Plant-dependent genotypic and phenotypic diversity of antagonistic rhizobacteria isolated from different Verticillium host plants

To study the effect of plant species on the abundance and diversity of bacterial antagonists, the abundance, the phenotypic diversity, and the genotypic diversity of rhizobacteria isolated from potato, oilseed rape, and strawberry and from bulk soil which showed antagonistic activity towards the soilborne pathogen Verticillium dahliae Kleb. were analyzed. Rhizosphere and soil samples were taken five times over two growing seasons in 1998 and 1999 from a randomized field trial. Bacterial isolates were obtained after plating on R2A (Difco, Detroit, Mich.) or enrichment in microtiter plates containing high-molecular-weight substrates followed by plating on R2A. A total of 5,854 bacteria isolated from the rhizosphere of strawberry, potato, or oilseed rape or bulk soil from fallow were screened by dual testing for in vitro antagonism towards VERTICILLIUM: The proportion of isolates with antagonistic activity was highest for strawberry rhizosphere (9.5%), followed by oilseed rape (6.3%), potato (3.7%), and soil (3.3%). The 331 Verticillium antagonists were identified by their fatty acid methyl ester profiles. They were characterized by testing their in vitro antagonism against other pathogenic fungi; their glucanolytic, chitinolytic, and proteolytic activities; and their BOX-PCR fingerprints. The abundance and composition of Verticillium antagonists was plant species dependent. A rather high proportion of antagonists from the strawberry rhizosphere was identified as Pseudomonas putida B (69%), while antagonists belonging to the Enterobacteriaceae (Serratia spp., Pantoea agglomerans) were mainly isolated from the rhizosphere of oilseed rape. For P. putida A and B plant-specific genotypes were observed, suggesting that these bacteria were specifically enriched in each rhizosphere.     

Impact of plant species and site on rhizosphere-associated fungi antagonistic to Verticillium dahliae kleb

Fungi with antagonistic activity toward plant pathogens play an essential role in plant growth and health. To analyze the effects of the plant species and the site on the abundance and composition of fungi with antagonistic activity toward Verticillium dahliae, fungi were isolated from oilseed rape and strawberry rhizosphere and bulk soil from three different locations in Germany over two growing seasons. A total of 4,320 microfungi screened for in vitro antagonism toward Verticillium resulted in 911 active isolates. This high proportion of fungi antagonistic toward the pathogen V. dahliae was found for bulk and rhizosphere soil at all sites. A plant- and site-dependent specificity of the composition of antagonistic morphotypes and their genotypic diversity was found. The strawberry rhizosphere was characterized by preferential occurrence of Penicillium and Paecilomyces isolates and low numbers of morphotypes (n = 31) and species (n = 13), while Monographella isolates were most frequently obtained from the rhizosphere of oilseed rape, for which higher numbers of morphotypes (n = 41) and species (n = 17) were found. Trichoderma strains displayed high diversity in all soils, but a high degree of plant specificity was shown by BOX-PCR fingerprints. The diversity of rhizosphere-associated antagonists was lower than that of antagonists in bulk soil, suggesting that some fungi were specifically enriched in each rhizosphere. A broad spectrum of new Verticillium antagonists was identified, and the implications of the data for biocontrol applications are discussed.     

Bulk and rhizosphere soil bacterial communities studied by denaturing gradient gel electrophoresis: plant-dependent enrichment and seasonal shifts revealed

The bacterial rhizosphere communities of three host plants of the pathogenic fungus Verticillium dahliae, field-grown strawberry (Fragaria ananassa Duch.), oilseed rape (Brassica napus L.), and potato (Solanum tuberosum L.), were analyzed. We aimed to determine the degree to which the rhizosphere effect is plant dependent and whether this effect would be increased by growing the same crops in two consecutive years. Rhizosphere or soil samples were taken five times over the vegetation periods. To allow a cultivation-independent analysis, total community DNA was extracted from the microbial pellet recovered from root or soil samples. 16S rDNA fragments amplified by PCR from soil or rhizosphere bacterium DNA were analyzed by denaturing gradient gel electrophoresis (DGGE). The DGGE fingerprints showed plant-dependent shifts in the relative abundance of bacterial populations in the rhizosphere which became more pronounced in the second year. DGGE patterns of oilseed rape and potato rhizosphere communities were more similar to each other than to the strawberry patterns. In both years seasonal shifts in the abundance and composition of the bacterial rhizosphere populations were observed. Independent of the plant species, the patterns of the first sampling times for both years were characterized by the absence of some of the bands which became dominant at the following sampling times. Bacillus megaterium and Arthrobacter sp. were found as predominant populations in bulk soils. Sequencing of dominant bands excised from the rhizosphere patterns revealed that 6 out of 10 bands resembled gram-positive bacteria. Nocardia populations were identified as strawberry-specific bands.     

The rhizosphere effect on bacteria antagonistic towards the pathogenic fungus Verticillium differs depending on plant species and site

Rhizobacteria with antagonistic activity towards plant pathogens play an essential role in root growth and plant health and are influenced by plant species in their abundance and composition. To determine the extent of the effect of the plant species and of the site on the abundance and composition of bacteria with antagonistic activity towards Verticillium dahliae, bacteria isolated from the rhizosphere of two Verticillium host plants, oilseed rape and strawberry, and from bulk soil were analysed at three different locations in Germany over two growing seasons. A total of 6732 bacterial isolates screened for in vitro antagonism towards Verticillium resulted in 560 active isolates, among which Pseudomonas (77%) and Serratia (6%) were the most dominant genera. The rhizosphere effect on the antagonistic bacterial community was shown by an enhanced proportion of antagonistic isolates, by enrichment of specific amplified ribosomal DNA restriction analysis types, species and genotypes, and by a reduced diversity in the rhizosphere in comparison to bulk soil. Such an effect was influenced by the plant species and by the site of its cultivation. Altogether, 16S rRNA gene sequencing of 66 isolates resulted in the identification of 22 different species. Antagonists of the genus Serratia were preferentially isolated from oilseed rape rhizosphere, with the exception of one site. For isolates of Pseudomonas and Serratia, plant-specific and site-specific genotypes were found.     

Plant-Dependent Genotypic and Phenotypic Diversity of Antagonistic Rhizobacteria Isolated from Different Verticillium Host Plants

To study the effect of plant species on the abundance and diversity of bacterial antagonists, the abundance, the phenotypic diversity, and the genotypic diversity of rhizobacteria isolated from potato, oilseed rape, and strawberry and from bulk soil which showed antagonistic activity towards the soilborne pathogen Verticillium dahliae Kleb. were analyzed. Rhizosphere and soil samples were taken five times over two growing seasons in 1998 and 1999 from a randomized field trial. Bacterial isolates were obtained after plating on R2A (Difco, Detroit, Mich.) or enrichment in microtiter plates containing high-molecular-weight substrates followed by plating on R2A. A total of 5,854 bacteria isolated from the rhizosphere of strawberry, potato, or oilseed rape or bulk soil from fallow were screened by dual testing for in vitro antagonism towards Verticillium. The proportion of isolates with antagonistic activity was highest for strawberry rhizosphere (9.5%), followed by oilseed rape (6.3%), potato (3.7%), and soil (3.3%). The 331 Verticillium antagonists were identified by their fatty acid methyl ester profiles. They were characterized by testing their in vitro antagonism against other pathogenic fungi; their glucanolytic, chitinolytic, and proteolytic activities; and their BOX-PCR fingerprints. The abundance and composition of Verticillium antagonists was plant species dependent. A rather high proportion of antagonists from the strawberry rhizosphere was identified as Pseudomonas putida B (69%), while antagonists belonging to the Enterobacteriaceae (Serratia spp., Pantoea agglomerans) were mainly isolated from the rhizosphere of oilseed rape. For P. putida A and B plant-specific genotypes were observed, suggesting that these bacteria were specifically enriched in each rhizosphere.     

Successful strategy for the selection of new strawberry-associated rhizobacteria antagonistic to Verticillium wilt

In order to isolate and characterize new strawberry-associated bacteria antagonistic to the soil-borne pathogenic fungus Verticillium dahliae Kleb., rhizobacterial populations from two different strawberry species, Greenish Strawberry (Fragaria viridis) and Garden Strawberry (F. x ananassa) obtained after plating onto King's B and glycerol-arginine agar, were screened for in vitro antagonism toward V. dahliae. The proportion of isolates with antifungal activity determined in in vitro assay against V. dahliae was higher for the Garden Strawberry than for the Greenish Strawberry. From 300 isolates, 20 isolates with strong antifungal activity were selected characterized by physiological profiling and molecular fingerprinting methods. Diversity among the isolates was characterized with molecular fingerprints using amplified ribosomal DNA restriction analysis (ARDRA) and the more discriminating BOX-PCR fingerprint method. The physiological profiles were well correlated with molecular fingerprinting pattern analysis. Significant reduction of Verticillium wilt by bacterial dipping bath treatment was shown in the greenhouse and in fields naturally infested by V. dahliae. The relative increase of yield ranged from 117% (Streptomyces albidoflavus S1) to 344% (Pseudomonas fluorescens P10) in greenhouse trials, and 113% (Streptomyces albidoflavus S1) to 247% (Pseudomonas fluorescens P6) in field trials. Evaluation resulted in the selection of three effective biocontrol agents (Pseudomonas fluorescens P6, P10, and Streptomyces diastatochromogenes S9) antagonistic to the Verticillium wilt pathogen.     

Cultivation-independent analysis of Pseudomonas species in soil and in the rhizosphere of field-grown Verticillium dahliae host plants

Despite their importance for rhizosphere functioning, rhizobacterial Pseudomonas spp. have been mainly studied in a cultivation-based manner. In this study a cultivation-independent method was used to determine to what extent the factors plant species, sampling site and year-to-year variation influence Pseudomonas community structure in bulk soil and in the rhizosphere of two Verticillium dahliae host plants, oilseed rape and strawberry. Community DNA was extracted from bulk and rhizosphere soil samples of flowering plants collected at three different sites in Germany in two consecutive years. Pseudomonas community structure and diversity were assessed using a polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) system to fingerprint Pseudomonas-specific 16S rRNA gene fragments amplified from community DNA. Dominant and differentiating DGGE bands were excised from the gels, cloned and sequenced. The factors sampling site, plant species and year-to-year variation were shown to significantly influence the community structure of Pseudomonas in rhizosphere soils. The composition of Pseudomonas 16S rRNA gene fragments in the rhizosphere differed from that in the adjacent bulk soil and the rhizosphere effect tended to be plant-specific. The clone sequences of most dominant bands analysed belonged to the Pseudomonas fluorescens lineage and showed closest similarity to culturable Pseudomonas known for displaying antifungal properties. This report provides a better understanding of how different factors drive Pseudomonas community structure and diversity in bulk and rhizosphere soils.     

Quorum-sensing effects in the antagonistic rhizosphere bacterium Serratia plymuthica HRO-C48

The rhizosphere-associated bacterium Serratia plymuthica HRO-C48 is not only able to suppress symptoms caused by soil-borne pathogens but is also able to stimulate growth of plants. Detailed knowledge about the underlying mechanisms and regulation are crucial for the application in biocontrol strategies. To analyse the influence of N -acyl homoserine lactone (AHL)-mediated communication on the biocontrol activity, the AHL-degrading lactonase AiiA was heterologously expressed in the strain, resulting in abolished AHL production. The comparative analysis of the wild type and AHL negative mutants led to the identification of new AHL-regulated phenotypes. In the pathosystem Verticillium dahliae –oilseed rape, the essential role of AHL-mediated signaling for disease suppression was demonstrated. In vitro , the regulatory function of AHLs in the synthesis of the plant growth hormone indole-3-acetic acid is shown for the first time. Additionally, swimming motility was found to be negatively AHL regulated. In contrast, production of extracellular hydrolytic enzymes is shown to be positively AHL-regulated. HRO-C48 emits a broad spectrum of volatile organic compounds that are involved in antifungal activity and, interestingly, whose relative abundances are influenced by quorum sensing (QS). This study shows that QS is crucial for biocontrol activity of S. plymuthica and discusses the impact for the application of the strain as a biocontrol agent.     

Pseudomonas community structure and antagonistic potential in the rhizosphere: insights gained by combining phylogenetic and functional gene-based analyses

The Pseudomonas community structure and antagonistic potential in the rhizospheres of strawberry and oilseed rape (host plants of the fungal phytopathogen Verticillium dahliae) were assessed. The use of a new PCR-DGGE system, designed to target Pseudomonas-specific gacA gene fragments in environmental DNA, circumvented common biases of 16S rRNA gene-based DGGE analyses and proved to be a reliable tool to unravel the diversity of uncultured Pseudomonas in bulk and rhizosphere soils. Pseudomonas-specific gacA fingerprints of total-community (TC) rhizosphere DNA were surprisingly diverse, plant-specific and differed markedly from those of the corresponding bulk soils. By combining multiple culture-dependent and independent surveys, a group of Pseudomonas isolates antagonistic towards V. dahliae was shown to be genotypically conserved, to carry the phlD biosynthetic locus (involved in the biosynthesis of 2,4-diacetylphloroglucinol - 2,4-DAPG), and to correspond to a dominant and highly frequent Pseudomonas population in the rhizosphere of field-grown strawberries planted at three sites in Germany which have different land use histories. This population belongs to the Pseudomonas fluorescens phylogenetic lineage and showed closest relatedness to P. fluorescens strain F113 (97% gacA gene sequence identity in 492-bp sequences), a biocontrol agent and 2,4-DAPG producer. Partial gacA gene sequences derived from isolates, clones of the strawberry rhizosphere and DGGE bands retrieved in this study represent previously undescribed Pseudomonas gacA gene clusters as revealed by phylogenetic analysis.     

Impact of Plant Species and Site on Rhizosphere-Associated Fungi Antagonistic to Verticillium dahliae Kleb.

Fungi with antagonistic activity toward plant pathogens play an essential role in plant growth and health. To analyze the effects of the plant species and the site on the abundance and composition of fungi with antagonistic activity toward Verticillium dahliae, fungi were isolated from oilseed rape and strawberry rhizosphere and bulk soil from three different locations in Germany over two growing seasons. A total of 4,320 microfungi screened for in vitro antagonism toward Verticillium resulted in 911 active isolates. This high proportion of fungi antagonistic toward the pathogen V. dahliae was found for bulk and rhizosphere soil at all sites. A plant- and site-dependent specificity of the composition of antagonistic morphotypes and their genotypic diversity was found. The strawberry rhizosphere was characterized by preferential occurrence of Penicillium and Paecilomyces isolates and low numbers of morphotypes (n = 31) and species (n = 13), while Monographella isolates were most frequently obtained from the rhizosphere of oilseed rape, for which higher numbers of morphotypes (n = 41) and species (n = 17) were found. Trichoderma strains displayed high diversity in all soils, but a high degree of plant specificity was shown by BOX-PCR fingerprints. The diversity of rhizosphere-associated antagonists was lower than that of antagonists in bulk soil, suggesting that some fungi were specifically enriched in each rhizosphere. A broad spectrum of new Verticillium antagonists was identified, and the implications of the data for biocontrol applications are discussed.     

Endophytic and ectophytic potato-associated bacterial communities differ in structure and antagonistic function against plant pathogenic fungi

Differences between endophytic and ectophytic bacterial communities with stress on antagonistic bacteria, were studied by comparing the composition of communities isolated from the rhizosphere, phyllosphere, endorhiza and endosphere of field-grown potato plants using a multiphasic approach. Terminal restriction fragment length polymorphism analysis of 16S rDNA of the bacterial communities revealed discrete microenvironment-specific patterns. To measure the antagonistic potential of potato-associated bacteria, a total of 2648 bacteria were screened by dual testing of antagonism to the soilborne pathogens Verticillium dahliae and Rhizoctonia solani. Composition and diversity of bacterial antagonists were mainly specific for each microenvironment. The rhizosphere and endorhiza were the main reservoirs for antagonistic bacteria and showed the highest similarity in their colonisation by antagonists. The most prominent species of all microenvironments was Pseudomonas putida, and rep-PCR with BOX primers showed that these isolates showed microenvironment-specific DNA fingerprints. P. putida isolates from the rhizosphere and endorhiza gave nearly identical fingerprints confirming the high similarity of bacterial populations. The phlD gene, involved in the production of the antibiotic 2,4-diacetyl-phloroglucinol, was found only among Pseudomonas isolates from the rhizosphere and endorhiza. Evaluation of the bacterial isolates for biocontrol potential based on fungal antagonism and physiological characteristics resulted in the selection of five promising isolates from each microenvironment. The most effective isolate was Serratia plymuthica 3Re4-18 isolated from the endorhiza.     

Bacterial Antagonists to Verticillium dahliae Kleb.

Bacteria were isolated from the rhizosphere of Verticillium dahliae hosts and from environments. A total of 1394 bacterial isolates were screened for their ability to inhibit in vitro the growth of the phytopathogenic fungi V. dahliae ; 15% (203 of the isolates) showed antifungal effects. Seventeen isolates were selected and determined for further investigations, seven different species were identified. Several of the bacterial species listed, e.g. Erwinia herbicola, Pseudomonas chlororaphis, Pseudomonas paucimobilis and Xanthomonas maltophilia have not been reported previously as bacterial antagonists of V. dahliae. Bacillus subtilis, Pseudomonas fluorescens and Xanthomonas maltophilia are strong antagonists. We proved that lytic enzymes and siderophores are involved in the inhibition of growth. Ultrastructural and morphological changes were induced in Verticillium dahliae by the antagonistic bacteria.     

Colonization and persistence of a plant growth-promoting bacterium Pseudomonas fluorescens strain CS85, on roots of cotton seedlings

Pseudomonas fluorescens CS85, which was previously isolated from the rhizosphere of cotton seedlings, acts as both a plant growth-promoting bacterium and a biocontrol agent against cotton pathogens, including Rhizoctonia solani, Colletotrichum gossypii, Fusarium oxysporum f sp. vasinfectum, and Verticillium dahliae. Strain CS85 was labeled separately with luxAB and gusA. The labeled strains were stably maintained and had high levels of expression of the marker genes, luxAB and gusA, after successive transfers on nonselective medium, long-term preservation, and after recovery from soil. The labeled strains displayed similar biocontrol characteristics (e.g., antibiosis, effects of growth-promotion and disease-control) to the original strain. The labeled strains colonized all surfaces of the young plant root zones, such as roots hairs and lateral roots, although the distribution of the labeled strains on the root surfaces was not uniform. Moreover, the population densities of the labeled strains on the root surface were stably maintained at high levels during the first 2 weeks of plant growth in the native soil, so that about 10(7)-10(8) CFU/g root were detected, then decreased gradually. Nevertheless, approximately 10(6) CFU/g root of the labeled strains were observed on the root surfaces 35 d after planting.     

A screening strategy of fungal biocontrol agents towards Verticillium wilt of cotton

Three hundred and seventy-three fungal isolates were obtained from the endorhiza, rhizosphere, and bulk soil of field-grown cotton plants. One hundred and five of them produced obvious inhibition zones against Verticillium dahliae Kleb., so they were selected as antagonists towards this pathogen. An assessment system was established to evaluate these 105 antagonists for their biocontrol potential and plant growth-promoting potential. Their biocontrol potential was assessed according to their in vitro antagonistic activity against V. dahliae and activities of fungal cell wall degrading enzymes including protease, cellulase, and chitinase. Their plant growth-promoting potential was assessed according to their in vitro activities of solubilizing phosphate and fixing nitrogen. Thirty-three antagonists received at least three points of the total value of assessed biocontrol potential and plant growth-promoting potential and were tested for their biocontrol efficacy and growth-promoting effect on cotton under greenhouse conditions. Twelve of them achieved positive biocontrol efficacy ranging from 8.58% to 69.78%; the conventional correlation coefficient of the biocontrol efficacy of these antagonists with their assessed biocontrol potential was 0.926. By using the screening strategy developed in this study, Fusarium oxysporum strain By125, Nectria haematococca Bx247, and Phomopsis sp. By231 were identified as potential BCAs for controlling Verticillium wilt in cotton, for they achieved biocontrol efficacy of 63.63–69.78% towards this disease and increased biomass by 18.54–62.63% under greenhouse conditions. The present study also demonstrated that the endorhiza of field-grown cotton plants may be a richer source of potential BCAs against Verticillium wilt than the rhizosphere and bulk soil.     

Biocontrol treatments confer protection against Verticillium dahliae infection of potato by inducing antimicrobial metabolites

Verticillium wilt, caused by Verticillium dahliae Kleb., is a serious potato (Solanum tuberosum L.) disease worldwide, and biocontrol represents a promising eco-friendly strategy to reduce its impact. We used extracts from Canada milk vetch (CMV) and a set of four V. dahliae-antagonistic bacterial strains to coat potato seeds at planting and examined the degree of protection provided against V. dahliae as well as accumulation of soluble phenolics as markers for induced resistance. All tested treatments were effective in reducing disease severity, and CMV showed the highest level of protection. In this treatment, flavonol-glycoside rutin was a highly abundant compound induced in potato tissues, with levels two to three times higher than those detected in noninoculated controls and V. dahliae-inoculated plants. We investigated dose-dependent effects of rutin on V. dahliae growth and sporulation in vitro and in planta. The effect of rutin on mycelial growth was inconsistent between disk assay and amended medium experiments. On the other hand, significant reduction of V. dahliae sporulation in vitro was consistently observed starting at 300 and 100 μM for isolates Vd-9 and Vd-21, respectively. We successfully detected 2-protocatechuoylphloroglucinolcarboxylic acid (2-PCPGCA) using ultra-performance liquid chromatography tandem mass spectrometry, indicating that V. dahliae dioxygenally oxidizes quercetin. Quercetin, as an aglycone, is freed from the sugar moiety by glucosidases and rhamnosidases produced by the fungus and is a substrate for quercetinases. The occurrence of quercetinases in V. dahliae provides a background to formulate a hypothesis about how by-product 2-PCPGCA may be interfering with potato defenses.     

Bulk and Rhizosphere Soil Bacterial Communities Studied by Denaturing Gradient Gel Electrophoresis: Plant-Dependent Enrichment and Seasonal Shifts Revealed

The bacterial rhizosphere communities of three host plants of the pathogenic fungus Verticillium dahliae, field-grown strawberry (Fragaria ananassa Duch.), oilseed rape (Brassica napus L.), and potato (Solanum tuberosum L.), were analyzed. We aimed to determine the degree to which the rhizosphere effect is plant dependent and whether this effect would be increased by growing the same crops in two consecutive years. Rhizosphere or soil samples were taken five times over the vegetation periods. To allow a cultivation-independent analysis, total community DNA was extracted from the microbial pellet recovered from root or soil samples. 16S rDNA fragments amplified by PCR from soil or rhizosphere bacterium DNA were analyzed by denaturing gradient gel electrophoresis (DGGE). The DGGE fingerprints showed plant-dependent shifts in the relative abundance of bacterial populations in the rhizosphere which became more pronounced in the second year. DGGE patterns of oilseed rape and potato rhizosphere communities were more similar to each other than to the strawberry patterns. In both years seasonal shifts in the abundance and composition of the bacterial rhizosphere populations were observed. Independent of the plant species, the patterns of the first sampling times for both years were characterized by the absence of some of the bands which became dominant at the following sampling times. Bacillus megaterium and Arthrobacter sp. were found as predominant populations in bulk soils. Sequencing of dominant bands excised from the rhizosphere patterns revealed that 6 out of 10 bands resembled gram-positive bacteria. Nocardia populations were identified as strawberry-specific bands.     

Establishment of introduced antagonistic bacteria in the rhizosphere of transgenic potatoes and their effect on the bacterial community

In a field release experiment, rifampicin resistant mutants of two antagonistic plant-associated bacteria were used for seed tuber inoculation of transgenic T4 lysozyme expressing potatoes, transgenic control potatoes and non-transgenic parental potatoes. The T4 lysozyme tolerant Pseudomonas putida QC14-3-8 was originally isolated from the tuber surface (geocaulosphere) of T4 lysozyme producing plants and showed in vitro antibacterial activity to the bacterial pathogen Erwinia carotovora ssp. atroseptica. The T4 lysozyme sensitive Serratia grimesii L16-3-3 was originally isolated from the rhizosphere of parental potatoes and showed in vitro antagonism toward the plant pathogenic fungus Verticillium dahliae. The establishment of the inoculated bacteria in the rhizosphere and geocaulosphere of the different plant lines was monitored over one growing season to assess the effect of T4 lysozyme produced by transgenic potato plants on the survival of both inoculants. Both introduced isolates were able to colonize the rhizo- and geocaulosphere of transgenic plants and non-transgenic parental plants, and established in the rhizosphere at levels of ca. log(10) 5 colony forming units g(-1) fresh weight of root. During flowering of plants, significantly more colony counts of the T4 lysozyme tolerant P. putida were recovered from transgenic T4 lysozyme plants than from the transgenic control and the parental line. At this time, the highest level of T4 lysozyme (% of total soluble protein) was detected. Effects of the inoculants on the indigenous microbial community were monitored by analysis of PCR-amplified fragments of the 16S rRNA genes of the whole bacterial community after separation by denaturing gradient gel electrophoresis (DGGE). At any sampling time, the DGGE pattern of rhizosphere and geocaulosphere communities did not show differences between the inoculated and non-inoculated potatoes. Neither of the introduced strains became a dominant member of the bacterial community. This work was the first approach to assess the establishment of plant growth promoting rhizobacteria and potential biocontrol agents on transgenic plants.     

Quorum-sensing signaling is required for production of the antibiotic pyrrolnitrin in a rhizospheric biocontrol strain of Serratia plymuthica

One mechanism that bacteria have adopted to regulate the production of antimicrobial compounds is population-density-dependent LuxRI-type quorum sensing (QS), exploiting the production of N-acyl homoserine lactone (AHL) autoinducer signals. In biocontrol bacteria, most known cases involve the AHL control of phenazine antibiotics production by rhizospheric pseudomonads. This work is the first to demonstrate that phenazines are not the only group of biocontrol-related antibiotics whose production is regulated by QS systems. Strain HRO-C48 of Serratia plymuthica isolated from the rhizosphere of oilseed rape and described as a chitinolytic bacterium, which protects crops against Verticillium wilt, was also shown to produce wide-range antibiotic pyrrolnitrin and several AHLs, including N-butanoyl-HSL, N-hexanoyl-HSL and N-3-oxo-hexanoyl-HSL (OHHL). The genes splI and splR, which are analogues of luxI and luxR genes from other Gram-negative bacteria, were cloned and sequenced. The mutant AHL-4 (splI::miniTn5) was simultaneously deficient in the production of AHLs and pyrrolnitrin, as well as in its ability to suppress the growth of several fungal plant pathogens in vitro. However, pyrrolnitrin production could be restored in this mutant by introduction of the splIR genes cloned into a plasmid or by addition of the conditioned medium from strain C48 or OHHL standard to the growth medium.     

A study on microbial diversity in different cultivars of Brassica napus in relation to its wilt pathogen,Verticillium longisporum

Oilseed rape (Brassica napus) is one of the major oilseed crops in the world but is vulnerable to attack by many pathogens and insect pests. In addition to the host plant genotype, micro-organisms present in the rhizosphere and within plant tissues affect the susceptibility to plant pathogens. While rapid progress has been achieved concerning the concept of plant resistance genes, information on the role of the microbial community in plant protection is less apparent. We have studied the endophytic bacterial populations present in different tissues of oilseed rape and also analysed several cultivars (Express, Libraska, Maluka and Westar), which differ in their susceptibility to the wilt pathogen Verticillium longisporum. The population diversity was studied using agar plating assay, fatty acid methyl ester analysis and functional characterisation of isolated strains. Our work shows that already in the seeds there exists diversity in populations as well as in the total microbial load between two of the four tested cultivars. About 50% of the strains isolated from cultivars Express and Libraska showed moderate to strong direct inhibition of V. longisporum. The diversity of the endophytic flora isolated from oilseed rape and its implications in crop protection are discussed.