Application of filamentous phages in environment: A tectonic shift in the science and practice of ecorestoration

Abstract Theories in soil biology, such as plant–microbe interactions and microbial cooperation and antagonism, have guided the practice of ecological restoration (ecorestoration). Below‐ground biodiversity (bacteria, fungi, invertebrates, etc.) influences the development of above‐ground biodiversity (vegetation structure). The role of rhizosphere bacteria in plant growth has been largely investigated but the role of phages (bacterial viruses) has received a little attention. Below the ground, phages govern the ecology and evolution of microbial communities by affecting genetic diversity, host fitness, population dynamics, community composition, and nutrient cycling. However, few restoration efforts take into account the interactions between bacteria and phages. Unlike other phages, filamentous phages are highly specific, nonlethal, and influence host fitness in several ways, which make them useful as target bacterial inocula. Also, the ease with which filamentous phages can be genetically manipulated to express a desired peptide to track and control pathogens and contaminants makes them useful in biosensing. Based on ecology and biology of filamentous phages, we developed a hypothesis on the application of phages in environment to derive benefits at different levels of biological organization ranging from individual bacteria to ecosystem for ecorestoration. We examined the potential applications of filamentous phages in improving bacterial inocula to restore vegetation and to monitor changes in habitat during ecorestoration and, based on our results, recommend a reorientation of the existing framework of using microbial inocula for such restoration and monitoring. Because bacterial inocula and biomonitoring tools based on filamentous phages are likely to prove useful in developing cost‐effective methods of restoring vegetation, we propose that filamentous phages be incorporated into nature‐based restoration efforts and that the tripartite relationship between phages, bacteria, and plants be explored further. Possible impacts of filamentous phages on native microflora are discussed and future areas of research are suggested to preclude any potential risks associated with such an approach.

thal, and influence host fitness in several ways, which make them useful as target bacterial inocula. Also, the ease with which filamentous phages can be genetically manipulated to express a desired peptide to track and control pathogens and contaminants makes them useful in biosensing. Based on ecology and biology of filamentous phages, we developed a hypothesis on the application of phages in environment to derive benefits at different levels of biological organization ranging from individual bacteria to ecosystem for ecorestoration. We examined the potential applications of filamentous phages in improving bacterial inocula to restore vegetation and to monitor changes in habitat during ecorestoration and, based on our results, recommend a reorientation of the existing framework of using microbial inocula for such restoration and monitoring. Because bacterial inocula and biomonitoring tools based on filamentous phages are likely to prove useful in developing cost-effective methods of restoring vegetation, we propose that filamentous phages be incorporated into nature-based restoration efforts and that the tripartite relationship between phages, bacteria, and plants be explored further. Possible impacts of filamentous phages on native microflora are discussed and future areas of research are suggested to preclude any potential risks associated with such an approach.

K E Y W O R D S
bioremediation, biosensors, ecological theory, filamentous phages, microbial ecology and fitness, restoration ecology

| INTRODUC TI ON
Theories in soil biology guide the efforts to restore vegetation in degraded habitats. Natural attenuation-banning any activity that results in environmental degradation-is useful only in those ecosystems that are in the early stages of degradation. Currently, a vast majority of degraded ecosystems show altered abiotic and biotic components and lowered resilience. Consequently, assisted restoration practices, such as planting native species, replacing or treating contaminated soil, and managing water resources, represent the only land restoration options for most of the degraded ecosystems.
The Society of Ecological Restoration defines ecorestoration as "the process of assisting the recovery of an ecosystem that has been degraded, damaged or destroyed" (SER, 2004). Restoration practices developed on the principles of plant-microbe mutualism and microbial cooperation, synergism, and antagonism have facilitated ecorestoration (Heneghan et al., 2008;Perring et al., 2015;Young, Petersen, & Clary, 2005).
Over 65% of the earth's surface has been degraded or contaminated, which has resulted in the loss of its potential to benefit human society-which is why ecorestoration is held to be a global need by the United Nations. Restoration practices have site-specific goals, such as to repair environmental damage after deforestation or overharvesting, to stabilize soil after mining, to restore the productivity of saline soils after heavy irrigation, and to remediate soils contaminated as a result of industrial activity. However, major goals of all land restoration continue to be the restoration of biodiversity, revival of ecosystem services for socio-economic security, and improved resilience of ecosystems to future environmental change.
Restoration may be voluntary, undertaken to improve the quality of life, or mandatory, a legislative directive to ensure sustainable development.
Degraded lands need specialized restoration efforts rather than conventional soil amelioration methods, because such lands are often exposed to multiple sources of stress such as high levels of contaminants, toxins, and pathogens (Perring et al., 2015). Traditionally, soil scientists use agronomic practices and chemical amendments to improve soil fertility (Filiberto & Gaunt, 2013), although different sources of stress need source-specific efforts to improve soil and plant health. The traditional methods commonly used for treating farmlands or small patches of degraded lands are not only too costly for restoring vast stretches of degraded ecosystems but also of limited efficacy in controlling pathogens and biological toxins and coping with changes in the environment (Figure 1). In contrast, microbial inocula (free-living, associative, and endosymbiotic) repair normal biological processes affected by degradation, ameliorate contaminated soils, and control phytopathogens in degraded habitats.
Microbial inocula are thus an ecologically sound option to speed up revegetation and revive ecosystem services under diverse environmental regimes (Tables 1-3). Some bacterial genera, such as Bacillus, Bradyrhizobium, Enterobacter, and Pseudomonas, have been widely used as inocula and even commercial formulations have been developed for use with economically and ecologically important plants (Table 1). Microbes may possess multiple traits that may be deployed in combating both abiotic and biotic sources of stress; however, restoration ecologists select bacteria with specific traits to tackle the most serious environmental challenges (Rau et al., 2009;Sharma, Mishra, Rau, & Sharma, 2015). Alternatively, consortia of microbes may also be developed to deal with multiple challenges, but their efficacy is reduced because members of such consortia differ in their environmental and nutritional requirements, and the use of consortia continues to face unpredictable challenges.
As a result of advances in molecular and genomic studies, bacterial viruses (bacteriophages or simply phages) have emerged as one of the key elements governing the structure and functioning of microbial communities (Kauffman et al., 2018). Phages play an important role in developing a resilient microbial community (Koskella & Brockhurst, 2014;Silveira & Rohwer, 2016) and in driving adaptation, competition, and antagonism in bacteria and thereby influence the evolution of bacteria and the assembly of microbial community (Rodriguez-Valera et al., 2009;Mai-Prochnow et al., 2015;Shapiro, Williams, & Turner, 2016) (Figure 2). However, applying these theories of microbial ecology to ecorestoration has not received due attention.
The growing knowledge of the ecology of filamentous phages from diverse bacterial genera and environmental settings makes them an important biological resource for environmental restoration (Fauquet, Mayo, Maniloff, Desselberger, & Ball, 2005;Rakonjac, 2012;Henry et al., 2015;Szekely & Breitbart, 2016;Mai-Prochnow et al., 2015). More than sixty filamentous phages have been reported from terrestrial and aquatic ecosystems in temperate and tropical regions (Fauquet, Mayo, Maniloff, Desselberger, & Ball, 2005;Rakonjac, Bennett, Spagnuolo, Gagic, & Russel, 2011). Metagenomic analyses show a high frequency of filamentous phages in such contaminated environments as industrial wastewater and sewage disposal sites, which need ecorestoration (Alhamlan, Ederer, Brown, Coats, & Crawford, 2013;Cantalupo et al., 2011). A recent and exhaustive metavirome study also showed the prevalence of filamentous phages in many diverse environments including soils and sediments, saline water and freshwater, and air and food (Szekely & Breitbart, 2016). Bacteria associated with higher animals, insects, corals, and even people (as gut bacteria) also harbor filamentous phages (Weynberg, Voolstra, Neave, Buerger, & van Oppen, 2015), and they have been reported from bacterial genera associated with ecologically important plant families (Brassicaceae, Poaceae, Rutaceae, and Solanaceae) widely employed in environmental restoration (Berg, Marten, & Ballin, 1996;Tseng, Lo, Lin, Pan, & Chang, 1990). Despite emerging evidence on the prevalence of filamentous phages, current knowledge of the ecology of filamentous phages in soil and plants and of their potential application in restoration continues to be limited because of the challenges in purifying and identifying the phages and in assaying their activities.
Filamentous phages are useful for manipulating bacteria for environmental applications because the phages are stably produced in their bacterial hosts and are easy to manipulate using genetic and chemical methods-however, they remain underexploited in current practice. A chronic infection by a filamentous phage induces longterm changes in the host physiology, which is desirable for developing microbial inocula.
Besides a relatively persistent relationship with the bacterial host, filamentous phages also show high host specificity up to the level of a strain, which qualifies them as a stable biomarker of their host (Henry et al., 2015;Lin et al., 1999 Finally, we identify priority research areas to realize the potential environmental benefits of filamentous phages and to prevent possible risks in their environmental applications. F I G U R E 2 An unsolved problem related to exploiting bioremediation potential of Pseudomonas in phenol contaminated field environment highlighting the potential of filamentous phage to provide solutions and path of research to arrive at them (based on Goldstein, Mallory, & Alexander, 1985;Mrozik, Miga, & Piotrowska-Seget, 2011). FP, filamentous phage.

| P OTENTIAL ARE A S FOR THE APPLI C ATI ON OF FIL AMENTOUS PHAG E S IN ENVIRONMENTAL RE S TOR ATION FOR SUS TAINAB LE DE VELOPMENT
Environmental restoration is acceptable to ecological economists as a tool for ensuring human well-being and developing a sustainable society, which is characterized by improved soil health, reduced negative impacts of industrial activity, and lower poverty (Lei, Pan, & Lin, 2016;Martin, 2017;Millennium Ecosystem Assessment, 2005;Sachs & Reid, 2006;Tallis, Kareiva, Marvier, & Chang, 2008). In fact, the policy to encourage environmental restoration proved promising in helping people to escape the poverty trap in China (Cao, Zhong, Yue, Zeng, & Zeng, 2009). Poverty traps represent a vicious circle formed due to a complex interaction between the poverty and environmental degradation, in which "poverty leads to environmental degradation, and environmental degradation then deepens poverty" (Tallis et al., 2008). Poverty forces the native people to engage in unsustainable exploitation of natural resources, which degrades the environment and reduces the resource base for the poor people.
Environmental degradation makes the land unproductive, therefore, reduces the income of native people. In this context, ecological restoration programs, which take into account the livelihood of the native people, also restore ecosystem goods and services besides economic and social development. In Changting County of China, the ecological restoration resulted in reduced soil erosion (68.3%), increased vegetation cover (75%), and species number (6 times) accompanied with increased employment (12.4%) and net income (11.2%) of native people (Cao et al., 2009 in a bioreactor using an automated controlling system, whereas in the treatment on the ground, we may add organic matter (compost) or fertilizers to the soil, with tillage (land farming) or without tillage (soil biopiles). In situ methods, on the other hand, involve little or no disturbance to soil structure and rely on either natural attenuation by natural physico-chemical and biological processes or on assisted restoration through enhanced microbial activity. Microbial activity can be enhanced by injecting nutrients, water, chemicals, and even air, using underground pipes, to the contaminated site, that is to the saturated soil zone (biosparging) or to the unsaturated zone (bioventing; Azubuike et al., 2016). Bioventing may be combined with vacuum-enhanced pumping for treating the contaminants in saturated and unsaturated zones (bioslurping). We may also either introduce specific bacteria to enrich the target bacteria (bioaugmentation) or add specific nutrients to stimulate the activity of targeted bacteria (biostimulation; Malhotra, Mishra, Karmakar, & Sharma, 2017).
In situ and ex situ soil treatments involve costly and labor-intensive physicochemical methods, and copious use of water puts additional pressure on existing water resources. Also, the use of chemicals to control pathogens adds to the cost, pollutes soil and water, and harms even useful microbes. To avoid these adverse ef- or the lack of real-time biomonitoring tools for tracking the inocula, pathogens, and contaminants-and both can be countered by using filamentous phages to modify the ecophysiology of their bacterial hosts suitably and as ultrasensitive biosensors for real-time biomonitoring. Filamentous phages carry genes or influence the expression of bacterial genes that help the bacterial hosts to adapt to abiotic and biotic stresses (Shapiro et al., 2016) by developing tolerance to microbial toxins and such abiotic sources of stress in the environment as salinity, desiccation, high temperatures, and high levels of contaminants (Secor et al., 2015;Shapiro et al., 2016;Yu et al., 2015). Filamentous phages may also make their bacterial hosts less virulent or lower our dependency on pesticides to control pathogens, thereby contributing to sustainable restoration ( However, filamentous phages should also enable PGPR to outcompete native bacteria in colonizing the soil and the rhizosphere. To achieve this goal, we recommend co-inoculation with filamentous phages and bacteria for ecorestoration (Figures 3 and 4). To this end, we first need to identify the filamentous phages that can bring about the desired changes in the biology and ecology of target bacteria to improve their efficacy as inoculants. Secondly, we need to identify filamentous phages-or modify them-to develop biosensors to track the inocula and the contaminants through time and space. Research in these areas will contribute to making microbial technologies both sustainable and effective.

| Bacteria as restoration inocula: Potential targets for modulation by filamentous phages
The bacteria used in commercial formulations as inocula for such environmental applications as improving soil health and promoting plant growth serve as potential targets for research involving filamentous phages. and Thiobacillus. To make these formulations more effective, we suggest that these genera be used as potential targets for exploring the benefits of filamentous phages, although filamentous phages for Pseudomonas have already been reported.

Ps29
• Increase dark coloration and pigmentation Yamada et al. (2007) φRSM   Therefore, research on filamentous phages to infect these bacterial genera deserves higher priority.

| Bacteria with known filamentous phages potentially useful in restoration
Filamentous phages have been reported from many bacterial genera potentially useful in ecorestoration. These genera include  Cf1c -X. campestris pv. Citri • Variation in gene structure and sequence Kuo et al. (1991) f1, c2 -Enterobacteria sp.
• Loss of cell viability and reduction in rates of RNA and protein synthesis Kuo et al. (2000) (Addy et al., 2012b;Ahmad et al., 2014;Derbise et al., 2007;Jian et al., 2013;Kuo et al., 2000;Waldor & Mekalanos, 1996;Whiteley et al., 2001;Yu et al., 2015; Table 2). These genera include species that cause diseases in plants and animals, show bioremediation activity, and promote plant growth. The filamentous phage-mediated ecological fitness of host bacteria has been investigated in selected species for pathogenicity, survival, colonization, multiplication, and distribution in a given ecological niche (Table 5). However, other species that may be potentially useful in bioremediation and restoration of vegetation are yet to be fully exploited.
Most bacterial genera also include species, which have as-  for treating industrial wastewater (Liu et al., 2016), and a protocol has also been developed for their mass multiplication.  (Fujio & Kume, 1991;Marteinsson, Birrien, Raguenes, Costa, & Prieur, 1999). These bacterial genera and species have a high potential for processing and bioremediation of wastes even at temperatures as high as 65-84°C.
Enterobacter, Pseudoaltermonas, and Vibrio species are not only significant for bioremediation but also for promoting plant growth.
Enterobacter sp. RNF 267 promotes the growth of coconut palms (Cocos nucifera) and maize (George, 2013), and inoculation of green gram (Vigna radiata) with Enterobacter EG-ER-1 and KG-ER-1 together with Bradyrhizobium sp. increased nodulation (Gupta et al. 2003). P. shioyasakiensis and V. sagamiensis SMJ18 tolerate not only salt and heavy metals (As, Cu, and Zn) but also show multiple traits that promote plant growth: They can fix nitrogen; solubilize phosphates; and produce IAA-, siderophores, and ACC (1-aminocyclopropane-1-carboxylate deaminase). Spartina maritima inoculated with V. sagamiensis SMJ18 shows more efficient photosynthesis, greater intrinsic water-use efficiency, and lower metal uptake-which is why the combination of V. sagamiensis and S. maritima has been recommended for ecorestoration of polluted estuaries.
As most of the above-mentioned bacterial genera can be commercialized and filamentous phages of these genera are known (Table 2), co-inoculation with bacteria and filamentous phages needs to be tested for environmental use (Figures 3 and 4).

| Phytopathogenic bacteria particularly useful in ecorestoration as targets of research on filamentous phages
Filamentous phages of bacterial phytopathogens also provide an opportunity to improve assisted phytoremediation as part of ecorestoration (Table 3; Figure 4). Filamentous phages have been characterized for six of the world's ten most serious bacterial phytopathogens. These six pathogens belong to four genera, namely

| FIL AMENTOUS PHAG E S TO BOOS T ENVIRONMENTAL COMPE TITIVENE SS OF BAC TERIAL INO CUL A
In many bacteria, filamentous phages influence the expression of phenotypic traits and trigger the appropriate ecophysiological mechanisms that help the bacteria to adapt better to sources of stress in the environment. Sometimes, a phage infection triggers a high level of cellular organization to prevent the host cells from being exposed to the sources of stress in the outside environment (  Yu et al., 2015). As discussed earlier, these genera are also important in bioremediation and in promoting plant growth. Filamentous phages therefore have the potential to improve the ecological and evolutionary potential of bacterial inocula so that the bacteria survive environmental stress, evolve in the changing environment, and contribute to the growth of plants (Figure 3).

| Phages and microbial adaptation
Filamentous phages influence the growth of their bacterial hosts to increase the adaptive potential of the hosts ( Such phage-mediated phenotypic and ecophysiological changes in bacterial hosts are immensely useful in ecorestoration. These changes in bacterial inocula will help the bacterial hosts to adapt to stress from abiotic sources, to maintain effective bacterial populations, and to perform their desired ecological functions (Arora, F I G U R E 5 Potential significance of filamentous phages to develop efficient biomonitoring system for tracking and management of targeted inoculated strain, pathogens, and contaminants Tiwari, & Singh, 2014;Gopalakrishnan et al., 2015;Lahav, 1962;Malusá, Sas-Paszt, & Ciesielska, 2012).
Other evidence shows that filamentous phages enhance the adaptive potential of bacterial hosts by influencing specific phenotypic traits or biological processes (Table 5). In E. coli HB11, infection from fd phage increases the total lipid content, which helps the host to resist freeze-fracture stress (Bayer & Bayer, 1986  Besides these mechanisms, filamentous phages also trigger highly structured organization of bacterial populations or communities (biofilm, for example), which protects their members from several sources of environmental stress ( Table 5)

: Suspensions of
Pseudomonas aeruginosa cells infected with Pf filamentous phage become more viscous, which helps the host cells to aggregate and adhere together to form a biofilm, which enables the assembly to survive desiccation and offers protection from aminoglycoside antibiotics and toxic chemicals (Secor et al., 2015;Webb et al., 2004).
A biofilm consisting of multiple species is a cross-species communication network that enables the constituent species to use nutrients including C more effectively when they are in short supply (Flemming et al., 2016). Based on the evidence discussed here, we suggest that (a) filamentous phages of target bacteria be isolated and analyzed to develop ecologically competitive bacterial inocula and (b) bacteria used in commercial inocula and potential PGPR be used as hosts to isolate suitable filamentous phages and the adaptive potential of such phages-bacteria co-inoculation be tested.
Infection of E. coli (K38, GM1, JM1) and V. cholerae by filamentous phages not only increases the ability of the hosts to tolerate toxins but also makes the hosts resistant to infection by other (homo-or heterologous) phages. In E. coli, infection by f1 filamentous phage leads to irreversible changes in the membrane protein that serves as a common receptor for colicin and phages (Table 5; Boeke et al., 1982;Sun & Webster, 1986;Zinder, 1973). Therefore, we propose the use of filamentous phages to develop ecologically competitive bacterial inoculants that can tolerate toxins and resist other phages in the soil. In E. coli K38, infection by filamentous phage triggers a phenotypic change in the membrane, which makes it more sensitive to deoxycholate, promotes leakage of β-lactamase, and increases the number of defective pilli on the host cell. Due to these changes, E.
coli K38 tolerates colicins but shows a reduced frequency of conjugation (Boeke et al., 1982). Cells of Vibrio cholerae infected with CTXф develop heteroimmunity against lambdoid phages and show a divergence in phage repressors and their cognate operators (rstR-og-2; Kimsey & Waldor, 1998). These changes confer a competitive advantage on the infected cells in countering attack by other bacterial species (Davies & Davies, 2010;Feldgarden & Riley, 1998). Thus, filamentous phages have the potential to protect their hosts from biotic sources of stress as well, both chemical and viral, which are common features of the ecosystem in which the inoculants find themselves.

| Phages to improve colonizing abilities of bacteria
Bacterial inocula used in remediation should not only survive, by competing successfully with other microbes, but also thrive, colonizing the contaminated sites to provide the desired ecological benefit.
In these bacterial hosts, filamentous phages either carry the virulence gene(s) (Addy et al., 2012b;Derbise & Carniel, 2014;Waldor & Mekalanos, 1996) or regulate the expression of virulence factors (Addy, Askora, Kawasaki, Fujie, & Yamada, 2012a, b;Ahmad et al., 2014). For example, N. meningitidis and Y. pestis are transformed into virulent strains capable of causing epidemics after receiving the toxin gene from Nf or MDA and from YpfΦ phage, respectively (Bille et al., 2005;Derbise et al., 2007). The filamentous phage CTXΦ transfers to V. cholerae O395 the gene ctxAB, which encodes the cholera toxin (Waldor & Mekalanos, 1996). Other strains of V. cholerae, namely N16961 and 395, develop into potential pathogens after they receive the gene vibrio pathogenicity island (VPI) from the filamentous phage VPIΦ (Li et al., 2003): That potential is realized if a helper phage, namely fs2, infects V. cholerae O1 thereby converting the host into a virulent strain. The phage-mediated transfer of rstC gene into V. cholerae O1 increases the production of the cholera toxin and triggers the multiplication of the resident CTXΦ phage, thus making the host highly virulent (Nguyen et al., 2008).
Filamentous phages can also convert pathogens into their superinfective forms by changing the phenotypic traits associated with virulence. For example, Xf2 infection converts Xanthomonas campestris pv oryzae N5850 into a highly virulent phytopathogen by increasing the production of extracellular polysaccharide (Kamiunten & Wakimoto, 1981). Virulent pathogenic variants of X. campestris pv citri evolve after infection by CF1c phage (Kuo et al., 1991), and infection by the phage PE226 transfers into Ralstonia solanacearum SL341 the genes responsible for producing toxins and thus widens the host range of the pathogen (Askora, Kawasaki, Usami, Fujie, & Yamada, 2009). Similarly, Pseudomonas aeruginosa PAO1 evolves into a superinfective phenotype after infection with the phage Pf4 (Rice et al., 2009;Webb et al., 2004).
Other plant-associated bacteria, such as E. coli (Bayer & Bayer, 1986), Enterobacteria (Kuo et al., 2000), P. aeruginosa (Secor et al., 2015), and R. solanacearum (MAFF106603 and MAFF106611; C319 and Ps29), also evolve into infective phenotypes if they are infected by their specific filamentous phages (Addy et al., 2012b;Yamada et al., 2007;  shown that these bacteria are also native soil bacteria, which promote plant growth and remediate the environment. For example, E. coli has been reported from soils from seven geo-climatic zones of India, and inoculating Zea mays with E. coli enhances nutrient uptake and plant growth (Nautiyal & Shono, 2010). In fact, plants exert niche-specific selection pressure on the organisms associated with them; for example, strains of E. coli associated with plants are a distinct phenotype and make an independent phylogroup different from that purified from mammalian hosts (Méric, Kemsley, Falush, Saggers, & Lucchini, 2013 Filamentous phage-infected P. aeruginosa shows high potential to colonize different habitats because of its ability to form biofilms, which have a liquid crystalline organizational structure (Rice et al., 2009;Secor et al., 2015). The biofilm is not an inert structure but a cooperative and interactive network that develops into an ecologically cohesive microbial community, which can rapidly colonize the target site (Hengzhuang, Wu, Ciofu, Song, & Høiby, 2011, 2012Høiby et al., 2011;Høiby, Bjarnsholt, Givskov, Molin, & Ciofu, 2010). In fact, Pf1-infected P. aeruginosa PAO1 strains outcompete the noninfected strains in forming a biofilm and also form a cohesive group for exchanging genes-exchanges from which the noninfected strains are excluded (Whiteley et al., 2001). Such gene exchanges within a biofilm help the bacterial hosts to develop into superinfective phenotypes that can adapt to and colonize new surfaces effectively (Rice et al., 2009;Webb et al., 2004). Lytic phages specific to native bacteria can also create a niche for the bacterial inocula (Kuykendall & Hashem, 1998;van Elsas & van Overbeek, 1993). Therefore, a consortium of lytic phages, filamentous phages, and the target bacterial inocula should be tested for improved colonization by the bacterial inocula of the contaminated site (Figures 3, 4).

| Phages to promote community assembly and evolution
A bacterial strain with high genetic stability confers desirable benefits in terms of plant growth and soil health after inoculation; however, successive generations of the strain should also have the ability to diversify and adapt to the changing environment (Sharma, Mishra, Mohmmed, et al., 2011). Filamentous phage may promote genetic stability as well as genetic diversity in a bacterial population depending upon the relative proportions of the filamentous phages and their bacterial hosts.
Environmental stress promotes genetic instability in bacteria; as a result, desirable bacterial phenotypes progressively disappear from the population (Mohmmed, Sharma, Ali, & Babu, 2001;Rau et al., 2009;Sharma et al., 2005;Sharma, Mishra, Mohmmed, et al., 2011;Sharma, Mishra, Rau, & Sharma, 2011). Plasmids, which are extrachromosomal genetic elements, carry environmentally relevant gene(s) in bacteria and help them to adapt to specific niches or confront environmental challenges. Often, the desired bacterial phenotype is lost after inoculation because the plasmid that encodes ecologically competitive gene(s) is lost (Bergstrom, Lipsitch, & Levin, 2000;Hall et al., 2015;Hall, Wood, Harrison, & Brockhurst, 2016;Harrison & Brockhurst, 2012;Mohmmed et al., 2001). At a high phage-to-bacterium ratio, infection by the filamentous phage inhibits conjugation in E. coli population (K38 and TOP10F) and ensures genetic stability (Boeke et al., 1982;Lin et al., 2011;Table 5). However, the filamentous phage and the F − recipient bacterial cells compete for a common receptor (pilus) of F + donor bacterial cells. Therefore, the ratio of phage to F − recipient bacterial cells determines which of the two events will be more frequent: infection of F + donor bacterial cells by the filamentous phage or conjugation between F + and F − bacterial cells (Novotny, Knight, & Brinton, 1968;Ou, 1973;Wan & Goddard, 2012). The role of filamentous phages in preventing conjugation (Lin et al., 2011;Wan & Goddard, 2012) shows their potential in ecorestoration to maintain genetic stability of bacterial inocula. In fact, the role of protein g3p of a filamentous phage in hindering conjugation process has already been demonstrated. Although a filamentous phage infects and persists within its bacterial host, regular reacquisition by the host may be required to maintain such infected bacteria in a population in sufficient numbers. Also, prior knowledge of the right ratio of filamentous phages to bacterial cells that ensures genetic stability in target bacteria is a prerequisite to using filamentous phages as co-inoculants.
Independent studies on E. coli strains have confirmed the potential of M13 filamentous phage to trigger genetic heterogeneity in an isogenic population of E. coli (De Paepe et al., 2010) or to maintain genetic homogeneity in E. coli W6 population (Wan & Goddard, 2012; Table 5). Using quantitative analysis, Lin et al. (2011) showed that as the M13 filamentous phage-to-E. coli ratio increases, the conjugation frequency decreases: A lower ratio favors conjugation and gene exchange, whereas a high ratio lowers the frequency of conjugation. We suggest that strains of bacterial inoculants be examined for the filamentous phages associated with them and the optimum ratio of filamentous phages to their bacterial hosts be determined for triggering gene exchange and genome diversification in the host population.
However, it is important to ask a fundamental question: Are the filamentous phages that trigger genetic stability or promote genetic diversity in bacterial populations different for different bacterial species or is the difference due to the relative proportions of phages and bacteria? To answer this question, we must isolate filamentous phages from different bacterial genera or species from the natural environment and then analyze the impact of their relationships on the ecology of the bacterial hosts using well-designed laboratory studies. We may use bacterial species with known filamentous phages for identifying the optimal ratio of phages to bacteria for genetic stability and that for genetic diversity in bacterial populations. Such studies will guide in situ genetic engineering of bacterial inocula. Figure 6 outlines a suggested path of research for developing ecologically competitive phage-bacterium inocula based on the ecological impact of the phages-to-bacteria ratio.

| Phages as sensors
The success of restoration efforts depends on the tools available for detecting and controlling pathogens and contaminants. To evaluate and guide restoration, restoration ecologists monitor inocula, pathogens, and contaminants in time and space (Felici et al., 2008;Harvey, 1993;Lynch et al., 2004;van Elsas, Duarte, Rosado, & Smalla, 1998). However, to monitor multiple contaminants and pathogens in degraded environments, we need different physicochemical and biological methods. Because we employ a consortium of bacterial strains and species to tackle multiple contaminants and pathogens, monitoring a consortium (multiple targets) for survival, colonization, and performance also warrants the use of an array of biological methods.
Compared to the conventional monitoring tools, those based on filamentous phages can be more easily tailored for diverse targets and even for detecting a target when it is present in ultralow levels and that too in real time ( Figure 5). Tracking of organisms (bacteria, viruses, etc.) and biological materials (spores, toxins, proteins, and DNA) relies on different methods, which may be (a) microbiological (culture, colony counting, chemical and biological plate assays), be reused by regenerating the receptor surface, which makes them ideal for environmental use (Rakonjac et al., 2011;Singh, Poshtiban, & Evoy, 2013). In restoration ecology, such biosensors may serve as bioindicators (specific to bacteria, viruses) or as biomarkers (specific to pathogen-specific biomolecules such as DNA and protein or to a specific biological activity). Because they are robust, such biosensors may track different biomaterials in complex environmental samples both in vivo (on plant surfaces and inside tissues) and in vitro (Table 5).  (Huang et al., 2007;Lee, Song, Hwang, & Lee, 2013); an ME-filamentous phage biosensor also detected bacterial pathogen in ultralow numbers (S. typhimurium: 50 cfu/ml) on the surface of tomato (Li, Johnson, et al., 2010;; and another ME biosensor with modified JRB7 detected spores of B. anthracis (10 3 spores/ml) in vitro.
Electrochemical biosensors have used filamentous phages to detect chemical changes in a cellular environment: The sensor phages display target-specific peptides, which detect and report the analyte as a change in current (amperometric sensor), impedance (impedance sensor), and voltage potential (light-addressable potentiometric sensor). Modified M13 helper phage expresses an electrochemically active reporter, such as alkaline phosphatase, at the surface; the reporter measures the current flow (oxidation-reduction reaction) and detects the signal in an amperometric sensing system. An amperometric electrochemical biosensor with modified M13 detected E. coli TG1 at concentrations as low as 1 cfu/ml by monitoring the activity of the reporter enzyme (Neufeld, Mittelman, Buchner, & Rishpon, 2005). A filamentous phage-based imaging system also detects pathogen-related chemical changes (acid-base homeostasis) in optically diffuse tissue (Hilderbrand, Kelly, Niedre, & Weissleder, 2008); for example, phage M13 was modified to ligate a pH-responsive cyanine dye (HCyc-646) to pVIII and thus developed into a ratiometric probe. The engineered phage-based sensor can also use impedance spectroscopy, an electrochemical technique, for such applications.
These methods are highly sensitive and easy: For example, a biosensor with engineered M13 filamentous phage covalently attached to a gold electrode measures electrical impedance over a wide range of frequencies (in kHz) and can detect ~120 nanomolar prostatespecific membrane antigen at signal-to-noise ratios greater than 10.
Filamentous phage-based light-addressable potentiometric sensors (LAPS) represent another such biosensor, which is composed of a semiconductor-insulator base activated by directed light pulses.
These label-free biosensors detect target enzyme activity or cellular pH, redox condition, or ion gradients, and LAPS have proved flexible enough to modify covalently with as many as four different phages.
Optical sensors, which rely on either spectrometry-based or resonance-based sensing, have the potential to detect target biomolecules at ultralow levels. Spectrometry-based methods such as UV/Vis spectrometry, bio/chemiluminescence, fluorescence or phosphorescence spectrometry, and infrared spectrometry measure changes in intensity at a particular wavelength whereas resonancebased methods such as surface plasmon resonance (SPR), fluorescence resonance energy transfer (FRET), and colorimetry measure changes in chemical properties upon a change in the wavelength.
Opto-fluidic ring resonator (OFRR) integrates microfluidics and photonic sensing technologies to develop an ultrasensing platform for detecting the target at ultralow levels (as low as nanoliters or at concentrations of 10 pg/mm 2 ). Low cost, high sensitivity, and good reusability of filamentous phage-based OFRR biosensors make them a promising platform for detection of biomolecules in the environment (Table 6): An OFRR (a lab-on-a-chip device) comprising immobilized phage R5C2 on silicon microfluidics detected targeted protein/DNA at picomolar levels in real time Zhu, White, Suter, Zourob, & Fan, 2008). label-free polymer-nanowires-based FET transistors or chemiresistors as biosensors detected bacterial pathogens even at 1 cfu/ml and phages at 10 3 pfu/ml in untreated environmental samples (Lee et al., 2013 φXacF1-infected X. axonopodis pv. citri strains are characterized with lower levels of extracellular polysaccharide production, reduced twitching motility, slower growth rate, and a dramatic reduction in virulence. X. axonopodis pv. citri MAFF673010 and MAFF301080 Extreme reduction in virulence, failed to cause citrus canker in lemon even after 4 weeks of post-infection Ahmad et al. (2014) TABLE 6 (Continued) highly specific and highly sensitive, can be mass-produced cheaply, and can withstand harsh environments.

| Phages for biological control
Quick detection and control of phytopathogens facilitate plant recruitment and restoration of vegetation (Al-Karaki, 2013;Barea, 2015;Bashan, 1998;Sharma et al., 2005). Pseudomonas (P. putida, P. aeruginosa), E. coli (Hagens & Bläsi, 2003), and Ralstonia solanacearum (Yamada, 2013) (Goodridge, 2010;Lu & Koeris, 2011;Viertel, Ritter, & Horz, 2014) and also prevents undesirable changes in the structure and functioning of the microbial community due to the release of toxic waste from the lysed pathogenic cells. For example, a modified M13 phage delivers "addiction toxin" genes (Gef and ChpBK), which triggers programmed cell death in target bacteria in vivo (Table 6). Such "suicide" systems have shown their potential to control some environmentally significant bacteria, namely Pseudomonas (P. putida, P. aeruginosa) and E. coli (Hagens & Bläsi, 2003). Phage M13 has been modified to encode restriction endonuclease (BglII) for killing the target bacteria with efficiency comparable to that of lytic phages. Therefore, modified filamentous phages not only control the target bacteria but also minimize the risk of the toxins being released into soil. Considering these benefits, we believe that filamentous phage-based biocontrol of pathogens will also add to the efficacy of bacterial inocula in restoration of vegetation.  (Mao et al., 2010; (Yamada, 2013). Ecologists have also been interested in φRSM because R. solanacearum is a serious threat to phytorestoration programs (Zhang et al., 2017), for example to programs to grow tobacco for ecorestoration of nutrient-deficient and contaminant-rich soils.
In fact, degraded soils show greater abundance of not only R. solanacearum but also of pathogenic members of Pseudomonas, Erwinia, and Xanthomonas. Controlling R. solanacearum is a challenge because it survives as a latent infection in indigenous weeds for many years (Hayward, 1991;Wenneker et al., 1999). However, it would also be useful to isolate and engineer filamentous phages of other pathogenic bacteria and test the potential of these phages to control the pathogens of other wild plants. Such use of filamentous phages to lower pathogenicity, virulence, and spread of dreadful phytopathogens may mark a new milestone in restoration programs.
The increasing numbers of bacterial genera discovered to have an association with filamentous phages and the ease with which the phage genome can be modified to produce antimicrobial peptides represent an untapped but most promising opportunity for biocontrol of pathogens. Using suitably modified filamentous phages along with other biocontrol agents can revolutionize the integrated management of phytopathogens. However, these phages are at times unstable in their host populations, even when they do not result in the evolution of host resistance (Lerner & Model, 1981). This characteristic, and the loss of filamentous phages during molecular applications (Mai-Prochnow et al., 2015), is obstacles to their use as inhibitors of pathogenic bacteria.  Daniell, Davy, and Smith (2000) affect members of Ralstonia are useful in controlling phytopathogenic strains of the bacteria and in improving the efficacy of endophytic bacterial strains used in phytoremediation.

| Phages for remediation of contaminated sites
Restoration ecologists use microbial technologies for remediation of contaminated sites and for restoring ecosystem goods and services (UNCCD, 2013), and filamentous phages have been engineered to detect contaminants (physical, organic, and inorganic) and to remove or to reduce them in the environment. To assess the success of such efforts, it is necessary to examine the environment for the presence of such contaminants (Brown, 1997;Henry et al., 2015;Viertel et al., 2014; Table 3).
Phages display specific peptides on phage coat proteins (pVIII and pIII), which have been used for detecting, binding to, or decomposing the contaminants (Henry et al., 2015;Nambudripad, Stark, & Makowski, 1991;Petrenko & Makowski, 1993;Thiriot, Nevzorova, & Opella, 2005). Display technology can screen billions of potential toxicants and help in developing fusion phages that retain not only their infectivity and immunogenicity but also their degradation ability (Lerner, Benkovic, & Schultz, 1991). Such properties of modified phages make them potential co-inoculants with the host bacteria to extract specific pollutants from the environment. For remediation of contaminated soils, phage inoculation thus represents a cost-effective method of setting up a factory in situ to produce large quantities of contaminant-specific biomolecules for remediation of the soil environment. This environment-friendly approach may not be a permanent solution for bioremediation but it is worthwhile to test its potential to reduce the concentration of contaminants in the environment.
Modified filamentous phages also offer another method of separating and processing minerals (bioleaching or biomining) for restoring abandoned mines. For example, modified phages show specificity and selectivity in binding to environmental sources of sphalerite (ZnS, a major ore of zinc) and chalcopyrite (CuFeS 2, a major ore of copper; Table 7). These phages separate sphalerite efficiently despite the presence of such natural contaminants as silica (a waste mineral) and pyrite (Curtis et al., 2009). The modified phages can potentially mine minerals and metals selectively from natural ore and remediate abandoned mines by acting on waste and industrial scrap and thus contribute to economic and ecological security.
Filamentous phages VP12 and VP14 have been modified to display a specific peptide (DSQKTNPS on pVIII) that sequesters physical pollutants (fine silt and clay particles; Curtis et al., 2011;Curtis et al., 2013). In fact, the mechanism by which the peptide binds to F I G U R E 6 Outline of multi-branched course of research proposed with a cohesive vision for future actions for environmental application of filamentous phage (i) for improving inoculants for plant growth, (ii) for engineering filamentous phage for biosensing and bioremediation, and (iii) for preventing potential risks of filamentous phage application in the environment. FP, filamentous phage chalcopyrite has also been elucidated: The two phages sequester on their surface particles smaller than 45 µm in diameter under a wide range of pH (3-11) and cation concentrations. The potential of such phages to aggregate soil particles and thereby prevent erosion can be tapped as a novel strategy in soil management.
Phages have also been modified to display peptides (12-mer) specific to organic contaminants, namely 2,4,6-trinitrotoluene (TNT) and 2,4,6-trinitrobenzene (TNB; Table 7). Such modified phages can detect the target organic contaminants at ultralow levels (10 ng/ ml) even in heterogeneous environmental samples (Goldman et al., 2002). Modified M13 phages displaying antibodies detected TNT and its derivatives even at concentrations of 1 ng/ml (Goldman et al., 2003). The modified phages displaying specific antibodies are highly selective and sensitive and can detect even minute quantities of such contaminants as morphine at ultralow levels (5 ng/ml within 2 min) in environmental samples (Pulli et al., 2005). Therefore, co-inoculation of modified phages with plant growth-promoting bacteria will serve as a supplementary biotechnology to sequester and detoxify targeted contaminants and to speed up revegetation of degraded lands.

| PRI ORIT Y ARE A S OF RE S E ARCH TO MINIMIZE THE P OTENTIAL RIS K TO THE ENVIRONMENT FROM THE APPLIC ATI ON OF FIL AMENTOUS PHAG E S
So far, filamentous phages have been presented in a positive light.
However, as with invasive species and transgenic organisms, the potential risks from introducing filamentous phages that are foreign to degraded soils must not be overlooked. Even if a phage is native to the soil, phage inoculation may disturb the phage-to-bacteria ratio, which is crucial to many biological processes in soil (Meaden & Koskella, 2013;Reyes et al., 2010). Because bacterial inoculation aims to restore adversely affected soil processes in degraded lands, the risk from using modified or natural filamentous phages is minimal (Sharma et al., 2015;Sharma, Mishra, Mohmmed, & Babu, 2008;Sharma, Mishra, Rau, et al., 2011). Based on a cost-benefit analysis, we maintain that the benefits of filamentous phages outweigh the risks from deploying them in damaged ecosystems ( Figure 6). Also, the impact of inoculation with filamentous phages is hard to predict unless we have prior knowledge of their host range and the proportion of filamentous phage-infected bacteria in bacterial populations.
Based on the low density and diversity of bacterial communities in degraded environments, we believe that the immediate benefits of phages outweigh the possible risks (Figures 5and 6).
Expansion of the host range of nonnative or modified filamentous phages and the activation of unknown silent phages in bacterial populations in inoculated soils are other potential risks from phage application. Filamentous phages are highly host-specific and rarely extend their host range to include other strains, species, or genera (Hyman & Abedon, 2010;Piekarowicz et al., 2014). To preclude the possible risk of filamentous phages serving as helper phages for the multiplication of satellite phages (Rakonjac et al., 2011), it is essential to conduct prior experiments with native soils (Figure 6).
Alternatively, to limit the replication of nontargeted phages in soil, we may load the useful genes from filamentous phages into a suicide vector (Addy, Askora, Kawasaki, Fujie, & Yamada, 2014;Huber & Waldor, 2002;Martínez & Campos-Gómez, 2016;Pant et al., 2015). To ascertain the possible impact of introduced phages on the functioning of microbial communities, we need to generate relevant knowledge using ecologically relevant laboratory-and field-based studies in a multi-species environment (microcosm or mesocosm).
However, in the environment, selection may work against the multiplication of such mutants. Bacteria with mutated pili develop resistance to filamentous phages but show reduced fitness. It may be noted that the pilus helps a bacterium to adhere to suitable objects, move, colonize, and spread into the environment. At the same time, the phages-to-bacteria ratio affects the frequency of conjugation in bacterial populations. Therefore, we propose that the population dynamics of filamentous phages and their bacterial hosts in a given environment be examined before deploying filamentous phages for remediation and the impact of population dynamics on the frequency of conjugation and on the functioning of host bacteria be estimated to develop strategies for the use of filamentous phages.
Finally, we need to consider the costs and benefits of the environmental use of filamentous phages based on in vitro studies, which are characterized by a stable environment with few limiting factors.
It is possible that the benefits will be lower in the fluctuating natural environments with many limiting factors. However, even marginal improvements in the efficacy of bacterial inoculants due to filamentous phages are worthwhile and particularly important in restoring degraded lands. The costs and benefits are context dependent. The effect of a phage on the bacterial cell is likely to have bottom-up consequences on bacterial populations, on microbial communities, and on other associated organisms (plants and animals). Such effects of phage inoculation on interactions between organisms will have ecological costs in terms of its impacts on other functional groups of microbes such as associative cooperators, competitors, and predators. Therefore, we suggest that the ecological cost of the application of filamentous phages be estimated and used as the foundation for developing suitable methods for co-inoculation with bacteria and filamentous phages ( Figure 6).

| CON CLUS IONS
Theories related to the ecology of soil microbes have guided microbe-assisted environmental restoration programs. Applying such theories to phage-bacterium interactions will improve microbial inoculation technologies for revegetation of degraded lands. Growing knowledge of the ecology of filamentous phages of different bacterial genera in diverse environmental settings shows the potential of phages as an emerging bioresource suitable for environmental applications. Co-inoculation of bacteria with filamentous phages will increase the ecological and evolutionary potential of microbial communities in degraded lands because filamentous phages will help bacterial inocula to colonize degraded sites subjected to multiple abiotic and biotic sources of stress. Further, the ease with which the genome of filamentous phages can be manipulated to express a range of peptides and proteins makes such phages a real-time sensing tool for environmental restoration. Sensors based on filamentous phages can detect inocula, pathogens, and contaminants in environmental samples at ultralow levels and will not only contribute to more informed decisions related to restoration but also save time and resources. We recommend that restoration ecologists exploit filamentous phages to enhance the ecophysiological capabilities of host bacteria and to find new filamentous phages and understand their ecological relevance. Researchers in environmental remediation will benefit from studying the ecology of filamentous phages to shape bacterial populations for a given purpose; in turn, the exercise of shaping bacterial populations will help the researchers to understand the ecology of phage better. The priority research areas identified in this review will help to realize the potential of phagebacterium co-inoculation in environmental restoration and, at the same time, minimize the possible risks from deploying phages. Such use of filamentous phages can usher a tectonic shift in the science and practice of ecorestoration.

ACK N OWLED G M ENTS
We acknowledge the anonymous reviewers and editors for their critical comments and constructive suggestions that helped us to improve the manuscript. We thank Yateendra Joshi and R. Geeta for copy-editing the MS. We acknowledge Inderjit Singh for his critical comments and constructive suggestions. We thank Aakansha Tyagi in extending support while copy-editing the MS. We also Fellowship.

CO N FLI C T O F I NTE R E S T
Authors declare no conflict of interest.

AUTH O R ' S CO NTR I B UTI O N
Conceived the hypothesis: RSS, VM, SK. Analyzed the literature/ data: SK, VM, RSS, PK. Wrote the paper: RSS, SK, VM, PK.

DATA ACCE SS I B I LIT Y
All the data used for developing the proposals and recommendations presented in this paper were sourced from published studies, and appropriate references are provided.