Scientia Horticulturae

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1 Scientia Horticulturae 125 (2010) Contents lists available at ScienceDirect Scientia Horticulturae journal homepage: Dissemination of Phytophthora cactorum, cause of crown rot in strawberry, in open and closed soilless growing systems and the potential for control using slow sand filtration F. Martínez a, S. Castillo b, E. Carmona b, M. Avilés b, a Departamento de Ciencias Agroforestales, Universidad de Huelva, Ctra. Palos s/n, 21819, Palos de la Frontera, Huelva, Spain b Departamento de Ciencias Agroforestales, Universidad de Sevilla, EUITA, Ctra. Utrera s/n, Sevilla 41013, Sevilla, Spain article info abstract Article history: Received 5 June 2009 Received in revised form 25 March 2010 Accepted 13 May 2010 Keywords: Phytophthora cactorum Crown rot Strawberry Hydroponics Closed growing system Pathogen dissemination Disease control Slow sand filtration Soil-borne fungi Closed (recirculating) growing systems provide a greater potential for the dispersal of water-borne plant pathogens and disease expression compared to open (run-to-waste) systems. Here we studied the effects of three soilless growing systems (open, closed, and closed with slow sand filtration) on the dispersion of Phytophthora cactorum propagules and the severity of the crown rot disease in strawberry (Fragaria ananassa Duch.). The plant-growth medium used was coir fiber. The three growing systems showed the same density of P. cactorum propagules in the water drained from the growing media. However, propagules of this pathogen were not detected by the baits in the filtered solution recovered from slow sand filtration. In all systems Phytophthora propagules dispersed from the inoculated plant to adjacent uninoculated plants. At the end of the first crop no differences in the severity of crown rot were found between the different systems of crop culture. However, at the end of the second crop cycle, crown rot in the closed soilless system without slow sand filtration was more severe than in the other two systems. These results demonstrated that the commercial potential of slow sand filtration to prevent propagule dispersal and hence suppress crown rot in strawberry crops grown in a closed culture system Elsevier B.V. All rights reserved. 1. Introduction Soilless growing systems offer many advantages. Not only do they contribute to improve crop yields and quality but they also conserve energy and water and reduce the application of crop protection products. In addition, soilless systems reduce the risk of crop damage from soil-borne pathogens through separation from infested soil (Paranjpe et al., 2008). Nevertheless, the development of these systems has not eliminated root diseases entirely and some pathogens, e.g. Pythium spp. and Phytophthora spp. continue to pose a serious threat in recirculating irrigation systems (Thinggaard and Andersen, 1995; Strong et al., 1997). To reduce the spread of soil-borne pathogens in recirculating systems, it is important to disinfest the nutrient solution (Garibaldi et al., 2004). A number of active techniques have been used for this purpose. These include ultraviolet radiation treatment (Runia, 1994a); heat treatment (Runia et al., 1997); ozonification (Runia, 1994b); the application of surfactants (Stanghellini and Miller, 1997), wetting agents (Minuto et al., 2000), and biocontrol agents (Stanghellini and Miller, 1997; Guetsky et al., 2001). Corresponding author. Tel.: ; fax: address: aviles@us.es (M. Avilés). In addition, passive techniques, such as slow sand filtration (SSF) (Wohanka, 1995), have been developed to remove pathogens from recirculating nutrient solutions. In passive techniques, mechanical, chemical and biological properties may interact. Passive methods require less energy and chemical compounds than active techniques. However, passive disinfection is selective as it does not remove all microbes from systems (Postma et al., 1999). The surviving microflora, a proportion of which may be antagonistic to important pathogens, contribute to disinfestation of the recirculating hydroponic solution in closed soilless systems with SSF (Van Os, 2004). Hultberg et al. (2008) report a selective effect of the filter to increasing the populations of biosurfactant-producing pseudomonads in closed soilless systems. The purpose of SSF is to provide both physical and biological mechanisms for selective filtration of pathogen propagules in an environment which retains and ideally, enhances, the resident antagonistic microflora but to prevent the dispersion of soil-borne pests and diseases (Postma et al., 1999; Wohanka, 1995; Wohanka et al., 1999). Thus, SSF contributes to the suppression of certain diseases (Postma et al., 1999; Van Os et al., 1999; Van Os and Postma, 2000). The type of soilless growing system has a significant effect on the microbiological environment around the roots. Closed systems with SSF are reported to exhibit a lower density of copiotrophic (Langer et al., 2004) bacteria and fungi than open systems (Martínez /$ see front matter 2010 Elsevier B.V. All rights reserved. doi: /j.scienta

2 F. Martínez et al. / Scientia Horticulturae 125 (2010) et al., 2005a). While several studies have demonstrated that SSF removes Pythium spp. and Phytophthora spp. propagules (Wohanka, 1995; Wohanka et al., 1999), other report that this kind of filtration and ultraviolet radiation significantly reduce root rot caused by Phytophthora cryptogea (Garibaldi et al., 2003). P. cactorum is a soil-borne pathogen that infests many herbaceous and woody species (Erwin and Ribeiro, 1996). Strawberry crown rot caused by P. cactorum was first reported in northern Germany in 1952 (Nienhaus, 1960). It has since plagued strawberry cultivation throughout Europe and in many other parts of the world. This microorganism has been reported in Huelva (Spain) (Santos et al., 2002) and has been introduced into soilless growing systems by infected, but latent, cold store plants (Huang et al., 2004) and via contaminated irrigation water (Thinggaard and Andersen, 1995; Strong et al., 1997). Certified pathogen-free strawberry plants do not necessarily guarantee the absence of P. cactorum due to the potential for latent infections and the lack of commercially robust detection techniques (Tello-Marquina et al., 1996). Soil-borne pathogens spread rapidly and disease epidemics can occur, particularly under certain conditions. Nevertheless, in some closed hydroponic systems little or no disease occurs, despite the presence of zoosporic pathogens. This suppression is thought to be related to microbiological activities (McPherson et al., 1995; McPherson, 1998; Postma et al., 2000a,b). McPherson (1998) found that P. cryptogea infection in tomato was partially suppressed in closed soilless growing systems. Moreover, when suppression did occur, it developed slowly and it was lost or reduced when the system was switched to run-to-waste (open soilless systems) and replenished with fresh rather than recirculating solution. This was considered to be due to a number of factors which created an adverse environment for the motile zoospores, thus reducing the infection potential. Here we examined the potential for spread of P. cactorum using open and closed (recirculating) systems of crop culture in a coir fiber substrate and also evaluated the use of SSF to prevent propagule dissemination via the recirculating hydroponic solution and the severity of crown rot in strawberry plants. 2. Materials and methods 2.1. Experiment design The assays were performed in Huelva (SW Spain) in a tunneltype methacrylate greenhouse in a randomized complete block design with three replicates. The assay was repeated twice. Each assay lasted 2 years (two crop cycles) in the same growth medium. Strawberry plug plants were used. The plants were then transplanted to three growing systems at the beginning of each crop cycle. The typical crop cycle in SW Spain begins in October and ends in May. The following soilless growing systems were tested: open (O), closed (C) and closed with slow sand filtration (CSSF). For all systems, non-inoculated rows of plants were available as controls. The experimental design involved 3 blocks 3 systems 2 rows (inoculated and non-inoculated) = 18 experimental units (Fig. 1). Each experimental unit consisted of one continuous crop row 6 m long. Each row had 65 hanging plants of the Camarosa variety. Plant density was 11 plants/m 2. The growth medium used was washed coir fiber (Bas Van Buuren B.V., Maasland, Netherlands). The plants were irrigated with a drip system (1 drip/plant, flow of 2.3 L/h). The growth medium was placed in containers called Hanging Bed- Pack (Polygal Plastic Industries, Ramat Hashofet, Israel). In the CSSF, the drained water was pumped into the sand filter to maintain a water layer of 35 cm, thereby obtaining a final flow rate of L/(m 2 h). The filter was built as described by Van Os et Fig. 1. Scheme of an assay layout (from overhead). Legend: O = open, C = closed and CSSF = closed with slow sand filtration; I = inoculated row; NI = non-inoculated row. al. (1999). One filtering layer of 100 cm (sand size ) and two drain layers of 10 and 15 cm (sand size 2 3 and 8 12 mm, respectively) were used. The three replicates for the inoculated and non-inoculated rows of the three systems were independent and fed by separate means. Weekly nutritional analysis of drainage water allowed regular adjustment to ensure a balanced nutritional regime between the different treatments. Each experimental unit had either two or three independent storage tanks: (1) nutrient solution storage tank (storage tank in which the nutrient solution was mixed with the drain water from the C and CSSF systems and subsequently stored); (2) drain water tank (storage tank of drain water from the strawberry crop); (3) filtered solution tank (storage tank of effluents from the sand filter only in the CSSF system) (Fig. 2). The content of the drain water tank was pumped into the nutrient solution tank to be mixed before being passed through the sand filter in the CSSF system. The mixture was passed through the sand filter and stored in the filtered storage tank. The filtered solution was applied to the strawberry crop. The sand filter was activated by passing the nutrient solution through it 1 month before transplanting the strawberry plants. Tap water was used to prepare the nutrient solutions. The last 12 plants at the end of each row were artificially inoculated before nutrient solution run-off (Fig. 2). The drained solution comprised leachates from non-inoculated and inoculated plants in each crop row. Inoculation was performed 2 weeks after transplantation and during the two crop cycles. P. cactorum from inoculated plants can infect other plants via nutrient solution and plant plant contact. The P. cactorum strains were previously isolated from a naturally infected strawberry plant grown in the field. P. cactorum isolates were previously evaluated by a pathogenicity test. The most virulent isolates were PC1 and PC5-8. A crown incision, 2 mm deep and 5 mm wide, was made in each plant. This incision was then filled with two plugs of V8 medium (Dhingra and Sinclair, 1995) from each isolate (PC1 and PC5-8). The incision was then covered with parafilm Dispersion of P. cactorum Water samples were taken regularly (every 15 days) from collection tanks 2 and 3 (see Fig. 2) for monitoring the proportion of

3 758 F. Martínez et al. / Scientia Horticulturae 125 (2010) Table 1 Percent recovery of P. cactorum in closed, open and closed with SSF growing systems over two cropping cycles. Growing system % baits with P. cactorum in drain water a First crop cycle b Second crop cycle b Closed Open Closed with SSF Analysis of variance was performed with transformed data (% baits infested + 0.5) 1/2. a Average of the 11 sample dates per crop cycle. b System not significant. samples were taken every 1.20 m from the row (6 locations per row) for the three soilless systems. Fifty subsamples (3 g growth medium in 6 ml distilled water) were analyzed using the host bait technique. Data are expressed as percentage of infested baits. For multifactorial ANOVA, several sampling dates were considered as independent factor Severity of disease In the artificially inoculated crop rows disease severity was measured at the end of the crop cycle in non-inoculated plants. Disease severity in the crown was measured in all uninoculated plants using the following seven-point scale: 1 = totally white crown tissue; 2 = few brown speckles; 3 = small patch of necrosis; 4 = more than 50% necrotic crown width; 5 = dead plant at the end of bioassay; 6 = dead plant 1 month before the end of crop cycle; 7 = dead plant 2 months before the end of crop cycle; 8 = dead plant 3 months before the end of the crop cycle. Positive infection by P. cactorum was determined by isolation of the pathogen from the crown of symptomatic plants at the end of the each crop cycle through isolation onto PARP-V8 (Ferguson and Jeffers, 1999). The mycelium and sporangia of P. cactorum were identified by microscopy Data analysis Fig. 2. Scheme of soilless growing systems: O = open, C = closed and CSSF = closed with slow sand filtration. Legend: (1) nutrient solution storage tank (storage tank in which the nutrient solution was mixed with the drain water from the C and CSSF systems and subsequently stored); (2) drain water storage tank (storage tank of drain water from the strawberry crop); (3) filtered solution storage tank (storage tank of effluents from the sand filter); (4) sand filter; (5) the last 12 plants from each row were artificially inoculated. host baits infested with P. cactorum. Analyses were repeated on 11 sampling dates during the first crop cycle and 11 days during the second cycle. The concentrations of propagules were evaluated using a host baits technique using immature carnation petals. Infested baits were plated in PARP-V8, a semi-selective medium (Ferguson and Jeffers, 1999). The presence of both mycelium and sporangia of P. cactorum on the baits was estimated by microscopy. Each count consisted of 10 carnation petals per sample from the drain water and filtered solution tanks of the CSSF and also from the drain water tank of the O and C systems. The recovery of P. cactorum from growth medium was estimated at the end of the first crop cycle. Growth medium with pieces of root Data from two assays for each crop cycle were pooled for final analysis after finding no significant system assay interaction in a preliminary analysis of variance. The following variables were analyzed by multifactorial ANOVA: the percentage of P. cactorum infested baits in drain water and growing medium for the three soilless systems, and in the drain water and filtered solution for the CSSF system, as well as the severity of crown rot in the three soilless systems. Statistically significant means were compared by Tukey s whole significant difference test (P 0.05). 3. Results The percentage of P. cactorum infested baits in the drain water tank from the three soilless systems revealed no significant effect of the system (Table 1), though the host baits did show a significant Table 2 Percent recovery of P. cactorum in closed growing system with SSF over two cropping cycles. Source of samples % baits with P. cactorum a First crop cycle Drain water 17.1 a 34.2 a Filtered solution 00.0 b 00.0 b Second crop cycle Values followed by different letters within each column, are significantly different based on Tukey s test at (P 0.05). a Average of the 11 sample dates per crop cycle.

4 F. Martínez et al. / Scientia Horticulturae 125 (2010) Table 3 Percent recovery of P. cactorum from the growing medium (coir fiber) in the three growing systems at the end of the first crop cycle. Distance (m) a (0 1) 0 b (1 2) 0 b (2 3) 0 b (3 4) 0 b (4 5) 0.7 b (5 6) 20.2 a P. cactorum % infested baits Each value is the mean of three systems for three replicates. Values followed by different letters within each column, are significantly different based on Tukey s test at (P 0.05). a Distance (m) measured from the beginning of the line (end opposite to zone of inoculated plants). Table 4 Crown rot severity a in uninoculated plants in the three soilless growing systems at the end of each cropping cycle. Growth system Crown rot severity First crop cycle b Closed 2.3 a 2.9 a Open 1.9 a 1.0 b Closed with SSF 1.8 a 1.9 b Second crop cycle b a Crown rot severity was scored based on a symptom severity scale where: 1 = totally white crown tissue, 2 = few brown speckles, 3 = small match of necrosis, 4 = more than 50% necrotic crown width, 5 = dead plant at the end of bioassay, 6 = dead plant 1 month before the end of crop cycle, 7 = dead plant 2 months before the end of crop cycle and 8 = dead plant 3 months before at the end of the crop cycle. b Values followed by different letters within each column, are significantly different based on Tukey s test P difference between the non-disinfected drain water and the drain water disinfected by SSF (Table 2). The percentage of P. cactorum infested baits in the coir substrate revealed no significant effect of the system (data not shown). For all systems, dispersal of P. cactorum in the coir substrate was detected between the inoculated plants and the adjacent uninoculated plants at the end of the first crop cycle (Table 3). In the first crop cycle, no significant differences in disease severity between the systems were observed at the end of the crop cycle (Table 4). However, at the end of the second crop cycle, significant differences in the severity of crown rot were detected between the closed system and the other two systems (Table 4). Crown rot was not observed in the non-inoculated rows of plants (control) in the three systems at any time in any crop cycle. 4. Discussion P. cactorum propagules were removed by SSF. This filtration method has also been reported to remove these propagules in other works (Martínez et al., 2005b, 2006). In this regard, propagules of oomycetes (Pythium spp. and Phytophthora spp.) are removed by SSF (Runia et al., 1997; Van Os et al., 1999; Wohanka, 1995; Wohanka et al., 1999) and this filtering technique shows high efficacy against other phytopathogenic fungi and bacteria (Waechter-kristensen et al., 1997). Slow filtration is a process in which a number of factors determine efficacy; i.e. pathogen, filtration rate, grain size, filter medium, specific surface, temperature, microbial life and preferential channelling (Van Os and Postma, 2000). SSF did not affect the percentage of infested baits for P. cactorum in the drain water tanks of the systems tested. However, our results confirm that SSF is an effective method for disinfesting recirculating nutrient solution in closed soilless systems. López-Medina et al. (2006) proposed that the combination of SSF and closed soilless growing systems does not affect yield. Despite the removal of propagules by SSF, in the first crop cycle no significant differences in severity of crown rot between the systems were observed. This observation is attributed to an effective dispersion of P. cactorum propagules through the crop row either through root to root contact or through the movement of zoospores in the free water. In the second crop cycle, we detected an increase in the severity of crown rot in C, as expected. It was noted that O and CSSF had less disease than C. However, no significant differences between the O and CSSF systems were detected. This finding is consistent with the observation that SSF removes P. cactorum propagules. SSF had no effect in the first crop cycle, but it reduced the severity of the disease in the second. In the second crop cycle the dispersion of P. cactorum propagules through the crop row either through root to root contact or through the movement of zoospores in the free water may have been reduced by the high microbial activity of the reused growth medium. This high microbial activity is possibly associated with high accumulation of plant exudates and plant decomposition residues from the first crop cycle (Martínez et al., 2009). Hardy and Sivasithamparam (1991) proposed that the lytic activity of a large population of microorganisms suppresses or kills Phytophthora spp. propagules. In this regard, the transmission of P. parasitica was very low when potting mix was not pasteurized in an ebb-and-flow subirrigation system (Strong et al., 1997). 5. Conclusions Closed (recirculating) growing systems provide a greater potential for the dispersal of water-borne plant pathogens than open (run-to-waste) ones. 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