AUCTORES
Globalize your Research
Review Article | DOI: https://doi.org/
*Corresponding Author:
Citation:
Copyright: © 2018. Shilpa Deshpande Kaistha. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Received: 30 November -0001 | Accepted: 01 January 1970 | Published: 01 January 1970
Keywords: Bacteriophages, Phage Treatments, Phytopathogen, Bioformulations, Biocontrol Agents.
Bacterial phytopathogens are responsible for the devastating loss of economical crop worldwide and tremendous efforts are being made to devise ways of controlling global food loss to pathogens using a myriad of strategies. The use of bacteriophages as biopesticides is gaining popularity due to their several advantages over conventional control measures. Recent developments in nanosciences and bioformulation technology are making it possible to convert laboratory based research efforts into commercial products which can be effective, beneficial and environmental friendly in the agricultural fields. This mini-review examines the recent developments in the field of phage treatments for control of phytopathogens and challenges that need to be circumvented for it to be a preferred therapy option.
Bacteriophages or viruses which specifically target and lyse bacterial cells, have been extensively studied as biocontrol agents since their discovery in the 1915 by Twort and d’Herelle in 1917 [1]. Bacteriophages or phages are nanosize, obligate intracellular lytic agents with high degree of host specificity which makes them ideal for targeting bacterial pathogens [2]. Recent surge in the technological developments in genetic engineering and nanosciences has burgeoned novel applications for phages in the detection and biocontrol of pathogens in clinical medicine, food industry as well as plant diseases [3,4]. Phage bioformulations as biopesticides for protection against plant pathogenic bacterial species causing destructive and economically devastating diseases is being widely explored as a viable option in integrated disease management [5].
Bacteriophages are nanometer size biological entities composed of DNA genome encapsulated in an icosahedral protein coat/capsid classified in the Order Caudovirales [2]. The three main phage families within the Order include family Myoviridae with rigid contractile tails; Podoviridae with short contractile tails and Siphoviridae with flexible long tails. The phages typically infect via its capsid or tail proteins interacting with specific proteins receptors on the host surface. Upon successful host contact, the phage genome is injected into the host cytosol where it commences its productive lifecycle. Phages are typically classified as virulent/lytic phages or lytic and lysogenic/temperate phages. Temperate phage may choose a lysogenic intermediate stage in their life cycle and integrate their genome into host DNA. The Host cells in its subsequent divisions perpetuate the phage genome. Eventually, the phage genome may excise from the host DNA and pursue a productive replication strategy wherein multiple copies of viral genome and outer capsid and structural proteins are synthesized. Several copies of the infectious phage are assembled and eventually host cell is lysed with the release of several virion copies in the environment. Temperate phages are usually not a choice for phage based biocontrol as they may show properties such as superinfection immunity that is the lysogenized host bacteria may show immunity to infection by the same phage type. Additionally, temperate phages may encode for host bacteria virulent factors and must be tested before bing considered as a candidate for phage based biopesticide [6]. Typically hence, isolation and characterization of lytic phages are preferred for the development of biopesticide formulations [5,7].
Phage based biopesticide systems
Phage treatments have numerous advantages over conventional phytopathogen control agents.
1) Phages are self replicating and auto dosing agents: The phages thrive on specific hosts and once the host is eliminated, they are automatically self-limited unlike chemical or other cellular biological agents. The number of phages in the environment hence remains regulated due to auto dosing phenomenon. Moreover, due their prokaryotic host specificity they do not affect eukaryotic cells [6].
2) Conventional chemical treatments for biocontrol of plant pathogens include the use of copper biocides as well as antibiotics [8]. A major issue with the prevalent measures is the development of copper and antibiotic resistance and its horizontal transfer to the environmental microbiota. The widespread use of high inhibitory concentrations required to be effective are also a cause for bioaccumulation of toxic levels in the food trophics [9]. On the contrary, the host specificity of bacteriophages ensures that they do not affect beneficial plant growth promoting microorganisms or other biological species. They are thus environmentally friendly, non toxic with no side effects and with no carbon foot print [5]. Moreover, with the current practices of organic farming, the use of phage biopesticides becomes a highly suitable countermeasure as bacteriophages are naturally persisting in the soil.
3) Phage treatments can be custom developed for the phytopathogens: Development of phage treatments formulations is relatively fast and economical in comparison to other antimicrobials. Phages formulation as cocktails or in conjunction with antibiotics, and chemical drugs has been extensively reported [10]. Such formulations have shown synergistic biocontrol activity [11] and addresses concerns regarding monophage resistance in host cells [12].
4) Phages show high degree of genetic plasticity and can be engineered to express different types of drugs and enzymes which can increase their efficacy as biocontrol agents [13].
5) Phages and phage based lytic enzymes provide a means of controlling biofilm forming phytopathogens that show high resistance to conventional treatments [14]. The use of phage and phage derived lytic products are also effective in mitigating biofilm and exopolysaccharide producing phytopathogens that are several fold more resistant to antimicrobial treatments in comparison to freeliving counterparts [15].
6) They can be administered to the fields through different applications and can be typically combined synergistically with different treatments and chemicals. Phage (108-10 pfu) sprays on leafs, phyllospheric treatments as well as soil applications have been effectively implemented in field trials [16].
7) Phage maybe developed for used as diagnostics as biosensors in addition to prophylactic, therapeutic or combinatorial measures for phytopathogen control [17].
The scientific literature is replete with studies regarding the isolation and characterization of phages against major crop phytopathogens affecting Solanaceae, citrus fruit plants amongst others [18]. Some of the successful phage treatments have been developed against bacterial phytopathogens such as Erwinia amylovora, Dickeya, Agrobacterium tuefacients, Pseudomonas syringe pv. Tomato, Xanthomonas campestric pv. Vasicatoria, Xanthomonas oryzae pv. Oryzae, Xanthomonas arborocola pv. Pruni, Xyllela, Pectobacterium and Ralstonia solanacearum [5,19] (Table 1). Although several phages have been isolated and tested in vitro for their biocontrol potential, all have not been successful in field applications and the following challenges have come forth in the studies conducted so far:
1) Poor persistence of the phages upon application onto the phyllosphere or rhizospheric soil due to physicochemical factors such as temperature, UV light, pH, rain, dessication as well phytochemicals produced by plants which requires careful optimization of dosage and frequency of applications [5,20–22].
2) Host resistance to phages due to mechanisms such as abortive infections, host receptor editing as well as biofilm formation related exopolysacharide capsule formations and phage immunity due to CRISPR and restriction modification systems [23].
3) Poor transport of phage application to the phytopathogen: Phytopathogens infecting vascular tissue (vascular pathogens) or intercellular mesophyll tissue (apoplastic pathogens) may not be accessible to topical phage applications [24].
4) Legislation governing the use of phage formulations in the agriculture fields [25]. For eg. EU regulations (1107/2009 EC) require that any changes in phage formulation would require re registration hampering the custom made phage cocktail formulation module as biopesticide.
Table: Bacteriophages as biopesticides for some bacterial pathogen mediated plant disease | φRSL1 (Jumbophage Myoviridae) φRSM3 (Inovirus type filamentous), φRSB1, References | |||
Bacterial Wilt of tomato, tobacco and eggplant | Ralstonia solanacearum, R. pseudosolanacearum, R. syzygii subsp indonesiensis (Ralstonia complex) [39,40] | |||
Soft Rot of potato, lettuce | Soft rot Enterobacteriaceae (SRE) Dickeya dadantii; Pectobacterium LIMEstone1Phage treated seed potatoes (MOI 100) shoed 10% tuber maceration compared to 40% in untreated [41,42] | |||
Bacterial blight of leek, citrus cancer disease in kiwi fruit Pseudomonas syringae | Phages vB_PsyM_KIL1, vB_PsyM_KIL2, vB_PsyM_KIL3, and vB_PsyM_KIL3b.(phage cocktail) [43,44] | |||
Fire blight of apple and pear Erwinia amylovora | fEa1337-26 & fEa2345 withPantoea agglomerans as a carrier reduced reduced severity by 84 and 96% respectively [5,27,45] | |||
Bacterial spot disease of pepper, tomato; Black rot of cabbage | Xanthomonas campestris pv. vesicatoria | Bacteriophage XCCSPC211 reduced disease when used with non-pathogenic Xanthomonas. Not effective with Cu biocide [30,33] | ||
Strategies to overcome phage application challenges
Phage application has been studied using foliar application on the phyllosphere and using soil drenching method based on the nature of the phytopathogen and disease caused [5]. The strategies developed to effective circumvention of challenges to the phage treatments application as biopesticides include:
Enhancing phage persistence on plants: Optimizations of phage application strategies are crucial to the success of phages as biopesticides. Phyllosphere applications are less effective upon exposure to the ultraviolet A and B spectra of radiations in the sunlight. One practical approach to solve this problem is the application of the phage after sunset. The frequency of the phage application also depends on the phytopathogen and varies from daily to weekly applications [22]. Treatments of phage prior, on the day of phytopathogen infections and post infection have been conducted in greenhouse trials with the most effective treatments being with co-inoculation (Schnabel, E. L. et. al. 1999). Soil drenching (108 pfu/ml) of OmniLytic Inc Phage active against X. perforans strain 97-2 was detectable on tomato leaf tissue at 104 pfu/g for up to 7 days, suggesting that soil applications may be more effective in comparison to direct phyllospheric usage which were undetectable in 1- 2 days [26].
The use of UV protectants in order to counter the inhibitory effects of visible and UV rays of sun, desiccation as well as phytochemicals in nature has been studied [16]. Erwinia amylovora bacteriophage Y2 used for the treatment of fire blight of Rosacea species was applied using natural sunscreen protective compounds such as extracts from carrot, red pepper, and beetroot, casein and soy peptone in solution, and purified substances such as astaxanthin, aromatic amino acids, and Tween 80 which increased the phage half-life by 50% without affecting its viability [27].
Use of phage non-pathogenic host carrier systems to ensure phage numbers and viability. This is particularly effective for prophylactic treatments wherein the pathogen host is unlikely to be present. The strategy is also highly effective in low nutritive conditions for phage survival.
Tobacco wilt incidence was shown to be reduced with the coapplication of avirulent Ralstonia solanacearum with phage [28, 29]. Xanthomonas campestris var campestris, causative agent of black rot of cabbage was controlled using a combination treatment of non pathogenic Xanthomonas with bacteriophage XcpSFC211 significantly prevented disease incidence in field trials [30].
Enhancing phage fitness- genetically engineered phages
Synthetic biology has made it possible to create tailor made phage systems capable of effective and targeted removal of pathogen [17]. The genetically engineered T7 phage expressing the endolysin DspB from Actinobacillus actinomycetemcomitans (T7DspB) capable of inhibiting biofilm formation in E. coli TB1 was developed [31]. Schmerer 2017 tested the efficacy of the engineered phage for control of long term biofilms in comparison to wild type phages to show that Phage T7DspB is an efficient tool for biofilm disposal. The phage product is now being commercialized by the company EnBiotix, Inc. (Cambridge, MA, USA). Engineered phages with enhanced antibiotic activity, anti quorum sensing molecules, targeted delivery of antimicrobial agents have been developed [32].
Overcoming host resistance: Unlike other antimicrobial chemicals, the phage population itself can overcome host resistance against phages, as it is a self-evolving system. Flaherty, 2001 showed the presence of H-mutants of phages which evolved to form broad host ranges against Xanthomonas campestris pv. Pelargonii [33]. Qiao, 2010 showed that evolving phage mutants of Ps. syringae phi 2954 which did not require the host protein glutaredoxin 3 for successful infection of the pathogen [34]. Phage resistance to abortive infection mutation via synthesis of pseudo ToxI RNA antitoxin preventing ToxN toxic activity in P. atrosepticum by phi TE [23]. Recently, reports of an anti CRISPR (Acr) proteins which bind to the CRISPR mechanisms in bacteria and hence allow phages to infect host cells despite the CRISPR mediated resistance are in preprint form [35]. In some studies, attenuation of bacterial pathogenicity is reported following development of phage resistance [35].
Phage cocktail formulations which may include a mixture of naturally occurring phage with range of host specificity to genetically engineered phages to overcome the host receptor diversity is a popular strategy used to overcome the problem of phage induced bacterial resistance.
A meta-analysis of phage host specificity representing 12,000 distinct experimental infection assays which explored the phage bacterium nestedness versus modular interactions (Flores, 2011). The copmplexicity of phage bacterium interactions were highlighted which were varied depending on taxanomic groups, habitats and biogeographical regions (Flores 2011, 2013). Phages with high degree of host specificity were likely to infect less resistant bacteria where as those with a broad host rage were capable of arresting the growth of highly resistant bacteria.
Increasing shelf life of phage biopesticides
Commercial agricultural phage formulations are concentrated phage formulations that require dilution with irrigation water prior to application in the greenhouse or field. Phage formulations are typically spray dried which may be diluted to suitable concentrations prior to applications. The encapsulation of phages in microcapsules or nanoparticle formulations is being explored for their sustained release in the environment (Malik et al, 2017).
Commercialization of Phage treatments
Commercialization of Phage treatments was intiated by Omnilytics, a US based company for their phage based biopesticide Agriphage for protection of bacterial speck of tomato and peppers which was approved by the US Environmental Protection Agency (Omnilytics, 2006). Enviroinvest (Hungarian) registered Erwiphage for the control of blight of apple caused by Erwinia amylovora. APS biocontrol developed Biolyse (phage based wash solution) for protection of soft rot of potato [5]. Agriphage (EPA registeration #67986-1) from Omnilytics comprises of mixture of bacteriphages active against Xanthomonas campestris pv. vescicatoria and Pseudomonas syringae. A number of patents have been given for bacteriophage application in the food industry [36,37]. In 2006, ListShield was the first phage product to be approved by US Food and Drug administration (FDA) for meat and poultry industry against food pathogen Listeria monocytogenes (Intralytics Inc). The company has two other phage products which can be sprayed on foods for protection from Salmonella (Salmofresh) and E. coli OH157:H7 (EcoShield). A fouth product ShigaShield has received GRAS status for protection of food from Shigella. Several patents and commercial products that are available confirm the acceptability of phage based biopesticide for use in the food and agriculture industry for pathogen control.
Challenges and Outlook
Increased incidence of environmental pollution due to industrialized plant disease control methods require an urgent and thorough overhauling in agricultural practices with a major thrust in encouraging the use of alternative pathogen control measures. Academic research in the area of phage based phytopathogen control has proven its efficacy, non toxicity and hence suitability to field applications in nature. However, its in field application and long term studies are required to address concerns regarding phage mdiated horizontal gene transfer, phage resistance as well as unpredicatible phage infection kinetics [38]. Field trials will also bring forth problems and challenges in its phage delivery systems and its optimization. Careful monitoring of phage persistence in agricultural fields may bring forth studies that clear doubts regarding the safety of introducing natural phage cocktails and genetically engineered phages into the environment. Awareness regarding a phage based integrated management systems for phytopathogen for farmers worldwide and the commercial availability of phage formulations as biopesticides is a strategy, which requires concerted effort from industry and government bodies.
This material is based on the research supported by ‘Minor Research Project Grant’ received from Chhatrapati Shahu Ji Maharaj University, Kanpur, India.
Clearly Auctoresonline and particularly Psychology and Mental Health Care Journal is dedicated to improving health care services for individuals and populations. The editorial boards' ability to efficiently recognize and share the global importance of health literacy with a variety of stakeholders. Auctoresonline publishing platform can be used to facilitate of optimal client-based services and should be added to health care professionals' repertoire of evidence-based health care resources.
