Integrating Biological Control Agents for Enhanced Management of Apple Scab ( Venturia inaequalis ): Insights, Risks, Challenges, and Prospects

: Apple scab incited by the ascomycete Venturia inaequalis poses a significant threat to apple cultivation, necessitating a reassessment of existing disease management strategies. Attempts to manage apple scab include diverse approaches like developing disease forecasting models and the extensive application of synthetic chemical fungicides. However, the efficacy of these methods is compromised by inconsistencies, environmental concerns, and the pathogen’s resistance, necessitating the exploration of alternative sustainable strategies. Addressing the challenges associated with apple scab management, this review strongly supports a shift towards the integration of biological control agents (BCAs). Emphasising the transformative synergy between BCAs and their bioactive secondary metabolites, we highlight their efficacy in advancing precision disease control through innovative and sustainable solutions. The review effectively presents a strong justification for the integration of BCAs and their by-products into apple scab management, offering insights into associated benefits, risks, and challenges while outlining promising prospects. Ultimately, it is expected to drive the adoption of environmentally conscious practices for effective apple scab management.


Introduction
Apple scab incited by the ascomycete Venturia inaequalis (anamorph Spilocea pomi Fr.) poses a significant threat to apple cultivation and is notably challenging and expensive to manage [1].This disease has emerged as a major concern in apple-growing regions worldwide, particularly in temperate areas characterized by cool and moist spring weather [2].Despite continuous research efforts, apple scab remains remarkably resilient, emerging as the most economically damaging pathogen on apple trees globally [3].The disease causes substantial economic damage on apple trees, affecting the vitality of leaves and fruits, and leading to both immediate and indirect losses [3][4][5].Its detrimental effects extend to tree vigour, yield, and fruit quality, making it a paramount concern for growers [3,6].Even a minor scab lesion renders an apple non-marketable, and the incidence of scab invariably results in significant losses [7].Since the emergence of synthetic fungicides in the 1940s, fungicides have become the sole means to control apple scab [4].Despite numerous attempts over decades to establish biocontrol strategies, it was only recently that a few of them have successfully reached the stage of commercial viability [1].

Overview of Apple Scab (Venturia inaequalis)
The precise time of emergence of scab on cultivated apples is unknown [2].The initial documented report of apple scab was published by Fries in Sweden in 1819 [6].However, the earliest indication of scab's incidence dates back to 1600, observed in a painting by Michelangelo Caravaggio titled 'The Supper at Emmaus' held at the National Gallery in London [2].The Simple Sequence Repeat (SSR) analysis of V. inaequalis samples from 28 orchards across five continents suggests a Central Asian origin, and its current global prevalence reveals significant genotypic diversity in almost all orchards [8].
Apple scab is caused by a pathogenic fungus V. inaequalis, representing the teleomorph or saprophytic state, and S. pomi Fr., representing the anamorph or parasitic state [6].The manifestation of V. inaequalis infection on apple trees is a complex process linked to various factors.The fungus primarily targets young plant tissue and exhibits enormous adaptability [7].It is most noticeable and severe on leaves and fruits, but it can also be seen on sepals, petals, young shoots, and bud scales [5].In early spring, the initial signs of apple scab become apparent as dark green velvety spots on the lower surfaces of expanding leaves.As these lesions advance on the upper surfaces of mature leaves, they grow, leading to leaf splintering, swelling, scaliness, and eventual leaf drop [6].Late-season infections, initially inconspicuous, may become apparent post-storage, leading to the phenomenon of pin-point scab [7].Despite extensive research on its biology and control, apple scab remains a significant challenge, resulting in substantial economic consequences.The extent of losses varies with disease severity and prevailing weather conditions, establishing apple scab as a critical limitation to apple production on a global scale [7].

Infection Mechanism
V. inaequalis employs a sophisticated infection mechanism that adapts to the host's phenology and environmental conditions [2].It thrives as a successful pathogen by accumulating specific genes that facilitate infection and reproduction without causing significant harmful effects to the host [2].Investigations on V. inaequalis infection mechanism have revealed a unique infection strategy.Unlike typical host cell penetration, the fungus resides in the subcuticular space before sporulation, effectively neutralizing the host's defence mechanisms, possibly through specialized effectors [9].Genetic evidence strongly supports the secretion of effectors by V. inaequalis.Several candidate effector genes have been identified, characterized by various features common to effector genes in filamentous fungi [9][10][11].Additionally, Thakur et al. [12] identified crucial genes in V. inaequalis associated with biological processes such as metabolism, transport, and response to stimuli.
The disease cycle consists of two phases: the sexual or primary phase occurring mainly in winter, and the asexual or secondary phase which takes place during the vegetative period in spring/summer (Figure 1) [6,13].
During the dormant phase of the host tree, V. inaequalis strategically resides on leaves and fruit debris as pseudothecial initials or conidia in twig lesions.It adapts to temperature fluctuations to precisely synchronize with the developmental stage of the host tree, thus maximizing its possibility for future colonization [2,13].As host plants break dormancy in early spring, heterothallic mating between different mating types takes place in the debris to initiate sexual reproduction.This process involves the fusion of a male hypha (antheridium) and a female receptive hypha (trichogyne) [5,13].Subsequently, fertilization leads to the development of a pseudothecium within a dense mat of fungal mycelium (stroma), where asci and ascospores are formed.The diploid stage is brief, with meiosis giving rise to haploid ascospores within the pseudothecium [13].The negatively geotropic nature of pseudothecium development causes the ostiole to face the air when leaves fall.This positioning increases the likelihood of ascospores being dispersed upward in spring, aiding their anemochoric dissemination [2].The pathogen strategically discharges ascospores when host susceptibility is at its highest, optimizing the likelihood of successful establishment in the host canopy, and, thus, ensuring efficient disease progression [2].When ascospores land on susceptible host tissue, in conditions of sufficient humidity, they germinate and attach to the leaf surface through adhesive mucilage, which acts as a bonding agent, allowing the spore to firmly adhere to the leaf and establish an infection site [7].
to the velvety appearance of scab lesions [13].The primary spread of the disease occurs within the canopy of the infected tree.Conidia are released by the pathogen and dispersed through water films, either locally or via splash-dispersion during rainfall events.This mode of dispersion allows the conidia to reach nearby susceptible host tissue, contributing to the spread and establishment of the disease within the orchard [2].This leads to secondary infections during the growing season [13].Susceptibility to V. inaequalis varies with the age of the host tissue.While young tissues of apple plants rely on a combination of quantitative trait loci (QTLs) and resistance genes (R genes), mature tissues exhibit ontogenic resistance [9].This resistance develops as the leaves and fruit mature and is complete when leaves are fully expanded.Infections primarily occur on unfolding leaves and internodes between them, with ontogenic resistance developing faster on the ventral side of the leaves during expansion.The basis of this resistance is not yet understood, but it becomes inactive as leaves senesce in late seasons [2,9,14,15].This phenomenon is observed in all apple cultivars and has never been overcome by the pathogen [9].

Management Strategies of Apple Scab
The introduction of synthetic fungicides in the late 1940s marked a turning point in the management of apple scab.Most apple cultivars are susceptible, and over time, fungicides became the predominant method for disease control, with limited exploration of alternative strategies on a commercial scale [7].The heavy reliance on numerous fungicide applications results in substantial expenses for growers, coupled with undesirable The pathogen utilizes enzymatic hydrolysis of the cuticle, facilitated by enzymes like cutinase, to penetrate the host, forming nutrient-absorbing stromata which, in turn, lead to the development of characteristic lesions [9].The single-celled, uninucleate conidia produced from these lesions on conidiophores emerge through the leaf cuticle, contributing to the velvety appearance of scab lesions [13].The primary spread of the disease occurs within the canopy of the infected tree.Conidia are released by the pathogen and dispersed through water films, either locally or via splash-dispersion during rainfall events.This mode of dispersion allows the conidia to reach nearby susceptible host tissue, contributing to the spread and establishment of the disease within the orchard [2].This leads to secondary infections during the growing season [13].
Susceptibility to V. inaequalis varies with the age of the host tissue.While young tissues of apple plants rely on a combination of quantitative trait loci (QTLs) and resistance genes (R genes), mature tissues exhibit ontogenic resistance [9].This resistance develops as the leaves and fruit mature and is complete when leaves are fully expanded.Infections primarily occur on unfolding leaves and internodes between them, with ontogenic resistance developing faster on the ventral side of the leaves during expansion.The basis of this resistance is not yet understood, but it becomes inactive as leaves senesce in late seasons [2,9,14,15].This phenomenon is observed in all apple cultivars and has never been overcome by the pathogen [9].

Management Strategies of Apple Scab
The introduction of synthetic fungicides in the late 1940s marked a turning point in the management of apple scab.Most apple cultivars are susceptible, and over time, fungicides became the predominant method for disease control, with limited exploration of alternative strategies on a commercial scale [7].The heavy reliance on numerous fungicide applications results in substantial expenses for growers, coupled with undesirable environmental consequences [7,16].To reduce dependence on fungicides, there is a huge concern to incorporate less-toxic control methods.This shift would not only mitigate costs for growers but also address environmental concerns associated with extensive fungicide use in apple cultivation [7,17].Typically, management practices employed to control apple scab focus on disrupting the disease cycle, specifically targeting its critical reproductive structures (e.g., spore germination).

Sanitation Practices
This approach involves minimizing overwintering inoculum through the mechanical removal of fallen leaves, ensuring meticulous orchard sanitation to control primary inoculum and, thereby, reduce the severity of scab infections [3].Cultural control and sanitation practices play a pivotal role in mitigating scab infection, encompassing measures such as leaf shredding, pruning, burning, or burying leaves in the soil, along with the strategic application of a 5% urea solution to accelerate the decomposition process [6,8].
While cultural control proves indispensable in managing apple scab, it comes with inherent challenges.These include labour-intensive practices, limited efficacy, timing sensitivity, potential pathogen resurgence, weather dependency, long-term commitment requirements, and challenges related to varietal susceptibility [5][6][7].Additionally, these inoculum reduction measures may be costly and impractical for certain operations [6].
MacHardy et al. [2] revealed that the introduction of horticultural practices in apple production has unintentionally facilitated the spread of V. inaequalis.Practices such as herbicide-treated strips, repeated mowing, and the use of semi dwarfing and dwarfing rootstocks, along with modern pruning techniques, create pathways for ascospores to reach the tree canopy more effectively [2].These changes enhance ascospore dispersal efficiency, resulting in an increased number of successful infections.Initially, the impact of these modifications on scab epidemiology went unnoticed due to simultaneous advancements in fungicides, application strategies, and technology, which helped manage the challenges posed by these horticultural changes [2].
Additionally, overhead irrigation systems that promote prolonged leaf wetness have been linked to the increased disease severity of apple scab [18].In contrast, drip irrigation systems that deliver water directly to the roots without wetting the foliage insure dry leaves which are less susceptible to infection and can help reduce disease development [5,18].

Mixed Cultivars
Implementing mixed-cultivar orchards, mimicking natural diversity, shows promise in reducing scab incidence [2].Carisse and Bernier [1] emphasized the significant potential for reducing disease severity by taking advantage of cultivar susceptibility to V. inaequalis through a thoughtful orchard design.This was demonstrated in the work of Blaise and Gessler [19], where a careful consideration of cultivar selection and arrangement resulted in a substantial reduction in disease severity.By strategically mixing cultivars within or between rows, barriers to inoculum spread are created, offering a promising avenue for reducing susceptible tissue and enhancing disease control [19].
While often notable, the reduction in scab observed in mixed-cultivar orchards is typically insufficient for effective disease control in commercial production [8].However, the most significant concern in implementing mixtures in commercial horticulture is the potential emergence and rapid build-up of a scab 'super race'.This combines virulence factors that can overcome most or all of the resistance genes in the host cultivars present in the mixture, rendering it ineffective as a mean for scab management [8,20].

Breeding for Resistance
Many commercially important apple cultivars are at risk of susceptibility to V. inaequalis [21,22].Unfortunately, the slow and challenging transfer of disease resistance alleles from wild sources to apple cultivars with desirable fruit quality has led to a limited number of scab-resistant cultivars in commercial production [11].The current strategy for cultivating scab-resistant cultivars involves incorporating Rvi resistance (R) genes into apple cultivars to enhance their defence mechanisms against V. inaequalis [20].A total of 20 R genes against V. inaequalis, numbered from Rvi1 to Rvi20, have been documented, with the majority discovered in wild Malus accessions and landraces [20,23].Several commercial varieties show significant susceptibility to scab due to either a lack of known Rvi genes or a compromised effectiveness against specific races of scab [20].The primary resistance gene used in breeding programs against apple scab has been Rvi6, which originated from the Japanese crab-apple Malus floribunda (Sieb.)sel.821 [24].Modern scab-resistant apple cultivars primarily rely on the Rvi6 gene to enhance resistance against apple scab [25,26] (Table 1).Unfortunately, several Rvi genes, including the widely utilized Rvi6 gene, have succumbed to new virulent strains of V. inaequalis [27].The erosion of Rvi6 has been observed in various varieties, including its origin, M. floribunda [20].This breakdown of some of the Rvi genes was initially observed in Germany and later reported in various countries around the world [24,[26][27][28][29].Despite ongoing breeding efforts over the past decades, the majority of commercially grown scab-resistant apple varieties still rely on the Rvi6 resistance [30].Additionally, resistance gene pyramiding, involving the cultivation of cultivars with multiple resistance genes for durable resistance, has been explored [26].While some combinations show positive results in field trials, their long-term effectiveness and commercial viability remain uncertain without direct empirical evidence [31].
Beyond resistance concerns, the widespread adoption of scab-resistant cultivars (such as Topaz, Prima, and Florina) in commercial orchards is limited due to worries about lower fruit quality and the uncertain durability of resistance [32].Moreover, these challenges include the existence of multiple races of V. inaequalis [6] and the emergence of virulent races even in certain wild Malus species or genotypes like 'Golden Delicious' that remains highly susceptible despite carrying the Rvi1 gene [6,20].This situation raises concerns about fruit quality and poses obstacles for growers to replace existing trees due to cost and time considerations [3].Hence, despite offering some protection against scab, resistant cultivars still face limitations such as less effectiveness against evolving pathogen strains, vulnerability to genetic uniformity, limitations in horticultural traits, longer development times, and an unclear long-term environmental impact [3].Balancing resistant varieties with other tools is crucial in apple scab management programs.

Scab Forecasting Models
Considering multiple factors like weather conditions, disease history, and the unique characteristics of different apple varieties, forecasting models play a crucial role in apple scab management [47,48].Two prominent models come to the forefront.The Mills and LaPlante [49], a foundational framework, focuses on scab infection periods using a combination of leaf wetness duration and temperature to comprehend and predict scab onset.Several global studies were conducted to validate Mills' table and warning system, revealing some disagreements with the model [50][51][52].Building upon this, the revised model by MacHardy and Gadoury [50], known as 'Mills/a -3', addresses a three-hour discrepancy observed in Mills' studies.This curve, along with data from field weather stations, is used to assess infection risk.Additionally, the integration of sophisticated models, like the MacHardy/Gadoury ascospore maturity model, into forecasting tools, along with the implementation of web-based Decision Support Systems (DSSs), has proven indispensable in offering real-time advice in horticultural extension services [47,53].
Despite notable advancements, challenges persist, particularly with discrepancies observed in the outcomes of forecasting tools [47,53].While models utilizing weather data demonstrate accuracy in predicting infection periods, the ultimate effectiveness of control strategies depends on the availability of potent curative fungicides [54].

Chemical Fungicides
Over the past seven decades, apple scab management has heavily relied on weekly fungicide applications, with commercial plantations typically undergoing 10 to 30 treatments throughout the growing season [55].V. inaequalis has been classified by the Fungicide Resistance Action Committee (FRAC) as a high-risk pathogen [56], signifying a substantial likelihood of fungicide resistance development in this pathogen [57].Many different active ingredients with diverse modes of action have been deployed for apple scab management (Table 2).The primary objective is to effectively inhibit the initial ascospore-driven primary infections that occur during early spring [58].
Historically, several multisite fungicides such as Dodine, Captan, sulphur, copper, and Benzimidazole fungicides played a crucial role in apple scab control.However, fungicide resistance gradually emerged over time, restricting their effectiveness in managing apple scab [57,59].Consequently, single-site fungicides targeting mitochondrial respiration or ergosterol biosynthesis, including quinone outside inhibitors (QoIs), sterol demethylation inhibiting fungicides (DMIs), and the newer generation II of Succinate dehydrogenase inhibitors (SDHIs), emerged as viable alternatives for the control of this pathogen [55,60].Nevertheless, the specific modes of action of these single-site fungicides pose a significant risk for resistance development [60,61].The mechanism underlying DMI fungicide resistance in V. inaequalis involves the overexpression of the CYP51A1 gene, along with efflux mechanisms and point mutations [62,63].Resistance to DMI fungicides has been reported globally [59,60,[64][65][66].Resistance to QoIs in V. inaequalis is primarily attributed to a target site mutation in the cytochrome b (cyt b) gene, known as G143A [57,[67][68][69].SDHI resistance in V. inaequalis, detected shortly after market introduction, was primarily observed in trial sites and associated with specific target site mutations (B-T258I, C-N85S, and C-H151R).Notably, resistance to SDHI fungicides in V. inaequalis was observed as individual cases and did not show an increase in frequency [57].Nevertheless, it is important to note that the high intrinsic activity and target specificity of SDHI fungicides significantly raise the risk of resistance development, particularly as newer SDHI fungicides are increasingly relied upon, leading to a reduction in available alternatives for diversifying the chemical fungicide options [61].

Biological Control
Biological control is gaining increasing attention as an alternative means for plant disease management [76].The availability of diverse products with different modes of action will play a crucial role in scab control and help mitigate the risk of over-reliance on individual products or synthetic fungicides.There is an urgent need to develop alternative products, specifically by exploring biocontrol options and by making multiple biocontrol products available that target distinct stages of the pathogen life cycle [8].

Insight into Integration of Biocontrol in Apple Scab Management
The biological control of plant pathogens involves inhibiting the diseases or their causative pathogens using living organisms or their specialized metabolites [76,77].This encompasses both living and non-living agents, offering environmentally friendly and specific disease management [77].Basically, biocontrol involves tipping the ecological balance in favour of the introduced agent to hinder pathogen development [3].By utilizing natural antagonists like bacteria or fungi, biological control serves as an eco-friendly alternative to synthetic fungicides, potentially posing fewer environmental risks.
Integrating biological control agents (BCAs) and their by-products into scab disease management represents a transformative approach in sustainable agriculture [58].Despite the potential benefits, there has been limited adoption of biocontrol methods for managing apple scab [78].While BCAs and their products are often compared to fungicides, such fundamental comparisons are unjustified due to the living nature of BCAs and their gradual pathogen-suppressive effects [79].Numerous studies have revealed that applying BCAs to a crop can achieve disease control levels comparable to or nearly equivalent to those achieved by fungicides [78,80].Instead of replacing fungicides, it is more appropriate to consider BCAs as a central component of an integrated pest management program [79].By BCAs-integration with other management approaches, their impact in agriculture can be significantly enhanced [48].
The first approach commonly employed for identifying novel BCAs against V. inaequalis involves screening several microbial libraries from the environment that might exhibit antagonistic potential against the pathogen [76].Subsequently, the selection process may identify microorganisms capable of inhibiting V. inaequalis through in vitro studies.Once potential antagonists are identified, understanding the conditions influencing their inhibitory effects becomes crucial, ensuring their effectiveness across diverse environmental conditions [81].
In the initial screening for potential antagonists, in vitro studies are preferred over in vivo studies due to logistical and cost constraints [3].The advantage of the in vitro approach lies in its ability to efficiently screen a large and diverse collection of strains for their antimicrobial or mycoparasitic activities [76].This makes in vitro experiments a more manageable method for screening potential BCA candidates [3].However, as noted by Collinge et al. [76], in vitro screening has limitations and can be misleading, with results not precisely indicating an efficacy on plant level.Notably, Fiss et al. [82] discovered significant antagonistic activity in a yeast strain (H25) against the apple scab pathogen in seedling assays, despite weak initial inhibitory effects in basic in vitro screenings.While the in vivo approach is a compromise, it offers a more realistic assessment under simulated field conditions, enabling a reasonable level of throughput and improved selection of promising BCA candidates [76].In general, a comprehensive approach that combines both in vitro and in vivo methods might provide a more robust evaluation of BCAs against V. inaequalis [83].This dual strategy leverages the advantages of high-throughput screening in vitro while ensuring selected candidates undergo realistic assessments in simulated field conditions [76,83].
Establishing a reliable and standardized method for assessing BCAs that considers specific stages in the pathogen's life cycle is essential for evaluating their impact on the scab pathogen.Most screening tests directed at traditional criteria like leaf rheology [84,85] and inhibition of mycelium and conidia production [78] have been considered unreliable or impractical [3].Philion [3] argued that screening techniques targeted at the vegetative phase of V. inaequalis are useless because tests concentrated on mycelium inhibition may not conclusively demonstrate efficiency in inhibiting pseudothecia formation.The key criterion suggested for BCAs' selection is the in vitro inhibition of ascospore production [3].By focusing on the in vitro inhibition of ascospore production, potential antagonistic agents that have the capability to suppress or reduce the formation and release of ascospores by the pathogen have been evaluated [3,16,86,87].This ascospore inhibition is essential for disrupting the pathogen's reproductive cycle and reducing its overall population size, ultimately leading to decreased disease severity [3].
Antagonists can alleviate pathogen development through multiple key mechanisms including niche or nutrient competition, parasitism, and antibiosis [58,77].These mechanisms can act independently or synergistically [58].While considerable efforts have been dedicated to elucidating the biological activity exhibited by beneficial microorganisms against plant pathogens, recent findings suggest that a synergistic interplay of various molecules, such as hydrolytic enzymes and antibiotics, contributes to their effectiveness [58,80,[88][89][90].Recognizing the crosstalk between potential antagonists and fungal pathogens highlights the potential for a more informed and rational application of these agents in agriculture [58].
BCAs demonstrate versatile effectiveness in managing plant diseases, both pre-and post-harvest [7].Particularly, in apple scab control, BCAs strategically applied after harvest and before leaf fall influence the overwintering phase and subsequent pathogen development [91,92].This tailored approach not only contributes to a comprehensive disease management paradigm throughout the orchard's life cycle but also highlights the remarkable adaptability of BCAs to various crop growth stages [91].
The history of apple scab biocontrol stretches back to several decades, with key contributions from pioneers like Cinq-Mars [93] and Ross [94] who explored microbial control strategies.Cinq-Mars focused on antagonists and culture filtrates, highlighting antibiosis and competition against V. inaequalis.Ross expanded these efforts, introducing microbial antagonism against pseudothecial development.Subsequent studies have highlighted the importance of using beneficial antagonists to reduce the overwintering pseudothecia in apple scab control strategies [86,95,96].Heye [84] identified Athelia bombacina Pers.as an effective basidiomycete inhibiting pseudothecia formation.These approaches are preventive and can be seamlessly incorporated into existing management schemes.Nonetheless, integrating all or some of these methods is more intricate than the straightforward use of fungicides.
The effectiveness of BCAs is intricately tied to specific environmental conditions and the unique characteristics of the antagonist [97].Different agents and target pathogens have unique preferences and dynamics, necessitating the consideration of specific conditions.For instance, Carisse and Bernier [1] indicate that for the optimal efficacy of Microsphaeropsis isolates as biocontrol agents, certain conditions such as temperatures between 20 and 25 • C, pH around 4, and a minimum of 8 h of light are favourable.In contrast, Ouimet et al.'s [81] study on Ophiostoma sp.demonstrated a high inhibitory effect on V. inaequalis (100%), and this antagonist remains effective regardless of temperature (5-25 • C) and pH variations (5-7), adapts to different carbon sources, and is not hindered by light.Therefore, it is essential to consider the specific characteristics of the BCAs and their favourable environmental conditions.

BCAs as Sustainable Alternatives
In recent years, there has been a significant increase in the interest in the biological control of plant pathogens [89].This can be attributed, partially, to public concerns about the use of potentially hazardous chemical plant protection agents (PPAs) [98].Additionally, biological control is increasingly seen as a viable approach for managing diseases that cannot be fully or adequately controlled by other means [76].The primary objective of biocontrol is to offer supplementary tools for disease management, featuring modes of action distinct from conventional chemical PPAs [99].Moreover, the likelihood of farmers adopting new products is higher when these products can be easily incorporated into existing production systems with minimal disruption [100].By incorporating BCAs into an integrated pest management program, including sanitation, host resistance and reduced fungicide rates, the efficient utilization of available resources can be ensured.This approach not only results in safer production practices, but also maximizes the effectiveness of disease management [101].
Microbial-based biopesticides stand out as prime candidates to replace or complement synthetic PPAs, contributing to the promotion of sustainable agri-food production [89,102].
However, the availability of microbial biofungicides remains limited in comparison to conventional chemical synthetic fungicides [89].A few biofungicides that have obtained official registration globally are primarily marketed for specific niches, notably high-value crops where there is a demand for PPA-free products [4].These biofungicides typically comprise a single strain of an antagonist, limiting their effectiveness to a narrow range of diseases and limiting their broader application [4].Considerable efforts have been made to manage apple scab using microbial biocontrol agents [103][104][105].However, despite numerous reports in the literature, there are, so far, no commercially available BCAs specifically designed to control apple scab [17,20,106].
A holistic strategy for managing apple scab involves applying a BCA in autumn to disrupt the overwintering structures of the pathogen (i.e., pseudothecia formation) [7].Several reports demonstrated that lower ascospore numbers were correlated with reduced disease severity in the subsequent spring [7].This strategy has been comprehensively studied [7,16,93,94,108,112]. Additionally, certain fungal members, such as A. bombacina and Coniothyrium sp., contribute to leaf decomposition and, thus, interfere with pseudothecia development.These fungi accelerate the breakdown of senescent leaves while also exhibiting antifungal properties [3,[112][113][114].On the other hand, extensive research has been conducted on the disease management of summer epidemics, driven by conidia produced by the biotrophic mycelia that develop underneath the cuticle of apple leaves [38,82,103,105].
Recently, Caffier et al. [115] explored the potential of using hybridization and host adaptation to limit the impact of V. inaequalis on apple trees.V. inaequalis also causes scab disease in other Rosaceae hosts such as Pyracantha and Loquat [115,116].These authors tested field isolates from different hosts and the progenies resulting from crosses between isolates from Pyracantha and apple for pathogenicity.The results revealed a strict host specificity between apple and Pyracantha isolates, with most causing disease on Loquat.Progeny resulting from crosses between V. inaequalis f.sp.pyracantha and V. inaequalis f.sp.pomi were unable to infect apple.The study suggests a potential biocontrol strategy, similar to sterile insect approaches, involving the introduction of Pyracantha isolates to reduce V. inaequalis populations in apple orchards without the use of chemicals.This strategy entails introducing Pyracantha isolates in autumn to produce hybrid ascospores incapable of causing apple disease in spring, with the aim of reducing the primary inoculum in apple-producing regions.However, experimental evaluation in orchards is necessary, along with an assessment of the risk posed by hybrids that may be pathogenic to apple or other Rosaceae hosts.[34]

Modes of Action of BCAs against V. inaequalis
BCAs and plant pathogens can engage in various interactions, employing diverse mechanisms of action.These mechanisms can be broadly categorized into direct antagonism against the pathogen, including parasitism, antibiosis, and competition; and indirect biocontrol activities, such as the induction of systemic resistance and promotion of plant growth [77,89].In many instances, the effective control of disease by BCAs is attributed to their utilization of multiple mechanisms.For instance, T. harzianum has been demonstrated to suppress various plant pathogens by employing a combination of mechanisms including antibiosis, cell wall degrading enzymes, and the induction of host resistance [77,121].The viability of biological control for disease reduction relies on understanding its mechanisms.This knowledge is crucial for creating conditions supporting treatment efficacy.Identifying how biocontrol agents suppress a pathogen helps anticipate resistance and prevent interference from other ecosystem components [88].

Indirect Biocontrol Activities against V. inaequalis
A commonly observed mechanism associated with the protection of plants by BCAs involves the induction of host defence pathways [80].Two main types of induced resistance exist: systemic acquired resistance (SAR), activated by pathogen infection and requiring salicylic acid, and induced systemic resistance (ISR), triggered by beneficial microorganisms [122].This mechanism stems from BCAs releasing elicitors, such as proteins, antibiotics, and volatiles, ultimately leading to the activation of genes in the salicylic acid pathway or the jasmonic acid/ethylene pathway [80].Bolar et al. [123] integrated genes encoding antifungal proteins, endochitinase, or exochitinase from the biocontrol fungus T. atroviride into 'Marshall McIntosh' apple plants.The resulting plants were evaluated for resistance to V. inaequalis.The study found that disease resistance correlated with the expression level of each enzyme when expressed individually, with endochitinase proving more effective than exochitinase.The persistent expression of fungal chitinases in apple lines was associated with enhanced resistance against V. inaequalis.Particularly, a significant positive correlation was observed between the level of endochitinase expression and scab resistance.
On the other hand, various other compounds have been investigated as defence elicitors to enhance disease management and resistance against apple scab (Table 4) [38] including chitosan [124] and fructans [38].Plant defence inducers, including harpins, and salicylic acid derivatives demonstrated efficacy against V. inequalis under field conditions [125,126].While these inducers showed a reduction in leaf and fruit scab when applied at different growth stages, their efficacy was markedly enhanced when used in combination with fungicides (Boscalid + Pyraclostrobin).In addition, Rusevski et al. [54] explored the biofungicidal potential of Vacciplant, a biofungicide containing laminarin as its active ingredient, for the biological control of apple scab.Laminarin, derived from brown seaweed, is a polysaccharide recognized for its antifungal and defence-inducing properties [126].The study revealed comparable efficacy to the standard fungicide Captan, showing promising results in decreasing infection levels on the leaves and fruits of two apple varieties.

Direct Biocontrol Activities against V. inaequalis 6.2.1. Competition
Competition in the context of biological control refers to niche overlap, where two or more microbial populations simultaneously demand the same resources [127].Competition is a widely utilized mechanism in biocontrol.It manifests in various ways, including reducing inoculum potential through nutrient competition, increasing saprotrophic competition during substrate colonization, and decreasing the actual pathogen density during survival or growth phases [127].BCAs utilize strategies like sequestering iron through the production of iron-binding siderophores, aiming to reduce the availability of iron to other organisms [80].This is particularly significant in the context of the effectiveness of biological control against pathogenic fungi, as these fungi heavily rely on iron (Fe 3+ ) for their growth and virulence [128].In their study, Miliute et al. [119] found that sterile culture filtrates from Pantoea species inhibited the growth of V. inaequalis and produced siderophores.They attributed the inhibition of V. inaequalis mycelial growth to these biochemical traits.Padder et al. [17] also attributed the mechanism of induced iron starvation as one of the possible reasons for the antifungal behaviour of Bacillus and Pseudomonas sp.against V. inaequalis.They also observed that iron chelation through this mechanism does not impact plant development, as plants can grow at significantly lower iron levels than the invading pathogenic microflora.While competition for nutrients and space plays a major role in biocontrol, appropriate methods are lacking to separate these two mechanisms of action [127].

Mycoparasitism
Mycoparasitism involves a series of steps in microbial interaction, including close contact with the pathogen, mutual recognition, the release of lytic enzymes, penetration, and active development inside the host [128].Through mechanisms like haustoria formation or invading the pathogen's mycelium, mycoparasites absorb nutrients from the host for their survival and growth [106].While mycoparasites weaken the host gradually, they do not completely eradicate it.Applying mycoparasites when the pathogen is first detected is crucial, as these biocontrol agents depend on a host for their survival [106].
Compared to synthetic chemical fungicides, mycoparasites exhibit a slower onset of action and require sufficient time to colonize and eliminate the pathogen.Consequently, when rapid action is imperative, biofungicides employing direct antibiosis should be prioritized [106].
Benyagoub et al. [58] offer valuable insights into the cellular mechanisms of mycoparasitism, specifically focusing on the interactions between the antagonistic fungus Microsphaeropsis sp. and the apple pathogen V. inaequalis.Through ultrastructural and cytochemical analyses, they showed a sequence of events in the mycoparasitic process, including attachment, penetration, fungal host structural response, and active multiplication of the mycoparasite.The antagonist, Microsphaeropsis sp., penetrated V. inaequalis hyphae by disrupting the osmiophilic coating layer.This disruption likely caused significant metabolic changes, exposing the underlying wall layers to mechanical pressure and potential enzymatic attack.

Antibiosis
Antibiosis stands out as the most extensively studied mechanism, as it provides a direct means for evaluating candidates based on their inhibitory activity [78].Antibiosis is specifically characterized by the secretion of volatile and/or non-volatile products that inhibit or restrict the growth of the target pathogen [81].It constitutes a chemically diverse group of organic, low-molecular-weight compounds synthesized by microorganisms [90].Over the past decades, a multitude of studies has unequivocally shown that various metabolites, including antibiotics, enzymes, proteins, and volatiles produced by antagonistic bacteria and fungi, play crucial roles in controlling a wide array of plant pathogens [90].The identification of specific secondary metabolites considered potential biological control agents against V. inaequalis has roots dating back several decades.
Early research led by Cinq-Mars [93] involved isolating microorganisms from apple leaves.Among these, Penicillium species, especially, demonstrated the production of antibiotics inhibiting V. inaequalis mycelial growth.In 1953, Ross [94] isolated 13 saprophytic fungi from apple leaf litter.These fungi were found to produce antibiotic substances in liquid culture, leading to complete or partial inhibition of V. inaequalis mycelial growth.Notably, only Oospora lactis and Penicillium species exhibited the secretion of toxic metabolites with activity against the pathogen.However, their fungitoxic effects were found to be lower than that of the organo-mercurial fungicide ('Tag') used for comparison.Later, Simard et al. [129] evaluated some fungal isolates for their antibiotic properties against V. inaequalis.Among the organisms isolated from apple leaf litter, 34 were identified to impede the germination of conidia, as determined by the sprayed-plate test.Remarkably, twelve of these isolates belonged to the genus Penicillium, consistent with the findings previously reported by Cinq-Mars [93].In 1984, Cullen and Andrews [130] investigated the potential of Chaetomium globosum and Athelia bombacina for apple scab control, revealing that C. globosum produced essential antibiotics.Antibiotic production varied among C. globosum strains, with higher levels positively correlating with increased antagonism against V. inaequalis on seedlings.Chetomin, tentatively identified in culture extracts, was observed using thin-layer chromatography and ultraviolet absorbance.In 1997, Ouimet et al. [78] identified Aureobasidium sp., Phoma sp., and Ophiostoma sp. as strong inhibitors, suppressing over 80% of V. inaequalis mycelial growth.Meanwhile, Ophiostoma sp.exhibited a prolonged inhibitory effect lasting beyond 58 days.However, the impact of metabolites released by the fungi results in an inhibitory effect of less than 5%.Recently, Padder et al. [17] isolated and characterized bacterial endophytes with antifungal properties from apple germplasm.They reported a 100% suppression of scab disease using bacterial cell-free supernatants.The observed impact on disease severity and incidence at all concentrations was linked to the presence of antimicrobial compounds, including but not limited to phenazines, ammonia, pyrrolnitrin, hydrocyanic acid, and pyoluterorin.In their study, Ebrahimi et al. [41].investigated the antifungal properties of various endophytic fungi against V. inaequalis in vitro.The study aimed to determine the release of metabolites from Aureobasidium sp. and C. globosum.Notably, the volatile organic compounds produced by specific isolates, including A. microstictum 7F2 and C. globosum 2S1, along with others, were found to completely prevent the mycelial growth of V. inaequalis.
Desmyttere et al. [131] conducted a comprehensive investigation into the antifungal properties of B. subtilis lipopeptides against V. inaequalis.Fengycin displayed potent antifungal activity comparable to the chemical fungicide tebuconazole, particularly against sensitive strains.Surfactin and mycosubtilin, either alone or in combinations, displayed varying effectiveness.In a similar study, Leconte et al. [110] explored the efficacy of B. subtilis lipopeptides in combating apple scab.Their in vitro tests on three lipopeptide families and their mixtures revealed varying levels of antagonistic activity against V. inaequalis.Notably, Fengycin exhibited significant inhibition among the three.Orchards trials revealed a reduction in scab incidence by 70%, with the mycosubtilin/surfactin mixture demonstrating better reproducibility.Combining these potent antifungal molecules in orchard treatments against V. inaequalis is likely to result in a synergistic effect against the pathogen [110].
Considering chitin as a main component of fungal cell wall, several studies have particularly focused on identifying antagonistic BCAs with chitinolytic properties [123,[132][133][134][135].This interest has led to the exploration of diverse chitinolytic microorganisms as potential biological control agents effective against various fungal plant pathogens [90,119,123].Taking a genetic approach, Bolar et al. [132] sought to enhance apple resistance against apple scab by introducing genes from Trichoderma harzianum encoding endochitinase and exochitinase.The simultaneous expression of these enzymes resulted in a synergistic reduction in disease severity, with minimal effects on plant vigour.Later, Miliute et al. [119] extended the exploration by identifying chitinase-producing endophytes from apple plants, specifically Pantoea species and the Pseudomonas fluorescens group.These endophytes exhibited the ability to inhibit the mycelial growth of V. inaequalis.Three isolates of Pantoea spp.demonstrated inhibitory effects, with one exhibiting additional trait, including siderophore production and hydrogen cyanide generation.
Chitosan, a deacetylated derivative of chitin, has emerged as a particularly promising compound due to its well-documented antifungal activity [124,125,136,137].The potential synergies resulting from combining chitosan with BCAs was investigated by DeGenring et al. [124].They found that chitosan, when applied pre-harvest, reduced the incidence and severity of apple scab, sooty blotch, flyspeck, and rust.The combination of chitosan with a biopesticide was even more effective in disease suppression.
In line with the ongoing efforts to advance scab management strategies, Hossain et al.

Biological Control of Apple Scab Using Botanicals
Plant extracts offer promising alternatives for pest and pathogen control [139,140].They have long been utilized as natural pesticides due to their cost-effectiveness, ease of preparation, and eco-friendly properties with no resistance risks [141].Plant extracts contain low-molecular-weight secondary metabolites, produced in response to stress, that serve as potential alternatives for controlling apple scab without harm to non-target organisms [139,142].Various promising plant extracts have demonstrated effective control against apple scab (Table 5).Bengtsson et al. [140] investigated the impact of Yucca schidigera extract in controlling V. inaequalis, comparing it with the chemical resistance inducer acibenzolar-S-methyl (ASM).Yucca extract and ASM significantly reduced apple scab symptoms and sporulation in seedling assays, exhibiting similar control efficacy to sulphur.Yucca extract and sulphur inhibited conidial germination in vitro, while ASM did not.Yucca extract inhibited conidial germination by 98% to 100%, sulphur exhibited inhibition ranging from 72% to 97%, and ASM showed inhibition of 5% to 25%.Yucca extract primarily acted fungitoxically, inhibiting pre-penetration and penetration, while ASM hindered the subsequent infection stages.Gene expression studies on apple seedlings suggested that Yucca extract may influence plant defence, with the upregulation of PR1 and PR8 genes resembling levels observed after ASM treatment.Additionally, essential oils like those from Thymus vulgaris and other extracts are well-suited for addressing plant pathogen control, including apple scab, owing to their antimicrobial and antifungal properties [143,144].However, the effectiveness of extracts and essential oils may vary under different circumstances, especially in field conditions.Therefore, a well-developed formulation is crucial to maintaining consistency and ensuring reliable performance during field trials [144,145].

Risks and Challenges of Utilizing BCAs in Apple Scab Management
Introducing new microorganisms to leaf and fruit surfaces, while common, presents potential risks and both positive and negative outcomes [97].The complex interactions between these introduced microorganisms and the existing balance in the plant environment need careful consideration due to the potential for unexpected effects [151,152].
Concerns arise from the potential misidentification of microorganisms during screening processes, leading to the inadvertent selection of harmful strains, including those that may pose threats to both plants and humans [77].In the context of commercialization, screening for BCAs presents a substantial challenge, as it involves identifying organisms suitable for commercial development while ensuring the exclusion of potential pathogens [3].Instances of initially identified BCAs turning out to be possible human pathogens highlight the need for accurate identification to mitigate risks [77].Trichothecium roseum Link.have demonstrated pathogenic traits under certain conditions, affecting both apple orchards and other plant species [3].Effectively navigating this challenge is crucial for ensuring that selected BCAs not only prove effective in pest management but also pose minimal risks to agriculture, the environment, and human health when deployed on a commercial scale [76,77].The integration of genomic sequencing has emerged as a possible solution [77], enhancing the identification process and thereby minimizing the potential hazards associated with introducing new microorganisms into diverse ecosystems.
Another associated risk with BCAs is the production of harmful metabolites or mycotoxins [3,76].A case in point is the ascomycete C. globosum, which initially showed promise in effectively controlling V. inaequalis within the phyllosphere.However, despite its efficacy, the production of toxins including chaetomin led to its eventual abandonment as a commercially viable BCA [76, 130,153].This case highlights the delicate balance required in selecting BCAs, emphasizing the necessity of a thorough evaluation not only for their efficacy in pest control but also for their potential to produce harmful substances that could have adverse consequences on both the target species and the surrounding environment [76].This risk calls for a comprehensive understanding of the metabolic pathways and secondary metabolite profiles of potential BCAs to ensure their safety and effectiveness in practical applications.
Furthermore, establishing a reliable method for assessing BCAs, particularly in the context of apple scab, is complicated by difficulties in the in vitro production of fungal asexual and sexual structures (e.g., conidia or pseudothecia) [3].The difficulty in replicating these structures outside their natural environment hampers the precision of BCA evaluations.In response to this challenge, alternative parameters, with mycelium being one such focus, have been explored [3].However, relying on mycelium alone in evaluating the efficacy may potentially lead to misleading conclusions [3].This emphasizes the continual need to refine and diversify evaluation methods for a comprehensive understanding of BCAs' efficacy and potential against V. inaequalis.
The effectiveness of specific BCAs against plant pathogens can be influenced by various factors, including environmental conditions, the timing of treatment, the season of application, the nature or method of treatment, and the frequency of application [1,81,154].BCAs in orchards encounter challenges in colonizing apple leaves under cold conditions, adapting to industrial production and employing multiple modes of action against V. inaequalis [1,2].The efficacy of BCAs during colder periods, particularly in the autumn, represents a substantial hurdle, demanding the development of strategies to enhance their effectiveness under these challenging conditions [3,7].In colder climates characterized by harsh winter conditions, the conidia and mycelia of V. inaequalis cannot withstand the low temperatures and freezing [155][156][157].Consequently, V. inaequalis overwinters within scabbed leaves as pseudothecia [155].The ability of BCAs to establish a robust presence on apple leaves in cold climates is crucial for their success in scab management, necessitating innovative approaches to address these specific environmental challenges [158].
On the other hand, biological products encounter challenges in achieving widespread adoption due to inconsistencies in their performance observed in both in vitro and in vivo assays [159].Ouimet et al. [78] explored the effect of fungal antagonists on the in vitro inhibition of mycelial growth of V. inaequalis and found that Ophiostoma sp.emerged as a potent inhibitor and completely prevented mycelial growth across various environmental conditions, including temperature, pH, and light variations.The same BCA reduced ascospores production up to 88.7% in a single trial [86].However, subsequent trials conducted by Carisse et al. [108] presented a contrasting outcome.Despite the recorded successes of the inhibitory potential of this antagonist, the Ophiostoma sp.isolate showed a notable failure to inhibit ascospore production.Across all tests, the average ascospore inhibition was merely 8.2%.Additionally, the slow commercial adoption of BCAs for plant disease management also stems from issues related to host specificity [154] as exemplified by the study on Epicoccum purpurascens and T. roseum [3].Despite their reported antagonistic activities in other plant-pathogen systems, these fungi showed no antagonistic ability against apple scab in the specific conditions of the study.This emphasizes the importance of understanding the context-specific interactions between BCAs and their target pathogens to ensure their effectiveness in practical applications.
To fully harness the potential of biocontrol, a shift towards comprehensive, fieldoriented research and the optimization of commercial formulations is essential [77].Regrettably, the transition from laboratory efficacy to real-world field success remains a challenge for many biological agents [128,154].The discrepancy in effectiveness is often attributed to the limitations imposed by physiological and ecological factors inherent in field conditions [128].Genetic engineering and molecular methods offer new ways to improve biocontrol agent selection and evaluation [128].Manipulating the genetic makeup of these agents optimizes their performance, ensuring a more seamless transition from controlled laboratory conditions to the complex and dynamic environments found in actual fields [128,160].These molecular approaches provide a valuable toolset to finetune the characteristics of biocontrol agents, potentially overcoming the hurdles posed by physiological and ecological factors that hinder their success in real-world applications.
Another significant challenge with BCAs is ensuring consistent efficacy and addressing limited shelf life, attributed to the variability of BCAs and external environmental factors [77].Köhl et al. [103] identified Cladosporium cladosporioides (H39) as a promising biocontrol agent, demonstrating significant reduction of V. inaequalis sporulation in orchard conditions.However, challenges in maintaining efficacy and shelf life hindered commercial success [104].To address these challenges, there is a critical need to advance the development of new formulations of BCAs that boast a higher degree of stability, efficiency, and survival [89,128,161,162].This necessitates the integration of novel biotechnological practices to enhance the overall performance and reliability of BCAs in practical agricultural applications [154].Fenta et al. [128] suggested the development of both dry and liquid formulations to enhance the efficacy of biocontrol and extending its shelf life.These formulations offer practical solutions for the challenges faced in commercial applications, ensuring that biocontrol agents remain effective and viable under diverse packaging conditions.By leveraging cutting-edge biotechnological approaches, working towards creating formulations that are more resilient, adaptable, and effective across a broader range of environmental conditions can be achieved [89,154,161].This proactive approach is crucial for unlocking the full potential of biological control in plant disease management, ensuring that BCAs can provide consistent and reliable results in various agricultural settings [154,162].As the field of biotechnology continues to evolve, it partly holds the key to overcome the challenges hindering the widespread commercial use of BCAs and establishing them as integral components of sustainable agriculture [89,128,162].
The commercial viability of microbial BCAs hinges on their efficacy at defined doses, adherence to industrial production standards, and practical benefits for growers [7].The upscaling of a specific BCA to the commercialization stage is an expensive and multi-step process [154].Initial steps include the isolation of the microorganism in pure culture or its enrichment, followed by accurate identification and characterization processes.Subsequent stages involve the development of a suitable formulation, mass production, efficacy testing of the product, inspection of storage stability, registration of the product, and subsequent marketing efforts to make the BCA available to end-users [154].The complex nature of these processes underlines the significant challenges and resource investments associated with bringing a BCA from research and development stages to practical, commercially viable application [76].For instance, Microsphaeropsis ochracea demonstrated a robust 80-90% efficacy in reducing ascospore production in the field, making it a promising biofungicide [163].However, challenges related to commercialization, including significant time and financial investments, coupled with grower acceptance, have impeded its progress into widespread use [164].Despite extensive research and numerous reports on the biological control of plant pathogens, the global availability of registered biofungicides remains rather limited [4,89,128,165].The majority of these biofungicides are commercialized for specific niches, particularly in high-value crops where there is a demand for pesticide-free products [4].This targeted commercialization reflects the challenges associated with scaling up and registering biofungicides for broader agricultural applications.

Future Trends and Conclusions
In the forthcoming years, the outlook for biological control in apple scab management is highly promising.Ongoing advancements in research tools, such as next-generation sequencing and functional genomics, are providing detailed insights into the specialized metabolic pathways of potential fungal and bacterial antagonists.This deeper understanding opens new avenues for the development of more potent BCAs against V. inaequalis.The evolution of the omics approach holds great potential for integrating biocontrol into apple scab disease management.A comprehensive understanding of the molecular processes underlying the biocontrol activity of BCAs against plant pathogens lays the groundwork for subsequent experiments using functional genetics approaches.
Generally, addressing challenges related to adoption, susceptibility to environmental factors, and enhancing the efficacy and persistence of microbial biocontrol agents is imperative for sustainable disease management.Overcoming these challenges requires united efforts to develop innovative strategies and bridge existing gaps in our understanding.Genetic engineering and genomic sequencing offer avenues to enhance the specificity and effectiveness of BCAs.By identifying and modifying key genes or traits, BCAs can be customized to more effectively counter specific pathogens, withstand adverse environmental conditions, and improve their overall performance.Moreover, a deeper understanding of the ecological dynamics within orchard ecosystems can guide the development of strategies to optimize the application and persistence of BCAs under varying conditions.Integrating this knowledge with innovative biotechnological approaches holds promise for creating more adaptable BCAs that can survive in adverse orchard environments.
In conclusion, despite extensive research on microbial antagonists, the formulation of BCAs for managing apple scab epidemics remains elusive.Additional research and innovative strategies are essential to fill these gaps and enhance our understanding, ultimately paving the way for the successful management of scab outbreaks.The primary goal of biocontrol research is to provide supplementary tools for disease management and the successful integration of BCAs into existing production systems.Moving forward, continued research and collaborative efforts will be key to realizing the full potential of biocontrol in apple scab management.

Table 1 .
Commercially available apple cultivars and their reactions to apple scab.

Table 2 .
Some common fungicides used to manage apple scab and their modes of action.

Table 3 .
Summary of notable antagonists and their usage in apple scab management.

Table 4 .
Commercially available biopesticides and their actions against apple scab.

Table 5 .
Some botanicals and their actions against apple scab.