Genotypic and phenotypic diversity of phlD-containing Pseudomonas strains isolated from the rhizosphere of wheat

Production of 2,4-diacetylphloroglucinol (2,4-DAPG) in the rhizosphere by strains of fluorescent Pseudomonas spp. results in the suppression of root diseases caused by certain fungal plant pathogens. In this study, fluorescent Pseudomonas strains containing phlD, which is directly involved in the biosynthesis of 2,4-DAPG, were isolated from the rhizosphere of wheat grown in soils from wheat-growing regions of the United States and The Netherlands. To assess the genotypic and phenotypic diversity present in this collection, 138 isolates were compared to 4 previously described 2, 4-DAPG producers. Thirteen distinct genotypes, one of which represented over 30% of the isolates, were differentiated by whole-cell BOX-PCR. Representatives of this group were isolated from eight different soils taken from four different geographic locations. ERIC-PCR gave similar results overall, differentiating 15 distinct genotypes among all of the isolates. In most cases, a single genotype predominated among isolates obtained from each soil. Thirty isolates, representing all of the distinct genotypes and geographic locations, were further characterized. Restriction analysis of amplified 16S rRNA gene sequences revealed only three distinct phylogenetic groups, one of which accounted for 87% of the isolates. Phenotypic analyses based on carbon source utilization profiles revealed that all of the strains utilized 49 substrates and were unable to grow on 12 others. Individually, strains could utilize about two-thirds of the 95 substrates present in Biolog SF-N plates. Multivariate analyses of utilization profiles revealed phenotypic groupings consistent with those defined by the genotypic analyses.     

Differential Ability of Genotypes of 2,4-Diacetylphloroglucinol-Producing Pseudomonas fluorescens Strains To Colonize the Roots of Pea Plants

Indigenous populations of 2,4-diacetylphloroglucinol (2,4-DAPG)-producing fluorescent Pseudomonas spp. that occur naturally in suppressive soils are an enormous resource for improving biological control of plant diseases. Over 300 isolates of 2,4-DAPG-producing fluorescent Pseudomonas spp. were isolated from the rhizosphere of pea plants grown in soils that had undergone pea or wheat monoculture and were suppressive to Fusarium wilt or take-all, respectively. Representatives of seven genotypes, A, D, E, L, O, P, and Q, were isolated from both soils and identified by whole-cell repetitive sequence-based PCR (rep-PCR) with the BOXA1R primer, increasing by three (O, P, and Q) the number of genotypes identified previously among a worldwide collection of 2,4-DAPG producers. Fourteen isolates representing eight different genotypes were tested for their ability to colonize the rhizosphere of pea plants. Population densities of strains belonging to genotypes D and P were significantly greater than the densities of other genotypes and remained above log 6.0 CFU (g of root)⁻¹ over the entire 15-week experiment. Genetic profiles generated by rep-PCR or restriction fragment length polymorphism analysis of the 2,4-DAPG biosynthetic gene phlD were predictive of the rhizosphere competence of the introduced 2,4-DAPG-producing strains.     

Differential ability of genotypes of 2,4-diacetylphloroglucinol-producing Pseudomonas fluorescens strains to colonize the roots of pea plants

Indigenous populations of 2,4-diacetylphloroglucinol (2,4-DAPG)-producing fluorescent Pseudomonas spp. that occur naturally in suppressive soils are an enormous resource for improving biological control of plant diseases. Over 300 isolates of 2,4-DAPG-producing fluorescent Pseudomonas spp. were isolated from the rhizosphere of pea plants grown in soils that had undergone pea or wheat monoculture and were suppressive to Fusarium wilt or take-all, respectively. Representatives of seven genotypes, A, D, E, L, O, P, and Q, were isolated from both soils and identified by whole-cell repetitive sequence-based PCR (rep-PCR) with the BOXA1R primer, increasing by three (O, P, and Q) the number of genotypes identified previously among a worldwide collection of 2,4-DAPG producers. Fourteen isolates representing eight different genotypes were tested for their ability to colonize the rhizosphere of pea plants. Population densities of strains belonging to genotypes D and P were significantly greater than the densities of other genotypes and remained above log 6.0 CFU (g of root)(-1) over the entire 15-week experiment. Genetic profiles generated by rep-PCR or restriction fragment length polymorphism analysis of the 2,4-DAPG biosynthetic gene phlD were predictive of the rhizosphere competence of the introduced 2,4-DAPG-producing strains.     

Wheat Cultivar-Specific Selection of 2,4-Diacetylphloroglucinol-Producing Fluorescent <Emphasis Type="Italic">Pseudomonas</Emphasis> Species from Resident Soil Populations

An emerging body of evidence indicates a role for plant genotype as a determinant of the species and genetic composition of the saprophytic microbial community resident to the rhizosphere. In this study, experiments were conducted to determine the capacity of five different wheat cultivars to enhance resident populations and support introduced strains of 2,4-diacetylphloroglucinol (2,4-DAPG)-producing fluorescent pseudomonads, a group of bacteria known to provide biological control of several soilborne diseases. When soils were cropped with three successive 28-day growth cycles of wheat, the 2,4-DAPG-producing strains were consistently recovered from the rhizosphere of the cultivar Lewjain, and commonly were present at populations higher than those recovered from other wheat cultivars. Based on restriction fragment length polymorphism and sequence analyses of phlD, a key gene involved in 2,4-DAPG production, two previously undefined phlD+ genotypes, referred to as genotypes PfZ and PfY, were discovered. Wheat cultivar Lewjain was the primary source of genotype PfY while cultivar Penawawa yielded the majority of genotype PfZ. Based on 16S rDNA sequence analysis, both new phlD genotypes were classified as P. fluorescens. Comparison of the rhizosphere competence of 2,4-DAPG-producing P. fluorescens Q2-87 (genotype B) and P. fluorescens LR3-A28 (genotype PfY) showed that both strains persisted at similar populations in the rhizosphere of all cultivars tested over a 30 day period when introduced as a seed inoculant. However, when strain LR3-A28 was applied as a soil inoculant, this strain was recovered at higher populations from the rhizosphere of wheat cultivar Lewjain than from the rhizospheres of two other cultivars. No cultivar effects were shown for strain Q2-87. Collectively, these results add further to evidence indicating a degree of specificity in interactions between plant cultivars and specific members of the saprophytic microbial community. Furthermore, as 2,4-DAPG-producing fluorescent Pseudomonas spp. have a central role in the spontaneous reduction in severity of take-all disease of wheat in response to continuous wheat monoculture, we postulate that the use of specific cultivars, such as Lewjain, which possess a superior capacity to enhance resident soil populations of these bacteria may have potential to reduce the length of the monoculture period required to induce natural suppressiveness of soils toward this disease.     

Antagonistic activity among 2,4-diacetylphloroglucinol-producing fluorescent Pseudomonas spp

Strains of fluorescent Pseudomonas spp. that produce 2,4-diacetylphloroglucinol (2,4-DAPG) differ in their ability to colonize roots. In this study, we screened 47 2,4-DAPG-producing strains representing17 distinct genotypes for antagonistic activity associated with the production of bacteriocins. Upon induction, over 70% of the strains inhibited the growth of other isolates in vitro. Greenhouse assays indicated that populations of sensitive strains in wheat rhizosphere soil declined more rapidly in the presence of antagonists than when introduced alone. Antagonism can influence the ability of biocontrol agents to establish and maintain effective population densities in situ.     

Enrichment and genotypic diversity of phlD-containing fluorescent Pseudomonas spp. in two soils after a century of wheat and flax monoculture

Fluorescent Pseudomonas spp. producing the antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG) play a key role in the suppressiveness of some soils to take-all of wheat and other diseases caused by soilborne pathogens. Soils from side-by-side fields on the campus of North Dakota State University, Fargo, USA, which have undergone continuous wheat, continuous flax or crop rotation for over 100 years, were assayed for the presence of 2,4-DAPG producers. Flax and wheat monoculture, but not crop rotation, enriched for 2,4-DAPG producers, and population sizes of log 5.0 CFU g root(-1) or higher were detected in the rhizospheres of wheat and flax grown in the two monoculture soils. The composition of the genotypes enriched by the two crops differed. Four BOX-PCR genotypes (D, F, G, and J) and a new genotype (T) were detected among the 2,4-DAPG producers in the continuous flax soil, with F- and J-genotype isolates dominating (41 and 39% of the total, respectively). In contrast, two genotypes (D and I) were detected in the soil with continuous wheat, with D-genotype isolates comprising 77% of the total. In the crop-rotation soil, populations of 2,4-DAPG producers generally were below the detection limit, and only one genotype (J) was detected. Under growth-chamber and field conditions, D and I genotypes (enriched by wheat monoculture) colonized the wheat rhizosphere significantly better than isolates of other genotypes, while a J-genotype isolate colonized wheat and flax rhizospheres to the same extent. This study suggests that, over many years of monoculture, the crop species grown in a field enriches for genotypes of 2,4-DAPG producers from the reservoir of genotypes naturally present in the soil that are especially adapted to colonizing the rhizosphere of the crop grown.     

Assessment of Genotypic Diversity of Antibiotic-Producing Pseudomonas Species in the Rhizosphere by Denaturing Gradient Gel Electrophoresis

The genotypic diversity of antibiotic-producing Pseudomonas spp. provides an enormous resource for identifying strains that are highly rhizosphere competent and superior for biological control of plant diseases. In this study, a simple and rapid method was developed to determine the presence and genotypic diversity of 2,4-diacetylphloroglucinol (DAPG)-producing Pseudomonas strains in rhizosphere samples. Denaturing gradient gel electrophoresis (DGGE) of 350-bp fragments of phlD, a key gene involved in DAPG biosynthesis, allowed discrimination between genotypically different phlD⁺ reference strains and indigenous isolates. DGGE analysis of the phlD fragments provided a level of discrimination between phlD⁺ genotypes that was higher than the level obtained by currently used techniques and enabled detection of specific phlD⁺ genotypes directly in rhizosphere samples with a detection limit of approximately 5 × 10³ CFU/g of root. DGGE also allowed simultaneous detection of multiple phlD⁺ genotypes present in mixtures in rhizosphere samples. DGGE analysis of 184 indigenous phlD⁺ isolates obtained from the rhizospheres of wheat, sugar beet, and potato plants resulted in the identification of seven phlD⁺ genotypes, five of which were not described previously based on sequence and phylogenetic analyses. Subsequent bioassays demonstrated that eight genotypically different phlD⁺ genotypes differed substantially in the ability to colonize the rhizosphere of sugar beet seedlings. Collectively, these results demonstrated that DGGE analysis of the phlD gene allows identification of new genotypic groups of specific antibiotic-producing Pseudomonas with different abilities to colonize the rhizosphere of sugar beet seedlings.     

Role of 2,4-diacetylphloroglucinol-producing fluorescent Pseudomonas spp. in the defense of plant roots

Plants have evolved strategies of stimulating and supporting specific groups of antagonistic microorganisms in the rhizosphere as a defense against diseases caused by soilborne plant pathogens owing to a lack of genetic resistance to some of the most common and widespread soilborne pathogens. Some of the best examples of natural microbial defense of plant roots occur in disease suppressive soils. Soil suppressiveness against many different diseases has been described. Take-all is an important root disease of wheat, and soils become suppressive to take-all when wheat or barley is grown continuously in a field following a disease outbreak; this phenomenon is known as take-all decline (TAD). In Washington State, USA and The Netherlands, TAD results from the enrichment during monoculture of populations of 2,4-diacetylphloroglucinol (2,4-DAPG)-producing Pseudomonas fluorescens to a density of 10 (5) CFU/g of root, the threshold required to suppress the take-all pathogen, Gaeumannomyces graminis var. tritici. 2,4-DAPG-producing P. fluorescens also are enriched by monoculture of other crops such as pea and flax, and evidence is accumulating that 2,4-DAPG producers contribute to the defense of plant roots in many different agroecosystems. At this time, 22 distinct genotypes of 2,4-DAPG producers (designated A - T, PfY and PfZ) have been defined by whole-cell repetitive sequence-based (rep)-PCR analysis, restriction fragment length polymorphism (RFLP) analysis of PHLD, and phylogenetic analysis of PHLD, but the number of genotypes is expected to increase. The genotype of an isolate is predictive of its rhizosphere competence on wheat and pea. Multiple genotypes often occur in a single soil and the crop species grown modulates the outcome of the competition among these genotypes in the rhizosphere. 2,4-DAPG producers are highly effective biocontrol agents against a variety of plant diseases and ideally suited for serving as vectors for expressing other biocontrol traits in the rhizosphere.     

Genotypic and phenotypic analyses reveal distinct population structures and ecotypes for sugar beet‐associated Pseudomonas in Oxford and Auckland

Fluorescent pseudomonads represent one of the largest groups of bacteria inhabiting the surfaces of plants, but their genetic composition in planta is poorly understood. Here, we examined the population structure and diversity of fluorescent pseudomonads isolated from sugar beet grown at two geographic locations (Oxford, United Kingdom and Auckland, New Zealand). To seek evidence for niche adaptation, bacteria were sampled from three types of leaves (immature, mature, and senescent) and then characterized using a combination of genotypic and phenotypic analysis. We first performed multilocus sequence analysis (MLSA) of three housekeeping genes (gapA, gltA, and acnB) in a total of 152 isolates (96 from Oxford, 56 from Auckland). The concatenated sequences were grouped into 81 sequence types and 22 distinct operational taxonomic units (OTUs). Significant levels of recombination were detected, particularly for the Oxford isolates (rate of recombination to mutation (r/m) = 5.23 for the whole population). Subsequent ancestral analysis performed in STRUCTURE found evidence of six ancestral populations, and their distributions significantly differed between Oxford and Auckland. Next, their ability to grow on 95 carbon sources was assessed using the Biolog? GN2 microtiter plates. A distance matrix was generated from the raw growth data (A₆₆₀) and subjected to multidimensional scaling (MDS) analysis. There was a significant correlation between substrate utilization profiles and MLSA genotypes. Both phenotypic and genotypic analyses indicated presence of a geographic structure for strains from Oxford and Auckland. Significant differences were also detected for MLSA genotypes between strains isolated from immature versus mature/senescent leaves. The fluorescent pseudomonads thus showed an ecotypic population structure, suggestive of adaptation to both geographic conditions and local plant niches.     

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.     

Characterization of native fluorescent Pseudomonas isolates associated with alfalfa roots in Uruguayan agroecosystems

Establishment of alfalfa crops is continuously threatened by seedling diseases caused by soilborne pathogens. The use of plant beneficial bacteria as inoculants is a feasible and environmentally friendly means to control soil pathogens. Identifying effective plant growth-promoting strains to use on local crops under local environmental conditions requires the screening of large collections of native isolates. A collection of 738 rhizospheric fluorescent Pseudomonas isolates was obtained from alfalfa plants from three agroecological regions representative of Uruguayan agricultural systems. The isolates were evaluated for in vitro pathogen inhibition, biosurfactant production, phosphate solubilization and the presence of genes involved in antibiotic synthesis. Isolates with strong in vitro antagonistic activity toward Pythium debaryanum were more abundant in alfalfa plants established in a previously natural ecosystem while biosurfactant producers were less abundant in that location. A subset of isolates was selected for genotypic characterization by rep-PCR using BOX primers. Twenty-four genotypes were defined, sixteen from a single geographical origin and eight composed of isolates from multiple origins. Genotypic profiles correlated well with phenotypic traits. A subset of isolates was assayed to determine their ability to protect alfalfa against P. debaryanum damping-off and to promote plant growth. Five native Pseudomonas isolates showed significant effects on alfalfa by increasing plant biomass and/or protecting from pathogen infection. Plant growth promoting isolates from each location were genotypically similar. Our work contributes to the knowledge of the phenotypic and genotypic diversity of rhizospheric fluorescent pseudomonads of forage legumes and the frequency of plant growth promoting traits associated with this group of bacteria in different agricultural systems.     

Phylogenetic diversity of fluorescent pseudomonads in agricultural soils from Korea

AIMS: To identify and compare the relative diversity and distribution of genotypes of culturable fluorescent pseudomonads from soils. METHODS AND RESULTS: Analysis of 160 isolates from seven soil samples using randomly amplified polymorphism DNA methods revealed 53 genotypes, which were subsequently identified by their 16S ribosomal DNA sequences. Phylogenetic analyses of the 53 genotypes along with 43 fluorescent pseudomonad type strains separated the genotypes into 10 distinct clusters that included two phylogenetic groups that were not represented by previously described type strains. CONCLUSIONS: The diversity of genotypes that was obtained from the soil samples was highly variable among the different soils and appeared to be associated with different soil management practices that also influence plant yields. SIGNIFICANCE AND IMPACT OF THE STUDY: The identification and phylogenetic analysis of these genotypes offers opportunities for study of phenotypic traits that may be associated within taxonomically related groups of fluorescent pseudomonad species and how these groups vary in relation to soil management practices.     

Biogeography and Degree of Endemicity of Fluorescent Pseudomonas Strains in Soil

Fluorescent Pseudomonas strains were isolated from 38 undisturbed pristine soil samples from 10 sites on four continents. A total of 248 isolates were confirmed as Pseudomonas sensu stricto by fluorescent pigment production and group-specific 16S ribosomal DNA (rDNA) primers. These isolates were analyzed by three molecular typing methods with different levels of resolution: 16S rDNA restriction analysis (ARDRA), 16S-23S rDNA intergenic spacer-restriction fragment length polymorphism (ITS-RFLP) analysis, and repetitive extragenic palindromic PCR genomic fingerprinting with a BOX primer set (BOX-PCR). All isolates showed very similar ARDRA patterns, as expected. Some ITS-RFLP types were also found at every geographic scale, although some ITS-RFLP types were unique to the site of origin, indicating weak endemicity at this level of resolution. Using a similarity value of 0.8 or more after cluster analysis of BOX-PCR fingerprinting patterns to define the same genotypes, we identified 85 unique fluorescent Pseudomonas genotypes in our collection. There were no overlapping genotypes between sites as well as continental regions, indicating strict site endemism. The genetic distance between isolates as determined by degree of dissimilarity in BOX-PCR patterns was meaningfully correlated to the geographic distance between the isolates' sites of origin. Also, a significant positive spatial autocorrelation of the distribution of the genotypes was observed among distances of <197 km, and significant negative autocorrelation was observed between regions. Hence, strong endemicity of fluorescent Pseudomonas genotypes was observed, suggesting that these heterotrophic soil bacteria are not globally mixed.     

Conservation of the 2,4-diacetylphloroglucinol biosynthesis locus among fluorescent Pseudomonas strains from diverse geographic locations.

The broad-spectrum antibiotic 2,4-diacetylphloroglucinol (PHL) is a major determinant in the biological control of a range of plant pathogens by many fluorescent Pseudomonas spp. A 4.8-kb chromosomal DNA region from Pseudomonas fluorescens Q2-87, carrying PHL biosynthetic genes, was used as a probe to determine if the PHL biosynthetic locus is conserved within PHL-producing Pseudomonas strains of worldwide origin. The phl gene probe hybridized with the genomic DNA of all 45 PHL-producing Pseudomonas strains tested, including well-characterized biocontrol strains from the United States and Europe and strains isolated from disease-suppressive soils from Switzerland, Washington, Italy, and Ghana. The PHL producers displayed considerable phenotypic and genotypic diversity. Two phenotypically distinct groups were detected. The first produced PHL, pyoluteorin, and hydrogen cyanide and consisted of 13 strains from almost all locations sampled in the United States, Europe, and Africa. The second produced only PHL and HCN and consisted of 32 strains from the U.S. and European soils. Analysis of restriction patterns of genomic DNA obtained after hybridization with the phl gene probe and cluster analysis of restriction patterns of amplified DNA coding for 16S rRNA (ARDRA) and randomly amplified polymorphic DNA (RAPD) markers indicated that the strains that produced both PHL and pyoluteorin were genetically highly similar. In contrast, there was more diversity at the genotypic level in the strains that produced PHL but not pyoluteorin. ARDRA analysis of these strains indicated two clusters which, on the basis of RAPD analysis, split into several subgroups with additional polymorphisms. In general, the occurrence of phenotypically and genotypically similar groups of PHL producers did not correlate with the geographic origin of the isolates, and highly similar strains could be isolated from diverse locations worldwide.     

Pseudomonas prosekii sp. nov., a Novel Psychrotrophic Bacterium from Antarctica

During Czech expeditions at James Ross Island, Antarctica, in the years 2007–2009, the bacterial diversity of the genus Pseudomonas was studied. Twelve fluorescent Pseudomonas strains were isolated from various samples and were subjected to a detailed taxonomic study. A polyphasic approach included genotypic and phenotypic analyses. The genotypic analysis involved sequencing of rrs, rpoB and rpoD genes, DNA–DNA hybridization (DDH) studies as well as manual ribotyping using HindIII endonuclease. The phenotypic characterization included conventional tests as well as biotyping using the Biolog system, protein profiling by SDS-PAGE, and MALDI-TOF MS analysis. Our taxonomic study revealed that all isolates belonged to the same Pseudomonas species with psychrotrophic growth not exceeding 37 °C. The cultures showed a unique position among the phylogenetically related pseudomonads. DDH experiment between the proposed type strain of the antarctic isolates and the closest neighbour P. arsenicoxydans CCM 8423^T showed only 40.9–50.1 % similarity, thus confirming that the characterized strains do not belong to the P. arsenicoxydans species. According to the results obtained we propose the name P. prosekii sp. nov. for this novel Pseudomonas taxon with type strain AN/28/1^T (=CCM 7990^T and LMG 26867^T).     

Assessment of genotypic diversity of antibiotic-producing pseudomonas species in the rhizosphere by denaturing gradient gel electrophoresis

The genotypic diversity of antibiotic-producing Pseudomonas spp. provides an enormous resource for identifying strains that are highly rhizosphere competent and superior for biological control of plant diseases. In this study, a simple and rapid method was developed to determine the presence and genotypic diversity of 2,4-diacetylphloroglucinol (DAPG)-producing Pseudomonas strains in rhizosphere samples. Denaturing gradient gel electrophoresis (DGGE) of 350-bp fragments of phlD, a key gene involved in DAPG biosynthesis, allowed discrimination between genotypically different phlD(+) reference strains and indigenous isolates. DGGE analysis of the phlD fragments provided a level of discrimination between phlD(+) genotypes that was higher than the level obtained by currently used techniques and enabled detection of specific phlD(+) genotypes directly in rhizosphere samples with a detection limit of approximately 5 x 10(3) CFU/g of root. DGGE also allowed simultaneous detection of multiple phlD(+) genotypes present in mixtures in rhizosphere samples. DGGE analysis of 184 indigenous phlD(+) isolates obtained from the rhizospheres of wheat, sugar beet, and potato plants resulted in the identification of seven phlD(+) genotypes, five of which were not described previously based on sequence and phylogenetic analyses. Subsequent bioassays demonstrated that eight genotypically different phlD(+) genotypes differed substantially in the ability to colonize the rhizosphere of sugar beet seedlings. Collectively, these results demonstrated that DGGE analysis of the phlD gene allows identification of new genotypic groups of specific antibiotic-producing Pseudomonas with different abilities to colonize the rhizosphere of sugar beet seedlings.     

Exploiting Genotypic Diversity of 2,4-Diacetylphloroglucinol-Producing Pseudomonas spp.: Characterization of Superior Root-Colonizing P. fluorescens Strain Q8r1-96

The genotypic diversity that occurs in natural populations of antagonistic microorganisms provides an enormous resource for improving biological control of plant diseases. In this study, we determined the diversity of indigenous 2,4-diacetylphloroglucinol (DAPG)-producing Pseudomonas spp. occurring on roots of wheat grown in a soil naturally suppressive to take-all disease of wheat. Among 101 isolates, 16 different groups were identified by random amplified polymorphic DNA (RAPD) analysis. One RAPD group made up 50% of the total population of DAPG-producing Pseudomonas spp. Both short- and long-term studies indicated that this dominant genotype, exemplified by P. fluorescens Q8r1-96, is highly adapted to the wheat rhizosphere. Q8r1-96 requires a much lower dose (only 10 to 100 CFU seed⁻¹ or soil⁻¹) to establish high rhizosphere population densities (10⁷ CFU g of root⁻¹) than Q2-87 and 1M1-96, two genotypically different, DAPG-producing P. fluorescens strains. Q8r1-96 maintained a rhizosphere population density of approximately 10⁵ CFU g of root⁻¹ after eight successive growth cycles of wheat in three different, raw virgin soils, whereas populations of Q2-87 and 1M1-96 dropped relatively quickly after five cycles and were not detectable after seven cycles. In short-term studies, strains Q8r1-96, Q2-87, and 1M1-96 did not differ in their ability to suppress take-all. After eight successive growth cycles, however, Q8r1-96 still provided control of take-all to the same level as obtained in the take-all suppressive soil, whereas Q2-87 and 1M1-96 gave no control anymore. Biochemical analyses indicated that the superior rhizosphere competence of Q8r1-96 is not related to in situ DAPG production levels. We postulate that certain rhizobacterial genotypes have evolved a preference for colonization of specific crops. By exploiting diversity of antagonistic rhizobacteria that share a common trait, biological control can be improved significantly.     

Effect of sorghum seedlings, and previous crop, on soil fluorescent Pseudomonas spp.

Hypotheses in which sorghum seedlings [Sorghum bicolor (L.) Moench] of different genotypes will differentially modify soil microorganisms and will affect subsequent planting of wheat (Triticum aestivum L.) seedlings, were tested. Wheat cultivar Lewjain, and sorghum genotypes Redlan and RTx433, were planted into soils previously planted with wheat or sorghum in growth chamber experiments. Total culturable fungi and oomycetes, and fluorescent Pseudomonas spp. numbers (cfu) were determined. Pseudomonads were screened for hydrogen cyanide (HCN) production, for the presence of the phlD gene for 2,4-diacetylphloroglucinol production (Phl) and for a region of the operon involved in phenazine-1-carboxylic acid (PCA) production. Pasteurized soils were inoculated with rifampicin-marked strains of Pseudomonas fluorescens then planted with Lewjain, Redlan and RTx433 to assess rhizosphere and soil colonization. Effects of plant species, sorghum genotype and previous crop on culturable fungi and oomycetes, and pseudomonad numbers (cfu g^?1 soil) were statistically significant. Soils planted with RTx433 or Lewjain had greater numbers of fungal cfu than soils planted with Redlan. When Lewjain seedlings were grown in soil previously planted with RTx433, there were greater numbers of fungal cfu than when Lewjain was planted into Redlan soil. Wheat planted into wheat soil resulted in statistically significantly fewer numbers of pseudomonads than when planted into sorghum soil. Overall, percentages of HCN-producing pseudomonads increased, especially when wheat seedlings were planted in wheat soil. For most treatments, percent of isolates with Phl declined, except when Redlan was planted into Redlan soil, which resulted in increased Phl isolates. When rifampicin-marked P. fluorescens isolates were applied to pasteurized soil, sorghum seedlings sustained rhizosphere and soil populations similar to those on wheat. Sorghum genotypes may differ in associations with soil microorganisms, suggesting that they may differentially affect numbers of fluorescent pseudomonads in cropping systems.     

Contribution of phlA and some metabolites of fluorescent pseudomonads to antifungal activity

Fluorescent Pseudomonas species are an important group of PGPR that suppress fungal root and seedling disease by production of antifungal metabolites such as 2,4-diacetylphloroglucinol (2,4-DAPG), pyoluteorin, pyrolinitrin, siderophores and HCN. The compound 2,4-DAPG is a major determinant in biocontrol of plant pathogens. A 7.2 kbp chromosomal DNA region, carrying DAPG biosynthetic genes (phlA, phlC, phlB, phlD, phIE and phlF). Detecting the ph1 genes make them an ideal marker gene for 2,4-DAPG-producing fluorescent pseudomonad's. In this study we detected ph1A gene (that convert MAPG to 2,4-DAPG) using PCR assay with primers phlA-1r and phlA- f that enabled amplification of phlA sequences from fluorescent pseudomonad's from ARDRA group 1 and 3. We could detect phlA gene in P. fluorescens strains CHAO, Pf-44, Pf-1, Pf-2, Pf-3, Pf-17, Pf-62 and Pf-64, native isolates of Iran. The efficacy of this method for rapid assay characterizing rhizosphere population of 2,4-DAPG producing bacteria from soil of different area of Iran is in progress. We used a collection of 48 fluorescent pseudomonas strains in vitro, with known biological control activity against some soil born phytopathogenic fungi such as, Macrophomina phaseoli, Rhizoctonia solani Vericillium dahlia, Phytophthora nicotiana, Pythium spp. and Fusarium spp. and the potential to produce known secondary metabolites such as protease. Strains Pf-1, Pf-2, Pf-3, Pf-17, Pf-33 and Pf-44 showed the best antifungal activity against all fungi used in this study. Thirty-eight of 48 strains produced protease. The ability to rapidly characterize populations of 2,4-DAPG producers will greatly enhance our understanding of their role in the suppression of root disease.     

Genotyping antibiotic producing fluorescent pseudomonads to select effective rhizobacteria for the management of major vanilla diseases

The genotypic diversity that occurs in antagonistic microorganisms provides an enormous resource for improving biological control of plant diseases. In this study, the diversity of 2,4-diacetylphloroglucinol (DAPG) and phenazine-1-carboxylic acid (PCA) producingPseudomonas sp. from different vanilla plantations of three different regions were studied. Out of eighty-five isolates of rhizobacteria from native soils of vanilla plantations, forty-four were positive to PCR amplification of a 560 bp fragment, which is specific of the genusPseudomonas. Among the forty-four isolates, twenty-one exhibited more antimicrobial activity against fungal pathogensviz., Fusarium oxysporum f. sp.vanillae (Tucker) Gordon,Phytophthora meadii McRae andColletotrichum vanillae underin vitro conditions. Twenty-one and sevenPseudomonas showed positive amplification for DAPG and phenazine respectively. An analysis of the level of biodiversity with DAPG and PCA producers at the species level was performed by comparing the restriction patterns of the genus specific 16S–23S ribosomal DNAs (rDNAs) amplified by PCR. This allowed us to cluster the isolates into four different amplified rDNA restriction analysis (ARDRA) groups. Eventually, it was found that some of the DAPG and PCA producers, which originated from different locations, fall under the same cluster. Bacterisation of vanilla plants with antibiotic producing fluorescent pseudomonads isolates resulted in a significant reduction of major diseases in vanilla both in glasshouse and field conditions.