Tunisia boasts a rich and diverse range of forage and pasture biodiversity, with over 960 pasture legume species, including 336 that are unique to the Mediterranean region (Ibidhi et al., 2018; Ferchichi et al., 2021). However, due to increasing purchasing power, the demand for ruminant products is expected to rise significantly in the coming years, making high levels of forage crop production imperative. The country’s livestock sector heavily relies on forage crops, particularly alfalfa (Medicago sativa), which covers an area of approximately 12,410 hectares (Guiza et al., 2022). Alfalfa plays a vital role in supporting livestock nutrition and ensuring food security.

Presently, the Tunisian livestock sector is grappling with a persistent shortage of forage. Like many other regions globally, it faces the challenge of climate change and its associated environmental stresses, among which soil salinization and pathogen infections are particularly important for alfalfa productivity (Djebali, 2008; Arraouadi et al., 2011; Badri et al., 2021). Furthermore, the interaction between salinity and Phoma medicaginis infection can exacerbate the adverse effects of these stresses on alfalfa plants (Castell-Miller et al., 2007; Kaiwen et al., 2020; Maiza et al., 2021). The coexistence of these stresses creates a more hostile environment for the crop to thrive. Under saline conditions, reduced water uptake and compromised nutrient absorption weaken the plant’s defenses against strain Phoma medicaginis 8 (Pm8) infection, making the plant more susceptible to disease progression (Djébali et al., 2013). Therefore, the increasing prevalence of salinity stress and P. medicaginis infection poses a significant threat to alfalfa production in Tunisia.

Despite its significance as a forage legume, there has been limited work on varietal selection for this species in Tunisia, with only two registered local varieties (Gabès and El Hamma) (Jabri et al., 2021; Maiza et al., 2021). Therefore, it is advisable to initiate new varietal selection efforts for alfalfa, utilizing diverse germplasm, to develop new varieties with robust agronomic performance under the combined constraints of salinity and P. medicaginis infection in the context of climate change.

Moreover, the socio-economic implications of the shortage of forage in Tunisia, particularly its impact on the livestock sector and food security, warrant further exploration and discussion.

As a result, diverse studies focusing on enhancing alfalfa have led to the development of cultivars showcasing superior attributes such as increased yield, enhanced stress tolerance, and improved forage quality (Badri et al., 2021). A significant challenge in alfalfa enhancement lies in pinpointing the genetic underpinnings responsible for variations within the species across various agronomic traits (Sakiroglu and Brummer, 2017). Addressing these implications is essential for devising effective strategies to mitigate the challenges faced by farmers and ensure sustainable agricultural practices.

Furthermore, a comparative analysis of alfalfa production and management strategies such as cropping systems-, and soil-related management practices (Thivierge et al., 2023) in other regions facing similar environmental stresses could provide valuable insights and lessons learned that can inform the development of tailored solutions for Tunisia and similar ecogeographic regions (Tlahig et al., 2020). By examining approaches adopted in different contexts, we can identify best practices and adapt them to the Tunisian agricultural landscape, thereby enhancing the resilience and productivity of the alfalfa sector.

Alfalfa is an autotetraploid species with a chromosome count of 32 (2n = 4x = 32) and a genome size ranging from 800 to 1000 Mb. Several challenges affect its genetic improvement because of various factors such as intra-varietal variation, environmental influences, and the complex life cycle of the plant (Liu et al., 2023). Due to these hindrances, conventional breeding in alfalfa has been difficult and only moderately efficient (Annicchiarico et al., 2015a). The progression of genomic technology could play a pivotal role in allowing association mapping studies that could accelerate and strengthen crop improvement (Zhang et al., 2020).

Genomic investigations, including those based on genotyping-by-sequencing (GBS) markers, have enabled the detection of QTLs that influence various important agricultural characteristics in alfalfa. These traits encompass biomass yield, tolerance to factors such as aluminum, drought, and salt, as well as the ability to withstand freezing and maintain biomass yield during drought stress (Filho et al., 2023). Genome-wide association studies (GWAS) offer a potent strategy for deciphering genomic regions involved in targeted traits by scrutinizing the association between genome-wide single-nucleotide polymorphism (SNP) markers and phenotypic variation with exceptional precision compared to other approaches (Porter and O’Reilly, 2017; Larkin et al., 2019; Lin et al., 2023). Indeed, the GWAS approach enables the detection of more than one distinct allele for each locus in the studied accessions, and it would allow for a much more precise identification of candidate genes potentially involved in the genetic determination of the agronomic traits of interest (Liu et al., 2023).

Previously, GWAS has been employed in alfalfa to pinpoint loci markers associated with forage yield (Sakiroglu et al., 2012), drought resistance (Zhang et al., 2015), salt tolerance (Yu et al., 2016), forage quality (Sakiroglu and Brummer, 2017; Biazzi et al., 2017), and resistance to Verticillium wilt (Yu et al., 2017). No information is available for candidate genes underlying possible alfalfa tolerance to combined stresses of P. medicaginis infection and salinity.

The current work aimed to (i) characterize the response of alfalfa accessions to the combination of salinity and P. medicaginis infection, and (ii) identify the genetic determinants of tolerance to these constraints in this species through GWAS, to predict breeding values using SNP markers with allele dosage in breeding populations of alfalfa.

Analyzing the morpho-physiological variation in alfalfa plants under stress conditions has been regarded as a reliable method for assessing its stress tolerance. This process represents a pivotal phase in the development of forthcoming breeding programs (Annicchiarico, 2006; Annicchiarico et al., 2022). Salinity and Phoma medicaginis infection are major issues for alfalfa that affect its growth and productivity. Previous studies have demonstrated that the alfalfa response to salinity varies greatly among accessions (Badri et al., 2021; Jabri et al., 2021).

This study represented an unprecedented example of exploring the morphological variation of alfalfa in response to the simultaneous presence of biotic and abiotic stressors. Our findings revealed a remarkable range of morpho-physiological variation within the examined collection of alfalfa genotypes when subjected to simultaneous stressors (salt and strain Pm8 infection) and, overall, the current results demonstrated significant genetic variation for most of the recorded traits. These findings are in accordance with the conclusions drawn by Annicchiarico et al. (2022) on the same population grown under drought stress. Badri et al. (2023) tested the effect of P. medicaginis on the variation in agronomic traits of different Medicago species, reporting that most of the variation in the measured parameters was explained by the infection treatment. Zaidi et al. (2021) demonstrated that the number of healthy leaves (recorded among the infection parameters of alfalfa genotypes) varied significantly among the genotypes infected by P. medicaginis, which is consistent with the current observations.

In this study, the highest heritability was found for NHL, suggesting that this trait is largely governed by genetic effects. Badri et al. (2023) found that most evaluated traits in infected Medicago species had high heritability values, suggesting their potential as useful criteria for identifying P. medicaginis-tolerant lines. Djaman et al. (2020) found a moderate heritability for the length of main stem in alfalfa, owing to the environmental effects affecting this character besides the genetic ones. The positive correlations observed among phenotypic traits in alfalfa accessions under combined stress of salinity and P. medicaginis infection suggest that these conditions influence multiple traits similarly, leading to correlated responses. The increase in chlorotic and chlorotic necrotic leaves may indicate an enhanced immune response to fungal infection, implying a complex interaction between abiotic and biotic stressors. This interaction may increase plant susceptibility to fungal infection or worsen the effects of saline stress on plant physiology. Additionally, the correlations between phenotypic traits may indicate adaptive strategies employed by plants to mitigate environmental stress damage, such as increasing chlorotic leaves.

In this study, GWAS allowed to identify 110 SNP markers associated with seven morphological traits of alfalfa grown under combined salinity and P. medicaginis infection. To our knowledge, this was the first attempt to locate genes regulating morpho-physiological traits in alfalfa under simultaneous abiotic (salinity) and biotic (strain Pm8 infection) stresses. Liu and Yu (2017) identified 42 SNPs significantly associated with salt tolerance using 198 alfalfa accessions with four physiological traits (dry weight, plant height, leaf chlorophyll content and stomatal conductance). Only 10 SNPs have been identified in a panel of 179 lines of alfalfa for Verticillium wilt tolerance (Yu et al., 2017). Several research studies used diploidized models and the reference genome of M. truncatula to identify putative genes in alfalfa under stress (Biazzi et al., 2017; Medina et al., 2020; Lin et al., 2021).

In this study, 24 putative genes have been linked to homologue molecular and biological responses to combined stresses. The GWAS for the length of the main stem showed a close association of this character with the gene MTR_3g112450, which encodes a peptide transporter necessary for the organic nitrogen (N) supplies and mediated N use efficiency under salt stress (Fang et al., 2017). Similarly, Wang et al. (2022) demonstrated the high expression and regulation of the peptide transporter family induced in alfalfa grown in salinity stress. Overall, the peptide transporter plays a pivotal role in our study to alleviate salt stress in alfalfa by facilitating the uptake of organic nitrogen compounds, which contribute to osmotic adjustment, antioxidant defense, nutrient uptake, and gene regulation (Chen et al., 2021). This coordinated response not only boosts the elongation of the main stems but also enhances the plant’s ability to withstand and recover from salt-induced damage (Wang et al., 2022).

Putative candidate genes that were linked to SNPs located in the first chromosome encode for the Na⁺/H⁺ exchanger, and were associated with the number of ramifications, which plays an important role for alfalfa salt tolerance (Guiza et al., 2022). The Na⁺/H⁺ exchanger, located in both the plasma and vacuolar membranes, plays a pivotal role in plants by actively expelling excess sodium ions (Na⁺) from the cytosol or sequestering them into the tonoplast. This mechanism is essential for mitigating the harmful effects of sodium toxicity (Yang et al., 2005). A study conducted by Li et al. (2011) showed the effective expression of SsNHX (Na⁺/H⁺ exchanger) in transgenic alfalfa, which could grow in high concentrations of NaCl (up to 400 mM). In addition, Lei et al. (2015) reported that alfalfa genotypes tolerant to salinity maintain moderately more stable expression levels of genes related to Na⁺/K⁺ transport and Na⁺/H⁺ exchanger. However, tonoplast and plasma membrane antiporters actively remove sodium ions from the cytosol. This process is powered by the proton-motive force, which is produced by the H⁺-ATPase in the plasma membrane, as well as the H⁺-ATPase and H⁺-pyrophosphatase in the vacuolar membrane. Notably, research has demonstrated that Na⁺/H⁺ exchanger was identified within the late endosome/vacuolar compartment, and it has been suggested that its functions may encompass sodium transport, control of water movement, regulation of vesicle volume, and possibly play a role in osmo-tolerance (Silva and Gerós, 2009). Our finding suggested that Na⁺/H⁺ exchanger gene holds significant importance in enhancing the proliferation of ramification in alfalfa under salt stress conditions. Its pivotal role lies in facilitating the exchange of nitrogen and hydrogen ions, crucial for mitigating the detrimental effects of salinity on plant growth (Li et al., 2018). This gene ensures the maintenance of optimal nutrient levels and pH balance within plant cells (Guiza et al., 2022), promoting the development of a greater number of ramifications in alfalfa even amidst challenging salt stress environments.

GWAS identified the presence of five common markers associated with both AFW and ADW. Among these markers, MTR_1g053065 was linked to a homologous of the transcription initiation factor IIB, which was found to be involved in pollen and endosperm development in Arabidopsis (Dubos et al., 2010). Similarly, Xu et al. (2019) noted that the comparative transcriptome analysis of five alfalfa genotypes grown under cold stress revealed a gene encoding TFIIB (transcription factor IIB). This marker was differentially expressed in Brassica napus under drought stress using GWAS mapping (Khanzada et al., 2020). In addition, the putative electron transporter was significantly associated with ADW and AFW. MTR_4g058015 is also involved in the regulation of Na⁺/K⁺ in alfalfa under salt stress (Li et al., 2010), and was also reported in salt tolerance of rice (Patishtan et al., 2018). Thus, the essential role of TFIIB in our study resulted in the regulation of ionic transport and osmotic adjustment leading to the enhancement of nutrient acquisition by alfalfa grown under salinity.

Furthermore, the association analysis for the ADW revealed that this trait was highly associated with the putative genes encoding for F-box/RNI superfamily protein on chromosome 7. Lechner et al. (2006) noted that F-box protein is a crucial element within the ubiquitin ligase complex, facilitating the ubiquitination process of specific target proteins. This molecular mechanism plays a pivotal role in regulating cellular processes associated with plant defense to fungi infection. This may explain the high significance of the marker encoding this gene in the current study, as it mediates the degradation of specific proteins involved in susceptibility pathways. Through targeted protein degradation, the F-box protein regulates key factors that contribute to the virulence of P. medicaginis or the susceptibility of alfalfa. Similarly, a candidate gene encoding F-box protein was identified in alfalfa, using GWAS, as a negative regulator of resistance to root rot caused by Aphanomyces spp (Bonhomme et al., 2014).

The plant NBS_LRR gene family contains a large class of disease resistance genes (Yu et al., 2020). In this study, a LRR receptor-like kinase family protein was positively associated with AFW and a LRR and NB-ARC domain disease resistance protein was linked to a marker located in chromosome 8 for NCNL. These genes encode proteins that are involved in plant disease resistance (Li et al., 2019). GWAS revealed multiple QTLs related to Verticillium wilt resistance on alfalfa chromosome 8 (Yu et al., 2017). These results imply that alfalfa resistance can be improved by stacking major R genes/QTLs for multiple pathogens associated with AFW trait (Fuchs, 2017).

The annotations of the homologue gene MTR_4g062300 (S4.1_23118171) associated with NCNL pertain to hypothetical proteins with unknown functions. Earlier research has revealed that genes responsible for these hypothetical proteins were notably upregulated either at the transcript or translation level following prolonged exposure to high salt levels. This suggests that certain hypothetical proteins play a role in enhancing salt tolerance in forage plants, as demonstrated by Lin et al. (2021).

Five genes associated with the NCL trait were found to be located on M. truncatula chromosomes 1, 2, 4 and 8. Among them, one encodes the protein involved in DUO pollen-like protein. DUO acquired sperm lineage-specific expression in the common ancestor of land plants leading to sperm with distinct morphologies (Warman et al., 2020). The other one encodes for fatty acid hydroxylase superfamily protein, which has a metabolism control function (Marchler-Bauer et al., 2017). Preserving membrane integrity and fluidity is a crucial aspect of stress adaptation. This involves the collaboration between membrane components and the lipid composition to ensure the resilience of the membrane structure when facing challenging conditions. In this study, under combined stress, alfalfa cell membrane fluidity significantly decreased by the appearance of chlorosis spots on leaves, which is a symptom of salinity stress. As a result, the normal physiological function of membrane-bound proteins is altered (Liu et al., 2015). Previous studies reported the presence of the same putative gene using the GWAS approach in maize grown at low temperatures (Zhang et al., 2020), as well as in wheat under salt and drought stresses (Urbanavičiūtė et al., 2021).

A glycosyl hydrolase family 16 xyloglucan endotrans-glycosylase was found to be linked to the marker S8.1_12523990 associated to NHL. Recent studies have shown that xyloglucan endotrans-glycosylase, a prominent cell wall modifying enzyme, causes cell expansion, with loosening/reinforcing cell walls, particularly in response to environmental stress (Panahabadi et al., 2022). They demonstrated that glycosyl hydrolase family 16 xyloglucan endotrans-glycosylase was included in brassinolide hormone biosynthesis and pathways. Besides, Konkolewska et al. (2023) mentioned that the glycosyl hydrolase family 16 xyloglucans are intricate hemi-cellulosic polysaccharides that undergo biosynthesis within the Golgi apparatus before being transported to the cell wall of alfalfa. These molecules play essential roles in processes such as cell growth and expansion, energy metabolism, and signaling. Our finding suggested that the glycosyl hydrolase family 16 xyloglucan endotrans-glycosylase plays a crucial role in maintaining the number of healthy leaves in alfalfa during fungal infection by enhancing cell wall integrity, activating defense responses, regulating leaf morphology, and promoting nutrient transport.

Understanding stress tolerance mechanisms and genes in alfalfa helps identifying suitable genotypes for regions with high level of salinity and P. medicaginis infection, ensuring forage quality and quantity. Most traits assessed in the current study showed high heritability due to genetic factors, suggesting that the five alfalfa genotypes identified as tolerant to the combined stresses could be a valuable resource for the breeding. Using GWAS, we identified a total of 24 significant markers associated with seven traits. Notably, markers such as S8.1_12523990, S4.1_23118171, and S8.1_2278244 were linked to traits related to cell wall rigidity and cellular metabolism. We also found a marker associated with ionic transport. Some markers were linked to the NBS_LRR gene family, which plays a role in biomass and infection parameters, possibly contributing to alfalfa disease resistance.

The validation of the identified markers and genes through functional studies and biochemical assays, coupled with the exploration of gene overexpression techniques, represents a crucial step in marker-assisted selection (MAS). This process offers potential avenues for future research, such as investigating gene editing techniques to manipulate key genes associated with combined stress tolerance. Similarly, the most promising combined-tolerant alfalfa genotypes, containing numerous candidate genes for target traits, could be selected as parents in future crosses aimed at producing genetically superior alfalfa varieties with possibly multiple candidate genes for salinity tolerance, thereby allowing for sustained productivity even in saline-affected soils. Besides, resistance to P. medicaginis infection could mitigate crop losses and reduce the need for chemical interventions. However, ensuring the stability and adaptability of stress-tolerant traits across different environmental conditions and geographic regions will be essential for the widespread adoption of new alfalfa varieties

The original contributions presented in the study are included in the article/supplementary materials. Further inquiries can be directed to the corresponding author.

WM: Data curation, Formal analysis, Software, Validation, Writing – original draft, Writing – review & editing. CJ: Conceptualization, Data curation, Methodology, Validation, Writing – review & editing. NJ: Data curation, Methodology, Validation, Writing – review & editing. NM: Methodology, Validation, Writing – review & editing. AG: Methodology, Validation, Writing – review & editing. NZ: Methodology, Validation, Writing – review & editing. GB: Methodology, Validation, Writing – review & editing. EK: Methodology, Validation, Writing – review & editing. ND: Resources, Validation, Writing – review & editing. LP: Resources, Validation, Writing – review & editing. MH: Conceptualization, Validation, Writing – review & editing. PA: Resources, Validation, Writing – review & editing. MS: Formal analysis, Software, Validation, Writing – review & editing. NL: Conceptualization, Funding acquisition, Validation, Writing – review & editing. MB: Conceptualization, Data curation, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Validation, Writing – original draft, Writing – review & editing.