
==== Front
J Ind Microbiol Biotechnol
J Ind Microbiol Biotechnol
jimb
Journal of Industrial Microbiology & Biotechnology
1367-5435
1476-5535
Oxford University Press

39210514
10.1093/jimb/kuae031
kuae031
Review
Environmental Microbiology
Jimb/4
AcademicSubjects/SCI01150
AcademicSubjects/SCI00540
Phytopathological management through bacteriophages: enhancing food security amidst climate change
https://orcid.org/0000-0002-0806-6907
Ul Haq Ihtisham Department of Physical Chemistry and Technology of Polymers, Silesian University of Technology, 44-100 Gliwice, Poland
Joint Doctoral school, Silesian University of Technology , 44-100 Gliwice, Poland
Postgraduate Program in Technological Innovation, Federal University of Minas Gerais, Belo Horizonte 31270-901 MG, Brazil
Department of Bioscience, COMSATS University Islamabad, Islamabad 44000, Pakistan

Khan Mehtab Department of Biology, University of Moncton, Moncton, NB E1A 3E9, Canada

https://orcid.org/0000-0001-9692-5961
Khan Imran Department of Molecular Signal Processing, Leibniz Institute of Plant Biochemistry, 06120 Halle, Germany
Department of Plant and Microbial Biology, North Carolina State University, Raleigh, NC 27695-7612, USA

Correspondence should be addressed to: Imran.Khan@ipb-halle.de
2024
29 8 2024
29 8 2024
51 kuae03106 8 2024
28 8 2024
11 9 2024
© The Author(s) 2024. Published by Oxford University Press on behalf of Society of Industrial Microbiology and Biotechnology.
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs licence (https://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial reproduction and distribution of the work, in any medium, provided the original work is not altered or transformed in any way, and that the work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Abstract

 

The increasing global population and climate change pose significant challenges to agriculture, particularly in managing plant diseases caused by phytopathogens. Traditional methods, including chemical pesticides and antibiotics, have become less effective due to pathogen resistance and environmental concerns. Phage therapy emerges as a promising alternative, offering a sustainable and precise approach to controlling plant bacterial diseases without harming beneficial soil microorganisms. This review explores the potential of bacteriophages as biocontrol agents, highlighting their specificity, rapid multiplication, and minimal environmental impact. We discuss the historical context, current applications, and prospects of phage therapy in agriculture, emphasizing its role in enhancing crop yield and quality. Additionally, the paper examines the integration of phage therapy with modern agricultural practices and the development phage cocktails and genetically engineered phages to combat resistant pathogens. The findings suggest that phage therapy could revolutionize phytopathological management, contributing to global food security and sustainable agricultural practices.

One-Sentence Summary

The burden of plant diseases and phage-based phytopathological treatment.

Graphical Abstract

Graphical Abstract

Plant diseases
Climate change
Phages
Phage-based phytopathological management
Phage cocktails
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pmcIntroduction

In recent times, the human population has been observed to increase continuously. It is expected to reach approximately 9.6 billion in the next 25 years, which is expected to cause a scarcity of agricultural and food resources (Nawaz et al., 2023). To address the needs of an expanding food supply, it is anticipated that crop production will need to increase by approximately 70%–80% through enhanced efficiency in agricultural operations. The crops are already under the influence of climate change, phytopathogens, and technological gaps that decrease the production pace (Kumar et al., 2022). The emerging outbreaks of phytopathogens with widespread incidence and consequences, such as yield and ecological losses, have significantly threatened the agriculture and food supply chain (Singh et al., 2023). It has been estimated that these phytopathogens cause a US$220 billion annual loss in crop yield (Singh et al., 2023). The widespread phytopathogens are Xanthomonas campestris pv. Campestris (Svircev et al., 2018), Pantoea stewartii (Jones et al., 2012), Ralstonia solanacearum (Askora et al., 2017), Dickeya solani (Czajkowski et al., 2015), X. campestris pv. Vesicatoria (Balogh et al., 2003), Xylella fastidiosa (Das et al., 2015), Pectobacterium carotovorum ssp (Lim et al., 2013), and Erwinia amylovora (Boulé et al., 2011). Climate change significantly altered geographic distribution, pathogen life cycles, host-pathogen interactions, and increased vector activity, which play a crucial role in plant pathogen expansion, also described as a triangle of plant diseases (Lahlali et al., 2024). Phytopathogens previously confined to tropical or subtropical regions are moving into areas that were once too cold for survival, affecting the local plant species that may not resist these new threats (Pagán & García-Arenal, 2018). Warmer temperatures can lead to more intense disease outbreaks as several pathogens thrive in warmer conditions, leading to faster reproduction rates and increased virulence. Moreover, the soil microbial flora may lose their efficiency due to existing ecological balances triggered by climate change (Castroverde et al., 2015). This implies that the decrease in the world's food supply and natural plant biodiversity is significantly put at risk by climate change-induced amplified risk of intensification of plant diseases, which triggers the search for new chemicals for phytopathogens. Recently, phage therapy has gained recognition as a sustainable and effective alternative to chemical-based plant protection agents (Ahmed & Li, 2023). Phage therapy employs bacteriophage viruses, typically identified as phages that use bacteria to replicate and occasionally enter the lytic cycle (Akhwale et al., 2019). They are precise, targeting only their host bacteria without affecting other microorganisms or the host organism itself, which makes them an intriguing tool for combating phytopathogens (Lin et al., 2017). Mostly phages such as vB_DsoM_LIMEstone1 (Czajkowski et al., 2015), 8AS1 phage (McKenna et al., 2001), 8RSL1 phage (Fujiwara et al., 2011), PP1 phage (Lim et al., 2013), and vB_PsyM_KIL (Rombouts et al., 2016) have a tail structure affixed to the capsid that is used to attach or inject their genetic material into the host bacterium (Fig. 1) (Taslem Mourosi et al., 2022).

Fig. 1. The illustration depicts the phage lysis mechanism, highlighting a bacteriophage's lytic and lysogenic cycles. The lytic cycle involves phage adsorption, infection, replication and assembly, and bacterial cell lysis, while the lysogenic cycle involves phage DNA integration, prophage replication, and lysogenic to lytic transition. The same phage does not reinfect lysogenic phages due to hyper infectious immunity, and it depicts the potential for a lysogenic cycle to restrain or lead to the lytic cycle upon specific triggers. Copyrights (2023), MDPI (Liang et al., 2023).

The initial reports of phage-based control of phytopathogens date back to the mid-20th century. One of the early mentions of the potential use of phages in plant pathology was by M. Kutateladze and N. Adamia in 1971 (Korniienko et al., 2022). Phages are crucial in regulating bacterial populations in natural ecosystems and offer promising applications in agriculture. Their ability to evolve alongside bacteria also provides a dynamic approach to tackling emerging resistant bacterial challenges (Garvey, 2022). Phage therapy already shown promising results against bacterial diseases in several economically important plants such as potatoes, tomato, radish, lettuce, grapefruit, citrus, cherry trees, mushrooms, and leek (Balogh et al., 2008; Fujiwara et al., 2011; Kim et al., 2011; Lim et al., 2013; Das et al., 2015; Rombouts et al., 2016; Gašić et al., 2018; Rabiey et al., 2020).

Herein, we aimed to highlight phytopathogenic diseases, the influence of climate change, and phage-based chemicals for phytopathogens to curb the burden of phytopathogenic diseases.

Plant Diseases: As an Economic Threat and Influence of Climate Change

Plant diseases are closely associated with significant losses in agriculture, leading to reduced crop yields and diminished quality, affecting food security, economic stability, and the health of ecosystems (Singh et al., 2023). The highly reported bacterial diseases in plants include fire blight caused by E. amylovora, affecting apples, pears, and other members of the Rosaceae family. Bacterial spot caused by X. campestris pv. vesicatoria, affecting tomatoes and peppers. Bacterial wilt is caused by R. solanacearum, affecting a wide range of hosts, including tomatoes, potatoes, and bananas. Citrus canker caused by X. citri subsp. citri, affecting citrus trees. Bacterial blight of rice caused by X. oryzae pv. Oryzae (Murillo & Sesma, 2001; Aćimović et al., 2015). These diseases can significantly impact crop yield and quality, necessitating effective management and control strategies. It led to an uncommon expansion in the agronomically significant bacterial diseases that were generally connected with the lessening agricultural yield. The expanded human populace worldwide entreated huge harvest agrarian yields to meet their food prerequisites; it is now below the required estimate (Hemathilake & Gunathilake, 2022). In recent times, the human population is observed to increase continuously. It is expected to reach approximately 9.6 billion in the next 25 years, which is expected to cause agricultural and food resource scarcities (Nawaz et al., 2023).

Plant diseases are the major culprit behind the diminished agriculture production (Yuliar et al., 2015). Furthermore, climate change also significantly influences plant disease dynamics since it affects the entire triangle of the plant diseases involved in disease cause and spread (Singh et al., 2023). The pathogen's distribution in soil includes geographical range, niche preference, virulence, and, importantly, their interactions with environmental factors, plant, and soil microbial flora. Since bacterial diseases are also influenced by temperature, climate change-induced temperature change may affect the disease dynamics (Trivedi et al., 2020). Additionally, the evolutionary progressions and physiological behaviors in plant hosts and pathogens are strongly influenced by the indirect effect of climate change. The disease susceptibility in plants is increased by water stress in forest trees due to prolonged drought causes; in some cases, such conditions also facilitate the incidence of new diseases (Ryu et al., 2018)

The Conventional Strategies for Phytopathological Management and Its Decline

A combination of cultural, biological, chemical, and physical methods is often used along with an attempt to develop resistant plant varieties (Collinge et al., 2022). The main approaches include the practice of crop rotation, proper irrigation, and sanitation to prevent pathogen spread by using fungicides and pesticides and natural predators or antipathotic agents, and integrated pest management (IPM) strategies. Bactericides are chemicals specifically designed for treating and eliminating bacteria in plants (Karlsson Green et al., 2020). Chemical treatments may harm beneficial organisms like pollinators and natural predators of nuisances. Exposure to chemical pesticides in direct contact or through the consumption of residues on food products may pose health risks for farmers, workers, and consumers (Pathak et al., 2022). Prolonged exposure to certain chemicals may have long-term health effects, including cancer and neurological disorders. Chemical and physical controls can be expensive, especially for small-scale farmers or in developing countries (Anaduaka et al., 2023). Over time, chemical use is likely to negatively affect soil quality and fertility and reduce agrobiodiversity and beneficial organisms (Schütte et al., 2017).

Antibiotics to treat plant diseases are chosen, considering their ability to fight specific pathogens and their safety for plants, people, and the environment. They are typically applied as foliar sprays or through irrigation systems (Ding et al., 2023). However, most antibiotics are not used explicitly for any particular plant pathogenic bacteria, which leads the bacteria to develop resistance. They also kill the natural bacterial flora in the soil, which is beneficial for plant growth and nutrient cycling (Svircev et al., 2018). The imprudent use of antibiotics in agriculture lead to developing antimicrobial resistance (Svircev et al., 2018).

Physical control is also one of the strategies to avoid plant diseases; it's done using solar heat for soil sterilization, effective management of pathogens, or application of controlled temperature to plant material and seed so that these organisms can be killed without harm to a plant. In the fight against plant diseases, it is also essential to adapt irrigation practices to limit excessive moisture, which can stimulate the growth of bacteria, and install covers or screens to protect plants from vectors that may contaminate them (Saeed et al., 2021). Bacteria, fungi, and phages are frequently used as biocontrol agents to control plant diseases. These biocontrol substances are environmentally friendly and can be employed more effectively than chemical pesticides. Depending on the target bacteria disease and crop, their use may differ between soil treatment and foliar spraying (Bonaterra et al., 2022). Bacteria-based control of bacterial diseases in plants, involving mechanisms such as competition for resources, production of antimicrobials, and introduction of plant defense response, relies on natural inhibitors against harmful bacteria. Bacillus, Pseudomonas, and Streptomyces species are commonly considered beneficial bacteria (Bonaterra et al., 2022). These biocontrol agents may treat the plants’ seed, soil, or surface. Fungi-based control of plant bacterial diseases, also known as myco-bactericides, involves using certain fungi to combat bacterial pathogens (Shafi et al., 2017).

In the beginning, the agricultural productivity was significantly boosted by conventional phytherapeutics however, gradually, pathogen resistance and climate change induced plant disease considerably reduce their efficacies. The rapid evolution in phytopathogens is triggered by strong selective pressure with the frequent use of chemical pesticides and antibiotics. The genetic adaptability of phytopathogens is another reason behind the reduce efficacy of these conventional antimicrobials for phytopathogens, which enable pathogens altered their target site of chemicals and detoxifying them. Additionally, the environmental factors, including sunlight, temperature, and moisture easily degrade chemicals and conventional antimicrobials resulting into reduction of efficacies. On the other hand, ecological imbalances is observed due to the use of disruption of ecosystem which may impede the beneficial microbial flora in soil. The failure and inefficacies of current chemicals and conventional antimicrobials implies need of phytopathological management strategies to control the plant diseases. However, factors such as phage host interactions, environmental conditions, and regulatory frameworks can have a bearing on the effectiveness and applicability of this strategy compared with traditional strategies (Zalewska-Piątek, 2023).

The Importance of Phage Therapy and Its Therapeutic Breadth

The historical journey of phage therapy, from its early 20th-century origins to its resurgence amidst the global antibiotic resistance crisis, underscores its enduring relevance and potential. The scientists “d'Herelle and Twort” recognized the existence of bacteriophages at the start of the 20th century and believed they were armed with therapeutic potential (Pirnay, 2020). The initial trials of prospective phages were applied in medicine as alternative therapeutic options against human diseases (Bhargava et al., 2021). Since their inception, phage studies have primarily focused on bacteria. However, recent research has opened up several new therapeutic possibilities (Marchi et al., 2023) such as vaccines diagnostics (Hussain et al., 2021), antimicrobial activity against clinically important pathogens (Mondal et al., 2023) (Mutti et al., 2023), immunomodulator (Howden et al., 2023), and anticancer agents (Ragothaman & Yoo, 2023). The various applications of bacteriophage research contribute to the United Nations Sustainable Development Goals, particularly SDG-3 (Good Health and Well-being) and SDG-6 (Clean Water and Sanitation) (Bisen et al., 2024). Phage research has led to the development of FDA-approved products across various industries, including food. Notable examples include ListShield™, EcoShield™, and SalmoFresh™, all produced by Intralytix Inc (Ranveer et al., 2024). Additionally, phages are also approved as food processing and storage products (Wang & Zhao, 2022; Liu et al., 2023). In recent years, phage research has increasingly concentrated on advancing phage biomanufacturing technologies in laboratory settings and industrial scales to ensure a high yield of phages (João et al., 2021; Ranveer et al., 2024). As scientific knowledge and technological advancements progress, integrating phage therapy into modern medical practices and environmental management strategies shows great promise. Ongoing research and development are crucial to fully realizing the potential of phages, ensuring their effective use in tackling critical health and environmental issues such as phytopathogens (Rabiey et al., 2020; Korniienko et al., 2022; Ahmed & Li, 2023).

Phage Therapy for Phytopathological Management

The first report of phage as a phytopathological management strategy was considered against potato tuber rot caused by E. carotovora subsp. Atroseptica, later, phages started to be used regularly as a biocontrol strategy in crops (Moore, 1926). The most commonly used phage proteins for lysing bacterial cells are lysins and holins. Holins perforate the host cytoplasmic membrane, creating a pathway for endolysins, ultimately destroying the bacterial cell wall (Pollenz et al., 2022). With the discovery of antibiotics, phage therapy was overshadowed, resulting in a decrease in concern for phage-based control (Villalpando-Aguilar et al., 2023). However, with time, the emergence of antimicrobial resistance in bacteria again complicates management of bacterial diseases. Among different phytopathological management strategies, phages were considered a priority in controlling plant diseases due to advantages such as fewer chances of resistance, co-evolving with host bacterium, and environmental compatibility (Aminov, 2010). Phage once got stance to be used as a biocontrol strategy and successfully applied for the treatment of several deadly plant pathogenic bacteria, including Pseudomonas spp., Xanthomonas spp., Pectobacterium spp., Ralstonia spp., Clavibacter michiganensis, and Agrobacterium tumefaciens (Buttimer et al., 2017). Therefore, phage therapy research accelerated the control of phytopathogens and resulted onto several phage-based biocontrol products, reflecting their concealed immense potential. Still, companies are investing in phages to make them commercially viable (Rabiey et al., 2020). Agriphage (Registered in USA-EPA Reg. No. 67986-1), with the United States Environmental Protection Agency (Liu et al., 2023), was successfully used to manage bacterial spots in tomatoes caused by X. campestris pv. vesicatoria or Ps. syringae pv. Recently, Agriphage has been formulated to treat the citrus canker disease as well in several plants, including grapefruit, orange, tangelo, kumquat, and tangerine (Https://www.certisbio.com/products/bacteriophages/agriphage-cc, n.d.). Although, citrus canker disease is caused by another phytopathogen called X. citri subsp. Citri, which suggests the therapeutic breadth of Agriphage products. For the treatment of C. michiganensis subsp. Michiganensis, another phage formulation developed known AgriPhage CMMTM holds registration under USA-EPA Registration Number 67986-6). Commercial organic growers are also allowed to use Agriphage as this product has been certified by the Organic Materials Review Institute (Buttimer et al., 2017). Similarly, Erwiphage is another critical phage-based biocontrol product, using fire blight disease in apples caused by E. amylovora. Moreover, for the treatment of potato tubers, a Scottish company named APS Biocontrol Ltd formulated Biolyse, which is effective against Enterobacteriacea-induced plant diseases (Branston., 2012). Additionally, phages can be genetically modified to enhance their ability to target specific bacteria, improve their efficacy in several ways, increase the phage therapy breadth (Liu et al., 2022) (Chen et al., 2019), and combat antibiotic-resistant phytopathogens. Enhancements to the phage's lytic capabilities are achievable through genetic modifications, such as the overexpression of endolysins that degrade bacterial cell walls or the incorporation of anti-CRISPR genes to neutralize bacterial defense mechanisms (Nazir et al., 2023). Phage genetic engineering for phytopathological management involves numerous biotechnological techniques that enable the phages to target and control plant pathogens effectively (Pires et al., 2016). Modifying the receptor-binding proteins (RBPs) on phages can expand or change their host specificity (Chen et al., 2019). The genes responsible for RBPs also can be swapped between different types of phages. Some phages have wide host ranges, such as IP008; thus, their long-tail fiber genes can be shifted to other phages that have the potential to kill phytopathogens. Previously, the long tail fiber genes were successfully shifted to the T2 phage to increase its host range (Mahichi et al., 2009). Another innovative approach involves engineering phages to carry genes that trigger plant immune responses, enhancing their natural defense mechanisms against pathogens. Ensuring the safety and efficacy of these genetically engineered phages involves controlled expression systems and rigorous field trials. These trials are essential for refining therapy protocols and ensuring the phages do not adversely affect nontarget organisms or human health (Łobocka et al., 2021).

Phages Employed for Phytopathological Management

The novel phytopathological management strategies focus on phage-based biocontrol methods (Fig. 2). Phages have attracted the attention of agri-researchers due to their high specificity, safety, environmentally friendly behavior, less resistance development, compatibility with IPM, and cost-effective production, which made them ideal tools for phytopathological management. The phages such as 8PD10.3 and 8PD23.1 (Okabe & Goto, 1963), vB_DsoM_LIMEstone1 and vB_DsoM_LIMEstone2 (Czajkowski et al., 2015), D1 phages (Adriaenssens et al., 2012), 8AS1 phage (McKenna et al., 2001), 8RSL1 phage (Fujiwara et al., 2011), PP1 phage (Lim et al., 2013), Stsc1 and Stsc3 phages (Goyer, 2005), vB_PsyM_KIL (Rombouts et al., 2016), and phages 8Ea1337-26 and 8Ea 2345 (Boulé et al., 2011) showed promising results in phytopathological management of various economically significant plant diseases. Additionally, phage cocktails that employ multiple phages in a single application have considerably high inhibitory potential against phytopathogens (Farooq et al., 2022).

Fig. 2. The schematic illustration of using phages in biocontrol-based phytopathological strategy. The biocontrol-based phytopathological strategy using phages starts with bacterial selection and phylogenetic analysis, incorporating geographical and isolation data to understand strain distribution. Phages are collected from infected areas, tested for host range, and correlated with bacterial phylogeny. Lytic phages safe for agriculture are selected based on genetic data. Phage mixtures targeting different receptors minimize resistance. In vertical farming, plants are grown in controlled hydroponic greenhouses with LED lighting and sensor-based monitoring. Hyperspectral sensors with machine learning detect early diseases, notifying farmers via smartphone for plant removal and biopesticide treatment. Unmanned automated vehicles (UAVs) with hyperspectral sensors detect field diseases, providing reports for targeted treatment. Figure copied with the permission of (Holtappels et al., 2021).

The cocktails of phages are widely regarded as attractive antibacterials, notable for their unique specificity and rapid multiplication characteristics. A mixture of phages with a broad range of host activities indicates a variety of receptors, enhancing the effectiveness of these antibacterial treatments and reducing the likelihood of resistance development (Yang et al., 2020). Genetically engineered phage cocktails can serve as natural biocontrol agents for various bacterial diseases, effectively targeting resistant pathogens without damaging the host plant and other microbial flora (Farooq et al., 2022). Researchers also employed more than one phage to control plant diseases. The phage cocktail was used in PP16/F002, PP101/F152, PP47/F157, and Q51/F018 to successfully reduce the soft rot diseases on potato tuber caused by Pe. carotovorum ssp. carotovorum, Pe. wasabiae (Bugaeva et al., 2021). Also, using two phages together in a single application is possible. Adriaenssens et al showed that the combination of two related phages such as vB_DsoM_LIMEstone1 and vB_DsoM_LIMEstone2 is effective against rotting and blackleg disease in potatoes caused by deadly phytopathogen called Dickeya dianthicola (Adriaenssens et al., 2012). On the other hand, the phage cocktail comprised of 8D1, 8D2, 8D3, 8D4, 8D5, 8D7, 8D9, 8D10, and 8D11 was able to reduce 30%–70% soft rot infection in potatoes caused by D. solani (Buttimer et al., 2017). A single phage 8AS1 was enough to reduce the surface lesions of the scab. A single co-inoculated 8RSL1 phage with host bacterium R. solanacearum in soil did not allow it to cause bacterial wilt, while the control plant showed typical symptoms of bacterial wilt (Fujiwara et al., 2011). Similarly, A P1 cocktail, composed of six phage isolates, can potentially eliminate 98% of R. solanacearum from potatoes and tomatoes by being directly applied as a soil drench (Farooq et al., 2022). The phage PP1 was reported to control the disease development in lettuce plants caused by Pe. carotovorum ssp. carotovorum in the greenhouse trials of phage PP1.

The 8Ea1337-26 phage reduced the 54% infection of potted apple tree blossoms caused by E. amylovora (Buttimer et al., 2017). Xylella fastidiosa is a widely known phytopathogen of several plants including olive, oleander, and almond and difficult to control; however, successfully eradicated with an engineered cocktail of four lytic phages (Ahern et al., 2014) (Farooq et al., 2022). Das et al. reduce the growth of X. fastidiosa in grapevines by pre and post-inoculation Sano, Salvo, Prado, and Paz used as a cocktail (Das et al., 2015). Phage therapy also showed promising results in reducing the soft rot disease caused by Pe. carotovorum ssp in lettuce. carotovorum (Lim et al., 2013). The combination of two phages, 8Ea1337-26 and 8Ea 2345, decreases the symptoms of infected detached pear tree blossoms by 84% and 96% caused by E. amylovora. These applications show the potential of phages to provide a biocontrol strategy against bacterial diseases in agriculture, offering an alternative to chemical pesticides and addressing the issue of antibiotic resistance.

Phages, being naturally occurring and biodegradable organisms, do not leave behind harmful residues like chemical pesticides. Consequently, they are environmentally safe and suitable for organic farming and ecosystems, where chemical substances are restricted or undesirable (Nawaz et al., 2023). Generally, bacteria develop resistance to phages at a slower rate than chemical antibiotics. Phage therapy can also be integrated into disease management strategies, resulting in a more effective IPM approach.

The Breadth of Phage Cocktails in Phytopathological Management

The phage cocktails as an antibacterial biocontrol strategy offer remarkable advantages, with the most important being minimal resistance development and wide therapeutic breadth. The phage cocktails are formulated with phages having various receptors, strong adsorption, short latency, and huge burst size; however, polyvalent phages are accounted for in phage cocktails as they can infect multiple bacterial strains belonging to the same species (Ross et al., 2016). Additionally, synthetic cocktails can be developed using genetically engineered phages. These phages are usually considered effective for pathogens within mixed populations (Nair & Khairnar, 2019). The technological advances, including CRISPR-Cas-mediated genome engineering, rebuilding/refactoring phage genome in vitro, and whole-genome synthesis from synthetic oligonucleotides, involved in the genetic modification of phages play central role in increasing phage efficiency (Pires et al., 2017). Erwinia amylovora phage Y2 can be genetically modified to enhance its effectiveness by integrating bacterial luxAB fusion or the E. amylovora phage L1 depolymerase gene into its genome, boosting its lethal capability. Additionally, genetically modifying the phage receptor binding proteins could expand its range of host targets (Kering et al., 2019). Several strategies, including phage–bacterial infection networks, have been practiced to optimize the phage cocktail by simplifying phage identification of the broadest host range (Molina et al., 2021). Using computational approaches such as Islander and TIGER (Yang et al., 2020) to formulate therapeutic cocktails has successfully overcome the shortcomings associated with traditional methods. It identifies and charts integrative genetic elements within bacterial genomes, revealing the bacterial hosts, the complete sequence, and the sequence at each end of the prophage (Farooq et al., 2022). Previously, safe phage therapeutic cocktails has been successfully developed through sequences of prophages that facilitate the engineering of their genomes (Mageeney et al., 2020). These technological advances have the potential to provide novel insights into synthetic biology, hastening the development of therapeutic mixtures that can effectively attack various phytopathogens (Farooq et al., 2022). However, the phage cocktail formulations mentioned above are not practical for preventing the emergence of new phage-resistant phytopathogens because of the ongoing battle between phages and bacterial pathogens. Furthermore, the complexity of plant-pathogen systems makes it unlikely to develop a single multidimensional cocktail for all bacterial phytopathogens. Constant surveillance of phytopathogens and modification of phage cocktail formulations is necessary in addressing newly emerging phage-resistant phytopathogens. Additionally since, environmental factors also affect the therapeutic efficacy of phages; suitable, protective formulations compatible with phages should be designed/implemented (Kering et al., 2020; Liu et al., 2023). The protective formulations employing skim milk (Zhang et al., 2020), surfactants, proteins, and amino acids, in combination with polymers (Colom et al., 2017), sodium alginate biopolymer matrix (Gonzalez-Menendez et al., 2018) can significantly maintain the therapeutic efficacy of phage cocktails for a more extended period. The phage formulation with pregelatinized cornflour, sucrose, casecrete, and powdered skim milk successfully reduced the bacterial spot-on tomato symptoms. In the field experiment, casecrete showed promising results, while skim milk showed good results in the greenhouse experiment. Thirty six hours after application, the phage populations formulated with casecrete were over 1,000 times higher than those of the nonformulated phages (Balogh et al., 2003).

Methods to Evaluate Phages for Phytopathological Management

The best method for employing phage therapy to prevent plant disease involves several key steps. Initially, it's crucial to identify the specific bacterial pathogen causing the disease. Following this, appropriate phages targeting and killing the pathogen are selected (Lin et al., 2017). These phages can then be applied to the plants or the soil through irrigation systems, sprays, or seed coatings. This targeted approach ensures minimal impact on nontarget microbes and promotes sustainable agricultural practices. Phages can be applied alone without introducing additional bacteria (Vu & Oh, 2020). The timing of phage application can vary; they can be used preventatively before infection occurs or as a treatment after detecting bacterial disease. Biological and environmental features must be taken into account during the handling of phages as they are susceptible, as survival of phages is reduced prominently due to ecological stresses when applied on the leaf surface (Iriarte et al., 2007).

Similarly, reports indicate that phages are most effective when the host is inoculated less than 1 hr before phage application (Ly-chatain, 2014). The effectiveness of phage therapy diminishes when administered before pathogen inoculation. This is believed to be due to the phages being unreachable once the pathogen enters the intercellular spaces of the plant's vascular system (Jones et al., 2012; Buttimer et al., 2017). Phage-based phytopathological management requires many phages near their host bacterium to attach to hosts before environmental factors diminish their populations (Jones et al., 2012). Key factors to enhance phage-bacterium interactions include the target bacterium's population density and accessibility, optimal timing of phage application, phage ability to infect and replicate in the environment, and phage density at the interaction site (Jones et al., 2012).

Phage can be directly applied on the surface of infected plant flowers coated with seeds or, it can be directly introduced into the soil, tailored to the crop's specific needs and the phytopathogen's nature (Elhalag et al., 2018; Vu & Oh, 2020). However, the phage application on the leaf surface may be affected by moisture/humidity (Gayder et al., 2023) like conventional pesticides (Ho et al., 2015). In dry conditions, the lack of water can cause desiccation, leading to the denaturation of proteins and nucleic acids within the phage (Fister et al., 2016). This structural damage impairs/diminishes the phage's ability to bind to and infect bacterial hosts.

Additionally, moisture is necessary for the diffusion of phages through their environment to encounter and infect bacteria (Fister et al., 2016). Phages can also be directly applied to fresh-cut fruits and vegetables, such as melon slices (Ranveer et al., 2024). The direct application of phages on plant surfaces is performed through phage spray, mainly used on aerial parts of plants, such as bacterial spots, blights, and cankers. For example, potato tubers are sprayed with phage suspension approximately 120 min before planting (Adriaenssens et al., 2012). The spray base method potentially has application breadth since it covers a large area and directly encounters pathogens (Eski et al., 2022). A phage-based spray containing 90% lactose remained sustainable for 1 year at 20°C at 60% relative humidity (Zhang et al., 2020).

Phages are mixed with stabilizers and other additives to increase the phage activity and viability. Using equipment similar to that used for pesticide application, the phage spray is distributed evenly across the plant. This might involve backpack sprayers for small applications or agricultural sprayers for large-scale farms. Multiple doses of spraying are necessary to eradicate the pathogens, particularly persistent pathogens.

To avoid seed-borne bacterial infections, phage can be coated on plant seeds during the soaking before planting. Using this strategy, disease spread from seed to plant is significantly reduced (Kimmelshue et al., 2019). It has been reported that phage coating on seeds increased the germination rate by up to 95% (Holtappels et al., 2021). The phage applied to fruits and vegetables has increased shelf life due to fewer chances of spoilage (Jagannathan et al., 2022). Figure 3 is a schematic illustration of phage therapy application and evaluation.

Fig. 3. The schematic illustration of phage application and its evaluation. The first part shows phage application methods and factors affecting phage efficiency. The second part shows how to evaluate phage therapy's efficiency against pathogens.

Current Achievements and Future Prospects in Phage-Based Phytopathological Management

In treating bacterial infections, phage therapy is a promising but complex alternative and complement to antibiotics. Compared with other treatment strategies, pyrethroid therapy offers several advantages and benefits for managing plant diseases, making it an attractive option in specific contexts (Lin et al., 2017). Compared to broad-spectrum chemical pesticides that can harm many organisms, including healthy soil microbes and insects, this specificity is a significant advantage (Sieiro et al., 2020; Stefani et al., 2021; Chung et al., 2023). Chemical pesticides and fungicides have a broader range of activities that affect more diverse organisms, including nontarget microorganisms and beneficial species. The chemicals are mainly used for killing or inhibiting the development of pathogens by different mechanisms, such as cell membrane breaks and inhibition of protein synthesis. Typically, the development of bacterial resistance to phages will occur slower than with chemical antibiotics.

Compared with traditional chemical agents, this dynamic nature of phages helps manage the resistance problem (Wright et al., 2019; Hasan & Ahn, 2022). The effectiveness of phage therapy may continue to be sustained over time because of this evolutionary arms race. Plants, humans, and animals are generally safe from phages because they're aimed at a specific type of bacterium that doesn't have any infectious effects on Eukaryotic cells. This is a considerable advantage compared to some chemical treatments, which may harm species other than the target species, such as humans (Podlacha et al., 2021; Jagannathan et al., 2022).

The phage biomanufacturing processes focus on the properties of the target product and associated impurities, and this understanding is vital to differentiate phages by size and shape (João et al., 2021). The phage variability in composition, stability, and biological activity makes the manufacturing processes very complex, which requires a thorough characterization of the manufacturing process. Applying theoretical approaches and simulation models in practical experiments makes phage production easy. Since phages are reproduced in the host bacterium cell, which can be genetically modified to yield a high output of phages (Chen et al., 2019; Łobocka et al., 2021), additionally, phages can be modified as a whole or any of its fragments. However, not every phage is modified with a single modification strategy due to some limitations, such as low transfection efficiency.

Despite the possible benefits, phage therapy has to confront several practical and ecological challenges that may reduce its efficiency and applicability in controlling bacterial infections within plants (Pires et al., 2020). Maintaining the phage potency and increasing its stability in the environment is one of the most confronted challenges in translating the knowledge of phage therapy from bench to phage-based products. Compared with other treatment strategies, such as antibiotics and copper compounds, phages are more sensitive to environmental factors such as light, temperature, humidity, and ultraviolet (Beales, 2004). Additionally, there are many factors (phyllosphere and rhizospheric) that affect phage interaction, including the concentration of target bacteria and its location, the concentration of phages and their capability to replicate in the environment, moisture content, pH, temperature, soil type, organic matter, and sunlight (Zhuang & Jin, 2003). In the rhizosphere, phages can be trapped in biofilms and adsorbed to clay (due to montmorillonite and kaolinite minerals), and acidic soil can inactivate the phage viability. The production and regulatory approval of phage-based products is complex and time-consuming, requiring rigorous quality control to ensure purity, potency, and safety, which can be challenging for large-scale production​ (Bretaudeau et al., 2020). The cost and accessibility of developing and producing phage-based products can also be prohibitive, limiting their use, especially among small-scale farmers​ (Svircev et al., 2018). Lastly, while phages are considered safe and environmentally friendly, their long-term ecological impacts are not fully understood. Addressing these limitations requires ongoing research and development, interdisciplinary collaboration, and supportive regulatory frameworks to fully realize the potential of phage therapy in managing plant diseases.

Conclusion and Standing of Phage Therapy

The field of phage therapy and phage-based applications is rapidly evolving, with ongoing research and development efforts to expand the range and efficacy of phage-based biocontrol. The benefits of this type of biocontrol method can be exploited if we modify aspects like the formulation of phage cocktails and use methods and integrate them with current agricultural practices. The power of phages can lead to a paradigm shift in managing plant diseases, allowing it to be implemented more sustainably and effectively. The evolution and dynamics of this field can be seen in the potential for genetically engineered phages and advanced bio-manufacturing systems. Specific plant pathogens will be able to be targeted with high precision by custom phages designed using technologies such as CRISPR-Cas. Substantial efforts have resulted in several breakthroughs, including protective formulations that improve phages’ viability, stability, and shelf life, mainly where they are used under various environmental conditions. Phage production has been simplified by merging theoretical approaches, such as simulation models, with practical experimentation. A mathematical model, developed by incorporating variable infection parameters dependent on bacterial growth rates, aids in forecasting the system's behavior and the effects of isolated or collective process variables. To foster global acceptance and use, developing international standards and protocols for phage therapy in agriculture will be helpful. Technological advances make phage production methods more cost-effective for farmers worldwide, making them profitable. This market will likely grow while more products are now made available to treat different crops and bacterial diseases due to increased awareness and the proven efficacy of phage therapy. Despite the possible benefits, phage therapy has to confront several practical and ecological challenges that may reduce its efficiency and applicability in controlling bacterial infections within plants.

Acknowledgments

All the authors of the manuscript thank and acknowledge their respective universities and institutes.

Author Contributions

I.U.H, wrote manuscript M.K., provided edits, I.K., I.U.H., and reviewed the manuscript.

Funding

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.
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