Indigenous bacteria with antagonistic and plant-growth-promoting activities improve slow-filtration efficiency in soilless cultivation

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<ul><li><p>Indigenous bacteria with antagonistic and plant-growth-promoting activities improve slow-filtrationefficiency in soilless cultivation</p><p>F. Dniel, P. Rey, M. Chrif, A. Guillou, and Y. Tirilly</p><p>Abstract: In tomato soilless culture, slow filtration allows one to control the development of diseases caused by patho-genic microorganisms. During the disinfecting process, microbial elimination is ensured by mechanical and biologicalfactors. In this study, system efficacy was enhanced further to a biological activation of filter by inoculating thepozzolana grains contained in the filtering unit with 5 selected bacteria. Three strains identified as Pseudomonas putidaand 2 as Bacillus cereus came from a filter whose high efficiency to eliminate pathogens has been proven over years.These 5 bacteria displayed either a plant growth promoting activity (P. putida strains) or antagonistic properties (B. cereusstrains). Over the first months following their introduction in the filter, the bacterial colonisation of pozzolana grainswas particularly high as compared to the one observed in the control filter. Conversely to Bacillus spp. populations,Pseudomonas spp. ones remained abundant throughout the whole cultural season. The biological activation of filter unitvery significantly enhanced fungal elimination with respect to the one displayed by the control filter. Indeed, the 6-month period needed by the control filter to reach its best efficacy against Fusarium oxysporum was shortened for thebacteria-amended filter; in addition, a high efficacy filtration was got as soon as the first month. Fast colonization ofpozzolana grains by selected bacteria and their subsequent interaction with F. oxysporum are likely responsible for filterefficiency. Our results suggest that Pseudomonas spp. act by competition for nutrients, and Bacillus spp. by antibiosisand (or) direct parasitism. Elimination of other fungal pathogens, i.e., Pythium spp., seems to differ from that ofFusarium since both filters demonstrated a high efficacy at the experiment start. Pythium spp. elimination appears tomainly rely on physical factors. It is worth noting that a certain percentage of the 5 pozzolana-inoculated bacteriafailed to colonise the filter unit and were, thus, driven to the plants by the nutrient solution. Their contribution to theestablishment of a beneficial microbial community in the rhizosphere is discussed.</p><p>508Key words: Pythium spp., Fusarium oxysporum, Bacillus cereus, Pseudomonas putida.</p><p>Rsum : La filtration lente permet de lutter contre le dveloppement des maladies en cultures hors-sol de tomate. Du-rant le processus de dsinfection, llimination des micro-organismes est assure par des facteurs mcaniques et biolo-giques. Pour amliorer lefficacit du systme, au cours de la prsente tude, nous avons biologiquement activ unfiltre en inoculant les grains de pouzzolane contenus dans la colonne filtrante avec 5 bactries slectionnes. Trois dessouches identifies comme tant Pseudomonas putida et 2 autres comme tant Bacillus cereus proviennent dun filtredont lefficacit leve pour liminer les microorganismes a t vrifie pendant plusieurs annes. Les souches deP. putida montrent une activit favorisant la croissance de plantes et celles de B. cereus des proprits antagonistes.Ds les premiers mois suivant leur introduction dans le filtre, la colonisation des grains de pouzzolane par les bactriestait particulirement leve en comparaison de celle du tmoin. A linverse des populations de Bacillus spp., celles dePseudomonas spp. furent particulirement abondantes tout au long de la saison culturale. Compar au filtre tmoin,lactivation biologique de la colonne filtrante induit une augmentation significative du niveau dlimination fongique.La priode de 6 mois ncessaire au tmoin pour atteindre son niveau maximal defficacit contre Fusarium oxysporumest raccourcie dans le cas du filtre ensemenc en bactries ; en effet, une trs grande efficacit est observe ds le pre-mier mois de filtration. Une colonisation rapide des grains de pouzzolane par les bactries slectionnes et leurs inte-ractions avec les F. oxysporum sont certainement responsables de lefficacit du systme. Ces observations suggrentune action par comptition nutritive dans le cas des Pseudomonas spp., et par antagonisme (antibiose et/ou parasitisme)pour les Bacillus spp. Llimination des autres champignons, i.e., Pythium spp., diffre de celle dcrite ci-dessus carune efficacit leve a t obtenue dans les 2 filtres ds le premier mois dexprimentation. Les facteurs physiques sont</p><p>Can. J. Microbiol. 50: 499508 (2004) doi: 10.1139/W04-034 2004 NRC Canada</p><p>499</p><p>Received 14 November 2003. Revision received 26 March 2004. Accepted 31 March 2004. Published on the NRC Research PressWeb site at http://cjm.nrc.ca on 8 September 2004.</p><p>F. Dniel, P. Rey,1 and Y. Tirilly. Laboratoire de Biodiversit et Ecologie Microbienne, ESMISAB, Universit de BretagneOccidentale-Brest, Technople Brest-Iroise, 29280, Plouzan, France.M. Chrif. Laboratoire de Phytopathologie, Institut National Agronomique de Tunisie, 43 Avenue Charles Nicolle, 1082 CitMahrajne, Tunis, Tunisie.A. Guillou. CATE, Station Exprimentale de Vzendoquet, 29250 Saint-Pol-de-Lon, France.</p><p>1Corresponding author (e-mail: patrice.rey@univ-brest.fr).</p></li><li><p>certainement responsables de llimination des Pythium spp. On peut noter qu un certain pourcentage des bactriesinocules na pas colonis le support dans la colonne filtrante, mais a t vhicul par la solution nutritive jusquauxplantes. Leur contribution ltablissement dune microflore bnfique dans la rhizosphre est discute.</p><p>Mots cls : Pythium spp., Fusarium oxysporum, Bacillus cereus, Pseudomonas putida.</p><p>Dniel et al.</p><p>Introduction</p><p>In soilless cultivation, the water, which comes fromsources such as lakes, rivers, and wells, is generally colo-nized by numerous bacteria and fungi, some of which arepathogenic to plants (Stanghellini and Rasmussen 1994).Once introduced, these microorganisms are easily spreadthrough the greenhouse by recirculated solutions. Closed hy-droponic systems minimize pollution by reusing the run-off,however, they concomitantly increase the risks of pathogenattacks by using water contaminated with pathogenic micro-organisms (McPherson et al. 1995; Van Os 1999). Findingmethods that prevent such disinfection has become a majorchallenge.</p><p>Several effective methods, such as heat treatment,ozonization, ultraviolet radiation, and chlorination, havebeen proposed for the disinfection of nutrient solutions(Ehret et al. 2001; Goldberg et al. 1992; Rey et al. 2001;Runia 1995; Steinberg et al. 1994). Using ultraviolet irradia-tion on recirculating solution has been proven to controlPythium spp.induced root rot in tomato and cucumberplants (Postma et al. 2001; Zhang and Tu 2000). Unfortu-nately, this active method affects the total microflora by de-stroying not only the target pathogen, but also nontargetmicroorganisms. (Zhang and Tu 2000). Similarly, Poncet etal. (2004) demonstrated that chlorine reduced bacterial di-versity in the rhizosphere. Postma et al. (2000) looked at therole of natural microflora in suppressing certain diseases bycomparing systems with and without their originalmicroflora. In fact, natural microflora have often shown acertain ability to suppress diseases (Berger et al. 1996; Chenet al. 1998). Tu et al. (1999) observed that a large bacterialpopulation in the rhizosphere can limit the extent of Pythiumroot rot, which led them to speculate about the involvementof resident bacteria in disease biosuppression. Generally, ac-tive disinfecting methods are unable to preserve nonpatho-genic microflora (McPherson et al. 1995) because theynegatively affect the suppressing potential of naturalmicroflora against certain pathogens, such as Pythiumspp. and Phythophthora spp.</p><p>To prevent this undesirable effect, the attention of re-searchers, over the last decade, has been directed on a prom-ising method for soilless cultivation, the slow-filtrationtechnique. During the disinfection process, the nutrient solu-tion flows slowly through a filter unit, which is filled withdifferent substrates, such as fine sand, rockwool flocks, orpozzolana grains. This passive method eliminates pathogenswithout destroying the natural microflora (Van Os andPostma 2000). Among the pathogens eliminated atsubstantial rates with this technique are zoosporic fungi (e.g.,Phytophthora spp.), bacteria (e.g., Xanthomonas campestris),nematodes, and even viruses (Ehret et al. 2001; Van Os et al.1999). Analysis of the total microflora has identified a clear</p><p>change in the bacterial community after the nutrient solutionhas passed through the filter unit (Postma et al. 1999). Inter-estingly, slow filtration keeps a part of the natural microfloraalive; it has been proven harmless to specific groups of bac-teria. Mechanical and biological factors are thought to be re-sponsible for the effectiveness of the system. However, untilnow, experiments conducted to improve system effective-ness have focused on determining flow rates through the fil-ter unit and on the nature and optimal depth of substrates infilter tubes (Wohanka et al. 1999). Brand and Wohanka(2001) showed that the formation of bacterial microcoloniesor biofilms on substrates is a key factor in enhancing effi-ciency. Brand (2000) isolated and identified a large numberof these bacteria, and showed that the dominating genus,Pseudomonas, contributed to more than 50% of all isolates.Of the other isolates, 10.2% were identified were assignedas Bacillus.</p><p>This study was designed to optimize biofiltration using se-lected bacteria. A new filter system often needs severalmonths to reach peak efficiency; our aim was to shorten thistime by inoculating the filter with specific bacteria. To dothis, bacteria were isolated from an effective filter, identi-fied, screened for their efficiency against fungal pathogens,and then a second, new, filter was inoculated with the fivemost abundant bacteria. The development of bacteria onpozzolana grains in the filter was also studied, and the effi-ciency of our procedure was assessed against a control filter.</p><p>Materials and methods</p><p>FiltersTwo filter units were used: one was inoculated with five</p><p>selected bacteria at the beginning of the cultivation season,and the other one served as a control. Each filter consisted ina plastic pipe (220 cm long, with an inner diameter of40 cm), and was filled with pozzolana grains (14 mm diam-eter), which acted as the filtering medium. Nutrient solution(Kemira, France) flowed through a layer of pozzolana 100-cm thick, which was deposited above three successive layersof graded gravel (28, 816, and 1632 mm) that had anoverall thickness of 40 cm. The upper water layer was regu-lated by a float switch, placed 4050 cm above thepozzolana surface. The filtration rate ranged from 100150 Lh1m2. The filter units were set, at room temperature,in 2 separate areas of an experimental tomato greenhouse atroom temperature. For each filter, the same nutrient solutionfed plants throughout the cultivation season.</p><p>Identification of bacteria used to inoculate filtersBacteria were selected from the top layer of pozzolana</p><p>grain from a three-year-old filter, chosen because of itshighly efficient elimination of fungi, such as Pythiumspp. and Fusarium oxysporum and bacteria, such as total</p><p> 2004 NRC Canada</p><p>500 Can. J. Microbiol. Vol. 50, 2004</p></li><li><p>bacteria microflora and fluorescent Pseudomonas, from thenutrient solution (Rey et al. 1999).</p><p>To isolate bacteria, 7 g of pozzolana grains were washedthree times with 63 mL of a solution of physiological water(0.85% NaCl) and Tween 80. Pozzolana grains were thensonicated in 63 mL of the same solution for 90 s at the high-est setting, using a VibraCell (Bioblock Scientific, Illkirch,France) bench sonicator. The solution sample was platedwith a spiral plater on plate-count agar (PCA) plates. We se-lected the three most abundant bacteria, and chose two oth-ers for their typical features; all of them were purified andidentified to the species level using biochemical tests, suchas catalase and oxydase activities and growth on selectivemedium, for example, King B. Pseudomonas was identifiedwith API 20 NE galleries, and Bacillus with API 20 E andAPI 50CH (API galleries, BioMrieux, France). For eachisolate, the profiles issued from API tests were analyzedwith APILAB software (BioMrieux, France), and identifi-cation down to the species level was expressed as a percent-age of probability.</p><p>Effect of the five selected bacteria on plant growthPlants were inoculated with the five selected bacteria us-</p><p>ing the following procedure.</p><p>Plant materialTomato seeds (Lycopersicon esculentum Mill. cv. Prisca)</p><p>were sterilized by immersing them in 70% ethanol for5 min, soaking them in 5% aqueous sodium hypochlorite for10 min, and rinsing them thoroughly three times in steriledistilled water. They were then placed on water-soaked filterpaper (Whatmann No. 1) in Petri dishes, and kept in the darkat 25 C. One week later, all the germinated seeds were indi-vidually transferred to sterile glass tubes (20 150 mm) thatcontained 12 mL of the nutrient solution described byHoagland and Arnon (1938). A sterile piece of filter paper(90 180 mm), perforated in its centre, was placed in eachtube to provide a solid support for the growing roots.Plantlets were grown under a 16-h light (at 25 C) : 8-h dark(at 16 C) photoperiod in a Sanyo growth cabinet (Antony,France). Light was provided by Mazdafluor Incandia 830tubes (Mazdafluor, France), which had an intensity of40 molm2s1.</p><p>Plant inoculation with bacteriaEach bacterium was precultured in tubes that contained</p><p>9 mL of nutrient broth, for 18 to 24 h at 30 1 C. Theywere then grown in 250-mL Erlenmeyer flasks, which werefilled with 125 mL of nutrient broth. The cultures wereincubated for 24 h on a rotary shaker (120 r/m) in the dark, at30 1 C. Three-week-old plantlets were inoculated bypouring 1 mL of each bacterial strain in the plant nutrientsolution at 2 different concentrations: 106 CFU(colony form-ing units)mL1 and 109 CFUmL1. For each experiment, 18to 23 plantlets were used. Control plants were treated with1 mL of distilled water. Root systems and shoots of plantswere weighed 3 weeks after inoculation with bacteria. Statis-tical analyses were performed using Duncans test at 95%confidence. The statistical program used for analysis wasStatGraphics, release 4.0 Manugistic Inc.).</p><p>Effect of the five selected bacteria on fungal growth invitro</p><p>This study used the dual-culture technique. The selectedbacteria were tested against the fungal species frequently de-tected in soilless tomato cultivation. Some of these fungi arepathogenic, such as Colletotrichum coccodes, F. oxysporumf.sp. radicis lycopersici (FORL), Pythium group F, Pythiumirregulare, and Rhizoctonia solani; others are nonpatho-genic, such as Acremonium sp., Aspergillus ochraceus,F. oxysporum, Pythium oligandrum, and Trichoderma viride.To produce bacterial inoculum, each bacterium wasprec...</p></li></ul>

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