Lentil (Lens culinaris Medik.) is one of the major food legumes grown widely in the world. It is relatively tolerant to drought and grown in all five continents of the world in regions facing erratic rainfall and temperature patterns. Global production of lentil is about 6.66 million tons, primarily contributed by Canada (34.86%), India (19.22%), followed by Australia (15.14%), Turkey (6.74%) and Russian Federation (3.91%) (FAO, 2024) (https://www.fao.org/faostat/en/#data/QCL). Lentil is considered a rich plant protein source (Samaranayaka, 2017). In recent years, lentil has gained prominence not only as a staple pulse but also as a functional food due to its high nutritional value and associated health benefits (Alexander et al., 2024). It has been incorporated into different world cuisines throughout the globe and also mentioned as folk medicine of many ethnicities of the world to treat different illnesses. Presently, there has been a surge of interest in the possible health benefits of dietary polyphenols as antioxidants that are responsible for the black colour of food (Sanodiya et al., 2022). Lentil is an important pulse crop of northern and central India, valued for its protein-rich grains, essential minerals, vitamins and nutritious livestock feed (Dixit et al., 2017). Lentil is increasingly gaining attention as a functional food. It is rich in essential macro and micronutrients, phytochemicals, secondary metabolites and minerals. Lentils also contain various bioactive compounds. Among various legumes, lentil has been found to have higher free radical scavenging and antioxidant activities (Pal et al., 2020). These constituents provide several health benefits, including anti-carcinogenic, blood sugar-lowering (hypoglycemic), cholesterol-lowering (hypocholesterolemic) and blood pressure-reducing effects (Faris and Attlee, 2017; Dewan et al., 2024). Seed coat colour diversity in lentil, ranging from green, brown and grey to black and pink (Coco et al., 2022) is primarily governed by the accumulation of polyphenolic compounds, which influence antioxidant capacity, nutritional quality and stress adaptation (Mirali et al., 2017). Among these, black-seeded lentils are particularly rich in phenolic compounds and have attracted increasing research attention in recent years due to their enhanced nutraceutical potential (Paranavitana et al., 2021).
       
Traditionally cultivated Indian lentils are mainly microsperma types with brown seed coat and pink/orange cotyledons, early duration, low biological yield, short stature and pilosae traits. However, macrosperma types with profuse foliage and large seed size also cover significant acreages in central India and adjoining states (Bhartiya et al., 2015). Brown small seeded lentil is grown widely under varied agro-climatic conditions in India. However, region specific, small black seeded lentil widely appreciated for its taste and nutritional value in NW Himalayan regions in Uttarakhand state of India. Black lentil is nutritionally and functionally superior due to its higher protein and fiber content, it exhibits consistently greater antioxidant capacity, likely due to its richer flavonoid profile and dark seed-coat pigments. These characteristics make black lentil a promising functional food with enhanced potential for combating oxidative stress and supporting long-term health benefits (Chatzimitakos et al., 2023; Multescu et al., 2024).
       
The black seeded lentil is being cultivated in the region over generations owing to the preference of local communities. This unique type of lentil also generates higher revenues to farmers as compared to the brown seeded lentil owing to its superior nutritional, nutraceutical value, unique taste, culinary uses and adaptability to the fragile Himalayan agro-ecosystem. However, despite their importance, these indigenous black-seeded landraces remain largely unexplored and underutilized in systematic breeding programmes, with very limited information available on their genetic variability and trait-specific performance under hill conditions. This highlights a critical research gap in terms of scientific evaluation and characterization of these valuable genetic resources. This gene pool is expected to harbour promising genetic variants, particularly for adaption to harsh agro-climatic conditions (rainfed, poor fertility and altitudinal variations), abiotic (drought and temperature variations), biotic (wilt) stresses and also for the higher nutritional and nutraceutical properties. Among biotic stresses, wilt caused by Fusarium oxysporum f. sp. lentis is one of the most devastating diseases of lentil, leading to significant yield losses under diverse agro-ecologies including the Himalayan region. The occurrence of multiple races of Fusarium oxysporum in the Indian subcontinent significantly hampers the lentil production (Hiremani and Dubey, 2018). The development and identification of wilt-resistant genotypes is therefore crucial for ensuring stable production and sustainability of lentil cultivation in this fragile ecosystem. Therefore, the objectives of the present investigation were: (i) to assess the extent of genetic variability among indigenous black lentil accessions for agro-morphological traits, (ii) to identify promising genotypes with superior performance and resistance against wilt disease for their potential use in breeding programmes.

Plant material and experimental sites
 
Seventy indigenous black lentil accessions collected from diverse altitudinal range were evaluated along with 3 checks VL Masoor 125, VL Masoor 126 and VL Masoor 148 (Table 1a) at ICAR-VPKAS, Almora (NHZ) (29^o36'N, 79^o40'E and 1250 m above mean sea level) in India during the Rabi 2022-23 and 2023-24.


       
The experimental site represents rainfed mid hill conditions of NW Himalayan region of India. Climate of the location is sub-temperate, with an annual average temperature ranging from 11.5^oC to 23^oC and an average annual precipitation of approximately 1152 mm. Agro-climatic details of the experimental site are presented as Table 1b.


 
Conduction of experiment
 
The experiment was conducted in an augmented block design during Rabi 2022-23 and 2023-24, in which 3 check varieties were repeated in each block along with accessions under study. Each plot consisted of 1 row of 3 m (i.e. plot size =0.69 m²) with one plot per treatment/accession. As far as crop husbandry practices are concerned, standard agronomic practices were followed to raise a healthy crop. The sowing of crop was done manually in a well-prepared levelled seedbed under rainfed condition. The inorganic fertilizer dose included basal application of 20 kg N: 40 kg P: 20 kg K at the time of ploughing and land preparation. Line sowing was done and row-to-row spacing was maintained 23 cm whereas  plant-to-plant spacing within rows was fixed at 2-3 cm. Weed control was performed manually twice at 45 and 75 days after sowing. The crop was harvested at physiological maturity and observations on growth and yield traits were recorded as per standard procedures.

Data recording
 
Data on 8 quantitative traits and 11 qualitative traits were recorded using standard lentil descriptors (IBPGR, 1985) developed by International Board for Plant Genetic Resources, Rome (http://hdl.handle.net/10568/72887). Observations on all the traits were recorded at specific growth stages as Table 1c.



Disease screening under field and sick-plot conditions
 
Seventy-three black-seeded lentil accessions including VL Masoor 125 and VL Masoor 148 as susceptible and resistant checks, respectively (Arya and Kushwaha, 2019) from varied altitudinal range of Uttarakhand hills were evaluated for Fusarium wilt incidence under field during 2022-23 and 2023-24. The pathogen causing wilt was identified based on characteristic field symptoms (yellowing, wilting and vascular discoloration in tap root in wilted plant) associated with Fusarium oxysporum f. sp. lentis.
       
Initial screening was conducted under natural field conditions (natural infestation) during 2022-23 to identify resistant accessions. Based on preliminary screening under field conditions 34 resistant accessions were shortlisted and further evaluated during 2023-24 under artificially created disease screening environment i.e. wilt sick plot conditions ensuring high and uniform disease pressure. The experimental plot size was 3 m×0.50 m. A susceptible cultivar was planted along the borders and after every five accessions as infector to enhance inoculum load and ensure effective disease spread. Observation on wilt disease incidence was recorded at weekly intervals from seedling stage up to pod-filling stage. Disease severity was assessed using a 1-9 rating scale as described by Bayaa and Erskine, (1990).  Based on disease reaction, accessions were classified into five categories: highly resistant, resistant, moderately resistant, moderately susceptible and susceptible (Table 2). The following formula was used to calculate wilt disease incidence (Mayee and Datar, 1986):


       
The disease response of test entries was compared with resistant (VL Masoor 148) and susceptible (VL Masoor 125) checks to validate disease pressure and screening efficiency.

Statistical analysis
 
Pooled data of 8 quantitative traits recorded during winter season 2022-23 and 2023-24 was subjected to the one-way ANOVA for augmented block design Fisher’s LSD test (Steel et al., 1997), descriptive statistics multivariate (PCA, cluster analysis) and correlation analysis using XL STAT software (version 2024.3.0 1423). The cluster analysis was carried out involving Euclidean distance of the distance metric and variables based on Ward’s method (Ward, 1963).

Agro-morphological characterization of black seeded lentil accessions
 
Analysis of variance (ANOVA) revealed significant (P<0.05) differences among the black lentil genotypes for all quantitative traits except pod length and 100-seed weight, indicating the presence of substantial genetic variability for most of the agronomic traits evaluated. The descriptive statistics conducted on the basis of 8 quantitative traits revealed variability for phenological traits viz., days to 50% flowering (87-98 days), days to maturity (142-151 days) among the accessions. The relatively long duration of phenological traits indicates the predominance of late-maturing black lentil germplasm adapted to the traditional cropping systems of the Uttarakhand Himalayas.
       
Similarly, yield traits viz., pod length (1.0-1.16 cm), primary branches (2-4), plant height (21-36 cm), number of pods per plant (25-74), 100 seed weight (1.49-2.46 g) and grain yield (18.12-142.03 g m^-2) also exhibited wide variability indicating ample scope for selection of high-yielding genotypes from hill black lentil gene pool.
       
Further, as per the 11 qualitative traits, hill accessions exhibited considerable early plant vigour (good-48/very good-25), presence of seedling pigmentation (72), erect growth habit (61), light green (25)/green leaf colour (48), absent-34/slight-39 pubescence on leaflets, small leaflet size (56), purple flower (57), rudimentary tendril (52), medium biomass score (63), lodging resistance (60) and pod pigmentation (51) (Table 3). The predominance of vigorous, erect, lodging-resistant accessions with desirable plant architecture and pigmentation traits indicates natural adaptation to the Himalayan production environment.


 
Multivariate analysis of quantitative traits
 
PCA with eigen values and variances due to first 3 principal components is presented in Table 4.  Among the 8 principal components (PCs), only 2 components had an eigen value > 1 exhibited overall variability of 60.12% among the accessions. Thus, provides a good approximation of the variation present in the original dataset (Fig 1). Maximum variability of 43.61% was shown by PC1 followed by PC2 (16.50%) and PC3 (11.22%), respectively which altogether accounted for 71.34% of the total phenotypic variance among accessions. Days to maturity and 100-seed weight contributed to all three principal components, with loadings of 0.676, -0.282 and -0.353 for days to maturity and 0.249, 0.724 and -0.258 for 100-seed weight in PC I, PC II and PC III, respectively. In contrast, days to 50% flowering exhibited a strong negative loading in PC II (-0.827) and a comparatively lower negative loading in PC III (-0.243), indicating that these components primarily differentiated the accessions based on earliness and seed size.




       
In the cluster analysis, 73 accessions were classified into 2 major clusters based on the genetic distance calculated on 8 agro-morphological traits (Fig 2). The result showed that cluster I comprised 28 accessions, however, cluster II contained 45 accessions. Inter cluster variance was found maximum in cluster II (319.50) as compared to cluster I (198.34) indicated greater phenotypic divergence among the accessions grouped in Cluster II, suggesting a broader genetic base. The clustering pattern was found to be clearly differentiated in the biplot between PC I and PC II with slight overlapping. Accessions of cluster I were found to possess more plant height (30 cm), number of pods per plant (62), primary branches (4) and high grain yield per plot (>75 g) and greater wilt resistance than those in Cluster II. These findings indicate that Cluster I contained agronomically superior accessions, while the higher diversity observed in Cluster II provides a valuable reservoir of novel alleles for broadening the genetic base in black lentil breeding programmes.

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Correlation analysis
 
The Pearson coefficient of correlation among the studied 8 quantitative traits are given in Table 5. The significant positive correlation was observed between grain yield per plot and plant height, number of primary branches per plant, number of pods per plant and days to maturity  that can be considered as criteria for selection of high yielding genotypes under rainfed conditions. Similarly, days to maturity also had highly significant positive correlation with plant height, number of primary branches per plant and number of pods per plant, indicating the potential for developing genotypes with longer durations to attain elevated grain yield in rainfed hill conditions of Uttarakhand.


 
Screening of wilt disease under field and sick-plot conditions
 
The field screening was conducted to identify resistant accessions by considering the variable timing of symptom development across accessions and the irregular distribution of the disease in the field. Furthermore, uniform inoculum load in the field across all the plants of test accessions was ensured to discriminate between resistant and susceptible genotypes. Based on the screening scores against wilt under natural field conditions during 2022-23, 6 accessions (8.22%) exhibited highly resistant, 19 accessions (26.03%) resistant, 20 accessions (27.40%) moderately resistant, 7 accessions (9.59%) moderately susceptible and 21 accessions (28.77%) susceptible reaction against wilt under field conditions. The wilt incidence varied from less than 1% in highly resistant accessions to over 50% in susceptible ones, showed significant diversity in response to wilt. The shortlisted 34 accessions were further evaluated under field and wilt sick plot condition during 2023-24. Among shortlisted 34 entries, none of entries exhibited highly resistant reaction i.e. PDI <1%. Whereas 5 entries viz., VM2 (IC0201723), VM 5 (IC0430437), VM8 (IC0014167), VM17 (IC0241602) and VM 23 (IC0053230) exhibited resistant reaction (2-10% wilt incidence), 24 accessions exhibited moderately resistant (10-20% wilt incidence) and remaining 5 accessions exhibited susceptible reaction (>50% wilt incidence). A higher proportion of accessions falling under moderately resistant and susceptible categories indicated the occurrence of partial resistance in the accessions for wilt disease. Based on the consistency of disease reaction of accessions during 2022-23 (field condition) and 2023-24 (field and sick plot condition) only 4 accessions viz., VM2 (IC0201723), VM 5 (IC0430437), VM8 (IC0014167), VM17 (IC0241602) were found to be promising against wilt disease in Uttarakhand hills. Therefore, they may be considered for further validation  and use as donor parents in lentil improvement programmes targeting wilt resistance. The Uttarakhand Himalayan region has a long history of lentil cultivation, primarily local landraces that have evolved through centuries of farmer-led selection under diverse and challenging agro-climatic conditions (Bhartiya et al., 2017). These landraces, especially the black-seeded lentil with orange cotyledons, are valued not only for their adaptation to low-input hill farming systems but also for their unique taste, superior nutritional profile and higher market value (Bhartiya et al., 2015; Langyan et al., 2022). These landraces, cultivated by small and marginal farmers under organic, rainfed conditions, are conserved in-situ and represent a unique gene pool that remains underutilized and insufficiently characterized for its agronomic and adaptive traits.
       
The wide variability observed in the present study for both phenological and yield-related traits is consistent with earlier reports in pulse crops, where landraces from heterogeneous environments exhibit higher genetic diversity due to natural and farmer-mediated selection (Erskine, 2009; Kumar et al., 2013). This variability can be attributed to the diverse altitudinal gradients, temperature fluctuations and moisture stress conditions prevalent in the Uttarakhand hills, which act as natural selection pressures.
       
In the present study, the black lentil accessions from Uttarakhand were predominantly small-seeded (<2.5 g/100 seeds), a trait commonly associated with better adaptation to rainfed and moisture-limited conditions (Salehi et al., 2008). Moreover, small seed size has been shown to enhance stability under drought stress compared to larger-seeded types that are typically more cold-tolerant (Erskine, 2009; Bhartiya et al., 2015; Zeroual et al., 2023). Furthermore, Large-seeded lentils reported to exhibit significant variations in flowering duration and yield traits in Indian conditions (Parihar et al., 2025). Thus, the predominance of small-seeded types in the present study indicated selection pressure towards drought escape and stability under rainfed hill conditions, highlighting their importance in breeding programmes targeting climate resilience (Bakoulopoulou et al., 2025).
       
The majority of accessions also exhibited early plant vigour, which contributes to escaping terminal drought, mitigating salinity effects and improving weed competitiveness, thereby contributing to resilience of lentil in fragile hill agro-ecosystem (Erskine et al., 2009; Kumar et al., 2013). Another key adaptive feature observed was pigmentation in the stem and pods in most of the accessions, indicative of anthocyanin accumulation. Low temperature stress considered  an important factor in increasing anthocyanin production (Ahmed et al., 2015) which significantly upregulates the expression of genes involved in anthocyanin biosynthesis, leading to increased anthocyanin accumulation, which enhances the plant’s tolerance to cold conditions (Zhang et al., 2019). The accessions also exhibited erect growth habit, medium biomass, resistance to lodging, rudimentary tendrils and small green leaflets with slight or no pubescence. These traits collectively enhance the resilience of crop to withstand the region’s low-input, low-fertility and water-deficient conditions. These findings are in agreement with earlier studies emphasizing the role of plant architecture in improving resource-use efficiency and stress tolerance in pulses (Dwivedi et al., 2023). Lodging resistance is a complex trait governed by both genetic factors and environmental conditions. Reduced lodging, often linked to lower biomass production, allows these landraces to thrive in the region’s low-fertility and water-deficient soils (Noor et al., 2024). Plants with excessive biomass are more susceptible to lodging due to the accumulation of fibre-rich stems (Ball et al., 2006). In contrast, moderate biomass production as observed in many of the studied accessions, serves as an effective adaptation under the hill region’s resource-poor soils, enabling plants to maintain structural strength while conserving nutrients and moisture (Noor et al., 2024). Therefore, selection for moderate biomass and lodging resistance could be a key breeding strategy for improving yield stability under hill conditions. Identifying and utilizing such adaptive traits through genetic variability studies is crucial for selecting superior genotypes aimed at improving seed yield and overall performance under stress conditions (Akhtar et al., 2015).
       
The results from PCA further confirmed the presence of substantial trait variation within the black lentil gene pool. The PCA in this study revealed that over 71% of the total phenotypic variation could be explained by the first three principal components, simplifying the process of trait-based selection for breeders. Traits such as grain yield per plot, number of pods per plant, pod length, number of primary branches, plant height and days to maturity were positively correlated and contributed significantly to overall variability.  These traits, therefore, offer reliable selection criteria for variety development for NW Himalayan hill region of India.
       
In addition to agronomic performance, resistance to wilt disease remains a major biotic constraint in lentil cultivation. Conventional disease control through chemical fungicides is increasingly limited due to pathogen resistance, environmental concerns and health hazards (Bahri et al., 2024). Hence, the identification of host plant resistance is imperative. In this regard, four accessions namely VM2, VM5, VM8 and VM17, consistently exhibited resistance to wilt across two seasons and under both field and wilt-sick plot conditions. Notably, accession VM17 (IC0241602) showed the lowest percent disease incidence (6.9%) along with high grain yield and associated traits such as number of pods per plant, primary branches per plant. The promising accessions identified here can play a key role in developing climate-resilient, high-yielding lentil cultivars suited to the unique agro-ecologies of the Himalayan region and beyond.

The black-seeded lentil landraces of the Uttarakhand Himalayas possess key adaptive traits such as small seed size, early vigour, anthocyanin pigmentation and lodging resistance, making them well-suited to low-input, rainfed hill environments. The significant variation among the genotypes showed wide variability for the observed traits. This study has effectively characterized the agro-morphological diversity of black lentil hill landraces and identified accessions combining high yield with wilt resistance. Accession VM17 (IC0241602) had the lowest disease incidence (6.9%) alongside increased grain yield and related traits, including the number of pods per plant and primary branches per plant. These promising lines hold significant potential for use in breeding programs aimed at developing resilient, high-yielding lentil varieties suited to diverse agro-climatic regions of India.

The authors gratefully acknowledge the support of the Indian Council of Agricultural Research (ICAR), New Delhi, for this institute-funded research conducted at ICAR-VPKAS, Almora.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.