water research 46 (2012) 4902e4917

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Review Plant viruses in aqueous environment e Survival, water mediated transmission and detection

Natasa Mehle a,b, Maja Ravnikar a,b,* a National Institute of Biology, Vecna pot 111, 1000 Ljubljana, Slovenia b Centre of Excellence for Biosensors, Instrumentation and Process Control, Velika pot 22, 5250 Solkan, Slovenia article info abstract

Article history: The presence of plant viruses outside their plant host or insect vectors has not been Received 16 April 2012 studied intensively. This is due, in part, to the lack of effective detection methods that Received in revised form would enable their detection in difficult matrixes and in low titres, and support the search 12 July 2012 for unknown viruses. Recently, new and sensitive methods for detecting viruses have Accepted 15 July 2012 resulted in a deeper insight into plant virus movement through, and transmission Available online 23 July 2012 between, plants. In this review, we have focused on plant viruses found in environmental waters and their detection. Infectious plant pathogenic viruses from at least 7 different Keywords: genera have been found in aqueous environment. The majority of the plant pathogenic Plant virus viruses so far recovered from environmental waters are very stable, they can infect plants Water via the roots without the aid of a vector and often have a wide host range. The release of Survival such viruses from plants can lead to their dissemination in streams, lakes, and rivers, Transmission thereby ensuring the long-distance spread of viruses that otherwise, under natural Irrigation conditions, would remain restricted to limited areas. Hydroponic The possible sources and survival of plant viruses in waters are therefore discussed. Due to the widespread use of hydroponic systems and intensive irrigation in horticulture, the review is focused on the possibility and importance of spreading viral infection by water, together with measures for preventing the spread of viruses. The development of new methods for detecting multiple plant viruses at the same time, like microarrays or new generation sequencing, will facilitate the monitoring of environmental waters and waters used for irrigation and in hydroponic systems. It is reasonable to expect that the list of plant viruses found in waters will thereby be expanded considerably. This will emphasize the need for further studies to determine the biological significance of water-mediated transport. ª 2012 Elsevier Ltd. All rights reserved.

Contents

1. Introduction ...... 4903 2. The origin and presence of plant viruses in environmental waters ...... 4903 3. The survival of plant viruses in waters ...... 4904 4. Infection of plants with contaminated water ...... 4907

* Corresponding author. National Institute of Biology, Vecna pot 111, 1000 Ljubljana, Slovenia. Tel.: þ386 59 232 801; fax: þ386 1 25 73 847. E-mail address: [email protected] (M. Ravnikar). 0043-1354/$ e see front matter ª 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.watres.2012.07.027 water research 46 (2012) 4902e4917 4903

5. Water as an effective route for spreading of viruses ...... 4907 5.1. The spreading of viruses in irrigation systems by water or nutrient solutions ...... 4908 6. Preventing plant viruses from spreading with water ...... 4909 6.1. The elimination of plant viruses from water ...... 4909 7. Methods for detecting plant viruses in water ...... 4910 7.1. Concentration ...... 4910 7.2. Biological assays ...... 4911 7.3. Detection of viral protein ...... 4911 7.4. Observation of viral particles ...... 4911 7.5. Detection of nucleic acids ...... 4911 7.6. Future development of methods for monitoring plant viruses in water ...... 4912 8. Conclusions ...... 4912 Acknowledgements ...... 4913 References ...... 4913

1. Introduction water-transmissible plant pathogens throughout the whole crop, increasing the chance of epidemics if not managed In certain cases, such as floods, earthquakes, pollution intensively (Stanghellini and Rasmussen, 1994a; Schnitzler, (Szewzyk et al., 2000; Casteel et al., 2006; Abraham, 2011) and 2004; Stewart-Wade, 2011). Plants will be repeatedly inocu- terrorist attacks (Khan et al., 2001; Gu¨ nter, 2006; Gleick, 2006; lated with viruses, independently of whether the initial source Costigliola and Quagliata, 2010; Ping, 2010; Knutsson et al., of water harbours the viruses or whether they enter the water 2011), water can be an important source of human, animal along the path of distribution. Therefore, the risk of dissemi- and plant infection by pathogenic microbes. The pollution of nation and required management of plant viruses, often environmental waters (for different definitions of term ‘envi- resulting in crop losses, has to be evaluated before recircu- ronmental waters’ see Hamstead (2007)), such as rivers, lakes, lating used water (Bandte et al., 2009). sea, tap and irrigation water, with human and animal viruses In this review we will discuss the presence and survival has received considerable attention (see reviews, e.g., Rudolfs of plant viruses in aqueous environments, the possibility of et al., 1950; Gerba and Schaiberger, 1975; Labelle and Gerba, water mediated transmission, how to minimize the risk of 1980; Szewzyk et al., 2000; Griffin et al., 2003; Scott et al., viruses spreading through irrigation systems, and the 2004; Toze, 2006; Reynolds et al., 2008). It is only 30 years methods adapted for detecting plant viruses in the aqueous since plant viruses were first shown to be present in envi- environment. Our objectives are to highlight the knowledge ronmental waters in considerable amounts (Koenig, 1986). that is the basis of our current understanding of plant viruses Since then, many questions have been raised concerning the in water systems and to emphasis the need for additional survival and spread of plant viruses by water, especially in the research to attack this issue of global importance. light of increased irrigation and use of hydroponic systems in agriculture. Agricultural irrigation consumes enormous quantities of 2. The origin and presence of plant viruses water; in developing countries, irrigation often exceeds 80 in environmental waters percent of total water use (Riley et al., 2011). Irrigation water can be sourced from surface water supplies such as ponds, The first results of studies on plant viruses in environmental lakes, rivers, and reservoirs and, as such, can harbour disease- waters were reviewed by Koenig (1986). Their possible sources causing microorganisms, including several plant viruses. were proposed to be the roots of infected plants growing in an Additionally, crop production in soilless cultures, using open ecological niche near the water, injured or decaying plant or closed hydroponic systems, has been increasing world- material, and sewage. As discussed later, a number of plant wide. In a closed system, the nutrient solution is recovered, viruses present in vegetables or fruits can pass through the replenished, and recycled following its delivery to the root alimentary tract as infective virions, to be released into system. In an open system, the nutrient solution is not sewage water that could find its way into environmental replenished or recycled, although it may be recovered waters. Additionally, the surface wash-out of locally scattered (Stanghellini and Rasmussen, 1994a). Soilless culture has and infected decaying plant residues and of the related soil provided an alternative to plant growers who face soil-related surface layer, including animal faeces, could bring the plant problems such as nematodes, pathogens, and nutrient viruses into waters. Virus-containing seeds and virus- imbalance (Stanghellini and Rasmussen, 1994a; Schnitzler, transmitting nematodes, zoospores or, more so, resting 2004). Water is becoming more costly and harder to access sporangia of fungi, may also be washed away by running soil in many countries, therefore it is necessary to use its sources water or drainage water. more responsibly, e.g., use of recycled water for irrigation in The pioneer report on the release to drainage water of commercial plant nurseries and greenhouses. On the other Tobacco necrosis virus (TNV) and Tobacco mosaic virus (TMV) hand, the use of circulating nutrient solutions in hydroponic from infected roots of Cleome spinosa and tobacco plants, systems holds the potential for rapid and effective spread of respectively, is more than 50 years old (Yarwood, 1960). In 4904 water research 46 (2012) 4902e4917

1969, van Dorst reported viruses in irrigation waters (cited in Infectious TNV, a member of the genus Necrovirus, was Pares et al. (1992)). However, the first observations on the confirmed to be present in ditches, streams and canals in presence of plant viruses in rivers and lakes, date from the Germany (Koenig et al., 1989; Yi et al., 1992), in rivers Cam, 80’s, when only a few research groups in Europe were focused Thames, Avon and in its tributaries, and in Esthwaite Water in on the problem (Koenig, 1986). During the last ten years there England (Tomlinson et al., 1983). Infectious Cucumber mosaic has been an increase in reports on the presence of plant virus (CMV), a member of the genus Cucumovirus, was isolated viruses in waters. Infectious plant pathogenic viruses from at from waters of Italian river Bradano (Piazzolla et al., 1986). least 7 different genera (Carmoviruses, Cucumoviruses, Dia- Carnation ringspot virus (CRSV), a member of the genus Dia- thoviruses, Tobamoviruses, Necroviruses, Potexviruses and nthovirus, was recovered from several German ditches and Tombusviruses) have been found in canals, rivers, streams, streams and from Oker Aue canal (Yi et al., 1992; Koenig et al., ponds, lakes and oceans (Table 1). 1988, 1989). Tobamoviruses like TMV and Tomato mosaic virus (ToMV) are Apart from viruses known to be plant pathogens, novel known to be extraordinarily stable and widespread RNA infectious viruses from genus Carmovirus (Yi et al., 1992), viruses that infect many different plant species (Hollings and Potexvirus (Koenig and Lesemann, 1985; Bu¨ ttner and Nienhaus, Huttinga, 1976). TMV was recovered from the rivers Henne in 1989), Tombusvirus (Yi et al., 1992; Koenig and Lesemann, 1985; Germany (Koenig and Lesemann, 1985), Lato in Italy (Piazzolla Gallitelli et al., 1989; Koenig et al., 2004)andTobamovirus (Koenig et al., 1986), Danube and Sava in former Yugoslavia (Tosi c and and Lesemann, 1985) have been isolated from environmental Tosi c, 1984), and from two forest ditches and a brook in waters. Their natural hosts and importance in nature are Croatia (Plese et al., 1996). Infectious ToMV was isolated from unknown. They are usually named after the river or place waters draining forest stands in Croatia (Plese et al., 1996), in where first isolated, e.g., Sikte waterborne virus (SWBV), Ahlum Germany Northrhine-Westfalia (Bu¨ ttner and Nienhaus, 1989), waterborne virus (AWBV) and Weddel waterborne virus (WWBV) in central New York and in the high peaks region of the (Yi et al., 1992), Neckar river virus (NRV; Tombusvirus Neckar; Adirondack Mountains (Jacobi and Castello, 1991). Infective Koenig and Lesemann, 1985), Lato river virus (LRV) (Gallitelli ToMV virions were detected also in clouds and fog (Castello et al., 1989), Havel river virus (HaRV) (Koenig et al., 2004). et al., 1995). ToMV RNA was detected even in < 500 to Additionally, Pepper mild mottle virus (PMMoV) was detected approximately 140,000 year old glacial ice subcores from drill in seawater (Rosario et al., 2009b), river water (Hamza et al., sites in Greenland (Castello et al., 1999) which may be an 2011) and, finally, using new generation sequencing, many abiotic reservoir (Rogers et al., 2004). Since many European other RNA and also DNA of known or suspected terrestrial forests stand on what was once agricultural land, the viruses plant viral pathogens were confirmed to be present in marine recovered in forest trees, soils and waters may have originated environments (Culley et al., 2006), in a fresh water lake from infected crop residues (Fillhart et al., 1998). Viruses iso- ecosystem (Djikeng et al., 2009) and in reclaimed water lated in forest ecosystems with no prior history of agriculture (Rosario et al., 2009a). The biological significance of these may be indigenous or introduced by some other mechanism recent findings remains to be established. (Fillhart et al., 1998). Castello et al. (1995) proposed several possible routes for the spreading of ToMV, such as spreading, in clouds, from agricultural areas to the mountain forests and 3. The survival of plant viruses in waters from the forest ecosystem, via water, back to agricultural areas. It was therefore not unexpected to find ToMV in seven The majority of plant viruses recovered from environmental out of nine water samples of different origins collected across waters are stable (remain infectious). The longevity of the sap Slovenia (Boben et al., 2007). The samples were taken from infectivity in vitro (LIV) for ToMV, TMV, CRSV, TBSV, CaRMV different sources around the state: one from irrigation water and PVX is between 50 and 3000 days. However some of these in a ToMV-infested greenhouse, one from a water sample have been regarded as rather unstable viruses, according to from a gravel pit and five from rivers. their LIVs, e.g. between 7 and 28 days for TNV and between 1 Besides tobamoviruses, several other infectious plant and 10 days for CMV (data on LIVs are from the database of viruses that cause diseases in agricultural crops, e.g., potex- Brunt et al. (1996) and onwards). virus Potato virus X (PVX), tombusviruses Tomato bushy stunt Experimental conditions may account for differences in virus (TBSV) and Carnation Italian ringspot virus (CIRV), have survival of viruses in vitro. For example, the LIV for TMV from been isolated from waters draining the forest area database Brunt et al. (1996 onwards) is 3000 days; in contrast, Northrhine-Westfalia (Bu¨ ttner et al., 1987; Bu¨ ttner and Park et al. (1999) reported a longevity in vitro of only five days. Nienhaus, 1989). Infectious TBSV was also isolated from Yarwood (1960) demonstrated that TNV is inactivated more lakes and rivers worldwide (see review of Koenig (1986)). Other rapidly in a dry than in a humid environment. The half-life of members of the genus Tombusvirus, e.g. Petunia asteroid mosaic TNV in a water suspension was estimated at about 46 and virus (PeAMV) and Grapevine Algerian latent virus (GALV), were 80 h. There are only a few data on the survival of plant viruses recovered from German ditches and streams (Koenig, 1988; in water or nutrient solutions under greenhouse conditions. Koenig et al., 1989; Yi et al., 1992). PeAMV was also recovered ToMV and PMMoV were shown to remain infectious in from sea water taken 200 m off the north-eastern beach of the nutrient solution for at least six months, independently of Isle of Helgoland (Fuchs et al., 1996). Carnation mottle virus storage medium and temperature (Pares et al., 1992). In these (CarMV), a member of the genus Carmovirus, was recovered experiments, purified ToMV and PMMoV were suspended in from the German river Oker (Koenig and Lesemann, 1985) and nutrient solution or in 0.06 M phosphate buffer, to final was detected in Baltic Sea water (Kontzog et al., 1988). concentrations of 80 and 20 mgml 1 respectively, and stored in water research 46 (2012) 4902e4917 4905

a glasshouse and at 4 C. Infectivity was tested by observing greatly decreases the inactivation rate of human and animal symptoms on the leaves of mechanically inoculated test viruses in waters (Gerba and Schaiberger, 1975; Labelle and plants of Nicotiana glutinosa. Gerba, 1980). Adsorption to clay particles or organic matter Aggregation and/or adsorption to solid materials such as protects plant viruses against inactivation in water, probably clays and silicates, or to organic debris, bacteria, or algae, by physical or chemical interactions (Piazzolla et al., 1986;

Table 1 e Infective plant pathogenic viruses found in environmental waters, such as canals, rivers, streams, ponds, lakes and oceans. Plant viruses Country of Origin of water Concentration of Detection methods Reference virus finds sample water sample

Carmoviruses: Carnation mottle Germany River Oker Ultracentrifugation Biological assays, Koenig and virus (CarMV) (200e300 ml) followed by EM and Lesemann, 1985 immunodiffusion test Weddel waterborne Germany Ditches and streams Ultracentrifugation Biological assays, followed Yi et al., 1992 virus (WWBV), in agricultural areas by immuno EM, Ahlum waterborne immunodiffusion test, virus (AWBV) SDS-PAGE and Northern blot hybridization Cucumoviruses: Cucumber mosaic Italy River Bradano Centrifugation Biological assay, followed Piazzolla virus (CMV) at 5000 g (1 l) by immuno EM et al., 1986 Dianthoviruses: Carnation ringspot Germany Oker Aue canal; Ultracentrifugation Biological assays, followed Koenig virus (CRSV) (60 ml) by EM, SDS-PAGE, Northern et al., 1988 blot-hybridization, dot blot hybridization Germany Ditches and drainage Ultracentrifugation Biological assays, followed Koenig et al., 1989 canals in grapevine by immuno EM, growing area immunodiffusion test Germany Ditches and streams in Ultracentrifugation Biological assays, Yi et al., 1992 agricultural areas and in followed by EM and a stream which had no immunodiffusion test direct contact with fields Necroviruses: Tobacco necrosis England Rivers Cham, Thames, Adsorption of Biological assays, followed Tomlinson virus (TNV) Avon and its tributaries, pre-filtered water by immuno EM et al., 1983 (Chenopodium lake Esthwaite to adsorbent filter (60 l), necrosis virus) further concentration of eluents by ultracentrifugation Germany Ditches and drainage Ultracentrifugation Biological assays, followed Koenig canals in grapevine by immuno EM, et al., 1989 growing area immunodiffusion test Germany Ditches and streams in Ultracentrifugation Biological assays, Yi et al., 1992 agricultural areas followed by EM and immunodiffusion test Potexviruses: Potato virus X (PVX) Germany Ponds and creeks in a Ultrafiltration (50 l) Biological assays, Bu¨ ttner and forest area followed by EM and Nienhaus, 1989 (Northrhine-Westfalia) immunoprecipitation test Tobamoviruses: Tobacco mosaic Former River Danube and Sava Ultracentrifugation EM and biological assays, Tosi c and virus (TMV) Yugoslavia at 60,000 g (1 l of followed by Tosi c, 1984 filtered water) immunodiffusion test Germany River Henne Ultracentrifugation Biological assays, Koenig and (200e300 ml) followed by EM and Lesemann, 1985 immunoprecititation test Italy River Lato Centrifugation Biological assay, followed Piazzolla at 5000 g (1 l) by immuno EM et al., 1986 Croatia Two forest ditches and Ultracentrifugation Biological assays, followed Plese et al., 1996 one brook (250 ml)/centrifugation by immunodiffusion test, at 4000 g/no EM and light microscopy

(continued on next page) 4906 water research 46 (2012) 4902e4917

Table 1 e (continued) Plant viruses Country of Origin of water Concentration of Detection methods Reference virus finds sample water sample

Tomato mosaic Germany Ponds and creeks in Ultrafiltration (50 l) Biological assays, Bu¨ ttner and virus (ToMV) a forest area followed by EM and Nienhaus, 1989 (Northrhine-Westfalia) immunoprecipitation test USA Streams and lakes from Adsorption to EM and biological assays, Jacobi and forest stands in central Zeta Plus 50 S followed by EM, Castello, 1991 New York and in the membrane filter (20 l), immunodiffusion test, ELISA Adirondack Mountains further concentration of eluents by ultracentrifugation USA Cloud (summit of Ultracentrifugation ELISA, RT-PCR-blot Castello et al., 1995 Whiteface (30e290 ml) hybridization, sequencing, Mountain), fog biological assays (coast of Maine) Croatia Two forest brooks Ultracentrifugation Biological assays, followed Plese et al., 1996 (250 ml) by immunodiffusion test, EM and light microscopy Slovenia Gravel pit in Ivanci, rivers Concentration by RT-real time PCR, ELISA, Boben et al., 2007 Drava, Vipava and Krka using CIM monolithic biological assays, chromatographic immuno EM supports (1 l) (in some cases concentration of water sample was not necessary) Tombusviruses: Tomato bushy England Rivers Thames, Adsorption of Biological assays, Tomlinson et al., stunt virus (TBSV) Trent, Cam, pre-filtered followed by immuno EM, 1983; Tomlinson Esthwaite Water water to adsorbent immunodiffusion and and Faithfull, 1984; filter (60 l), immunoprecipitation tests further concentration of eluents by ultracentrifugation Germany River Elbe, Mosel Ultracentrifugation Biological assays, Koenig and (200e300 ml) followed by EM and Lesemann, 1985 immunodiffusion test Germany Ponds and creeks in a Ultrafiltration (50 l) Biological assays, Bu¨ ttner and forest area followed by EM and Nienhaus, 1989 (Northrhine-Westfalia) immunodiffusion test Carnation Italian Germany Ponds and creeks in a Ultrafiltration (50 l) Biological assays, Bu¨ ttner et al., 1987; ringspot forest area followed by EM and Bu¨ ttner and virus (CIRV) (Northrhine-Westfalia) immunodiffusion test Nienhaus, 1989 Petunia asteroid mosaic Germany Ditches and drainage Ultracentrifugation Biological assays, Koenig, 1988; virus (PeAMV) canals in a grapevine followed by immuno EM, Koenig et al., 1989 growing area immunodiffusion test Baltic sea 200 m off the northeastern Ultracentrifugation Biological assays, Fuchs et al., 1996 beach of the Isle (400 ml of filtered water) followed by serological test of Helgoland Grapevine Germany Ditches and streams in Ultracentrifugation Biological assays, Yi et al., 1992 Algerian latent agricultural areas followed by immuno EM, virus (GALV) immunodiffusion test, Northern blot hybridization Sikte waterborne Germany Ditches and streams in Ultracentrifugation biological assays, Yi et al., 1992 virus (SWBV) agricultural areas followed by immuno EM, immunodiffusion test, SDS-PAGE and Northern blot hybridization Neckar river Germany River Neckar Ultracentrifugation Biological assays, Koenig and virus (NRV) (200e300 ml) followed by EM and Lesemann, 1985 immunodiffusion test Havel river virus Germany River Havel, Ultracentrifugation Biological assays, Koenig et al., 2004 (HaRV) small stream (500 ml) followed by immuno EM, in a woody area RT-PCR and sequencing water research 46 (2012) 4902e4917 4907

Koenig, 1986). Piazzolla et al. (1986) suggested that sediments grew into SBMV-infected plants (Teakle and Morris, 1981). The exert a protective action on the relatively unstable CMV. The rate of infection was higher in seeds with cracked coats than strength of adsorption of plant viruses to inorganic or organic in those with intact coats. matter is expected to vary with different viruses, different Many plant viruses can infect plants through roots with the adsorbing materials and environmental conditions, such as aid of vectors, either nematodes or fungi. For several viruses, pH, salts, and the presence of other materials. Adsorption, however, vectorless transmission via the roots is the most desorption, and re-adsorption appear to occur continuously likely route (reviewed by Koenig (1986) and Mandahar (1990)). under natural conditions (Koenig, 1986). Virus infection of roots can occur even under sterile condi- The stability of several plant viruses, like that of several tions and is more effective in sand than in liquid media, human viruses, is extremely high (Carter, 2005; Reynolds suggesting that the viruses enter the plants through damaged et al., 2008). TBSV has been shown to retain its infectivity root hairs or small wounds that appear during root growth. after ingestion and subsequent exposure at 37 C to the The transmission rate of some viruses through roots can enzymes of the human alimentary tract, bile salts and wide increase in the presence of fungi. Transmission of Red clover fluctuations in pH (from about 3.0 to 8.8) (Tomlinson et al., necrotic mosaic virus (RCNMV) through roots has been observed 1982). Zhang et al. (2006) found 35 different plant viruses in to occur in the absence of fungus, but to increase in the human faeces, the most abundant being PMMoV. They presence of zoospores of Olpidium brassicae (Koenig, 1986). provided evidence that the faecal PMMoV was viable and Virus infections contracted through the roots are often could infect a host plant. Additionally, they demonstrated that restricted to the roots or, if not, become systemic in the shoots PMMoV can survive standard food processing, for example the only with delay and in unpredictable amounts. The infections manufacture of hot chilli sauce and powdered chilli. In their are occasionally symptomless, but may be more severe than study, the amount of faecal PMMoV appeared to be signifi- those initiated in leaves. cantly higher than the virus load found in food, based on a dry The infection rate and severity of the symptoms depend on weight basis. There is no evidence for active replication of the virus concentration in waters, as shown for ToMV (Pares PMMoV in human gut, but it is possible that this increase et al., 1992). Development of symptoms depends on environ- could be the result of the digestive reduction of food, e.g., mental factors such as temperature. In growth chamber there may be fewer inhibitors or released viral RNA may be experiments, the effects of temperature on root- or foliar- more easily accessed for the detection. Further, PMMoV was ToMV inoculated peppers were similar (Schuerger and detected in wastewater and in seawater samples collected Hammer, 1995). Moderate or severe symptoms failed to near point sources of secondary treated wastewater off south- develop on either root- or foliar-inoculated plants incubated at eastern Florida (Rosario et al., 2009b) and in river water in 18 C but were observed on those incubated at 24 Corat32C. Germany (Hamza et al., 2011). They suggested that PMMoV is a promising new indicator of faecal pollution in surface water. Viruses that are less stable may not persist “free” in waters 5. Water as an effective route for spreading or survive passage through the alimentary tract of vertebrates, of viruses but may survive in fungal resting spores or in seeds. Some viruses are known to survive in seeds. This can be particularly The majority of viruses so far been detected in environmental important for viruses that are hosted in annual plants, and for waters, such as carmo-, tombus-, diantho-, tobamo- and those using invertebrate vectors, such as nematodes, which potex-viruses, lack an aerial vector that would ensure their normally move slowly, therefore natural dispersal of virus- spread over long distances. They are however reproduced in infected seeds by wind or water may be a factor in easier infected plants in high concentrations and are released transport and dissemination of a virus (Matthews, 1992). The abundantly from infected roots. They are very stable, can association of viruses with long-lived resting fungal spores has infect plants via the roots without the aid of a vector, often been reviewed by Campbell (1996). Melon necrotic spot virus have a wide host range and, once established in a plant, (MNSV) attaches to the outer covering of the aquatic zoospores spread readily to neighbouring plants by mechanical contact of the chytrid fungus Olpidium bornovanus and can remain of leaves or through the soil via roots. Dissemination in viable in the soil for up to several years (Campbell, 1996). streams, lakes, and rivers may ensure the long distance spread of viruses that would otherwise remain restricted to limited areas (Koenig, 1986, 1988; Mandahar, 1990). Due to the 4. Infection of plants with contaminated findings of infectious ToMV particles in clouds and fog, water Castello et al. (1995) suggested that atmospheric spread of infectious plant viruses, without invertebrate vectors, could The presence of viruses in waters can have epidemiological be a significant long-distance transport mechanism for stable importance only if they can enter the plant, the most acces- plant viruses. Furthermore, human activities favour the sible pathway being through the roots (Mandahar, 1990). distribution of several viruses by sewage waters and by However, the possibility of viral infection through leaves or spreading liquid manure on fields (Koenig, 1986). The through other the upper part of plants when using sprinkler spreading of viruses by means of water is nowadays one of the irrigation systems, for example should also not be neglected. main concerns in agriculture and horticulture where irriga- Infection with water-borne viruses can also occur at the time tion is practiced, as summarized below. of plant germination. Bean seeds that had been soaked in Several viral diseases have been observed in hydroponic SBMV infected tap water before planting in autoclaved soil production, caused by, e.g., Lettuce big-vein associated virus 4908 water research 46 (2012) 4902e4917

(LBVaV), Mirafiori lettuce big-vein virus (MLBVV) and Tomato a hydroponic system. The importance of using waters for chlorotic spot virus (TCSV) in lettuce crops (Tomlinson and irrigation in the dissemination of BNYVV, the causal agent of Faithfull, 1980; Rosales et al., 2004; Colariccio et al., 2004, rhizomania, has been reviewed by Koenig (1988). The 2005; Navarro et al., 2005); MNSV, Zucchini yellow mosaic virus proposed model envisages that the virus-containing resting (ZYMV), Cucumber green mottle mosaic virus (CGMMV) and spores of diseased sugar beet, Pythium betae, percolate through Cucumber leaf spot virus (CLSV) in cucumbers (Wong et al., 1992; the soil into drains and are then transported to ditches and Stanghellini and Rasmussen, 1994a,b; Rosner et al., 2006); canals. When the water from these ditches is used for spray- Potato virus Y (PVY) and Pepino mosaic virus (PepMV) in different ing or irrigation, BNYVV can spread to neighbouring fields. tomato cultivars (Crescenzi et al., 2005; Fakhro et al., 2011). Viruses from infected roots, released into nutrient solution Other viral pathogens that attach to vegetables and orna- in a hydroponic system, can infect other plants, again through mentals are listed by Schnitzler (2004). Evidence for survival of the roots, without any contact or mechanical spread from the virus in a water environment and its potential for plant upper plant parts and, as is assumed, without the help of infection via the root system was, however, demonstrated for vectors. This has been demonstrated for ToMV infecting only a few of them, mainly because such experiments are very tomato (Pares et al., 1992), Pelargonium flower break virus (PFBV) long and complex (see 5.1). infecting pelargonium plants (Krczal et al., 1995) and EU In commercial hydroponic systems the viruses are spread, genotype of PepMV infecting tomato (Schwarz et al., 2010). not only directly by the nutrient solution, but, as in other These experiments are described in detail below. Bu¨ ttner et al. horticultural production systems, through leaf or root contact (1995) indicated that other viruses, like Tomato spotted wilt virus between adjacent plants, by aerial vectors, tools, clothes and (TSWV), can also be transmitted in hydroponic systems. hands of workers contaminated during crop handling. Additionally, similar transmission of viruses in hydroponic Rigorous experimental conditions to confirm the spreading systems, using artificially inoculated nutrient solution, has viruses specifically by nutrient solution is therefore necessary been demonstrated for other combinations, such as ToMV for a better understanding of impact and remedying of viruses infecting pepper (e.g., Schuerger and Hammer, 1995) and on crops grown in hydroponic systems. Maize white line mosaic virus (MWLMV) infecting roots of maize seedlings (Louie et al., 1992). Transmission of TMV in hydro- 5.1. The spreading of viruses in irrigation systems by ponics may occur via root-tip grafts (Park et al., 1999). water or nutrient solutions Pares et al. (1992) studied the spreading of ToMV in a recirculating nutrient solution, using a nutrient film tech- Some viruses are spread effectively in irrigation systems with nique of soil-less culture. ToMV was found in the nutrient the help of aquatic zoospores of fungal vectors from the solution three days after leaf inoculation of tomato plants, at Chytridiomycetes family, and fungal transmission is probably a level that could increase to a concentration readily detect- several orders of magnitude more effective than vectorless able by electron microscopy after a concentration step. transmission (Campbell, 1996). Pathogenic viruses that have Healthy tomato plants, with no contact with infected plants, been shown to be fungal-transmitted in irrigation systems by became systemically infected as early as ten days after Olpidium spp., are LBVaV and MLBVV in lettuce, TNV in bean planting into nutrient solution containing infectious virions. and cucumber, Cucumber necrosis virus (CNV), CLSV, and MNSV Almost all the tomato plants that became infected developed in cucumber, and PepMV in tomato (Tomlinson and Faithfull, symptoms in the shoots. The authors pointed out that the 1980; Tomlinson and Thomas, 1986; Stanghellini and results of earlier studies of Paludan (1985) are inconsistent Rasmussen, 1994a; Campbell, 1996; Navarro et al., 2005; with theirs, e.g., that many tomato plants infected with ToMV Alfaro-Fernandez et al., 2010). For example, growing lettuce through roots do not become infected systemically. However, crops hydroponically in recirculated nutrient solutions such differences in results can be due to the different provides an ideal environment for production and release of concentration of viruses in nutrient solution. The report of viruliferous zoospores of O. brassicae and their transfer from Pares et al. (1992) describes the first attempt to quantify virus plant to plant, often resulting in total infection with lettuce leakage from roots into nutrient solution, by counting parti- big-vein diseases (LBVaV and MLBVV) (Jones, 2004). In the cles observed under an electron microscope. They clearly experiment of Alfaro-Fernandez et al. (2010) it was shown that demonstrated that ToMV can be released from plant roots, the EU genotype of PepMV was transmitted to tomato plants survive in nutrient solution, infect other plants through roots (cv. Marmande) with a transmission rate of 8% when irrigated without root contact, and produce symptoms in those plants. with the drainage water collected from PepMV-infected plants Krczal et al. (1995) confirmed the transmission of PFBV in whose roots contained the fungal culture of Olpidium viru- geraniums by recirculating nutrient solution in a hydroponic lentus. However, vector-free drainage water obtained from system. Different cultivars of geranium plants were raised on irrigating the plants infected with PepMV was not successful rockwool cubes, and no direct root or leaf contact between in transmission of the virus to healthy tomato plants. Thus, inoculated and healthy plants occurred during their experi- PepMV was not transmitted in the irrigation water alone ment. The nutrient solutions were recirculated three times without the presence of the vector. This contrasts with studies a day by plunger pumps and distributed by drip irrigation. To of Schwarz et al. (2010) that showed that PepMV distributed prevent virus transmission by insects, all plants were pro- through a recirculating hydroponic system caused the infec- tected by a closed cover of nylon fabric. Contamination of the tion of healthy plants (see below). Additionally, in this recirculating nutrient solution was detected in ultra- experiment, the common root pathogen of tomato, Pythium centrifuged samples by ELISA two weeks after starting the aphanidermatum, delayed infection with PepMV in hydroponic system. The virus concentration in nutrient water research 46 (2012) 4902e4917 4909

solution increased continuously up to the fourth week, fol- time PCR (Lo´ pez et al., 2006; Agindotan et al., 2007; Ling et al., lowed by some fluctuations until the end of their trial. 2007; Kogovsek et al., 2008; Gutierrez-Aguirre et al., 2009b). Systemic infection of originally healthy plants was detected Appropriate hygiene measures, e.g., disinfection of tools and for the first time after seven weeks, and after 15 weeks all workers’ clothing, and removal of crop residues, are required. plants were infected. Additional prophylactic measures, such as decontamination Infectious PepMV particles of the EU genotype can be of pots and tables by chemical disinfectants, should be released into nutrient solutions from infected tomato roots routinely applied between crop rotations. Since plant viruses and can infect originally healthy tomato plants of cvs. Castle are known to be spread by water, several additional preven- Rock and Hildares without direct root contact (Schwarz et al., tive measures are proposed in irrigation systems. 2010). In their climate-chamber experiments the nutrient Monitoring of irrigation water source and recycled nutrient solution was collected weekly from PepMV-infected plants solutions is recommended. Since the concentrations of plant and supplied to the roots of healthy tomato plants while, the viruses present in such waters are known to be low, water irrigation in their greenhouse experiments, nutrient solution samples need to be concentrated prior to analysis and very was continuously circulated by a pump at a flow rate of about sensitive diagnostic methods are needed (suitable diagnostic 2lmin 1. In the former experiments, PepMV was transmitted methods are described in Section 7). The risk of viral spread by to roots of healthy tomato seedlings of cv. Castle Rock through irrigation waters can be reduced by decontamination of the the nutrient solution within one to five weeks, while ELISA water if it is pumped from drainage channels or from the tests of leaves and green fruits remained negative for PepMV vicinity of dumps. Decontamination of fresh water supplies is throughout the 11-week cultivation period. In addition it was recommended if surface water from an agronomically intense shown that roots of three week old seedlings appeared to be area is used for mixing nutrient solutions or for irrigation. more effectively infected through the nutrient solution than Additionally, when cultivating in closed hydroponic systems, roots of five week old seedlings. In greenhouse experiments, the grower has to decontaminate the recirculated nutrient the roots of originally healthy tomato plants of two cultivars, solution to prevent viral or other microbial epidemics. At the cv. Hildares and cv. Castle Rock, tested positive for PepMV end of the season, recirculating nutrient delivery systems after two and six weeks respectively after starting root inoc- should be decontaminated. The risk of viral spread by water ulation. Subsequently a rapid spread from the roots into the cannot be eliminated completely, but it is possible to mitigate young leaves and developing fruits was found within one the impact on the yield by planting intrinsically virus- week. PepMV was only occasionally detected in the older resistant plants (e.g., Pategas et al., 1989; Strange and Scott, leaves. In the case of cv. Hildares, 11 weeks after starting the 2005), or plants that have been rendered resistant by genetic experiment, 17% of old leaves, 75% of young leaves and 100% modification or by classical breeding. of mature fruits sampled tested positive for PepMV. In If a disease outbreak occurs, infected plants should be contrast only 27% of mature fruits of cv. Castle Rock were eradicated, together with subsequent sanitation of horticul- infected with PepMV 13 weeks after root inoculation. The tural equipment, tools, workers’ clothing and greenhouse differences between the two cultivars may be due, not only to structures (Schnitzler, 2004). Recirculating nutrient delivery genetic factors, but also to environmental factors, since the systems should be decontaminated to prevent recontamina- experiments with different cultivars were performed in tion of disease-free transplants. It is not sufficient to limit different years. None of the infected plants showed any plant removal only to symptomatic plants in recirculating symptoms on fruits, leaves or other organs, and only plants of hydroponic systems, because virulent virions may be released cv. Castle Rock recorded significant yield losses when infected into nutrient solutions prior to plant removal, and thus via contaminated nutrient solution. In these experiments, the transported to other sections of the recirculating systems. nutrient solution contained PepMV particles below the limits Eradication of symptomatic plants is only admissible if the of detection of ELISA and IC-RT-PCR. However, the results crop is near maturity and subsequent secondary spread of indicate that, even with very low viral titres, effective trans- the pathogen is not a major concern However, eradication of mission of PepMV to healthy tomato plants is possible the viral pathogen at the end of the season is necessary through the nutrient solution. (Schuerger and Hammer, 1995).

6.1. The elimination of plant viruses from water 6. Preventing plant viruses from spreading with water The possibility of removing or inactivating microbial contaminants, including viruses, in order to deliver water that Viral diseases can cause great economic losses, e.g. TSWV, is safe for human consumption has been discussed by several that affects tomatoes, occasionally leads to losses of up to authors, e.g., Khan et al. (2001), Reynolds et al. (2008) and Riley 100% (Rosello´ et al., 1996). Because viruses when present in et al. (2011). The management of diseases caused by plant their plant hosts cannot be controlled by chemical treatment, pathogens in irrigation or recycled water has been reviewed crop protection must rely on genetic resistance or disease extensively by Schnitzler (2004), Hong and Moorman (2005) avoidance. and Stewart-Wade (2011), however only a few of the The risk of virus spread in agriculture and horticulture can methods described were shown to be suitable for plant virus be reduced by regular monitoring of seeds and seedlings, elimination and are therefore pointed out in next paragraph. using the very sensitive diagnostic methods that are neces- Additionally, since some viruses spread effectively in hydro- sary, especially for diagnosis of latent infection, such as real ponic systems with the help of aquatic zoospores of fungal 4910 water research 46 (2012) 4902e4917

vectors, these must also be treated, if such vectors are thought The most widely used method for diagnosing plant viruses is to be present in the water (Tomlinson and Faithfull, 1980; the enzyme-linked immunosorbent assay (ELISA) (Clark and Tomlinson and Thomas, 1986; Jones, 2004; Stewart-Wade, Adams, 1977). Its sensitivity depends primarily on the 2011). Each approach has its advantages and disadvantages quality of the antiserum. However, even with good quality and each requires a certain level of grower education. None of antisera, the sensitivity of the double-antibody-sandwich them are simple or provide once-only treatments (Hong and ELISA (DAS-ELISA) procedure is limited to detecting plant Moorman, 2005). viruses down to 1e10 ng ml 1 (Bergmayer, 1986), while the Rosner et al. (2006) found that 4 ppm of hypochlorite for reverse transcriptase polymerase chain reaction (RT-PCR) 30 min eradicates CLSV from recycled waters; however the usually enables a further 100- to 1000-fold increase in sensi- required incubation time and concentration of the disinfec- tivity (Candresse et al., 1998). Using a relatively new important tant depend on the particular virus to be inactivated. Heat diagnostic method in plant virology, real-time PCR (RT-qPCR) treatment at 95 C for 10 s, or ultra-violet (UV) radiation with (Mumford et al., 2000; Boonham et al., 2000; Lo´ pez et al., 2006; a dose of 200 mJ cm 2 were both effective in disinfecting water Agindotan et al., 2007; Ling et al., 2007; Kogovsek et al., 2008;  against ToMV, and ozonisation to a redox value of 754 mV was Gutierrez-Aguirre et al., 2009b; Cepin et al., 2010), concentra- effective in disinfecting water against CGMMV (Runia, 1995). tions as low as 10 genome copies of viruses per reaction can be Heating recycled water to 95 C needs a large infrastructure reliably detected (Gutierrez-Aguirre et al., 2009b). and is costly. It kills pathogens and beneficial microbes indiscriminately and may have undesirable effects on some 7.1. Concentration chemical compounds. It requires the water to be cooled before use (Stewart-Wade, 2011). Disadvantages of UV light, whose A variety of methods have been used for concentrating plant effect is to alter DNA/RNA and thereby kill these organisms, viruses in water samples. Tomlinson et al. (1983) and are e.g., cost, necessity of effective filtering of water prior to Tomlinson and Faithfull (1984) clarified 60 l samples of water treatment, and generation of ozone and/or free radicals that by passing them through pre-filters, adsorbed the viruses to may inhibit the grow of plants downstream of the treatment a Zeta-Plus adsorbent filter, and eluted them with a beef (Stewart-Wade, 2011). Acidic electrolyzed oxidizing water, extract into a smaller volume followed by concentration by produced by electrolysis of deionised water containing a low ultracentrifugation. A similar procedure, but without prior concentration of a salt, has a high oxidation-reduction clarification, was used by Jacobi and Castello (1991), and large potential, low pH, contains hypochlorous acid, and can volumes of water samples (50 l) were concentrated by ultra- display strong virucidal activity (Kim et al., 2000; Stewart- filtration alone by Bu¨ ttner et al. (1987) and Bu¨ ttner and Wade, 2011). This method is simple and has several advan- Nienhaus (1989). Ultracentrifugation of much smaller, non- tages, e.g., no phytotoxicity, no health or environmental pre-filtrated water samples was used by Koenig and hazard, but its efficacy in water is unknown (Stewart-Wade, Lesemann (1985), Koenig et al. (1988, 1989, 2004), Castello 2011). Krczal et al. (1995) demonstrated that, using slow sand et al. (1995), Plese et al. (1996),andGosalvez et al. (2003). filtration, PFBV infection was retarded by six weeks and that Similarly, by using ultracentrifugation, Tosi c and Tosi c (1984) the number of infected plants was reduced to about one third and Fuchs et al. (1996) concentrated pre-filtrated water of those in the unfiltered unit. Liu et al. (1999) used 30K and 5K samples. Plant viruses in water samples can also be concen- Daulton pore-size ultrafiltration membranes to remove 99% trated by polyethylene glycol (PEG) precipitation (Gosalvez and 100% of the ToMV, respectively, from greenhouse waste- et al., 2003; Hamza et al., 2011), or even recovered from sedi- water. However, the elimination of microorganisms from ments of low-speed centrifuged water samples (Piazzolla et al., recycled irrigation water using ultra filtration systems is 1986; Plese et al., 1996). The latter shows that viruses may be probably not practicable due to high installation costs and adsorbed to larger particles, in which case prefiltration would pumping and downstream processing costs, and rapid clog- remove most of the virus (Piazzolla et al., 1986; Koenig, 1986). ging of the expensive filters (Stewart-Wade, 2011). A method The procedures described above are time consuming and involving sedimentation, that is appropriate also for virus laborious. Additionally, the use of small volumes of water elimination, is reviewed by Stewart-Wade (2011). It is based on samples can lead to non-representative testing (Riley et al., production of positive and negative ions, via an electric 2011). Branovic et al. (2003) developed the new method for current, which then attract negatively and positively charged human virus concentration using Convective Interaction contaminants in recycled irrigation water. The resulting Media (CIM) monolithic chromatographic supports. They did reaction products coagulate and precipitate, forming sludge. not demonstrate weather they were able to concentrate viral The method is simple, safe and easy to use, inexpensive to run particles or viral RNA only. Concentration of viral particles and is not affected by variations in the water. It is, however, was proven for the first time for plant virus ToMV (Kramberger expensive to install and the sludge need to be removed et al., 2004). Up to now, the CIM technology was shown to be regularly. useful for concentration of several human virus particles from water samples (Gutierrez-Aguirre et al., 2009a, 2011; Kovac et al., 2009). The viruses are bound by a CIM monolith, then 7. Methods for detecting plant viruses in eluted with a high salt buffer. ToMV, diluted considerably water below the sensitivity of ELISA, was concentrated by several orders of magnitude in this one-step procedure and main- The concentration of viruses in water samples can be low, so tained its infectivity after the concentration process. It has their detection requires an appropriate concentration step. since been shown to be appropriate for concentrating ToMV water research 46 (2012) 4902e4917 4911

from rivers (Boben et al., 2007). In comparison with methods Henning, 1997) and rapid serological tests for on-site detec- described previously for concentrating plant viruses from tion (Danks et al., 2007; Salomone and Roggero, 2002). water, the CIM procedure is a much faster and more efficient Serological methods are suitable for direct and indirect for concentrating highly diluted plant viruses than the previ- detection of plant viruses in water samples. First, they were ously described methods. used for characterising viruses isolated on test plants (indirect Following concentration, a variety of methods can be used determination of the presence of plant viruses in waters), e.g., for virus detection. Use of at least two methods is recom- immunodiffusion and/or immunoprecipitation tests mended for reliable confirmation of virus presence in water (Tomlinson et al., 1983; Tomlinson and Faithfull, 1984; Tosi c samples. and Tosi c, 1984; Koenig and Lesemann, 1985; Koenig et al., 1989; Bu¨ ttner and Nienhaus, 1989; Bu¨ ttner et al., 1987; Yi 7.2. Biological assays et al., 1992; Plese et al., 1996) and later ELISA (Jacobi and Castello, 1991). Castello et al. (1995) and Boben et al. (2007) A biological assay detects only infectious viral particles, while have shown that plant viruses can be also detected directly other methods are unable to discriminate between infectious from concentrated water samples by ELISA. and non-infectious viruses. The biological assay is used to determine the host range of a virus but, for reliable charac- 7.4. Observation of viral particles terization, other specific diagnostic methods should be used. Mechanical inoculation of leaves is not always very effective Individual plant viruses can be seen only under the electron in achieving infection. Although, in theory, one virus particle microscope (EM). EM directly shows the considerable detail of is sufficient to infect a cell, in practice a large number of their morphology and inner particle structure, and thus helps particles, usually more than 500, are required to give rise with virus recognition. The method most used for sample prepa- certainty to infection of one cell with formation of a visible ration is negative contrasting. Elongated virus particles can be lesion (Dijkstra and De jager, 1998). This may be due to the fact readily distinguished from surrounding cell debris, but small that only a small proportion of epidermis cells have been isometric particles, unless present in large numbers, may be wounded to such an extent that virus particles can enter the hard to detect because of their similarity to small cell organ- cytoplasm (Dijkstra and De jager, 1998). elles, such as ribosomes. Methods that combine the possibility Plant viruses have been recovered by rubbing the water to visualise the type of viral particle using EM with the spec- sample obtained after concentration on several different ificity of serological tests are called immunoelectron micros- species of test plants (Jacobi and Castello, 1991; Plese et al., copy, e.g., immunosorbent EM (ISEM) and decoration EM. 1996) or on a few test plants only, e.g. Chenopodium quinoa (Francki et al., 1985; Dijkstra and De jager, 1998; Bos, 1999). (Tomlinson et al., 1983; Tomlinson and Faithfull, 1984; In particular cases, plant viruses can be detected directly Piazzolla et al., 1986; Koenig et al., 1989; Fuchs et al., 1996), C. from concentrated water samples by EM (Tosi c and Tosi c, 1984; quinoa and Nicotiana clevelandii (Koenig and Lesemann, 1985), Jacobi and Castello, 1991). EM has been used mainly as an Chenopodium amaranticolor, Chenopodium murale, Nicotiana indirect method for classification the viral particles that caused tabacum cv. Samsun and N. glutinosa (Tosi c and Tosi c, 1984), C. the lesions on test plants after inoculation with concentrated quinoa, Nicotiana benthamiana and N. tabacum var. Xanthi-nc water samples (Koenig and Lesemann, 1985; Koenig et al., 1988; (Bu¨ ttner and Nienhaus, 1989). Biological assay was used as Bu¨ ttner et al., 1987; Bu¨ ttner and Nienhaus, 1989; Jacobi and a method for detection and for the isolation and propagation Castello, 1991; Plese et al., 1996). Additionally, light micros- of viruses that is needed for further characterization copy, as one of the confirmatory methods, was used to inves- (Tomlinson et al., 1983; Bu¨ ttner et al., 1987; Bu¨ ttner and tigate viral cell inclusions (Plese et al., 1996). Further specific Nienhaus, 1989; Piazzolla et al., 1986; Koenig and Lesemann, tests needed for virus identification, such as immuno EM, were 1985; Koenig et al., 1988, 1989, 2004) or as a test of infectivity then selected after the first screening tests, (Tomlinson et al., (Castello et al., 1995; Boben et al., 2007). 1983; Tomlinson and Faithfull, 1984; Piazzolla et al., 1986; Koenig et al., 1989; Yi et al., 1992; Koenig et al., 2004). 7.3. Detection of viral protein 7.5. Detection of nucleic acids The sizes of viral coat proteins were determined by means of sodium dodecyl sulphate polyacrylamide gel electrophoresis With the rapid development of molecular techniques, it is after purification of viruses propagated on test plants infected now possible to obtain substantial information about viral with water samples (Koenig et al., 1988; Yi et al., 1992). nucleic acids. Such nucleic acid based techniques have proved However, the most widely used methods for detection of viral to be very suitable for detecting and identifying plant viruses. coat protein are serological methods. All serological techniques Northern blot and Northern blot and dot hybridization tests are based on antibodyeantigen reactions between a specific have been used to identify viruses from water samples antiserum generated against the antigen of interest, e.g., viral previously purified from test plants (Koenig et al., 1988; Yi coat protein. The most commonly applied immunoassay is et al., 1992). Methods that amplify the nucleic-acid signal, ELISA (Dijkstra and De jager, 1998; Hampton et al., 1990). The e.g., PCR, RT-PCR and qPCR, are nowadays most widely used to method has been made suitable for plant viruses by Clark and detect low concentrations of nucleic acids. Adams (1977). Several other traditional serological methods are Castello et al. (1995, 1999) detected ToMV in cloud, fog and available for plant virus diagnosis, e.g., immunodiffusion, glacial ice samples by RT-PCR, and amplification products immunoprecipitation, tissue printing (Krzymowska and were sequenced. Unknown viruses, found in environmental 4912 water research 46 (2012) 4902e4917

waters, were characterised by RT-PCR and sequencing (Koenig 2009; Roossinck et al., 2010). Its use for studying microbial et al., 2004). Gosalvez et al. (2003) developed RT-PCR for populations in a sample by analyzing the nucleotide sequence detection of MNSV in water samples. Samples from a water content has been applied to a wide range of environmental source pool of a hydroponic culture or from recirculating samples, including viral metagenomes from sea water nutrient solution were concentrated by ultracentrifugation or (Breitbart et al., 2002; Angly et al., 2006; Culley et al., 2006), PEG precipitation and followed by RT-PCR analysis. The fresh water lakes (Djikeng et al., 2009) and reclaimed water method was shown to be much more sensitive (10 ng ml 1 (Rosario et al., 2009a). Next-generation sequencers have the detection limit) than ELISA (160 mgml 1 detection limit). ability to process millions of sequence reads in parallel Boben et al. (2007) developed a quantitative RT qPCR (Mardis, 2008; Pop and Salzberg, 2008; Aw and Rose, 2011; method for both detecting and quantifying ToMV in irrigation Studholme et al., 2011). Additionally, a bioinformatic method waters. This enabled some environmental water samples to has been developed to analyze sequence data rapidly, with be tested ToMV-positive in concentrations down to minimal post-processing (Segerman et al., 2011). 4.2 10 10 mg ml 1, without the concentration step. The Finally, the possibility of simplifying the whole procedure sensitivity of the method was further improved by using CIM of molecular diagnostics is currently being explored, with the monolithic chromatographic columns to concentrate the aim of providing a simple way to confirm the presence of water samples. The overall procedure is simple, rapid and viruses on-site. This would reduce the time from sampling to highly sensitive, and could be adopted for other plant, human result and accelerate the adoption of measures to prevent or animal viruses. It is suitable for monitoring irrigation and risks. For this, special emphasis has to be given to speeding other water samples. Quantitative RT qPCR was used to the analytical procedure and making it portable. CIM tech- quantify PMMoV in concentrated samples of seawater nology, in combination with RT-qPCR detection using the (Rosario et al., 2009b) and river water (Hamza et al., 2011). portable Smart Cycler real time PCR thermocycler, has been adapted to field requirements (Gutierrez-Aguirre et al., 2011). 7.6. Future development of methods for monitoring The model viruses used were rotaviruses from water samples, plant viruses in water but the method can be adapted to detecting plant viruses. The next steps should be the design of user-friendly, on-site Diagnostic procedures for monitoring environmental waters devices for CIM-based concentration and for molecular should have the following characteristics: high specificity and detection (Gutierrez-Aguirre et al., 2011). Isothermal amplifi- sensitivity, high and rapid throughput, and low cost. The cation procedures, such as loop-mediated isothermal ampli- methods currently used for viral disease diagnosis are, in fication (LAMP) (James et al., 2010; Nemoto et al., 2010; general, designed to detect one or a few (multiplex PCR/qPCR) Tsutsumi et al., 2010; Bekele et al., 2011) enable target ampli- viruses at the same time, but multiple detection of plant fication without the need for expensive thermocyclers. In pathogens in a single step would be useful. This would not addition, LAMP amplification products can be easily detected only decrease handling time, but also reduce the amounts of in a lateral flow device (James et al., 2010), simplifying the consumables and reagents needed, resulting in lower costs. methodology even more, since LAMP is a rapid and simple This could be achieved by using, for example, microarrays, technique that is less prone to inhibitors present in the new generation sequencing or xMap detection technologies. sample (Bekele et al., 2011). Microarrays are one of the new emerging methods in plant virology (Boonham et al., 2007) currently being developed for potato (Boonham et al., 2003), cucumber (Lee et al., 2003), and 8. Conclusions other (Bystricka et al., 2005; Kostrzynska and Bachard, 2006) plant pathogens. Up to 30,000 DNA probes can be arrayed onto Several plant viruses have been found in drainage and other a single glass microscope slide (Boonham et al., 2003). The environmental waters, such as canals, rivers, streams, ponds, probes are gene sequences from each of the viruses to be lakes and oceans. The majority that have so far been detected detected. The microarray is exposed to fluorescence labelled in waters are very stable and can survive outside host cells for DNA from the sample to be tested, and scanned using a long time. They often have a wide host range and are a microarray scanner to reveal the presence of any of the reproduced in infected plants in high concentrations. They targets of interest (Boonham et al., 2007). Although some can be released into drainage waters from infected roots and attempts have been made to simplify the methods (Bystricka from injured or decaying plant material. Due to the stability of et al., 2005), the microarray technology is still expensive for several viruses in the alimentary tract of vertebrates, sewage, common practice. The Luminex xMAP platform has been dung and liquid manure are also important sources of viruses. designed to combine the serological and molecular method- Drainage waters and sewages often flow into the rivers, lakes ologies (Van der Vlugt et al., 2011). In serological applications, and streams used for irrigation purpose. this technology is closely related to the ELISA, but the test can Soilless culture has provided an alternative to plant be performed in less than 2 h without compromising speci- growers facing soil-related problems such as nematodes, ficity or sensitivity. Multiplex Luminex tests for several pathogens, and nutrient imbalances, but the use of circulating combinations of plant viruses in different crops are now nutrient solution in hydroponic systems holds the potential under development (Van der Vlugt et al., 2011) and others for rapid spread of water-transmissible plant pathogens have shown that a metagenomic diagnostic technique throughout the whole crop. Although numerous plant viruses utilizing next-generation sequencing can be used as a rapid have been detected in aqueous environments, their survival in and universal diagnostic tool in plant virology (Adams et al., waters and the possibility of their direct transmission through water research 46 (2012) 4902e4917 4913

the nutrient solution are still unknown. This is due to the long Adams, I.P., Glover, R.H., Monger, W.A., Mumford, R., duration of the complicated and extensive experiments Jackeviciene, E., Navalinskiene, M., Samuitiene, M., needed. However, this knowledge is necessary for effective Boonham, N., 2009. Next-generation sequencing and metagenomic analysis: a universal diagnostic tool in plant prevention of disease spreading, with consequent huge virology. Molecular Plant Pathology 10 (4), 537e545. economic losses. In addition, virus transfer from horticultural Agindotan, B.O., Shiel, P.J., Berger, P.H., 2007. Simultaneous to native plants was already proven (Jones, 2009). In the detection of potato viruses, PLRV, PVA, PVX and PVY from research on the barley and cereal yellow dwarf viruses in dormant potato tubers by TaqMan real-time RT-PCR. Journal grasslands of the western US, the findings indicated that both of Virological Methods 142 (I1e2), 1e9. biotic and abiotic factors have influence on virus ecology and Alfaro-Fernandez, A., Cordoba-Selles, M.D.C., Herrera- epidemiology (Power et al., 2011). Vasquez, J.A., Cebrian, M.D.C., Jorda, C., 2010. Transmission of Pepino mosaic virus by the fungal vector Olpidium virulentus. In the 80s and 90s, the presence of plant viruses in Journal of Phytopathology 158, 217e226. concentrated water samples was monitoring using mechan- Angly, F.E., Felts, B., Breitbart, M., Salamon, P., Edwards, R.A., ical inoculation of test plants, followed by relatively insensi- Carlson, C., Chan, A.M., Haynes, M., Kelley, S., Liu, H., tive diagnostic methods. All plant viruses cannot be Mahaffy, J.M., Mueller, J.E., Nulton, J., Olson, R., Parsons, R., transmitted by mechanical inoculation to test plants; in Rayhawk, S., Suttle, C.A., Rohwer, F., 2006. The marine addition, they are host-specific. Nowadays more sensitive viromes of four oceanic regions. PLoS Biology 4 (11), 2121e2131. e368. diagnostic methods are available, but they are usually specific Aw, T.G., Rose, J.B., 2011. Detection of pathogens in water: from for one virus or one group of viruses, thus requiring several phylochips to qPCR to pyrosequencing. Current Opinion in reactions in order to identify all the viruses present. New Biotechnology. http://dx.doi.org/10.1016/j.copbio.2011.11.016. methods being developed for the simultaneous detection of ISSN 0958-1669. multiple plant viruses, like microarrays or new generation Bandte, M., Pestemer, W., Bu¨ ttner, C., Ulrichs, C., 2009. Ecological sequencing, will facilitate the monitoring of environmental aspects of plant viruses in tomato and pathogen risk e waters and waters used for irrigation and in hydroponic assessment. Acta Horticulturae 821, 161 168. Bekele, B., Hodgetts, J., Tomlinson, J., Boonham, N., Nikolic, P., systems. As a result, the list of plant viruses found in waters is Swarbrick, P., Dickinson, M., 2011. Use of a real-time LAMP likely to lengthen significantly. The biological significance of isothermal assay for detecting 16SrII and XII phytoplasmas in the results of such developments will require much further fruit and weeds of the Ethiopian Rift Valley. Plant Pathology research. In irrigation waters, the viruses are usually present 60, 345e355. in concentrations below the detection limit of classical Bergmayer, H.U., 1986. Antigens and antibodies 2. In: methods, although sufficient to infect plants. The develop- Bergmayer, J., Grassl, M. (Eds.), Methods of Enzymatic ment of highly sensitive diagnostic methods is thus neces- Analysis, third ed. VCH Verlagsgesellschaft, Weinheim, pp. 474e481. sary. 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