Spatio-temporal distribution and diversity of bacterial endophytes in apple (Malus domestica Borkh.) and their antagonistic potential against Alternaria mali

Introduction

The term endophyte (Greek: endon = within and phyton = plant) was first introduced by Anton de Bary in 1866 to describe microorganisms that inhabit internal plant tissues without causing apparent harm to their host. Since then, the concept has expanded to encompass phylogenetically diverse bacterial and fungal communities that colonize healthy plant tissues for part or all of their life cycle (Hallmann et al., 1997; Rosenblueth and Martinez-Romero, 2006) [18,36] . These endophytic microorganisms establish intricate associations with their host plants, ranging from commensalistic to mutualistic interactions, and play a pivotal role in enhancing plant growth, stress tolerance, and resistance to biotic challenges. Plant endophyte interactions are governed by a complex interplay of factors, including host genotype, tissue specificity developmental stage, environmental conditions, and geographical location (Arnold and Lutzoni, 2007; Qawasmeh et al., 2012) [5,33] . Seasonal fluctuations in temperature, humidity, and host physiological activity further influence endophytic population dynamics, resulting in highly diverse and temporally dynamic microbial communities (Ek-Ramos et al., 2013) [15] . Consequently, endophytic assemblages exhibit pronounced spatial and temporal variation, emphasizing the need for systematic studies to understand their distribution patterns and functional attributes in perennial crops. Apple (Malus domestica Borkh.) is one of the most economically important temperate fruit crops worldwide and represents a complex perennial agroecosystem that harbours diverse microbial communities. Apple production is severely constrained by several diseases, among which Alternaria leaf blotch, caused by Alternaria mali, is a major concern due to its destructive impact on foliage, fruit quality, and yield. Currently, disease management relies heavily on chemical fungicides, which remain the most commonly adopted strategy for protecting apple orchards (Karacic et al., 2024) [21] . However, prolonged fungicide use has led to the development of pathogen resistance, accumulation of pesticide residues on fruits, environmental pollution, and disruption of beneficial microbial communities, thereby raising serious concerns regarding sustainability and food safety (Chowdappa et al., 2013) [13] . These limitations necessitate the exploration of eco-friendly and biologically sustainable alternatives. In this context, bacterial antagonists particularly endophytic bacteria functioning as biological control agents (BCAs) have gained considerable attention as promising tools for disease management in perennial fruit crops, including apple (Pane and Zaccardelli, 2015; Shoaib et al., 2019) [31,41] . Endophytic bacteria inhabit internal plant tissues, share ecological niches with pathogens, and effectively suppress disease through mechanisms such as nutrient competition, antibiosis, volatile production, and induction of systemic resistance. Among the diverse bacterial BCAs, Bacillus spp. are regarded as exceptionally promising due to their Gram-positive nature, ability to form endospores, and tolerance to adverse environmental conditions, making them suitable for formulation, storage, and field application (Kukreti et al., 2023; Ankitha et al., 2023) [3,23] . Several endophytic bacterial taxa, including Bacillus velezensis, Paenibacillus spp., and Bacillus pseudomycoides, have demonstrated strong antagonistic activity against a wide range of phytopathogens. Bacillus velezensis, described by (Ruiz-Garcia et al. 2005) [38] , promotes plant growth through enhanced nutrient acquisition and phytohormone production while suppressing fungal pathogens via bioactive secondary metabolites such as lipopeptides, polyketides, and extracellular enzymes (Kim et al., 2017) [22] . Similarly, Paenibacillus spp. are known to produce hydrolytic enzymes, form protective biofilms, and emit volatile organic compounds that inhibit fungal growth (Timmusk and Wagner, 1999; El-Deeb et al., 2013) [16,43] . Bacillus pseudomycoides has also exhibited notable biocontrol efficacy against both soil-and foliar-borne pathogens (Yanti et al., 2018) [47] . Previous studies have extensively characterized B. velezensis strains A6 and P42, originally isolated from pomegranate and rice, respectively, revealing their ability to produce a wide array of antimicrobial secondary metabolites, including fengycin, iturin, bacillomycin, and surfactin (Amruta et al., 2018; Prasanna et al., 2021) [2,32] . These strains exert antagonistic effects through siderophore production, hydrogen cyanide, volatile and non-volatile compounds, and lytic enzymes, while also inducing systemic resistance via activation of key defense-related enzymes in host plants (Shabanamol et al., 2017; Kukreti et al., 2023) [23,40] . In addition to disease suppression, such endophytes enhance plant growth by improving nutrient uptake and modulating phytohormone levels (Rana et al., 2011; Manivannan et al., 2012) [28,34] . Although host specificity of endophytes remains debated, with some studies suggesting hostrestricted benefits (Long et al., 2008) [26] and others demonstrating cross-host colonization (Sessitsch et al., 2005; Ma et al., 2011) [27,39] , non-host endophytes offer significant advantages due to their ecological adaptability and broad-spectrum disease suppression (Byregowda et al., 2022) [11] . Such traits are particularly valuable in perennial orchard systems, where environmental variability and long-term sustainability are critical considerations (Choudhary et al., 2023) [12] . Despite growing interest in plant-associated microbiomes, comprehensive investigations on the spatio-temporal distribution and tissue-specific diversity of bacterial endophytes in apple remain limited, particularly under the temperate Himalayan conditions of the Kashmir Valley. Elucidating these patterns is essential for identifying ecologically adapted and functionally efficient endophytes that can be harnessed for sustainable orchard management. Therefore, the present study was undertaken to investigate the seasonal and tissue-specific distribution and diversity of bacterial endophytes associated with apple cultivars grown at two distinct locations in the Kashmir Valley and to evaluate the antagonistic potential of selected endophytic bacteria, including Bacillus cereus and Bacillus Wildmannii against Alternaria mali using in vitro dual culture, volatilome assays.

Materials and Methods

Collection of Plant Samples

Healthy and asymptomatic plant samples of apple (Malus domestica Borkh.) were collected from two high-density cultivars, Gala Red Lum and Oregon Spur, cultivated at two geographically distinct experimental sites in the Kashmir Valley, India: the Sher-e-Kashmir University of Agricultural Sciences and Technology of Kashmir (SKUAST-K), Shalimar, Srinagar, and the ICAR-Central Institute of Temperate Horticulture (CITH), Budgam. These locations were selected to represent contrasting agro-climatic conditions and orchard management regimes prevailing in the region. Sampling was conducted during three distinct phenological seasons spring, summer, and autumn-to capture seasonal variability in bacterial endophytic communities. Tissue sampling was performed in accordance with seasonal physiological availability. During the spring season (April), flowers, leaves, and twigs were collected; in the summer season (late June to early July), fruits, leaves, and twigs were sampled; while during autumn (September), sampling was confined to leaves and twigs. All samples were collected exclusively from apparently healthy, disease-free apple trees to minimize the likelihood of pathogen contamination. Immediately following collection, samples were placed in sterile polyethylene bags, appropriately labelled with cultivar, location, season, and tissue type, and transported to the laboratory under cooled and aseptic conditions. Samples were processed at the earliest possible time for the isolation of bacterial endophytes using standard microbiological protocols.

Surface Sterilization of Plant Tissues

Plant samples were thoroughly washed under running tap water to remove adhering debris, and tissues exhibiting visible surface injuries were discarded. Surface sterilization was carried out following the protocol of Araujo et al. (2001) [4] with minor modifications. Briefly, tissues were sequentially immersed in 70% (v/v) ethanol for 1 min, followed by sodium hypochlorite solution (2.5% available chlorine) for 4 min, then again in 70% ethanol for 30 s, and finally rinsed three times with sterile distilled water. The efficacy of surface sterilization was confirmed by plating 100 µL aliquots of the final rinse water onto nutrient agar (NA) plates. Plates were incubated at 25 ± 1°C for 24 h and monitored for microbial growth to ensure the absence of epiphytic contamination.

Isolation of Bacterial Endophytes

Surface-sterilized plant tissues were aseptically sectioned into small fragments, airdried under sterile conditions, and placed onto nutrient agar plates. The plates were incubated at 37°C for 48 h and monitored periodically for the emergence of bacterial colonies. Colonies originating from within the plant tissues were considered endophytic in nature and selected for further purification.

Purification and Maintenance of Bacterial Isolates

Emerging bacterial colonies displaying distinct morphological features were purified through repeated streaking on fresh nutrient agar plates until homogeneous and morphologically consistent colonies were obtained. Pure cultures were maintained on nutrient agar slants at 4°C for short-term preservation and periodically sub-cultured to maintain viability. Each isolate was assigned a unique identification code based on its location, season, cultivar, and tissue of origin.

Morphological and Microscopic Characterization

Morphological characterization of purified isolates was performed based on colony characteristics observed on nutrient agar plates after 24-48 h of incubation, including colony shape, colour, margin, elevation, and surface texture. Microscopic characterization was carried out using Gram staining to determine Gram reaction and cellular morphology, following standard procedures (Boone et al., 2005; Barik, 2015) [6,10] .

Grouping of Bacterial Endophytes into Morphotypes

Based on combined morphological and microscopic characteristics, bacterial isolates were grouped into distinct morphotypes. Isolates exhibiting similar colony morphology, Gram reaction, and cellular features were assigned to the same morphotype, whereas those with distinct traits were categorized separately. These morphotypes were treated as operational taxonomic units (OTUs) for subsequent diversity analysis. Morphotyping minimized redundancy among phenotypically similar isolates and facilitated robust estimation of endophytic bacterial diversity.

Assessment of Spatial and Temporal Distribution

The spatial and temporal distribution of bacterial endophytes was assessed by categorizing isolates according to location (SKUAST-K and CITH), season (spring, summer, autumn), cultivar, and tissue type. The frequency and distribution of isolates and morphotypes within each category were documented to evaluate patterns of endophytic colonization.

Diversity Analysis of Bacterial Endophytes 2.8.1 Shannon-Wiener Diversity Index

Bacterial endophyte diversity was quantified using the Shannon-Wiener diversity index (H′), which integrates both morphotype richness and evenness, and was calculated using the formula:

where pᵢ represents the relative abundance of the ith morphotype, calculated as the proportion of isolates belonging to that morphotype within a given location-season combination.

Spatial and Temporal Variation Analysis

Spatial variation in endophytic diversity was evaluated by comparing Shannon index values between locations, while temporal variation was assessed by comparing diversity indices across seasons within each location.

Multivariate Analysis of Bacterial Community Composition

Differences in bacterial endophyte community composition were assessed using the Jaccard dissimilarity index based on presence-absence data of morphotypes. Pairwise dissimilarity matrices were generated and visualized through heat map analysis. Principal Coordinates Analysis (PCoA) was employed to ordinate samples and visualize clustering patterns across locations and seasons. Multivariate analyses were conducted using PAST software (version 4.0) and the R statistical environment (vegan package).

Statistical Analysis and Data Visualization

All diversity indices and multivariate analyses were computed using PAST and R software. Graphical outputs were generated to facilitate interpretation of spatial and temporal patterns in bacterial endophyte diversity.

In Vitro Evaluation of Antagonistic Activity 2.11.1 Dual Culture Assay

The antagonistic activity of bacterial endophytes against Alternaria mali was assessed using the dual culture technique. A 5 mm mycelial disc from the actively growing margin of a 7-day-old A. mali culture grown on potato dextrose agar (PDA) was placed on one side of a PDA plate, while 48 h-old bacterial cultures grown on nutrient agar were streaked on the opposite sides of the plate. Each treatment was replicated three times (Latha et al., 2009) [24] . Plates were incubated at 28 ± 1 °C. Control plates contained the pathogen alone. Mycelial growth was measured after complete colonization in control plates, and percent inhibition was calculated using the formula described by Vincent (1947) [45] :

Where C = growth in control, T = growth in treatment.

Volatile Organic Compound (VOC) Assay

The inhibitory effect of volatile organic compounds (VOCs) produced by bacterial endophytes against A. mali was evaluated using a sealed plate assay. A 5 mm agar plug from an actively growing 7-day-old A. mali culture was placed on PDA in the base plate, while the corresponding lid plate containing nutrient agar was streaked with the bacterial isolate. The two plates were immediately sealed together using a double layer of parafilm to create a closed system and incubated at 28 ± 1°C until maximum growth was observed in control plates (Rouissi et al., 2013) [37] . Percent inhibition was calculated using the Vincent (1947) [45] formula as described above.

Results and Discussion

Isolation of Endophytic Bacteria from Apple Cultivars

The bacterial endophytic micro-organisms were isolated from two cultivars of apple Gala Red Lum and Oregon Spur, collected from two locations, namely SKUAST-K, Shalimar and ICAR-CITH, Budgam, during three different seasons. Plant tissues such as leaves, flowers, fruits and twigs were used for the isolation of bacterial endophytes. (Table 1). Based on colony morphology, a total of 111 bacterial endophytic isolates were obtained during the course of the study.

Morphological characterization of endophytic bacterial isolates

The bacterial isolates had varying morphological characters.

Colony colour varied between cream, creamy white, yellow, white and light yellow. Colony margin was of entire and erose. Whereas the elevation was observed to be of convex, raised and flat. And based on the opacity the cultures were of opaque, transparent and translucent in their nature.

Plate 2: Colony morphology of bacterial endophytic isolates

Representative bacterial endophytes 3.3 Microscopic observation of endophytic bacterial isolates

Microscopic observation was carried out through Gram staining under 100 X oil immersion to study the differences among bacterial isolates. The isolates were found to be of both Gram positive and Gram negative with shape varied between cocci, rod and coccobacilli.

Plate 3: Microscopic characteristics of bacterial endophytic isolates

Gram negative 3.4 Season-wise, Location-wise, Cultivar-wise and Tissuewise Distribution of Endophytic Bacteria

The season-wise, location-wise, cultivar-wise and tissue-wise distribution of bacterial endophytes isolated from apple is presented in Table 2.

Endophytes diversity across seasons:The distribution pattern of bacterial endophytic microorganisms showed a considerable variation in their population across different seasons. The highest population of bacterial endophytic microflora (44) was observed during spring season followed by summer (39) and autumn (28). Location-wise comparison revealed that SKUAST-K yielded a higher number of isolates (62) compared to ICAR-CITH (49).

Endophytes diversity across plant parts:

The diversity in endophytes, regardless of plant tissues was astonishingly very high, evident from (Table 2), showing highest (47) numbers of endophytic bacteria confined to apple leaf samples followed by twigs (36). Whereas apple flowers (15) and fruits (13) samples showed a comparatively lower degree of diversity out of total bacterial endophytes. Overall, the results clearly demonstrate that the recovery of bacterial endophytes in apple cultivars is strongly influenced by season, tissue type, location, and cultivar, with spring season and leaf tissues being the most favourable sources for endophyte isolation.

Grouping of Endophytic Bacterial Isolates into Morphotypes

Based on colony morphology, pigmentation, margin, elevation, opacity, Gram reaction and cell shape, the bacterial endophytic isolates were grouped into five distinct morphotypes (M1-M5). Isolates showing similar cultural and microscopic characteristics were grouped under a single morphotype to reduce redundancy and facilitate diversity analysis. Each morphotype was considered as an Operational Taxonomic Unit (OTU) for the calculation of diversity indices.

Alpha Diversity of Bacterial Endophytes across Locations and Seasons

Alpha diversity of bacterial endophytes was assessed using morphotypes as Operational Taxonomic Units (OTUs) to evaluate within-sample diversity. The Shannon diversity index (H ′) revealed noticeable variation across locations and seasons (Table 3). At ICAR-CITH, the Shannon diversity index remained 0.000 during spring, summer and autumn, indicating a highly uniform bacterial endophyte community with no detectable variation across seasons. In contrast, SKUAST-K consistently exhibited measurable bacterial endophyte diversity during all three seasons. The highest Shannon diversity index at SKUAST-K was recorded during summer (0.308), followed by autumn (0.231), while the lowest value was observed during spring (0.116).

Overall, Shannon diversity index values were consistently higher at SKUAST-K compared to ICAR-CITH, suggesting a greater richness and evenness of bacterial endophytes at SKUAST-K. These results clearly indicate that spatial location and seasonal variation significantly influenced the alpha diversity of bacterial endophytes associated with apple.

Beta Diversity Analysis: Spatial and Temporal Variation in Bacterial Endophyte Communities

Beta diversity analysis was carried out using the Jaccard dissimilarity index based on presence-absence data of bacterial morphotypes to evaluate differences in community composition across locations and seasons. The heat map (Figure 5) illustrates the degree of similarity and dissimilarity among bacterial endophyte communities.

Variations in colour intensity represent differences in community composition, with similar colour patterns indicating greater similarity and contrasting patterns indicating higher dissimilarity. The observed variation in colour distribution among samples clearly suggests that bacterial endophyte community composition varied across both spatial (location) and temporal (seasonal) scales, demonstrating pronounced heterogeneity.

Principal Coordinate Analysis (PCoA) Based on Jaccard Dissimilarity

To further examine spatial and seasonal variation in bacterial endophyte community structure, Principal Coordinate Analysis (PCoA) based on Jaccard dissimilarity was performed (Figure 6). The PCoA plot revealed distinct clustering patterns of bacterial endophyte communities, indicating differences in community composition among samples collected from different locations and seasons. The separation of clusters along the principal coordinate axes suggests that both location and season exerted a significant influence on the structure of bacterial endophyte communities associated with apple.

In vitro evaluation of bacterial endophytes against

Alternaria mali

The antagonistic potential of bacterial endophytes isolated from apple was evaluated against Alternaria mali, the causal agent of Alternaria leaf blotch disease, under in vitro conditions using the dual culture technique.

Antagonistic effect of bacterial endophytes on mycelial growth of Alternaria mali

A total of 111 bacterial endophytes were screened for their ability to suppress the mycelial growth of A. mali by the dual culture assay. The mycelial growth of the pathogen varied significantly in the presence of different bacterial isolates, indicating variability in their antagonistic potential. Among all the tested endophytes, isolate I-38 exhibited the maximum suppression of fungal growth, recording the lowest mean mycelial growth of 11.3 mm. This was followed by isolates I-50 (12.5 mm). The remaining isolates were comparatively less effective in restricting the growth of A. mali. Statistical analysis revealed significant differences among treatments at the 5% level of significance (Table 4 Plate 4).

Percent Inhibition of Mycelial Growth of A. mali

The antagonistic efficacy of bacterial endophytes was further expressed in terms of percent inhibition of mycelial growth of A. mali. The percent inhibition varied significantly Samong the 111 isolates tested. Consistent with the mycelial growth results, isolate I-38 (87.3%) recorded the highest inhibition of the pathogen, followed by isolates I-50 (86.0%) (Table 4 Plate 4). These isolates demonstrated strong antagonistic activity against A. mali under in vitro conditions. The observed inhibition may be attributed to the production of antifungal metabolites, competition for nutrients and space, or the secretion of cell wall degrading enzymes by the bacterial endophytes. The volatile metabolite assay demonstrated considerable variation among the selected bacterial endophytes in their ability to suppress the mycelial growth of Alternaria mali under in vitro conditions. The extent of pathogen growth differed significantly in response to volatile compounds released by the bacterial isolates (Table 5, Plate 5). Among the tested endophytes, isolate I-38 resulted in the lowest mycelial growth of A. mali, indicating strong antifungal activity mediated through volatile metabolites.

This was followed by isolate I-50 which also caused substantial reduction in fungal growth. The observed differences among treatments were statistically significant at the 5% level.

In vitro Inhibition of Alternaria mali by Volatile Metabolites

The inhibitory effect of volatile metabolites produced by bacterial endophytes on A. mali was further expressed in terms of percent inhibition of mycelial growth. Significant variation in volatile-mediated inhibition was observed among the bacterial isolates evaluated (Table 5 Plate 5).

Isolate I-38 exhibited the highest percent inhibition of A. mali due to volatile metabolite production, followed by isolate I-50. These results suggest that volatile metabolite production plays an important role in the antagonistic interaction between certain bacterial endophytes and A. mali.

Plate 5: Volatile test of bacterial endophytes against Alternaria mali

Discussion

The present investigation revealed a rich and ecologically structured assemblage of bacterial endophytes associated with apple cultivars, as evidenced by the recovery of 111 isolates across tissues, seasons, and locations, confirming apple as a stable reservoir of endophytic bacterial diversity. The perennial woody nature of apple and the presence of longlived internal tissues likely provide favourable niches for sustained endophytic colonization, as also reported in earlier studies (Miliute et al., 2015; Liu et al., 2017) [25,30] [9,14] . Morphotype-based classification revealed substantial phenotypic heterogeneity among isolates, supporting its ecological relevance as a practical proxy for diversity assessment under culturedependent conditions (Hughes et al., 2001; Martins et al., 2019) [20] . Higher alpha diversity recorded at SKUAST-K, particularly during spring, reflects favourable microclimatic conditions that support coexistence of multiple endophytic taxa, whereas reduced alpha diversity at ICAR-CITH indicates strong environmental filtering and site-specific constraints, as reported in orchard ecosystems under restrictive conditions (Martins et [46] . Furthermore, beta diversity analysis based on Jaccard dissimilarity, supported by PCA ordination and heatmap visualization, revealed pronounced community turnover across locations, seasons, and tissues, highlighting the dynamic and context-dependent nature of apple-associated endophytic bacterial communities (Martins et al., 2019; Trivedi et al., 2020; Berg et al., 2021) [44] . The fungal pathogen isolated from diseased apple leaves was morphologically and pathologically confirmed as Alternaria mali, exhibiting diagnostic cultural and microscopic characteristics and fulfilling Koch's postulates, consistent with classical and contemporary descriptions of the pathogen and its role as the causal agent of Alternaria leaf blotch in apple (Roberts, 1924; Simmons, 2007; Filajdic and Sutton, 1995; Harteveld et al., 2014) [17,19,35,42] . Collectively, these findings demonstrate that bacterial endophyte diversity and ecological distribution in apple are governed by a complex interplay of host traits, environmental factors, and temporal dynamics, providing a strong ecological foundation for the targeted exploitation of endophytic bacteria in sustainable apple disease management.

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