Differential Response of Wheat Cultivars to Pseudomonas brassicacearum and Take-All Decline Soil

2,4-Diacetylphloroglucinol (DAPG)-producing Pseudomonas spp. in the P. fluorescens complex are primarily responsible for a natural suppression of take-all of wheat known as take-all decline (TAD) in many fields in the United States. P. brassicacearum, the most common DAPG producer found in TAD soils in the Pacific Northwest (PNW) of the United States, has biological control, growth promoting and phytotoxic activities. In this study, we explored how the wheat cultivar affects the level of take-all suppression when grown in a TAD soil, and how cultivars respond to colonization by P. brassicacearum. Three cultivars (Tara, Finley, and Buchanan) supported similar rhizosphere population sizes of P. brassicacearum when grown in a TAD soil, however they developed significantly different amounts of take-all. Cultivars Tara and Buchanan developed the least and most take-all, respectively, and Finley showed an intermediate amount of disease. However, when grown in TAD soil that was pasteurized to eliminate both DAPG producers and take-all suppression, all three cultivars were equally susceptible to take-all. The three cultivars also responded differently to the colonization and phytotoxicity of P. brassicacearum strains Q8r1-96 and L5.1-96, which are characteristic of DAPG producers in PNW TAD soils. Compared with cultivar Tara, cultivar Buchanan showed significantly reduced seedling emergence and root growth when colonized by P. brassicacearum, and the response of Finley was intermediate. However, all cultivars emerged equally when treated with a DAPG-deficient mutant of Q8r1-96. Our results indicate that wheat cultivars grown in a TAD soil modulate both the robustness of take-all suppression and the potential phytotoxicity of the antibiotic DAPG.

Rhizosphere Competence of Wild-Type and Genetically Engineered Pseudomonas brassicacearum Is Affected by the Crop Species

2,4-Diacetylphloroglucinol (2,4-DAPG)-producing Pseudomonas brassicacearum Q8r1-96 is a highly effective biocontrol agent of take-all disease of wheat. Strain Z30-97, a recombinant derivative of Q8r1-96 containing the phzABCDEFG operon from P. synxantha (formerly P. fluorescens) 2-79 inserted into its chromosome, also produces phenazine-1-carboxylic acid. Rhizosphere population sizes of Q8r1-96, Z30-97, and 2-79, introduced into the soil, were assayed during successive growth cycles of barley, navy bean, or pea under controlled conditions as a measure of the impact of crop species on rhizosphere colonization of each strain. In the barley rhizosphere, Z30-96 colonized less that Q8r1-96 when they were introduced separately, and Q8r1-96 out-competed Z30-96 when the strains were introduced together. In the navy bean rhizosphere, Q8r1-96 colonized better than Z30-97 when the strains were introduced separately. However, both strains had similar population densities when introduced together. Strain Q8r1-96 and Z30-97 colonized the pea rhizosphere equally well when each strain was introduced separately, but Z30-97 out-competed Q8r1-96 when they were introduced together. To our knowledge, this is the first report of a recombinant biocontrol strain of Pseudomonas spp. gaining rhizosphere competitiveness on a crop species. When assessing the potential fate of and risk posed by a recombinant Pseudomonas sp. in soil, both the identity of the introduced genes and the crop species colonized by the recombinant strain need to be considered.

Colonization of Pythium oligandrum in the Tomato Rhizosphere for Biological Control of Bacterial Wilt Disease Analyzed by Real-Time PCR and Confocal Laser-Scanning Microscopy

It recently has been reported that the non-plant-pathogenic oomycete Pythium oligandrum suppresses bacterial wilt caused by Ralstonia solanacearum in tomato. As one approach to determine disease-suppressive mechanisms of action, we analyzed the colonization of P. oligandrum in rhizospheres of tomato using real-time polymerase chain reaction (PCR) and confocal laser-scanning microscopy. The real-time PCR specifically quantified P. oligandrum in the tomato rhizosphere that is reliable over a range of 0.1 pg to 1 ng of P. oligandrum DNA from 25 mg dry weight of soil. Rhizosphere populations of P. oligandrum from tomato grown for 3 weeks in both unsterilized and sterilized field soils similarly increased with the initial application of at least 5 × 105 oospores per plant. Confocal microscopic observation also showed that hyphal development was frequent on the root surface and some hyphae penetrated into root epidermis. However, rhizosphere population dynamics after transplanting into sterilized soil showed that the P. oligandrum population decreased with time after transplanting, particularly at the root tips, indicating that this biocontrol fungus is rhizosphere competent but does not actively spread along roots. Protection over the long term from root-infecting pathogens does not seem to involve direct competition. However, sparse rhizosphere colonization of P. oligandrum reduced the bacterial wilt as well as more extensive colonization, which did not reduce the rhizosphere population of R. solanacearum. These results suggest that competition for infection sites and nutrients in rhizosphere is not the primary biocontrol mechanism of bacterial wilt by P. oligandrum.

Host Crop Affects Rhizosphere Colonization and Competitiveness of 2,4-Diacetylphloroglucinol-Producing Pseudomonas fluorescens

Strains of Pseudomonas fluorescens producing the antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG) are biocontrol agents which play a key role in the suppressiveness of some soils against soilborne pathogens. We evaluated the effect of the host plant genotype on rhizosphere colonization by both indigenous and introduced 2,4-DAPG-producing P. fluorescens. First, population densities of indigenous 2,4-DAPG producers in the rhizospheres of alfalfa, barley, bean, flax, lentil, lupine, oat, pea, and wheat grown in a Fusarium wilt-suppressive Puget silt loam were determined. Population densities differed among the various crops and among pea cultivars, with lentil and oat supporting the highest and lowest densities of 2,4-DAPG producers, respectively. Second, to determine the interactions among 2,4-DAPG producers in the rhizosphere, a Shano sandy loam was inoculated individually and with all possible combinations of P. fluorescens Q8r1-96 (genotype D), F113 (genotype K), and MVP1-4 (genotype P) and sown to wheat or pea, and the rhizosphere population dynamics of each strain was monitored. All three strains were similar in ability to colonize the rhizosphere of wheat and pea when introduced alone into the soil; however, when introduced together in equal densities, the outcome of the interactions differed according to the host crop. In the wheat rhizosphere, the population density of strain F113 was significantly greater than that of Q8r1-96 in the mixed inoculation studies, but no significant differences were observed on pea. The population density of strain Q8r1-96 was greater than that of MVP1-4 in the mixed inoculation on wheat, but the opposite occurred on pea. In the wheat rhizosphere, the population of MVP1-4 dropped below the detection limit (log 3.26 CFU g-1 of root) in the presence of F113; however, on pea, the population density of MVP1-4 was higher than that of F113. When all three strains were present together, F113 had the greatest density in the wheat rhizosphere, but MVP1-4 was dominant in the pea rhizosphere. Finally, eight pea cultivars were grown in soil inoculated with either MVP1-4 or Q8r1-96. The effect of the pea cultivar on rhizosphere colonization was dependent on the bacterial strain inoculated. Rhizosphere population densities of MVP1-4 did not differ significantly among pea cultivars, whereas population densities of Q8r1-96 did. We conclude from these studies that the host crop plays a key role in modulating both rhizosphere colonization by 2,4-DAPG-producing P. fluorescens and the interactions among different genotypes present in the same rhizosphere.

Interactions Between Strains of 2,4-Diacetylphloroglucinol-Producing Pseudomonas fluorescens in the Rhizosphere of Wheat

Strains of fluorescent Pseudomonas spp. that produce the antibiotic 2,4-diacetylphoroglucinol (2,4-DAPG) are among the most effective rhizobacteria controlling diseases caused by soilborne pathogens. The genotypic diversity that exists among 2,4-DAPG producers can be exploited to improve rhizosphere competence and biocontrol activity. Knowing that D-genotype 2,4-DAPG-producing strains are enriched in some take-all decline soils and that P. fluorescens Q8r1-96, a representative D-genotype strain, as defined by whole-cell repetitive sequence-based polymerase chain reaction (rep-PCR) with the BOXA1R primer, is a superior colonizer of wheat roots, we analyzed whether the exceptional rhizosphere competence of strain Q8r1-96 on wheat is characteristic of other D-genotype isolates. The rhizosphere population densities of four D-genotype strains and a K-genotype strain introduced individually into the soil were significantly greater than the densities of four strains belonging to other genotypes (A, B, and L) and remained above log 6.8 CFU/g of root over a 30-week cycling experiment in which wheat was grown for 10 successive cycles of 3 weeks each. We also explored the competitive interactions between strains of different genotypes inhabiting the same soil or rhizosphere when coinoculated into the soil. Strain Q8r1-96 became dominant in the rhizosphere and in nonrhizosphere soil during a 15-week cycling experiment when mixed in a 1:1 ratio with either strain Pf-5 (A genotype), Q2-87 (B genotype), or 1M1-96 (L genotype). Furthermore, the use of the de Wit replacement series demonstrated a competitive disadvantage for strain Q2-87 or strong antagonism by strain Q8r1-96 against Q2-87 in the wheat rhizosphere. Amplified rDNA restriction analysis and sequence analysis of 16S rDNA showed that species of Arthrobacter, Chryseobacterium, Flavobacterium, Massilia, Microbacterium, and Ralstonia also were enriched in culturable populations from the rhizosphere of wheat at the end of a 30-week cycling experiment in the presence of 2,4-DAPG producers. Identifying the interactions among 2,4-DAPG producers and with other indigenous bacteria in the wheat rhizosphere will help to elucidate the variability in biocontrol efficacy of introduced 2,4-DAPG producers and fluctuations in the robustness of take-all suppressive soils.

Rhizobium population dynamics in the pea rhizosphere of rhizobial inoculant strain applied in different formulations

The effect of inoculant formulation on the population dynamics of rhizobia in the pea rhizosphere was investigated using a streptomycin-resistant mutant of Rhizobium leguminosarum bv. viceae NITRAGIN128C56G (128C56G strR). The isolate was formulated into liquid, peat powder, and granular peat carriers, and was tested on pea at field sites near Saskatoon, Saskatchewan, and Beaverlodge, Alberta, in 1996 and 1997. The liquid and peat powder formulations were applied to seed while the granular inoculant was applied to soil. In three out of four site years, population dynamics were similar among formulations: an initial decline or lag period lasting 2–5 days followed by an increase to approximately 105 colony-forming units (CFU)/seedling by 14–28 days after planting (DAP) and, where sampled, a continuing increase from 107 to 108 CFU/plant at 63 DAP. In these same site years, nodule number (not determined at Beaverlodge in 1997) and nodule occupancy at 60 days were not significantly different among formulations. In contrast, soil populations of 128C56G strR from the liquid formulation declined to near zero by 28 DAP at Beaverlodge in 1996, when soil moisture was excessive in spring because of high rainfall. Populations increased in this treatment after this time, but remained significantly lower than the populations of the other two formulations throughout the sampling period. Pea seed yields were not significantly different among treatments in either year at Beaverlodge, but were significantly higher with granular inoculant than the noninoculated control in Saskatoon. Within inoculated treatments at Saskatoon, there were no significant differences in grain yield.Key words: Rhizobium leguminosarum, rhizosphere, population dynamics.

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

ABSTRACT 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)-producingPseudomonas 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 Population Density of Pseudomonas fluorescens on Production of 2,4-Diacetylphloroglucinol in the Rhizosphere of Wheat

The role of antibiotics in biological control of soilborne pathogens, and more generally in microbial antagonism in natural disease-suppressive soils, often has been questioned because of the indirect nature of the supporting evidence. In this study, a protocol for high pressure liquid chromatography/mass spectrometry is described that allowed specific identification and quantitation of the antibiotic 2,4-diacetylphloroglucinol (Phl) produced by naturally occurring fluorescent Pseudomonas spp. on roots of wheat grown in a soil suppressive to take-all of wheat. These results provide, for the first time, biochemical support for the conclusion of previous work that Phl-producing fluorescent Pseudomonas spp. are key components of the natural biological control that operates in take-all—suppressive soils in Washington State. This study also demonstrates that the total amount of Phl produced on roots of wheat by P. fluorescens strain Q2-87, at densities ranging from approximately 105 to 107 CFU/g of root, is proportional to its rhizosphere population density and that Phl production per population unit is a constant (0.62 ng/105 CFU). Thus, Phl production in the rhizosphere of wheat is strongly related to the ability of the introduced strain to colonize the roots.