Effects of Pseudomonas Putida Wcs358r and Its Genetically

Soil Biology & Biochemistry 34 42002) 1021±1025 www.elsevier.com/locate/soilbio

Effects of Pseudomonas putida WCS358r and its genetically modi®ed phenazine producing derivative on the Fusarium population in a ®eld experiment, as determined by 18S rDNA analysis

P. Lee¯anga,*, E. Smita, D.C.M. Glandorfb, E.J. van Hannenc, K. Wernarsa

aDepartment MGB, National Institute of Public Health and the Environment, P.O. Box1, 3720 BA Bilthoven, The Netherlands bDepartment of Plant Ecology and Evolutionary Biology, Section of Plant Pathology, Utrecht University, P.O. Box80084, 3508 TB Utrecht, The Netherla nds cDepartment of Microbial Ecology, Center for Limnology, Netherlands Institute of Ecology, P.O. Box1299, 3600 BG Maarssen, The Netherlands Received 11 May 2001; received in revised form 11 February 2002; accepted 13 February 2002

Abstract We measured effects of Pseudomonas putida WCS358r and its genetically modi®ed phenazine producing derivative on the Fusarium population in the soil of a wheat ®eld in the Netherlands. We used 18S rDNA analysis to study the Fusarium population through a strategy based on screening clone libraries by using ampli®ed ribosomal DNA restriction analyses 4ARDRA). After screening a total of 1000 clones, 70 clones had a Fusarium-like ARDRA pattern. Phylogenetic analysis showed that 51out of 70 of these clones cluster in a monophyletic group together with the Fusarium isolates that were also obtained from the experimental ®eld, suggesting that these clones originated from fungal strains belonging to the genus Fusarium. Both the introduced Pseudomonas parental strain and the GMM inhibit the development of the Fusarium and probably account for the higher diversity of Fusarium-like clone types at day 40 compared to day 13 and 27. While the antagonistic properties of P. putida WCS358r appear to suppress the Fusarium population, the introduced genetic modi®cation does not seem to play a great additional role. q 2002 Elsevier Science Ltd. All rights reserved.

Keywords: Genetically modi®ed microorganisms; Pseudomonas putida; Fusarium; Ampli®ed ribosomal DNA restriction analyses; 18S rDNA; Wheat rhizosphere; Soil

The biological interactions and competition between 4Whipps et al., 1994). Recently Glandorf et al. 42001) microorganisms in the rhizosphere may lead to suppression described a ®eld experiment in which a GMM was released. of plant pathogens 4Weller, 1988). While soil-borne fungi A Psuedomonas putida isolate was genetically modi®ed play an important role in the functioning of the soil eco- with a gene cluster coding for the production of system by decomposing organic matter, some of these fungi phenazine-1-carboxylic acid 4Thomashow et al., 1990) to are also responsible for a wide range of plant diseases increase its biocontrol properties towards plant pathogenic 4Alabouvette, 1990). These pathogenic fungi can be repressed fungi such as Gaeumannomyces graminis var. tritici. How- by compounds produced by some bacteria 4Lemanceau and ever, preliminary data suggested this P. putida WCS358r < Alabouvette, 1991; Maplestone and Campbell, 1989). When phz was also inhibiting the growth of certain soil fungi like these bacteria are isolated and reintroduced under experi- Fusarium 4Glandorf et al., 2001). While some Fusarium mental conditions to study suppression of pathogenic fungi oxysporum strains are pathogenic, there are also large popu- 4Schippers et al., 1987) the results often are inconsistent lations of non-pathogenic F. oxysporum in soil 4Edel et al., 4Weller, 1988; Weller and Tomashow, 1994). To enhance 1997) 4Larkin and Fravel, 1998). Glandorf et al. 42001) used their antagonistic properties bacteria can be genetically plate counts on Komada medium and it was shown that the modi®ed 4Phillips and Streit, 1998), for instance to produce Fusarium population was slightly affected in vitro by the P. additional fungal inhibiting compounds. There are concerns putida WCS358r < phz. However, plate count data from about possible adverse effects on non-target organisms and fungi are dif®cult to interpret 4Cooke and Rayner, 1984) fungi in particular are a neglected group in terms of impact and therefore the aim of this work was to assess the feasi- studies of genetically modi®ed microorganisms 4GMM) bility and applicability of a molecular based strategy to monitor the Fusarium population in soil 4Egger, 1992). * Corresponding author. Tel.: 131-30-2742051; fax: 131-30-2744434. We examined rhizosphere samples for changes in the E-mail address: paula.lee¯[email protected] 4P. Lee¯ang). Fusarium population from a ®eld trial in which P. putida

Table 1 Band sizes 4kb) of various ARDRA patterns of Fusarium isolates and environmental clones after restriction with the enzyme TaqI

Bands size 4kb) # Fusarium oxysporum 4pattern A) Fusarium ambrosium 4pattern B) Clone 1Clone 2 Clone 3 Clone 4

0.7 0.7 0.7 0.7 0.6 0.6 0.6 0.4 0.4 0.4 0.3 0.3 0.3 0.2 0.2 0.2 0.2 0.10.1

WCS358r < phz was introduced. In parallel to the studies originate from Fusarium species. Seventy clones had on bacterial ecology 4Felske et al., 1997), we studied Fusar- ARDRA patterns identical to pattern A of the Fusarium ium community structure by using PCR ampli®cation of isolates 4Table 1), while no clones of the library matched 18S rDNA. The experiments were carried out in a ®eld pattern B. The sequence of a 0.5 kb fragment of these 70 located at De Uithof, Utrecht, The Netherlands in 1997 clones was determined. The 18S rDNA sequences obtained 4Glandorf et al., 2001). We plated untreated soil on a Fusar- were screened against sequences in GenBank/EMBL using ium speci®c medium. Ten grams of rhizosphere soil of Blast to ®nd fungi with 18S rDNA sequences homologous to untreated wheat plants was shaken in 5 ml of 10 mM the clones 4Altschul et al., 1990). Most of the clones 466)

MgSO4 for 30 s. Serial dilutions were plated onto Komada had high similarities to sequences from a variety of cultured medium 4Komada, 1975) for the speci®c isolation of Fusar- fungal species in Genbank with homologies ranging from ium spp. Plates were incubated at 20 8C for 1week. Fungi 0.96 to 0.99 4Table 2). Four sequences matched with a with various colony morphologies were selected. The fungal clone sequence from the database 4Smit et al., fungi were morphologically identi®ed as Fusarium at 1999) and were not used for further analysis. The number the Centraalbureau voor Schimmelcultures 4CBS), Baarn, of clones and the species diversity increases from day 13 to The Netherlands. In total we performed ampli®ed ribosomal 40 4Table 2). DNA restriction analyses 4ARDRA) on twelve different Of the isolates the 1.5 kb fragment was sequenced by Fusarium species, eight soil isolates and four isolates using the primers EF4, Fung5, NS4, NS5 4White et al., obtained from CBS. The ARDRA patterns of eleven 1990) and EF3. Sequence differences were limited and the Fusarium species appeared to be identical 4pattern A), number of bases that differed between species pairs ranged only one Fusarium ARDRA pattern was different 4pattern between 0 and 13 of the approximately 1500 bases that were B) 4Table 1). sequenced. The experimental treatments consisted of non-coated The 18S rDNA sequences from the different Fusarium seeds 4Control), coated seeds with the parental strain P. isolates were submitted to Genbank. Accession numbers putida WCS358r 4Parental strain) and coated seeds with were: Gibberella avenacea AF141946, Fusarium cerealis P. putida WSC358r < phz modi®ed strains 4GMM8). At AF141947, Fusarium culmorum AF141948, Fusarium equi- 13, 27 and 40 days after sowing, wheat rhizosphere samples seti AF141949, Fusarium merismoides aggreg. AF141950, were collected from six plots of each treatment and were F. oxysporum AF141951, Nectria haematococca aggreg. pooled to minimize soil heterogeneity and to reduce the AF141952, Gibberella pulicaris AF149875. number of samples. Rhizosphere suspensions were obtained A phylogenetic tree was constructed by maximum like- and total DNA was extracted and puri®ed as described in lihood analysis 4PAUPp: version 4.0b4a, David L. Swofford, Smit et al. 41997). PCR speci®c for fungal 18S-rDNA was Laboratory of Molecular Systematics, Smithsonian Insti- performed on the DNA of the 12 Fusarium isolates and tute, Washington, DC, USA). The alignment construction directly on the DNA extracted from wheat rhizosphere as was based on secondary structure using Dedicated Com- described in Glandorf et al. 42001) by using primers: EF3 parative Sequence Editor 4De Rijk and De Wachter, and EF4 4Smit et al., 1999). PCR products from the wheat 1993). The analysis was performed with 100 random- rhizosphere were puri®ed and cloned into the pGEMT- addition-sequence replicates. Nucleotide frequencies and vector as described in Smit et al. 41999). After the transfor- transition to transversion ratios were estimated from the mation, approximately 130 white clones were picked for data. Nucleotide substitution rates were assumed to follow each treatment to construct an 18S rDNA gene library. a gamma distribution with shape parameter 0.5 with We used the ARDRA technique to screen approximately setting according to the HKY model 4Hasegawa et al., 1000 clones with an insert of the expected size. The 1985). As a swapping algorithm, tree-bisection-reconnection ARDRA patterns of the different clones were analyzed 4TBR) was used. The tree was rooted by using Aspergillus with the Biogene 4V96.15) software package 4Vilber ¯avus as an outgroup. A preliminary phylogenetic tree was Lourmat, France) and clones with patterns similar to those made of the 66 clone sequences in order to distinguish of the Fusarium isolates were selected and were expected to presumptive Fusarium-like clones from non-Fusarium P. Lee¯ang et al. / Soil Biology & Biochemistry 34 <2002) 1021±1025 1023

Table 2 Fungal species containing 18S rDNA homologous to that of the 66 clones from the ®eld experiment analyzed using Blast

Day 13 Day 27 Day 40

Control Parent GMM Control Parent GMM Control Parent GMM

Nectria haematococca aggr. 4I, 4 154 311124 VIII, IX)a Tolypocladium cylindrosporum 1 4II) Gibberella pulicaris 4III) 1 1 Keissleriella cladophila 1 Leptosphaeria maculans 1 1 Spizellomyces acuminatus 2 Meliolina sydowiana 1 Verticillium lecanii 4IV) 1 Fusarium culmorum 4X, XI) 12 Fusarium merismoides 11 aggr.4VII) Westerdykella dispersa 1 Pestalosphaeria sp. 12 Fusarium cerealis 1 Neocosmospora vasinfecta 13 Fabrella tsugae 11 Fusarium equiseti 4V) 1 Rhizoctonia zeae 1 Tolypocladium cylindrosporum 1 Chaetomium elatum 1 Geosmithia putterillii 1

a The different Fusarium-like clone types recognized after phylogenetic analysis 4Fig. 1) are given between brackets. clones 4results not shown). The clones were grouped into 11 or formae speciales. However, the value of ITS as a phylo- different clone type groups based on the assumption that a genetic tool for studying Fusarium has also been shown to difference of more than three different basepairs is large be limited 4O'Donnell, 1992). The mitochondrial small enough to presume that these sequences originate from subunit 4mtSSU) rDNA and the EF-1a coding region and different species 4I±XI). introns 4O'Donnel et al., 1998) might provide better phylo- Phylogenetic analysis of the 18S rDNA sequences of the genetic markers for resolving relationships within Fusarium cultured eight Fusarium isolates revealed high homology species in future studies. among the different species 4not shown). The sequences of Of the 1000 clones that we originally analyzed, 51 some isolates, such as F. oxysporum, Gibberella pulicaris, appeared to belong to the genus Fusarium 4Fig. 1). This Gibberella avenacea and N. haematococca aggreg., differed indicates that the Fusarium population is only a very by less than two bases. Since these species could not be small part of the total community in this soil. This is a distinguished on the basis of their 18S rDNA sequences, signi®cant ®nding since Fusarium is considered to be abun- only one representative of this group was used for phylo- dantly present in soil and rhizosphere. Culture based tech- genetic analysis. niques might overestimate the Fusarium population size. We constructed the maximum likelihood phylogenetic On day 13, we recovered four clone types that clustered tree containing Fusarium isolates and one representative within the Fusarium group 4Table 3). Clone type I was of each clone type and a number of closest relatives dominant and sequences of these clones were highly homo- obtained from the Blast search 4Fig. 1). Phylogenetic analy- logous to the N. haematococca aggreg. 4Table 2). Verticil- sis showed that a number of different species could not be lium lecanii and Geosmithia putterillii clustered close to distinguished from one another based on their 18S rDNA Fusarium. All clones found on day 27 were type I, but on sequence. The majority of the sequences was almost day 40 eight different clone types were found. In Table 3, completely homologous 496±99%) to known cultured results of the phylogenetic analysis are presented in a way fungal species. This is in contrast to molecular studies on that the data from the different treatments can be distin- bacteria, which have revealed sequences from completely guished. The majority of the clones belong to type I, and unknown, uncultured bacterial taxa 4Hugenholtz et al., in time diversity is increasing. Using all the data from Table 1998). This demonstrates and con®rms the limited resolu- 3, the percentage of Fusarium clones was calculated for tion of the 18S rDNA to distinguish closely related species. each of the treatments: control 49%, parental strain 19% Therefore researchers often use ITS regions to type species and GGM 27%. This suggests that the antagonistic properties 1024 P. Lee¯ang et al. / Soil Biology & Biochemistry 34 <2002) 1021±1025

Fig. 1. The neighbor-joining tree showing the phylogenetic position of the Fusarium-like clones found on day 13, 27 and 40. Between brackets the number of clones of each type is given. of P. putida WCS358r seem to have a suppressive effect on mediated disease 4Schippers et al., 1987). Competition the Fusarium population, whereas the genetic modi®cation between fungi and bacteria is likely to play a key role in does not seem to play a great additional role. It is known the establishment of certain species on the root surface. The that bacteria producing siderophores can suppress Fusarium results obtained in this study might very well be indirectly

Table 3 Number of the Fusarium-like clone types discernable after phylogenetic analysis from the samples of the three treatments in the ®eld trial at the different sampling days

Clone type Day 13 Day 27 Day 40

Control Parent GGM Control Parent GGM Control Parent GGM

I 3 144 311625 II 1 III 1 IV 11 V 1 VI VII 1 VIII 1 IX 1 1 X 1 XI 1 P. Lee¯ang et al. / Soil Biology & Biochemistry 34 <2002) 1021±1025 1025 related to the introduced strains. The possibility exists that Hugenholtz, P., Goebel, B.M., Pace, N.R., 1998. Impact of culture inde- other species are suppressed allowing Fusarium strains to pendent studies on the emerging phylogenetic view of bacterial diver- colonize the roots. sity. Journal of Bacteriology 180, 483±488. Komada, H., 1975. Development of a selective medium for quantitative We successfully used molecular techniques to study the isolation of Fusarium oxysporum from natural soils. Research Plant Fusarium population in a ®eld experiment. Both the Protection Resistance 8, 114±125. introduced parent and the genetically modi®ed P. putida Larkin, R.P., Fravel, D.R., 1998. Ef®cacy of various fungal bacterial WCS358r were found to suppress certain Fusarium species biocontrol organisms for control of Fusarium wilt of tomato. 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