u003c/em\u003e was the dominant fungal genus (74%), with \u003cem\u003eF. oxysporum\u003c/em\u003e being the most prevalent species. Pathogenicity tests confirmed 82% of isolates as pathogenic. Notably, pathogenic isolates were also recovered from asymptomatic plants. Aggressiveness analysis classified isolates into distinct clusters, identifying a highly aggressive \u003cem\u003eF. proliferatum\u003c/em\u003e isolate. Crucially, this work provides the first report of bean leafroll virus (BLRV; \u003cem\u003eLuteovirus phaseoli\u003c/em\u003e) and alfalfa mosaic virus (AMV; \u003cem\u003eAlfamovirus AMV\u003c/em\u003e) infecting chickpea in Argentina. BLRV was detected in 36% of fields and AMV in 9%. These findings demonstrate that CYS etiology is complex, resulting from the interaction of \u003cem\u003eFusarium\u003c/em\u003e species and viruses, providing the essential knowledge required to design effective integrated management strategies.\u003c/p\u003e","manuscriptTitle":"Etiology of Chickpea Yellowing Syndrome (CYS) in Argentina: The Role of Fungal and Viral Pathogens","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-13 10:51:25","doi":"10.21203/rs.3.rs-8534319/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision","date":"2026-02-25T22:06:19+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2026-01-09T05:22:06+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-09T03:22:14+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-08T09:00:02+00:00","index":"","fulltext":""},{"type":"submitted","content":"European Journal of Plant Pathology","date":"2026-01-06T13:41:28+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"european-journal-of-plant-pathology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ejpp","sideBox":"Learn more about [European Journal of Plant Pathology](http://link.springer.com/journal/10658)","snPcode":"10658","submissionUrl":"https://www.editorialmanager.com/ejpp/default2.aspx","title":"European Journal of Plant Pathology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"c32faddd-450d-413d-aa24-250caff7a46a","owner":[],"postedDate":"January 13th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-23T16:00:45+00:00","versionOfRecord":{"articleIdentity":"rs-8534319","link":"https://doi.org/10.1007/s10658-026-03215-4","journal":{"identity":"european-journal-of-plant-pathology","isVorOnly":false,"title":"European Journal of Plant Pathology"},"publishedOn":"2026-03-22 15:58:06","publishedOnDateReadable":"March 22nd, 2026"},"versionCreatedAt":"2026-01-13 10:51:25","video":"","vorDoi":"10.1007/s10658-026-03215-4","vorDoiUrl":"https://doi.org/10.1007/s10658-026-03215-4","workflowStages":[]},"version":"v1","identity":"rs-8534319","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8534319","identity":"rs-8534319","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}