| Assessing efficacy of ultra-filtration and slow filtration in soil-less production by molecular detection | 7 | Assessing efficacy of ultra- filtration and slow filtration in soil-less production by molecular detection of Pythium oligandrum and Bacillus subtilis as model organisms Gautier CALMIN 1, Guillaume DENNLER 1, Lassaad BELBAHRI 1, Andreas WIGGER 2, François LEFORT 1* Ms. received 14th june 2007, accepted 20th november 2007 T Abstract Greenhouse crops are mainly grown on artificial substrates in soil-less systems. Re-circulating nutrient systems offer an advantageous alternative for controlling nutrient solutions leaching from greenhouses into the environment, which repre - sents an environmental issue in greenhouse production areas. However, the potential for the rapid spread of plant pathogens, is the main hindrance to the general adoption of re-circulating nutrient systems by the greenhouse industry. Here we assessed by real time PCR the efficacy of filtration for eliminating oomycete and bacterial pathogens, artificially added in the re-circulating nutrient solution, by using a tangential ultra -filtration system, a slow sand filter (SSF) and a slow Rockwool granule filter (SRF). The oomycete Pythium oligandrum and the bacterium Bacillus subtilis were used as model organisms because of their easy culture, their lack of pathogenicity and their similarity with common oomycete and bacte - rial plant pathogens. Using real time PCR and species specific oligonucleotides showed that ultra -filtration and SRF filter were highly effective in removing P. oligandrum and B. subtilis , while the SSF, although completely removing P. oligandrum , was less effective for B. subtilis removal. Real time PCR showed to be a much quicker alternative to classical counting on agar plate culture. Keywords : recycled nutrient solution, real time PCR, Bacillus subtilis , Pythium oligandrum . T Introduction vironmental pollution caused by the green house in - dustry, and questioning about the sustainability of For environmental and economic reasons, recycling such an activity has been resulting in the promotion irrigation runoff is becoming a common practice in of closed re-circulating nutrient systems. Although greenhouse crop production. Most greenhouse crops re-circulation of nutrient solutions proved to be eco - are grown on artificial substrates in soil -less systems nomically rewarding, possible contamination of irri - because of the improved control over water, aeration, gation tanks and re-circulating systems by important nutrition and root distribution. These systems were plant pathogens such as Pythium and Phytophthora developed as drain-to-waste or open systems, in species, is listed among the main limitations. which excess nutrient solutions were allowed to Contamination of re-circulating nutrient solutions drain to the soil and groundwater. Concern about en - could also be a potential way for contaminating fruits 1 Group Plant and Pathogens, Research Institute Earth, Nature and Landscape, Geneva Institute of Technology, Architecture and Landscape, School of Engineering of Lullier, University of Applied Sciences of Western Switzerland, 150 Route de Presinge, 1254 Jussy, Switzerland. 2 Section Culture Maraîchère, Centre d’Enseignement Professionnel de Lullier, 150 Route de Presinge, 1254 Jussy, Switzerland. * Correponding author. E-mail: [email protected] | ARCHIVES DES SCIENCE S | Arch.Sci . (2008) 61:7-16 | | 8 | Gautier CALMIN et al. Assessing efficacy of ultra-filtration and slow filtration in soil-less production by molecular detection | and vegetables with food borne pathogens or fungal In the present study, using specifically designed real toxins (Powell et al. 2002; Steele and Odumeru time PCR, we assessed the efficacy of ultra -filtration 2004). and SSF filtration on tomato and eggplant green - house soil -less cultures for removing microorganism Development of closed systems required effective (Pythium oligandrum, Bacillus subtilis ) artifi - disinfection of plant nutrient solutions (Ehret et al. cially added in the circulating nutrient solution. 2001) and several active and passive methods have been tested in the past years: UV treatment, heat treatment, ozonization, membrane filtration, such as T Materials and methods ultra -filtration or slow filtration (Runia 1995). Ultra -filtration (membrane filtration ) may remove up Greenhouse trials to 6 logs of bacteria, viruses or parasites (Taylor and Wiesner 1999). On the other hand, slow sand filtra - Experimental trials on closed soil -less systems were tion (SSF) techniques, involving filtration through a carried out at the Vegetable Crop section of the Pro - sand bench, present advantages of low installation fessional Training Centre of Lullier (Jussy, Switzerland). and maintenance costs, but do not totally eliminate Tomato plants, hyb. Temptation (Enza Zaden BV, The the microflora. It appears however that Pythium and Netherlands), 30 days old were grown on Rockwool Phytophthora could be totally removed by SSF cubes (Rockwool, Mid-Glamorgan, UK) and transplant - (Runia 1995), while bacterial removal was less effec - ed on Rockwool substrate (Rockwool) during the year tive at 80 to 98.8% of total bacteria (Wohanka et al. 2005. Eggplant plants, hyb. Combo (Rijk Zwaan Zaad - 1999). teelt en Zaadhandel BV, The Netherlands) and hyb. Fig. 1: Schematic representation of the filtration units used in this study. A. Biofiltration unit. B. Ultrafiltration unit. | ARCHIVES DES SCIENCE S | Arch.Sci . (2008) 61:7-16 | | Assessing efficacy of ultra-filtration and slow filtration in soil-less production by molecular detection Gautier CALMIN et al. | 9 | Cristal (Semillas Fito S.A., Spain) were grown in a sim - ilar system, in which Rockwool was replaced by coco peat (type BLG 943 DUO , 100 cm x 20 cm x 12 cm , weight 2,4 kg , Biogrow, Montescot, France ). Plants were maintained on concrete benches with a drip irrigation system (one water emitter for each plant, flow rate of 2 L/h) in a soil -less culture. General information on wa - ter, fertilizers and the nutrient solution adopted dur - ing the trials was reported by Garibaldi et al. (2003). Slow-filtration The slow Rockwool filtration (SRF) system was con - Fig.2: A: Slow sand filter (SSF). structed as described by Wohanka et al. (1999) with Grodan granules of hydrophile type 012-519 (Gro - dan, Hedehusene , Denmark ). The slow Rockwool fil - The slow sand filter (SSF) was a BIO AQUA FILTER ter (SRF) is schematically described in Fig. 1A and supplied by Rossat SA (Payerne, Switzerland) and is shown on Fig. 2C. The drain water was pumped into shown on Fig. 2A. The BIO AQUA FILTER is based on the Rockwool filter to maintain a water layer of 35 the use of quartz particles (Vasilgrit quality 25/16 cm. Air forced by pressure into the filter enhances (0,4-0,8mm) and Vasilgrit quality 16.14.1 (0,6/1,2 efficiency of the system. The effluent flow rate from the mm), Sifraco, France). The drain water was pumped filter was adjusted by a flow-meter located between into the coral filter. The effluent flow rate from the the filter and the disinfected water storage tank, ob - filter was adjusted by a water valve located between taining a final flow rate of 40 L.m -2 .h -1 . the filter and the disinfected water storage tank, ob - taining a final flow rate of 100 L.m -2. h-1 . Both slow fil - ters were allowed to ripe during 4 weeks of func - tioning after starting before the experiment. Fig.2: C. Slow rockwool filter (SRF). Ultra-filtration The ultra -filtration system was supplied by Membratec SA (Sierre, Switzerland). The pilot ultra - filtration unit adopted is schematically described in Fig. 1B and shown on Fig. 2B. The capillary ultra -fil - tration membrane (HYDRAcap, Hydranautics, USA) has a capillary configuration with inside diameter of 0.8 mm and outside diameter of 1.2 mm. Molecular weight cut-off of the HYDRAcap capillary membrane is 100.000-150.000 Daltons. The fiber polymer is polyethersulfone, modified to maintain a hydrophilic property. The flow pattern is inside out (feed water enters bore of the capillary). Fig.2: B. Ultrafiltration filtering station | ARCHIVES DES SCIENCE S | Arch.Sci . (2008) 61:7-16 | | 10 | Gautier CALMIN et al. Assessing efficacy of ultra-filtration and slow filtration in soil-less production by molecular detection | Isolates, culture conditions and inoculation Sampling of the nutrient solution before filtration was performed by collecting 1 L samples from the raw Pythium oligandrum Drechsler, Strain No. 1133 water buffer tank (Fig1) for each installation (SSF, (Provided by Yves Tirilly, Ecole Supérieure de SRF and ultrafiltration). Filtered nutrient solution Microbiologie et Sécurité Alimentaire, Université de samples (1 L) were taken by a deviation on the fil - Bretagne occidentale), was grown on V8 medium or tered water from the filtrate water buffer tank (Fig on pea broth medium at 24°C in the dark and regu - 1) for each installation (SSF, SRF and ultrafiltration). larly sub-cultured. For the production of inoculum, P. Samples (5 L ) were concentrated by passing through oligandrum was cultured in a liquid pea broth a 47 mm diameter Durapore ® 0.22 µ m mesh mem - medium (Le Floch et al. 2003) . Plastic bottles con - brane. Bacteria present on the filter were then re - taining 150 ml of culture medium autoclaved at 121 leased by vortexing the filter in sterile water (2 ml) °C for 15 min were inoculated with 4 disks of P. oli - and serial dilutions plated on agar plates in 90 mm gandrum (10 mm in diameter), then incubated in diam. Petri dishes. Bacteria colonies were checked the dark for 14 days at 25 °C. Mycelial mats were then visually for colony shape and morphology. removed and fragmented into distilled water using a Waring blender (Le Floch et al. 2003). Plate counting Bacillus subtilis subsp. spizizenii (strain DSMZ 6405) used in this study was obtained from the DSMZ Counting was carried out on Petri plates after (Deutsche Sammlung von Mikroorganismen und spreading diluted samples on V8 medium supple - Zellkulturen GmbH: Braunscheig, Germany) and mented with ampicillin, pimaricin and rifampicine for maintained on Luria-Bertani (LB) medium.