Efficacy of electrolytically-derived disinfectant against dispersal of Fusarium oxysporum and Rhizoctonia solani in hydroponic tomatoes

Scientia Horticulturae 234 (2018) 116–125

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Efficacy of electrolytically-derived disinfectant against dispersal of Fusarium oxysporum and Rhizoctonia solani in hydroponic tomatoes

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Marlon Hans Rodrigueza,b, Martina Bandtea, , Thomas Gaskina, Gerhard Fischerc, Carmen Büttnera a

Division Phytomedicine, Faculty of Life Science, Humboldt-Universität zu Berlin, Lentzeallee 55, D-14195 Berlin, Germany Universidad Francisco de Paula Santander, Facultad de Ciencias Agrarias y del Ambiente, GICAP, Cúcuta, Colombia c Universidad Nacional de Colombia, Facultad de Ciencias Agrarias, Bogota, Colombia b

A R T I C L E I N F O

A B S T R A C T

Keywords: Sanitation Free chlorine Vascular wilt Potassium hypochlorite Nutrient solution

Demand for conservation and recycling of water has increased significantly. Therefore irrigation water used for horticultural or agricultural purposes needs to be treated before being reused to eradicate plant pathogens and thereby reducing the risk of pathogen dispersal and losses due to disease. The economically important fungal plant pathogens Fusarium oxysporum (Synder and Hans) and Rhizoctonia solani (Kühn) were selected to examine the efficacy of nutrient solution treatment by electrolytic disinfection to prevent the dispersal of these pathogens in the hydroponic production of tomatoes (Solanum lycopersicum Mill.). First, we determined the efficacy of the disinfectant to inactivate F. oxysporum and R. solani in vitro. The electrolytically generated potassium hypochlorite (KClO) was tested at five concentrations of free chlorine (0.2, 0.5, 0.8, 1.0, 2.0 mg/L) in nutrient solutions of pH 5.5, 6.0 and 6.5 with four contact times (5, 30, 60, 120 min). Best sanitation was achieved in nutrient solution at pH 6.0. In vitro, F. oxysporum required 2 mg/L at 30 min for complete inactivation whereas chlorination had only a minimal effect on viability of R. solani. Subsequent trials under practical conditions applied the disinfectant via a new sensor-based disinfection procedure. Potassium hypochlorite solution produced on site and injected into a recirculating nutrient solution once a week for 60 min at a free chlorine concentration of 0.5 mg/L (ORP 780 mV) inhibited the dispersal of F. oxysporum and R. solani during the entire test period of 16 weeks. In contrast all tomato test plants irrigated with untreated nutrient solution became infected with F. oxysporum and a third of them additionally with R. solani. At the applied dose no plant damage occurred. Thus, the treatment proved to be effective and applicable to prevent dispersal of fungal pathogens by nutrient solution under simulated field conditions.

1. Introduction Tomato (Solanum lycopersicum Mill.) is considered one of the most economically important vegetable crops in the world. Production is currently around 130 million tons, of which 88 million are destined for the fresh market and 42 million are processed (Anonymous, 2016). In the European Union, the tomato also holds the number one position among vegetables, with 16.6 million tons, representing 12% of global production. Tomatoes are characterized by a high water requirement. Particularly in arid and semi-arid areas irrigation is required for both the quantity and quality of tomato production. Several sources of water can be used for irrigation purposes: municipal water, groundwater, water collected from roofs and paved surfaces, run-off water and surface water from ponds, lakes, streams and rivers. Some of these sources pose a high risk of disseminating plant pathogens. Whereas ⁎

groundwater and municipal water tend not to harbor plant pathogens, run-off water collected by channels and stored in ponds or tanks poses a high risk for dispersal of plant pathogens (Moorman et al., 2014). Pathogens may be introduced to run-off water directly from crops in cultivated fields or natural vegetation surrounding the fields. Numerous species of zoosporic organisms, fungi, bacteria and viruses have been found in surface water and recirculating nutrient solution (Hong and Moorman, 2005; Hong et al., 2014; Mehle and Ravnikar, 2012). The latter greatly facilitates the spread of waterborne pathogens within crops, since pathogens washed or leached from the crop can accumulate in the holding tank and be delivered back to the crop repeatedly with each irrigation cycle. Due to this potential risk, physical and chemical disinfection methods are used in greenhouse facilities to minimise the occurrence and spread of plant pathogens (Stewart-Wade, 2011). Recently

Corresponding author. E-mail address: [email protected] (M. Bandte).

https://doi.org/10.1016/j.scienta.2018.02.027 Received 15 June 2017; Received in revised form 12 February 2018; Accepted 13 February 2018 0304-4238/ © 2018 Elsevier B.V. All rights reserved.

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extensive investigations. In vitro studies were carried out to ascertain dose-effect relations of the disinfectant. In vivo studies were conducted to elucidate the efficacy and suitability under simulated field conditions

Raudales et al. (2014) summerised established water treatments to control plant pathogens including the mode of action of each technology. Currently the grower can choose from physical treatments such as filtration, heat, and ultraviolet (UV) radiation or chemical water treatments such as bromine, chlorine, chlorine dioxide, ozone, hydrogen peroxide, and ionized copper or silver. Chlorine has been used successfully in the disinfection of public water, seeds and in post-harvest processes (Van Haute et al., 2015). To treat irrigation water sodium hypochlorite or calcium hypochlorite are commonly used as they are easy to apply, relatively persistent and inexpensive (Fisher et al., 2014). When hypochlorite is introduced to water it reacts to form free chlorine species of hypochlorous acid (HOCl) and hypochlorite (OCl − ) ions, which oxidise organic materials including any pathogens present in the water (Zheng et al., 2016). This chemical compound destroys pathogens through penetration of the cell wall, damaging proteins and membranes and disrupting metabolic processes (Fisher et al., 2014). HOCl predominates at a solution of pH below 7.5 and is a much stronger sanitizer than hypochlorite which predominates at a pH greater than 7.5 (De Hayr et al., 1994). The contact time and dose required for the chlorination of irrigation water to eradicate plant pathogens varies with species and life stage (Cayanan et al., 2009; Hong and Richardson, 2004; Scarlett et al., 2016). Furthermore the effectiveness of chlorine is influenced by water-soluble fertilizer and organic matter (OM) as it is readily oxidised by OM, and it reacts with the nitrogen to form chloramines (Hong et al., 2003). Chloramines are more stable but have a lower biocidal effect with only 4% the disinfection efficacy of hypochlorous acid (White, 2010). Electrolysed oxidised water has drawn significant attention in the food industry as a non-thermal method of sanitation and microbial inactivation (Rahman et al., 2016), and is a promising technology for the treatment of irrigation water (Elmer et al., 2014). It is a solution with disinfecting properties which is generated by passing a dilute salt solution (commonly sodium or potassium chloride) through an electrolytic cell. This application constitutes a sustainable and green method, which has several advantages compared to other sanitation techniques including cost effectiveness, ease of application, on-the-spot production, human safety and protection of the environment. It should be emphasized that neither transport nor storage of hazardous substances is required and the disinfecting effect can be adjusted according to the particular on-site chlorine demand. The efficacy of a sensor-based disinfection with electrolytically-derived potassium hypochlorite to inhibit the dispersal of plant viruses in tomato crops was recently determined for Pepino mosaic virus (Bandte et al., 2016). The present study was undertaken to evaluate the efficacy of the potassium hypochlorite treatment of nutrient solution in eliminating two common undesirable fungal plant pathogens in tomato production, Fusarium oxysporum f. sp. lycopersici (Synder and Hans) and Rhizoctonia solani (Kühn). F. oxysporum causes a highly destructive disease leading to extensive crop losses in both field and protected tomatoes, and remains a major limiting factor for tomato production (McGovern, 2015). The fungus is soil-borne and causes vascular wilt by infecting plants through the roots and spreading internally through the cortex to the vascular tissue. It can survive in the form of mycelium and chlamydospores in substrate and plant debris for longer periods of time. This persistence of resting spores combined with the limited range of effective fungicides complicates disease management. R. solani also ranks among the most important soil-borne fungal pathogens (Cao et al. 2004). It is a species complex composed of a diverse assemblage of soil fungi that vary with respect to host specificity and morphology (Bartz et al., 2010). These fungi can cause diseases in more than 500 genera of plants. Typical symptoms such as seedling damping-off, root necrosis, basal stem cankers, and fruit rot, result from the colonization of plant tissues by fungal hyphae or sclerotia present in the substate (Jones et al., 2014). To evaluate the efficacy of controlling fungal pathogens and the suitability of the sensor-based disinfection system we conducted

2. Material and methods 2.1. Plant pathogens and inoculum Conidia and mycelial fragments of F. oxysporum f. sp. lycopersicum (DSM-62059, Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) were prepared by growing the culture on “Synthetic Low Nutrient Agar” (SNA, Nirenberg, 1976) at 22 °C, under an 8 h light: dark regime, for 10 days. Plates were flooded with high-purity water and scraped with a sterile rod to detach spores. The resulting suspension was added to the liquid medium and incubated at 30 °C for 10 days, shaking at 120 rpm. To remove large mycelial fragments the spore suspension was filtered through two layers of cheesecloth. R. solani (PM-5, collection of the Division Phytomedicine, Humboldt-Universität zu Berlin, Berlin, Germany) was cultured on “Potato Dextrose Agar” (PDA) at 22 °C under an 8 h light:dark regime for 12 days. The inoculum was prepared by placing colonised PDA plugs (5 × 5 mm) in 100 ml of Potato dextrose broth (PDB) and incubated at 30 °C for 12 days in the dark on an orbital shaker. Mycelium was harvested and homogenized in a blender (Clatronic, model SM2452) for 30 s with high-purity water. Suspensions of all fungal propagules were quantified using a hemocytometer and diluted with sterile deionised water to obtain 106 propagules/mL prior to testing the chlorine treatment. The viability of these propagules was checked on PDA and determined in colony forming units (CFU)/ml; the average value over all tests was 91%. 2.2. Potassium hypochlorite solution Hypochlorite was produced on-site in a single chamber brine electrolysis plant (nt-BlueBox mini; newtec Umwelttechnik GmbH; Berlin, Germany) as described by Schuch et al. (2016). A direct current of 10 A with a voltage of 13 V was applied with titanium electrodes to a brine solution containing potassium chloride (KCl) and fresh water leading to the formation of chlorine (Cl2). In turn Cl2 was disproportionated to hypochlorous acid (HClO) and Cl− in the presence of hydroxyl ions (OH-) in an aqueous solution. The potassium hypochlorite (KClO) solution produced by the device contained 36.6 mg of free chlorine/L. The content of free chlorine in this electrolytically generated stock solution as well as in the working solutions was checked manually using a handheld apparatus (Pocket Colorimeter II, Hach Lange GmbH, Germany). Following the manufactors instructions the measurement was carried out at 528 nm in a volume of 10 mL with a photometric precision of ± 0.0015 Abs. 2.3. In vitro experiments The antifungal efficacy of the potassium hypochlorite solution was tested in vitro on F. oxysporum and R. solani. It was tested in nutrient solution at three different pH values representinga horticulturally-relevant range: 5.5, 6.0, and 6.5. The nutrient solution consisted of tap water and a stock solution of macronutrients (calcium nitrate 1.7 mmol/l, magnesium sulfate 2.6 mmol/l, potassium nitrate 3.3 mmol/l, monopotassium phosphate 0.4 mmol/l, ammonium nitrate 0.4 mmol/l and 10 mg/l Fe EDTA 13%) and micronutrients according to Göhler and Molitor (2002). The pH value was adjusted to 6.0, the electrical conductivity (EC) value to 1.8. Testing covered a range of different concentrations (0, 0.2, 0.5, 0.8, 1.0 and 2.0 mg free chlorine/L displayed by the electrolytic processed potassium hypochlorite) and contact times (5, 30, 60, and 120 min). These doses can be expected not to cause plant damage in practical use. Contact times were achieved by using 0.01 M sodium thiosulfate 117

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Fig. 1. Schematic view of experimental set-up. Nutrient solution was supplied continuously with recirculation (pump power 400 l/h at 50 Hz). Each Tank (A and B) provided the solution to the 13 plants that were positioned in two rows. A root barrier (1626 mesh/cm2) hampered root contact between infected donator plants and healthy plants. The pruning management, spacing and the bushy habitus of the tomato variety “Hoffmanns Rentita” prevented the transmission of the fungal pathogens. The experiment was performed twice.

placed on SNA and PDA respectively, as described above. Addition of antibiotics (100 mg Penicillin G, 10 mg Chlortetracycline and 50 mg Streptomycin sulphate per litre of nutrient media) to PDA facilitated microscopic evaluation. Surface disinfection was applied with a solution of 2% commercial bleach (6.15% NaOCl) for 2 min. Incubation was carried out at 22 °C, under an 8 h light: dark regime, for 10 days. F. oxysporum and R. solani isolates were identified microscopically based on morphological characteristics. At the end of the in vivo trial stem sections of each tomato plant were tested in a similar manner. All tests were perfomed with three replicates. Samples of treated and untreated nutrient solution were taken weekly to assess contamination with F. oxysporum and R. solani. Samples of 100 ml each were used to detect these fungal pathogens. Twenty subsamples each of 10 μl were transferred to a haemocytometer to count characteristic conidia under the microscope. Subsequently 10 subsamples of 2 ml each were transferred to Petri dishes with SNA and PDA (see 2.1), incubated at 22 °C under an 8 h light: dark regime for 10 days and evaluated by counting CFU. The disinfectant generated by the electrolytic disinfector (see 2.2) was added automatically to the nutrient solution using a DosaCompact dosing system (Dosatronic GmbH, Ravensburg, Germany). This complete measuring and control system included an electrode to meassure free chlorine (Edelmetall-Elektrode M12, Dinotec GmbH, Maintal, Germany) and a magnet membrane dosage pump (KMS-MF, Dosatronic GmbH, Ravensburg, Germany) with a maximum injection rate of 50 strokes per minute and a stroke volume of 0.09 ml per stroke. The target concentration of 0.2 and 0.5 mg free chlorine per L respectively for 60 min weekly was controlled and recorded online by the dosing system. The content of free chlorine was checked photometrically by hand as described above, to monitor the accuracy of the sensor-based measurement and injection. The experimental design to evaluate treatment efficacy in vivo is shown schematically in Fig. 1. Thirteen plants were positioned in two channels which were supplied with nutrient solution via a 400 L tank. The channels were constantly flushed with this nutrient solution at a

pentahydrate to neutralize the oxidation effect of the disinfectant. The analyses were carried out each in a volume of 11 ml. Each nutrient solution was first adjusted to the required pH value, supplied with the particular amount of the disinfectant, fed with the pathogen suspension and incubated separately at room temperature. In addition to untreated nutrient solution, high-purity water served as a further control. After an incubation of 5, 30, 60 and 120 min, sodium thiosulfate was added to stop the disinfection reaction. Propagule residues such as mycelia, conidia, and sclerotia were counted using the haemocytometer in triplets of each treated solution. Additionally viable propagule numbers were determined by plating. Aliquots of each treatment were transferred to 5 plates each with the nutrient medium and incubated at 25 °C for 7 days, after which propagule numbers were estimated for each plate by colony counts. Furthermore, all plates were preserved for a further 20 days for assessment of viable plant pathogens. The experiment was carried out twice with five replicates per treatment. The degradation of free chlorine was estimated at a concentration of 2.0 mg/L. The nutrient solution and high-purity water (control) containing the particular pathogen were adjusted to pH 5.5, 6.0 and 6.5 respectively. The content of free chlorine was quantified manually in each triplicate after 0, 5, 10, 30, 60, and 120 min using as described above. 2.4. In vivo experiments In vivo trials were carried out with the small bush tomato cv. 'Hoffmanns Rentita' in greenhouse compartments (22 °C, 16 h photoperiod). Seeds were sown in perlite and transferred to rockwool cubes (10 × 10 × 7 cm3) 15 days after sowing (das). Tomato plants infected with F. oxysporum f. sp. lycopersici and R. solani were obtained by immersion of the roots in the respective fungal solution (1 × 106 propagules/mL) for 2 min prior transfer to rockwool cubes. To confirm an infection with a particular fungal pathogen 12 sections (2 mm long) of each tomato plant root were taken weekly and 118

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continuous flow rate of 84 L h − 1. One storage tank was subjected to the disinfection procedure, while the other tank with only nutrient solution acted as the non-treated control. Root contact between healthy (test) and infected (control) plants was prevented by a root barrier (1626 mesh/cm2). Cultivation lasted for 16 weeks and followed commercial practices: 22 °C, 16 h photoperiod, and relative humidity 30–60%. The nutrient solution (see 2.3) had a pH value of approx. 6.0. The composition and pH of the nutrient solution was measured weekly in the laboratory and corrected when necessary. The experiment was repeated once. 2.5. Data collection and analyses The data on pathogen viability was converted into logarithmic data (CFU mL−1 + 2) to meet assumptions of Anova. Data were analysed using a completely randomised model with the arrangement 3 × 6 × 4 (pH, dose and time) for viability and an arrangement of 4 × 2 × 4 (pH, solution and time) for free chlorine concentration according to each pathogen. The Anova showed highly significant differences (P < 0.05) except where noted. Mean values were compared to Tukey's test (P = 0.05) using procedure GML of SAS software version 9.2 (SAS Institute, Cary, NC). Plants in in vivo experiments were assessed individually on a weekly basis for F. oxysporum and R. solani infection. Number and fresh weight of tomato fruits were determined per plant. Fruits were harvested at skin color gauge 9 (OECD, 2003) every week after 56 days. Fruits with a diameter < 40 mm, severe discoloration or growth cracks were defined unmarketable. Data on tomato fruits were subjected to two-way analysis of variance. Means were compared by Tukey’s t-test at significance level α = 0.05. Significant differences are represented by different letters. 3. Results 3.1. Free chlorine in nutrient solution Fig. 2. Free chlorine profile of electrolytically generated potassium hypochlorite (2 mg free chlorine/L) in high purity water and nutrient solution dependent of the presence of plant pathogens. Top: high purity water: pathogen-free - R. solani-⬜ and F. oxysporum -⬥. Below: nutrient solution at pH 5.5: pathogen-free -⬣, R. solani-⬜ and F. oxysporum-⬥.

No degradation of free chlorine was observed in high purity water within 120 min. However, the presence of fungal pathogens appeared to cause a rapid disinfectant consumption in high purity water. During the first five minutes of contact time the fungal pathogens F. oxysporum and R. solani caused a reduction from 2.0 to 0.600 ± 0.010, and 1.603 ± 0.015 mg free chlorine/L high purity water, respectively (Fig. 2). By contrast, the concentration of free chlorine in nutrient solution was drastically reduced from 2.0 to 0.123 ± 0.006 mg free chlorine/L within five minutes in which the presence of fungal pathogens did not account for an additional effect.

could be observed at the lower application rates. For instance at pH 6.5 only a small reduction could be achieved (Fig. 3a). Although the viability of R. solani declined as the chlorine concentration and contact time increased, it was not possible to eradicate the pathogen under tested conditions (Fig. 3c, d). Even after an application of 2 ppm free chlorine for 120 min only a slight linear reduction could be observed, independent of the pH value. For the complete inactivation of R. solani 18 mg free chlorine/L nutrient solution, adjusted to pH 6.0, was required for 30 min (data not shown).

3.2. Efficacy of treatment on viability of fungal pathogens in vitro The viability of the fungal pathogens showed no statistical difference between pH 5.5 and 6.0. Therefore only dose-effect relations considering the pH values 6.0 and 6.5 are illustrated. As expected, the efficacy of electrolytically generated potassium hypochlorite to sanitise nutrient solution varied between pathogens with application rate, contact time, and pH value of the nutrient solution (Fig. 3). In all cases, propagules of the fungal plant pathogens in untreated nutrient solution retained their viability over the period of 120 min. As the free chlorine concentration increased, the number of inactivated conidia of F. oxysporum and mycelia of R. solani increased. Inactivated pathogens showed degraded cells (Fig. 4) and did not proliferate on nutrient media. Nevertheless relatively high chlorine concentrations and contact times were required for a complete inactivation of fungal plant pathogens. Complete inhibition of F. oxysporum germination occurred only at 2 ppm for 30 min in a nutrient solution of pH 6.0 (Fig. 3b). Independent of pH value, little or no effect of contact time

3.3. Effect of treatment on plant growth and fruit yield None of the plants supplied with treated nutrient solution showed phytotoxic foliar damage and/or growth differences. Infected control and test plants developed characteristic symptoms when cultivated in treated or untreated nutrient solution. Thus, F. oxysporum-infected tomato plants showed asymmetrical chlorosis on the leaves and wilt. Finally, the leaves dried and dropped prematurely; the vascular tissue of root and stem presented the typical reddish discoloration caused by plugged water-conducting tissues. R. solani-infected plants exhibited rot of stem bases and roots. The fruit yield of plants infected with these fungal plant pathogens was reduced (Table 1). However, the values do not account for time of infection nor whether the reduction is due to a single or mixed 119

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Fig. 3. Effect of the disinfectant at doses of 0.2 mg free chlorine/L -○, 0.5 mg free chlorine/L -▾, 0.8 mgfree chlorine/L -△, 1.0 mg free chlorine/L -⬛, and 2.0 mg free chlorine/L -⬜ on viability of plant pathogenic fungi at different pH of the nutrient solution. a. F. oxysporum (pH 6.5); b. F. oxysporum (pH 6.0); c. R. solani (pH 6.5) and d. R. solani (pH 6.0). Control, particular fungal pathogen in nutrient solution without any sanitation -⬤. The vertical bars represent standard errors (n: 10).

ORP 570 ± 13 mV) had no inhibitory effect on these pathogens and resulted in all plants becoming infected. Likewise the untreated nutrient solution enabled the dispersal of the fungal pathogens and the infection of test plants. In both experiments infection of all test plants (A05-A13) with F. oxysporum occurred during the third and eighth cultivation week. In contrast only 4 out of 9 test plants (A06, A08, A09, and A13) in the first run and 3 out of 9 test plants (A08, A09, and A11) in the second run were infected with R. solani. Infection with this pathogen occurred during the fifth and eleventh cultivation week. Interestingly, all R. solani-infected control plants became quickly infected with F. oxysporum although no mixed infection could be detected in control plants initially infected with F.oxysporum.

infection. Control plants as well as test plants cultivated in untreated nutrient solution exhibited the lowest yield (Table 1). The fruit of test plants only weighed half of that of control plants. The yield of control plants was slightly, but not significantly higher when nutrient solution was sanitized. In both experimental runs treatment with potassium hypochlorite significantly increased the number of fruits/plant of infected control as well as test plants (Table 2). However, a high percentage (about 40%) of unmarketable fruits emerged. In test plants, treatment of the nutrient solution successfully decreased this amount to less than 5%, whereas it had no effect on the amount of unmarketable fruits of infected control plants.

3.4. Effect of sanitising treatment on pathogen dispersal 4. Discussion The first time F.oxysporum and R. solani were isolated from untreated nutrient solution was five and six weeks after experimental set up, respectively. Mycelium of R. solani and microconidia of the F. oxysporum were first observed by light microscopy and quantified using a haemocytometer 14 weeks after experimental set up. The nutrient solution of both tanks was contaminated continuously and naturally by the infected control plants (source plants). These plants were not cured by the chlorine treatment and remained infected throughout the experiment. Treatment with the disinfectant, however, inhibited the dispersal of both fungal pathogens in all experimental series. None of the tomato plants supplied with nutrient solution treated weekly with 0.5 mg free chlorine/L for 60 min (pH 6.0 ± 0.3 and ORP 780 ± 31 mV) were infected with F. oxysporum or R. solani (Fig. 5), whereas the dose of only 0.2 mg free chlorine/L (pH 6.0 ± 0.2 and

Viability of fungal plant pathogens was affected after applying KClO-solutions considering different concentration of free chlorine and contact time. Selected dosage realigned previous studies using sodium hypochlorite (NaClO) solution as a disinfectant (Hong et al., 2003; Cayanan et al., 2009). As expected the sensitivity of the pathogens to chlorine varied according to species. The fungal pathogen F. oxysporum was shown to be killed by the disinfectant, whereas R. solani survived the treatment and showed only a small decrease in viability. Nevertheless, Cayanan et al. (2009) reported control with doses of 12 ppm (12 mg/L) at 10 min exposure. However, the application of more than 2.0 mg free chlorine/L is not advisable due to the likely risk of phytotoxicity in crop plants. The free chlorine concentration of 2 mg/L and contact time of 30 min found here to inactivate the fungal pathogen F. 120

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Fig. 4. Effect of free chlorine obtained from the disinfectant on fungal plant pathogen propagules. a. F. oxysporum no sanitation. b. F. oxysporum (2 mg free chlorine/L at 30 min). c. R. solani no sanitation. d. R. solani (18 mg free chlorine/L at 30 min). Optical microscope, magnification 400x.

oxysporum was lower buttook longer than the optimal treatment regimens reported by Cayanan et al. (2009) and Fisher et al. (2014) to control this pathogen. These authors suggested treatments with 8 mg chlorine/L for 10 min and 14 mg chlorine/L for 6 min, respectively. Fisher et al. (2014) illustrated that it is often possible to reduce the required contact time and dosage when pH was lowered to 6. This is likely to be due to the increased availability of hypochlorous acid (HOCl) as an oxidizing agent under acidic conditions. Efficacy of hypochlorous acid (HOCl) in killing microorganisms is about 40–80 times greater than hypochlorite which predominates as the pH increases (De Hayr et al., 1994) and at pH 7.0 approximately 73% of chlorine is HOCl but this increases to 96% when the pH drops to 6.0 (Nakayama and Bucks, 1986). Every horticultural operation is different and sanitation protocols using chlorine are greatly influenced by nutrient solution and irrigation water with a wide pH range of 6.0–7.5 (Fisher, 2014). Sanitation efficacy can easily be optimised by moderate acidification of the

Table 1 Yield of tomato plants during 10 harvest weeks dependent on an infection with F. oxysporum and R. solani. The data represent mean values (infected control plants n = 4, test plants n = 9). First and second run (January to May 2016). Control: no sanitation, sanitation: 0.5 mg free chlorine/L for 60 Min, weekly. Comparisons were calculated using Tukey-test. Values followed by different letters differ significantly from each other (p < 0.05). Values with the prefix ± represent the standard deviation. Run

1

2

Tomato plants

R. solani and F. oxysporum infected plants Test plants R. solani and F. oxysporum infected plants Test plants

Yield/plant [Kg] Control

Sanitation

0.46 ± 0.06 b

0.61 ± 0.09 b

0.65 ± 0.27 b 0.54 ± 0.10 b

1.38 ± 0.16 a 0.62 ± 0.09 b

0.65 ± 0.23 b

1.37 ± 0.14 a

Table 2 Number of tomato fruits and amount of unmarketable fruits during 10 harvest weeks, obtained from plants irrigated with nutrient solution disinfected weekly with KClO at 0.5 mg free chlorine/L for 60 min. The data represent mean values (infected control plants n = 4, test plants n = 9). First and second run (January to May 2016). Control: no sanitation, sanitation: 0.5 mg free chlorine/L for 60 min, weekly. Unmarketable fruit: diameter < 40 mm, discoloration or cracks. Comparisons were calculated using Tukey-test. Values followed by different letters differ significantly from each other (p < 0.05). Values with the prefix ± represent the standard deviation. Run

1 2

Tomato plants

R. solani and F. oxysporum-infected control plants Test plants R. solani and F. oxysporum-infected control plants Test plants

Mean Fruit/plant [No.]

Unmarketable fruits [%]

Control

Sanitation

Control

Sanitation

10.25 ± 1.26 b 12.75 ± 1.50 ab 10.25 ± 1.26 b 13.5 ± 1.29 ab

15.11 27.56 15 ± 29 ±

39.02 41.18 34.15 37.04

40.44 4.84 36.30 3.45

121

± 1.45 ab ± 2.30 a 2.12 ab 1.87 a

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Fig. 5. Dispersal of F. oxysporum and R.solani by nutrient solution in an NFT-system and infection of tomato plants within a 16 week survey dependent on a treatment sanitising the nutrient solution. Initially F. oxysporum-infected (plant A03, A04, B03 and B04), R. solani (plant A01, A02, B01 and B02) and non-infected (plant A05 to A13 and B05 to B13) tomato plants are cultivated in a NFT-system using recirculating nutrient solution. Plants index by B are supplied with treated nutrient solution (0.5 mg KClO/L for 60 min a week, (ORP 780 mV) whereas plants index by A are provided with untreated nutrient solution. Plants were tested weekly for a natural infection with the pathogens. The first time each pathogen was detected in a particular plant is indicated by a dark line. Survey 1 (top) and Survey 2 (below).

reduction of free chlorine by plant pathogens in high purity water was about 30% and 70% for R. solani and F. oxysporum respectively, at the same chlorine concentration and contact time. This difference most probably arose from the reaction of HOCl with the different structural components of these individual pathogens. Studies with NaClO also indicate that the affect on the free chlorine concentration is pathogendependent (Cayanan et al., 2009; Hong, 2001; Hong and Richardson, 2004). Deborde and Gunten (2008) showed that numerous inorganic and organic micro pollutants can react with hypochlorous acid and be transformed to complex Cl forms. The drastic reduction of free chlorine content in nutrient solution require a fast and precise measurement technique to avoid inappropriate dosing with the disinfectant. Therefore, an exclusively colorimetric measurement is not adequate due to

nutrient solution. As for F. oxysporum, significant improvements in sanitation efficacy of the KClO solution only occurred at pH 6.0 and 6.5. Nutrient solutions with a pH value of 6.0 are widely regarded as the most common in hydroponic production of horticultural crops. The free chlorine concentration is strongly affected by inorganic elements in the nutrient solution. Fertiliser solutions normally contain ions of iron, sulphur, boron, ammonium and other elements that will interact with hypochlorous acid (HOCl), thereby reducing its effectiveness as a disinfectant. In the first five minutes of contact time the reduction in free chlorine was 94% in nutrient solutions. This effect was probably related to the ammonium sulfate content of the nutrient solution. Ammonium can react with HClO, converting into complex Clforms as observed by Meador and Fisher (2013). In contrast, the 122

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sufficient to prevent the dispersal of PepMV. For reliable avoidance of the dispersal of the fungal pathogens tested here, 0.5 mg free chlorine/L for 1 h weekly is required, much less than in the in vitro experiments. These differences in the dosage are presumably related to i) the pathogen titer present in an artificial and natural inoculum and ii) the reservoir effect. The number of spores is extremely high in artificial suspensions compared to naturally contaminated nutrient solution. Already Raudales et al. (2014) point to observed discrepancies between pathogen mortality and disease incidence reviewing measures to control waterborne microbes in irrigation. Furthermore it can be expected that chloramines contribute to the sanitation efficacy. Although, these chemical compounds have a weaker sanitising effect than hypochlorite, they are quite stable and presumably provide a disinfection reservoir. Such a residual activity may protect the nutrient solution against recontamination for a certain time, keeping the concentration of plant pathogens in the nutrient solution below the infectivity threshold. Likewise, potassium chloride proved good for sanitation purposes as firstly potassium is a major plant nutrient and secondly, because the solution generated from potassium chloride induces less phytotoxicity than magnesium chloride (Buck et al., 2003). Phytotoxicity is of major concern when using chlorine in irrigation water and concentrations above 2.0 mg/L can lead to stunted growth, root, and leaf damage (Cayanan et al., 2009). Although Pedrero et al., (2010) reported no crop damage in fields irrigated with reconditioned waste water that contained more than 5 mg/L chlorine. Finally, it must be mentioned that chlorous disinfectants react rapidly with any oxidisable material including various molecules, elements, particles, and organisms in the nutrient solution. These reactions lower the concentration of free chlorine available to come into contact with, be absorbed by, and inactivate microorganisms. Therefore a greater amount of chlorine species is required in water with higher organic and inorganic content (Hong et al. 2003). Thus, relying on injection rate settings is insufficient. Monitoring the composition of the nutrient solution in order to determine the chlorine content to be injected, is a major prerequisite to ensure sanitation and to avoid overdosage and under-feeding. In addition, the disproportionation of chlorine monoxide into chlorate and chloride which is promoted by high temperature, UV radiation, and heavy metal ions, has to be considered. Industry quality control and official food surveillance showed that residues of chlorate were found in different vegetables (Gil et al., 2016). Chlorate levels between 0.01 and 0.92 mg/kg were found in about 20% of 600 samples of products of plant origin (KaufmannHorlacher et al., 2014). It is assumed that these residues are caused by the application of chlorine-containing disinfectants during post-harvest processes. Recently, Dannehl et al. (2016) reported on a highly significant correlation between the chlorate-accumulation in tomatoes and the application of hypochlorite as a disinfectant for hydroponic systems, although they classified the consumption of those tomatoes as harmless because maximum residue levels for chlorate (EFSA, 2015) were not exceeded. Nevertheless, we demonstrate here that with the use of ORP sensor-controlled dosing application of hypochlorite can be optimised, and combined with following the recommendations of Schuch et al. (2016) for the generation of electrolytically-derived hypochlorite, this should keep risks of chlorate accumulation low.

the limited reading time. In addition to free chlorine our control unit recorded the real-time ORP during the disinfection process. The effective ORP of 780 mV that we identified is similar to that reported by Lang et al (2008) who applied sodium hypochlorite to kill Pythium aphanidermatum and P. dissotocum. Our in vivo experiments tried to simulate the conditions at a production site. The recirculating nutrient solution was infested naturally using infected tomato plants and not artificially, by addition of a pure solution of the respective pathogens, as in most previous studies (Machado et al., 2013; Mehle et al., 2014). Thus, the initial concentration of the pathogens continuously released from infected plants is much lower. However, this continuous release is more likely to deliver inoculum when conditions are optimal for infection, and, depending on the stability and viability of the pathogens. However inoculum may build up to yield a high level of infection pressure over time. Although F. oxysporum may not live long in water there have been a number of reports of dissemination in either surface water or closed hydroponic systems. For instance tomato plants grown by NFT became infected when F. oxysorum f. sp. lycopersici was introduced into the nutrient solution, and the disease severity clearly correlated with the initial inoculum density (Evans, 1979; Vanachter et al., 1983). Two F. oxysporum infected plants were found to be sufficient to infect tomato plants throughout our entire system via the circulating nutrient solution, with the first infections detected in the first three weeks after setting up with R. solani-infected source plants. Initial fungal infection may lead to reduced competitive fitness of plants and reduced vitality making plants more susceptible to fungal pathogens. F. oxysporum f. sp. lycopersici is able to penetrate through either the differentiated tissues of the mature root or young tissues of the apical zone leading to vascular infection of the tomato plant (Olivain and Alabouvette, 1999). Typical symptoms of wilt disease can first be expected 15–20 days after artificial inoculation by the standard root dip method where roots are wounded before incubation in a conidial suspension (Nirmaladevi et al., 2016). Surprisingly, none of the F. oxysporum-infected source plants exhibited an infection with R. solani, even though fungal propagules were a permanent inoculum in the nutrient solution for the entire investigation period of 16 weeks. Dissemination of other formae speciales of F. oxysporum in closed hydroponic systems may differ. For instance, f. sp. radicis lycopersici did not lead to any infection in tomato when spore suspensions were introduced to recirculating nutrient solutions (Jenkins and Averre, 1983). Perhaps these differences are not attributed to a single forma speciales but were due to technical aspects of the experimental set up. Rattink (1990) described that large amounts of fungal spores that settled to the bottom of tanks were not transported during irrigation cycles. As shown repeatedly, recycling of water for irrigation purposes presents a potential high risk of exposure to various harmful fungal, oomycete, bacterial and viral plant pathogens in the presence of one or few infected source plants (Ivors and Moorman, 2014; Lamichhane and Bartoli, 2015; Schwarz et al., 2010; Stewart-Wade, 2011; Wick et al., 2014). Thus, there is a huge need to reduce the potential inoculum load in irrigation water. Several approaches for controlling pathogen dissemination are commercially available, but they generally entail large investments, require maintenance and may represent a substantial energy cost (Moorman et al., 2014). For instance, disinfection of recycled irrigation water by pasteurization has been practiced for some time in Europe. Unfortunately, it requires heating up the water or nutrient solution to 95 °C for 30 s to eliminate bacterial, fungal and viral pathogens (Newman, 2004). We tested the efficacy of a low concentration potassium hypochlorite solution produced by an electrolytic disinfector under practical conditions and focused our attention on the prevention of severe fungal pathogen dispersal. Recently the disinfection system was found to be suitable for inactivating the viral plant pathogen Pepino mosaic virus (PepMV) (Bandte et al., 2016). Electrolytic disinfection of nutrient solutions with only 0.2 mg free chlorine for 1 h per week was shown to be

5. Conclusion In vitro studies confirmed the antimicrobial value of electrolyticallyderived potassium hypochlorite (KClO) on fungal plant pathogens. The disinfectant seems to be suitable to contribute to phytosanitary measures in crop production, in particular disinfection of nutrient solution and irrigation water. It is important to note, that the efficacy of the disinfectant was affected by the presence of inorganic elements such as ammonium and ions of iron, sulphur and boron in nutrient solutions. The huge reduction of the main antimicrobial compound hypochlorous acid (HOCl) – up to 90% – has to be considered in disinfection regimes, 123

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in manuallyoperated as well as sensor-based units. otherwise the desired phytosanitary effect may not be achieved. Overdosing leads to phytotoxic effects or can cause the accumulation of chlorate and perchlorate in the plant, which may result in unmarketability. Hence, regular assessments are needed andthe ORP should be used as a complementary tool to pH value and free chlorine monitoring to control the sanitation efficacy of electrolytically-derived potassium hypochlorite in nutrient solutions. In vivo studies confirmed that even a few infected plants in a hydroponic system can act as an inoculum source and lead to an efficient and rapid dispersal of fungal pathogens via the nutrient solution. The presented automated sanitising treatment, however, was found to inhibit the dispersal of the economically important pathogens F. oxysporum f. sp. lycopersici and R. solani. A pH value of 6.0 with ORP 780mV appeared to be optimal for obtaining optimal sanitationyet at the minimum dose. In the frame of an integrated plant protection program, electrolytic water disinfection has to be aimed at keeping the fungal inoculum as low as possible to inhibit or at least to decrease dispersal. Cultivation with a periodic treatment will result in a low infection pressure and keeps the required dosage low in regard to chlorine concentration. This minimizes the risk of a phytotoxic reaction and the accumulation of undesirable byproducts such as chlorates. Finally sanitation of irrigation water by the electrolytic disinfection of recirculating nutrient solution enables resource-conserving production without sacrificing yield losses in vegetable production.

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