Indigenous bacteria with antagonistic and plant-growth-promoting activities improve slow-filtrationefficiency in soilless cultivation
F. Dniel, P. Rey, M. Chrif, A. Guillou, and Y. Tirilly
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.
508Key words: Pythium spp., Fusarium oxysporum, Bacillus cereus, Pseudomonas putida.
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
Can. J. Microbiol. 50: 499508 (2004) doi: 10.1139/W04-034 2004 NRC Canada
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.
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.
1Corresponding author (e-mail: email@example.com).
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.
Mots cls : Pythium spp., Fusarium oxysporum, Bacillus cereus, Pseudomonas putida.
Dniel et al.
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.
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.
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
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.
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 path