Mngement of Bcteri to Improve Slow Filtrtion Efficiency in Tomto Soilless Culture P. Rey 1, F. Déniel 1, A. Guillou 2 nd S. Le Quillec 3 1 Lbortoire de Biodiversité et Ecologie Microbienne, ESMISAB, Université de Bretgne Occidentle-Brest, 2928 Plouzné, Frnce 2 Sttion expérimentle de Vézendoquet, CATE, 2925 Sint-Pol-de-Léon, Frnce 3 CTIFL, Centre de Crquefou, 35, llée des Spins, 4447 Crquefou, Frnce Keywords: Bcillus cereus, Fusrium oxysporum, Pseudomons fluorescens, Pythium spp. Abstrct In tomto soilless culture, slow filtrtion llows one to control the development of diseses cused by pthogenic micro-orgnisms. In this study, columns of two filter units were inoculted with 5 selected bcteri (3 strins of Pseudomons putid nd 2 of Bcillus cereus). One of the two filters hd been inoculted yer before the other, but both series of nlyses were conducted on the sme yer. Fungl elimintion (Fusrium oxysporum nd Pythium spp.) ws usully successful t bout 98 to % ll over the culturl seson fter one yer nd two yers of filtering by the two systems. Bcteri popultions were generlly more numerous onto the different lyers of pouzzoln grins in the two-yer-old filter thn in the one-yer-old filter. High biologicl ctivity ws detected t 4-cm depth from the top in the two-yerold filter; in the other one it ws found only t 2 cm. This high colonistion of the filtering medium by bcteri coincided with the removl of fungi from the solution flowing through the columns. Therefore, the biologicl ctivtion of filter units constitutes relible method to stndrdise slow filtrtion efficcy. INTRODUCTION In soilless cultures, wter supply is one of the min sources for the introduction of pthogenic microorgnisms since reservoir nd surfce wters like rivers re frequently contminted by pthogens, i.e. Pythium spp., Fusrium oxysporum (Stnghellini nd Rsmussen, 1994). Moreover, the equipment of greenhouses with «closed» systems to minimise pollution by re-using the run-off solution mkes them t risk of pthogen spred into the recycled nutrient solution (McPherson et l., 1998; Vn Os, 1999). Thus, preventing pthogenic infections through pproprite disinfection of nutrient solutions hs become mjor chllenge. In the lst decde, mong the systems which hve been proposed for disinfecting nutrient solutions, the slow filtrtion technique hs roused considerble interest (Ehret et l., 21; Rey et l., 1999; Runi, 1995; Wohnk, 1995). Despite the long history of this type of filtrtion in the drinking wter industry, its introduction in horticulturl production systems is recent. During the disinfection process, nutrient solutions flow slowly through filter unit filled with different filtering supports: snd, rockwool flocks or pouzzoln grins. Mechnicl nd biologicl fctors such s micro-orgnisms with ntgonistic properties re thought to be responsible for the system effectiveness. Different studies hve evidenced the elimintion, t substntil efficiency rtes, of zoosporic fungi, e.g. Phytophthor spp, pthogenic bcteri nd even viruses (Ehret et l., 21; Vn Os et l., 1999). But, ccording to Déniel et l. (24) nd Clvo-Bdo et l. (23) there is limittion to the benefits of this disinfecting technique: indeed, full efficiency is often reched only fter vrible period of time, which my extend to 6 months. This length of time cn be shortened by biologicl ctivtion of the filter unit chieved through inocultion with selected bcteri. After selection by reserchers of our group of 5 bcteri (3 Pseudomons putid strins nd 2 Bcillus cereus ones) from filter whose efficiency to eliminte pthogens hd been proven over yers, these bcteri were introduced in new column. The 6-month period needed by the control filter to rech its Proc. IC on Greensys Eds.: G. vn Strten et l. Act Hort. 691, ISHS 25 349
best efficcy ginst Fusrium oxysporum ws shortened for the bcteri-mended filter becuse it demonstrted high filtrtion power over the first month of use. Fungl removl coincided with the development of n ctive microbil popultion on the pouzzolne grins contined in the filter unit (Déniel et l., 24). This led us to pursue our investigtions by ssessing the efficcy of one-yer nd two-yer-old filters; both were inoculted with the 5 bcteri previously selected just before strting the filter unit. Moreover, microbil colonistion of pouzzoln grins ws followed throughout the culturl seson by smpling some of them from the upper surfce lyer; on the other hnd, the colonistion within the whole filter units ws exmined t the end of culturl seson. MATERIAL AND METHODS Filter Unit Two filter units were used for these investigtions; ech of them consisted of 22-cm-long plstic pipe (4 cm in inner dimeter) filled with pouzzoln grins (2- to 4- mm in dimeter) nd grvel. Wter flew through 11-cm-thick lyer of pouzzoln grins lid t the top of three lyers of grded grvel (16-32, 8-16, 2-8 mm); the overll thickness ws 4 cm. The upper wter lyer ws regulted by flot switch t 4-5 cm bove the pouzzolne surfce. The filtrtion rte rnged within 1-15 L.h -1.m -2. The filter units were set within 2 independent comprtments of n experimentl tomtosoilless greenhouse t room temperture. One should note tht the roots of tomto plnts were grown in plstic slbs filled with coco-fiber substrte. Inocultion of Filter with Bcteri In previous study, 5 selected bcteri isolted from pouzzolne grins of very efficient filter hd been identified s P. putid (strins L2, L4 nd L5) nd B. cereus (strins L1 nd L3) (Déniel et l., 24). Prior to their inocultion to filter units, these bcteri were, first, pre-cultured for 18 to 24 h t 3 ± 1 C in tubes filled with 9 ml of Tryptic Soy Broth (TSB). For ech bcterium, 1 ml of pre-culture ws poured in 25-mL Erlenmeyer flsks filled with 15 ml of TSB. Three Erlenmeyer flsks were used for strin L1, 18 for L2, 3 for L3, 11 for L4 nd 5 for L5; the number of Erlenmeyers used per strin corresponded to the estimted proportion of ech bcterium t isoltion. The cultures were incubted for 24 h on rotry shker (12 rpm) in the drk t 3 ± 1 C. Inocultion ws mde by pouring the content of ll the Erlenmeyer flsks; the totl volume of inoculum ws 12 L. The first inocultion ws performed t the end of Februry (strt of culturl seson), nd the second one two weeks lter. Though one of the filters hd been inoculted yer before the other, both series of nlysis were conducted on the sme yer. Nutrient Solution Smpling To test the effectiveness of slow filtrtion, smples of the nutrient solution were collected in triplicte from April to September just before it flew through the filter; ech time, three other smples were lso tken from the filter effluent. Detection of potentil fungi focused on Pythium spp. nd Fusrium oxysporum becuse both re keycomponents of roots nd nutrient solution microflor in soilless greenhouses. The concentrtion of Pythium spp. nd Fusrium oxysporum ws determined on nutrient solution smples filtered through.45-µm membrne. Filters were plted on selective medi nmed CMA-PARP for Pythium spp. nd Komd for F. oxysporum. Pythium thlles were counted fter incubtion of the pltes for 48 h t 25 C in the drk, wheres F. oxysporum propgules were counted 5 nd 7 dys fter incubtion under the sme conditions. Results were expressed in percent of eliminted micro-orgnisms. 35
Assessment of Pouzzoln Grins-plted Bcteri Colonies Between Mrch nd September some grins of pouzzoln were monthly collected from the upper lyer to study the bcteril popultions responsible for their colonistion. The following specific medi were used for bcteril popultion counts: Plte Count Agr (PCA) for mesophylic erobe bcteri, ntibiotics-mended Glucose-Agr (GA) for Bcillus spp., Cetrimide-Fucidine-Cefloridine (CFC) for Pseudomons spp. nd King B for fluorescent Pseudomons. These counts were expressed in Colony Forming Unit (CFU) per pouzzoln grm. At the end of culturl seson (November), other pouzzoln grins from both filter units were tken every 1 cm to count bcteri popultions. Dt significnce ws ssessed with the Lest Significnt Difference (LSD) test t 95% of confidence. The sttisticl progrm used for nlysis ws SttGrphics softwre, relese 4., developed by Mnugistic Inc., Rockville, USA. RESULTS Elimintion of Fungi Contined in Nutrient Solution fter Slow Filtrtion The re-circulting nutrient solutions used in the greenhouse of concern here were regulrly invded by different fungi, in prticulr Pythium spp. nd F. oxysporum. Over our 6-month experiment (April - September) their elimintion by slow filtrtion through filters mended with selected bcteri demonstrted the technique efficcy (Tbles 1 nd 2). Indeed, with the two-yer-old filter, F. oxysporum ws eliminted t bout 99%. Tble 2 highlights tht very similr percentges were obtined with the one-yer-old filter, the lowest vlue being 97.6% in My. Pythium spp. ws lso eliminted by both filters t high rte (Tble 1). The one-yer-old filter hd removl of 98% t lest, except in April where it ws only 87%. Evolution of Bcteril Popultions on Pouzzoln Grins t the Surfce of the Two Filter Units All the smples of pouzzoln grins collected t the top of filter were highly colonised by vrious bcteri belonging to the mesophylic erobe microflor (Fig. 1). Among the popultions detected in both filters, the lrgest ones were found over the first two months in the one-yer-old filter, then they decresed nd levelled off from June to September. From My to September, bcteri popultions were more importnt in the two-yer-old filter thn in the other one (Fig. 1). Bcillus spp., Pseudomons spp. nd fluorescent Pseudomons were lso detected on pouzzoln grins. With the exception of Mrch for the one-yer-old filter, Figure 2 shows the lck of significnt difference bout popultions of Pseudomons spp. between both filters; the sme profile ws obtined with fluorescent Pseudomons (dt not shown). Conversely to other bcteri, Bcillus spp. regulrly colonised more pouzzoln grins between June nd September (dt not shown). Bcteril Popultions on Pouzzoln Grins Inside the Two Filters Units A typicl verticl profile ws found with respect to the distribution of bcteri within ech filter. The upper 2-cm-pouzzoln grin lyer of the one-yer-old filter ws hevily colonised by the mesophylic erobe microflor (Fig. 3), Pseudomons spp., fluorescent Pseudomons nd Bcillus spp.; then, these popultions decresed nd levelled off from 3 to 1 cm. A similr profile ws got with the two-yer-old filter with high colonistion of the upper 4- to 5-cm of pouzzoln grins followed with lower nd stble numbers of bcteri in the other lyers (Fig. 3). It is worth noting tht, t ech lyer of the filter units, the bcteri popultions were significntly lrger in the two-yerold filter thn in the other one. DISCUSSION The present study demonstrted tht biologicl ctivtion of slow filter units with 5 selected bcteri (3 P. putid strins nd 2 B. cereus strins) resulted in very high 351
elimintion (generlly bout 98 to %) of fungi such s F. oxysporum nd Pythium spp. ll over the culturl seson. The similr results obtined over the first nd second yers fter filter inocultion with bcteri indicte tht the monitoring of micro-orgnisms within the column constitutes relible method of control to stndrdise slow filtrtion efficcy. In previous study, Deniel et l. (24) demonstrted tht, over the first month following the introduction of P. putid nd B. cereus strins in filter unit, the bcteril colonistion of pouzzoln grins ws lwys high s compred to control filter with no prior mendment by bcteri. In this experiment where both units hd been mended with selected bcteri, the pouzzoln grins t the top of both filters were colonised with mesophylic erobe bcteri, Bcillus spp. nd Pseudomons spp.; some popultions, e.g. Pseudomons spp., were of quite like size. However, t the end of the culturl seson, smpling of pouzzoln grins t different depths within the filter units showed generlly more numerous bcteri in the two-yer-old filter thn in the other one. High biologicl ctivity ws detected t 2-cm-depth in the one-yer-old filter, nd 4 cm in the other. This fst nd high colonistion of the filtering medium by n ctive microbil popultion coincides with the removl of fungi from the solution flowing through the filter units. During this experiment the 5 inoculted bcteri were driven to the plnts by the nutrient solution through lekge process (dt not shown). These bcteri re nonpthogenic for plnts nd promote the growth of young tomto plntlets (Déniel et l., 24). However, their effect on the root system is unknown. Are they responsible for generting suppressive potentil in soilless cultures like other micro-orgnisms (Postm et l., 2)? The question is still open. However, ccording to McPherson et l. (1995), the use of «pssive» method, (slow filtrtion) s opposed to «ctive» ones (het tretment, ozonistion nd UV rdition) is highly recommended to fvour this beneficil microflor in soilless cultures. ACKNOWLEDGMENTS Finncil support for this reserch ws provided by the Brittny nd Pys de l Loire Councils (GIS-LBIO progrm) nd the French Reserch Deprtment ( Direction de l Technologie n 1B419). We thnk M. Euzen for excellent technicl ssistnce nd Dr M.P. Friocourt for criticl nd helpful discussion of this work. Literture Cited Clvo-Bdo, L.A., Pettitt, T.R., Prsons, N., Petch, G.M., Morgn, J.A.W. nd Whipps, J.M. 23. Sptil nd temporl nlysis of the microbil community in slow snd filters used for treting horticulturl irrigtion wter. Appl. Environ. Microb. 69:2116-2125. Déniel, F., Rey, P., Chérif, M., Guillou, A. nd Tirilly, Y. 24. Indigenous bcteri with ntgonistic- nd plnt growth promoting-ctivities improve slow filtrtion efficiency in soilless culture. Cn. J. Microbiol. 5: 499-58. Ehret, D.L., Alsnius, B., Wohnk, W., Menzies, J.G. nd Utkhede, R. 21. Disinfesttion of recirculting nutrient solutions in greenhouse horticulture. Agronomie. 21:323-339. McPherson, G.M., Hrrimn, M.R. nd Pttison, D. 1995. The potentil for spred of root diseses in recirculting hydroponic systems nd their control with disinfection. Med. Fc. Lndbouww. Univ. Gent. 6/2b:371-379. Postm, J., Willemsen-de-Klein, M.J. nd Vn Elss, J.D. 2. Effect of the indigenous microflor on the development of root nd crown rot cused by Pythium phnidermtum in cucumber grown in rockwool. Phytopthology. 9:125-133. Rey, P., Picrd, K., Déniel, F., Benhmou, N. nd Tirilly, Y. 1999. Development of n IPM system in soilless culture by using slow snd filtrtion nd biocontrol gent, Pythium oligndrum. IOBC wprs Bulletin. 22:25-28. Runi, W.Th. 1995. A review of possibilities for disinfection of recircultion wter from soilless cultures. Act Hort. 382:221-229. 352
Stnghellini M.E. nd Rsmussen, S.L. 1994. Hydroponics, solution for zoosporic pthogens. Plnt Dis. 78:1129-1138. Vn Os, E.A. 1999. Closed soilless growing systems: sustinble solution for dutch greenhouse horticulture. Wter Sci. Technol. 39:15-115. Vn Os, E.A., Amsing, J.J., vn Kuik, A.J. nd Willers, H. 1999. Slow snd filtrtion: potentil method for the elimintion of pthogens nd nemtodes in recirculting nutrient solutions from glsshouse-grown crops. Act Hort. 481:519-526. Wohnk, W. 1995. Disinfection of recirculting nutrient solutions by slow snd filtrtion. Act Hort. 382:246-255. Tbles Tble 1. Efficiency of bcteri-mended filters to eliminte Pythium spp. Filters mended with bcteri April My June July August Sept One-yer-old Influent solution 87 62 72 13 15 11 Effluent solution % elimintion 1 11 87 4 96 2 98.2 Two-yer-old Influent solution 153 12 152 125 127 128 Effluent solution % elimintion 1 1 99.2 2 98.7 1 Pythium spp. thlles were counted on selective medi coded CMA-PARP fter 48-h incubtion of the pltes t 25 C in the drk. Results re expressed in percent of eliminted micro-orgnisms. Tble 2. Efficiency of bcteri-mended filters to eliminte Fusrium oxysporum. Filters mended with bcteri April My June July August Sept One-yer-old Influent solution 3692 1842 758 4 3 217 Effluent solution 42 45 15 45 7 % elimintion 1 98.9 97.6 98 98.9 99.7 Two-yer-old Influent solution 125 9667 12 8333 8667 1719 Effluent solution % elimintion 1 7 47 99.5 87 99.3 1 15 99.8 12 99.3 1 F. oxysporum thlles were counted on selective medi coded Komd fter 48-h incubtion of the pltes t 25 C in the drk. Results re expressed in percent of eliminted micro-orgnisms. 353
Figurese 1, 1, g 1 yer old filter 2 yers old filter 1, de f bcd bc de e cd b 1, C.F.U./g of pouzzoln 1, 1, 1, 1, 1, Mrch April My June July August September Fig. 1. Evolution of mesophylic erobe bcteri popultions on pouzzoln grins tken from the upper surfce lyer of 1- nd 2-yer-old filters both mended with bcteri. Results were expressed in Colony Forming Unit (CFU) per pouzzoln grm. Columns with sme letter re not significntly different t p =.5% (LSD test). 1, b 1 yer-old filter 1, 2 yers old filter 1, C.F.U./g of pouzzoln 1, 1, 1, 1, 1, 1, Mrch April My June July August September Fig. 2. Evolution of Pseudomons spp. popultions on pouzzoln grins tken from the upper surfce lyer of 1- nd 2-yer-old filters both mended with bcteri. Results were expressed in Colony Forming Unit (CFU) per pouzzoln grm. Columns with sme letter re not significntly different t p =.5% (LSD test). 354
C.F.U./g of pouzzoln 5 1 15 2 25 3 1 bc e 2 bc e 3 e 4 f Pouzzoln lyer (cm) 5 6 7 8 bc bc cd d 9 1 bc bc One yer-old filter Two yer-old filter 11 b Fig. 3. Colonistion by mesophylic erobe bcteri popultions of pouzzoln grins tken t different depths in the 1- nd 2-yer-old filter units both mended with bcteri. Results were expressed in Colony Forming Unit (CFU) per pouzzoln grm. Columns with sme letter re not significntly different t p =.5% (LSD test). 355
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