Presence of multiple spondyloarthritis (SpA) features is important but not sufficient for a diagnosis of axial spondyloarthritis: data from the SPondyloArthritis Caught Early (SPACE) cohort.

OBJECTIVES: Concerns have been raised about overdiagnosis of axial spondyloarthritis (axSpA). We investigated whether patients with chronic back pain (CBP) of short duration and multiple SpA features are always diagnosed with axSpA by the rheumatologist, and to what extent fulfilment of the Assessment of SpondyloArthritis International Society (ASAS) axSpA criteria is associated with an axSpA diagnosis. METHODS: Baseline data from 500 patients from the SPondyloArthritis Caught Early cohort which includes patients with CBP (≥3 months, ≤2 years, onset <45 years) were analysed. All patients underwent full diagnostic workup including MRI of the sacroiliac joints (MRI-SI) and radiograph of sacroiliac joints (X-SI). For each patient, the total number of SpA features excluding sacroiliac imaging and human leucocyte antigen B27 (HLA-B27) status was calculated. RESULTS: Before sacroiliac imaging and HLA-B27 testing, 32% of patients had ≤1 SpA feature, 29% had 2 SpA features, 16% had 3 SpA features and 24% had ≥4 SpA features. A diagnosis of axSpA was made in 250 (50%) of the patients: 24% with ≤1 SpA feature, 43% with 2 SpA features, 62% with 3 SpA features and 85% with ≥4 SpA features. Of the 230 patients with a positive ASAS classification 40 (17.4%) did not have a diagnosis of axSpA. HLA-B27 positivity (OR 5.6; 95% CI 3.7 to 8.3) and any (MRI-SI and/or X-SI) positive imaging (OR 34.3; 95% CI 17.3 to 67.7) were strong determinants of an axSpA diagnosis. CONCLUSIONS: In this cohort of patients with CBP, neither the presence of numerous SpA features nor fulfilment of the ASAS classification criteria did automatically lead to a diagnosis axSpA. Positive imaging was considered particularly important in making a diagnosis of axSpA.

EULAR recommendations for the use of imaging in the diagnosis and management of spondyloarthritis in clinical practice.

A taskforce comprised of an expert group of 21 rheumatologists, radiologists and methodologists from 11 countries developed evidence-based recommendations on the use of imaging in the clinical management of both axial and peripheral spondyloarthritis (SpA). Twelve key questions on the role of imaging in SpA were generated using a process of discussion and consensus. Imaging modalities included conventional radiography, ultrasound, magnetic resonance imaging, computed tomography (CT), positron emission tomography, single photon emission CT, dual-emission x-ray absorptiometry and scintigraphy. Experts applied research evidence obtained from systematic literature reviews using MEDLINE and EMBASE to develop a set of 10 recommendations. The strength of recommendations (SOR) was assessed by taskforce members using a visual analogue scale. A total of 7550 references were identified in the search process, from which 158 studies were included in the systematic review. Ten recommendations were produced using research-based evidence and expert opinion encompassing the role of imaging in making a diagnosis of axial SpA or peripheral SpA, monitoring inflammation and damage, predicting outcome, response to treatment, and detecting spinal fractures and osteoporosis. The SOR for each recommendation was generally very high (range 8.9-9.5). These are the first recommendations which encompass the entire spectrum of SpA and evaluate the full role of all commonly used imaging modalities. We aimed to produce recommendations that are practical and valuable in daily practice for rheumatologists, radiologists and general practitioners.

Use of Arabidopsis thaliana to study mechanisms of control of Verticillium wilt by Pseudomonas fluorescens PICF7.

Verticillium wilt (VW), caused by Verticillium dahliae Kleb., is an important disease in many crops and its effective management has proven difficult. Among the various disease control measures to be implemented, the use of microbial antagonists (biological control agents, BCAs) constitutes an environmentally-friendly approach fitting criteria of modern sustainable agriculture. Pseudomonas fluorescens PICF7 was isolated from root tissues of nursery--propagated olive plants. Selection of this strain was based on in vitro growth inhibition of V. dahliae, colonizing ability of olive roots, endophytic lifestyle, and control of the highly-virulent defoliating (D) pathotype of V. dahliae in olive planting stocks. The mode of action by which PICF7 controls VW in olive is as yet unknown; moreover, to uncover potential biocontrol mechanisms poses additional difficulties in this pathosystem because the target is a tree. Therefore we used the model plant Arabidopsis thaliana to study: i) if PICF7 colonizes the rhizosphere of A. thaliana; ii) disease symptoms caused by V. dahliae in A. thaliana; iii) control of VW by PICF7 in different accessions and mutants of A. thaliana; and iv) if motility, antibiosis and/or siderophores are involved in control of V. dahliae by PICF7. Diverse bioassays were conducted and in all of them both the BCA and the pathogen were introduced in the rhizosphere of A. thaliana. Both D and non-defoliating isolates of V. dahliae caused disease symptoms in A. thaliana. PICF7 colonized and persisted in the rhizosphere of different Arabidopsis accessions and could control the D pathotype in some of them. PICF7 mutants affected in antibiosis significantly lost their ability to control VW in A. thaliana. We conclude that the model plant A. thaliana is useful to unravel interactions between this BCA and V. dahliae.

Is it Useful to Repeat Magnetic Resonance Imaging of the Sacroiliac Joints After Three Months or One Year in the Diagnosis of Patients With Chronic Back Pain and Suspected Axial Spondyloarthritis?

OBJECTIVE: To investigate the value of repeated magnetic resonance imaging (MRI) of the sacroiliac (SI) joints in diagnosing chronic back pain patients in whom axial spondyloarthritis (SpA) is suspected and to examine determinants of positive MRI findings in SI joints. METHODS: Patients with chronic back pain (duration 3 months-2 years, age ≥16 years, age at onset <45 years) with ≥1 SpA feature who were included in the Spondyloarthritis Caught Early cohort underwent visits at baseline, at 3 months, and at 1 year. Visits included an evaluation of all SpA features and repeated MRI of SI joints. MRI-detected axial SpA positivity (according to the definition from the Assessment of SpondyloArthritis international Society) was evaluated by 2 or 3 well-trained readers who were blinded with regard to clinical information. The likelihood of a positive MRI finding at follow-up visits (taking into consideration contributing factors) was calculated by generalized estimating equation analysis. RESULTS: Of the 188 patients, 38.3% were male, the mean ± SD age was 31.0 ± 8.2 years, and the mean ± SD symptom duration was 13.2 ± 7.1 months. Thirty-one patients (16.5%) had positive MRI findings in the SI joints at baseline. After 3 months and after 1 year, the MRI results had changed from positive to negative in 3 of 27 patients (11.1%) and 11 of 29 patients (37.9%), respectively, which was attributable in part to the initiation of anti-tumor necrosis factor therapy. Status changes from negative to positive were seen in 5 of 116 patients (4.3%) after 3 months and in 10 of 138 patients (7.2%) after 1 year. HLA-B27 positivity and male sex were independent determinants of the likelihood of a positive MRI scan at any time point (42% in HLA-B27+ men and 6% in HLA-B27- women). If the baseline results were negative, the likelihood of a positive scan at follow-up was very low (≤7%). CONCLUSION: MRI-detected status changes in the SI joints were seen in a minority of the patients, and both male sex and HLA-B27 positivity were important predictors of MRI positivity. Our findings indicate that conducting MRI scans after 3 months or after 1 year in patients with suspected early axial SpA is not diagnostically useful.

Systemic resistance induced by rhizosphere bacteria.

Nonpathogenic rhizobacteria can induce a systemic resistance in plants that is phenotypically similar to pathogen-induced systemic acquired resistance (SAR). Rhizobacteria-mediated induced systemic resistance (ISR) has been demonstrated against fungi, bacteria, and viruses in Arabidopsis, bean, carnation, cucumber, radish, tobacco, and tomato under conditions in which the inducing bacteria and the challenging pathogen remained spatially separated. Bacterial strains differ in their ability to induce resistance in different plant species, and plants show variation in the expression of ISR upon induction by specific bacterial strains. Bacterial determinants of ISR include lipopolysaccharides, siderophores, and salicylic acid (SA). Whereas some of the rhizobacteria induce resistance through the SA-dependent SAR pathway, others do not and require jasmonic acid and ethylene perception by the plant for ISR to develop. No consistent host plant alterations are associated with the induced state, but upon challenge inoculation, resistance responses are accelerated and enhanced. ISR is effective under field conditions and offers a natural mechanism for biological control of plant disease.

Are Additional Tests Needed to Rule Out Axial Spondyloarthritis in Patients Ages 16-45 Years With Short-Duration Chronic Back Pain and Maximally One Spondyloarthritis Feature?

OBJECTIVE: To investigate whether HLA-B27 testing and imaging of the sacroiliac joints are needed in patients with ≤1 spondyloarthritis (SpA) feature, referred to a secondary care setting, after medical history collection, clinical examination, and measurement of acute phase reactants. METHODS: Baseline data from patients in the Spondyloarthritis Caught Early (SPACE) cohort visiting the rheumatology outpatient clinic of 5 centers across Europe (with back pain ≥3 months, ≤2 years, onset at ages <45 years) were used. All patients underwent a full diagnostic work-up: magnetic resonance imaging (MRI) and radiographs of the sacroiliac joints, HLA-B27 testing, and assessment of all other SpA features. Patients were diagnosed according to the treating rheumatologist and classified according to the Assessment of SpondyloArthritis international Society (ASAS) axial SpA criteria. RESULTS: Of the 354 patients, 133 (37.5%) showed 0 or 1 SpA feature after medical history collection, physical examination, and measurement of acute phase reactants (38 without SpA features, 95 with 1 SpA feature). Of the patients with ≤1 SpA feature, 18.4% (with 0 SpA features) and 17.9% (with 1 SpA feature) were diagnosed with axial SpA according to the rheumatologist after additional investigations (HLA-B27 testing and sacroiliac joint imaging). Additionally, 4 of 38 patients (10.5%) without SpA features fulfilled the ASAS axial SpA criteria (all according to the imaging arm only: 2 as MRI+/modified New York criteria (mNY)+, 1 as MRI+/mNY-, and 1 as MRI-/mNY+). Of the 95 patients with 1 SpA feature, 22 (23.2%) fulfilled the ASAS axial SpA criteria (all according to the imaging arm only: 3 as MRI+/mNY+, 15 as MRI+/mNY-, and 4 as MRI-/mNY+). CONCLUSION: In these patients in a secondary care setting with ≤1 SpA feature, axial SpA could not be ruled out without sacroiliac joint imaging and/or HLA-B27 testing.

No role for bacterially produced salicylic Acid in rhizobacterial induction of systemic resistance in Arabidopsis.

ABSTRACT The role of bacterially produced salicylic acid (SA) in the induction of systemic resistance in plants by rhizobacteria is far from clear. The strong SA producer Pseudomonas fluorescens WCS374r induces resistance in radish but not in Arabidopsis thaliana, whereas application of SA leads to induction of resistance in both plant species. In this study, we compared P. fluorescens WCS374r with three other SA-producing fluorescent Pseudomonas strains, P. fluorescens WCS417r and CHA0r, and P. aeruginosa 7NSK2 for their abilities to produce SA under different growth conditions and to induce systemic resistance in A. thaliana against bacterial speck, caused by P. syringae pv. tomato. All strains produced SA in vitro, varying from 5 fg cell(-1) for WCS417r to >25 fg cell(-1) for WCS374r. Addition of 200 muM FeCl(3) to standard succinate medium abolished SA production in all strains. Whereas the incubation temperature did not affect SA production by WCS417r and 7NSK2, strains WCS374r and CHA0r produced more SA when grown at 33 instead of 28 degrees C. WCS417r, CHA0r, and 7NSK2 induced systemic resistance apparently associated with their ability to produce SA, but WCS374r did not. Conversely, a mutant of 7NSK2 unable to produce SA still triggered induced systemic resistance (ISR). The possible involvement of SA in the induction of resistance was evaluated using SA-nonaccumulating transgenic NahG plants. Strains WCS417r, CHA0r, and 7NSK2 induced resistance in NahG Arabidopsis. Also, WCS374r, when grown at 33 or 36 degrees C, triggered ISR in these plants, but not in ethylene-insensitive ein2 or in non-plant pathogenesis- related protein-expressing npr1 mutant plants, irrespective of the growth temperature of the bacteria. These results demonstrate that, whereas WCS374r can be manipulated to trigger ISR in Arabidopsis, SA is not the primary determinant for the induction of systemic resistance against bacterial speck disease by this bacterium. Also, for the other SAproducing strains used in this study, bacterial determinants other than SA must be responsible for inducing resistance.

Microbial Antagonism at the Root Level Is Involved in the Suppression of Fusarium Wilt by the Combination of Nonpathogenic Fusarium oxysporum Fo47 and Pseudomonas putida WCS358.

ABSTRACT Two biological control agents, nonpathogenic Fusarium oxysporum Fo47 and Pseudomonas putida WCS358, were evaluated for suppression of Fusarium wilt of flax grown in nutrient solution and for suppression of the population density and metabolic activity of the causal organism F. oxysporum f. sp. lini strain Foln3GUS on root surfaces. Due to the presence of an introduced gusA reporter gene construct in Foln3GUS, the pathogen expressed beta-glucuronidase activity that was related to its carbon metabolism. At a Fo47 to Foln3GUS inoculum ratio of 100:1, both the population density of the pathogen and the beta-glucuronidase activity on and in flax roots were reduced by the nonpathogenic strain, and Fusarium wilt was suppressed. At a Fo47 to Foln3GUS inoculum ratio of 10:1, Fo47 decreased the severity of Fusarium wilt to a smaller extent and it also reduced beta-glucuronidase activity without reducing the density of Foln3GUS on flax roots. At a nonpathogenic to pathogenic Fusarium strains ratio of 10:1, the addition of P. putida WCS358 further suppressed Fusarium wilt and the density of the pathogen at the root level, whereas a mutant of WCS358 deficient in pseudobactin production had no significant effect. Iron availability to WCS358 on flax roots, assessed by ice-nucleation activity conferred from a transcriptional fusion (pvd-inaZ) of an ice-nucleation reporter gene to an iron-regulated promoter, was sufficiently low to allow pseudobactin production. P. putida WCS358 did not reduce the severity of Fusarium wilt of flax when inoculated without Fo47, and it did not improve disease suppression achieved by high inoculum doses of Fo47 (a Fo47 to Foln3GUS ratio of 100:1). Together, these data provide evidence that (i) suppression of Fusarium wilt of flax by Fo47 is related to reductions in the population density and metabolic activity of the pathogen on the root surface; (ii) WCS358 can enhance the biological control activity of Fo47, but this enhancement depends on the population of Fo47 relative to the pathogen; and (iii) pseudobactin contributes to suppression of Fusarium wilt by the combination of Fo47 and WCS358 on roots in which conditions are conducive to pseudobactin production by the bacterium.

Antagonistic Effect of Nonpathogenic Fusarium oxysporum Fo47 and Pseudobactin 358 upon Pathogenic Fusarium oxysporum f. sp. dianthi.

Pseudobactin production by Pseudomonas putida WCS358 significantly improves biological control of fusarium wilt caused by nonpathogenic Fusarium oxysporum Fo47b10 (P. Lemanceau, P. A. H. M. Bakker, W. J. de Kogel, C. Alabouvette, and B. Schippers, Appl. Environ. Microbiol. 58:2978-2982, 1992). The antagonistic effect of Fo47b10 and purified pseudobactin 358 was studied by using an in vitro bioassay. This bioassay allows studies on interactions among nonpathogenic F. oxysporum Fo47b10, pathogenic F. oxysporum f. sp. dianthi WCS816, and purified pseudobactin 358, the fluorescent siderophore produced by P. putida WCS358. Both nonpathogenic and pathogenic F. oxysporum reduced each other's growth when grown together. However, in these coinoculation experiments, pathogenic F. oxysporum WCS816 was relatively more inhibited in its growth than nonpathogenic F. oxysporum Fo47b10. The antagonism of nonpathogenic F. oxysporum against pathogenic F. oxysporum strongly depends on the ratio of nonpathogenic to pathogenic F. oxysporum densities: the higher this ratio, the stronger the antagonism. This fungal antagonism appears to be mainly associated with the competition for glucose. Pseudobactin 358 reduced the growth of both F. oxysporum strains, whereas ferric pseudobactin 358 did not; antagonism by pseudobactin 358 was then related to competition for iron. However, the pathogenic F. oxysporum strain was more sensitive to this antagonism than the nonpathogenic strain. Pseudobactin 358 reduced the efficiency of glucose metabolism by the fungi. These results suggest that pseudobactin 358 increases the intensity of the antagonism of nonpathogenic F. oxysporum Fo47b10 against pathogenic F. oxysporum WCS816 by making WCS816 more sensitive to the glucose competition by Fo47b10.

Effect of pseudobactin 358 production by Pseudomonas putida WCS358 on suppression of fusarium wilt of carnations by nonpathogenic Fusarium oxysporum Fo47.

Nonpathogenic Fusarium oxysporum Fo47b10 combined with Pseudomonas putida WCS358 efficiently suppressed fusarium wilt of carnations grown in soilless culture. This suppression was significantly higher than that obtained by inoculation of either antagonistic microorganism alone. The increased suppression obtained by Fo47b10 combined with WCS358 only occurred when Fo47b10 was introduced at a density high enough (at least 10 times higher than that of the pathogen) to be efficient on its own. P. putida WCS358 had no effect on disease severity when inoculated on its own but significantly improved the control achieved with nonpathogenic F. oxysporum Fo47b10. In contrast, a siderophore-negative mutant of WCS358 had no effect on disease severity even in the presence of Fo47b10. Since the densities of both bacterial strains at the root level were similar, the difference between the wild-type WCS358 and the siderophore-negative mutant with regard to the control of fusarium wilt was related to the production of pseudobactin 358. The production of pseudobactin 358 appeared to be responsible for the increased suppression by Fo47b10 combined with WCS358 relative to that with Fo47b10 alone.

Siderophore receptor PupA as a marker to monitor wild-type Pseudomonas putida WCS358 in natural environments.

For application of genetically engineered fluorescent Pseudomonas spp., specific markers are required for monitoring of wild-type Pseudomonas strains and their genetically modified derivatives in natural environments. In this study, the specific siderophore receptor PupA of plant growth-promoting Pseudomonas putida WCS358 was used as a marker to monitor wild-type strain WCS358. After introduction into natural soil and rhizosphere environments, strain WCS358 could be recovered efficiently on a medium amended with 300 microM pseudobactin 358. Although low population densisties of indigenous pseudomonads (less than or equal to 10(3)/g of soil or root) were recovered on the pseudobactin 358-amended medium, subsequent agglutination assays with a WCS358-specific polyclonal antiserum enabled accurate monitoring of populations of wild-type strain WCS358 over a range of approximately 10(3) to 10(7) CFU/g of soil or root. Genetic analysis of the background population by PCR and Southern hybridization revealed that natural occurrence of the pupA gene was limited to a very small number of indigenous Pseudomonas spp. which are very closely related to P. putida WCS358. The PupA marker system enabled the study of differences in rhizosphere colonization among wild-type strain WCS358, rifampin-resistant derivative WCS358rr, and Tn5 mutant WCS358::xylE. Chromosomally mediated rifampin resistance did not affect the colonizing ability of P. putida WCS358. However, Tn5 mutant WCS358::xylE colonized the radish rhizosphere significantly less than did its parental strain.

Agglutination, adherence, and root colonization by fluorescent pseudomonads.

Two fractions of agglutination activity towards fluorescent pseudomonads were detected in root washes of potato, tomato, wheat, and bean. High-molecular-mass (>10 Da) components in crude root washes agglutinated only particular saprophytic, fluorescent Pseudomonas isolates. Ion-exchange treatment of the crude root washes resulted in preparations of lower-molecular-mass (10 to 10 Da) fractions which agglutinated almost all Pseudomonas isolates examined. Also, components able to suppress agglutination reactions of pseudomonads with the lower-molecular-mass root components were detected in crude root washes of all crops studied. Pseudomonas isolates were differentially agglutinated by both types of root components. The involvement of these two types of root components in short-term adherence and in colonization was studied in potato, tomato, and grass, using Pseudomonas isolates from these crops. Short-term adherence of isolates to roots was independent of their agglutination with either type of root components. With agglutination-negative mutants, the high-molecular-mass components seemed to be involved in adherence of Pseudomonas putida Corvallis to roots of all crops studied. Short-term adherence to roots of four Pseudomonas isolates could be influenced by addition of both crude and ion-exchange-treated root washes, depending on their agglutination phenotype with these root wash preparations. Potato root colonization by 10 different isolates from this crop, over a period of 7 days, was not correlated with their agglutination phenotype. Agg mutants of P. putida Corvallis were not impaired in root colonization. It is concluded that the root agglutinins studied can be involved in short-term adherence of pseudomonads to roots but do not play a decisive role in their root colonization.

Analysis of the pmsCEAB gene cluster involved in biosynthesis of salicylic acid and the siderophore pseudomonine in the biocontrol strain Pseudomonas fluorescens WCS374.

Mutants of Pseudomonas fluorescens WCS374 defective in biosynthesis of the fluorescent siderophore pseudobactin still display siderophore activity, indicating the production of a second siderophore. A recombinant cosmid clone (pMB374-07) of a WCS374 gene library harboring loci necessary for the biosynthesis of salicylic acid (SA) and this second siderophore pseudomonine was isolated. The salicylate biosynthesis region of WCS374 was localized in a 5-kb EcoRI fragment of pMB374-07. The SA and pseudomonine biosynthesis region was identified by transfer of cosmid pMB374-07 to a pseudobactin-deficient strain of P. putida. Sequence analysis of the 5-kb subclone revealed the presence of four open reading frames (ORFs). Products of two ORFs (pmsC and pmsB) showed homologies with chorismate-utilizing enzymes; a third ORF (pmsE) encoded a protein with strong similarity with enzymes involved in the biosynthesis of siderophores in other bacterial species. The region also contained a putative histidine decarboxylase gene (pmsA). A putative promoter region and two predicted iron boxes were localized upstream of pmsC. We determined by reverse transcriptase-mediated PCR that the pmsCEAB genes are cotranscribed and that expression is iron regulated. In vivo expression of SA genes was achieved in P. putida and Escherichia coli cells. In E. coli, deletions affecting the first ORF (pmsC) diminished SA production, whereas deletion of pmsB abolished it completely. The pmsB gene induced low levels of SA production in E. coli when expressed under control of the lacZ promoter. Several lines of evidence indicate that SA and pseudomonine biosynthesis are related. Moreover, we isolated a Tn5 mutant (374-05) that is simultaneously impaired in SA and pseudomonine production.

Flagella of a plant-growth-stimulating Pseudomonas fluorescens strain are required for colonization of potato roots.

The role of motility in the colonization of potato roots by Pseudomonas bacteria was studied. Four Tn5-induced flagella-less mutants of the plant-growth-stimulating P. fluorescens WCS374 appeared to be impaired in their ability to colonize growing potato roots.

Polymerase chain reaction for verification of fluorescent colonies of Erwinia chrysanthemi and Pseudomonas putida WCS358 in immunofluorescence colony staining.

The potential of polymerase chain reaction (PCR) for verifying the identity of colonies stained by the immunofluorescence colony-staining (IFC) procedure was investigated. Using primers directed against conserved sequences of the pectate lyase-genes coding for isozymes PLa, PLd and PLe of Erwinia chrysanthemi, the authors confirmed the identity of 96% of 20 fluorescent target colonies, punched from IFC-stained samples with pure cultures. In pour plates with mixtures of Erw. chrysanthemi and non-target colonies from potato peel extracts, the identity of 90% of 113 target colonies was confirmed. Using primers directed against sequences of the ferric-pseudobactin receptor gene pupA of Pseudomonas putida WCS358, the identity of 96% of 22 target colonies was confirmed in IFC-stained samples with pure cultures. In pour plates with mixtures of Ps. putida WCS358 and non-target bacteria from compost extracts, the identity of 59% of 108 fluorescent colonies was confirmed by PCR. It was shown that components from non-target bacteria lowered the threshold level of PCR for Ps. putida WCS358 100-fold.

Effect of genetically modified Pseudomonas putida WCS358r on the fungal rhizosphere microflora of field-grown wheat.

We released genetically modified Pseudomonas putida WCS358r into the rhizospheres of wheat plants. The two genetically modified derivatives, genetically modified microorganism (GMM) 2 and GMM 8, carried the phz biosynthetic gene locus of strain P. fluorescens 2-79 and constitutively produced the antifungal compound phenazine-1-carboxylic acid (PCA). In the springs of 1997 and 1998 we sowed wheat seeds treated with either GMM 2, GMM 8, or WCS358r (approximately 10(7) CFU per seed), and measured the numbers, composition, and activities of the rhizosphere microbial populations. During both growing seasons, all three bacterial strains decreased from 10(7) CFU per g of rhizosphere sample to below the limit of detection (10(2) CFU per g) 1 month after harvest of the wheat plants. The phz genes were stably maintained, and PCA was detected in rhizosphere extracts of GMM-treated plants. In 1997, but not in 1998, fungal numbers in the rhizosphere, quantified on 2% malt extract agar (total filamentous fungi) and on Komada's medium (mainly Fusarium spp.), were transiently suppressed in GMM 8-treated plants. We also analyzed the effects of the GMMs on the rhizosphere fungi by using amplified ribosomal DNA restriction analysis. Introduction of any of the three bacterial strains transiently changed the composition of the rhizosphere fungal microflora. However, in both 1997 and 1998, GMM-induced effects were distinct from those of WCS358r and lasted for 40 days in 1997 and for 89 days after sowing in 1998, whereas effects induced by WCS358r were detectable for 12 (1997) or 40 (1998) days. None of the strains affected the metabolic activity of the soil microbial population (substrate-induced respiration), soil nitrification potential, cellulose decomposition, plant height, or plant yield. The results indicate that application of GMMs engineered to have improved antifungal activity can exert nontarget effects on the natural fungal microflora.

Repeated introduction of genetically modified Pseudomonas putida WCS358r without intensified effects on the indigenous microflora of field-grown wheat.

To investigate the impact of genetically modified, antibiotic-producing rhizobacteria on the indigenous microbial community, Pseudomonas putida WCS358r and two transgenic derivatives were introduced as a seed coating into the rhizosphere of wheat in two consecutive years (1999 and 2000) in the same field plots. The two genetically modified microorganisms (GMMs), WCS358r::phz and WCS358r::phl, constitutively produced phenazine-1-carboxylic acid (PCA) and 2,4-diacetylphloroglucinol (DAPG), respectively. The level of introduced bacteria in all treatments decreased from 10(7) CFU per g of roots soon after sowing to less than 10(2) CFU per g after harvest 132 days after sowing. The phz and phl genes remained stable in the chromosome of WCS358r. The amount of PCA produced in the wheat rhizosphere by WCS358r::phz was about 40 ng/g of roots after the first application in 1999. The DAPG-producing GMMs caused a transient shift in the indigenous bacterial and fungal microflora in 1999, as determined by amplified ribosomal DNA restriction analysis. However, after the second application of the GMMs in 2000, no shifts in the bacterial or fungal microflora were detected. To evaluate the importance of the effects induced by the GMMs, these effects were compared with those induced by crop rotation by planting wheat in 1999 followed by potatoes in 2000. No effect of rotation on the microbial community structure was detected. In 2000 all bacteria had a positive effect on plant growth, supposedly due to suppression of deleterious microorganisms. Our research suggests that the natural variability of microbial communities can surpass the effects of GMMs.