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Bacteriophages: A Potential Next Generation Biocontrol Tool for Plant Disease Management

Bacteriophages or the phages are the viruses that parasitize bacteria. The introduction of bacteriophages to the world began one hundred and three years ago in 1915 when F. W. Twort reported that he observed some „glassy transformation‟ on cultures of micrococci. However after two years, in 1917, Felix d‟ Herelle, a Canadian bacteriologists working at the Pasteur Institute in Paris, also published his independent discovery of bacteriophages (Summers, 2006). The term bacteriophages was given by Felix d‟Herelle from „bacteria‟ and the Greek word „phagein‟ which means „to eat‟ referring to the remarkable ability of the bacteriophages to cause lysis of growing bacterial cultures (Ceyssens, 2009).


Introduction
Bacteriophages or the phages are the viruses that parasitize bacteria. The introduction of bacteriophages to the world began one hundred and three years ago in 1915 when F. W. Twort reported that he observed some "glassy transformation" on cultures of micrococci. However after two years, in 1917, Felix d" Herelle, a Canadian bacteriologists working at the Pasteur Institute in Paris, also published his independent discovery of bacteriophages (Summers, 2006). The term bacteriophages was given by Felix d"Herelle from "bacteria" and the Greek word "phagein" which means "to eat" referring to the remarkable ability of the bacteriophages to cause lysis of growing bacterial cultures (Ceyssens, 2009).
Since its discovery in the early 20 th century, the bacteriophages were widely used as therapeutic agents against human and animal bacterial diseases. Experiments regarding the use of phages as biocontrol agents in agriculture soon followed similar trend. However, in spite of the popular and promising success of the early phage therapy, it did not prove to be reliable and effective means for the control of bacterial diseases. The widespread success and availability of broad spectrum antibiotics, inconsistent results

ISSN: 2319-7706 Volume 7 Number 09 (2018)
Journal homepage: http://www.ijcmas.com The biocontrol potential of the bacteriophages has been known to man since its discovery a century ago. Bacteriophage therapy or the use of bacteriophages for the management of bacterial diseases is a concept with a controversial past and hence, still an uncommon practice especially in the agricultural sector. However, in the present decade there has been a renewed interest and several successful researches owing to the better understanding of the phage biology and ecology by using modern techniques like the next generation sequencing and metagenomics. Moreover, the bacteriophages are a potential solution to mitigate the existing challenges like the emergence and re-emergence of pathogenic bacteria, development of multi drug resistant bacteria, environmental hazards due to over use of chemicals etc. This article gives an overview on the bacteriophages and their potential to utilize them in the field of agriculture with an aim to build up the interest of further research on it, especially in India to handle the bacterial diseases organically. on the efficiency of phage therapy probably due to lack of knowledge regarding the bacteriophages and credibility problems caused by lack of quality control and properly controlled studies led to the decline of phage therapy trials both in the field of medicine and agriculture (Jones et al., 2007). Gill and Hyman, (2010) listed out three major factors that led to the failure of early phage therapy: inappropriate phage choice, poor phage preparation and phage decay prior to application.
The most frequent management tactic for the control of bacterial diseases is the use of antibiotics. However, the major problem of most antibiotics is that they are broad spectrum in their mode of action. The indiscriminant targeting of bacteria by broad spectrum antibiotics causes generation of resistance to a wide range of bacterial population. Moreover, overuse of antibiotics has led to the spread of R-plasmids and multidrug resistant (MDR) strains. This has led to re-evaluation and re-thinking of the use of phages as biological control agents (BCA) (Wu et al., 2013). In the present decade however, better understanding of the phages, phage-host interactions, better isolation, characterization, propagation as well as delivery has become possible with the help of modern biotechnological tools. This has led to a great spike in phage therapy research all over the world. USA, Japan, China and European countries play a lead role in the phage therapy research as well as field level application. In India however, it is still an uncommon practice. The present article gives an overview of the bacteriophages and the recent trends in their application in the field of agriculture.

Phage biology and classification
Bacteriophages are the most abundant organisms on Earth. It has been estimated that there are approximately 10 30 bacterial cells in the biosphere and about 10 31 phages in the planet. This makes the viral to bacterial ratio to be around 5-10: 1 (Hatfull, 2008). Phages are natural parasites of the bacteria and are found ubiquitously in nature. It is believed that at least a single type of phage, more likely more than one can infect every strain of bacteria (Keen, 2015). As calculated by viral ecologists, globally there are about 10 23 phage infections per indicating the dynamic nature of the phage population apart from its huge numbers (Hatfull and Hendrix, 2012) Bacteriophages are polyphyletic in origin as they are extremely heterogeneous in their structural, physiochemical and biological properties (Ackermann, 2005). They show a great diversity in their morphology as well as genomics.

Phage morphology
Like all viruses, a bacteriophage particle or virion is composed of a single or double stranded (ss or ds) DNA or RNA which is encapsulated inside a protein or a lipoprotein coat. The morphology of the bacteriophage plays a major role in their classification. The main foundation for the present phage classification was given by Bradley in 1967. Bradley reported six different morphological types of phages, exemplified by phages T4, λ, T7, ΦX174, MS2 and fd. The International Committee on the Taxonomy of Viruses (ICTV) uses virion morphology and nucleic acid composition as a basis for the classification. The latest report (10th) ICTV has been published in the year 2017 (https://talk.ictvonline.org/ictvreports/ictv_online_report).
There are a total of 19 morphotypes of bacteriophages according to ICTV, amongst which the best studied and a vast majority (96%) belongs to the order Caudovirales having an icosahedral or elongated head and a tail (Latin Cauda means tail). They are known to infect both Eubacteria and Archaea and are probably as ancient as 3.5 billion years old (Ackermann, 2005;Ackermann, 2009b). The tailed phages are monophyletic in origin possessing related morphologic, physiochemical and physiological properties. The most distinguishing characteristics are that they contain dsDNA, have icosahedral or elongated heads, the tails are elongated and possess some fixation structures like base plates, spikes and fibers and they do not have envelope (Ackermann, 2009b).
The order Caudovirales is divided into 3 families which are as follows (Ackermann, 2009b):

Myoviridae
The tail of myoviruses is composed of a neck, a contractile sheath and a central tube. They are larger in size than other groups and are highly evolved. Around 25% of the tailed phages belong to this family.

Siphoviridae
The tails are simple, non-contractile, flexible or rigid tubes. They are the most numerous of the tailed phages. It comprises of around 61% of the tailed phages.

Podoviridae
The tails of podoviruses are short and noncontractile. They are more related to Siphoviridae than Myoviridae. Podoviruses comprises of approximately 14.5% of the tailed phages.
Use of electron microscopy is one of the major tools for the characterization of bacteriophages, mainly the unknown phages as it is based on visualization and morphological identification of the viral particles. Ruska in 1940 (cited by Ackermann, 2009a) published the first electron micrographs of bacteriophages in which the lysis caused by coliphages to an E. coli cells were photographed. The discovery of the negative staining technique by Brenner and Horne in 1959 can be regarded as a landmark as it greatly boosted its use for virus detection. As reported by Ackermann and Prangishvili (2012), around 6300 prokaryote viruses have been described morphologically since the discovery of negative staining in 1959, amongst which 6196 were bacterial and 88 archaeal viruses.

Phage genomics
According to Ackermann (2009b), the phage genomes include double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), single-stranded RNA (ssRNA), and doublestranded RNA (dsRNA). The vast majority of the phages contain dsDNA whereas; phages containing dsRNA are very rare. All the DNA phages contain a single molecule of DNA and several phages have lipid containing envelops or internal vesicles (Ackermann, 2005). The phage diversity is also reflected in their genome sizes. The phage genome size are reported to range from 2,435bp in Leuconostoc phage L5 (Hatful, 2008) to 497,513bp in Bacillus megaterium phage G (Donelli et.al., 1975). Hatful, 2008 reports that the distribution of genome sizes of the phages are not uniform. The largest peak of which is seen at 30-50kbp interval (approximately 50% of all phages) followed by the group whose genomes are smaller than 10kbp (about 20% of total) and those in 100-200kbp interval (6% of total). The tailed phages having more than 200bp are classified as jumbo phages. Yuan and Gao (2017) suggested that the jumbo phages possesses several novel characters not seen in other phages with smaller genomes. These jumbo phages have evolved from the phages with smaller genomes by acquiring some additional functional genes which in turn reduces its dependence on host bacteria.
Due to their relatively small genome sizes and simplicity of isolation, the complete genomes of the bacteriophages can be sequenced quite easily. Some major events in history regarding the sequencing of phage genomes are depicted in Table 1.

Phage life-cycles and their impact in phage based biocontrol
The bacteriophages can be considered as parasites of bacteria as they rely on their host bacteria for the completion of their life cycle. They can undergo two different types of life cycles: the lytic and the lysogenic cycle. The phages gets adsorbed to the host bacterium at some specific receptor sites like protein receptors (structural proteins interacting with peptidoglycan layer, specific and non-specific porins forming membrane channels, enzymes, substrate receptors with high affinity, transport proteins responsible for secretions), or lipopolysaccharide (LPS) receptors in case of gram negative bacteria etc. (Rakhuba et al., 2010). In lytic cycle, after the adsorption of the phage particle on the host bacterium, only the nucleic acid of the phage particle is penetrated inside the host cell. This results in switching off the protein mechanism of the host bacterium in the favor of the phages resulting in production phage proteins and nucleic acid. Hence, a large number of progeny phages are formed weakening the host, thus resulting in lysis and death of the host cell. The bacteriophages which show the lytic cycle are also widely known as virulent phages. Some examples of virulent phages are Coliphage T4, Coliphage T2 etc. (Adams, 1959;Orlova, 2012). As mentioned by Orlova (2012), as many as 50-200 new phages can be released from a single phage particle after completion of the lytic cycle. The word "lysogeny" on the other hand means "generating lysis" (Lwoff, 1953). A lysogenic infection or life cycle can be characterized by incorporation of the phage DNA into the genome of the host bacterium and thus replication of the phage DNA along with the replication of the host (Orlova, 2012). A lysogenic bacterium can undergo many cell divisions without losing its lysogenic property. Lysis of a lysogenic bacterium spontaneously or in response to certain environmental factors is accompanied by the release of many mature phage particles. However, if a lysogenic bacterium is disrupted, no infectious particles are known to be released (Lwoff, 1953;Adams, 1959;Brathwaite, 2015). The prokaryote which harbors latent phages is known as lysogenic and the latent form of the phage is known as prophage (Adams, 1959;Campbell, 2006). The bacteriophages that show a latent state are known as temperate or lysogenic phages such as Coliphage λ, Mu-1 etc.).
Selection of the right kind of phage is the key for successful phage based biocontrol mechanisms. Only the virulent phages are capable of causing lysis of their host bacterial cells and hence are of prime interest in phage therapy. The lytic phages have a narrow host range i.e. they are known to infect only specific bacterial species (Doss et al., 2017). Some phages are known to infect only a few strains of one species of bacteria, while some others may be species specific as well as genus specific (Gill and Hyman, 2010). This is a boon as the phages are target specific and does not infect the other beneficial bacteria of the biosphere. However, due to their high specificity, it is more likely that a phage cocktail is required against a particular bacterial disease to counter all the strains of the bacterial pathogen. To use the phages for bio-control purpose, it is desirable to perform the host range analysis and choose the phages that allow productive infection on all strains of the pathogen genus or species being targeted (Buttimer et al., 2017). Apart from that, some lytic phages are also capable of transduction i.e. bacterial gene transfer with the aid of phages (Klumpp et al., 2008). For successful phage based bio-control, the lysogenic and transducing phages should be eliminated during selection.

Application of phages in plant disease management
Although a vast majority of plant diseases are of fungal origin, the bacterial plant pathogens are highly devastating and are responsible to cause major economic losses every year. Management of the plant pathogenic bacteria has been a challenging issue because of several factors like lack of effective bactericides, the antibiotics being environmentally hazardous as well as noneconomic and phyto toxicity of the copper compounds rapid development of antibiotic resistant strains of bacteria, pathogen variability, high probability of mutation gene transfer, high mutation rates resulting in bacteria overcoming plant genetic resistance, (Jones et al., 2007;Balogh et al., 2010). The bacteriophage based biocontrol strategies are a potential alternative to antibiotics and can effectively solve the challenges of bacterial disease management. There are several advantages of the use of bacteriophages in bacterial disease management. Some of them are listed below (Jones et al., 2007;Buttimer et al., 2017;Wu et al., 2017):

Bactericidal agents
Once infected by an obligately lytic phage, bacteria will not regain their viability. Narrow host range: Many phages are strain specific or infects a few strains of the target bacterium A phage cocktail is required against a particular bacterial disease to counter all the strains of the bacterial pathogen.

2.
High production cost: Constant study and improvisation of the phage based bio pesticide is required from time to time.
Once the protocols have been standardized, it does not take much time and effort to carry out similar studies. Moreover, molecular detection techniques can be used easily now days with a reasonable price and a short time frame.

3.
Temperate and transducing phages: The temperate and transducing phages can convert susceptible bacteria to a virulent one by horizontal gene transfer (HGT).
Correct identification and selection required to eliminate the temperate and transducing phages when using them for bio control purposes.
Simultaneous treatment of phage PE204 with R. solanacearum of the rhizosphere of tomato completely inhibited bacterial wilt. Pre-treatment was not effective but post treatment delayed disease development. Bae et al., (2012) Potato Twelve lytic bacteriophages specific to R. solanacearum were isolated and characterized. More than 80% of the potato plants could be protected using phage cocktail and the same cocktail could kill 98% of the live bacteria spiked in the sterilized soil one week after application. Wei et al., (2017)

Dickeya solani Soft Rot Potato
The bio assays and field trials of two closely related and specific bacteriophages, vB_DsoM_LIMEstone1 and vB_DsoM_LIMEstone2 revealed that the phages reduced soft rot of inoculated tubers and also produced a potato crop with higher yields.
Adriaenssens et al., A total of nine bacteriophages specific to Dickeya solani were isolated from soil in Poland. Bioassays with the phages ΦD1, ΦD2, ΦD3, ΦD4, ΦD5, ΦD7, ΦD9, ΦD10, ΦD11 reduced the disease incidence of soft rot by up to 30-70% on co-inoculated potato slices with pathogen and phage.

Pseudomonas tolassi
Brown blotch of mushroom Mushroom The surfaces of mushroom caps were inoculated with both pathogenic bacteria and their phages. Formation of blotches was completely blocked by co-incubated phages. Kim et al., (2011)

Auto 'dosing'
Phages are self-sustainable, self-replicating and self-limiting. They replicate as long as the host bacterium is present in the environment and degrade quickly in the absence of its host.

Minimal disruption to micro biota
Phages are target specific and do not harm the beneficial bacteria present in nature. Moreover, phages are prokaryotic viruses and hence, are completely harmless to the eukaryotes.

Narrower potential for inducing resistance
Phage resistance occurs in a small population size as they have a very narrow host range. Moreover, some phages use specific receptors (EPS, LPS, flagella, pilli etc.) in bacteria that are essential for survival in the infected hosts.
Mutations of the bacteria leading to resistance to the phages frequently results in loosing of virulence.

Lack of cross-resistance with antibiotics
The mechanisms used by the phages to infect bacteria completely differ from the mechanisms involved in antibiotic resistance.

Rapid discovery
Phages are abundant in nature and can be isolated from wherever the host bacterium is present like soil, water, plant surface, animals etc.

Formulation and application versatility
Phage based products are relatively easy and inexpensive to produce. Phages can be blended with creams, impregnated into solids, and applied as liquid preparations and hence, diverse formulations can be made.

Prevent the formation of biofilms
Due to their mode of action on the bacteria, bacteriophages are known to prevent the formation of these biofilms and hence can be used as a prophylactic measure to control the biofilm forming bacteria.
There are very few limitations of using phages as bio control agents which are listed in Table 2 along with their probable remedies: Owing to its multiple advantages, numerous successful research works have been carried out in the present decade all over the world which itself is an evidence that bacteriophage have a great potential to be used as bio control agents. A selected summary of the phage based bio control experiments with respect to some important plant pathogenic bacteria since the year 2010 is shown in Table 3.
The true potential of the bacteriophages have only been understood in the present decade after more than a hundred years of its discovery. Bacteriophage therapy or phage based biocontrol is an exciting rediscovered field of bacterial disease management. Presently, although the use of phage based biocontrol is emerging, but is still an uncommon practice in the field of agriculture. Bacteriophages have several qualities which makes them potential biocontrol candidates. Moreover, the phage based biocontrol strategies will also help to overcome the challenge of antibiotic resistance up to a great extent. They are natural components and hence can be fitted in organic farming strategies. They can also be used as a component of the integrated management strategies as they can be combined with chemicals and other biocontrol agents. Scientific research for the development of phage based biocontrol strategies using modern biotechnological tools for isolating, characterizing, engineering, manufacturing and delivering phages will play a major role in shaping the future of phage based biocontrol practices.