Banana (Musa spp.) is one of the most widely cultivated fruit crops in tropical and subtropical regions and serves as an important source of food, nutrition and income for millions of people worldwide. India is the largest producer of banana, with Tamil Nadu being one of the leading banana-growing states in terms of productivity. Owing to its year-round production, high consumer demand and economic importance, banana plays a significant role in the horticultural sector and contributes substantially to the livelihood of farming communities (INDIASTAT, 2024).
       
Despite its commercial importance, banana productivity is considerably affected by several biotic stresses, among which Sigatoka leaf spot is one of the most destructive foliar diseases (Shaikh et al., 2016). The disease, caused by Pseudocercospora musae, reduces the functional photosynthetic leaf area through the development of chlorotic and necrotic lesions, ultimately leading to decreased photosynthesis, poor bunch development, premature fruit ripening and substantial yield losses (Jiménez  et al., 2025). The severity of the disease varies with cultivar, environmental conditions and crop management practices, making the identification of tolerant cultivars an important component of integrated disease management.
       
Plant resistance to foliar pathogens is governed not only by genetic factors but also by several anatomical, physiological and biochemical characteristics that influence host pathogen interactions. Leaf anatomical traits such as stomatal characteristics, leaf thickness and leaf architecture may affect pathogen penetration and colonization, while physiological attributes including chlorophyll content, photosynthetic efficiency and plant water status influence the capacity of the host to withstand disease-induced stress. Similarly, biochemical compounds such as phenolic constituents constitute an important component of the plant defence system by restricting pathogen establishment and limiting disease progression (George et al., 2022).
       
Previous investigations have independently evaluated physiological responses, anatomical characteristics or biochemical constituents in banana cultivars under different environmental conditions. However, comprehensive information integrating these traits with field-level Sigatoka leaf spot resistance across commercially important banana cultivars remains limited. A better understanding of the association between leaf structural and physiological attributes and disease response would facilitate the identification of useful phenotypic markers for resistance screening and support banana improvement programmes.

Therefore, the present investigation was undertaken to evaluate the anatomical, physiological, morphological and biochemical characteristics of fifteen banana cultivars and to determine their association with Sigatoka leaf spot resistance under field conditions. The findings are expected to provide useful information for the identification of tolerant cultivars and contribute to breeding strategies aimed at developing banana varieties with improved resistance to Sigatoka leaf spot.

The experiment was conducted during 2025-2026 at the college orchard, SRM College of Agricultural Sciences, SRMIST, Baburayanpettai, Chengalpattu District, Tamil Nadu, India (12.9853° N, 79.9698° E). Fifteen banana (Musa spp.) cultivars representing different genomic groups were evaluated under field conditions (Table 1). The experiment was laid out in a Randomized Block Design (RBD) with three replications. Uniform, healthy and disease-free sword suckers were planted at a spacing of 2.0 x 2.0 m and maintained under the recommended package of practices throughout the experimental period. Observations on leaf anatomical, physiological, morphological, biochemical and disease parameters were recorded at the appropriate growth stage. Chlorophyll index was measured on the third fully expanded leaf from the top using a SPAD chlorophyll meter (Dennis et al., 2023). Chlorophyll a, b, total chlorophyll and carotenoid contents were calculated according to the method of Arnon 1949. Stomatal density was expressed as stomata mm^-2 following the procedure described by (Pathoumthong et al., 2023). Stomatal length and breadth were measured from microscopic images using an ocular micrometer. Three measurements were recorded from each sample and the mean values were expressed in micrometres (µm). Stomatal distribution was assessed by examining both adaxial and abaxial leaf surfaces.


       
Leaf thickness was measured using a digital vernier caliper. Measurements were expressed in millimetres (mm) (Madail et al., 2022). Leaf area was estimated non-destructively using the equation:
 

Leaf area (cm²) = Leaf length x Leaf breadth x 0.80

 
Where, 0.80 represents the Correction factor proposed by (Kumar et al., 2002). Leaf angle was measured as the angle formed between the pseudostem and the petiole of the third fully expanded leaf using a protractor and expressed in degrees (°). Relative water content was calculated according to Barrs and Weatherley (1962) using the following equation:
 

  

       
Total phenol content was estimated as per Singleton and Rossi (1965). Disease severity was assessed under natural field conditions using a 0-6 disease rating scale based on the percentage of infected leaf area (Raghavendra et al., 2025). Disease Severity Index (DSI) was calculated using the following formula:
  

 

 
Where,
n = Number of leaves in each disease grade.
b = Disease grade (0-6).
N = Total number of leaves assessed.
 
Statistical analysis
 
The experimental data were subjected to analysis of variance (ANOVA) appropriate for a Randomized Block Design (RBD) using OPSTAT statistical software (CCS Haryana Agricultural University, Hisar, India).

Photosynthetic pigments
 
Amruthapani recorded the highest chlorophyll a (5.70 mg g^-1 FW) and total chlorophyll (9.00 mg g^-1 FW), whereas Njalipoovan exhibited the lowest chlorophyll a (1.74 mg g^-1 FW). Chlorophyll b was highest in Njalipoovan (3.79 mg g^-1 FW) and lowest in Grand Naine (0.91 mg g^-1 FW). Carotenoid content also varied significantly among cultivars, with Kadali recording the highest concentration (3.58 mg g^-1 FW), while Grand Naine exhibited the lowest value (1.94 mg g^-1 FW) (Table 2).


       
Photosynthetic pigments are essential for light harvesting, energy transfer and maintenance of photosynthetic activity. Chlorophyll a serves as the primary reaction centre pigment, whereas chlorophyll b broadens the spectrum of absorbed light by transferring excitation energy to chlorophyll a. Carotenoids perform a dual role by assisting in light harvesting and protecting the photosynthetic apparatus against photooxidative damage through quenching of reactive oxygen species generated under environmental and biotic stress (Meghwal  et al., 2024; Surendar et al., 2013a).
       
Despite significant variation in pigment concentration, the present investigation demonstrated that higher chlorophyll or carotenoid content alone did not consistently correspond to lower Sigatoka leaf spot severity (Arora et al., 2008). The observations in the present study indicate that photosynthetic pigments contribute to maintaining physiological activity during disease stress but are not independent determinants of resistance.
       
The maintenance of chlorophyll during pathogen infection is advantageous because it prolongs photosynthetic competence and delays premature leaf senescence. Nevertheless, successful resistance against Pseudocercospora musae depends on the coordinated interaction of structural barriers, physiological resilience and biochemical defence mechanisms. Similar variability in photosynthetic pigments among banana cultivars has been reported by previous investigators, who attributed these differences to genotype-dependent regulation of chloroplast development and stress adaptationm (Niaz et al., 2024).
       
Significant variation in stomatal density was observed among the banana cultivars evaluated (Table 3), indicating differences in leaf anatomical characteristics that may influence host–pathogen interactions. Stomata regulate gaseous exchange and transpiration and play a central role in maintaining plant water relations. However, they also represent natural openings through which several foliar pathogens may initiate infection or establish favourable conditions for colonization. Consequently, stomatal density has frequently been investigated as an anatomical trait associated with disease susceptibility (de Morais et al., 2023). In the present investigation, variation in stomatal density alone did not consistently explain the observed differences in Sigatoka leaf spot severity among cultivars. This suggests that stomatal density should not be considered an isolated determinant of resistance but rather one component of a complex defence system involving leaf structural characteristics, physiological responses and biochemical defence mechanisms. These findings support the concept that anatomical traits influence disease development by modifying the physical and physiological environment of the leaf, while effective resistance ultimately depends upon the coordinated action of multiple defence-related attributes.


 
Stomatal length, breadth and distribution
 
Variations in stomatal dimensions influence stomatal conductance, transpiration rate and gaseous exchange, thereby affecting the physiological status of the plant under both normal and stress conditions (Table 3).
       
Besides regulating gas exchange, stomata play an important role in plant–pathogen interactions because they constitute natural openings on the leaf surface. The size and distribution of stomata may influence the establishment of favourable microenvironments for fungal pathogens and consequently affect disease development. However, their contribution to disease resistance is often indirect and depends on their interaction with other structural and physiological characteristics (de Morais et al., 2023).
       
The present investigation revealed considerable variability in stomatal dimensions among the evaluated cultivars. Nevertheless, cultivars with larger or smaller stomata did not consistently exhibit corresponding differences in Sigatoka leaf spot severity. This indicates that stomatal morphology alone cannot explain resistance and should be interpreted together with stomatal density, leaf structural characteristics and biochemical defence responses. The predominantly hypostomatic nature of banana leaves may further influence disease development by restricting stomata mainly to the abaxial surface. Such an arrangement modifies the leaf microenvironment and may affect moisture retention, pathogen establishment and subsequent lesion development. Therefore, stomatal morphology should be regarded as one component of an integrated resistance mechanism rather than an independent selection criterion.
 
Leaf thickness
 
Leaf thickness varied significantly among the evaluated banana cultivars (Table 4), demonstrating substantial differences in leaf structural characteristics. Thicker leaves generally possess a well-developed mesophyll and greater mechanical strength, enabling them to withstand environmental stress and potentially restrict pathogen penetration (Harilalnaik et al., 2021). Increased lamina thickness may provide an effective physical barrier against fungal invasion by enhancing tissue compactness and delaying pathogen colonization (Madail et al., 2022).


       
In the present study, cultivars with greater leaf thickness did not consistently record lower disease severity. This observation suggests that structural protection provided by thicker leaves may contribute to disease tolerance but does not independently determine resistance. Rather, leaf thickness appears to function synergistically with physiological and biochemical defence mechanisms in limiting disease progression (Wang et al., 2025).
       
Relative water content (RWC) differed significantly among the evaluated banana cultivars, indicating variation in their ability to maintain leaf water status under field conditions (Table 5). Cultivars with higher RWC retained greater cellular hydration, reflecting improved physiological stability and water balance. Maintenance of leaf water status is essential for sustaining photosynthesis, enzymatic activity and cellular metabolism, particularly under biotic stress. Infection by P. musae progressively reduces the functional photosynthetic area, which can adversely affect transpiration, carbon assimilation and overall plant vigour. Therefore, cultivars capable of maintaining higher relative water content are expected to better tolerate disease-induced physiological stress (Surendar et al., 2013b).


       
In the present investigation, variation in RWC did not completely correspond with disease severity across all cultivars, suggesting that water status alone was insufficient to explain differences in resistance.
 
Total phenol content
 
Significant variation in total phenol content was observed among the fifteen banana cultivars (Table 6), reflecting differences in their biochemical defence potential. Virupakshi recorded the highest phenol content (58.98 mg GAE g^-1 FW), followed by Amruthapani (58.04 mg GAE g^-1 FW), whereas Monthan exhibited the lowest value (33.90 mg GAE g^-1 FW).


       
Phenolic compounds constitute one of the primary defence mechanisms employed by plants against invading pathogens. Increased phenolic accumulation is frequently associated with enhanced resistance to fungal diseases (Bashmil et al., 2021).
       
Although cultivars with higher phenol content generally tended to exhibit lower disease severity, the relationship was not absolute in the present investigation. Virupakshi accumulated the highest phenolic concentration but did not express the greatest field tolerance. Conversely, Dwarf Cavendish and Njalipoovan exhibited comparatively lower disease severity through a favourable combination of anatomical, physiological and biochemical characteristics rather than exceptionally high phenol concentration alone.
       
The present findings suggest that phenolic compounds contribute substantially to defence against Pseudocercospora musae; however, effective resistance is achieved through coordinated interactions among structural barriers, physiological resilience and biochemical responses. Therefore, total phenol content should be considered an important component of integrated resistance screening rather than a single diagnostic marker.
 
Disease severity index
 
Among the cultivars, Dwarf Cavendish recorded the lowest Disease Severity Index (DSI), followed by Njalipoovan, indicating comparatively higher levels of field tolerance (Table 6, Plate 1). In contrast, Poovan exhibited the highest disease severity, reflecting its greater susceptibility to Pseudocercospora musae. The remaining cultivars displayed intermediate responses, suggesting a continuous spectrum of resistance rather than distinct resistant and susceptible groups.


       
The observed variation in disease severity highlights the influence of genetic background on host response to Sigatoka leaf spot. However, comparison of disease scores with the measured anatomical, physiological and biochemical traits revealed that no single characteristic consistently accounted for the observed differences among cultivars. For example, cultivars possessing higher chlorophyll concentration or greater phenolic content did not invariably exhibit lower disease severity. Likewise, favourable anatomical traits such as increased leaf thickness or altered stomatal characteristics alone were insufficient to explain the observed resistance pattern. These findings indicate that field tolerance is governed by the cumulative interaction of multiple plant characteristics rather than by any individual trait (Kimunye et al., 2020).

The study revealed that resistance to Sigatoka leaf spot could not be attributed to any single trait; rather, it was associated with the coordinated interaction of multiple structural, physiological and biochemical characteristics.

Among the evaluated cultivars, Dwarf Cavendish and Njalipoovan exhibited comparatively lower disease severity and may serve as valuable genetic resources for resistance screening and future banana improvement programmes. Conversely, susceptible cultivars identified in the study may be useful as reference materials in disease resistance research.
 

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.